Ionizable lipids for nucleic acid delivery

Abstract

To provide ionizable lipids capable of transfecting living cells with a genetic material.SOLUTION: There is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with a structured lipid, a sterol, and a stabilizer. In the formula, R1 contains an amino group. These compounds are particularly useful in T cell transfection, the formulation and in vivo and ex vivo delivery of nucleic acid and protein therapeutics for cancer therapy, and in the preparation of vaccines.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 868,900, filed June 29, 2019, and U.S. Provisional Patent Application No. 63 / 009,042, filed April 13, 2020.

[0002]

[0003] Field

[0004] The disclosed subject matter relates generally to ionizable lipids, and in particular to ionizable lipids capable of transfecting genetic material into living cells. [Background technology]

[0003]

[0006] Related Prior Art

[0007] The number of nucleic acid treatment strategies for disease continues to grow, even for diseases that do not have an underlying genetic cause. Each nucleic acid therapeutic has a different morphology, chemistry, and charge, and typically requires a different delivery modality.

[0004]

[0008] Lipofection has been investigated as a means to alter the genetic properties of cells through lipid-mediated gene delivery since at least 1987. Over the years, such lipids have been improved to suit more conditions. Cationic lipids such as DODAC, DOTMA, DDAB, and DOTAP were used in the 1990s but proved to be too toxic to be considered for clinical applications.

[0005]

[0009] A clinically relevant approach has been the development of ionizable lipids for human use. Examples of ionizable lipids include 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA (see, e.g., U.S. Pat. No. 8,158,601), and 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA). DLin-MC3-DMA or "MC3" is used in the approved drug Onpattro™ patisiran, but has shelf life issues.

[0006]

[0010] There continues to be a need for more options for clinically relevant transfection lipids. Summary of the Invention

[0007]

[0012] According to an embodiment of the present invention there is provided a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka]

[0013] In the formula, p is 0 or 1;

[0014] E 1 -O-δ 1 , -OC(O)O-δ 1 , -OC(O)-δ 1 , -OC(O)N(Q)-δ 1 , -OC(O)S-δ 1 , -C(O)N(Q)-δ 1 , -C(O)O-δ 1 , -N(Q)C(O)-δ 1 , -N(Q)C(O)O-δ 1 , -N(Q)C(O)S-δ 1 , and -N(Q)C(O)N(Q)-δ 1 Q is selected from H, or C 1 ~C 5 is alkyl; 1 is R 1indicates a bond connecting to the group;

[0015] R 1 teeth [ka] is selected from; where

[0016] R 3 and R 4 are each independently C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, and C 2 ~C 6 alkynyl; or R 3 and R 4 together contain oxygen (O) or up to two nitrogens (N), each independently C 1 ~C 6 Alkyl, cyclopropyl, OH, and C 1 ~C 3 may form a 4-6 membered ring optionally substituted with 1-2 substituents selected from an alkoxy group;

[0017] R 5 is C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl, C 3 ~C 6 cycloalkyl, and 2-hydroxyethyl groups;

[0018] R 6 H and C 1 ~C 6 alkyl groups;

[0019] a is 1, 2, 3, 4 or 5;

[0020] b and c are independently 0, 1, or 2;

[0021] c' is 1, 2, 3, 4, or 5;

[0022] d is 1 or 2;

[0023] e is 0, 1, or 2;

[0024] E 2 -OC(O)-δ 2 , -OC(O)O-δ 2 , -OC(O)N(Q)-δ 2 , -O-δ 2 , -OCH 2 CH 2 O-δ 2 , and -OC(O)(CH 2 ) 6 C(O)O-δ 2 Q is selected from H or C 1 ~C 5 is alkyl; 2 is R 2 indicates a bond connecting to the group;

[0025] R 2 teeth [ka] or a group of formula -(CH 2 ) g -[L 3 -(CH 2 )] h -R 9 where

[0026] L 1 and L 2 each independently represents a direct bond, -O-δ 3 , -CH 2 OC(O)-δ 3 , and -CH 2 O-δ 3 δ 3 is R 7 and R 8 indicates a bond connecting to the group;

[0027] R 7 and R 8 are each independently C 4 ~C 10 Alkyl, C 4 ~C 10 Alkenyl or C 4 ~C 10 is alkynyl;

[0028] f is 0, 1, 2, 3, 4, or 5;

[0029] L 3 teeth [ka] Selected from;

[0030] R 9 H and C 4 ~C 8 alkyl groups;

[0031] g is an integer ranging from 1 to 18;

[0032] h is 0, 1, 2 or 3;

[0008]

[0033] According to another embodiment of the present invention there is provided a compound of formula (II), or a pharma- ceutically acceptable salt thereof: [ka]

[0034]

[0035] In the formula, E 1 -OC(O)O-δ 1 , -OC(O)-δ 1 , -OC(O)N(Q)-δ 1 , and -OC(O)S-δ 1 Q is selected from H or C 1 ~C 5 is alkyl; 1 is R 1 indicates a bond connecting to the group;

[0036] R 1 teeth, [ka] Selected from;

[0037] Where:

[0038] R 3 and R 4 are each independently C 1 ~C 6alkyl group; or R 3 and R 4 together contain no more than two nitrogen atoms (N), and C 1 ~C 6 may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups;

[0039] R 5 is C 1 ~C 6 Alkyl, and C 3 ~C 6 cycloalkyl groups;

[0040] R 6 H and C 1 ~C 6 alkyl groups;

[0041] a is 1, 2, 3, or 4;

[0042] b and c are independently 0, 1, or 2;

[0043] c' is 2, 3, or 4;

[0044] d is 2;

[0045] e is 0 or 1;

[0046] E 2 -O-δ 2 , -OC(O)-δ 2 , -OCH 2 CH 2 O-δ 2 , and -OC(O)(CH 2 ) 6 C(O)O-δ 2 where δ 2 is R 2 indicates a bond connecting to the group;

[0047] R 2 teeth [ka] or a group of formula -(CH 2 ) g -[L 3 -(CH 2 )]h -R 9 where

[0048] L 1 and L 2 each independently represents a direct bond, -O-δ 3 , -CH 2 OC(O)-δ 3 , and -CH 2 O-δ 3 δ 3 is R 7 and R 8 indicates a bond connecting to the group;

[0049] R 7 and R 8 are each independently C 4 ~C 10 Alkyl, C 4 ~C 10 Alkenyl or C 4 ~C 10 is alkynyl,

[0050] f is 0, 1, 2, 3, 4, or 5;

[0051] L 3 teeth [ka] Selected from;

[0052] R 9 H and C 4 ~C 8 alkyl groups;

[0053] g is an integer ranging from 1 to 18;

[0054] h is 0, 1, or 2;

[0009]

[0055] According to another embodiment of the present invention there is provided a compound of formula (II), or a pharma- ceutically acceptable salt thereof: [ka]

[0056] E in the formula 1 -OC(O)O-δ 1 , -OC(O)-δ1 , -OC(O)N(Q)-δ 1 , and -OC(O)S-δ 1 Q is selected from H or C 1 ~C 5 is alkyl; 1 is R 1 represents a bond connected to a group,

[0057] R 1 teeth [ka] is selected from

[0058] where

[0059] R 3 and R 4 are each independently C 1 ~C 6 alkyl group; or R 3 and R 4 together contain no more than two nitrogen atoms (N), and C 1 ~C 6 may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups;

[0060] R 5 is C 1 ~C 6 alkyl, and cyclopropyl groups;

[0061] R 6 H and C 1 ~C 6 alkyl groups;

[0062] a is 1, 2, 3, or 4;

[0063] b is 0 or 1;

[0064] c is 0, 1, or 2;

[0065] c' is 2, 3, or 4;

[0066] d is 2;

[0067] e is 1;

[0068] E 2 -O-δ2 , -OC(O)-δ 2 , -OCH 2 CH 2 O-δ 2 , and -OC(O)(CH 2 ) 6 C(O)O-δ 2 where δ 2 is R 2 represents a bond connected to a group,

[0069] R 2 teeth [ka] or a group of formula -(CH 2 ) g -[L 3 -(CH 2 )] h -R 9 where

[0070] L 1 and L 2 are each direct bonds;

[0071] R 7 and R 8 are each independently C 4 ~C 10 alkyl groups;

[0072] f is 0 or 1;

[0073] L 3 teeth [ka] Selected from;

[0074] R 9 H and C 4 ~C 8 alkyl groups;

[0075] g is an integer ranging from 1 to 18;

[0076] h is 0, 1, or 2.

[0010]

[0077] According to another embodiment of the present invention there is provided a compound of formula (III), or a pharma- ceutically acceptable salt thereof: [ka]

[0078]

[0079] R 1 teeth, [ka] is selected from

[0080] During the ceremony,

[0081] R 3 and R 4 are each independently C 1 ~C 6 alkyl group; or R 3 and R 4 together contain no more than two nitrogen atoms (N), and C 1 ~C 6 may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups;

[0082] R 5 is C 1 ~C 6 alkyl, and cyclopropyl groups;

[0083] R 6 H and C 1 ~C 6 alkyl groups;

[0084] a is 1, 2, 3, or 4;

[0085] b is 0 or 1;

[0086] c is 0, 1, or 2;

[0087] c' is 2, 3, or 4;

[0088] d is 2;

[0089] e is 1;

[0090] E 2 -O-δ 2, -OC(O)-δ 2 , -OCH 2 CH 2 O-δ 2 , and -OC(O)(CH 2 ) 6 C(O)O-δ 2 where δ 2 is R 2 indicates a bond connecting to the group;

[0091] R 2 teeth [ka] or a group of formula -(CH 2 ) g -[L 3 -(CH 2 )] h -R 9 where

[0092] L 1 and L 2 are each direct bonds;

[0093] R 7 and R 8 are each independently C 4 ~C 10 alkyl groups;

[0094] f is 0 or 1;

[0095] L 3 teeth [ka] Selected from;

[0096] R 9 H and C 4 ~C 8 alkyl groups;

[0097] g is an integer ranging from 1 to 18;

[0098] h is 0, 1, or 2.

[0011]

[0099] According to an embodiment of the present invention, R 1 teeth,

[0100] [ka] It is one of the following.

[0012]

[0101] In a further embodiment, each R 2 is independent,

[0102] [ka] It is.

[0013]

[0103] In yet another embodiment of the present invention, 1 teeth, [ka] where δ 1 is R 1 The bond connecting the group is shown.

[0014]

[0104] In yet another embodiment of the present invention, 2 teeth, [ka] where δ 2 is R 2 4 depicts a bond connected to a group or a pharma- ceutically acceptable salt thereof.

[0015]

[0105] According to an embodiment of the present invention, there is provided a compound selected from the group consisting of the compounds set forth in Table 1, or a pharma- ceutically acceptable salt thereof. [Table 1] JPEG0007667327000022.jpg207149 JPEG0007667327000023.jpg207149 JPEG0007667327000024.jpg224149 JPEG0007667327000025.jpg192149

[0016]

[0106] Also provided according to embodiments of the present invention are compounds listed in Table 2. [Table 2] JPEG0007667327000027.jpg216149 JPEG0007667327000028.jpg216149 JPEG0007667327000029.jpg206149 JPEG0007667327000030.jpg225149 JPEG0007667327000031.jpg205149 JPEG0007667327000032.jpg195149 JPEG0007667327000033.jpg225149 JPEG0007667327000034.jpg206149 JPEG0007667327000035.jpg206149 JPEG0007667327000036.jpg206149 JPEG0007667327000037.jpg202149 JPEG0007667327000038.jpg211149 JPEG0007667327000039.jpg217149 JPEG0007667327000040.jpg188149 JPEG0007667327000041.jpg225149 JPEG0007667327000042.jpg215149 JPEG0007667327000043.jpg215149 JPEG0007667327000044.jpg225149 JPEG0007667327000045.jpg221149 JPEG0007667327000046.jpg198149 JPEG0007667327000047.jpg205149 JPEG0007667327000048.jpg91149

[0017]

[0107] The ionizable lipids of the present invention have asymmetric centers and exist as racemates, racemic mixtures, individual enantiomers, mixtures of enantiomers, individual diastereomers, and diastereomeric mixtures along with all possible isomers, such as tautomers, and mixtures thereof.

[0018]

[0108] In an embodiment of the invention, the experimental pKa of formulated lipid nanoparticles containing the lipids in Table 1 is calculated using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. The TNS assay procedure is described in Example 25.

[0019]

[0109] According to an embodiment of the present invention, there is provided a lipid-mix composition comprising any of the above compounds in combination with a structured lipid, a sterol, and a stabilizer, and at least one therapeutic agent.

[0020]

[0110] In embodiments, the structured lipid comprises one or more structured lipids selected from the group consisting of DSPC, DSPE, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the structured lipid is DSPC. In other embodiments, the structured lipid is DOPE.

[0021]

[0111] In embodiments, the stabilizer comprises one or more surfactants and a polymer-conjugated lipid.

[0022]

[0112] In an embodiment, the compound is present at about 10 Mol%-90 Mol%, the structural lipid is present at about 0-50 Mol%, the sterol is present at about 0-45 Mol%, the stabilizer is present at 0-10 Mol%, and the total mol% of all components is 100 mol%.

[0023]

[0113] In a further embodiment, the chemical compound is present at about 40 Mol% to 60 Mol%, the structured lipid is present at about 11 to 40 Mol%, and the total mol% of all components is 100 mol%.

[0024]

[0114] In an embodiment, the molar ratio of the compound to the remaining components is 30 Mol % to 70 Mol %.

[0025]

[0115] In an embodiment, the compound is present at 40 Mol%, DSPC is present at 20 Mol%, cholesterol is present at 37.5 Mol%, and polyoxyethylene (10) stearyl ether is present at 2.5 Mol%. In another embodiment, the compound is present at 40-47.5 Mol%, DSPC is present at 12.5 Mol%, cholesterol is present at 38.5-46 Mol%, and PEG-DMG 2000 is present at 1.5 Mol%. In yet another embodiment, the compound is present at 40-47.5 Mol%, DOPE is present at 12.5 Mol%, cholesterol is present at 38.5-46 Mol%, and PEG-DMG 2000 is present at 1.5 Mol%.

[0026]

[0116] In some embodiments, the lipid-mix composition further comprises a targeting moiety. In embodiments, the sterol is cholesterol. In embodiments, the therapeutic agent comprises one or more nucleic acids. In embodiments, the therapeutic agent comprises a polypeptide.

[0027]

[0117] In embodiments, the stabilizer is selected from the group consisting of PEG-DMG 2000, polyoxyethylene (10) stearyl ether, polyoxyethylene (40) stearate, polysorbate 80, polyoxyethylene (4) lauryl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (23) lauryl ether, and D-α-tocopherol polyethylene glycol 1000 succinate.

[0028]

[0118] According to one embodiment of the present invention there is provided a lipid-mix composition in the form of lipid particles.

[0029]

[0119] Also provided is the use of the compounds of the present invention to prepare a therapeutic agent for administering the therapeutic agent to cells ex vivo. In further embodiments, the therapeutic agent is a pharmaceutical preparation for use in the treatment of cancer. In some embodiments, the therapeutic agent is a pharmaceutical preparation for use in T cell engineering. In yet other embodiments, the therapeutic agent is a vaccine.

[0030]

[0120] In some embodiments, the therapeutic agent comprises a nucleic acid therapeutic. In these embodiments, the nucleic acid therapeutic is an mRNA, an siRNA, an miRNA, a guide RNA, a synthetic guide RNA, an artificial chromosome, a circular or linear DNA, a DNA minicircle, or an msDNA. In some of these embodiments, the mRNA is a self-replicating RNA molecule.

[0031]

[0121] According to an embodiment of the present invention there is provided a use of a lipid mix composition for the preparation of a vaccine. In an embodiment the vaccine relates to the prevention of a viral disease. In an embodiment the vaccine relates to a coronavirus infection.

[0032]

[0122] According to an embodiment of the present invention, there is provided a use of the lipid mix composition as described above for use in therapeutics or cancer vaccines. According to an embodiment of the present invention, there is provided a use of the lipid mix composition as described above for use in in vivo or ex vivo protein modulation.

[0033]

[0123] In embodiments, the mRNA is a self-replicating RNA molecule.

[0034]

[0124] According to an embodiment of the present invention, there is provided a use of the lipid mix composition described above in the preparation of a medicament for modulating human T cells, CAR-T, TCR, gene editing, or allogeneic T cells.

[0035]

[0125] Further provided is an embodiment of the use of the lipid mix composition of the present invention in the preparation of a medicament for modulating T cells, wherein the T cells are isolated from a patient or are T cells that have been specifically genetically engineered to be T cells or allogeneic T cells.

[0036]

[0126] In an embodiment, the lipid-mix composition further comprises a polypeptide. In an embodiment, the lipid-mix composition further comprises both a polypeptide and a nucleic acid.

[0037]

[0127] In an embodiment, the lipid mix composition further comprises a ribonucleoprotein.

[0038]

[0128] According to the present invention, there is provided a compound in which one of the hydrogens is replaced with a halogen. In an embodiment, the halogen is iodine or fluorine.

[0039]

[0129] According to an embodiment of the present invention, there is provided the above compound, or a pharma- ceutically acceptable salt thereof, wherein the experimental pKa of the nanoparticles is in the range of 5.6 to 7.1.

[0040]

[0130] According to an embodiment of the present invention, there is provided a pharmaceutical composition comprising the above compound and at least one pharma- ceutically acceptable carrier or excipient.

[0041]

[0131] Further features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0042] [Figure 1] 1 is a bar graph showing relative GFP expression levels in live pan-T cells mediated by mRNA lipid nanoparticles (LNPs) containing DLin-MC3-DMA, PNI76 or PNI121 in a CT10 composition with an N / P ratio of 10, where gene expression was analyzed by flow cytometry 48 hours after treatment. [Diagram 2] Graph showing percent GFP expression 48 h after treatment in isolated primary human T cells mediated by mRNA-LNPs containing lipid MC3 or PNI lipid 121 in a CT10 composition with an N / P ratio of 10. The X-axis represents six different donors. [Diagram 3] 1 is a bar graph showing quantification of eGFP expression determined by ELISA following treatment of CD4+ / CD8+ T cells with eGFP mRNA LNPs containing DLin-MC3-DMA, PNI 76, PNI 119, PNI 120, PNI 121, and PNI 122 in a CT10 composition with an N / P ratio of 10. [Figure 4] 1 is a bar graph showing GFP expression in previously cryopreserved isolated primary human T cells treated 4 days after activation with 500 ng of encapsulated mRNA per 125,000 cells with mRNA-LNPs containing DLin-MC3-DMA, PNI 127, PNI 328, PNI 329, or PNI 541 in a CT10 composition with an N / P ratio of 8. GFP MFI (first column), transfection efficiency (middle column), and viability (third column) were measured by flow cytometry 48 h after LNP addition. [Diagram 5]Two bar graphs are shown showing GFP expression in isolated primary human T cells mediated by mRNA-LNPs containing the ionizable lipid Dlin-MC3-DMA, or each of several novel ionizable lipids PNI lipids (PNI 321, 325, 328, 329, 336, 534, 535, 540, 541) in a CT10 composition and N / P ratio of 8, 3 days after T cell activation with 500 ng of encapsulated mRNA per 125,000 T cells. Transfection efficiency (top) and MFI (bottom) were measured by flow cytometry 48 h after LNP addition. [Figure 6] 1 is a bar graph showing expression levels of secreted and cytosolic recombinant human erythropoietin (EPO) as determined by ELISA following mRNA LNP-mediated transfection of pan-T cells compared to untreated control ("-LNP"), DLin-MC3-DMA, PNI 76, or PNI 121 in LM02 compositions of N / P10. Controls were normal human serum standards provided by the manufacturer. [Figure 7] 1 shows EPO expression levels in C56BL / 6 mice following iv administration of a 0.5 mg / Kg dose of recombinant human EPO-encoding mRNA LNPs containing the ionizable lipid DLin-MC3-DMA, PNI-76, PNI121, or PNI127 in the LM02 composition of N / P6. [Figure 8] Figure 1 shows EPO expression levels 6h (left) and 24h (right) in C56BL / 6 mice after iv administration of 1 or 3mg / Kg doses of recombinant human EPO-encoding mRNA LNPs containing ionizable lipids DLin-MC3-DMA, PNI 121, or PNI 127 using LM02 composition of N / P6. PBS was used as a negative control. [Figure 9]FIG. 13 is a scatter plot showing EPO expression levels (mIU / mL) 6h after administration of 5-moU EPO mRNA encapsulated in LM02 LNAPs (LNPs) containing 15 different ionizable lipid compounds (PNI 336, PNI 534, PNI 535, PNI 342, PNI 321, PNI 532, PNI 538, PNI 541, PNI 325, PNI 328, PNI 329, PNI 539, PNI 540, PNI 127) at an N / P ratio of 6 and a dose of 0.5mg / Kg. [Figure 10] Figure 1 shows various cytokine levels measured by ELLA™ Simple Plex™ automated ELISA (Protein Simple) in mice 48 h after iv administration of 1 mg / kg (mpk) of mRNA encoding 5moU rhEPO mediated by LNPs of N / P6 LM02 compositions containing ionizable lipids MC3, PNI 121, or PNI 127. PBS was the negative control. [Figure 11] Flow cytometry histograms of CD19 CAR positive T cells (CAR+ right of dotted line) 48 h after treatment with PNI 127, PNI 329, or PNI 328 containing CAR mRNA LNPs (CT10, N / 8, 500 ng encapsulated mRNA per 125,000 treated T cells). Controls were untreated human primary T cells. T cells were activated 3 days prior to treatment. [Figure 12] Scatter plot showing OVA-specific IgG antibody titers in serum from a group of four mice immunized with 5 μg of OVA mRNA LNP vaccine. Two immunizations were performed 10 days apart (day 1, day 10), followed by blood sampling and OVA-specific IgG titer measurement by ELISA two weeks after the second dose. A 50 μg dose of OVA protein was used as a positive control. 5 μg of OVA-encoding mRNA produced a similar IgG response as a 10-fold higher dose of OVA protein. [Figure 13]Scatter plot showing OVA-specific IgG antibody titers in serum from 4 individual mice immunized with 10 μg PBS control, 50 ug OVA protein, or 10 μg OVA mRNA LNP vaccine on days 1 and 10. OVA-specific IgG titers were performed by ELISA using blood samples taken 2 weeks after the second dose. 10 μg of OVA-encoding mRNA produced an IgG response equal to or greater than a 5-fold higher dose of OVA protein. [Figure 14] 1 is a graph showing TNS curves illustrating surface pKa measurements of LNPs containing various ionizable lipid compounds PNI 119, PNI 121, PNI 321, PNI 329, PNI 336, PNI 535, PNI 538, and PNI 540 (LM02 composition). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043]

[0146] Throughout the accompanying drawings, like features are designated with like reference numerals.

[0044]

[0147] In this disclosure, the word "comprises" is used in an open-ended sense to mean that the items preceding the word are included but not excluding items not specifically stated. In embodiments that include or may include a stated feature or variable or parameter, it is understood that alternative embodiments may consist of or consist essentially of such feature or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that there is more than one of that element, unless the context clearly requires that there is one, and only one, of that element.

[0045]

[0148] In this disclosure, recitation of numerical ranges by endpoints includes all whole numbers, integers, and all intermediate fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In this disclosure, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing a "compound" includes a mixture of substances.

[0046]

[0149] In this disclosure, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0047]

[0150] "Lipid" refers to a structurally diverse group of organic compounds that can be fatty acid derivatives or sterols, or lipids such as lipidoids (e.g., C12-200) or substances found in polymer-conjugated lipids, and are characterized by being insoluble in water but soluble in many organic solvents.

[0048]

[0151] "Lipid mix composition." A lipid mix composition refers to the type of components, the ratio of components, and the ratio of total components to nucleic acid payload. For example, a lipid mix composition of 40 Mol% ionizable lipids, 20 Mol% structural lipids, 17.5 Mol% sterols, and 2.5 Mol% stabilizer would be a lipid mix composition.

[0049]

[0152] As used herein, "N / P" is the ratio of moles of amine groups of the ionizable lipid to moles of phosphate groups of the nucleic acid. In an embodiment of the invention, the N / P ratio is 4-10, with a most preferred ratio of N / P 4-12. In one embodiment, the N / P ratio is 10. The nucleic acid component is combined with the lipid mix composition at a preconceived ratio, such as an amine (N) of the ionizable lipid to phosphate (P) of the nucleic acid of N / P 4, N / P 6, N / P 8, N / P 10, N / P 12, or another related specific N / P ratio, to form a lipid-nucleic acid particle, or LNP.

[0050]

[0153] "Lipid Particles". The present invention provides lipid particles made from the lipid mix composition described above. Lipid particles refer to the physical organization of the lipid mix composition and the therapeutic agent and between the components. Lipid nanoparticles are lipid particles. Lipid particles are generally spherical aggregates of lipids, nucleic acids, cholesterol and stabilizers. The positive and negative charges, ratios, and hydrophilic and hydrophobic properties determine the physical structure of the lipid particles in terms of size and orientation of the components. The structural organization of these lipid particles can result in an aqueous interior with minimal bilayers, as in liposomes, or can have a solid interior, as in solid nucleic acid-lipid nanoparticles. There can be single or multiple morphologies of phospholipid monolayers or bilayers. The lipid particles are between 1 and 1000 μm in size.

