Fast-off polyethylene glycol lipids

Abstract

The present disclosure provides PEG lipids and transfer vehicle compositions including the same. Incorporation of the PEG lipids of the present disclosure into transfer vehicles can provide one or more desirable properties, including low immune response and / or low immunogenicity, rapid shedding of the PEG lipids from the transfer vehicles, and / or degradability of the PEG lipids, e.g., via ester hydrolysis or cleavage.

Description

Background Technology

[0001] For decades, polyethylene glycol (PEG) lipids have been incorporated into lipid nanoparticles because they can help control nanoparticle size and reduce immune cell interactions and clearance. However, it is known that therapeutic agents containing PEG (PEGylated drugs) can elicit immune responses, including the production of anti-PEG antibodies, which can lead to accelerated blood clearance (ABC) and reduced or lost efficacy upon repeated administration. Conventionally, PEG lipids incorporated into lipid nanoparticles (LNPs) for gene delivery are thought to primarily elicit thymus-independent (TI) immune responses that do not result in the formation of PEG-specific antibodies. However, when stably anchored PEG lipids interact sufficiently with existing B cells, these B cells can produce anti-PEG IgM antibodies that can bind to LNPs and induce ABC upon subsequent repeated administration. Summary of the Invention

[0002] This disclosure provides PEG lipids and transfer media compositions comprising the same. In some embodiments, incorporating the PEG lipids of this disclosure into a transfer media provides one or more desired properties compared to a control PEG lipid (e.g., as described herein) or a composition comprising the same, including low immune response and / or low immunogenicity (e.g., reduced antibody binding capacity and thus reduced accelerated blood clearance (ABC)), rapid detachment of the PEG lipid from the transfer media, and / or degradability of the PEG lipid (e.g., via ester hydrolysis or cleavage).

[0003] The subject PEG lipid compound may include at least one lipid and a core portion having a linker portion connected to the PEG moiety. In some embodiments, the subject compound includes a connector and additional linker groups connecting the core portion to the PEG moiety and one or more lipids. In some embodiments, the subject compound includes a branched core portion.

[0004] In some embodiments, the transfer medium is lipid nanoparticles (LNPs). This application also provides LNPs comprising a subject PEG lipid and an ionizable lipid. In some embodiments, the LNP comprises a subject PEG lipid and a subject ionizable lipid (as described herein). This document also provides methods for preparing subject PEG lipid compounds and the use of said subject PEG lipid compounds in various transfer media, such as lipid nanoparticle formulations.

[0005] This disclosure provides PEG lipids and transfer mediator compositions comprising the same, wherein the PEG lipids are compounds having formula (I): (A – Z A ) n – B – YA – L – Y B – P (I) in: Y A Selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; P is the PEG portion; L is an optional connector; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; A is a lipid; Z A It is an optional connection part; and n is 1 or 2, where: When n is 2, B is the branched core part, and When n is 1, B is an optional connector.

[0006] In some embodiments of the compounds and compositions disclosed herein, the PEG lipid is a compound having formula II:

[0007] (II)

[0008] in: X 1 It is O or NH; X 2 It is a bond, O or NH; and P is the PEG portion; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; L is an optional connector; m is an integer selected from 1 to 10 (for example, m is an integer selected from 1 to 8, 1 to 6, or 2 to 6).

[0009] Z A It is an optional connection part; and

[0010] A is lipid.

[0011] In some embodiments of the compounds and compositions disclosed herein, the PEG lipid is a compound having the formula VA:

[0012] (VA)

[0013] in: P is the PEG portion; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; L is an optional connector; X 2 It is a bond, O or NH; X 1 It is O or NH; B is the core component of the branching process; Each Z A Independently, it is an optional connecting part; and Each A is an independent lipid.

[0014] In some embodiments of the compositions disclosed herein, the transfer medium further comprises an ionizable lipid having the formula LIV: , Formula (LIV) Or its pharmaceutically acceptable salt, wherein: It is an integer selected from 1 to 7; R a It is hydrogen or hydroxyl; R h It is hydrogen or C1-C6 alkyl; R 1 It is C1-C 30 Alkyl or R 1 ；

[0015] R 2 It is C1-C 30 Alkyl or R 2 ; R 1 and R 2 Selected independently from: –(CH2) q C(O)O(CH2) r C(R 8 (R) 9 (R) 10 ), –(CH2) q OC(O)(CH2)r C(R 8 (R) 9 (R) 10 ),as well as –(CH2) q OC(O)O(CH2) r C(R 8 (R) 9 (R) 10 ); in: q is an integer selected from 0 to 12. r is an integer selected from 0 to 6, where r appears at least once and is not 0; R 8 Is it H or R? 11 ; R 9 R 10 and R 11 Each is independently C1-C 20 Alkyl or C2-C 20 -Alkenyl; and Where (i) R 1 It is R 1 (ii) R 2 It is R 2 , or (iii) R 1 It is R 1 And R 2 It is R 2 .

[0016] In some embodiments of the compositions disclosed herein, the transfer medium further comprises ionizable lipids and RNA polynucleotides, such as cyclic RNA polynucleotides.

[0017] In some embodiments of the composition, the transfer medium is lipid nanoparticles (LNPs). Attached Figure Description

[0018] Figure 1 The zeta potential measurements in millivolts (mV) are depicted for LNP formulations including ionizable lipid 204 (Table 3) and different PEG lipids from Table 1 or 2A.

[0019] Figure 2 TNS pKa assays for LNP formulations containing different PEG lipids from Tables 1 and 2A were plotted.

[0020] Figures 3A-3BThe study shows systemic luminescence expression in Balb / C mice 5 days after administration of LNP (formulated with different combinations of ionizable lipids from Table 3 and PEG lipids from Table 1 or 2) for 3 or 4 weeks. Figures 3A-3B In the table, “lipid A”, “lipid B” and “lipid C” correspond to ionizable lipids 204, 138 and 160 in Table 3, respectively. Figure 3A LNP formulated with lipid A is provided. Figure 3B LNPs formulated with lipids B and C are provided.

[0021] Figures 4A-4B The changes in body weight (expressed as a percentage of body weight) in Balb / C mice treated with LNP (which encapsulates circular RNA expressing firefly luciferase and is formulated with different combinations of ionizable lipids from Table 3 and PEG lipids from Table 2A) at 0.5 mg / kg RNA for 4 consecutive weeks are shown. Figures 4A-4B In the table, "lipid B" and "lipid C" correspond to ionizable lipids 138 and 160 in Table 3, respectively. Figure 4B yes Figure 4A The continuation of.

[0022] Figures 5A-5B The anti-PEG IgM titers are shown in Balb / C mice 5 days after administration for 3 or 4 weeks with LNP (which encapsulates circular RNA and is formulated with different combinations of ionizable lipids from Table 3 and PEG lipids from Table 2A). PBS was used as a control. Figures 5A-5B In the table, “lipid A”, “lipid B” and “lipid C” correspond to ionizable lipids 204, 138 and 160 in Table 3, respectively. Figure 5A LNP formulated with lipid A is provided. Figure 5B LNPs formulated with lipids B and C are provided.

[0023] Figure 6 The anti-PEG IgG titers were measured in Balb / C mice 5 days after administration of LNPs containing circular RNA and formulated with different PEG lipids from Table 2A, administered 4 times weekly. PBS was used as a control.

[0024] Figures 7A-7B This indicates that 24 hours after treatment with LNP (which encapsulates circular RNA encoding the CD-19 chimeric antigen receptor (CAR) protein, formulated with different combinations of ionizable lipids and PEG lipids)... Figure 7A ) and 72 hours ( Figure 7B Balb / C mice showed depletion of B cells in peripheral blood at a certain time. PBS was used as a control. Figures 7A-7BIn the table, "lipid B" and "lipid C" correspond to ionizable lipids 138 and 160 in Table 3, respectively.

[0025] Figures 8A-8E This illustrates the expression of LNP in various spleen cell types in Balb / C mice after treatment with LNP (which encapsulates circular RNA encoding the Wasabi fluorescent reporter protein, formulated with different combinations of ionizable lipids from Table 3 and PEG lipids from Table 2A). The cell types shown in each of these figures include: CD45+ leukocytes (…). Figure 8A ), B220+ B cells ( Figure 8B ), CD3+ T cells ( Figure 8C CD11b+ myeloid cells ( Figure 8D ) and NK+ natural killer cells ( Figure 8E ).for Figures 8A-8E PBS was used as a control. Figures 8A-8E In the table, "lipid B" and "lipid C" correspond to ionizable lipids 138 and 160 in Table 3, respectively.

