Circular RNA compositions and methods
Disclosed herein are circular RNAs and transfer vehicles, along with related compositions and methods of treatment. The circular RNAs can comprise group I intron fragments, spacers, an IRES, duplex forming regions, and/or an expression sequence, thereby having the features of improved expression, functional stability, low immunogenicity, ease of manufacturing, and/or extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful in producing the precursor or circular RNAs, which have improved circularization efficiency and/or are compatible with effective circular RNA purification methods.
1. An ionizable lipid of Formula (1):
or a pharmaceutically acceptable salt thereof, wherein
each n is independently an integer from 2-15;
L 1 and L 3 are each independently —OC(O)—* or —C(O)O—*, wherein “*” indicates the attachment point to R 1 or R 3 ;
R 1 and R 3 are each independently a linear C 9 -C 20 alkyl, a linear or branched C 9 -C 20 alkenyl,
each optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and
R 2 comprises an optionally substituted five-membered heteroaryl selected from imidazolyl, pyrazolyl, or 1,2,4-triazolyl, wherein when R 2 comprises imidazolyl, the imidazolyl is not fused with a pyrimidinyl ring.
2. The ionizable lipid of claim 1 , wherein R 2 comprises an optionally substituted imidazole.
3. The ionizable lipid of claim 1 , wherein R 2 is selected from a group consisting of:
4. The ionizable lipid of claim 1 , wherein the ionizable lipid is represented by Formula (1-1) or Formula (1-2):
5. The ionizable lipid of claim 1 , wherein R 1 and R 3 are each independently selected from the group consisting of:
6. The ionizable lipid of claim 1 , wherein R 1 and R 3 are the same.
7. The ionizable lipid of claim 1 , selected from the group consisting of:
8. A pharmaceutical composition comprising a transfer vehicle, wherein the transfer vehicle comprises the ionizable lipid of claim 1 .
9. The pharmaceutical composition of claim 8 , wherein the transfer vehicle comprises a helper lipid and/or structural lipid.
10. The pharmaceutical composition of claim 9 , wherein the structural lipid binds to C1q, and/or promotes the binding of the transfer vehicle comprising said lipid to C1q compared to a control transfer vehicle lacking the structural lipid, and/or increases uptake of C1q-bound transfer vehicle into an immune cell compared to a control transfer vehicle lacking the structural lipid.
11. The pharmaceutical composition of claim 8 , wherein the transfer vehicle comprises a PEG-lipid.
12. The pharmaceutical composition of claim 11 , wherein the PEG-lipid is a modified PEG-lipid.
13. The pharmaceutical composition of claim 12 , wherein the modified PEG-lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, or a combination thereof.
14. A lipid nanoparticle (LNP) comprising an ionizable lipid, a helper lipid, and a structural lipid, wherein the ionizable lipid is a compound of Formula (1):
or a pharmaceutically acceptable salt thereof, wherein
each n is independently an integer from 2-15;
L 1 and L 3 are each independently —OC(O)—* or —C(O)O—*, wherein “*” indicates the attachment point to R 1 or R 3 ;
R 1 and R 3 are each independently a linear C 9 -C 20 alkyl, a linear or branched C 9 -C 20 alkenyl,
each optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and
R 2 comprises an optionally substituted five-membered heteroaryl selected from imidazolyl, pyrazolyl, or 1,2,4-triazolyl, wherein when R 2 comprises imidazolyl, the imidazolyl is not fused with a pyrimidinyl ring.
15. The LNP of claim 14 , wherein R 2 comprises an optionally substituted imidazole.
16. The LNP of claim 14 , wherein R 2 is selected from a group consisting of:
17. The LNP of claim 14 , wherein R 1 and R 3 are each independently selected from the group consisting of:
18. The LNP of claim 14 , wherein R 1 and R 3 are the same.
19. The LNP of claim 14 , wherein the ionizable lipid is selected from the group consisting of:
20. A pharmaceutical composition comprising: (1) a payload molecule; and (2) a LNP of claim 14 .
21. A method of delivering a therapeutic agent to a target cell, comprising administering a LNP of claim 14 .
22. The method of claim 21 , wherein the therapeutic agent is a RNA polynucleotide.
23. The method of claim 22 , wherein the RNA polynucleotide is linear or circular.
24. The method of claim 23 , wherein the target cell is a eukaryotic cell.
25. The method of claim 24 , wherein the eukaryotic cell is a hepatocyte, a hematopoietic cell, an epithelial cell, an endothelial cell, a lung cell, a bone cell, a stem cell, a mesenchymal cell, a neural cell, a photoreceptor cell, a retinal pigmented epithelial cell, a secretory cell, a cardiac cells, an adipocyte, a vascular smooth muscle cell, a cardiomyocyte, a skeletal muscle cell, a beta cell, a pituitary cell, a synovial lining cell, an ovarian cell, a testicular cell, a fibroblast, a B cell, a T cell, a reticulocyte, a leukocyte, a granulocyte, a tumor cell, an NK cell, an NKT cell, a macrophage, or a neutrophil.