[0051]

[0154] "Viability" when referring to cells in vitro means the ability to continue to grow, divide, and continue to grow and divide as is normal for a cell type or tissue culture line. Cell survival can be affected by harsh conditions or treatments. Cell viability is critical for ex vivo therapy or parenteral administration.

[0052]

[0155] "Ionizable lipid". The compounds of the present invention include ionizable lipid. As used herein, the term "ionizable lipid" refers to a lipid that is cationic or becomes ionizable (protonated) when the pH is lowered below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values ​​lower than the pKa, the lipid can bind to negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that become positively charged upon lowering the pH, and any of the many lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0053]

[0156] The ionizable lipid or compound is preferably present in the lipid composition according to other embodiments of the present invention in a ratio of about 30 to about 70 Mol% in some embodiments, about 30 Mol% in other embodiments, about 40 Mol% in other embodiments, and about 50 Mol% and about 60 Mol% in still other embodiments ("Mol%" refers to the percentage of the total number of moles of a particular component). In this paragraph, the term "about" refers to a range of plus or minus 5 Mol%. DODMA or 1,2-dioleyloxy-3-dimethylaminopropane is an ionizable lipid, as is DLin-MC3-DMA or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate.

[0054]

[0157] The lipid particles may be generated from lipid formulations that include the ionizable lipids of the present invention.

[0055]

[0158] Structured lipids, also known as "helper lipids" or "neutral lipids", are incorporated in the lipid formulations and lipid particles of the present invention in some embodiments. The lipid formulations and lipid particles of the present invention comprise one or more structured lipids at about 10-40 mol % of the composition. Suitable structured lipids support the formation of particles during manufacture. Structured lipids refer to any one of a number of lipid species that exist in either anionic, uncharged or neutral zwitterionic form at physiological pH. Exemplary structured lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0056]

[0159] Exemplary structured lipids include zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In one preferred embodiment, the structured lipid is distearoylphosphatidylcholine (DSPC).

[0057]

[0160] In another embodiment, the structured lipid is any lipid that is negatively charged at physiological pH.These lipids include phosphatidylglycerol, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids.Other suitable structured lipids include glycolipids (e.g., monosialoganglioside GM1).

[0058]

[0161] Stabilizing agents or stabilizing agentss are included in the lipid formulation embodiments to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or aid in the formation of hydrophobic-hydrophilic interactions between molecules. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (polyoxyethylene(40) stearate), and Brij™ S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol conjugated lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0059]

[0162] In some embodiments, the stabilizer comprises about 0.1-3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5-2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is present at 2.6-10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 mol% of the lipid mixture.

[0060]

[0163] Steroids are included in preferred lipid mix compositions for some applications, and lipid particles made therefrom include sterols, such as cholesterol and plant sterols. In some embodiments, cholesterol is present in the lipid mixes of the present invention at about 30-50 mol % of the final lipid mix. Alternatively, cholesterol is present at about 35-41 mol % of the final lipid mix. In other embodiments, no sterols are present.

[0061]

[0164] Nucleic acids. The lipid mix compositions and lipid particles of the invention are useful for systemic or localized delivery of nucleic acids. As used herein, the term "nucleic acid therapeutic" (NAT) is meant to include any oligonucleotide or polynucleotide whose delivery to a cell causes a desired effect. This definition includes diagnostic and research reagents that follow the same physical principles provided by the present invention. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, while longer fragments are called polynucleotides. In certain embodiments, the oligonucleotides of the invention are 20-50 nucleotides in length. In an embodiment of the invention, the oligonucleotides are 996-4500 nucleotides in length, as in the case of messenger RNA.

[0062]

[0165] The term "nucleic acid" also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single-stranded, double-stranded, or contain portions of both double-stranded and single-stranded sequences, as appropriate. mRNA can be modified or unmodified, base-modified, and can contain different types of capping structures, e.g., Cap1.

[0063]

[0166] As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers, such as 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA), linked by internucleotide phosphodiester bonds, e.g., 3'-5' and 2'-5', inverted bonds, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs. Polynucleotides have associated counterions such as H+, NH4+, trialkylammonium, Mg2+, Na+, and the like. Polynucleotides can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides can be composed of internucleotide, nucleobase, and / or sugar analogs.

[0064]

[0167] As used herein, the term "polypeptide" encompasses "oligopeptides" and "proteins," which in some embodiments are therapeutic agents, as well as their tertiary and quaternary structures. Oligopeptides generally consist of 2-20 amino acids. Polypeptides are single linear chains of any length of many amino acids held together by amide bonds. Proteins may consist of one or more types and include structural proteins, energy catalysts, albumins, hemoglobins, immunoglobulins, and enzymes.

[0065]

[0168] A "ribonucleoprotein" in some embodiments is a complex of a Cas9 protein and a guide RNA. In some embodiments, a ribonucleoprotein is a therapeutic agent as referred to in aspects of the present invention.

[0066]

[0169] Currently, nucleic acid therapeutics include deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes, ribonucleic acid, oligonucleotides and ribozymes for gene therapy targeting various diseases such as cancer, infectious diseases, genetic diseases and neurodegenerative diseases. As described herein, nucleic acid therapeutics (NATs) are incorporated into lipid particles during their formation together with the compounds of the present invention. More than one type of nucleic acid therapeutics may be incorporated in this way. They may be derived from natural sources or, more commonly, may be synthetic or culture grown. Examples of nucleic acid therapeutics include, but are not limited to, antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozymes, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-enriched DNA, pDNA or aptamers. Nucleic acid reagents are used to silence genes (e.g., with siRNA), express genes (e.g., with mRNA), edit genomes (e.g., with CRISPR / Cas9), and reprogram cells back to their organism of origin (e.g., ex vivo cell therapy to reprogram immune cells for cancer treatment; autologous or allogeneic delivery).

[0067]

[0170] The nucleic acid present in the lipid particles according to the present invention includes any form of nucleic acid known in the art. The nucleic acid used in the present invention can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viruses or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, guide RNAs, such as CRISPR-Cas9 gRNAs, ribozymes, microRNAs, mRNAs, and triplex-forming oligonucleotides. More than one type of nucleic acid can be incorporated into the lipid particles, for example mRNA and guide RNA, either together or in combination with different types of each, or with proteins.

[0068]

[0171] Plasmid DNA is the preferred nucleic acid to be formulated in embodiments of the present invention. Plasmids are separate DNA molecules that are separated from chromosomal DNA within a cell and can replicate independently. Plasmids range in size from less than 1000 nucleotides to tens of thousands of nucleotides. The most common form is a small circular double-stranded DNA. Plasmids can be synthesized and delivered to mammalian cells for therapeutic purposes. Synthetic plasmids are used as vectors in molecular cloning and serve to drive the replication of recombinant DNA sequences within a host organism. Plasmids can be introduced into cells by transformation using physical methods such as electroporation, or by lipid particle enhanced transfection using chemical means as in the present invention. These lipid mix compositions of the present invention have several advantages over physical approaches, such as i) high biocompatibility and low toxicity in cell and tissue systems, ii) relative ease of manufacture, iii) lipophilic matrices are less susceptible to the erosion phenomenon observed with polymeric systems, and iv) increased circulatory half-life in vivo due to their invisibility to the immune system.

[0069]

[0172] In some cases, the nucleic acid encodes an engineered receptor, e.g., a recombinant receptor, that specifically binds to a ligand, and a molecule involved in a metabolic pathway, or a functional portion thereof. Alternatively, the molecule involved in a metabolic pathway is a recombinant molecule that includes an exogenous entity. The engineered receptor and the molecule involved in a metabolic pathway can be encoded by one nucleic acid or two or more different nucleic acids. In some instances, a first nucleic acid can encode an engineered receptor that specifically binds to a ligand, and a second nucleic acid can encode a molecule involved in a metabolic pathway.

[0070]

[0173] "Therapeutic agent" as used herein includes the nucleic acid therapeutics described herein, the polypeptides described herein, as well as polysaccharides, salts, small molecules, inorganic ions and radionuclides.

[0071]

[0174] Lipid particles according to some embodiments of the present invention can be characterized by electron microscopy. Particles of the present invention having a substantially solid core have an electron-dense core as seen by electron microscopy. One such structure is disclosed by Cullis et al. in US Patent No. 9,758,795. Electron-dense is defined as the area-average electron density (as seen in 2-D cryo-EM images) of the inner 50% of the projected area of ​​the solid core particle is equal to or greater than x% (x=20%, 40%, 60%) of the maximum electron density at the periphery of the particle. Electron density is calculated as the absolute value of the difference of the image intensity of the region of interest from the background intensity of regions not containing nanoparticles.

[0072]

[0175] The lipid particles of the present invention can be sized using a device such as a Malvern™ Zetasizer™, which measures the size of particles in solution. The particles have an average particle diameter of about 15 to about 300 nm. Another term for lipid particles is "LNP," which stands for "lipid nanoparticle." In some embodiments, the average particle diameter is greater than 300 nm. In some embodiments, the lipid particles have a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the lipid particles have a diameter of about 50 to about 150 nm. Smaller particles generally exhibit increased in vivo circulation lifetimes compared to larger particles. Smaller particles have increased ability to reach tumor sites than larger nanoparticles. In one embodiment, the lipid particles have a diameter of about 15 to about 50 nm.

[0073]

[0176] Mixing. Lipid particles according to embodiments of the invention can be prepared by standard T-tube mixing techniques, turbulent mixing, trituration mixing, stirring-enhanced sequential self-assembly, or passive mixing of all components with self-assembly of the components into nanoparticles. A variety of methods have been developed to formulate lipid nanoparticles (LNPs) containing genetic drugs. For example, suitable methods are disclosed by Ansell, Mui and Hope in U.S. Pat. No. 5,753,613 and by Saravolac et al. in U.S. Pat. No. 6,734,171. These methods involve mixing preformed lipid particles with a nucleic acid therapeutic (NAT) in the presence of ethanol or mixing lipids dissolved in ethanol with an aqueous medium containing NAT, resulting in lipid particles with NAT encapsulation efficiencies of 65-99%. Both of these methods rely on the presence of ionizable lipids to achieve encapsulation of the NAT and stabilizers to inhibit aggregation and the formation of large structures. The properties of the resulting lipid particle system, such as size and NAT encapsulation efficiency, are sensitive to a variety of lipid mix composition parameters, such as ionic strength, lipid and ethanol concentrations, pH, NAT concentration, and mixing ratio. 1

[0177] Microfluidic two-phase droplet technology has been applied to produce monodisperse polymeric microparticles for drug delivery or larger vesicles for encapsulation of cells, proteins, or other biomolecules. The use of hydrodynamic flow focusing, a common microfluidic technique that provides rapid mixing of reagents, to create monodisperse liposomes of controlled size has been demonstrated.

[0074]

[0178] In general, parameters such as relative lipid and NAT concentrations during mixing, as well as mixing speed, are difficult to control using current formulation techniques, resulting in variability in the properties of the resulting NAT both within and between preparations. Automated micromixing instruments, such as the NanoAssemblr® instrument (Precision Nanosystems Inc, Vancouver, Canada), allow for rapid and controlled production of nanomedicines (liposomes, lipid nanoparticles, and polymeric nanoparticles). The NanoAssemblr® instrument achieves controlled molecular self-assembly of nanoparticles through microfluidic mixing cartridges that allow millisecond mixing of nanoparticle components at nanoliter, microliter, or larger scales, along with customization or parallelization. Rapid mixing on a small scale allows for reproducible control of particle synthesis and quality that is not possible with larger instruments.

[0075]

[0179] A preferred method includes equipment such as microfluidic mixing devices such as NanoAssemblr® Spark™, ​​Ignite™, Benchtop™, and NanoAssemblr® Blaze™ to achieve nearly 100% of the nucleic acid used in the formation process is encapsulated in the particles in one step. In one embodiment, the lipid particles are prepared by a process in which about 90 to about 100% of the nucleic acid used in the formation process is encapsulated in the particles.

[0076]

[0180] U.S. Patent Nos. 9,758,795 and 9,943,846 to Cullis et al. describe methods using small volume mixing techniques and novel formulations derived therefrom. U.S. Patent Application Publication No. 20160022580 to Ramsay et al. describes more advanced methods of formulating different materials using small volume mixing techniques and products. U.S. Patent No. 9,943,846 to Walsh et al. discloses a microfluidic mixer with different passages and wells for the components to be mixed. WO 2017117647 to Wild, Leaver and Taylor discloses a microfluidic mixer with disposable sterile passages. U.S. Patent No. 10,076,730 to Wild, Leaver and Taylor discloses a branched donut-shaped micromixing geometry and its application to micromixing. WO 2018006166 to Chang, Klaassen, Leaver et al. discloses a programmable automated micromixer and a mixing chip therefor. U.S. Design Nos. D771834, D771833, D772427, and D803416 by Wild and Leaver, and U.S. Design Nos. D800335, D800336, and D812242 by Chang et al. disclose mixing cartridges having microchannels and mixing geometries for mixer devices sold by Precision Nanosystems Inc.

[0077]

[0181] In an embodiment of the invention, a device for biological microfluidic mixing is used to prepare lipid particles according to an embodiment of the invention. The device includes first and second reagent streams that are fed into a microfluidic mixer and the lipid particles are collected at an outlet or emerge into a sterile environment.

[0078]

[0182] The first stream contains a therapeutic agent in a first solvent. Suitable first solvents include those in which the therapeutic agent is soluble and which are miscible with the second solvent. Suitable first solvents include aqueous buffers. Exemplary first solvents include citrate and acetate buffers or other low pH buffers.

[0079]

[0183] The second stream comprises a lipid mix material in a second solvent. Suitable second solvents include those in which the ionizable lipids according to embodiments of the present invention are soluble and miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, acids, and alcohols. Exemplary second solvents include aqueous ethanol 90%, or absolute ethanol.

[0080]

[0184] In one embodiment of the invention, a suitable device includes one or more microchannels (i.e., channels whose largest dimension is less than 1 millimeter). In one example, the microchannel has a diameter of about 20 to about 300 μm. In several examples, at least one region of the microchannel has one or more surfaces having a primary flow direction and at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the primary direction (e.g., zigzag herringbone mixer) as described in U.S. Pat. No. 9,943,846, or a diverging donut-shaped flow as described in U.S. Pat. No. 10,076,730. To achieve maximum mixing rates, it is advantageous to avoid excessive flow resistance in front of the mixing region. Thus, one example of the device has non-microfluidic channels with dimensions greater than 1000 μm to deliver fluids to a single mixing channel.

[0081]

[0185] Less automated micromixing methods and equipment, such as Zhang, Sh et al. 2 and Stroock A et al., U.S. Patent Application Publication No. 20040262223, and Jeffs, LB et al. 3 are also useful in making the lipid particle compositions of the present invention.

[0082]

[0186] The ionizable lipids of the present invention may be used to deliver therapeutic agents to cells in vitro or in vivo. In certain embodiments, the therapeutic agent is a nucleic acid delivered to a cell using the nucleic acid-lipid particles of the present invention. The nucleic acid can be an siRNA, miRNA, LNA, plasmid, replicon, mRNA, guide RNA, transposon, or a single gene. In other embodiments, the therapeutic agent delivered to one or more cells is a gene editing technique. Gene editing techniques are a group of scientific techniques that alter the DNA of an organism, allowing the addition, removal, or modification of genetic material at specific locations within the genome. There are several methods for genome editing, such as CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9), TALEN, and ZFN. 4 There is.

[0083]

[0187] In other embodiments, the therapeutic agent is an oligopeptide, polypeptide, or protein delivered to cells using the peptide-lipid particles of the present invention. In other embodiments, the therapeutic agent is a mixture of a nucleic acid and a protein component, such as Cas9. The methods and lipid mix compositions can be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.

[0084]

[0188] In certain embodiments, the present invention provides a method for introducing a nucleic acid into a cell (i.e., transfection). Transfection is a technique commonly used in molecular biology for the introduction of a nucleic acid therapeutic (or NAT) from the extracellular to the intracellular space for the purpose of transcription, translation and expression of the delivered gene(s). Transfection efficiency is generally defined as i) the percentage of the total treated population of cells that show positive expression of the delivered gene, as measured by live or fixed cell imaging (for detection of fluorescent proteins) and flow cytometry, or ii) the intensity or amount of protein expressed by the treated cell(s) analyzed by live or fixed cell imaging or flow cytometry, or iii) using protein quantification techniques such as ELISA or Western blot. These methods can be performed by contacting the particles or lipid mix compositions of the present invention with the cells for a sufficient time for intracellular delivery to occur.

[0085]

[0189] Exemplary applications include providing intracellular delivery to knock down or silence specific cellular targets in vitro and in vivo using well-known procedures. Alternatively, applications include delivery of DNA or mRNA sequences encoding therapeutically useful polypeptides. In this way, treatments for genetic diseases are provided by supplying missing or absent gene products. The methods of the present invention may be performed in vitro, ex vivo, or in vivo. For example, the lipid mix compositions of the present invention may also be used for in vivo delivery of nucleic acids to cells using methods known to those skilled in the art. In another example, the lipid mix compositions of the present invention may be used to deliver nucleic acids ex vivo to a sample of patient cells, which are then returned to the patient.

[0086]

[0190] The delivery of nucleic acid therapeutic agents by lipid particles of the present invention is described below.

[0087]

[0191] For in vivo administration, the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseously, intramuscularly, or intratumorally). In certain embodiments, the pharmaceutical compositions are administered intravenously, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical (skin, eye, mucosa), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0088]

[0192] For ex vivo applications, the pharmaceutical composition is preferably administered to a biological sample removed from an organism, after which the cells are washed and restored to the organism. The organism may be a mammal, in particular a human. This process is used, for example, for cell reprogramming, gene repair, and immunotherapy.

[0089]

[0193] In one embodiment, the present invention provides a method for regulating the expression of a target polynucleotide or polypeptide.These methods generally comprise contacting a cell with the lipid particles of the present invention that are combined with a nucleic acid that can regulate the expression of a target polynucleotide or polypeptide.As used herein, the term "regulate" refers to altering the expression of a target polynucleotide or polypeptide.Regulating can mean increasing or enhancing, or it can mean decreasing or reducing.

[0090]

[0194] In a related embodiment, the invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, the method comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing the siRNA, microRNA, or antisense oligonucleotide, and the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide, or its complement.

[0091]

[0195] In a related embodiment, the present invention provides a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is specifically selected from an mRNA, a self-amplifying RNA (SAM), a self-replicating DNA, or a plasmid comprising a nucleic acid therapeutic agent or its complement that encodes or expresses the underexpressed polypeptide.

[0092]

[0196] In embodiments, the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients.

[0093]

[0197] Methods for delivery of biologically active agents for the treatment of disease - Patents.com

[0198] In one embodiment, the compounds, compositions, methods and uses of the present invention are for delivering biologically active agents to liver cells (e.g., hepatocytes). In one embodiment, the compounds, compositions, methods and uses of the present invention are for delivering biologically active agents to tumors or tumor cells (e.g., primary tumors or metastatic cancer cells). In another embodiment, the compounds, compositions, methods and uses deliver biologically active agents to skin fat, muscle and lymph nodes (subcutaneous administration).

[0094]

[0199] In the case of delivery of biologically active agents to the liver or liver cells, in one embodiment, the compositions of the present invention are contacted with the liver or liver cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheterization, stent implantation) to facilitate delivery. In the case of delivery of biologically active agents to kidney or kidney cells, in one embodiment, the compositions of the present invention are contacted with the kidney or kidney cells of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent implantation) to facilitate delivery. In the case of delivery of biologically active agents to tumors or tumor cells, in one embodiment, the compositions of the present invention are contacted with the tumor or tumor cells of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent implantation) to facilitate delivery.

[0095]

[0200] For delivery of biologically active agents to the CNS or CNS cells, in one embodiment, the compositions of the invention are contacted with the CNS or CNS cells (e.g., brain cells and / or spinal cord cells) of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent implantation, osmotic pump administration (e.g., intramedullary or ventricular)) to facilitate delivery. For delivery of biologically active agents to the Peripheral Nervous System (PNS) or PNS cells, in one embodiment, the compositions of the invention are contacted with the PNS or PNS cells of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection) to facilitate delivery. For delivery of biologically active agents to the lung or lung cells, in one embodiment, the compositions of the invention are contacted with the lung or lung cells of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct intrapulmonary administration to lung tissue and cells) to facilitate delivery.

[0096]

[0201] For delivery of biologically active agents to the vasculature or vascular cells, in one embodiment, the compositions of the invention are contacted with the vasculature or vascular cells of a patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamping, catheterization, stent implantation) to facilitate delivery.

[0097]

[0202] For delivery of biologically active agents to skin or skin cells (e.g., dermal and / or follicular cells), in one embodiment, the compositions of the present invention are contacted with the patient's skin or skin cells (e.g., dermal and / or follicular cells) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct skin application, iontophoresis) to facilitate delivery. For delivery of biologically active agents to eye or ocular cells (e.g., macula, fovea, cornea, retina), in one embodiment, the compositions of the present invention are contacted with the patient's eye or ocular cells (e.g., macula, fovea, cornea, retina) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eye drops, implantation) to facilitate delivery. For delivery of a biologically active agent to the ear or cells of the ear (e.g., cells of the inner, middle and / or outer ear), in one embodiment, the compositions of the invention are contacted with the ear or cells of the ear (e.g., cells of the inner, middle and / or outer ear) of a patient, for example, by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection) as is generally known in the art to facilitate delivery. For delivery of a biologically active agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen presenting cells, including professional antigen presenting cells), in one embodiment, the compositions of the invention are delivered intramuscularly, after which immune cells can infiltrate the delivery site and process the delivered RNA and / or the encoded antigen produced by non-immune cells, such as muscle cells. Such immune cells can include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and / or bone marrow derived myeloid dendritic cells), monocytes (e.g., human peripheral blood monocytes), and the like (see, e.g., Geall, Andy et al., International Publication WO 2012 / 006372).

[0098]

[0203] Immunization. For purposes of immunization, the compositions of the invention are generally prepared as injectables, pulmonary or nasal aerosols, or in a delivery device (e.g., a syringe, nebulizer, sprayer, inhaler, skin patch, etc.), which can be used to administer the pharmaceutical composition to a subject, e.g., a human, for immunization.

[0099]

[0204] In accordance with the present invention, for immunization purposes, in some embodiments, the invention encompasses delivery of RNA encoding an immunogen that elicits an immune response that recognizes the immunogen and provides immunity against a pathogen, or against an allergen, or against a tumor antigen. Immunization against disease and / or infection caused by a pathogen is preferred.

[0100]

[0205] RNA is delivered with the lipid composition of the invention (e.g., formulated as liposomes or LNPs). In some embodiments, the invention utilizes LNPs in which RNA encoding an immunogen is encapsulated. Encapsulation within LNPs can protect the RNA from digestion by RNases. Encapsulation efficiency does not need to be 100%. The presence of external RNA molecules (e.g., on the outer surface of a liposome or LNP) or "naked" RNA molecules (RNA molecules not associated with a liposome or LNP) is acceptable. Preferably, for a composition comprising lipids and RNA molecules, at least half of the RNA molecules (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) are encapsulated in the LNP or complexed LNP.

[0101]

[0206] Some lipid nanoparticles may contain a lipid core (e.g., the composition may contain a mixture of LNPs and nanoparticles with lipid cores). In such cases, RNA molecules may be encapsulated by LNPs with aqueous cores and complexed with LNPs with lipid cores by non-covalent interactions (e.g., ionic interactions between negatively charged RNA and cationic lipids). Encapsulation and complexing with LNPs (whether with lipids or with aqueous cores) can protect the RNA from RNase digestion. The encapsulation / complexing efficiency does not have to be 100%. The presence of "naked" RNA molecules (RNA molecules not associated with liposomes) is acceptable. Preferably, in a composition comprising a population of LNPs and a population of RNA molecules, at least half of the population of RNA molecules (e.g., at least, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA molecules) are encapsulated within the LNPs or complexed with the LNPs.

[0102]

[0207] For delivery of RNA encoding an immunogen, the preferred range of LNP diameters is in the range of 60-180 nm, and in more particular embodiments in the range of 80-160 nm. The LNPs can be part of a composition comprising a population of LNPS, where the LNPS within the population can have a range of diameters. For compositions comprising a population of LNPS of different diameters, it is preferred that (I) at least 80% by number of the LNPS have a diameter in the range of 60-180 nm, e.g., in the range of 80-160 nm, and (ii) the mean diameter (by intensity, e.g., Z-average) of the population is ideally in the range of 60-180 nm, e.g., in the range of 80-160 nm; and / or the diameters within the plurality have a polydispersity index of less than 0.2. To obtain LNPS of the desired diameter(s), mixing can be performed using a process in which two feed streams of aqueous RNA solution are combined in a single mixing zone with one stream of ethanolic lipid solution, all at the same flow rate, e.g., in a microfluidic channel. See elsewhere for information on the NanoAssemblr® microfluidic mixer sold by Precision Nanosystems Inc., Vancouver, Canada.