[0026] Figures 9A-9E The figure illustrates the results of administering the LNP containing the hEPO oRNA payload disclosed herein to BL / 6 mice. Figure 9A and 9C The bar chart shows the expression of anti-PEG IgM in animals given LNP containing the PEG lipids of this disclosure, compared with the control of LNP containing DMG-PEG2k and PBS. Figure 9B and 9D The bar chart shows serum hEPO expression in animals given LNP containing the PEG lipids of this disclosure, compared to the control LNP containing DMG-PEG2k and PBS. Figure 9E The bar chart shows Figure 9D The data normalized the hEPO expression concentration of LNPs containing PEG lipids of this disclosure to LNPs containing DMG-PEG2k. Figures 9A-9E In each of the five columns, in a given mouse group, the measurements from left to right represent weeks 1, 2, 3, 4, and 5.

[0027] Figures 10A-10C The bar charts report the T cells expressing the tested LNP CD19 CAR protein payload in peripheral blood, spleen, and bone marrow samples from humanized mice.

[0028] Figure 11A-11C The bar charts report B cell depletion in humanized mice after administration of LNPs containing CD19 CAR protein payloads in peripheral blood, spleen, and bone marrow samples. Detailed Implementation

[0029] Among other things, this application also provides PEG lipid compounds and transfer media formulations comprising them. In some embodiments, the transfer media are lipid nanoparticles (LNPs) that can be used to deliver polynucleotides. LNPs comprising subject PEG and ionizable lipids (e.g., as described herein) are provided. Subject lipid nanoparticles may comprise RNA polynucleotides, particularly cyclic RNA polynucleotides (also known as circRNA or oRNA®).

[0030] In some implementations, the subject PEG lipid compound is rapidly detached because the entire PEG lipid detaches from the LNP without hydrolysis at the ester, compared to one or more of the control lipids in Table 1.

[0031] In some implementations, the subject PEG lipid compound is rapidly degraded because the PEG lipid is hydrolyzed at the ester site more quickly compared to one or more of the control lipids in Table 1.

[0032] In some embodiments, the subject PEG lipid compound exhibits a better immune response and / or immunogenicity compared to one or more of the control lipids in Table 1. In some embodiments, the subject PEG lipid exhibits one or more of the following compared to one or more of the control lipids in Table 1: a lower anti-PEG response, a lower anti-PEG immune response, and / or lower anti-PEG antibodies (e.g., lower anti-PEG IgM and lower anti-PEG IgG in serum).

[0033] In some embodiments, the subject PEG lipid compound provides desired or improved functional properties in LNP formulations compared to one or more of the PEG lipids disclosed in one or more of the following international patent publication numbers: WO2022 / 173531, WO2018 / 232120, WO2018 / 232120, WO2022 / 169787 and WO2020 / 227510.

[0034] In some implementations, the subject PEG lipid compound may provide desired or improved functional properties in LNP formulations compared to one or more control PEG lipids (e.g., the PEG lipids in Table 1).

[0035]

[0036] RNA therapy, along with related compositions and methods, is also disclosed herein. In some embodiments, RNA therapy allows for increased RNA stability, expression, and prolonged half-life, among other things.

[0037] In some embodiments, methods are provided herein that include administering the circular RNA polynucleotides provided herein into cells for therapeutic purposes or to produce useful proteins. In some embodiments, the method is advantageous in providing the production of desired polypeptides with a longer half-life than linear RNA within eukaryotic cells due to the resistance of the circular RNA to ribonucleases.

[0038] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit this disclosure. Each section may be applied to any aspect of this disclosure. In this application, unless otherwise stated, the use of "or" means "and / or".

[0039] 1. PEG lipids

[0040] This disclosure provides polyethylene glycol (PEG) lipid compounds.

[0041] In some embodiments, the PEG lipid compound includes at least one lipid linked to a core portion having a linker portion linked to a PEG portion. In some embodiments, the subject compound includes a core portion that is a linear linker connecting the core portion to both the PEG portion and the lipid portion. In some embodiments, the subject compound includes a branched lipid linked to the core portion. In some embodiments, the subject compound includes a branched core portion to which two or more lipid portions may be linked. The PEG lipid compound may include one, two, or more cleavable (e.g., susceptible to hydrolysis or enzymatic cleavage in vivo) linker groups.

[0042] In some implementations, the PEG lipid is a compound having formula I: (A – Z A ) n – B – Y A – L – Y B – P (I) in: Y A Selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; P is the PEG portion; L is an optional connector; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH; A is a lipid; ZA It is an optional connection part; and n is 1 or 2, where: When n is 2, B is the branched core part, and When n is 1, B is an optional connector.

[0043] In some embodiments of Formula I, n is 2 and B is a branched core moiety. In some embodiments, the core moiety is an optionally substituted branched C1-C6 alkylene group. In some embodiments, B comprises an optionally substituted branched C3-C6 alkylene group. In some embodiments, B comprises an optionally substituted C3-alkylene group. In some embodiments, B comprises an optionally substituted C4-alkylene group. In some embodiments, B comprises an optionally substituted C5-alkylene group. In some embodiments, B comprises an optionally substituted C6-alkylene group.

[0044] In some implementations of Equation I, the branched core portion B has Equation B-1:

[0045] (B-1)

[0046] Among them W 1 -W 3 They do not exist independently or are C1-C20 alkylene groups.

[0047] In some implementations of formula B-1, W 1 -W 3 Each is independently absent or C1-C6 alkylene groups. In some embodiments of formula B-1, W 1 -W 3 Each is independently absent or C1-C3 alkylene groups. In some embodiments of formula B-1, W 1 -W 3 Each is independent and does not exist independently, nor is it a C1-C2 alkylene group. In some embodiments of formula B-1, W 1 -W 3 Each exists independently, or is methylene.

[0048] In some implementations of formula B-1, W 1 -W 3 Since neither of them exists, B is: .

[0049] In some implementations of formula B-1, W 1 -W 3Two of them are absent, and the other is a C1-C20 alkylene, such as a C1-C6 alkylene, such as C1-alkylene, C2-alkylene, C3-alkylene, C4-alkylene, C5-alkylene, or C6-alkylene.

[0050] In some implementations, B is selected from one of the following branching core components: , , , and .

[0051] In some implementations of formula B-1, W 1 -W 3 One of them is absent, and the other two are C1-C20 alkylene compounds, such as C1-alkylene, C2-alkylene, C3-alkylene, C4-alkylene, or C5-alkylene.

[0052] In some implementations, B is selected from one of the following branching core components: , , , and .

[0053] In some implementations of formula B-1, W 1 -W 3 Each is independently a C1-C20 alkylene, for example, a C1-C6 alkylene, such as C1-alkylene, C2-alkylene, C3-alkylene, C4-alkylene, or C5-alkylene.

[0054] In some implementations, B is selected from one of the following branching core components: , , , , , , and .

[0055] In some embodiments of Formula I, n is 1 and B is a bond. In some embodiments of Formula I, n is 1 and B is a linear linker. In some embodiments, the linker comprises an optionally substituted linear C1-C6 alkylene. In some embodiments, B comprises an optionally substituted linear C3-C6 alkylene. In some embodiments, B comprises an optionally substituted linear C3-alkylene. In some embodiments, B comprises an optionally substituted linear C4-alkylene. In some embodiments, B comprises an optionally substituted linear C5-alkylene. In some embodiments, B comprises an optionally substituted linear C6-alkylene.

[0056] In some implementations of Formula I, Y A It is -OC(O)-. In some implementations, Y A It is -C(O)O-. In some implementations, Y A It is -OC(O)O-. In some implementations, Y A It is -OC(O)NH-. In some implementations, Y A It is -NHC(O)O-. In some implementations, Y A It is -NHC(O)NH-.

[0057] In some implementations of Formula I, A, Z A L, Y B P is defined below.

[0058] In some embodiments of Formula I, n is 1 and the PEG lipid is a compound having Formula II:

[0059] (II)

[0060] in: X 1 It is a bond, O or NH; X 2 It is a bond, O or NH; and P is the PEG portion; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; L is an optional connector; m is an integer selected from 1 to 10 (for example, m is an integer selected from 1 to 8, 1 to 6, or 2 to 6); Z A It is an optional connection part; and A is lipid.

[0061] In some implementations of Formula II, L or LY B Yes. In some embodiments, the connector (L) comprises a linear or branched alkylene group. In some embodiments, the alkylene group has 1 to 10 carbon atoms. In some embodiments, the alkyl chain has 2 to 8 carbon atoms. In some embodiments, the alkyl chain has 3 to 7 carbon atoms. In some embodiments, LY B Includes a connecting portion, which is -OC(O)-, -C(O)O-, or -OC(O)O-. In some embodiments, LY B Includes a connecting portion, which is -OC(O)NH-, -NHC(O)O-, or -NHC(O)NH-.

[0062] In some implementations of Formula II, Y B It does not exist. In some implementations of Formula II, L or LY B It does not exist.

[0063] In some embodiments of Formula II, L is selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 ynylene. In some embodiments of Formula II, L is an optionally substituted C1-C10 alkylene. In some embodiments of Formula II, L is an optionally substituted C1-C6 alkylene. In some embodiments of Formula II, L is an optionally substituted C1-C3 alkylene. In some embodiments of Formula II, L is methylene. In some embodiments of Formula II, L is ethylene.

[0064] In some implementations of Formula II, X 1 It is O. In some implementations of Formula II, X 1 It is NH.

[0065] In some implementations of Formula II, X 2 It is O. In some implementations of Formula II, X 2 It is NH. In some embodiments of Formula II, X 2 It is a key.

[0066] In some implementations of Formula II, X 1 It is O, and X 2 It is the key. In some implementations of Formula II, X 1 It is O, and X 2 It is NH. In some implementations, X 1 and X 2Each is O. In some implementations of Formula II, X 1 It is a key, and X 2 It is O. In some implementations of Formula II, X 1 It is NH, and X 2 It is the key. In some implementations of Formula II, X 1 It is NH, and X 2 It is O. In some implementations, X 1 and X 2 Both are NH.

[0067] In some embodiments of Formula II, the compound has Formula IIIA:

[0068] (IIIA)

[0069] Where s is an integer from 1 to 10.

[0070] In some embodiments of Formula IIIA, s is 1 to 8, for example, 1 to 6. In some embodiments of Formula IIIA, s is 2 to 8. In some embodiments of Formula IIIA, s is 3 to 7. In some embodiments of Formula IIIA, s is 1. In some embodiments of Formula IIIA, s is 2. In some embodiments of Formula IIIA, s is 3. In some embodiments of Formula IIIA, s is 4. In some embodiments of Formula IIIA, s is 5. In some embodiments of Formula IIIA, s is 6. In some embodiments of Formula IIIA, s is 7. In some embodiments of Formula IIIA, s is 8.

[0071] In some implementations of Formula IIIA, A and Z A As defined below.

[0072] In some embodiments of formula IIIA, the compound has formula IIIA-1:

[0073] (IIIA-1)

[0074] Where R 2 and R 3 Each is independently a C1-C15 alkyl or C2-C15 alkenyl.

[0075] In some embodiments of formula IIIA or IIIA-1, s is 2 to 8, for example 3 to 7. In some cases of formula IIIA or IIIB, s is 2, 3, 4, 5, 6, 7, or 8.

[0076] In some embodiments of formula IIIA-1, R 2and R 3 Each is independently a C1-C15 alkyl group, such as a C1-C12 alkyl group or a C4-C8 alkyl group. In some embodiments, R 2 and R 3 Each is independently a C4-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C5-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C6-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C7-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C8-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C9-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C10-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C11-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C12-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C13-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C14-alkyl chain. In some embodiments, R 2 and R 3 Each is an independent C15-alkyl chain.

[0077] In some embodiments of formula IIIA-1, R 2 and R 3 Each is independently a C2-C15 alkenyl group, such as a C2-C12 alkenyl group or a C4-C8 alkenyl group. In some embodiments, R 2 and R 3 Each is an independent C4-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C5-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C6-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C7-olefin chain. In some embodiments, R 2and R 3 Each is an independent C8-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C9-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C10-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C11-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C12-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C13-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C14-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C15-olefin chain.

[0078] In some embodiments of formula IIIA-1, R 2 and R 3 Same. In some embodiments of formula IIIA-1, R 2 and R 3 different.

[0079] In some embodiments of Formula II, the compound has Formula IIIB:

[0080] (IIIB)

[0081] Where r is an integer selected from 1 to 10 (for example, r is an integer selected from 2 to 8, or 1 to 6, or 3 to 7, for example, r is 1, 2, 3, 4, 5, 6 or 7).

[0082] In some embodiments of Formula II, the compound has Formula IIIC:

[0083] (IIIC)

[0084] Where r is an integer selected from 1 to 10 (e.g., 2 to 8, or 1 to 6, or 3 to 7, for example, r is 1, 2, 3, 4, 5, 6 or 7).

[0085] In some embodiments of Formulas IIIB and IIIC, r is an integer selected from 2 to 8 (e.g., 3 to 7). In some embodiments of Formulas IIIB and IIIC, r is an integer selected from 1 to 6. In some embodiments of Formulas IIIB and IIIC, r is 1. In some embodiments of Formulas IIIB and IIIC, r is 2. In some embodiments of Formulas IIIB and IIIC, r is 3. In some embodiments of Formulas IIIB and IIIC, r is 4. In some embodiments of Formulas IIIB and IIIC, r is 5. In some embodiments of Formulas IIIB and IIIC, r is 6. In some embodiments of Formulas IIIB and IIIC, r is 7. In some embodiments of Formulas IIIB and IIIC, r is 8.

[0086] In some embodiments of Formula II, the compound has Formula IIID:

[0087] (IIID)

[0088] Where q is an integer selected from 2 to 10.

[0089] In some embodiments of Formula IIID, q is an integer selected from 2 to 8 (e.g., 2 to 6). In some embodiments of Formula IIID, q is 2. In some embodiments of Formula IIID, q is 3. In some embodiments of Formula IIID, q is 4. In some embodiments of Formula IIID, q is 5. In some embodiments of Formula IIID, q is 6. In some embodiments of Formula IIID, q is 7. In some embodiments of Formula IIID, q is 8.

[0090] In some implementations of formulas IIIA-IIID, Z A It does not exist; A is -CH(R) 2 (R) 3 ); and R 2 and R 3 Each is independently a C1-C15 alkyl or C2-C15 alkenyl.

[0091] In some implementations of formulas IIIA-IIID, A is -CH(R) 2 (R) 3 ), and R 2 and R 3 Each is independently a C1-C15 alkyl group, such as a C1-C12 alkyl group or a C4-C8 alkyl group. In some embodiments, R 2 and R 3 Each is independently a C4-alkyl chain. In some embodiments, R 2 and R 3Each is independently a C5-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C6-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C7-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C8-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C9-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C10-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C11-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C12-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C13-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C14-alkyl chain. In some embodiments, R 2 and R 3 Each is an independent C15-alkyl chain.

[0092] In some implementations of formulas IIIA-IIID, A is -CH(R) 2 (R) 3 ), and R 2 and R 3 Each is independently a C2-C15 alkenyl group, such as a C2-C12 alkenyl group or a C4-C8 alkenyl group. In some embodiments, R 2 and R 3 Each is an independent C4-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C5-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C6-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C7-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C8-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C9-olefin chain. In some embodiments, R2 and R 3 Each is an independent C10-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C11-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C12-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C13-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C14-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C15-olefin chain.

[0093] In some implementations of formulas IIIA-IIID, A is -CH(R) 2 (R) 3 ), and R 2 and R 3 Same. In some embodiments of formulas IIIA-IIID, A is -CH(R) 2 (R) 3 ), and R 2 and R 3 different.

[0094] In some implementations of Formula II, LY B It does not exist.

[0095] In some embodiments of Formula II, the compound has Formula IVA:

[0096] (IVA)

[0097] Where t is an integer selected from 0 to 10 (for example, t is an integer selected from 2 to 8 or 2 to 6).

[0098] In some implementations of Formula IVA, A and Z A As defined below.

[0099] In some embodiments of formula IVA, the compound has formula IVB:

[0100] (IVB)

[0101] in: R 2 and R 3It is independently a C1-C15 alkyl or C2-C15-alkenyl (e.g., C1-C12 alkyl or C4-C8 alkyl).

[0102] In some implementations of formula IVB, R 2 and R 3 Each is independently a C1-C15 alkyl group, such as a C1-C12 alkyl group or a C4-C8 alkyl group. In some embodiments, R 2 and R 3 Each is independently a C4-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C5-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C6-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C7-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C8-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C9-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C10-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C11-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C12-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C13-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C14-alkyl chain. In some embodiments, R 2 and R 3 Each is an independent C15-alkyl chain.