[0103]

[0208] A mixture of lipids useful for forming a lipid composition (e.g., LNPS) for immunization includes a lipid of formula (I); cholesterol; and a stabilizer, such as PEG-DMG. The mixture may also include a neutral zwitterionic lipid, such as DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) or DSPE. In certain embodiments, the lipid compositions provided by the present invention (e.g., LNPS) have adjuvant activity, i.e., in the absence of an immunogen, such as a protein antigen or a nucleic acid (DNA or RNA), such as a nucleic acid encoding such an antigen.

[0104]

[0209] RNA molecules. After in vivo administration of the immunizing composition, the delivered RNA is released and translated in the cell to provide the immunogen in situ. In certain embodiments, the RNA is positive ("+") stranded and can be translated by the cell without the need for an intervening replication step such as reverse transcription. In certain embodiments, the RNA is a self-replicating RNA. A self-replicating RNA molecule (replicon) can produce multiple daughter RNAs by transcription from itself (through an antisense copy it generates) without the need for proteins when delivered to a vertebrate cell. Thus, a self-replicating RNA molecule is in certain embodiments: a (+) stranded molecule that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA thus produces multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can be translated into themselves to provide in situ expression of the encoded immunogen, or can be transcribed to provide additional transcripts of the same sense as the delivered RNA, which are translated to provide in situ expression of the immunogen. The overall result of repeated transcription of this sequence is an expansion in the numbers of the introduced replicon RNA, such that the encoded immunogen becomes the major polypeptide product of the host cell.

[0105]

[0210] One suitable system for achieving self-replication is to use alphavirus-based RNA replicons. These (+) strand replicons are translated after delivery to the cell to produce a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that creates a genomic (-) strand copy of the delivered (+) strand RNA. These (-) strand transcripts can themselves be transcribed to provide further copies of the parent (+) strand RNA, and also to provide subgenomic transcripts that code for immunogens. Translation of the subgenomic transcripts thus results in in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc.

[0106]

[0211] Mutant or wild-type viral sequences, such as the attenuated TC83 mutant of VEEV, can be used for the replicon. A preferred self-replicating RNA molecule thus encodes (I) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase can be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsPI, nsP2, nsP3 and nsP4. While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins in certain embodiments, the self-replicating RNA molecules of the invention do not encode alphavirus structural proteins. Thus, although a particular self-replicating RNA can trigger the production of its own genomic RNA copies in a cell, it does not generate virions containing the RNA. The alphavirus structural proteins necessary for perpetuation in wild-type virus are absent from the self-replicating RNA of the invention, and their place is taken by a gene(s) encoding the immunogen of interest, with the subgenomic transcript encoding the immunogen rather than the structural alphavirus virion proteins. Thus, a self-replicating RNA molecule useful in the present invention may have two open reading frames: one, e.g., the first (5') open reading frame, encoding a replicase; the other, e.g., the second (3') open reading frame, encoding an immunogen. In some embodiments, the RNA may have an additional (e.g., downstream) open reading frame, e.g., to encode an additional immunogen or to encode an accessory polypeptide. The self-replicating RNA molecule may have a 5' sequence compatible with the encoded replicase. The self-replicating RNA molecule may have a variety of lengths, but is typically about 5,000-25,000 nucleotides, e.g., 8,000-15,000 nucleotides, or 9,000-12,000 nucleotides in length. Thus, the RNA is longer than that seen in conventional mRNA delivery.In some embodiments, the self-replicating RNA is greater than about 2000 nucleotides in length, for example, about: 9000, 12000, 15000, 18000, 21000, 24000, or more nucleotides in length.

[0107]

[0212] The RNA molecule may have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to the 7-methylguanosine via a 5'-5' bridge. The 5' triphosphate may enhance RIG-I binding, thus aiding in the adjuvant effect. The RNA molecule may have a 3' poly-A tail and may also contain a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention for immunization purposes are typically single-stranded. Single-stranded RNA can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA formed during replication of single-stranded RNA or within secondary structures of single-stranded RNA.

[0108]

[0213] RNA molecules for immunization purposes can be conveniently prepared by in vitro transcription (IVT). IVT can use (cDNA) templates that are produced and propagated in bacteria in the form of plasmids or that are synthetically produced (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). As described by Hekele, Armin et al. in WO 2011 / 005799, the self-replicating RNA can contain one or more nucleotides with modified nucleobases (in addition to the 5' cap structure). For example, the self-replicating RNA can contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues. However, in some embodiments, the RNA does not contain any modified nucleobases and may also be free of modified nucleotides, i.e., all nucleotides of the RNA are standard A, C, G and U ribonucleotides (except for the 5' cap structure, which may contain 7' methylguanosine). In other embodiments, the RNA may include a 5' cap containing a 7' methylguanosine, and the first I, 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose. RNA used for immunization purposes in the present invention ideally contains only phosphodiester linkages between nucleosides, but in some embodiments contains phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. The present invention includes embodiments in which multiple RNA species, e.g., two, three, four or more RNA species, different types of RNA (e.g., mRNA, siRNA, self-replicating RNA, and combinations thereof) are formulated with the lipid compositions provided by the present invention.

[0109]

[0214] In some embodiments, the immunogenic RNA molecules used for immunization purposes in the present invention encode polypeptide immunogens. In these embodiments, after administration, the RNA is translated in vivo and the immunogens are capable of eliciting an immune response in the recipient. The immunogens may elicit an immune response against a pathogen (e.g., a bacterium, a virus, a fungus, or a parasite), but in some embodiments, an immune response against an allergen or a tumor antigen. The immune response may include an antibody response (usually involving IgG) and / or a cell-mediated immune response. Polypeptide immunogens typically elicit an immune response that recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments, the polypeptides may act as mimotopes to elicit an immune response that recognizes saccharides. The immunogens are typically surface polypeptides, such as adhesins, hemagglutinins, envelope glycoproteins, spike glycoproteins, and the like. The RNA molecules can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogens are expressed as separate polypeptides from a replicon, one or more of these may be provided with an upstream IRES or additional viral promoter elements. Alternatively, multiple immunogens may be expressed from a polyprotein encoding individual immunogens fused to a short autocatalytic protease (e.g., foot and mouth disease virus 2A protein) or as inteins. In certain embodiments, polypeptide immunogens (e.g., I, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunogens) may be used alone or together with an RNA molecule, such as a self-replicating RNA, encoding one or more immunogens (the same or different as the polypeptide immunogens).

[0110]

[0215] In some embodiments, the immunogen is selected from the group consisting of, but not limited to, SARS CoV-1, SARS-CoV-2, 9(Roujian Lu, Xiang Zhao, Juan Li et al. "Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus: Implications for Virus Origins and Receptor Binding" Lancet 2020 Feb 22;395(10224):565-574. doi:10.1016 / S0140-6736(20)30251-8. Epub 2020 Jan 30.) Useful immunogens for Neisseria meningitidis, but not limited to, include membrane proteins, such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins. Three useful polypeptide combinations have been described by Giuliani et al. 11 (2006) Proc Natl Head Sci USA 103(29):10834-9; useful polypeptide immunogens for Streptococcus pneumoniae are disclosed by Veja, Masignani et al. in WO 2009 / 016515, including, for example, the RrgB pilus subunit, beta-N-acetyl-hexosaminidase precursor (spr0057), spr0096, general stress protein GSP-781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA;

[0111]

[0216] Hepatitis viruses, where the immunogen can include hepatitis B surface antigen (HBsAg), hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus antigen; Rhabdoviruses, where the immunogen can include, but is not limited to, those derived from lyssaviruses (e.g., rabies virus) and vesiculoviruses (VSV); Caliciviridae, where the immunogen can include, but is not limited to, those derived from Norwalk virus (Norovirus), and Norwalk-like viruses, e.g., Hawaii virus and Snow Mountain virus; Avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus of porcine (TGEV); immunogens, where the immunogen can include oncoviruses, lentiviruses (e.g., HIV-I or HIV-2) or retroviruses, including those derived from spumaviruses; reoviruses, including those in which the immunogen is derived from orthoreovirus, rotavirus, orbivirus, or coltivirus; parvoviruses, including those in which the immunogen is derived from parvovirus B19; herpesviruses, including those in which the immunogen is derived from human herpesviruses, such as herpes simplex virus (HSV) (e.g., HSVI and 2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8); papovaviruses, including those in which the immunogen is derived from papillomaviruses and adenoviruses.

[0112]

[0217] In some embodiments, the immunogen elicits an immune response against a virus that infects the fish.

[0113]

[0218] Fungal immunogens can be derived from dermatophytes and other opportunistic fungi.

[0114]

[0219] In some embodiments, the immunogen elicits an immune response against a parasite of the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale. Thus, the present invention may be used to immunize against malaria. In some embodiments, the immunogen elicits an immune response against a parasite of the family Ploceidae, particularly those from the genera Lepeophtheirus and Caligus, such as sea lice, e.g., Lepeophtheirus salmonis or Caligus rogercresseyi.

[0115]

[0220] In some embodiments, the immunogen is an mRNA specific for a neoantigen in a cancer cell or solid tumor. (7) Peng, M., Mo, Y., Wang, Y. et al. Neoantigen vaccine: an emerging tumor immunotherapy. Mol Cancer 18, 128 (2019).

[0116]

[0221] In some embodiments, the immunogens include (a) cancer-testis antigens, such as NY-ESO-I, SSX2, SCPI, and RAGE, BAGE, GAGE, and MAGE family polypeptides, such as GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used to address, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) mutant antigens, such as p53 (associated with various solid tumors, such as colorectal, lung, head and neck cancer), p21 / Ras (associated with, for example, melanoma, pancreatic cancer, and colorectal cancer), CDK4 (associated with, for example, melanoma), MUMI (associated with, for example, melanoma), caspase-8 (associated with, for example, head and neck cancer), CIA (associated with, for example, melanoma), and / or IL-1 (associated with, for example, melanoma). 0205 (e.g., associated with bladder cancer), HLA-A2-R1701, beta-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin's lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLRFUT; (c) overexpressed antigens, such as galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT I (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast, colon cancer), and carcinoembryonic antigen (e.g., associated with breast cancer, lung cancer, and cancers of the gastrointestinal tract, such as colorectal cancer);(d) common antigens, e.g., melanoma-melanocyte antigens, e.g., MART-1 / Melan A, gp100, MCIR, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-I / TRPI, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-associated antigens, e.g., PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with prostate cancer; (f) tumor antigens selected from immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens such as E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29&BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, FGF-5, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-I, RCASI, SDCCAG16, TA-90(Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, etc.;

[0117]

[0222] Pharmaceutical compositions as vaccines. Pharmaceutical compositions of the invention, particularly those useful for immunization, may contain one or more small molecule immune enhancers. Pharmaceutical compositions of the invention may contain one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free and preservative-free vaccines can be prepared.

[0118]

[0223] The composition comprises an immunologically effective amount of the lipid composition (e.g., LNPS) described herein, as well as other components, as appropriate. By immunologically effective amount is meant an amount administered to an individual, either as a single dose or as part of a series of doses, that is effective for treatment (e.g., a prophylactic immune response against a pathogen). This amount will vary depending on the health and condition of the individual being treated, the age, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's evaluation of the medical situation, and other relevant factors. It is expected that this amount will fall within a relatively broad range that can be determined by routine testing.

[0119]

[0224] The compositions of the invention are generally expressed in terms of the amount of RNA per dose. A preferred dose has about 100 pg of RNA (e.g., 10-100 pg, e.g., about 10 pg, 25 pg, 50 pg, 75 pg, or 100 pg), although expression can be seen at much lower levels, e.g., about 1 pg / dose, about 100 ng / dose, about 10 ng / dose, about 1 ng / dose, etc. The invention also provides a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.) containing the pharmaceutical composition of the invention. The device can be used to administer the composition to a vertebrate subject.

[0120]

[0225] The LNP-formulated RNA and pharmaceutical compositions described herein are used in vivo to induce an immune response against an immunogen in a subject. The invention provides a method of eliciting an immune response in a vertebrate comprising administering an effective amount of the LNP-formulated RNA or pharmaceutical composition described herein. The immune response is preferably protective and preferably includes antibodies and / or cell-mediated immunity. The compositions can be used for both priming and boosting purposes. Alternatively, a prime-boost immunization schedule can be a mixture of RNA and the corresponding polypeptide immunogen (e.g., RNA prime, protein boost).

[0121]

[0226] The invention also provides lipid particles (LNPs) or pharmaceutical compositions for use in eliciting an immune response in a vertebrate. The invention also provides the use of the LNPs or pharmaceutical compositions in the manufacture of a medicament for eliciting an immune response in a vertebrate. By eliciting an immune response in a vertebrate by these uses and methods, the vertebrate may be protected against various diseases and / or infections, for example bacterial and / or viral diseases as mentioned above. Vaccines according to the invention may be either prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but are typically prophylactic. The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g. horse, cow, deer, goat, pig).

[0122]

[0227] The compositions of the invention are generally administered directly to a patient. Direct delivery may be achieved by parenteral injection (e.g. subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of a tissue. Alternative delivery routes include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection may be via a needle (e.g. a hypodermic needle), although needle-free injections may alternatively be used. A typical intramuscular dose is 0.5 ml. The invention may be used to induce systemic and / or mucosal immunity, preferably to elicit enhanced systemic and / or mucosal immunity. Dosing may be according to a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule.

[0123]

[0228] In a multiple dosing schedule, the various doses may be given by the same or different routes, e.g., a parenteral prime and a mucosal boost, a mucosal prime and a parenteral boost, etc. The multiple doses are typically administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, the multiple doses may be administered at approximately 6, 10 and 14 weeks of age, e.g., 6, 10 and 14 weeks of age, as often used in the World Health Organization's Expanded Programme on Immunization ("EPI"). In an alternative embodiment, two primary doses are administered about 2 months apart, e.g., about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g., about 6, 8, 10 or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, for example about 7, 8 or 9 weeks apart, followed by one or more booster doses about six months to one year after the third primary dose.

[0124]

[0229] Gene editing

[0230] Gene editing is a group of scientific techniques that can be used to change an organism's DNA by adding, removing, or modifying genetic sequences at specific locations in the genome. Several approaches to genome editing have been developed, including CRISPR ("clustered regularly interspaced short palindromic repeats") and CRISPR-associated protein 9 ("Cas9"). CRISPR-Cas9 was adapted from a genome editing system in bacteria that captures small pieces of DNA from invading viruses and creates DNA segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to "remember" the virus, so that if the virus attacks again, the bacteria can produce RNA segments from the CRISPR array to target the virus' DNA. The bacteria then use Cas9 or a similar enzyme to cut and separate the DNA, disabling the virus.

[0125]

[0231] The CRISPR-Cas9 system adapted for gene editing works similarly. A small piece of RNA is produced with a short "guide" sequence that attaches (binds) to a specific target sequence of DNA in the genome. This RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Cas9 is the enzyme used most often, but other enzymes (e.g. Cpf1) can also be used. Once the DNA has been cut, researchers can use the cell's own DNA repair mechanisms to alter the DNA by adding or deleting pieces of genetic material, or by replacing existing segments with customized DNA sequences.

[0126]

[0232] In an embodiment of the invention, the new ionizable lipids are utilized as part of, for example, the delivery of CRISPER-Cas based chimeric RNA polynucleotide sequences to modify organisms by manipulation of target sequences in genomic loci of interest. Other nucleic acid components can include guide sequences, tracr mate sequences and tracr sequences capable of hybridizing to target sequences in eukaryotic cells, such as those described by CHZHAN, Fen et al. in WO14204726.

[0127]

[0233] In embodiments, the CRISPR-Cas9 system is used to target genes in live cells by delivery of the CRISPR-Cas9 system to the appropriate location (i.e., to cells within an organ or tissue of interest). Preferred tissues are within the following organs: kidney; digestive system, including stomach, pancreas, duodenum, ileum and / or colon; lung; brain, particularly neurons, and / or cns in general; eye, e.g. retinal tissue; ear, e.g. inner ear; skin; muscle; bone; and / or liver in general.

[0128]

[0234] The genes that are subject to editing using ionizable lipids and compositions according to embodiments of the present invention are those associated with diseases.

[0129]

[0235] Pharmaceutical compositions according to 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" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient will generally be equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, but not limited to, one-half or one-third of such a dosage.

[0130]

[0236] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or additional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the type, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. For example, a composition may contain between 0.1 percent and 99 percent (w / w) of active ingredient.

[0131]

[0237] Pharmaceutical formulations may further comprise pharma- ceutically acceptable excipients, which as used herein include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, suitable for the particular dosage form desired. Techniques for preparing various excipients and compositions for formulating pharmaceutical compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006). Use of conventional excipient vehicles is contemplated in the present invention, except where such vehicles may be incompatible with a substance or its derivatives, for example, by producing some undesirable biological effect or otherwise interacting in a deleterious manner with other component(s) of the pharmaceutical composition.

[0132]

[0238] In some embodiments, the lipid particles may be increased and / or decreased in particle size. The change in particle size may help combat biological reactions such as, but not limited to, inflammation, or may increase the biological effect of the NAT delivered to a mammal by altering biodistribution. Size may also be used to determine the target tissue, with larger particles being cleared quickly and smaller ones reaching different organ systems.

[0133]

[0239] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, diluents, viscosity reducing agents, antioxidants, solubility enhancers, bulking agents, fillers, surfactants and / or emulsifiers, preservatives, buffers, lubricants, and / or oils. Excipients may be available in a variety of grades and may be of natural, synthetic, or semi-synthetic origin, animal-derived, vegetable-derived, biotechnological (recombinant), and / or mineral origin, and may be solid, semi-solid, liquid, or gaseous. Such excipients may optionally be included in the pharmaceutical formulations of the present invention.

[0134]

[0240] In some embodiments, exemplary mRNAs, plasmids or other NATs include human growth hormone, erythropoietin, ATP-binding cassette (ABC) transporter, alpha-1-antitrypsin, acid alpha glucosidase, arylsulfatase A, carboxypeptidase N, a-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase, iduronate sulfatase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase, alpha- ... Cetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase, beta-glucuronidase, BMPER-2, glucocerebrosidase, heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase, hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC), carbamoyl-phosphate synthase 1 (CPS) 1), argininosuccinate synthetase (ASS 1), argininosuccinate lyase (ASL), arginase 1 (ARGI), cystic fibrosis transmembrane conductance regulator (CFTR), survival of motor neuron (SMN), factor VIII, factor IX, transcription activator-like effector nucleases such as TALENS, zinc finger nucleases (ZFN), (CRISPR) associated protein 9 (Cas9), and self-replicating RNA, and low density lipoprotein receptor (LDLR).

[0135]

[0241] Other plasmids or nucleic acids can be applied to cell-based systems using the present invention in the context of research or screening scenarios. These include the introduction of genetic material for the purpose of inducing specific physiological or functional changes in cells, for example, in the reprogramming process for the generation of induced pluripotent stem cells. In this case, specific genes (known as Yamanaka factors) are introduced into patient-derived somatic cells to cause the reversal of the cells to a hepatocyte-like state. These enable the cells to divide indefinitely and become pluripotent (able to differentiate into many other downstream cell types), which can be used for both research and clinical applications. These and similar genetic manipulation steps are enhanced by the lipid particles of the present invention to improve the efficiency of processes commonly used when functioning with induced hepatocytes.

[0136]

[0242] The following describes representative lipid particles prepared with nucleic acids, their preparation methods, evidence of advantages, and methods of delivering therapeutic effects using them.

[0137]

[0243] The lipid mix composition of the lipid particles was generated by rapidly mixing a lipid-ethanol solution with an aqueous buffer in a microfluidic mixer designed to induce chaotic advection and provide a mixed environment controlled at an intermediate Reynolds number (24 < Re < 1000). The microfluidic flow path has a herringbone feature or is configured as shown by Wild, Leaver, and Taylor in International Publication No. 2017117647.

[0138]

[0244] The lipid particle size and "polydispersity index" (PDI) were measured by dynamic light scattering (DLS). PDI indicates the width of the particle distribution. It is a parameter calculated from cumulative analysis of the intensity autocorrelation function measured by (DLS) assuming a single particle size mode and a single exponential fit to the autocorrelation function. From a biophysical point of view, a PDI of less than 0.1 indicates that the sample is monodisperse. Particles produced by mechanical micromixers, such as the NanoAssemblr® Spark™ and NanoAssemblr® Benchtop (Precision Nanosystems Inc.), are substantially uniform in size assuming all other variables are neutral. A lower PDI indicates a more homogenous population of lipid particles. Spark™ instruments are used in a screening environment to identify lead compositions. Once a composition is selected, the lipid particles can be fine-tuned using the NanoAssemblr® Benchtop. Once the process parameters flow rate ratio and total flow rate are identified for a particular nanoparticle composition, the nanoparticle technology can be scaled up using the same process parameter values.

[0139]

[0245] In a preferred embodiment, the nucleic acid is a plasmid composed of double-stranded deoxyribonucleic acid. A plasmid is a genetic structure, typically a small circular strand of DNA, that resides in the cytoplasm of a cell (as opposed to the nucleus where traditional cytogenetics is applied) and can replicate independently of chromosomes. Plasmids can also be used to create novel cell or animal models for medical research. Plasmids are important tools in molecular biology and as emerging therapeutics due to their i) ease of manipulation and isolation, ii) ability to self-replicate for large-scale production, iii) long-term stability, and iv) functionality in a range of organisms and applications. In addition to an origin of replication (or none depending on the intended use), genetically engineered plasmids have restriction enzyme recognition sites that allow the circle to be disrupted and new genetic material to be introduced, and a selection marker, e.g., an antibiotic resistance gene. Plasmids can be from about 1000 bp to about 20 kilobase pairs (bp).

[0140]

[0246] As used herein, the term "about" is defined as meaning plus or minus 10% of the indicated number and is used to indicate that a desired target concentration may be, for example, 40 Mol%, but due to inconsistencies in mixing, the actual percentage may vary by + / - 5 Mol%.

[0141]

[0247] As used herein, the term "substantially" is defined as plus or minus 5% of the number indicated and is used to indicate that a desired target concentration may be, for example, 40 Mol%, but that due to inconsistencies in mixing, the actual percentage may vary by + / - 5 Mol%.

[0142]

[0248] As used herein, the term "nucleic acid" is defined as a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have a direct effect in restoring, correcting or altering physiological function, or to serve as a research reagent. In a preferred embodiment, the nucleic acid is an oligonucleotide. In a preferred embodiment, the therapeutic agent is a nucleic acid therapeutic agent, such as an RNA polynucleotide. In a preferred embodiment, the therapeutic agent is a double-stranded circular DNA (plasmid), linearized plasmid DNA, minicircle or msDNA (multiple copy single stranded DNA).

[0143]

[0249] In this disclosure, the word "comprise" is used in an open-ended manner to mean that the items preceding the word are included, but not excluding items not specifically stated. In embodiments that include or may include a stated feature or variable or parameter, it is understood that alternative embodiments may consist of, or consist essentially of, such feature or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that there is more than one of the element, unless the context clearly requires that there is one, and only one, of that element.

[0144]

[0250] In this disclosure, "transfection" refers to the transfer of nucleic acid into cells for the purpose of inducing expression of a particular gene(s) of interest in both laboratory and clinical settings, typically including nucleic acid and ionizable lipids for binding to structural lipids. LIPOFECTIN™ and LIPOFECTAMINE™ are well-established commercial transfection reagents sold by ThermoFisher Scientific. These research reagents contain permanently cationic lipids and are not suitable for in vivo or ex vivo use.

[0145]

[0251] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., all whole numbers, all integers, and all intermediate fractions (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In this disclosure, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. In this disclosure, the term "or" is generally used to include "and / or" unless the content clearly dictates otherwise.

[0146]

[0252] "Stabilizer" or "stabilizing agent" is a term used to identify a substance added to the ionizable lipids, structured lipids, and sterols that form the lipid composition according to the present invention. The stabilizer is non-ionic as described herein. Examples of non-ionic stabilizers include polysorbates (Tween), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), Myrj™ 52 (polyoxyethylene (40) stearate). Combinations of stabilizers, such as polysorbates and maltosides, alkyl polyglycosides (TBD), PEG-conjugated lipids, or other polymer-conjugated lipids, are also used in some embodiments.

[0147]

[0253] "Alkyl" means a straight or branched, non-cyclic or cyclic saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; representative saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; representative unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0148]

[0254] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0149]

[0255] "Alkynyl" refers to an alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0150]

[0256] The term "acyl" refers to a carbonyl group substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, and heteroaryl. For example, acyl includes (C 1 ~C 20 ) alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C 3 ~C 20 ) cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl), and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.).

[0151]

[0257] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, in which the ring atoms can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

[0152]

[0258] "Heterocycle" means a 3-7 membered monocyclic or 7-10 membered bicyclic heterocyclic ring, saturated, unsaturated, or aromatic, containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be bonded through any heteroatom or carbon atom. Heterocycles include heteroaryls, as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0153]

[0259] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms in a monocyclic ring, 1-6 heteroatoms in a bicyclic ring, or 1-9 heteroatoms in a tricyclic ring, selected from O, N, or S (e.g., carbon atoms and 1-3 monocyclic, 1-6 bicyclic, or 1-9 tricyclic heteroatoms N, O, or S, respectively), where any ring atom may be substituted. The heteroaryl groups described herein may also contain fused rings that share a common carbon-carbon bond. The term "alkylheterocycle" refers to a heteroaryl in which at least one ring atom is substituted with an alkyl, alkenyl, or alkynyl.