[0103] In some implementations of formula IVB, R 2 and R 3 Each is independently a C2-C15 alkenyl group, such as a C2-C12 alkenyl group or a C4-C8 alkenyl group. In some embodiments, R 2 and R 3 Each is an independent C4-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C5-olefin chain. In some embodiments, R 2 and R3 Each is an independent C6-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C7-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C8-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C9-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C10-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C11-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C12-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C13-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C14-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C15-olefin chain.

[0104] In some implementations of formula IVA-IVB, R 2 and R 3 The same. In some implementations of formula IVA-IVB, R 2 and R 3 different.

[0105] In some embodiments of Formula IVA or IVB, t is an integer selected from 2 to 8 (e.g., 2 to 6 or 3 to 7). In some cases of Formula IVA or IVB, t is 2, 3, 4, 5, 6, or 7. In some embodiments of Formula IVA or IVB, t is 0. In some embodiments of Formula IVA or IVB, t is 1. In some embodiments of Formula IVA or IVB, t is 2 or 3.

[0106] In some embodiments of any of Formula II, IIIA, IIIB, IVA, or IVB, m is an integer selected from 1 to 8 (e.g., 1 to 6 or 2 to 6). In some embodiments of any of Formula II, IIIA, or IIIB, m is 2, 3, 4, 5, or 6. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8.

[0107] On the other hand, this article provides compounds having the formula VA:

[0108] (VA)

[0109] in: P is the PEG portion; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; L is an optional connector; X 2 It is a bond, O, NH or CH2; X 1 It is O or NH; B is the core component of the branching process; Each Z A Independently, it is an optional connecting part; and Each A is an independent lipid.

[0110] In some embodiments of formula VA, the compound is not: Where r is an integer selected from 40 to 50; Where r is an integer selected from 40 to 50; or .

[0111] In some embodiments of formula VA, B comprises optionally substituted branched C1-C6 alkylene groups. In some embodiments, B comprises optionally substituted branched C3-C6 alkylene groups. In some embodiments, B comprises optionally substituted C3-alkylene groups. In some embodiments, B comprises optionally substituted C4-alkylene groups. In some embodiments, B comprises optionally substituted C5-alkylene groups. In some embodiments, B comprises optionally substituted C6-alkylene groups.

[0112] In some implementations of formula VA, the branched core portion B has formula B-1:

[0113] (B-1)

[0114] Among them W 1 -W 3 They do not exist independently or are C1-C20 alkylene groups.

[0115] In some implementations of formula B-1, W 1 -W 3 Each is independently absent or C1-C6 alkylene groups. In some embodiments of formula B-1, W 1 -W 3 Each is independently absent or C1-C3 alkylene groups. In some embodiments of formula B-1, W 1 -W 3 Each is independent and does not exist independently, nor is it a C1-C2 alkylene group. In some embodiments of formula B-1, W 1 -W 3 Each exists independently, or is methylene.

[0116] In some implementations of B-1, W 1 -W 3 Two of them are absent, and the other is a C1-C20 alkylene, such as a C1-C6 alkylene, such as C1-alkylene, C2-alkylene, C3-alkylene, C4-alkylene, or C5-alkylene.

[0117] In some implementations, B includes structure B-2: (B-2) Where a and c are independently 1, 2 or 3, and b is 0, 1, 2 or 3.

[0118] In some embodiments of Equation B-2, b is 0, a is 1, and c is 1. In some embodiments, b is 0, a is 2, and c is 2. In some embodiments, b is 0, a is 3, and c is 3.

[0119] In some implementations, B-2 is selected from one of the following branching core components: , and .

[0120] In some embodiments of formula B-2, a, b, and c are each independently 1, 2, or 3. In some embodiments, a, b, and c are each 2. In some embodiments, a, b, and c are each 1.

[0121] In some implementations of equation B-2, a, b, and c are 0 and B is: .

[0122] In some implementations, B is selected from the following branching core components: , , , , and .

[0123] In some implementations, B is selected from one of the following branching core components: , , , and .

[0124] In some embodiments, compounds having formula VA have formula VB:

[0125] (VB)

[0126] in: a and c are independently 1, 2, or 3, and b is 0, 1, 2, or 3; and L is selected from optionally substituted C1-C10 alkylene groups, optionally substituted C1-C10 heteroalkylene groups, optionally substituted C2-C10 alkenyl groups, and optionally substituted C2-C10 alkyne groups.

[0127] In some embodiments, compounds having the formula VA or VB include a linker Z that covalently links the lipid to the branched core moiety (B). A As described in this article, Z A It is an optional part, therefore in some cases, Z A The absence of this property allows one or more lipids (A) to be directly covalently linked to the branched core portion (B).

[0128] In some implementations of VA or VB, the connection part Z A It is an ether, ester, carbonate, amide, carbamate, urea, amine, acid anhydride, or disulfide group. In some embodiments, Z A Selected from -SS-, -C(O)OC(O)-, -NH-, -NHCONH-, -OCONH-, -NHCO-, -OC(O)O-, -OCO-, and -O-. In some embodiments, Z A It is -SS-. In some implementations, Z A It is -C(O)OC(O)-. In some implementations, Z A It is -NH-. In some implementations, Z A It is -NHCONH-. In some implementations, Z A It is -OCONH-. In some implementations, Z A It is -NHCO-. In some implementations, Z A It is -OC(O)O-. In some implementations, ZA It is -OCO-. In some implementations, Z A Yes -O-.

[0129] In some implementations of VA or VB, the connection part Z A Selected from -CONH-, -NHCO-, -CO2-, -OCO-, -O-, OCO2-, -OCONH-, -NHCOO-, -OPO(O)O-, maleimide-thiol conjugates, and triazoles. In some embodiments, Z A Yes -O-. In some implementations, Z A It is -CO2-. In some implementations, Z A It is -OCO-. In some implementations, Z A It's -OPO(O)O-.

[0130] In some implementations of VA or VB, L or LY B Yes. In some embodiments, the linker (L) comprises a linear or branched alkylene group. In some embodiments, the alkylene group comprises 1 to 10 carbon atoms. In some embodiments, the alkyl chain comprises 2 to 8 carbon atoms. In some embodiments, the alkyl chain comprises 3 to 7 carbon atoms. In some embodiments, L is selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenyl, and optionally substituted C2-C10 ynylene. In some embodiments, LY B Includes a connecting portion, which is -OC(O)-, -C(O)O-, or -OC(O)O-. In some embodiments, LY B Includes a connecting portion, which is -OC(O)NH-, -NHC(O)O-, or -NHC(O)NH.

[0131] In some implementations of VA or VB, L is absent. In some implementations of VA or VB, LY is absent. B It does not exist.

[0132] In some embodiments of formula VB, the compound has formula VC:

[0133] (VC)

[0134] in: X 1 It is O or NH; X 2 It is a bond, NH, O or CH2; R 2 and R3 Each is independently a C1-C15 alkyl or C2-C15-alkenyl (e.g., C8-C15 alkyl or C10-C15 alkyl); s is an integer between 2 and 100 (e.g., 20 to 50, 30 to 50, or 40 to 50); and R 0 It is hydrogen, optionally substituted alkyl, optionally substituted acyl or protecting group.

[0135] In some implementation schemes of VA, VB, or VC, X 1 It is O. In some implementations of VA, VB, or VC, X 1 It is NH.

[0136] In some implementation schemes of VA, VB, or VC, X 2 It is O. In some implementations of VA, VB, or VC, X 2 It is NH. In some implementations of formula VA, VB, or VC, X 2 It is a key.

[0137] In some implementations of the VC, R 2 and R 3 Each is independently a C1-C15 alkyl group, such as a C4-C15 alkyl group or a C4-C8 alkyl group. In some embodiments, R 2 and R 3 Each is independently a C4-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C5-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C6-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C7-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C8-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C9-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C10-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C11-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C12-alkyl chain. In some embodiments, R 2 and R 3Each is independently a C13-alkyl chain. In some embodiments, R 2 and R 3 Each is independently a C14-alkyl chain. In some embodiments, R 2 and R 3 Each is an independent C15-alkyl chain.

[0138] In some implementations of the VC, R 2 and R 3 Each is independently a C2-C15 alkenyl group, such as a C4-C12 alkenyl group or a C4-C8 alkenyl group. In some embodiments, R 2 and R 3 Each is an independent C4-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C5-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C6-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C7-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C8-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C9-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C10-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C11-alkene chain. In some embodiments, R 2 and R 3 Each is an independent C12-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C13-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C14-olefin chain. In some embodiments, R 2 and R 3 Each is an independent C15-olefin chain.