[0154]

[0260] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with a defined substituent group, including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, arylcarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted. Exemplary substituents include amino, alkylamino, dialkylamino, and cyclic amino compounds.

[0155]

[0261] "Halogen" means fluoro, chloro, bromo and iodo substituents.

[0156]

[0262] The terms "alkylamine" and "dialkylamine" refer to -NH(alkyl) and -N(alkyl) amines, respectively. 2 The term "hydroxyalkyl" refers to the -alkyl-OH radical. The term "alkylheterocycle" refers to an alkyl in which at least one methylene is replaced by a heterocycle.

[0157]

[0263] In some embodiments, the methods of the present invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, a protecting group within the context of the present invention is a group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is a group that reduces or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0158]

[0264] The present invention can further be described as a compound as shown in formula (I), (II) or (III), or a pharma- ceutically acceptable salt thereof, wherein the experimental pKa of the nanoparticles is in the range of 5.8 to 7.1.

[0159]

[0265] The compounds of the present invention can be prepared by known organic synthesis techniques, for example, as described in more detail in the Examples. The scope of the present invention can include various salts, hydrates, and solvates of the compounds. The compounds of the present invention can also include various pharmaceutically acceptable isotopes. The compounds of the present invention include various pharmaceutically acceptable substitutions, such as fluorinated or iodinated derivatives.

[0160]

[0266] The compounds of the present invention can be synthesized using a variety of possible synthetic routes which can be readily selected by one skilled in the art of organic synthesis.

[0161]

[0267] The phrase "pharmacologically acceptable" is used to describe compounds, materials, compositions, nanoparticle suspensions in solution or any other form, e.g., lyophilized, powdered, aerosol, or other dosage form, within the bounds of sound medical judgment, suitable for use in contact with tissues or cells of humans and animals with a reasonable benefit / risk ratio. The benefit / risk ratio may result from protecting the therapeutic entity, e.g., small molecule, nucleic acid, peptide, or protein, from degradation in an in vivo or ex vivo biological environment.

[0162]

[0268] The compounds may also be evaluated in one or more preclinical models known to those trained in the art to demonstrate therapeutic validation of pharma- ceutically viable cargoes, such as NATs, peptides and proteins, including, but not limited to, rodent models and non-human primates.

[0163]

[0269] The features and advantages of the present subject matter will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying drawings. As will be understood, the subject matter disclosed and claimed can be modified in various respects without departing from the scope of the claims. Accordingly, the drawings and following description are to be considered illustrative in nature, and not limiting, with the full scope of the present subject matter being set forth in the claims. EXAMPLES

[0164]

[0270] Overview:

[0271] All solvents and reagents were commercial products and were used as received unless otherwise noted. Temperatures are given in degrees Celsius. The structures of final starting materials, intermediates and final products are confirmed by standard analytical methods, e.g., MS or NMR. 1 H NMR spectra were obtained using an AVANCE NEO NanoBay Bruker 400MHz NMR spectrometer in CDCl. 3 Recorded in solution at 298 K. Chemical shifts are given in parts per million (ppm) relative to TMS (0.00), and the coupling constant J is 1The values ​​are given in Hertz (Hz) versus H. The following abbreviations are used to indicate signal patterns: s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet, dd = doublet of doublets, br s = broad singlet, dt = doublet of triplets. Unless otherwise stated, column purification was performed using an Isolera™ prime with an isocratic or gradient composition of the appropriate eluent.

[0165]

[0272] All final compounds were determined to be more than 85% pure (retention time RT, min) by analysis by reversed-phase UHPLC-MS using a Shimadzu Nexera UHPLC instrument, DAD and ELSD and an Acquity Peptide BEH C18 2.1 mm × 50 mm, 1.7 μm column and gradient, 10 mM ammonium bicarbonate in water (A) and acetonitrile:methanol 80:20 ratio (B). Gradient studies were performed at 0.8 mL / min linearly over 12 min from 80 to 100% B. The injection volume was 2 μL and the column temperature was ambient. Detection was performed in multimode by electrospray and atmospheric pressure chemical ionization (ESI and APCI) in positive and negative modes using a Shimadzu 2020 Single Quad mass spectrometer (Science Park, Singapore) and an evaporative light scattering detector (ELSD) except for PNI 76, 119, 120, 121, 122 and 127. Low-resolution MS data for PNIs 76, 119, 120, 121, 122 and 127 were recorded using a Bruker micrOTOF™ Time-of-Flight mass spectrometer with a positive electrospray ionization source on an Agilent™ 1200 HPLC. Sodium formate was used as a reference. Samples were introduced by flow injection via the HPLC with acetonitrile / water (0.1% formic acid) as the mobile phase.

[0166] Abbreviation

[0273] ACD-A = Anticoagulant citrate dextrose solution

[0274] AcOH = acetic acid

[0275] aq. = aqueous

[0276] cat.=catalyst

[0277] DCM = dichloromethane

[0278] DIPEA = N,N-diisopropylethylamine

[0279] DMAP = 4-dimethylaminodimethylaminopyridine

[0280] DMF = N,N-dimethylformamide

[0281] EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0282] EPO = erythropoietin

[0283] ESI = electrospray ionization

[0284] EtOAc = ethyl acetate

[0285] Et 2 O = diethyl ether

[0286] g = grams

[0287] h=time

[0288] Hz = Hertz

[0289] K = Kelvin

[0290] MC3=DLin-MC3-DMA

[0291] MeOH = methanol

[0292] mg = milligrams

[0293] MHz = Megahertz

[0294] min=minutes

[0295] mL = milliliters

[0296] mmol = millimolar

[0297] MS=mass spectrometry

[0298] NMR=nuclear magnetic resonance

[0299] Pet.=oil

[0300] ppm = parts per million

[0301] Satd.=saturated

[0302] TFA = trifluoroacetic acid

[0303] THF = tetrahydrofuran

[0304] TLC = Thin Layer Chromatography

[0305] TMS = tetramethylsilane

[0306] TNS = 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt

[0307] UHPLC = Ultra High Performance Liquid Chromatography

[0308] ℃=degree Celsius temperature

[0167] Example 1 Synthesis of 1,4-anhydroxylitol (1) [ka]

[0168]

[0309] In a 250 mL one-neck round-bottom flask equipped with a reflux condenser, add (±)-xylitol (20.0 g, 131.4 mmol) and add 10% aq. H 2 SO 4 (10 mL). The reaction mixture was heated at 135° C. for 6 h. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with aqueous saturated NaHCO 3 The solution was quenched with ethyl acetate and lyophilized to give crude (±)-1,4-anhydroxylitol (1, 14.5 g, 23.86 mmol, 82.0% yield) as a thick colorless oil which was used in the next step without further purification. 1 H (400MHz, MeOD) δ 4.16-4.03 (m, 4H), 3.82 (dd, 1H, J = 12.0, 4.0), 3.74 (dd, 1H, J =12.0, 4.0), 3.66 (d, 1H, J = 8.0).

[0169] Example 2

[0310] Synthesis of PNIs 121, 122, 127, 321, 325, 328, 329, 336, 538, 539, and 540 [ka]

[0170]

[0311] PNI 121: [ka]

[0312] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added DMAP (cat.), followed by addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) with N 2 The mixture was stirred at room temperature for 4 h. DCM (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added to the reaction mixture and stirred at room temperature for 4 h. TLC analysis of the reaction mixture shows complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by linoleic acid (7.2 mL, 23.3 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol). Then, dry DMF (20 mL) and dry CH 2 Cl 2 (10 mL) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL) and washed with water (3×20 mL). The organic layer was washed with brine and anhyd.Na 2 SO 4 The crude mixture was dried over ice and evaporated to dryness on a rotary evaporator to give 5% MeOH / CH 2 Cl 2 The crude product was purified by silica gel column chromatography using 1,2-dichloromethane as the eluent to give PNI 121 as a colorless oil (590 mg, 0.76 mmol) in 8% yield. 1 H (500MHz, CDCl 3) δ 5.42-5.31 (m, 9H), 5.13-5.11 (m, 1H), 4.33-4.23 (m, 3H), 4.20-4.16(m, 1H), 3.76 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 2.78 (apparently t,4H, J = 5.0 Hz), 2.40-2.32 (m, 8H), 2.26 (s, 6H), 2.06 (q, 8H, J = 15.0 Hz, 5.0Hz), 1.85-1.79 (m, 2H), 1.65-1.60 (m, 4H), 1.39-1.26 (m, 28H), 0.90 (apparently t, 6H, J = 7.5 Hz). C 47 H 82 NO 7 Molecular weight [M+H] + Calculated value: 772.6091. Measured value: 772.6126.

[0171]

[0313] PNI 122: [ka]

[0172]

[0314] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added DMAP (cat.), followed by addition of a solution of 5-(dimethylamino)pentanoic acid hydrochloride (1.68 g, 9.3 mmol) in dry DMF (10 mL) with N 2 The mixture was stirred at room temperature for 4 h. DCM (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added and the reaction was stirred at room temperature for 4 h. TLC analysis of the reaction mixture indicates complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by linoleic acid (7.2 mL, 23.25 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol). Next, dry DMF (20 mL) and dry DCM (10 mL) were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated on a rotary evaporator, diluted with ethyl acetate (50 mL) and washed with water (3×20 mL). The organic layer was washed with brine and anhydrous NaCl. 2 SO4 The crude mixture was dried on a rotary evaporator and evaporated to dryness to give a crude mixture that was purified by silica gel column chromatography using 5% MeOH / DCM as eluent to give PNI 122 as a colorless oil (912 mg, 1.16 mmol) in 12% yield. 1 H (500MHz, CDCl 3 ) δ 5.42-5.31 (m, 9H), 5.13-5.11 (m, 1H), 4.35-4.23 (m, 3H), 4.17 (dd,1H, J = 10.0 Hz, 5.0 Hz), 3.76 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 2.78 (apparently t, 4H, J = 5.0 Hz), 2.39-2.30 (m, 8H), 2.25 (s, 6H), 2.06 (q, 8H, J= 15.0 Hz, 5.0 Hz), 1.69-1.60 (m, 6H), 1.52 (p, 2H, J = 7.5Hz), 1.39-1.26 (m,28H), 0.90 (apparent t, 6H, J = 7.5 Hz). C 48 H 84 NO 7 Molecular weight [M+H] + Calculated value: 786.6248. Measured value: 786.6365.

[0173]

[0315] PNI 127: [ka]

[0174]

[0316] To a solution of (±)-1 (200 mg, 1.49 mmol) in dry DMF (2 mL) was added DMAP (cat.), followed by addition of a solution of 1,4-dimethylpiperidine-4-carboxylate hydrochloride (289 mg, 1.49 mmol) in dry DMF (3 mL) under N 2The mixture was stirred at room temperature under atmospheric pressure. DCM (5 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (571 mg, 2.98 mmol) was added and the reaction was stirred overnight. TLC analysis of the reaction mixture shows complete consumption of (±)-1. DMAP (cat.) was added to the reaction mixture, followed by linoleic acid (1.2 mL, 3.73 mmol) and EDCI hydrochloride (1.14 g, 5.96 mmol). Next, dry DMF (5 mL) and dry DCM (5 mL) were added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (20 mL) and washed with water (3×10 mL). The organic layer was washed with brine and anhydrous NaCl was added. 2 SO 4 The crude mixture was dried on a rotary evaporator and evaporated to dryness to give a crude mixture that was purified by silica gel column chromatography using 5% MeOH / DCM as eluent to give PNI 127 as a colorless oil (225 mg, 0.28 mmol) in 19% yield. 1 H (500MHz, CDCl 3 ) δ 5.42-5.31 (m, 9H), 5.14-5.12 (m, 1H), 4.34-4.27 (m, 2H), 4.24 (apparently d, 2H, J = 5.0 Hz), 3.76 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 2.78 (apparently t, 4H, J = 5.0 Hz), 2.69 (brs, 2H), 2.36-2.26 (m, 8H), 2.20-2.12 (m,3H), 2.06 (q, 8H, J = 15.0 Hz, 5.0 Hz), 1.66-1.60 (m, 6H), 1.38-1.29 (m, 28H),1.22 (s, 3H), 0.90 (apparent t, 6H, J = 7.5 Hz). C 49 H 84 NO 7 Molecular weight [M+H] + Calculated value: 798.6248. Measured value: 798.6157.

[0175]

[0317] PNI 321: [ka]

[0176]

[0318] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.50 g, 3.73 mmol) in dry DMF (10 mL) was added a solution of DMAP (0.046 g, 0.373 mmol) and 3-(dimethylamino)propionoic acid hydrochloride (0.573 g, 3.73 mmol) in dry DMF (2.5 mL). To this stirred solution was added dry DCM (10 mL) followed by EDCI hydrochloride (1.429 g, 7.46 mmol) and DIPEA (1.628 ml, 9.32 mmol). The reaction mixture was stirred at room temperature for 4 h, at which time 1 was completely consumed as shown by TLC. DMAP (0.091 g, 0.746 mmol) was added to the reaction mixture followed by linoleic acid (2.61 g, 9.32 mmol) in dry DMF (7.5 mL). Then EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (3.26 ml, 18.64 mmol) were added, followed by dry DCM (10 mL). The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2×50 mL). The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layers were washed with brine (2×50 mL). The organic layer was separated and washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 80% EtOAc in pet. ether to give PNI 321 (0.265 g, 0.327 mmol, 8.78% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42 - 5.30 (m, 9H), 5.15 - 5.11 (m, 1H), 4.34 - 4.16 (m, 4H), 3.76 (dd, 1H, J = 8.0, 4.0), 2.78 (apparent t, 4H, J = 6.0), 2.63 (apparent t, 2H, J = 6.0), 2.52 (apparent t, 2H, J = 6.0), 2.36 - 2.31 (m, 4H), 2.25 (s, 6H), 2.06 (q, 8H, J = 8.0), 1.64 - 1.61 (m, 4H), 1.38 - 1.26 (m, 28H), 0.91 - 0.88 (m, 6H). RT = 3.61 min. Purity 93.7%. C 46 H 80 NO 7 The ESI-MS of: m / z = 759 [M + H] + .

[0177] PNI 325:

Chem.

[0178]

[0319] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 700 mg, 5.22 mmol) and DMAP (63.8 mg, 0.522 mmol) in dry DMF (5 mL) at 25 °C, a solution of 1-methylpiperidine-4-carboxylic acid (747 mg, 5.22 mmol) in dry DMF (2.5 mL) was added under nitrogen atmosphere. Then, dry DCM (10 mL) and EDCI hydrochloride (2 g, 10.44 mmol) were added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h, when TLC analysis showed complete consumption of 1. A solution of DMAP (63.8 mg, 0.522 mmol) and linoleic acid (3659 mg, 13.05 mmol) in DMF (5 mL) was added to the reaction mixture. Then, EDCI hydrochloride (2 g, 10.44 mmol) was added, followed by dry DCM (10 mL). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. Completion of the reaction was confirmed by TLC analysis, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2×50 mL). The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layers were washed with brine (2×50 mL). The organic layer was separated and washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 325 (0.600 g, 0.765 mmol, 14.66% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42 - 5.30 (m, 9H), 5.11 (apparent s, 1H), 4.33 - 4.15 (m, 4H), 3.76 (d, 1H, J = 12.0), 2.83 (br s, 2H), 2.78 (apparent t, 4H, J = 6.0), 2.35 - 2.29 (m, 5H), 2.26 (s, 3H), 2.06 (q, 8H, J = 8.0), 2.01 - 1.89 (m, 4H), 1.81 - 1.75 (m, 2H), 1.68 - 1.59 (m, 4H), 1.40 - 1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0). RT = 3.63 min. Purity 97.4%. C 48 H 82 NO 7 ESI-MS of: m / z = 785 [M + H] + .

[0179] PNI 328:

Chem.

[0180]

[0320] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 700 mg, 5.22 mmol) and DMAP (63.8 mg, 0.522 mmol) in dry DMF (5 mL) at 25 °C, a solution of 1-methylpyrrolidine-3-carboxylic acid (674 mg, 5.22 mmol) in dry DMF (2.5 mL) was added under nitrogen atmosphere. Then, dry DCM (10 mL) was added, followed by EDCI hydrochloride (2 g, 10.44 mmol). The reaction mixture was stirred at room temperature for 4 h, whereupon TLC analysis showed complete consumption of 1. Then, a solution of DMAP (63.8 mg, 0.522 mmol) and linoleic acid (3659 mg, 13.05 mmol) in dry DMF (5 mL) was added to the above reaction mixture. Then, EDCI hydrochloride (4002 mg, 20.88 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2×50 mL). The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layers were washed with brine (2×50 mL). The organic layers were separated and washed with anhydrous Na 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 328 (0.290 g, 0.377 mmol, 7.22% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 5.41-5.30 (m, 9H), 5.12 (apparently s, 1H),4.34-4.19 (m, 4H), 3.76 (d, 1H, J = 12.0), 3.09-3.05 (m, 1H), 2.82-2.76 (m,5H), 2.71-2.51 (m, 3H), 2.36-2.32 (m, 7H), 2.14-2.03 (m, 10H), 1.68-1.60 (m,4H), 1.36-1.26 (m, 28H), 0.90 (apparently t, 6H, J = 6.0). RT= 3.62 minutes. Purity 93.1%. C 47 H80 NO 7 ESI-MS: m / z = 771 [M+H] + .

[0181] PNI 329: [ka]

[0182]

[0321] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.7 g, 5.22 mmol) and DMAP (0.064 g, 0.522 mmol) in dry DMF (15 mL) at 25 °C, a solution of 1,3-dimethylpyrrolidine-3-carboxylic acid (0.747 g, 5.22 mmol) in dry DMF (5 mL) was added under nitrogen atmosphere. Then, dry DCM (5 mL), EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (2.279 ml, 13.05 mmol) were added successively. The reaction mixture was stirred at room temperature for 4 h, when TLC analysis showed complete consumption of 1. To the reaction mixture was added a solution of DMAP (0.064 g, 0.522 mmol) and linoleic acid (3.66 g, 13.05 mmol) in dry DMF (5 mL). Then EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added followed by dry DCM (5 mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2×50 mL). The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layer was washed with brine (2×50 mL). The organic layer was separated and washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 80% EtOAc in pet. ether to give PNI 329 (0.090 g, 0.115 mmol, 2.199% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42 - 5.29 (m, 9H), 5.14 - 5.12 (m, 1H), 4.35 - 4.22 (m, 4H), 3.76 (dd, 1H, J = 8.0, 4.0), 3.01 (apparent d, 1H, J = 8.0), 2.78 (t, 4H, J = 6.0), 2.71 - 2.57 (m, 2H), 2.47 - 2.39 (m, 2H), 2.39 - 2.31 (m, 7H), 2.09 - 1.97 (m, 9H), 1.79 - 1.67 (m, 4H), 1.37 - 1.26 (m, 31H), 0.88 (apparent t, 6H, J = 6.0). RT = 3.78 min. Purity 91.6%. C 48 H 82 NO 7 The ESI - MS of: m / z = 785 [M + H] + .

[0183]

[0323] PNI 336:

Chem.

[0184]

[0324] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.7 g, 5.22 mmol) in dry DMF (10 mL) was added DMAP (0.064 g, 0.522 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 2-(1-methyl-1H-imidazol-4-yl)acetic acid (0.731 g, 5.22 mmol) in DMF (5 mL) followed by dry DCM (5 mL). Then EDCI hydrochloride (2.001 g, 10.44 mmol) and DIPEA (2.329 ml, 13.05 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, when TLC analysis showed complete consumption of 1. To the above mixture was added a solution of DMAP (0.064 g, 0.522 mmol) and linoleic acid (3.66 g, 13.05 mmol) in DMF (5 mL). Then EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added. Finally, dry DCM (5 mL) was added and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed the completion of starting material. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2×30 mL) and the combined aqueous layers were extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (2×50 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried over ice. The organic layer was filtered and concentrated under reduced pressure. The crude material was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 336 (0.37 g, 0.474 mmol, 9.08% yield) as a pale yellow liquid. 1 H (400MHz, CDCl 3) δ 7.45 (s, 1H), 6.88 (s, 1H), 5.42 - 5.30 (m, 9H), 5.11 - 5.09 (m, 1H), 4.37 - 4.20 (m, 4H), 3.75 (dd, 1H, J = 8.0, 4.0), 3.70 - 3.69 (m, 2H), 3.68 (s, 3H), 2.77 (t, 4H, J = 6.0), 2.33 (t, 4H, J = 8.0), 2.05 (q, 8H, J = 8.0), 1.64 - 1.59 (m, 4H), 1.38 - 1.26 (m, 28H), 0.89 (apparent t, 6H, J = 6.0). RT = 3.44 min. Purity 89.4%. C 47 H 77 N 2 O 7 The ESI - MS of: m / z = 782 [M + H] + .

[0185]

[0325] PNI 538:

Chem.

[0186]

[0326] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.5 g, 3.73 mmol) in dry DMF (10 mL) was added DMAP (0.046 g, 0.373 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 2-(1-methylpyrrolidin-3-yl)acetic acid (0.587 g, 4.10 mmol) in DMF (5 mL) followed by dry DCM (10 mL). EDCI hydrochloride (1.429 g, 7.46 mmol) and DIPEA (1.664 mL, 9.32 mmol) were added to the contents of the reaction flask. The reaction mixture was stirred at room temperature for 4 h, when TLC analysis indicated complete consumption of 1. Then a solution of DMAP (0.455 g, 3.73 mmol) and linoleic acid (2.61 g, 9.32 mmol) in DMF (5 mL) was added. To the reaction mixture, EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol) were added. Finally, dry DCM (10 mL) was added and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed consumption of starting material. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2×30 mL) and the combined aqueous layers were extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (2×50 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried over ice. The organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 538 (0.165 g, 0.210 mmol, 5.64% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.43 - 5.30 (m, 9H), 5.12 - 5.11 (m, 1H), 4.32 - 4.14 (m, 4H), 3.75 (dd, 1H, J = 12.0, 4.0), 2.83 - 2.73 (m, 5H), 2.65 - 2.50 (m, 3H), 2.46 - 2.44 (m, 2H), 2.37 - 2.82 (m, 8H), 2.18 - 2.03 (m, 10H), 1.55 - 1.42 (m, 4H), 1.40 - 1.24 (m, 28H), 0.90 (apparently t, 6H, J = 6.0). RT = 3.23 min. Purity 87.0%. C 48 H 82 NO 7 The ESI-MS of: m / z = 785 [M+H] + .

[0187]

[0327] PNI 539:

Chem.

[0188]

[0328] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25° C., a solution of 4-(pyrrolidin-1-yl)butanoic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL) was added under nitrogen atmosphere. Then, EDCI hydrochloride (1.715 g, 8.95 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 4 h, when TLC analysis showed complete consumption of 1. To the reaction mixture was added a solution of DMAP (0.109 g, 0.895 mmol) and linoleic acid (3.14 g, 11.18 mmol) in dry DMF (7.5 mL). Then EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2×40 mL). The aq. layer was extracted with EtOAc (2×35 mL) and the combined organic layer was washed with brine (2×50 mL). The organic layer was separated and washed with anhydrous Na 2 SO 4 The crude product was purified by column chromatography (Isolera™) on silica gel (100-200 mesh) with 70% EtOAc in pet. ether to give PNI 539 (0.5 g, 0.608 mmol, 13.58% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 5.42-5.30 (m, 9H), 5.13-5.11 (m, 1H), 4.33-4.14 (m, 4H), 3.75 (dd,1H, J = 12.0, 4.0), 2.78 (t, 4H, J = 6.0), 2.50-2.31 (m, (apparently t, 6H, J = 6.0). RT = 3.71 min. Purity 90.6%. C49 H 84 NO 7 ESI-MS: m / z = 799 [M+H] + .

[0189] PNI 540: [ka]

[0190]

[0329] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25° C., 2-(1-methylpiperidin-2-yl)acetic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL) was added under nitrogen atmosphere. EDCI (1.715 g, 8.95 mmol) and dry DCM (10 mL) were then added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To this reaction mixture was added a solution of DMAP (0.109 g, 0.895 mmol) and linoleic acid (3.14 g, 11.18 mmol) in DMF (7.5 mL). Then EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred under nitrogen atmosphere at 25° C. for 16 h. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2×40 mL). The aq. layer was extracted with EtOAc (2×35 mL) and the combined organic layer was washed with brine (2×50 mL). The organic layer was washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 75% EtOAc in pet. ether to give PNI 540 (0.41 g, 0.500 mmol, 11.18% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42-5.30 (m, 9H), 5.13-5.11 (m, 1H), 4.33-4.17 (m, 4H), 3.75 (dd,1H, J = 12.0, 4.0), 2.86 (br s, 2H), 2.78 (t, 4H, J = 6.0), 2.60 (br s, 1H),2.46-2.27 (m, 9H), 2.06 (q, 8H, J = 8.0), 1.77-1.61 (m, 10H), 1.40-1.25 (m,28H), 0.89 (apparently t, 6H, J = 6.0). RT = 3.79 minutes. Purity 97.4%. C 49 H 84 NO 7 ESI-MS: m / z = 799 [M+H] + .