[0139] In some implementations of the VC, R 2 and R 3 The same. In some implementations of the VC, R 2 and R 3 different.

[0140] In some embodiments of VC, s is an integer selected from 2 to 80 (e.g., 10 to 80, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 30 to 50, or 40 to 50). In some embodiments, s is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0141] In some implementations of the VC, R 0 It is hydrogen, optionally substituted alkyl, optionally substituted acyl, or protecting group. In some embodiments, R 0 It is hydrogen. In some implementations, R 0 It is an optionally substituted C1-C6 alkyl group. In some embodiments, R 0 It is an acyl group. In some embodiments, R 0 It is a protecting group. In some embodiments, R 0 It is a trialkylsilyl protecting group. In some embodiments, R 0 It is a diarylalkylsilyl protecting group. In some embodiments, R 0 It is a tetrahydropyranyl protecting group. In some embodiments, R 0 It is a benzyl protecting group.

[0142] As described herein, compounds having the formula VA-VC may have two lipid moieties (A).

[0143] In some implementations of VA or VB, A and Z A As defined below.

[0144] In some implementations, the PEG lipid is a compound having formula (VI): (VI) in: P is the PEG portion; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; L is an optional linker selected from the following: optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenyl, and optionally substituted C2-C10 ynylene. Y A Selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; m is an integer selected from 1 to 10; and R 2 and R 3 Each is independently a optionally substituted C1-C15 alkyl or optionally substituted C2-C15 alkenyl.

[0145] In some implementations, the PEG lipid is a compound having formula (VI): (VI) in: P is a formula The PEG portion; z is an integer selected from 1 to 200; R is either hydrogen or methyl; Y B It is selected from the following optional connection parts: -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; L is an optional linker selected from the following: optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenyl, and optionally substituted C2-C10 ynylene. Y A Selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O- and -NHC(O)NH-; m is an integer selected from 1 to 10; and R 2 and R 3 Each is an optional substitution of C1-C15 aliphatic compounds.

[0146] In some implementations of Formula VI, Y B It exists and is selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-. In some embodiments of formula VI, Y B It does not exist. In some implementations of formula VI, Y B It is -OC(O)-. In some implementations of formula VI, Y B It is -C(O)O-. In some implementations of formula VI, Y B It is -OC(O)O-. In some implementations of formula VI, Y B It is -OC(O)NH-. In some embodiments of formula VI, Y B It is -NHC(O)O-. In some embodiments of formula VI, Y B It is -NHC(O)NH-.

[0147] In some embodiments of Formula VI, L is present and selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenyl, and optionally substituted C2-C10 ynynyl. In some embodiments of Formula VI, L is absent. In some embodiments of Formula VI, L is an optionally substituted C1-C10 alkylene. In some embodiments of Formula VI, L is an optionally substituted C1-C10 heteroalkylene. In some embodiments of Formula VI, L is an optionally substituted C2-C10 alkenyl. In some embodiments of Formula VI, L is an optionally substituted C2-C10 ynynyl. In some embodiments of Formula VI, L is an optionally substituted C1-C9 alkylene. In some embodiments of Formula VI, L is an optionally substituted C3-C9 alkylene. In some embodiments of Formula VI, L is a C1-C9 alkylene. In some embodiments of Formula VI, L is a C3-C9 alkylene. In some embodiments of Formula VI, L is –(CH2)-. In some embodiments of Formula VI, L is –(CH2)2-. In some embodiments of Formula VI, L is –(CH2)3-. In some embodiments of Formula VI, L is –(CH2)4-. In some embodiments of Formula VI, L is –(CH2)5-. In some embodiments of Formula VI, L is –(CH2)6-. In some embodiments of Formula VI, L is –(CH2)7-. In some embodiments of Formula VI, L is –(CH2)8-. In some embodiments of Formula VI, L is –(CH2)9-.

[0148] In some implementations of Formula VI, Y A Selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-. In some embodiments of formula VI, Y A It is -OC(O)-. In some implementations of formula VI, Y A It is -C(O)O-. In some implementations of formula VI, Y A It is -OC(O)O-. In some implementations of formula VI, Y A It is -OC(O)NH-. In some embodiments of formula VI, Y A It is -NHC(O)O-. In some embodiments of formula VI, Y A It is -NHC(O)NH-.

[0149] In some embodiments of Formula VI, m is an integer selected from 1 to 10. In some embodiments of Formula VI, m is an integer selected from 1 to 8. In some embodiments of Formula VI, m is an integer selected from 1 to 6. In some embodiments of Formula VI, m is an integer selected from 2 to 6. In some embodiments of Formula VI, m is an integer selected from 1, 2, and 3. In some embodiments of Formula VI, m is an integer selected from 1 and 2. In some embodiments of Formula VI, m is 1. In some embodiments of Formula VI, m is 2. In some embodiments of Formula VI, m is 3. In some embodiments of Formula VI, m is 4. In some embodiments of Formula VI, m is 5. In some embodiments of Formula VI, m is 6. In some embodiments of Formula VI, m is 7. In some embodiments of Formula VI, m is 8. In some embodiments of Formula VI, m is 9. In some embodiments of Formula VI, m is 10.

[0150] In some implementations of Formula VI, R 2 and R 3 Each is independently an optional substitution of C1-C15 aliphatic compounds. In some embodiments of formula VI, R 2 and R 3 The same. In some implementations of Formula VI, R 2 and R 3 different.

[0151] In some implementations of Formula VI, R 2 It is an optional substitution of C1-C15 aliphatic compounds. In some embodiments of formula VI, R 2 It is an optionally substituted C1-C15 alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C1-C15 branched alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C1-C15 straight-chain alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C2-C15 alkenyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C2-C15 branched alkenyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C2-C15 straight-chain alkenyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C4-C11 alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted straight-chain C4-C11 alkyl group. In some embodiments of formula VI, R 2 It is an unsubstituted C4-C11 alkyl group. In some embodiments of formula VI, R 2It is an unsubstituted straight-chain C4-C11 alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted C6-C11 alkyl group. In some embodiments of formula VI, R 2 It is an optionally substituted straight-chain C6-C11 alkyl group. In some embodiments of formula VI, R 2 It is an unsubstituted C6-C11 alkyl group. In some embodiments of formula VI, R 2 It is an unsubstituted straight-chain C6-C11 alkyl group. In some embodiments of formula VI, R 2 It is an unsubstituted C5-C7 alkyl group. In some embodiments of formula VI, R 2 It is an unsubstituted straight-chain C5-C7 alkyl group. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)3CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)4CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)5CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)6CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)7CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)8CH3. In some embodiments of formula VI, R 2 It is optionally replaced by –(CH2)9CH3. In some embodiments of formula VI, R 2 It is an optional substitution –(CH2) 10 CH3.

[0152] In some implementations of Formula VI, R 3 It is an optional substitution of C1-C15 aliphatic compounds. In some embodiments of formula VI, R 3 It is an optionally substituted C1-C15 alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C1-C15 branched alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C1-C15 straight-chain alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C2-C15 alkenyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C2-C15 branched alkenyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C2-C15 straight-chain alkenyl group. In some embodiments of formula VI, R3 It is an optionally substituted C4-C11 alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted straight-chain C4-C11 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted C4-C11 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted straight-chain C4-C11 alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted C6-C11 alkyl group. In some embodiments of formula VI, R 3 It is an optionally substituted straight-chain C6-C11 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted C6-C11 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted straight-chain C6-C11 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted C5-C7 alkyl group. In some embodiments of formula VI, R 3 It is an unsubstituted straight-chain C5-C7 alkyl group. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)3CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)4CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)5CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)6CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)7CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)8CH3. In some embodiments of formula VI, R 3 It is optionally replaced by –(CH2)9CH3. In some embodiments of formula VI, R 2 It is an optional substitution –(CH2) 10 CH3.

[0153] In some implementations of Formula VI, R 2 and R 3 Each independently selected , , , , , , , , , , , and .

[0154] In some implementations, R 2 and R 3 Each independently selected , , , , , , , and .

[0155] In some implementations, Selected from , , , , , , , , and .

[0156] In some implementations of formula VI, P is a formula... The PEG portion and R is hydrogen or methyl. In some embodiments, P is... In some implementations, P is... .