[0191]

[0330] Example 3 Synthesis of PNI 342 and 541 [ka]

[0192]

[0331] Compound 2:

[0332] To a stirred solution of oleic acid (58 g, 205 mmol) in dry EtOH (580 mL) was added conc. 2 SO 4 (1.094 mL, 20.53 mmol) was slowly added and the reaction mixture was refluxed for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was cooled to 0 °C and saturated aqueous NaHCO 3 The solution was neutralized with 100 mL of DCM and extracted with DCM (3×250 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 After drying, filtration and concentration, ethyl oleate (2, 61.6 g, 198 mmol, 97% yield) was obtained as a colorless oil, which was used directly in the next step without further purification. 1 H (400MHz, CDCl 3) δ 5.39-5.31 (m, 2H), 4.13 (q, 2H, J = 8.0), 2.29 (t, 2H, J = 8.0),2.02 (apparently q, 4H, J = 8.0), 1.62 (p, 2H, J = 8.0),1.35-1.24 (m, 23H), 0.89 (t, 3H, J = 6.0). RT = 3.73 min. Purity 99.5%. C 20 H 39 O 2 ESI-MS: m / z = 311 [M+H] + .

[0193]

[0333] Compound 3:

[0334] A solution of diiodomethane (31.2 mL, 386 mmol) in toluene (120 mL) was stirred at −15° C. under nitrogen atmosphere. Diethylzinc (129 mL, 193 mmol, 1.5 M solution in toluene) was added dropwise to the reaction mixture at −15° C. for 30 min. Note: The internal temperature of the reaction mixture should be maintained below 0° C. Then, a solution of 2 (30 g, 97 mmol) in toluene (30 mL) was added dropwise to the above reaction mixture such that the internal temperature was maintained below 0° C. The reaction mixture was stirred at the same temperature for 15 min and allowed to warm to room temperature gradually over 30 min. After stirring at room temperature for 7 h, TLC analysis indicated the completion of the reaction. The reaction mixture was cooled to 0° C. and saturated aqueous NH 4 The mixture was quenched with Cl solution (100 mL). The organic layer was separated and the aq. layer was extracted with toluene (2×75 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 6% EtOAc in pet. ether to give 3 (28.05 g, 86 mmol, 89% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 4.13 (q, 2H, J = 8.0), 2.29 (t, 2H, J = 8.0), 1.63 (p, 2H, J =8.0), 1.38-1.24 (m, 25H), 1.19-1.08 (m, 2H), 0.89 (t, 3H, J = 6.0), 0.68-0.62(m, 2H), 0.59-0.54 (m, 1H), -0.34 (apparently q, 1H, J = 4.0).RT = 4.01 min. Purity 98.7%. C 21 H 41 O 2 ESI-MS: m / z = 325 [M+H] + .

[0194]

[0335] Compound 4:

[0336] LiOH (1.660 g, 69.3 mmol) was added to a stirred solution of 3 (15 g, 46.2 mmol) in EtOH (105 mL) and water (45 mL) and the reaction mixture was stirred at ambient temperature for 16 h. Upon completion of the reaction as indicated by TLC, the reaction mixture was concentrated in vacuo to give a residue. The residue was diluted with water (100 mL) and washed with MTBE (2×100 mL). The aq. layer was cooled to 0° C., acidified with 6N HCl and extracted with EtOAc (3×300 mL). The combined organic layers were washed with anhydrous NaCl. 2 SO 4 It was dried over water, filtered and concentrated to give 4 (13.05 g, 43.0 mmol, 93% yield) as a white solid. 1 H (400MHz, CDCl 3 ) δ 2.36 (t, 2H, J = 6.0), 1.64 (p, 2H, J = 8.0), 1.39-1.28 (m, 22H),1.19-1.10 (m, 2H), 0.89 (t, 3H, J = 6.0), 0.68-0.62 (m, 2H), 0.59-0.54 (m, 1H),-0.33 (apparently q, 1H, J = 4.0). RT = 2.91 min. Purity 97.7%. C 19 H 35 O 2 ESI-MS: m / z = 295 [MH] - .

[0195]

[0337] PNI 342: [ka]

[0196]

[0338] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.8 g, 5.96 mmol) in dry DMF (10 mL) was added DMAP (0.073 g, 0.596 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.916 g, 5.96 mmol) in dry DMF (3 mL), followed by dry DCM (10 mL) and then EDCI hydrochloride (2.287 g, 11.93 mmol). The reaction mixture was stirred at room temperature for 4 h and consumption of 1 was confirmed by TLC analysis. To the above reaction mixture was added a solution of DMAP (0.146 g, 1.193 mmol) and 4 (4.42 g, 14.91 mmol) in dry DMF (10 mL). EDCI hydrochloride (4.57 g, 23.86 mmol) was then added followed by dry DCM (10 mL) and the reaction mixture was stirred at room temperature for 16 h. TLC analysis indicated complete consumption of starting material and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water. The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layers were washed with brine (2×50 mL). The organic layer was separated and anhydrous Na 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 50% EtOAc in Pet. ether. The product was re-purified (Isolera™) using 10% acetone in DCM to give PNI 342 (0.19 g, 0.240 mmol, 4.03% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.38 - 5.35 (m, 1H), 5.16 - 5.12 (m, 1H), 4.34 - 4.14 (m, 4H), 3.80 - 3.74 (m, 1H), 2.66 - 2.58 (m, 2H), 2.55 - 2.50 (m, 2H), 2.36 - 2.31 (m, 4H), 2.25 (s, 6H), 1.62 (p, 4H, J = 8.0), 1.39 - 1.26 (m, 44H), 1.19 - 1.09 (m, 4H), 0.89 (apparent t, 6H, J = 8.0), 0.68 - 0.62 (m, 4H), 0.59 - 0.54 (m, 2H), -0.33 (apparent q, 2H, J = 4.0). RT = 5.03 min. Purity 94.7%. C 48 H 88 NO 7 The ESI-MS of: m / z = 791 [M+H] + .

[0197]

[0339] PNI 541:

Chem.

[0198]

[0340] To a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.6 g, 4.47 mmol) and DMAP (0.055 g, 0.447 mmol) in dry DMF (10 mL) at 25 °C, a solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.866 g, 4.47 mmol) in dry DMF (2.5 mL) was added. Then, EDCI hydrochloride (1.715 g, 8.95 mmol) and dry DCM (10 mL) were added. The reaction mixture was stirred at room temperature for 4 h, and TLC analysis showed complete consumption of 1. To this reaction mixture was added a solution of DMAP (0.109 g, 0.895 mmol) and 4 (3.32 g, 11.18 mmol) in dry DMF (7.5 mL). Then, EDCI hydrochloride (3.43 g, 17.89 mmol) and dry DCM (10 mL) were added to the reaction mixture. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in EtOAc (70 mL) and washed with water (2×40 mL). The aq. layer was extracted with EtOAc (2×35 mL) and the combined organic layer was washed with brine (2×50 mL). The organic layer was separated and anhydrous Na was added. 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 65% EtOAc in pet. ether to give PNI 541 (0.68 g, 0.819 mmol, 18.31% yield) as a colorless oil. 1 H (400MHz, CDCl 3) δ 5.35 (d, 1H, J = 4.0), 5.15 - 5.13 (m, 1H), 4.33 - 4.23 (m, 4H), 3.76(d, 1H, J = 12.0), 2.92 (br s, 2H), 2.61 - 2.39 (m, 4H), 2.36 (td, 4H, J = 8.0,4.0), 2.22 - 2.19 (m, 3H), 1.68 - 1.57 (m, 6H), 1.39 - 1.25 (m, 47H), 1.19 - 1.11 (m,4H), 0.89 (apparent t, 6H, J = 6.0), 0.71 - 0.62 (m, 4H),0.59 - 0.54 (m, 2H), -0.33 (q, 2H, 4.0). RT = 5.03 min. Purity 96.2%. C 51 H 92 NO 7 The ESI-MS of: m / z = 831 [M+H] + .

[0199] Example 4 Synthesis of PNI 535

Chem.

[0200]

[0341] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.70 g, 5.22 mmol) in dry DMF (5 mL) was added DMAP (0.064 g, 0.522 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.673 g, 5.74 mmol) in dry DMF (2.5 mL) followed by dry DCM (5 mL). EDCI hydrochloride (2.001 g, 10.44 mmol) and DIPEA (2.329 ml, 13.05 mmol) were then added. The reaction mixture was stirred at room temperature for 4 h, at which time TLC analysis indicated consumption of 1. To the reaction flask was added a solution of DMAP (0.064 g, 0.522 mmol) and 2-hexyldecanoic acid 5 (3.35 g, 13.05 mmol) in DMF (5 mL). Then EDCI hydrochloride (4.00 g, 20.88 mmol) and DIPEA (3.37 g, 26.1 mmol) were added. Finally, dry DCM (5 mL) was added and the reaction mixture was stirred at room temperature for 16 h. TLC analysis showed the completion of starting material. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2×30 mL) and the combined aq. layers were extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (2×50 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried over ice. The organic layer was separated, filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 60% EtOAc in pet. ether to give PNI 535 (0.43 g, 0.606 mmol, 11.60% yield) as a colorless liquid. 1 H (400MHz, CDCl 3) δ 5.35-5.32 (m, 1H), 5.12-5.08 (m, 1H), 4.34-4.20 (m, 4H), 3.80-3.73(m, 1H), 2.64-2.60 (m, 2H), 2.53-2.49 (m, 2H), 2.40-2.31 (m, 2H), 2.24 (s, 6H),1.60-1.53 ​​(m, 4H), 1.50-1.40 (m, 4H), 1.34-1.16 (m, 40H), 0.88 (apparently t, 12H, J = 8.0). RT = 3.08 min. Purity 97.9%.C 42 H 80 NO 7 ESI-MS: m / z = 711 [M+H] + .

[0201] Example 5 Synthesis of PNIs 119, 120, and 344 [ka]

[0202]

[0342] PNI 119: [ka]

[0203]

[0343] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added DMAP (cat.), followed by addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) with N 2 Added under atmosphere. CH 2 Cl 2 (25 mL) was added to the reaction mixture at room temperature. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added and the reaction was stirred at room temperature for 4 h. TLC analysis of the reaction mixture indicates complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by myristic acid (5.32 g, 23.3 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol) dissolved in dry DMF (20 mL) as solids. Then, dry CH2 Cl 2 (10 mL) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL) and washed with water (3×20 mL). The organic layer was washed with brine and anhyd.Na 2 SO 4 The crude mixture was dried on a rotary evaporator and evaporated to dryness to give PNI 119 as a colorless oil (780 mg, 1.17 mmol) in 13% yield. 1 H (500MHz, CDCl 3 ) δ 5.36 (d, 1H, J = 5.0 Hz), 5.13-5.11 (m, 1H), 4.33-4.28 (m, 2H),4.25 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 4.18 (dd, 1H, J = 10.0 Hz, 7.5 Hz), 3.75(d, 1H, J = 10.0 Hz), 2.39-2.32 (m, 6H), 2.28 (apparently t,2H, J = 5.0 Hz), 2.21 (s, 6H), 1.79 (p, 2H, J = 7.5 Hz), 1.65-1.59 (m, 4H),1.33-1.26 (m, 40H), 0.89 (apparent t, 6H, J = 7.5 Hz). C 39 H 74 NO 7 Molecular weight [M+H] + Calculated value: 668.5465. Measured value: 668.5466.

[0204]

[0344] PNI 120: [ka]

[0205]

[0345] To a solution of (±)-1 (1.25 g, 9.3 mmol) in dry DMF (15 mL) was added DMAP (cat.), followed by addition of a solution of 5-(dimethylamino)pentanoic acid hydrochloride (1.68 g, 9.3 mmol) in dry DMF (10 mL) with N2 The mixture was added at room temperature under atmospheric conditions. 2 Cl 2 (25 mL) was added to the reaction mixture. Finally, solid EDCI hydrochloride (3.56 g, 18.6 mmol) was added to the reaction mixture and stirred at room temperature for 4 h. TLC analysis of the reaction mixture shows complete consumption of (±)-1. Further, DMAP (cat.) was added to the reaction mixture, followed by myristic acid (5.31 g, 23.3 mmol) and EDCI hydrochloride (7.13 g, 37.2 mmol) as solids dissolved in dry DMF (20 mL). Then, dry DCM (10 mL) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (50 mL) and washed with water (3×20 mL). The organic layer was washed with brine and anhydrous Na 2 SO 4 The crude mixture was dried on a rotary evaporator and evaporated to dryness to give a crude mixture that was purified by silica gel column chromatography using 5% MeOH / DCM as eluent to give PNI 120 as a colorless oil (1.03 g, 1.51 mmol) in 16% yield. 1 H (500MHz, CDCl 3 ) δ 5.35 (d, 1H, J = 5.0 Hz), 5.12-5.11 (m, 1H), 4.32-4.23 (m, 3H),4.16 (dd, 1H, J = 10.0 Hz, 7.5 Hz), 3.75 (d, 1H, J = 10.0 Hz), C 40 H 76 NO 7 Molecular weight [M+H] + Calculated value: 682.5622. Measured value: 682.5589.

[0206]

[0346] PNI 344: [ka]

[0207]

[0347] To a stirred solution of (±)-1,4-anhydroxylitol (1, 0.500 g, 3.73 mmol) in dry DMF (5 mL) was added DMAP (0.046 g, 0.373 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.437 g, 3.73 mmol) in dry DMF (2.5 mL) and dry DCM (10 mL). EDCI hydrochloride (1.429 g, 7.46 mmol)) and DIPEA (1.623 ml, 9.32 mmol) were added and the reaction mixture was stirred at room temperature for 4 h. TLC analysis showed complete consumption of 1. Then a solution of DMAP (0.046 g, 0.373 mmol) and myristic acid (2.128 g, 9.32 mmol) in DMF (5 mL) was added to the above reaction mixture. Further, EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (3.25 ml, 18.64 mmol) were added. Finally, dry DCM (10 mL) was added to the reaction mixture and stirred at room temperature for 16 h. Completion of the reaction was confirmed by TLC analysis and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL) and washed with water (2×30 mL). The aq. layer was extracted with EtOAc (2×30 mL) and the combined organic layer was washed with brine (2×50 mL). The organic layer was separated and anhydrous Na was added. 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 344 (0.220 g, 0.336 mmol, 9.02% yield) as a colorless oil. 1 H (400MHz, CDCl 3) δ 5.38-5.34 (m, 1H), 5.13-5.11 (m, 1H), 4.34-4.16 (m, 4H), 3.76 (d,1H, J = 12.0), 2.65-2.61 (m, 2H), 2.54-2.50 (m, 2H), 2.36-2.31 (m, 4H), 2.25(s, 6H), 1.62 (p, 4H, J = 8.0), 1.35-1.22 (m, 40H), 0.88 (apparent t, 6H, J = 6.0). RT = 3.57 min. Purity 91.8%. C 38 H 72 NO 7 The ESI-MS of: m / z = 655 [M+H] + .

[0208] Example 6 Synthesis of PNI 534

Chemical formula

[0209]

[0348] To a stirred solution of (±)-1,4-anhydroxylitol (1, 1.0 g, 7.46 mmol) in dry DMF (5 mL) was added DMAP (0.091 g, 0.746 mmol) at room temperature under nitrogen atmosphere. To this mixture was added a solution of 3-(dimethylamino)propanoic acid hydrochloride (0.961 g, 8.20 mmol) in DMF (2.5 mL) followed by dry DCM (10 mL). Then EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, at which time TLC analysis indicated consumption of 1. To the reaction mixture was added a solution of DMAP (0.091 g, 0.746 mmol) and dodecanoic acid (3.73 g, 18.64 mmol) in dry DMF (5 mL). Then EDCI hydrochloride (5.72 g, 29.8 mmol) and DIPEA (4.82 g, 37.3 mmol) were added. Finally, dry DCM (10 mL) was added and the mixture was stirred at room temperature for 16 h. TLC analysis showed complete consumption of starting material. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The EtOAc layer was washed with water (2×30 mL) and the combined aq. layers were extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (2×50 mL) and anhyd.Na. 2 SO 4 The mixture was dried over ice. The organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 534 (0.230 g, 0.385 mmol, 5.16% yield) as a colorless oil. 1 H (400MHz, CDCl 3) δ 5.38-5.34 (m, 1H), 5.16-5.11 (m, 1H), 4.34-4.15 (m, 4H), 3.80-3.74(m, 1H), 2.64-2.59 (m, 2H), 2.53-2.49 (m, 2H), 2.36-2.31 (m, 4H), 2.24 (s, 6H),1.62 (p, 4H, J = 8.0), 1.36-1.21 (m, 32H), 0.88 (apparently t,6H, J = 6.0). RT = 1.85 min. Purity 85.6%.C 34 H 64 NO 7 ESI-MS: m / z= 599 [M+H] + .

[0210] Example 7 Synthesis of PNI 532 [ka]

[0211]

[0349] Compound 8:

[0350] To a stirred solution of 8-(tert-butoxy)-8-oxooctanoic acid (6, 11.36 g, 49.3 mmol) in dry DCM (100 mL) was added DMAP (0.524 g, 4.29 mmol) at room temperature under nitrogen atmosphere. (Z)-Non-2-en-1-ol (7, 6.1 g, 42.9 mmol) was added to the above mixture and stirring was continued for 15 min. The reaction was cooled to 0 °C and DCC (9.73 g, 47.2 mmol) was added. The reaction mixture was allowed to reach room temperature and stirred for 16 h. TLC analysis showed complete consumption of starting material. The precipitated urea was filtered through a celite pad and washed with DCM (2 × 50 mL). The combined filtrate was concentrated and the residue was purified by satd. aq. NaHCO 3 The organic layer was separated and washed with anhydrous Na 2 SO 4The solvent was removed under reduced pressure, and the residue was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 3% EtOAc in pet. ether to give (Z)-1-(tert-butyl) 8-(non-2-en-1-yl)octanedioate (8, 11.35 g, 29.8 mmol, 69.4% yield). 1 H (400MHz, CDCl 3 ) δ 5.68-5.61 (m, 1H), 5.56-5.49 (m, 1H), 4.63 (d, 2H, J = 4.0), 2.31(t, 2H, J = 8.0), 2.20 (t, 2H, J = 8.0), 2.10 (apparently q,2H, J = 8.0), 1.67-1.57 (m, 4H), 1.44 (s, 9H), 1.40-1.25 (m, 12H), 0.89 (t, 3H,J = 6.0). RT = 1.40 min. Purity 89.2%.C 21 H 38 O 4 ESI-MS of Na: m / z= 377 [M+Na] + .

[0212]

[0351] Compound 9:

[0352] TFA (10.00 mL, 130 mmol) was added dropwise to a stirred solution of 8 (5 g, 14.10 mmol) in dry DCM (50 mL) at room temperature under nitrogen atmosphere and stirring was continued for 16 h. TLC analysis indicated the presence of 8. Again, TFA (10.00 mL, 130 mmol) was added and the reaction mixture was refluxed for 16 h. The reaction did not go to completion and TLC analysis indicated the presence of 8. The reaction was stopped and the mixture was evaporated under reduced pressure. The crude material was purified by column silica gel (100-200 mesh) chromatography (Isolera™) using 30% EtOAc in Pet. ether to give (Z)-8-(non-2-en-1-yloxy)-8-oxooctanoic acid (9, 3.41 g, 11.43 mmol, 81% yield) as a brown oil and 600 mg of 8 was also recovered. 1 H (400MHz, CDCl 3) δ 5.68-5.61 (m, 1H), 5.56-5.49 (m, 1H), 4.63 (d, 2H, J = 4.0),2.37-2.30 (m, 4H), 2.10 (apparently q, 2H, J = 8.0), 1.68-1.60(m, 4H), 1.40-1.24 (m, 12H), 0.89 (t, 3H, J = 6.0).

[0213]

[0353] PNI 532 [ka]

[0214]

[0354] To a 100 mL two-neck round bottom flask containing a well-stirred solution of (±)-1,4-anhydroxylitol (1, 0.5 g, 3.73 mmol) in dry DMF (5 mL) was added a solution of DMAP (0.046 g, 0.373 mmol) and 3-(dimethylamino)propanoic acid hydrochloride (0.480 g, 4.10 mmol) in dry DMF (2.5 mL) at room temperature under nitrogen atmosphere. To this mixture was added dry DCM (10 mL) followed by EDCI (1.429 g, 7.46 mmol) and DIPEA (1.204 g, 9.32 mmol). The reaction mixture was stirred at ambient temperature for 4 h, whereupon TLC analysis indicated consumption of 1. DMAP (0.046 g, 0.373 mmol) was added to the above reaction mixture. A solution of 9 (2.78 g, 9.32 mmol) in dry DMF (5 mL) was added, followed by EDCI hydrochloride (2.86 g, 14.91 mmol) and DIPEA (2.409 g, 18.64 mmol). Finally, dry DCM (10 mL) was added and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (60 mL). The organic layer was washed with water (2×30 mL) and the aq. layer was extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (2×50 mL) and anhydrous NaCl was added. 2 SO 4The mixture was dried over ice. The organic layer was separated, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 70% EtOAc in pet. ether to give PNI 532 (0.270 g, 0.340 mmol, 9.12% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ5.68-5.61 (m, 2H), 5.55-5.49 (m, 2H), 5.37-5.34 (m, 1H),5.16-5.10 (m, 1H), 4.62 (d, 4H, J = 8.0), 4.33-4.10 (m, 4H), 3.79-3.73 (m, 1H),2.65-2.60 (m, 2H), 2.53-2.50 (m, 2H), 2.36-2.28 (m, 8H), 2.25 (s, 6H), 2.10 (q,4H, J = 8.0), 1.67-1.59 (m, 8H), 1.39-1.24 (m, 24H), 0.89 (apparent t, 6H, J = 6.0). RT = 2.24 min. Purity 97.2%. C 44 H 76 NO 11 ESI-MS: m / z = 795 [M+H] + .

[0215] Example 8 Synthesis of PNI 127, 573, 574, and 575 [ka]

[0216]

[0355] Compound 10:

[0356] In a 100 mL one-neck round bottom flask, a well-stirred solution of (±)-1,4-anhydroxylitol (1, 5.1 g, 38.0 mmol) in dry pyridine (25 mL), trityl chloride (10.07 g, 36.1 mmol) was added at room temperature under nitrogen atmosphere and stirring was continued for 16 h. After completion of the reaction as indicated by TLC, excess solvent was concentrated under reduced pressure. The residue was dissolved in DCM (100 mL) and water (200 mL). The organic layer was separated and the aq. layer was extracted with DCM (3×50 mL). The combined organic layers were washed with brine (3×100 mL) and anhydrous NaCl was added. 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography using 50% EtOAc in pet. ether to give (±)-10 (8.65 g, 22.93 mmol, 60.3% yield) as a white sticky solid. 1 H (400MHz, CDCl 3 ) δ 7.47-7.43 (m, 6H), 7.34-7.30 (m, 6H), 7.28-7.23 (m, 3H), 4.32 (brs, 1H), 4.28-4.23 (m, 3H), 3.77 (dd, 1H, J = 12.0, 4.0), 3.50 (dd, 1H, J =12.0, 4.0), 3.43 (dd, 1H, J = 8.0, 4.0), 3.14 (br s, 1H). RT = 2.66 min. Purity 99.9%. C 24 H 23 O 4 ESI-MS: m / z = 375 [MH] - .

[0217]

[0357] Compound 11:

[0358] To a 250 mL 3-neck round bottom flask was added a stirred solution of linoleic acid (6.55 g, 23.36 mmol) in dry DCM (50 mL), DMAP (2.85 g, 23.36 mmol), EDCI hydrochloride (17.91 g, 93 mmol) and DIPEA (9.38 ml, 53.7 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 15 min and a solution of (±)-10 (3.52 g, 9.34 mmol) in dry DCM (20 mL) was added. After stirring for 16 h, the reaction mixture was quenched with water (150 mL) and extracted with DCM (3×50 mL). The combined organic layers were washed with brine (3×75 mL) and anhydrous NaCl. 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% EtOAc in pet. ether to give (±)-11 (7.21 g, 7.84 mmol, 33.6% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 7.44-7.42 (m, 6H), 7.32-7.28 (m, 6H), 7.26-7.22 (m, 3H), 5.43-5.31(m, 9H), 5.12-5.10 (m, 1H), 4.35-4.31 (m, 1H), 4.26 (dd, 1H, J = 8.0, 4.0),3.74 (dd, 1H, J = 12.0, 4.0), 3.35 (apparently t, 1H, J =8.0), 3.16 (dd, 1H, J = 8.0, 6.0), 2.79 (t, 4H, J = 6.0), 2.36 (t, 2H, J =8.0), 2.16-1.99 (m, 10H), 1.65 (p, 2H, J = 8.0), 1.40-1.22 (m, 30H), 0.90 (t,6H, J = 6.0). RT = 3.95 min. Purity 98.1%.C 60 H 84 O 6 ESI-MS of Na: m / z= 923 [M+Na] + .

[0218]

[0359] compound 12

[0360] TFA (0.427 ml, 5.55 mmol) was added to a solution of (±)-11 (2.0 g, 2.219 mmol) and triethylsilane (1.772 ml, 11.09 mmol) in dry DCM (50 ml) under nitrogen at 0° C. The reaction mixture was stirred at 0° C. for 1 h, at which time TLC analysis indicated complete consumption of 11. The mixture was diluted with satd.aq.NaHCO 3 The mixture was quenched with anhydrous sodium hydroxide (50 mL) and extracted with DCM (2×50 mL). 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 10% EtOAc in pet. ether to give (±)-12 (1.15 g, 1.745 mmol, 79% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 5.43-5.29 (m, 9H), 5.11-5.09 (m, 1H), 4.51 (dd, 1H, J = 12.0, 8.0),4.30 (dd, 1H, J = 8.0, 4.0), 4.20-4.16 (m, 2H), 4.11-4.07 (m, 1H), 3.76 (dd,1H, J = 12.0, 4.0), 2.78 (t, 4H, J = 6.0), 2.37-2.30 (m, 4H), 2.06 (q, 8H, J =8.0), 1.69-1.62 (m, 4H), 1.40-1.25 (m, 28H), 0.90 (t, 6H, J = 6.0). RT = 2.29 min. Purity 92.5%. C 41 H 71 O 6 ESI-MS: m / z = 659 [M+H] + .

[0219]

[0361] PNI 127 (alternative method)

[0362] To a 100 mL two-necked round bottom flask containing a well-stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.970 g, 5.01 mmol) in dry DCM (30 ml), DMAP (0.556 g, 4.55 mmol) and EDCI hydrochloride (1.745 g, 9.10 mmol) were added under nitrogen atmosphere. The reaction mixture was stirred at 25° C. for 10 min, a solution of (±)-12 (3 g, 4.55 mmol) in dry DCM (15 mL) was added, and the reaction mixture was stirred at 25° C. for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (150 mL), washed with water (2×100 mL), brine (2×100 mL), and anhydrous NaCl. 2 SO 4 The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 3% MeOH in DCM to give PNI 127 (2.96 g, 3.71 mmol, 81% yield) as a pale yellow oil.

[0220]

[0363] PNI 574: [ka]

[0221]

[0364] To a 100 mL two-neck round bottom flask containing a stirred solution of 2-(4-methylpiperazin-1-yl)acetic acid (0.125 g, 0.789 mmol) in dry DCM (25 mL) were added DMAP (0.102 g, 0.789 mmol) and EDCI hydrochloride (0.348 g, 1.821 mmol) under nitrogen atmosphere. The reaction mixture was stirred at 25° C. for 10 min and a solution of (±)-12 (0.4 g, 0.607 mmol) in dry DCM (5 mL) was added. The mixture was stirred at 25° C. for 16 h and completion of the reaction was confirmed by TLC analysis. The reaction mixture was quenched with water (120 mL) and extracted with DCM (3×50 mL). The combined organic layers were washed with brine (3×75 mL) and anhydrous NaCl was added. 2 SO 4The mixture was dried over hexane, filtered and concentrated under reduced pressure. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 574 (0.090 g, 0.101 mmol, 16.70% yield) as a thick red oil. 1 H (400MHz, CDCl 3 ) δ 5.42-5.30 (m, 9H), 5.13 (s, 1H), 4.33-4.14 (m, 4H), 3.74 (d, 1H, J= 4.0), 3.27 (s, 2H), 2.78 (t, 4H, J = 6.0), 2.73-2.46 (m 8H), 2.39-2.26 (m,7H), 2.06 (q, 8H, J = 8.0), 1.66-1.58 (m, 4H), 1.49-1.26 (m, 28H), 0.90 (apparently t, 6H, J = 6.0). RT = 2.34 min. Purity 93.3%.C 48 H 83 N 2 O 7 ESI-MS: m / z = 800 [M+H] + .

[0222]

[0365] PNI 573: [ka]

[0223]

[0366] PNI 573 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 573 was carried out using quinuclidine-4-carboxylate hydrochloride (0.092 g, 0.592 mmol), DMAP (0.076 g, 0.592 mmol), EDCI hydrochloride (0.304 g, 1.593 mmol) in dry DCM (25 mL), and 12 (0.3 g, 0.455 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 573 (0.18 g, 0.226 mmol, 49.7% yield) as a pale yellow oil.1 H (400MHz, CDCl 3 ) δ 5.42-5.30 (m, 9H), 5.14-5.07 (m, 1H), 4.32-4.23 (m, 3H), 4.11 (dd,1H, J = 12.0, 8.0), 3.76 (dd, 1H, J = 12.0, 4.0), 3.24-3.05 (m, 6H), 2.77 (t,4H, J = 6.0), 2.33 (q, 4H, J = 8.0), 2.08-1.98 (m, 10H), 1.92 (apparently t, 4H, J = 8.0), 1.66-1.58 (m, 4H), 1.42-1.26 (m, 28H), 0.89 (t, 6H,J = 6.0). RT = 2.34 min. Purity 92.4%.C 49 H 82 NO 7 ESI-MS: m / z= 796 [M+H] + .

[0224]

[0367] PNI 575: [ka]

[0225]

[0368] PNI 575 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 143 was carried out using 1-methylpiperidine-3-carboxylic acid (0.085 g, 0.592 mmol), DMAP (0.076 g, 0.592 mmol), EDCI hydrochloride (0.304 g, 1.593 mmol) in dry DCM (25 mL), and 12 (0.3 g, 0.455 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 575 (0.265 g, 0.338 mmol, 74.2% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42-5.10 (m, 9H), 5.11 (s, 1H), 4.34-4.12 (m, 4H), 3.77 (d, 1H, J= 12.0), 2.99-2.59 (m, 6H), 2.40-2.13 (m, 7H), 2.11-1.84 (m, 11H), 1.80-1.52(m, 8H), 1.42-1.26 (m, 28H), 0.90 (apparent t, 6H, J = 6.0).RT = 2.48 min. Purity 93.2%. C 48 H 82 NO 7 ESI-MS: m / z = 785 [M+H] + .

[0226] Example 9 Synthesis of PNIs 576, 577, and 578 [ka]

[0227]

[0369] Compound 13:

[0370] Compound 13 was synthesized using a method similar to that used for the synthesis of 11. The synthesis of 13 was carried out using 4 (9.06 g, 30.5 mmol), DMAP (4.05 g, 33.2 mmol), EDCI hydrochloride (10.15 g, 53.1 mmol), DIPEA (14.28 mL, 80 mmol) in dry DCM (70 mL), and (±)-10 (5.0 g, 13.28 mmol) in dry DCM (30 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% EtOAc in pet. ether to give 13 (11.1 g, 11.89 mmol, 90% yield) as a colorless oil. 1 H (400MHz, CDCl 3) δ 7.46-7.44 (m, 6H), 7.34-7.24 (m, 9H), 5.44 (d, 1H, J = 4.0),5.14-5.12 (m, 1H), 4.37-4.33 (m, 1H), 4.28 (dd, 1H, J = 12.0, 4.0), 3.76 (dd,1H, J = 12.0, 4.0), 3.38 (dd, 1H, J = 12.0, 8.0), 3.19 (dd, 1H, J = 12.0, 8.0),2.39 (apparently t, 2H, J = 6.0), 2.18-2.02 (m, 2H), 1.67 (p,2H, J = 8.0), 1.50-1.24 (m, 46H), 1.19-1.13 (m, 4H), 0.92 (t, 6H, J = 6.0),0.71-0.65 (m, 4H), 0.62-0.57 (m, 2H), -0.30 (q, 2H, J = 6.0).

[0228]

[0371] Compound 14:

[0372] Compound 14 was synthesized using a method similar to that used for the synthesis of 12. The synthesis of 14 was carried out by reacting (±)-13 (11.0 g, 11.78 mmol) and Et 3 SiH (9.41 ml, 58.9 mmol) and TFA (1.489 mL, 11.78 mmol) were used. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 10% EtOAc in pet. ether to give 14 (5.5 g, 7.96 mmol, 67.5% yield) as a colorless oil. 1 H (400MHz, CDCl 3) δ 5.28-5.10 (m, 2H), 4.53-4.07 (m, 3H), 3.78-3.56 (m, 2H), 2.40-2.31(m, 4H), 1.68-1.59 (m, 4H), 1.38-1.10 (m, 44H), C 43 H 79 O 6 ESI-MS: m / z = 691 [M+H] + .

[0229]

[0373] PNI 576: [ka]

[0230]

[0374] PNI 576 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 576 was carried out using 1-cyclopropylpiperidine-4-carboxylic acid (0.108 g, 0.637 mmol), DMAP (0.085 g, 0.695 mmol), EDCI hydrochloride (0.222 g, 1.158 mmol) in dry DCM (15 mL), and 14 (0.4 g, 0.579 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 3% MeOH in DCM to give PNI 576 (0.45 g, 0.534 mmol, 92% yield) as a yellow oil. 1 H (400MHz, CDCl 3) δ 5.35 (s, 1H), 5.10 (s, 1H), 4.31-4.18 (m, 4H), 3.76 (d, 1H, J =12.0), 3.05-2.95 (m, 1H), 2.40-2.12 (m, 7H), 1.93-1.81 (m, 2H), 1.76-1.50 (m,8H), 1.42-1.26 (m, 44H), 1.19-1.05 (m, 4H), 0.89 (t, 6H, J = 6.0), 0.69-0.61(m, 4H), 0.59-0.54 (m, 2H), 0.48-0.33 (m, 4H), -0.33 (q, 2H, J = 6.0). RT =3.35 min. Purity 99.1%. C 52 H 92 NO 7 ESI-MS: m / z = 843 [M+H] + .

[0231]

[0375] PNI 577: [ka]

[0232]

[0376] PNI 577 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 577 was carried out using 1-ethylpiperidine-4-carboxylic acid (0.104 g, 0.658 mmol), DMAP (0.085 g, 0.658 mmol), EDCI hydrochloride (0.290 g, 1.519 mmol) in dry DCM (20 mL), and 14 (0.35 g, 0.506 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 577 (0.29 g, 0.349 mmol, 69.0% yield) as a thick, colorless oil. 1 H (400MHz, CDCl 3) δ 5.37-5.35 (m, 1H), 5.13 5.09 (m, 1H), 4.33-4.16 (m, 4H), 3.76 (d,1H, J = 12.0), 2.95-2.90 (m, 2H), 2.51-2.25 (m, 7H), 2.14-1.75 (m 6H), 1,70-1.55(m, 4H), 1.42-1.28 (m, 44H), 1.17-1.09 (m, 7H), 0.89 (t, 6H, J = 6.0),0.68-0.62 (m, 4H), 0.59-0.54 (m, 2H), -0.33 (q, 2H, J = 6.0). RT = 3.59 min. Purity 95.1%. C 51 H 92 NO 7 ESI-MS: m / z = 830 [M+H] + .

[0233]

[0377] PNI 578: [ka]

[0234]

[0378] PNI 578 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 578 was carried out using 2-(1-methylpiperidin-2-yl)acetic acid (0.096 g, 0.608 mmol), DMAP (0.078 g, 0.608 mmol), EDCI hydrochloride (0.290 g, 1.519 mmol) in dry DCM (20 mL), and 14 (0.35 g, 0.506 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 578 (0.32 g, 0.385 mmol, 76% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.35 (s, 1H), 5.13 (s, 1H), 4.32-4.16 (m, 4H), 3.76 (dt, 1H, J =8.0, 4.0), 2.81-2.18 (m, 12H), 1.77-1.51 (m, 10H), 1.45-1.21 (m, 44H),1.19-1.10 (m, 4H), 0.89 (t, 6H, J = 6.0), 0.68-0.61 (m, 4H), 0.59-0.54 (m, 2H),-0.33 (q, 2H, J = 4.0). RT = 3.61 minutes. Purity 99.7%. C 51 H 91 NO 7 ESI-MS of Na: m / z = 853 [M+Na] + .

[0235] Example 10 Synthesis of PNI 579 and 580 [ka]

[0236]

[0379] Compound 15:

[0380] Compound 15 was synthesized using a method similar to that used to synthesize 11. The synthesis of 15 was carried out using 2-hexyldecanoic acid (3.92 g, 15.27 mmol), DMAP (1.866 g, 15.27 mmol) in dry DCM (40 mL), followed by EDCI hydrochloride (6.37 g, 33.2 mmol), DIPEA (5.80 ml, 33.2 mmol), and (±)-10 (2.5 g, 6.64 mmol) in dry DCM (10 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 2% EtOAc in pet. ether to give 15 (5.12 g, 6.00 mmol, 90% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 7.47-7.45 (m, 6H), 7.33-7.22 (m, 9H), 5.29 (d, 1H, J = 4.0),5.09-5.07 (m, 1H), 4.29-4.24 (m, 2H), 3.75 (dd, 1H, J = 8.0, 4.0), 3.42 (dd,1H, J = 12.0, 8.0), 3.22 (dd, 1H, J = 12.0, 4.0), 2.43-2.34 (m, 1H), 2.22-2.15(m, 1H), 1.66-1.38 (m, 8H), 1.36-1.12 (m, 40H), 0.92-0.86 (m, 12H).

[0237]

[0381] Compound 16:

[0382] Compound 16 was synthesized using a method similar to that used to synthesize 12. The synthesis of 16 was carried out using (±)-15 (5.1 g, 5.98 mmol), triethylsilane (4.77 mL, 29.9 mmol), and TFA (1.151 mL, 14.94 mmol) in dry DCM (70 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 2% EtOAc in pet. ether to give (±)-16 (3.23 g, 5.29 mmol, 88% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 5.26 (d, 1H, J = 4.0), 5.16 (s, 1H), 4.29 (dd, 1H, J = 12.0, 4.0),4.21-4.17 (m, 1H), 3.80-3.74 (m, 2H), 3.58 (dd, 1H, J = 12.0. RT = 2.18 min. Purity 99.9%.C 37 H 71 O 6 ESI-MS: m / z = 611 [M+H] + .

[0238]

[0383] PNI 579 [ka]

[0239]

[0384] PNI 579 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 579 was carried out using 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.152 g, 0.786 mmol), DMAP (0.096 g, 0.786 mmol), EDCI hydrochloride (0.301 g, 1.571 mmol) in dry DCM (20 mL), and 16 (0.4 g, 0.655 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 4% MeOH in DCM to give PNI 579 (0.444 g, 0.592 mmol, 90% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 5.34 (d, 1H, J = 4.0), 5.11-5.10 (m, 1H), 4.32-4.27 (m, 3H),4.21-4.16 (m, 1H), 3.74 (dd, 1H, J = 12.0, 4.0), 2.69 (br s, 2H), 2.41-2.31 (m,5H), 2.22-2.14 (m, 4H), 1.63-1.41 (m, 10H), 1.30-1.22 (m, 43H), 0.88 (t, 12H, J= 8.0). RT = 2.66 min. Purity 97.4%. C 45 H 84 NO 7 ESI-MS: m / z = 751 [M+H] + .

[0240]

[0385] PNI 580: [ka]

[0241]

[0386]

[0387] PNI 580 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 580 was carried out using 4-(dimethylamino)butanoic acid hydrochloride (0.165 g, 0.982 mmol), DMAP (0.120 g, 0.982 mmol), EDCI hydrochloride (0.377 g, 1.964 mmol) in dry DCM (20 mL), and 16 (0.4 g, 0.655 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 4% MeOH in DCM to give PNI 580 (0.39 g, 0.539 mmol, 82% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 5.33 (d, 1H, J = 4.0), 5.10 (d, 1H, J = 4.0), 4.33-4.21 (m, 4H),3.75 (dd, 1H, J = 8.0, 4.0), 2.42-2.29 (m, 12H), 1.91-1.79 (m, 2H), 1.64-1.41(m, 8H), 1.34-1.20 (m, 40H), 0.88 (t, 12H, J = 6.0). RT = 2.49 min. Purity 98.7%. C 43 H 82 NO 7 ESI-MS: m / z = 724 [M+H] + .

[0242] Example 11 Synthesis of PNIs 581, 582, and 583 [ka]

[0243]

[0388] Compound 17:

[0389] To a stirred solution of 10 (2.0 g, 5.31 mmol) in dry DMF (100 mL) and dry THF (100 mL) in a 500 mL three-neck RBF at 0° C. under nitrogen atmosphere, NaH (1.062 g, 26.6 mmol) was slowly added and the reaction mixture was stirred at the same temperature for 15 min. Then, linoleyl bromide (5.25 g, 15.94 mmol) was added and the reaction mixture was slowly warmed to 25° C. and stirred for 7 hr. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water (250 mL) and extracted with EtOAc (3×120 mL). The combined organic layers were washed with brine (3×150 mL) and Na 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated to give the crude product, which was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 3% EtOAc in pet. ether to give 17 (2.4 g, 2.75 mmol, 51.7% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 7.49-7.45 (m, 6H), 7.30-7.20 (m, 9H), 5.42-5.31 (m, 8H), 4.22-4.18(m, 1H), 4.04 (dd, 1H, J = 8.0, 4.0), 3.91-3.86 (m, 1H), 3.84 (d, 1H, J = 4.0),3.73 (dd, 1H, J = 12.0, 4.0), 3.50-3.43 (m, 2H), 3.39-3.28 (m, 3H), 3.22 (dd,1H, J = 12.0, 8.0), 2.78 (t, 4H, J = 6.0), 2.06 (q, 8H, J = 8.0), 1.59 (p, 4H,J = 4.0), 1.43-1.23 (m, 32H), 0.91-0.88 (m, 6H). RT = 3.86 min. Purity 98.1%. C 60 H 88 O 4 ESI-MS of Na: m / z = 896 [M+Na] + .

[0244]

[0390] Compound 18:

[0391] In a 250 mL two-necked RBF, 17 (2.35 g, 2.69 mmol) and Et 3 To a stirred solution of SiH (2.149 ml, 13.45 mmol) was added TFA (0.427 mL, 5.55 mmol) at 0 °C and the reaction mixture was stirred at 0 °C for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with satd.NaHCO 3 The mixture was quenched with hexanes (60 mL) and extracted with DCM (2×50 mL). 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated to give the crude product, which was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 8% EtOAc in pet. ether to give 18 (1.46 g, 2.314 mmol, 86% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 5.42-5.30 (m, 8H), 4.13 (dd, 1H, J = 8.0, 4.0), 4.09-4.05 (m, 1H),3.96-3.92 (m 2H), 3.90-3.81 (m, 2H), 3.75 (dd, 1H, J = 12.0, 4.0), 3.64-3.59(m, 1H), 3.47-3.42 (m, 3H), 2.78 (t, 4H, J = 6.0), 2.06 (q, 8H, J = 8.0),1.62-1.48 (m, 4H), 1.40-1.26 (m, 32H), 0.90 (t, 6H, J = 6.0). RT = 2.63 minutes. Purity 99.9%. C 41 H 75 O 4 ESI-MS: m / z = 631 [M+H] + .

[0245]

[0392] PNI 581: [ka]

[0246]

[0393] To a stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid (0.120 g, 0.762 mmol) in dry DCM (20 mL) in a 100 mL two-neck RBF under nitrogen atmosphere, DMAP (0.072 g, 0.586 mmol) and EDCI hydrochloride (0.281 g, 1.466 mmol) were added and the reaction mixture was stirred for 10 min at 25° C. Then 18 (0.37 g, 0.586 mmol) in dry DCM (5 mL) was added to the reaction mixture and stirred for 16 h at 25° C. TLC showed majority unreacted starting material. To a stirred solution of 1,4-dimethylpiperidine-4-carboxylic acid hydrochloride (0.148 g, 0.762 mmol) in dry DCM (10 mL) again in a 100 mL two-neck RBF under nitrogen atmosphere, DMAP (0.072 g, 0.586 mmol) and EDCI hydrochloride (0.281 g, 1.466 mmol) were added and the reaction mixture was stirred at 25° C. for 10 min. To this mixture was then added the first reaction mixture and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as indicated by TLC, the solvent was evaporated to give a residue. The residue was dissolved in EtOAc (130 mL) and washed with water (2×100 mL), brine (2×100 mL). The organic layer was extracted with Na 2 SO 4 The mixture was dried over 100 ml, filtered and concentrated to give the crude product, which was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 4% MeOH in DCM to give PNI 581 (0.395 g, 0.501 mmol, 86% yield) as a brown oil. 1 H (400MHz, CDCl 3) δ 5.42-5.31 (m, 8H), 4.44-4.38 (m, 1H), 4.30-4.26 (m, 1H), 4.22-4.18(m, 1H), 4.07 (dd, 1H, J = 8.0, 4.0), 3.93 (s, 1H), 3.85 (d, 1H, J = 4.0), 3.75(d, 1H, J = 8.0), 3.60-3.54 (m, 1H), 3.45 (t, 2H, J = 6.0), 3.41-3.36 (m, 1H),3.26 (br s, 2H), 2.84-2.71 (m, 6H), 2.65 (s, 3H), 2.32-2.22 (m, 2H), 2.06 (q,8H, J = 8.0), 1.68-1.48 (m, 6H), 1.38-1.26 (m, 35H), 0.90 (t, 6H, J = 6.0). RT= 4.06 min. Purity 97.8%. C 49 H 88 NO 5 ESI-MS: m / z = 771.2 [M+H] + .

[0247]

[0394] PNI 582: [ka]

[0248]

[0395] PNI 582 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 582 was carried out using 1-methylpiperidine-3-carboxylic acid (0.113 g, 0.792 mmol), DMAP (0.097 g, 0.792 mmol), EDCI hydrochloride (0.304 g, 1.585 mmol) in dry DCM (20 mL), and 18 (0.4 g, 0.634 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 582 (0.43 g, 0.569 mmol, 90% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.42-5.30 (m, 8H), 4.38-4.34 (m, 1H), 4.23-4.14 (m, 2H), 4.08 (dd,1H, J = 12.0, 4.0), 3.92 (s, 1H), 3.83 (s, 1H), 3.77 (d, 1H, J = 12.0),3.58-3.53 (m, 1H), 3.45 (t, 2H, J = 8.0), 3.42-3.36 (m, 1H), 3.17 (br s, 1H),2.93 (br s, 1H), 2.78 (t, 4H, J = 8.0), 2.60-2.26 (m, 4H), 2.17-1.96 (m, 10H),1.87-1.74 (m, 2H), 1.67-1.49 (m, 6H), 1.38-1.26 (m, 32H), 0.90 (t, 6H, J =6.0). RT = 2.99 min. Purity 99.2%. C 48 H 86 NO 5 ESI-MS: m / z = 756.6 [M+H] + .

[0249]

[0396] PNI 583: [ka]

[0250]

[0397] PNI 583 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 583 was carried out using 2-(4-methylpiperazin-1-yl)acetic acid (0.125 g, 0.792 mmol), DMAP (0.097 g, 0.792 mmol), EDCI hydrochloride (0.304 g, 1.585 mmol) in dry DCM (20 mL), and 18 (0.4 g, 0.634 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 4% MeOH in DCM to give PNI 583 (0.415 g, 0.527 mmol, 83% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3) δ 5.41-5.30 (m, 8H), 4.41 (dd, 1H, J = 12.0, 4.0), 4.25-4.16 (m, 2H),4.09 (dd, 1H, J = 8.0, 4.0), 3.92 (s, 1H), 3.83 (d, 1H, J = 4.0), 3.76 (dd, 1H,J = 12.0, 4.0), 3.58-3.52 (m, 1H), 3.44 (t, 2H, J = 8.0), 3.41-3.36 (m, 1H),3.28 (s, 2H), 2.84-2.58 (m, 12H), 2.43 (s, 3H), 2.06 (q, 8H, J = 8.0),1.57-1.51 (m, 4H), 1.40-1.26 (m, 32H), 0.92-0.86 (m, 6H). RT = 2.68 min. Purity 97.9%. C 48 H 87 N 2 O 5 ESI-MS: m / z = 771.6 [M+H] + .

[0251] Example 12 Synthesis of PNI-76 [ka]

[0252]

[0398] PNI 76:

[0399] To a solution of (±)-19 (600 mg, 3.65 mmol) in dry DMF (10 mL) was added DMAP (cat.), followed by addition of a solution of 4-(dimethylamino)butanoic acid hydrochloride (1.56 g, 9.3 mmol) in dry DMF (10 mL) with N 2 Added under atmosphere. CH 2 Cl 2(10 mL) was added to the reaction mixture. Solid EDCI hydrochloride (1.39 g, 7.30 mmol) was then added to the reaction mixture and stirred at room temperature for 4 h. TLC analysis of the reaction mixture indicates complete consumption of (±)-19. Further, DMAP (cat.) was added to the reaction mixture followed by myristic acid (2.73 g, 11.98 mmol) and EDCI hydrochloride (3.50 g, 18.25 mmol) as solids dissolved in dry DMF (10 mL). Dry DCM (10 mL) was then added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, diluted with EtOAc (20 mL) and washed with water (3×10 mL). The organic layer was washed with brine and anhydrous Na 2 SO 4 The crude mixture was dried on a rotary evaporator and evaporated to dryness to give a crude mixture that was purified by silica gel column chromatography using 5% MeOH / DCM as eluent to give PNI 76 as a colorless oil (294 mg, 0.32 mmol) in 9% overall yield.