[0157] In some embodiments of Formula VI, z is an integer selected from 1 to 200. In some embodiments of Formula VI, z is an integer selected from 30 to 75. In some embodiments of Formula VI, z is an integer selected from 35 and 50. In some embodiments of Formula VI, z is 45. In some embodiments of Formula VI, z is 44. In some embodiments of Formula VI, z is 43. In some embodiments of Formula VI, z is 42. In some embodiments of Formula VI, z is 41. In some embodiments of Formula VI, z is 40. In some embodiments of Formula VI, z is 46. In some embodiments of Formula VI, z is 47. In some embodiments of Formula VI, the compound having Formula VI is part of a composition comprising a plurality of compounds having Formula VI, wherein z is an integer from 30 to 75, and wherein the compound having Formula VI in the composition has an approximately normal distribution of z within the range. In some embodiments, the compound having Formula VI is part of a composition comprising a plurality of compounds having Formula VI, wherein z is an integer from 35 to 50, and wherein the compound having Formula VI in the composition has an approximately normal distribution of z within the range. In some embodiments, the compound having formula VI is part of a composition comprising a plurality of compounds having formula VI, wherein z is an integer from 40 to 50, and wherein the compound having formula VI in the composition has an approximately normal distribution of z centered at z = 45.

[0158] In some implementations of formula VI, P is a formula... The PEG portion, where z is 40 to 50, and R is hydrogen; Y B L does not exist; Y A It is -OC(O)-; m is 1 to 9; and R 2 and R 3 Each is independently a C1-C15 alkyl group.

[0159] In some implementations of formula VI, P is a formula... The PEG portion, where z is 40 to 50, and R is methyl; Y B L does not exist; Y A It is -OC(O)-; m is 1 to 9; and R 2 and R 3 Each is independently a C1-C15 alkyl group.

[0160] In some implementations of formula VI, P is a formula... The PEG portion, where z is 40 to 50, and R is hydrogen; Y B It is -C(O)O-; L is a C1-C10 alkylene group; Y AIt is -OC(O)-; m is 1 to 4; and R 2 and R 3 Each is independently a C1-C15 alkyl group.

[0161] In some implementations of formula VI, P is a formula... The PEG portion, where z is 40 to 50, and R is methyl; Y B It is -C(O)O-; L is a C1-C10 alkylene group; Y A It is -OC(O)-; m is 1 to 4; and R 2 and R 3 Each is independently a C1-C15 alkyl group.

[0162] A. Lipids (A)

[0163] PEG lipid compounds having formulas I, II, IIIA-IIID, IVA, VA-VB can have A and Z. A The connected lipid portion, as defined below.

[0164] Each lipid (A) may be independently selected from optionally substituted C6-C30 alkyl, optionally substituted C6-C30 alkenyl, and optionally substituted C6-C30 heteroalkyl. The lipids may be linear or branched, and may be unsubstituted or substituted.

[0165] In some embodiments, each lipid (A) independently comprises a linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl group optionally substituted with one or more substituents selected from: oxo, halogenated, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic)(alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkyl Amino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyl sulfoxide, alkyl sulfoxide alkyl, alkyl sulfonyl, alkyl sulfonyl, and phosphonates.

[0166] In some embodiments, at least one lipid (A) comprises a C8-C30 linear or branched alkyl group. In some embodiments, at least one lipid (A) comprises a linear C8-C30 alkyl group. In some embodiments of formula VA or VB, at least one lipid (A) comprises decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, or eicosane. In some embodiments, at least one lipid (A) comprises a branched C8-C30 alkyl group. In some embodiments, the branched C8-C30 alkyl group comprises two lipid tails, wherein each lipid tail comprises a C7-C12 alkyl chain. In some embodiments, each lipid tail comprises a C7-alkyl chain. In some embodiments, each lipid tail comprises a C8-alkyl chain. In some embodiments, each lipid tail comprises a C9-alkyl chain. In some embodiments, each lipid tail comprises a C10-alkyl chain. In some embodiments, each lipid tail comprises a C11-alkyl chain. In some embodiments, each lipid tail comprises a C12-alkyl chain.

[0167] In some embodiments, at least one lipid (A) comprises a C8-C30 linear or branched alkenyl group. In some embodiments, at least one lipid (A) comprises a linear C8-C30 alkenyl group. In some embodiments of formula VA or VB, at least one lipid (A) comprises myristyl, palmyl, oleyl, or linoleyl.

[0168] In some embodiments, at least one lipid (A or AZ) A -) Selected from: –C10-C30 linear or branched alkyl groups; –C10-C30 linear or branched alkenyl groups; –(CH2)qC(O)O(CH2)rCH(R 1 (R) 2 ); –(CH2)qOC(O)(CH2)rCH(R 1 (R) 2 ); –(CH2)qOC(O)O(CH2)rCH(R 1 (R) 2 ); –(CH2)qOCH(R 1 (R) 2 ); –(CH2)qOC(O)CH(R 1 (R) 2 ); –(CH2)qC(O)OCH(R 1 (R) 2 );as well as –(CH2)qOC(O)OCH(R 1 (R) 2 ), in: q is an integer selected from 0 to 12. Each r is an integer selected independently from 0 to 6; R 1 Is it hydrogen or R? 3 ;and R 2 and R 3 Each is independently a C1-C12 alkyl or C2-C12-olefin.

[0169] In some embodiments, at least one lipid (A) comprises di-myristoyl-glycerol (DMG): .

[0170] In some embodiments, at least one lipid (A) comprises di-palmitoylglycerol (DPG): .

[0171] In some embodiments, at least one lipid (A) comprises di-stearoyl-glycerol (DSG): .

[0172] In some embodiments, at least one lipid (A) comprises a phospholipid. In some embodiments, at least one lipid (A) comprises di-myristoyl-phosphatidyl-ethanolamine (DMPE): .

[0173] In some embodiments, at least one lipid (A) comprises di-palmitoyl-phosphatidyl-ethanolamine (DPPE): .

[0174] In some embodiments, at least one lipid (A) comprises di-stearoyl-phosphatidyl-ethanolamine (DSPE): .

[0175] In some embodiments, at least one lipid (A) comprises dioleoylphosphatidylethanolamine (DOPE): .

[0176] In some embodiments, at least one lipid (A or AZ) A -) Selected from: , , , , , , and , Wherein: each t is independently an integer selected from 0 to 6. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6.

[0177] In some embodiments, at least one lipid (A or AZ) A -) Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0178] In some implementations, at least one lipid (A) is a sterol or cholesterol.

[0179] In some implementations, at least one lipid (A) is cholesterol: .

[0180] In some embodiments, at least one lipid (A) is β-sitosterol: .

[0181] In some embodiments, at least one lipid (A) is a stigmasterol: .

[0182] In some embodiments, at least one lipid (A) is lanosterol: .

[0183] In some implementations, at least one lipid (A) is 7-dehydrocholesterol: .

[0184] In some embodiments, at least one lipid (A) is a yeast sterol: .

[0185] In some embodiments, at least one lipid (A) is lanosterol: .

[0186] In some embodiments, at least one lipid (A) is rapeseed sterol: .

[0187] In some embodiments, at least one lipid (A) is a campesterol: .

[0188] B. Optional connection part Z A

[0189] In some embodiments, PEG-lipid compounds having formulas I, II, IIIA-IIID, IVA, VA-VB include a linker Z. A The connecting portion covalently links the lipid to the compound. As described herein, Z A It is an optional part, therefore in some cases Z A The absence of this property allows the lipid (A) to be directly covalently linked to the compound. It should be understood that the exemplary linked lipid moiety described herein is depicted as an A group in some embodiments and as AZ in some other embodiments. A - group.

[0190] In some implementations, the connection portion Z A It is an ether, ester, carbonate, amide, carbamate, urea, amine, acid anhydride, or disulfide group. In some embodiments, Z A Selected from -SS-, -C(O)OC(O)-, -NH-, -NHCONH-, -OC(O)NH-, -NHCO-, -OC(O)O-, -OC(O-), and -O-. In some embodiments, Z A It is -SS-. In some implementations, Z A It is -C(O)OC(O)-. In some implementations, Z A It is -NH-. In some implementations, Z A It is -NHCONH-. In some implementations, Z A It is -OCONH-. In some implementations, Z A It is -NHCO-. In some implementations, Z A It is -OC(O)O-. In some implementations, Z A It is -OCO-. In some implementations, Z A Yes -O-.

[0191] In some implementations, the connection portion Z A Selected from -CONH-, -NHCO-, -CO2-, -OC(O)-, -O-, OCO2-, -OCONH-, -NHC(O)O-, -OPO(O)O-, maleimide-thiol conjugates, and triazoles. In some embodiments, Z A Yes -O-. In some implementations, Z A It is -CO2-. In some implementations, Z A It is -OC(O)-. In some implementations, Z A It's -OPO(O)O-.

[0192] C. PEG portion P

[0193] In some embodiments, the PEG portion (P) comprises at least 10 ethylene monomer units, such as at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, 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, at least 95, or at least 100 ethylene glycol monomer units. In some embodiments, P comprises at least 100 ethylene glycol monomer units, such as at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, or at least 150 ethylene glycol monomer units. In some embodiments, P includes at least 150 ethylene glycol monomer units, such as at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, or at least 220 ethylene glycol monomer units (e.g., at least 12, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 220). In some implementations, P comprises 10, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 ethylene glycol monomer units, plus or minus 10%.

[0194] In some embodiments, P includes PEG blocks of 500 Da to 20 kDa (e.g., 500 Da to 10 kDa, 500 Da to 7 kDa, 500 Da to 5 kDa, or 500 Da to 2 kDa). In some embodiments, P includes PEG blocks of 1 kDa to 20 kDa (e.g., 1 kDa to 17.5 kDa, 1 kDa to 15 kDa, 1 kDa to 10 kDa, 5 kDa to 20 kDa, 5 kDa to 17.5 kDa, 5 kDa to 15 kDa, 5 kDa to 10 kDa, or 5 kDa to 7.5 kDa). In some embodiments, P includes PEG blocks of 1.5 kDa to 3.5 kDa (e.g., 1.5 kDa to 3 kDa, 1.5 kDa to 2.5 kDa, or 1.5 to 2 kDa). In some embodiments, P includes 500 Da, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa, 8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa, 10 kDa, 10.5 kDa, 11 kDa, 11.5 kDa, 12 kDa, 12.5 kDa, 13 kDa, 13.5 kDa, 14 kDa, 14.5 kDa, 15 kDa, 15.5 kDa, 16 kDa, 16.5 kDa, 17 kDa, 17.5 kDa, 18 kDa, 18.5kDa, 19 kDa, 19.5 kDa or 20 kDa + / - 10% PEG block.

[0195] In some implementations, P includes a PEG block.

[0196] In some implementations, P includes two or more PEG blocks.

[0197] In some implementations, P includes formula P1: P A – (T – P B ) u (P1) in: P A and P B Each is an independent PEG block; T is an optional linking group; and u is an integer selected from 1 to 6.

[0198] In some embodiments of formula P1, u is an integer selected from 1 to 3. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3.

[0199] In some implementations of formula P1, P A and P B It is the same PEG block. In some embodiments of formula P1, P A and P B These are different PEG blocks.

[0200] In some implementations, formula P1 does not include a linking group (T).

[0201] In some embodiments, formula P1 includes a linking group (T). Any suitable linking group may be used in a compound having formula P1. In some embodiments, T includes triazole, maleimide, -S-, -NH-, -CO-, -CONH-, -NHCO-, -CO2-, -OCO-, -O-, OCO2-, -OCONH-, -NHCOO-, -OPO(O)O-, or any combination thereof.

[0202] In some embodiments, formula P1 includes a linker group (T), which is a cleavable linker. The cleavable linker enables the compound to be biodegradable. Any convenient cleavable linker can be used in a subject macrocyclic compound comprising formula P1. In some embodiments, the cleavable linker can be cleaved by exposure to a stimulus. A non-exhaustive list of stimuli includes pH, temperature, light, redox changes, overexpressed enzymes, hypoxia, sound, magnetic force, electrical energy, and any combination thereof. In some embodiments, formula P1 includes a linker group (T), which is a cleavable linker comprising a group selected from: disulfides, hydrazones, vinyl ethers, imines, orthoesters, borates, amides, peptides, azo compounds, and any combination thereof.

[0203] In some implementations, the PEG lipid compound is selected from: , , , , , , and .

[0204] In some implementations, the PEG lipid compound is selected from: , , , , and .

[0205] In some implementations, the PEG lipid compound is selected from: , , , and .

[0206] In some implementations, the PEG lipid compound is .

[0207] In some implementations, the PEG lipid compound is .

[0208] In some implementations, the PEG lipid compound is .

[0209] In some implementations, the PEG lipid compound is .

[0210] In some implementations, the PEG lipid compound is .

[0211] Exemplary PEG lipid compounds of this disclosure are shown in Table 2A.

[0212]

[0213]

[0214] Further exemplary PEG lipid compounds of this disclosure are shown in Table 2B.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] In some embodiments of the compounds in Table 2B, z is selected from 1 to 200. In some embodiments, z is an integer selected from 30 to 75. In some embodiments, z is an integer selected from 35 and 50. In some embodiments, z is 45. In some embodiments, z is 44. In some embodiments, z is 43. In some embodiments, z is 42. In some embodiments, z is 41. In some embodiments, z is 40. In some embodiments, z is 46. In some embodiments, z is 47. All kinds of compounds in Table 2B having any of the above-mentioned “z” values ​​should be understood as being specifically considered herein as exemplary compounds of this disclosure (e.g., compound P131, where z = 45; P131, where z = 44; P131, where z = 43; ... etc.).

[0223] 2. Transfer medium

[0224] This disclosure provides transfer media formulations comprising the subject PEG lipids. Transfer media are particulate media or carriers that can be used to encapsulate and / or deliver a variety of therapeutic agents. Transfer media can be nanoparticles, micelles, liposomes, emulsions, and / or dendritic polymers. Typically, PEG lipids can be used as components of such transfer media, for example, as a coating that protects the particles during drug delivery. In some embodiments, the transfer media are lipid nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, polymer nanoparticles, metal nanoparticles, or nanogels.

[0225] In some implementations, the transfer medium encapsulation is selected from therapeutic agents such as contrast agents, chemotherapy drugs, polynucleotides, and genes.

[0226] Exemplary transfer media have now been described in more detail.

[0227] 3. Liposome formulations

[0228] In some embodiments, the formulation comprises the subject PEG-lipid in the liposome formulation. In these embodiments, the molar ratio of PEG-lipid is from about 0.1% to 100% of the total lipids. In some embodiments, the molar ratio of PEG-lipid is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the total lipids. In some embodiments, the PEG-lipid has formula (I) (as described herein). In some embodiments, the PEG-lipid has formula (II) (as described herein). In some embodiments, the PEG-lipid has formulas (IIIA-IIID) (as described herein). In some embodiments, the PEG-lipid has formulas (IVA-IVB) (as described herein). In some embodiments, the PEG-lipid has formulas (VA-VC) (as described herein). In some embodiments, the PEG-lipid has formula (VI) (as described herein).

[0229] In some embodiments, the liposome formulation includes accessory lipids, structural lipids, and a main PEG-lipid. In some embodiments, the molar ratio of accessory lipids:structural lipids:PEG-lipids in the liposome formulation is about 56:38:5. In some embodiments, the molar ratio of accessory lipids:structural lipids:PEG-lipids in the liposome formulation is about 50:45:5. In some embodiments, the molar ratio of accessory lipids:structural lipids:PEG-lipids in the liposome formulation is about 45:45:10. In some embodiments, the molar ratio of accessory lipids:structural lipids:PEG-lipids in the liposome formulation is about 60:38:2. In some embodiments, the molar ratio of accessory lipids:structural lipids:PEG-lipids in the liposome formulation is about 60:35:5. In some embodiments, the molar ratio of each of the auxiliary lipid, structural lipid, and PEG-lipid is within the stated values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%.

[0230] In some embodiments of the liposome formulation, the auxiliary lipid is a phospholipid (e.g., as described herein). In some embodiments, the phospholipid is hydrogenated soybean phosphatidylcholine (HSPC). In some embodiments of the liposome formulation, the structural lipid is cholesterol. In some embodiments, the liposome formulation comprises a molar ratio of approximately 56:38:5 of HSPC:cholesterol:PEG-lipid. In some embodiments, the molar ratio of each of HSPC, cholesterol, and PEG-lipid is within the stated values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%.

[0231] 4. Lipid nanoparticle formulations

[0232] In some embodiments, this disclosure provides lipid nanoparticles (LNPs) comprising the subject PEG lipid. The subject LNP formulation may also comprise one or more ionizable lipids, accessory lipids, structural lipids, and polynucleotides (as described below). In some embodiments, the LNP further comprises a fifth lipid or more additional lipids in addition to the aforementioned lipid components. In some embodiments, the LNP delivery medium further comprises one or more targeting portions covalently or non-covalently bound to the outer surface of the LNP delivery medium. In some embodiments, the targeting portion is a portion that binds to or otherwise facilitates the uptake of one or more specific cell populations.