[0253]

[0400] PNI 76 HCl salt

[0401] PNI 76 was dissolved in a minimum amount of anhyd. diethyl ether. HCl in ether was added until the pH was 3-5 (checked with pH paper). The solvent was evaporated and the mixture was washed with CH to remove traces of HCl. 2 Cl 2 The compound was then dissolved in a small amount of deionized water and lyophilized to obtain the HCl salt of PNI 76. 1 H (500MHz, CDCl 3) δ 5.38 (d, 1H, , J = 3.0 Hz), 5.22-5.19 (m, 1H), 5.07 (d, 1H, J = 5.0Hz), 4.61 (d, 1H, J = 10.0 Hz), 4.26 (dd, 1H, J = 10.0 Hz, 5.0 Hz), 4.21 (dd,1H, J = 10.0 Hz, 5.0 Hz), 4.13 (dd, 1H, J = 15.0 Hz, 5.0 Hz), 3.79 (d, 1H, J =10.0 Hz), 2.42-2.29 (m, 14H), 2.23 (apparently t, 2H, J = 7.5Hz), 1.84 (p, 2H, J = 6.3 Hz), 1.65-1.54 (m, 6H), 1.30-1.27 (m, 60H), 0.89 (apparent t, 9H, J = 7.5 Hz). 54 H 102 NO 9 Molecular weight [M+H] + Calculated value: 908.7555. Measured value: 908.7511.

[0254] Example 13 Synthesis of PNI 369 [ka]

[0255]

[0402] Compound 21:

[0403] To a stirred solution of 2-(dodecyloxy)ethanol (20, 3.7 g, 16.06 mmol) and triphenylphosphine (10.53 g, 40.1 mmol) in dry toluene (70 mL), carbon tetrabromide (13.31 g, 40.1 mmol) was added slowly and the reaction mixture was stirred at 60° C. for 16 h. After completion of the reaction as indicated by TLC analysis, the reaction mixture was concentrated under reduced pressure and the crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% EtOAc in Pet. ether to give 1-(2-bromoethoxy)dodecane (21, 4.55 g, 15.20 mmol, 95% yield) as a colorless oil. 1H (400MHz, CDCl 3 ) δ 3.74 (d, 2H, J = 6.0), 3.50-3.45 (m, 4H), 1.59 (p, 2H, J = 8.0),1.38-1.21 (m, 18H), 0.89 (t, 3H, J = 6.0). RT = 3.40 min. Purity 96.6%.

[0256]

[0404] Compound 22:

[0405] Compound 22 was synthesized using a method similar to that used to synthesize 17. The synthesis of 22 was carried out using 10 (1.0 g, 2.66 mmol), sodium hydride (0.531 g, 13.28 mmol, 60% dispersion in mineral oil), and 1-(2-bromoethoxy)dodecane (21, 2.337 g, 7.97 mmol) in dry DMF (10 mL) and dry THF (10 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% EtOAc in pet. ether to give 22 (1.1 g, 1.373 mmol, 51.7% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 7.46 (d, 6H, J = 8.0), 7.30-7.20 (m, 9H), 4.25-4.21 (m, 1H), 4.06-4.02(m, 2H), 4.00 (d, 1H, J = 4.0), 3.79-3.75 (m, 1H), 3.72-3.58 (m, 5H), 3.52-3.46(m, 3H), 3.41-3.28 (m, 5H), 3.22 (d, 1H, J = 8.0, 4.0), 1.60 (t, 2H, J = 6.0),1.48 (p, 2H, J = 8.0), 1.35-1.21 (m, 36H), 0.89 (t, 6H, J = 6.0). RT = 2.63 min. Purity 97.8%. C 52 H 80 O 6 ESI-MS of Na: m / z = 823.5 [M+Na] + .

[0257]

[0406] Compound 23:

[0407] Compound 23 was synthesized using a method similar to that used for the synthesis of 18. The synthesis of 23 was carried out by reacting 22 (1.1 g, 1.373 mmol) and Et 3 SiH (1.096 mL, 6.86 mmol) and TFA (0.264 mL, 3.43 mmol) were used. The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 20% ​​EtOAc in pet. ether to give 23 (0.58 g, 1.038 mmol, 76% yield) as a colorless oil. 1 H (400MHz, CDCl 3 ) δ 4.15-4.03 (m, 4H), 3.87-3.76 (m, 4H), 3.66-3.54 (m, 7H), 3.47-3.43(m, 4H), 1.61-1.54 (m, 4H), 1.36-1.20 (m, 36H), 0.90-0.87 (m, 6H). RT = 1.97 min. Purity 99.1%. C 33 H 67 O 6 ESI-MS: m / z = 559.5 [M+H] + .

[0258]

[0408] PN [ka]

[0259]

[0409] PNI 369 was synthesized using a method similar to that used to synthesize PNI 574. The synthesis of PNI 369 was carried out using 1,4-dimethylpiperidine-4-carboxylic acid (0.110 g, 0.698 mmol), DMAP (0.086 g, 0.698 mmol), EDCI hydrochloride (0.359 g, 1.879 mmol) in dry DCM (25 mL), and 23 (0.3 g, 0.537 mmol) in dry DCM (5 mL). The crude product was purified by silica gel (100-200 mesh) column chromatography (Isolera™) using 5% MeOH in DCM to give PNI 369 (0.21 g, 0.301 mmol, 56.0% yield) as a pale yellow oil. 1 H (400MHz, CDCl 3 ) δ 4.37-4.28 (m, 2H), 4.21-4.17 (m, 1H), 4.11-4.07 (m, 2H), 3.99 (d,1H, J = 4.0), 3.81-3.76 (m, 1H), 3.73-3.69 (m, 1H), 3.66-3.59 (m, 3H), 3.57-3.52(m, 4H), 3.46-3.41 (m, 4H), 2.89 (br s, 2H), 2.48-2.35 (m, 5H), 2.21 (d, 2H, J= 12.0), 1.86-1.71 (m, 2H), 1.60-1.52 (m, 4H), 1.37-1.20 (m, 39H), 0.88 (t, 6H,J = 8.0). RT = 2.13 min. Purity 98.5%.C 41 H 80 NO 7 ESI-MS: m / z= 698.6 [M+H] + .

[0260]

[0410] Example 14 Synthesis scheme of representative compounds [ka] JPEG0007667327000092.jpg191149 JPEG0007667327000093.jpg178149 JPEG0007667327000094.jpg191149 JPEG0007667327000095.jpg168149 JPEG0007667327000096.jpg69149

[0261] Example 15 Isolation and expansion of primary T cells from human whole blood

[0411] Unless otherwise stated, all reagents were purchased from STEMCELL Technologies, Vancouver, Canada.

[0262]

[0412] Lyophilized human IL-2 was reconstituted in sterile 1x PBS without calcium or magnesium in a biological safety cabinet to a concentration of 0.1 mg / ml. 50 μl of this human IL-2 was added to 50 mL of ImmunoCult-XF™ T Cell Growth Medium to generate the medium for T cells. 7-30 mL of whole human peripheral blood containing ACDA anticoagulant was placed in a sterile 50 mL polypropylene conical tube in a biological safety cabinet.

[0263]

[0413] Negative Selection Protocol. Blood was drawn from healthy human donors and mixed with ACD-A anticoagulant. Both CD4+ and CD8+ T cells were isolated using a pan-T cell negative selection kit, EasySep™ Direct Human T Cell Isolation Kit. Cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). On the day of isolation, cells were activated with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Cactivator.

[0264]

[0414] Positive Selection Protocol. Blood was drawn from healthy human donors and mixed with ACDA anticoagulant. A PBMC suspension was prepared using density gradient centrifugation with the use of Lymphoprep™. T cells were then positively selected from the PBMC suspension using the EasySep™ Human CD3 Pos Selection Kit II. Cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). On the day of isolation, cells were activated with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator.

[0265]

[0415] T cells were isolated from blood using the EasySep™ Direct Human T Cell Isolation Kit. First 50 μl / mL of Isolation Cocktail™ and then 50 μl / mL of EasySep™ RapidSpheres™ were added to the tube of blood. The blood was gently mixed and incubated at room temperature (RT) for 5 minutes. The tube was placed into an EasySep™ 50 Magnet™ device and incubated at RT for 10 minutes. The enriched cell suspension was pipetted into a new sterile 50 mL polypropylene tube and the RapidSpheres™ process was repeated.

[0266]

[0416] This doubly enriched cell suspension was pipetted into a new sterile 50 mL polypropylene conical tube and centrifuged for 10 min at 300 g and RT.

[0267]

[0417] The supernatant was removed and the cell pellet was resuspended in 10 mL PBS and spun again at 300 g for 10 min to wash residual supernatant from the cells. The supernatant was again removed and the cells were resuspended in pre-warmed complete T cell medium. Samples were taken and a trypan blue exclusion test for cell viability was performed (Thermo Fisher).

[0268] Example 16 T cell activation / proliferation

[0418] The scientific background of T cell activation is described in a 2003 paper by Trickett A. et al. 6 The cell suspension from Example 8 was diluted to 1E6 cells / ml in Complete T Cell medium (ThermoFisher) and the T cells were activated by adding 25 μl of either ImmunoCult™ Human CD3 / CD28 / CD2 Triple T Cell Activator™ or ImmunoCult™ Human CD3 / CD28 Binary T Cell Activator™ per mL of T Cell medium. Cell growth was monitored by daily cell counts under expansion. Cells were diluted with Complete T Cell medium to maintain a concentration of approximately 1E6 cells / mL. Around days 5, 6 or 7, the T cells entered the log phase of growth and rapid proliferation occurred.

[0269]

[0419] CD25 expression was assessed to ensure T cells were in log phase, which must be greater than 80% by flow cytometry, and cell proliferation was monitored by graphing total T cell numbers over time (not shown).

[0270] Example 17 Downstream processing and analysis of treated T cells: Flow cytometry and ELISA

[0420] Reagents were obtained from Stemcell Technologies unless otherwise stated. T cells were isolated from a single donor. 48H after lipid particle mRNA exposure, treated T cells were harvested by transferring the cell suspension to a pre-labeled 1.5mL tube and centrifuging at 300xG for 10 minutes at 4°C. The supernatant was removed and the pellet was resuspended in PBS. A volume of 0.5ul of BD Horizon™ Fixable Viability Stain 575V™ (BD Biosciences) was added and the mixture was incubated for 10 minutes at RT in the dark. This dye binds amines.

[0271]

[0421] The cells were centrifuged again as before, then washed twice with 1 mL dye buffer (BSA, BD Pharminigen) and the washed pellet was diluted in 100 μl BSA. The following antibodies were added in 2 μl volumes to each tube of treated cells: CD25, CD8, CD4, (PerCP-Cy™ 5.5 mouse anti-human CD25, BV786 mouse anti-human CD8 Clone RPA-T8, APC-Cy™ 7 mouse anti-human CD4 Clone SK3 (all BD Pharmingen)) with the exception of controls: eGFP only samples and viability controls had no antibody added. Single dye compensation tubes had only one antibody added per tube.

[0272]

[0422] The tubes were incubated at 4°C for 30 min, whereupon 400 μl of dye buffer was added and the cells were centrifuged again. The cells were washed once with 1 mL of dye buffer and spun down again as in step 1. The cell pellet was resuspended in 1 mL of dye buffer and added to a pre-labeled flow tube (Corning Falcon) with a cell strainer cap.

[0273]

[0423] Negative Selection Protocol. Blood was drawn from healthy human donors and mixed with ACDA anticoagulant. Both CD4+ and CD8+ T cells were isolated using the pan-T cell negative selection kit EasySep™ Direct Human T Cell Isolation Kit. Cells were maintained in ImmunoCult-XF™ T Cell Experimental Medium supplemented with human recombinant IL2 (Peprotech). On the day of isolation, cells were activated with the triple activator ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator.

[0274]

[0424] Freezing and thawing of human T cells. Blood was drawn from healthy human donors and mixed with ACDA anticoagulant. Both CD4+ and CD8+ T cells were isolated using a pan-T cell negative selection kit EasySep Direct Human T Cell Isolation Kit. Cells were cryopreserved using CryoStor® CS10 and stored in liquid nitrogen. Upon thawing, cells were maintained in ImmunoCult-XF™ T Cell Exp Medium supplemented with human recombinant IL2 (Peprotech). On the day of thawing, cells were activated with triple activator. After transfection, flow cytometry was used for the study of general eGFP expression levels as well as Median Fluorescence Intensity values.

[0275] Example 18 Microfluidic mixing of nucleic acid therapeutics (NATs) into lipid particles (LNPs)

[0425] N / P=10 was used for all these experiments unless otherwise noted. Lipid mix composition solutions were prepared in ethanol by mixing defined amounts of lipids (see Table 3) from individual lipid stocks in ethanol. A lipid mix solution concentration of 37.5 mM was used for NanoAssembl® SPARK™, and a lipid mix solution of 12.5 mM was typically used for NanoAssembl® Benchtop or Ignite™. [Table 3] JPEG0007667327000098.jpg192149 JPEG0007667327000099.jpg174149

[0276]

[0426] IL = ionizable lipid; Tween 80 = polysorbate 80; BRIJ™ L4 = polyoxyethylene (4) lauryl ether; BRIJ™ S10 = polyoxyethylene (10) stearyl ether; BRIJ™ S20 = polyoxyethylene (20) stearyl ether; BRIJ™ S35 = polyoxyethylene (23) lauryl ether; TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate; lipid H = equal ratio of Tween 20 / polysorbate 80 / tridecyl-D-maltoside; stabilizer = any stabilizer including PEG-DMG or as defined above in this category.

[0277]

[0427] The components of the lipid mix include ionizable lipids, structural lipids, cholesterol, and stabilizers. A low pH buffer (3-6) may be used. For ionizable amino lipids, the pH of the buffer is typically lower than the pKa of the lipid.

[0278]

[0428] Preparation of siRNA, messenger RNA or plasmid NAT is described below. Particle size observed was generally in the range of 50-200 nm for mRNA depending on lipid composition.

[0279]

[0429] Messenger RNA or plasmid NAT was diluted to the required concentration with sodium acetate buffer. Lipid-nucleic acid particle samples were then prepared by running both fluids using the NanoAssemblr® Spark instrument. Briefly, 10-20 μg of nucleic acid in a total volume of 3 μL of 100 mM sodium acetate buffer was mixed with 16 μL of 37.5 mM lipid mix solution as required by the N / P ratio (4, 6 or 10 in the illustrated example). The lipid-nucleic acid particles created in the instrument were immediately diluted with 48 μL of 1×PBS, pH 7.4, without Ca and Mg, in the aqueous output well. These nucleic acid-lipid particles were immediately collected in a microcentrifuge tube containing 96 μL of 1×PBS, pH 7.4, without Ca and Mg. Encapsulation efficiency was measured by a modified Ribogreen™ assay (QuAnti-iT RiboGreen™ RNA Assay Kit, Fisher). The particle attributes observed generally ranged in size from 60 to 200 nm for mRNA depending on lipid composition and production method.

[0280]

[0430] Lipid-based formulations were also produced on a larger instrument, the NanoAssemblr® Ignite™, for testing. Briefly, 350 μL of mRNA was diluted to the required concentration of 0.2-0.3 mg / mL using 100 mM sodium acetate buffer. Lipid particle samples were then prepared by running both fluids, i.e., nucleic acid in aqueous solvent and lipid mix in ethanol, at a flow ratio of 3:1 and a total flow rate of 12 ml / min. After mixing in the microfluidic device, lipid nucleic acid particle (LNAP) samples were diluted into RNAse-free tubes containing 3-40 volumes of phosphate buffered saline (PBS), pH 7.4. Finally, ethanol was removed by dialysis in PBS, pH 7, or using an Amicon™ centrifugal filter (Millipore, USA) at 3000 RPM, or using a TFF system. Once the required concentration was achieved, the lipid nucleic acid particles were filter sterilized using a 0.2 μm filter in aseptic conditions. The final encapsulation efficiency was measured by Ribogreen™ assay.

[0281]

[0431] Nucleic acid reagents. Messenger RNA or plasmid nucleic acid therapeutics (NATs) as described below were diluted to the required concentration using sodium acetate buffer. LNAP samples were then prepared by running both fluids using the NanoAssemblr® Spark instrument. Briefly, 10-20 μg of nucleic acid in a total volume of 32 μL of 100 mM sodium acetate buffer was mixed with 16 μL of 37.5 mM lipid mix solution as required by the N / P ratio (4, 6, 8, 10 in the illustrated example). The microfluidically mixed LNAPs created in the instrument were immediately diluted with 48 μL of 1×PBS, pH 7.4, without Ca++ and Mg++, in the aqueous output well. These LNAPs were immediately collected in a microcentrifuge tube containing 96 μL of the same buffer (pH 7.4). Encapsulation efficiency was measured by a modified Ribogreen™ assay (Quanti-iT RiboGreen™ RNA Assay Kit, Fisher). This information was used to establish the desired dosage.

[0282]

[0432] The nucleic acid therapeutic agent model reagents used in the following experiments were as follows:

[0433] Trilink Cleancap® eGFP mRNA: Cat.L-7601 (Trilink Biotechnologies, San Diego, CA); Trilink Cleancap® EPO mRNA: Cat.L-7209 (Trilink Biotechnologies); Millipore Sigma TagRFP Simplicon RNA Kit: Cat.SCR712 (contains both TagRFP RNA & B18R RNA) (Millipore Sigma Canada, Oakville Ontario); CD19 CAR plasmid with EGFP reporter was purchased from Creative Biolabs (Shirley, NY) and contains the T7 promoter (Mut)-signal peptide-scFv-CD8 hinge transmembrane-4-1BB-CD3zeta-T2A-eGFP reporter gene CAR cassette (2353bp) in pcDNA. The total size of this custom CD19 CAR plasmid DNA template was approximately 7649-7661bp.

[0283]

[0434] Unmodified CAR messenger RNA (mRNA) transcripts encoding the CD19 scFv-h(BB±-eGFP reporter gene cassette were synthesized by in vitro transcription using wild-type bases and capped using the Cleancap® AG method by Trilink Biotechnologies Inc. (cap 1). The unmodified CAR mRNA transcripts were enzymatically polyadenylated followed by DNase and phosphatase treatment. The final mRNA transcription products were silica membrane purified and packaged in a solution of 1 mM sodium citrate buffer (pH 6.4) at a concentration of 1 mg / mL. The custom CD19 CAR plasmid vector and the mRNA encoding the CD19 CAR were purchased from Creative Biolab and Trilink Biotechnologies Inc, respectively.

[0284]

[0435] OVA antigen is used as a model antigen to stimulate immune responses. 10This makes it useful for vaccine research. Usually, OVA antigens are used with sensitizers such as alum, but when OVA antigens are delivered in the form of mRNA, the premise is that sensitizers such as alum are not necessary and the mRNA itself can generate a response to the antigen by immune cells. Cleancap® OVA mRNA L-7610 and Cleancap® OVA mRNA (5moU) L-7210 were used.

[0285]

[0436] Plasmid preparation, custom made by GenScript USA Inc, Piscataway, NJ, pCX-EGFP plasmid of size 5514 nt containing ampicillin resistance, restriction enzyme HINDIII in ddH2O was used for this evaluation. The plasmid contained a GFP expression component that produces the target protein only when the plasmid is expressed in the cell.

[0286] Example 19

[0437] Comparative data on lipids active with eGFP mRNA LNPs in primary human T cells

[0438] Reagents were obtained from Abcam, Cambridge, UK unless otherwise stated. eGFP SimpleStep™ ELISA Kit was used to demonstrate mRNA delivery and activity in vitro. Assays were performed as directed by the eGFP SimpleStep ELISA Kit protocol. Briefly, frozen human T cells previously isolated from fresh human whole blood using a negative selection protocol were thawed and activated with triple activators. Ten days after activation, T cells were administered N / P10 mRNA LNPs encoding eGFP at 2 μg mRNA per 500,000 cells. 48 hours after treatment with mRNA LNPs, T cells were harvested and lysed for total eGFP content.

[0287]

[0439] To expose isolated and activated T cells (day 0) to formulated mRNA, LNPs containing 2 ug of CleanCap™ eGFP (Trilink Biotechnologies, San Diego, Calif.) mRNA were added to 500,000 T cells in 1 mL of complete T cell media along with 1 ug / mL of recombinant human ApoE4 ("ApoE") (Peprotech Inc., Montreal, Canada).

[0288]

[0440] The positive control was a standard lipid (DLin-MC3-DMA or "MC3" lipid data) using the Trio activation protocol.

[0289]

[0441] LNP was calculated based on previous Ribogreen™ assay results. T cells were counted by Trypan blue (Sigma) exclusion and diluted to 500,000 cells / mL. Briefly, in a 12-well plate, 1 mL aliquots were placed into each well. ApoE was added to each well to a final concentration of 1 ug / mL. Based on the calculation in step 1, the required amount of mRNA LNP was added (day 7) and the plate was incubated for 48 h.

[0290]

[0442] The lipid mix compositions were tested for their ability to induce transfection as measured by the median fluorescence intensity of labeled mRNA in T cells (measured by flow cytometry). Primary human T cells freshly isolated from human blood were selected by a negative selection method and treated with a dose of 2 μg of mRNA per 500,000 cells.

[0291]

[0443] The ionizable lipids, PNI 76, 119, 121 and MC3, were compared in a composition of 40 Mol % ionizable lipid, 20 Mol % structural lipid DSPC, 37.5 Mol % cholesterol, and 2.5 Mol % BRIJ™ S10, with an N / P ratio of 10.

[0292]

[0444] The transfection efficiency and amount of eGFP expressed for each lipid are shown in columns 5 and 6 of Table 4, respectively. Figure 1 shows the performance of LNPs containing either PNI 76 or PNI 121 for the ionizable lipid Dlin-MC3-DMA (MC3) in a CT10 composition with an N / P ratio of 10, and analyzed for gene expression by flow cytometry 48 hours after treatment for eGFP mRNA expression. It was found that PNI 76 had equal transfection efficiency to MC3, while PNI 121 was greater than MC3.

[0293]

[0445] In related experiments, primary human T cells from six different donors were isolated from fresh whole blood using a negative selection protocol, activated with triple activators, and then treated with mRNA LNPs 7 days after activation. Gene expression was analyzed by flow cytometry 48 hours after treatment, and the results are shown in Figure 2. The transfection efficiency of LNPs containing PNI 121 was found to be greater than that containing clinically confirmed MC3 in various donors. The viability of T cells was not affected by their treatment with PNI 121 compared to untreated cells (not shown).

[0294]

[0446] Furthermore, the level of eGFP expression was quantified by eGFP ELISA as shown in Figure 3. Human T cells previously isolated and frozen from fresh human whole blood using a negative selection protocol were thawed and activated with triple activators. Ten days after activation, T cells were administered CT10 mRNA LNPs containing DLin-MC3-DMA, PNI 76, PNI 119, PNI 120, PNI 121, and PNI 122 encoding eGFP at 2 μg mRNA per 500,000 cells, N / P10. After 48 hours, protein expression was measured by eGFP ELISA. All PNI novel lipids were found to mediate eGFP expression. Most notably, eGFP expression mediated by PNI 121 was greater than MC3.

[0295]

[0447] In a related experiment, GFP expression in previously cryopreserved isolated primary human T cells treated with mRNA encapsulated by mRNA-LNPs containing DLin-MC3-DMA, PNI 127, PNI 328, PNI 329, or PNI 541 in a CT10 composition with an N / P ratio of 8 at 500 ng per 125,000 cells 4 days after activation. In Figure 4, GFP MFI (first column), transfection efficiency (middle column), and viability (third column) were measured by flow cytometry 48 h after LNP addition.

[0296]

[0448] GFP MFI was measured by flow cytometry 48 h after LNP addition. T cells were isolated from whole blood using a negative isolation procedure (EasySep™ Human T Cell Isolation Kit, Stemcell Technologies). Three days after activation, T cells were administered mRNA-LNPs containing 500 ng of encapsulated mRNA per 125,000 cells. LNPs were of the CT10 composition of N / P8. The results are shown in Figure 5. Transfection efficiency (upper graph) and MFI (lower graph) were measured by flow cytometry 48 h after LNP addition. PNI 328, 329 and 541 show higher levels of protein expression (MFI) than MC3.

[0297]

[0449] Table 4 shows the quantitative results for additional experiments with MC3, PNI 76, PNI 119 and PNI 121 eGFP mRNA LNPS at CT10 of N / P8. [Table 4]

[0298] Example 20 Erythropoietin mRNA delivery and expression

[0450] Quantikine® IVD Human Epo ELISA 2 antibody sandwich assay was used to demonstrate mRNA delivery and activity in vitro. Reagents were obtained from Quantikine, Minneapolis, MN. The assay was performed as directed in the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol REF DEP00 Package Insert. Briefly, primary human T cells were isolated from fresh whole blood using a negative selection protocol and activated with triple activators. Seven days after activation, T cells were dosed with mRNA LNPs encoding EPO at 2 μg of mRNA per 500,000 cells and N / P10. Quantikine® Human Serum Control was used. T cells were harvested 48 hours after treatment with mRNA LNPs and lysed to obtain cytoplasmic EPO, and secreted EPO samples were taken from the culture supernatant. Results are shown in Figure 6 in mIU / mL. Table 5 shows the data from Figure 6 and the fold increase in EPO over MC3 for PNI 76 and 121. It was found that EPO expression mediated by LNPs containing PNI 121 was higher than those containing MC3. [Table 5]

[0299]

[0451] C56BL / 6 mice were administered iv doses of 0.5, 1 or 3 mg / Kg of recombinant human EPO-encoding mRNA LNPs containing ionizable lipids MC3, PNI 76, PNI 121 and PNI 127 using the LM02 composition of N / P6, and hEPO protein was measured using the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol. Results for the 0.5 mg / Kg dose are shown in the scatter plot of Figure 7. Results for the 1 and 3 mg / Kg doses are shown in Figure 8 as scatter plots of hEPO expression levels at 6h (left) and 24h (right).

[0300]

[0452] In another study, 5-moU EPO mRNA encapsulated in LM02 LNPs with 15 different ionizable lipids (PNI 336, PNI 534, PNI 535, PNI 342, PNI 321, PNI 532, PNI 538, PNI 541, PNI 325, PNI 328, PNI 329, PNI 539, PNI 540, PNI 127) was administered at a dose of 0.5 mg / Kg and N / P ratio of 6. hEPO protein was measured using the ProteinSimple® Ella platform and rhEPO microfluidic cartridge provided by the manufacturer. The results are shown in Figure 9, where several of the PNI compounds (PNI 328, 329, 539, 540, and 127) compare favorably with MC3. PNI-127, PNI-329, PNI 541 and PNI-565 are better than MC3 in terms of the amount of protein produced (higher MFI than MC3). Example 21 Characterization and Encapsulation of Lipid Nucleic Acid Particles or "LNPs"

[0453] After lipid particles were prepared as described above, particle size (hydrodynamic diameter of the particle) was determined by dynamic light scattering (DLS) using a Zetasizer™ Nano ZS™ (Malvern Instruments, UK). A He / Ne laser tuned to a wavelength of 633 nm was used as the light source. Data were measured from scattering intensity data taken in backscattering detection mode (measurement angle = 173). Measurements were the average of 10 measurements of 2 cycles each per sample. The Z-average size is reported as particle size and is defined as the harmonic intensity mean particle diameter.

[0301]

[0454] The physical characteristics of lipid nanoparticle (LNP) compositions containing compounds according to formula (I) and produced by NanoAssemblr® Spark and benchtop are shown in Tables 6 and 7 below. The size, change in size of LNP (PDI), encapsulation efficiency, and pKa are shown. These physical characteristics of LNP are important in terms of stability, biodistribution, and cell membrane crossing. Encapsulation was good in all formulations, with polydispersity (PDI) below 0.3. Size and PDI were measured using dynamic light scattering technology, and nucleic acid encapsulation efficiency was calculated by a modified QuantiT RiboGreen RNA assay.

[0302]

[0455] [Table 6]

[0456] [Table 7]

[0303] Example 22 hEPO / Cytokine Measurements by Simplex™ Automated ELISA

[0457] Blood from mice treated with hEPO mRNA LNPs was analyzed using the automated ELISA platform "ELLA" with Simplex™ antibody panel cartridges. Human-specific EPO and mouse-specific IL-5, IL-6, TNF-α, and IFN-γ were measured (reagents from Bio-techne). Briefly, 50 μL of sample reagent (diluted biological sample, quality control, or calibration point sample) was placed into each sample inlet and 1 mL of wash buffer was placed into the corresponding inlet of the cartridge. All immunoassay operations (e.g., start the system, run sample and split into channels, incubate sample, wash, rehydrate and run secondary antibody, wash, rehydrate and run streptavidin dye conjugate, incubate, wash, scan) were performed automatically. Raw signal levels (relative fluorescence units, RFU), mean signal values, standard deviations, and coefficients of variation (CV) for each glass nanoreactor (GNR) value are provided. The RFU values ​​are automatically backfitted by ELLA to yield analyte concentrations according to the analyte / sample using the manufacturer's predetermined calibration method. The results are shown in Figure 10.

[0304]

[0458] Quantikine® IVD Human Epo ELISA double antibody sandwich assay was used to verify mRNA delivery and activity in vivo. Reagents were obtained from Quantikine, Minneapolis, MN. The assay was performed as directed in the Quantikine® IVD® ELISA Human Erythropoietin Immunoassay protocol REF DEP00 Package Insert. Briefly, serum samples were analyzed for EPO expression analysis 6h and / or 24h after intravenous administration of 0.5, 1 or 3mg / kg doses of EPO-encoding mRNA and expressed in mIU / mL. Standard appropriately diluted serum, as well as horseradish peroxidase (HRP)-conjugated rabbit anti-EPO polyclonal antibody and TMB (tetramethylbenzidine) as substrate, were added to a 96-well microplate pre-coated with EPO antibody provided by the manufacturer and incubated for 20 minutes. Stop solution (sulfuric acid) was added to the wells, the plate was read at 450nm, and the concentration of EPO was calculated. The amount of color produced is directly proportional to the amount of conjugate bound to the EPO antibody, which is directly proportional to the amount of EPO in the sample or standard. A standard curve was generated by plotting absorbance versus the concentration of the standards provided. Quantikine® Human Serum Control (CEP 01, CEP 03) was used as an internal control.

[0305] Example 23 CD19 CAR expression mediated by mRNA-LNPs in isolated primary human T cells

[0459] mRNA containing IL with CT10 composition of N / P8 was assayed after in vitro exposure. CAR vector pcDNA3.1 anti-CD19-h(BB lambda)-EGFP-2nd-CAR(T7 Mut) 7661bp was purchased from Creative BioLabs, NY, USA.

[0306]

[0460] T cells were isolated from whole blood using a negative isolation procedure and T cell activation, and expansion was performed by triple activation with ImmunoCult™ Human T Cell Expansion Media as described above. As seen in Figure 11, CD19 CAR expression was greater in transfected T cells in vitro for PNI 328, PNI 329, and PNI 127 LNPs than PBS.

[0307] Example 24 In vivo delivery of Ova-encoding mRNA LNPs by intramuscular administration

[0461] LNPs of composition LM02b were used to encapsulate CleanCap® OVA WT mRNA [TriLink L7610] or CleanCap® OVA 5-moU mRNA [TriLink L7210] encoding the OVA antigen. A lipid mixture of ionizable lipids, DOPE, cholesterol, and PEG-DMG2000, all in ethanol, was mixed with a low PH buffer solution of OVA mRNA at an N / P ratio of 8 using a NanoAssemblr® Ignite™ microfluidic mixer. The LNPs were filtered using Amicon™ ultrafiltration technology and characterized for size, PDI, and encapsulation efficiency. Size and PDI were measured using dynamic light scattering technology, and nucleic acid encapsulation efficiency was calculated from QuantiT RiboGreen RNA assay. These OVA mRNA-LNPs had a hydrodynamic diameter of about 83 nm, a polydispersity index of about 0.1, and an encapsulation efficiency of about 97%.

[0308]

[0462] All animals were treated according to the Animal Care and Use Committee ethical protocols. For the experiment, 6-8 week old C57BL / 6 mice (n=4) were purchased from Envigo. On days 1 and 10, mice were immunized intramuscularly with 50 μL of Ova LNPs. Each mouse was vaccinated with a dose of 5 μg of OVA mRNA encapsulated in LNPs. A dose of 50 μg of OVA antigen was used as a positive control. At the defined time intervals, 110-130 μL of blood was collected by saphenous vein exsanguination technique and processed into serum. Two weeks after the second immunization, animals were euthanized and blood was collected by cardiac puncture. Blood samples were immediately processed into serum and stored at -80°C. Aliquots of serum samples were thawed and analyzed for Ova expression and IgG measurements using standard ELISA techniques at appropriate dilutions.

[0309]

[0463] For serum preparation, after whole blood collection, blood was allowed to clot for 15-30 minutes at room temperature. Clotted blood was removed by centrifuging the tubes at 1000-2000 x g for 10 minutes at 4 degrees Celsius. The supernatant was carefully removed and transferred to a sterile, screw-capped, clear polypropylene tube on ice.

[0310]

[0464] OVA ELISA of serum samples: Mice sera were collected by saphenous vein bleeding 6 h after vaccination. OVA antigen was measured using a standard sandwich ELISA using the Ovalbumin (OVA)-ELISA Kit abx150365 (Abbexa Biologics, Inc., Arlington, TX, USA) components according to the manufacturer's recommendations. Briefly, in a 96-well Abrexxa™ microplate pre-coated with antibody, standard appropriately diluted serum and biotin conjugate reagent were added to the wells and incubated for 1 h. Horseradish peroxidase (HRP) conjugate reagent was added and incubated for 20 min. Stop solution was added to each well and the absorbance at 450 nm was measured using a plate reader, from which the concentration of OVA protein was calculated.

[0311]

[0465] Anti-OVA-IgG ELISA of serum samples: Sera from immunized mice were collected 2 weeks after the second immunization. OVA-specific production of IgG in response to OVA-encoding mRNA of various LNPs was measured by ELISA as described above. Briefly, 96-well ELISA plates were pre-coated with OVA protein at a concentration of 2 μg protein per well in 100 mM sodium bicarbonate buffer (pH 9.6) overnight at 4°C. The pre-coated plates were then blocked with 10% FBS (BSA) in 7.4 buffered PBS-Tween-20™ (0.05%) v / v and incubated at 37°C for 2 h. Serum samples and immunoglobulin standards were appropriately diluted (1:8 to 1:1000) in 1% BSA-PBS, added to the 96-well plates, and incubated at RT for 2 h. For labeling, (HRP)-conjugated goat anti-mouse IgG (catalog #7076, Cell Signaling) was used at a dilution of 1:5,000 in PBS-Tween-10% FBS and incubated for 1 h. After the incubation period, horseradish peroxidase substrate (3,3',5,5'-tetramethylbenzide-TMB) was added. After 30 min incubation, a stop solution of 2N sulfuric acid was added and absorbance at 450 nm was determined using a plate reader. Results are shown in Figure 12 for PNI ID 325 and 539. Results for PNI 127 are shown in Figure 13.

[0312]

[0466] Referring to Table 7 and the figures, PNI 121, PNI 127, PNI 328, PNI 329, and PNI 540 are equivalent to MC3 in in vivo protein expression. PNI 121, PNI 127, PNI 328, PNI 329, and PNI 541 are better than MC3 in terms of the amount of protein produced in protein expression in human T cells (higher MFI than MC3).

[0313]

[0467] LNPs loaded with OVA antigens, including PNI 127, PNI 325, and PNI 539, were shown to effectively elicit immune responses similar to those of OVA antigen, indicating the utility of PNI lipids for vaccine applications. [Table 8]

[0314] Example 25 pKa studies

[0468] The effect of surface pKa on transgene expression following LNP transfection in LNPs derived from different ionizable lipids was examined.

[0315]

[0469] The pKa of each cationic lipid was determined using fluorescent probes for the conformational state of the protein. 5 The lipid nanoparticles were determined using a fluorescence-based assay of 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS, Sigma Aldrich). Empty lipid nanoparticles containing ionizable lipids of LM02 composition in distilled water at a concentration of 3.125 mM total lipid were formulated using a NanoAssemblr® Spark™ and then characterized on a Zetasizer™ using a 30x dilution with 1x PBS in a low volume cuvette. The lipid nanoparticles were further diluted 4x with distilled water to 0.781 mM total lipid. TNS was prepared as a 25 μM stock solution in distilled water. 6.4 μL of the diluted LNP sample at 0.781 mM total lipid was then mixed with 10 μL of diluted TNS to obtain 10 mM HEPES, 10 mM MES, 10 mM NH 4 The final volume was 250 μL containing a buffer containing OAc and 130 mM NaCl, where the pH ranged from 3 to 9 in 0.5 pH increments. Each well had a final concentration of 20 μM total lipid, 1 μM TNS, and 233.4 μL of buffer solution (total volume 250 μL).

[0316]

[0470] Samples were mixed thoroughly and fluorescence intensity was measured at room temperature in a BioTek™ Synergy™ H1 Hybrid Multi-Mode Monochromator™ Fluorescence Microplate Reader using excitation and emission wavelengths of 321 nm and 445 nm, respectively. A sigmoidal best-fit analysis was applied to the fluorescence data using GraphPad Prism™ software, and pKa was determined as the pH at half-maximal fluorescence intensity.

[0317]

[0471] The results are shown in FIG. 14 illustrating surface pKa measurements of LNPs incorporating ionizable lipid compounds PNI 119, PNI 121, PNI 321, PNI 329, PNI 336, PNI 535, PNI 538, and PNI 540 (LM02 composition). Lipids with lower pKa values ​​were found to be inactive in transgene expression in T cells. Lipids with higher pKa values ​​were found to be toxic to T cells. Lipids in the pKa range of 5.5-6.9 were found to be active in promoting transgene expression in T cells.

[0318] Example 26 Plasmid encapsulation

[0472] A pCX-EGFP plasmid of size 5514nt was used, custom made by GenScript USA Inc, Piscataway, NJ. Lipid particle preparation was as described above. PNI ionizable lipids PNI 121, PNI 127, PNI 328, PNI 329, and PNI 541 were tested for their ability to encapsulate plasmids suitable for mammalian expression (described above) using the NanoAssemblr® Ignite™ System with the LM02 composition of N / P6. All lipids tested produced acceptable LNPs with a mean hydrodynamic diameter of 110nm and a PDI of 0.2, with an encapsulation efficiency of greater than 80%.

[0319]

[0473] Although preferred embodiments have been described above and illustrated in the accompanying drawings, modifications may be made without departing from the disclosure as would be apparent to one skilled in the art, and such modifications are considered possible variations that fall within the scope of the disclosure.

[0320] References 1. Garg, S.; Heuck, G.; Ip, S.; Ramsay, E.,Microfluidics: a transformational tool for nanomedicine development and production. J Drug Target 2016, 24 (9), 821-835. 2. Zhang, S.-h.; Shen, S.-c.; Chen, Z.; Yun, J.-x.; Yao, K.-j.; Chen, B.-b.; Chen, J.-z., Preparation of solid lipid nanoparticles in co-flowing microchannels. Chemical Engineering Journal 2008,144 (2), 324-328. 3. JEFFS, LB, et al., A Scalable,Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA.Pharmaceutical Research 2005, 22 (3), 362-372. 4. Gaj, T.; Gersbach, CA; Barbas, CF,3rd, ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering. Trends inbiotechnology 2013, 31 (7), 397-405. 5. McClure, W. O.; Edelman, G. M.,Fluorescent Probes for Conformational States of Proteins. I. Mechanism ofFluorescence of 2-p-Toluidinylnaphthalene-6-sulfonate, a Hydrophobic Probe*.Biochemistry 1966, 5 (6), 1908-1919. 6. Trickett, A.; Kwan, Y. L., T cellstimulation and expansion using anti-CD3 / CD28 beads. J Immunol Methods 2003,275 (1-2), 251-5. 7. Lundstrom, K. Nanoparticle-baseddelivery of self-amplifying RNA. Gene Ther 27, 183-185 (2020). 8. Peng, M., Mo, Y., Wang, Y. et al.Neoantigen vaccine: an emerging tumor immunotherapy. Mol Cancer 18, 128 (2019). 9. Roujian Lu 1, Xiang Zhao 1, Juan Li 2,et al. “Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus:Implications for Virus Origins and Receptor Binding” Lancet 2020 Feb22;395(10224):565-574. 10. Morokata T, et.al, Immunology (1999)345-351. Giuliani et al. (2006) Proc Natl Head Sci USA103(29):10834-9

Claims

1. A compound of formula (I), or a pharma- ceutically acceptable salt thereof, in combination with a structured lipid, a sterol, and a stabilizer. 【Chemistry 1】 [In the formula, p is 0 or 1; E １ Ha-O-δ １ , -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , -OC(O)S-δ １ , -C(O)N(Q)-δ １ , -C(O)O-δ １ , -N(Q)C(O)-δ １ , -N(Q)C(O)O-δ1, -N(Q)C(O)S-δ １ , and -N(Q)C(O)N(Q)-δ １ Q is selected from H or C １ ~C ５ alkyl; １ is R １ represents a bond connecting to a group; R １ teeth 【Chemistry 2】 where: R ３ and R ４ are each independently C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, and C ２ ~C ６ alkynyl; or R ３ and R ４ together contain oxygen (O) or up to two nitrogens (N), each independently C １ ~C ６ Alkyl, cyclopropyl, OH, and C １ ~C ３ may form a 4-6 membered ring optionally substituted with 1 to 2 substituents selected from alkoxy groups; R ５ is C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Alkynyl, C ３ ~C ６ cycloalkyl, and 2-hydroxyethyl groups; R ６ is H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, 4 or 5; b and c are independently 0, 1, or 2; c' is 1, 2, 3, 4, or 5; d is 1 or 2; e is 0, 1, or 2; E ２ is -OC(O)-δ ２ , -OC(O)O-δ ２ , -OC(O)N(Q)-δ ２ , -O-δ ２ , -OCH ２ CH ２ O-δ ２ and -OC(O)(CH ２ ) ６ C(O)O-δ ２ and Q is H or C １ ~C ５ alkyl; ２ is R ２ represents a bond connecting to a group; R ２ teeth 【Chemistry 3】 or a group represented by the formula -(CH ２ ) ｇ - [L ３ - (CH ２ ) ] ｈ -R ９ where L １ and L ２ each independently represents a direct bond, -O-δ ３ , ~CH ２ OC(O)-δ ３ , and ~CH ２ O-δ ３ and δ ３ is R ７ and R ８ represents a bond connecting to a group; R ７ and R ８ are each independently C ４ ~C １０ Alkyl, C ４ ~C １０ Alkenyl or C ４ ~C １０ alkynyl; f is 0, 1, 2, 3, 4, or 5; L ３ teeth 【Chemistry 4】 is selected from R ９ is H and C ４ ~C ８ alkyl groups, g is an integer ranging from 1 to 18; h is 0, 1, 2, or 3. A lipid mix composition comprising:

2. A compound of formula (II), or a pharma- ceutically acceptable salt thereof, in combination with a structured lipid, a sterol, and a stabilizer. 【Chemistry 5】 [In the formula, E １ is -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , and -OC(O)S-δ １ Q is selected from H or C １ ~C ５ alkyl; １ is R １ represents a bond connecting to a group; R １ teeth 【Chemistry 6】 is selected from: R ３ and R ４ are each independently C １ ~C ６ alkyl group; or R ３ and R ４ together contain no more than two nitrogens (N), １ ~C ６ may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups; R ５ is C １ ~C ６ Alkyl, and C ３ ~C ６ cycloalkyl groups; R ６ is H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b and c are independently 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 0 or 1; E ２ Ha-O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ and -OC(O)(CH ２ ) ６ C(O)O-δ ２ where δ ２ is R ２ represents a bond connecting to a group; R ２ teeth 【Chemistry 7】 or a group represented by the formula -(CH ２ ) ｇ - [L ３ - (CH ２ ) ] ｈ -R ９ where L １ and L ２ each independently represents a direct bond, -O-δ ３ , -CH ２ OC(O)-δ ３ , and -CH ２ O-δ ３ and δ ３ is R ７ and R ８ represents a bond connecting to a group; R ７ and R ８ are each independently C ４ ~C １０ Alkyl, C ４ ~C １０ Alkenyl or C ４ ~C １０ alkynyl; f is 0, 1, 2, 3, 4, or 5; L ３ teeth 【Chemistry 8】 Selected from: R ９ is H and C ４ ~C ８ alkyl groups; g is an integer ranging from 1 to 18; h is 0, 1, or 2. A lipid mix composition comprising:

3. A compound of formula (II), or a pharma- ceutically acceptable salt thereof, in combination with a structured lipid, a sterol, and a stabilizer. 【Chemistry 9】 [In the formula, E １ is -OC(O)O-δ １ , -OC(O)-δ １ , -OC(O)N(Q)-δ １ , and -OC(O)S-δ １ Q is selected from H or C １ ~C ５ alkyl; １ is R １ represents a bond connecting to a group; R １ teeth 【Chemistry 10】 is selected from: R ３ and R ４ are C １ ~C ６ or R ３ and R ４ together contain no more than two nitrogens (N), １ ~C ６ may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups; R ５ is C １ ~C ６ alkyl, and cyclopropyl groups; R ６ is H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b is 0 or 1; c is 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 1; E ２ Ha-O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ and -OC(O)(CH ２ ) ６ C(O)O-δ ２ where δ ２ is R ２ represents a bond connecting to a group; R ２ teeth 【Chemistry 11】 or a group represented by the formula -(CH ２ ) ｇ - [L ３ - (CH ２ ) ] ｈ -R ９ where L １ and L ２ are each a direct bond; R ７ and R ８ are C ４ ~C １０ independently selected from alkyl groups; f is 0 or 1; L ３ teeth 【Chemistry 12】 Selected from: R ９ is H and C ４ ~C ８ alkyl groups; g is an integer ranging from 1 to 18; h is 0, 1, or 2. A lipid mix composition comprising:

4. A compound of formula (III), or a pharma- ceutically acceptable salt thereof, in combination with a structured lipid, a sterol, and a stabilizer. 【Chemistry 13】 [R １ teeth 【Chemistry 14】 is selected from: R ３ and R ４ are each independently C １ ~C ６ alkyl group; or R ３ and R ４ together contain no more than two nitrogens (N), １ ~C ６ may form a 5-6 membered ring optionally substituted with 1-2 substituents selected from alkyl groups; R ５ is C １ ~C ６ alkyl, and cyclopropyl groups; R ６ is H, and C １ ~C ６ alkyl groups; a is 1, 2, 3, or 4; b is 0 or 1; c is 0, 1, or 2; c' is 2, 3, or 4; d is 2; e is 1; E ２ Ha-O-δ ２ , -OC(O)-δ ２ , -OCH ２ CH ２ O-δ ２ and -OC(O)(CH ２ ) ６ C(O)O-δ ２ where δ is selected from ２ is R ２ represents a bond connecting to a group; R ２ teeth 【Chemistry 15】 or a group represented by the formula -(CH ２ ) ｇ - [L ３ - (CH ２ ) ] ｈ -R ９ where L １ and L ２ are each a direct bond; R ７ and R ８ are each independently C ４ ~C １０ alkyl groups; f is 0 or 1; L ３ teeth 【Chemistry 16】 Selected from: R ９ is H and C ４ ~C ８ alkyl groups; g is an integer ranging from 1 to 18; h is 0, 1, or 2. A lipid mix composition comprising:

5. A compound having any one of the following structures in combination with a structured lipid, a sterol, and a stabilizer: 【Chemistry 17】 【change】 【change】 【change】 【change】 or a pharma- ceutically acceptable salt thereof A lipid mix composition comprising:

6. A compound having any one of the following structures in combination with a structured lipid, a sterol, and a stabilizer: 【Chemistry 18】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 or a pharma- ceutically acceptable salt thereof A lipid mix composition comprising:

7. 7. The lipid mix composition of any one of claims 1 to 6, wherein the structured lipid comprises one or more structured lipids selected from the group consisting of DSPC (distearoylphosphatidylcholine), DSPE (distearoyl-phosphatidylethanolamine), DPPC (dipalmitoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine), and DOPE (dioleoyl-phosphatidylethanolamine).

8. A lipid mix composition according to any one of claims 1 to 7, wherein the compound is present at 10 Mol% to 90 Mol%, the structured lipid is present at 0 to 50 Mol%, the sterol is present at 0 to 45 Mol%, the stabilizer is present at 0 to 10 Mol%, and the total mol% of all components is 100 mol%.

9. 9. The lipid mix composition of claim 8, wherein the chemical compound is present at 40 Mol% to 60 Mol%, the structured lipid is present at 11 to 40 Mol%, and the total mol% of all components is 100 mol%.

10. The lipid mix composition of claim 8, wherein the molar ratio of the compound to the remaining components is from 30 Mol% to 70 Mol%.

11. A lipid-mix composition according to any one of claims 1 to 10, wherein the composition is in the form of lipid particles.

12. A lipid mix composition described in any one of claims 1 to 11, wherein the experimental pKa of nanoparticles of a compound or a pharma- ceutically acceptable salt thereof described in any one of claims 1 to 6 is in the range of 5.6 to 7.

1.

13. A lipid mix composition according to any one of claims 1 to 12, wherein the compound is present at 40 Mol%, DSPC (distearoylphosphatidylcholine) is present at 20 Mol%, cholesterol is present at 37.5 Mol% and polyoxyethylene (10) stearyl ether is present at 2.5 Mol%.

14. A lipid mix composition according to any one of claims 1 to 12, wherein the compound is present at 40-47.5 Mol%, DSPC (distearoylphosphatidylcholine) is present at 12.5 Mol%, cholesterol is present at 38.5-46 Mol% and PEG-DMG 2000 is present at 1.5 Mol%.

15. 13. A lipid mix composition according to any one of claims 1 to 12, wherein the compound is present at 40-47.5 Mol%, DOPE (Dioleoyl-phosphatidylethanolamine) is present at 12.5 Mol%, cholesterol is present at 38.5-46 Mol% and PEG-DMG 2000 is present at 1.5 Mol%.

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