[0233] The formation of the lipid nanoparticles (LNPs) described herein can be achieved by any method known in the art. For example, as described in U.S. Patent Publication No. US2012 / 0178702 A1, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Semil et al. (2010) Nat. Biotechnol. 28:172-176; Jayaraman et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of these are incorporated herein by reference in their entirety).

[0234] In one embodiment, the LNP formulation can be prepared by methods described, for example, in International Patent Publication Nos. WO 2011 / 127255 or WO2008 / 103276, the contents of each of which are incorporated herein by reference in their entirety.

[0235] In one embodiment, the LNP formulation described herein may include a polycationic composition. As a non-limiting example, the polycationic composition may be a composition selected from Formulas 1-60 of U.S. Patent Publication No. US2005 / 0222064 A1, the contents of which are incorporated herein by reference in their entirety.

[0236] In one embodiment, the lipid nanoparticles can be formulated by the methods described in U.S. Patent Publication No. US2013 / 0156845 A1 and International Patent Publication Nos. WO2013 / 093648 A2 or WO2012 / 024526 A2, each of which is incorporated herein by reference in its entirety.

[0237] In one embodiment, the lipid nanoparticles described herein can be prepared in a sterile environment using the system and / or method described in U.S. Patent Publication No. US2013 / 0164400 A1, the entire contents of which are incorporated herein by reference.

[0238] In one embodiment, the LNP formulation may be formulated in nanoparticles, such as the nucleic acid-lipid particles described in U.S. Patent No. 8,492,359, which is incorporated herein by reference in its entirety.

[0239] Nanoparticle compositions may optionally include one or more coatings. For example, nanoparticle compositions may be formulated in coated capsules, films, or tablets. Capsules, films, or tablets comprising the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0240] In some embodiments, the lipid nanoparticles described herein can be synthesized using methods including microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to, slit interdigitated micromixers, including but not limited to slit interdigitated micromixers and / or staggered herringbone micromixers (SHM) manufactured by Precision Nanosystems (Vancouver, BC, Canada), Microinnova (Allerheiligen bei Wildon, Austria) (Zhigaltsev, IV et al. (2012) Langmuir. 28:3633-40; Belliveau, NM et al. Mol. Ther. Nucleic. Acids. (2012) 1:e37; Chen, D. et al. J. Am. Chem. Soc. (2012) 134(22):6948-51; each of which is incorporated herein by reference in its entirety).

[0241] In some embodiments, the LNP generation method including SHM further includes mixing at least two input streams, wherein mixing occurs via microstructure-induced chaotic convection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern, causing swirling flow and folding of the fluids around each other. The method may also include surfaces for fluid mixing, wherein the surfaces change orientation during fluid circulation. Methods for generating LNPs using SHM include those disclosed in U.S. Patent Publications US2004 / 0262223 A1 and US2012 / 0276209 A1, each of which is incorporated herein by reference in its entirety.

[0242] In one embodiment, the lipid nanoparticles may be formulated using a micromixer, such as, but not limited to, a slit interdigitated microstructure mixer (SIMM-V2), a standard slit interdigitated micromixer (SSIMM), a Caterpillar (CPMM), or an Impinging-jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany. In one embodiment, the lipid nanoparticles are produced using microfluidic technology (see, Whitesides (2006) Nature. 442: 368-373; and Abraham et al. (2002) Science. 295: 647-651; each of which is incorporated herein by reference in its entirety). As a non-limiting example, controlled microfluidic formulations include passive methods for driving flow in microchannels at low Reynolds number mixing-stabilized pressures (see, for example, Abraham et al. (2002) Science. 295: 647-651; which is incorporated herein by reference in its entirety).

[0243] In one embodiment, the polynucleotides (e.g., circRNAs) of this disclosure can be formulated in lipid nanoparticles created using a micromixer chip, such as, but not limited to, those from Harvard Apparatus (Holliston, MA), Dolomite Microfluidics (Royston, UK), or Precision Nanosystems (Van Couver, BC, Canada). The micromixer chip can be used to rapidly mix two or more fluid flows using splitting and recombination mechanisms.

[0244] In some embodiments, the LNP of this disclosure includes a molar ratio of ionizable lipids between about 40% and about 60%, a molar ratio of accessory lipids between about 3.5% and about 14%, a molar ratio of structural lipids between about 28% and about 50%, and a molar ratio of subject PEG-lipids between about 0.5% and about 5%, including all endpoints. In some embodiments, the total molar percentage of ionizable lipids, accessory lipids, structural lipids, and PEG-lipids in the LNP is 100%.

[0245] In some embodiments, the molar ratio of ionizable lipids in the LNP is about 40 to about 60% of the total lipids present in the LNP. In some embodiments, the molar ratio of ionizable lipids in the LNP is about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60% of the total lipids present in the LNP. All values ​​include all endpoints.

[0246] In some embodiments, the molar ratio of accessory lipids in the LNP is from about 3.5% to about 14% of the total lipids present in the LNP. In some embodiments, the molar ratio of accessory lipids in the LNP is from about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, or about 14% of the total lipids present in the LNP. In some embodiments, the accessory lipid is DSPC. In some embodiments, the accessory lipid is DOPE. All values ​​include all endpoints.

[0247] In some embodiments, the molar ratio of structural lipids in the LNP is from about 28% to about 50% of the total lipids present in the LNP. In some embodiments, the molar ratio of structural lipids in the LNP is about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the total lipids present in the LNP. In some embodiments, the structural lipid is cholesterol. All values ​​include all endpoints.

[0248] In some embodiments, the molar ratio of the subject PEG-lipid in the LNP is about 0.1% to about 5% of the total lipids present in the LNP. In some embodiments, the molar ratio of the subject PEG-lipid in the LNP is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, 3.4%, about 3.5%, about 4.0%, about 4.5%, or about 5% of the total lipids present in the LNP. In some embodiments, the PEG lipid has formula (I) (as described herein). In some embodiments, the PEG lipid has formula (II) (as described herein). In some embodiments, the PEG lipid has formula (III) (as described herein). In some embodiments, the PEG lipid has formula (IV) (as described herein). In some embodiments, the PEG lipid has formula (V) (as described herein). In some embodiments, the PEG lipid has formula (VI) (as described herein). In some embodiments, the PEG lipid has formula (VII) (as described herein). In some embodiments, the PEG lipid has formula (VIII) (as described herein). In some embodiments, the PEG lipid has formula (VI) (as described herein). All values ​​include all endpoints.

[0249] In some embodiments, the molar ratio of ionizable lipids:auxiliary lipids:structural lipids:PEG-lipids in the LNP is approximately 45:9:44:2. In some embodiments, the molar ratio is approximately 50:10:38.5:1.5. In some embodiments, the molar ratio is approximately 41:12:45:2. In some embodiments, the molar ratio is approximately 62:4:33:1. In some embodiments, the molar ratio is approximately 53:5:41:1. In some embodiments, the molar ratio of each of the ionizable lipid, auxiliary lipid, structural lipid, and PEG-lipid is within the stated values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%.

[0250] In one embodiment, the lipid nanoparticles may have a size of about 10 to about 100 nm, such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 80 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 80 nm, about 3 ... Diameters of about 40 to 90 nm, about 40 to 100 nm, about 50 to 60 nm, about 50 to 70 nm, about 50 to 80 nm, about 50 to 90 nm, about 50 to 100 nm, about 60 to 70 nm, about 60 to 80 nm, about 60 to 90 nm, about 60 to 100 nm, about 70 to 80 nm, about 70 to 90 nm, about 70 to 100 nm, about 80 to 90 nm, about 80 to 100 nm, and / or about 90 to 100 nm. In one embodiment, the lipid nanoparticles may have a diameter of about 10 to 500 nm. In one embodiment, the lipid nanoparticles may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm. Each possibility represents a separate embodiment of this disclosure.

[0251] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

[0252] In some embodiments, the lipid nanoparticles described herein may have a size from less than 0.1 µm to up to 1 mm, such as, but not limited to, less than 0.1 µm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm. Diameters of 450 μm, 475 μm, 500 μm, 525 μm, 550 μm, 575 μm, 600 μm, 625 μm, 650 μm, 675 μm, 700 μm, 725 μm, 750 μm, 775 μm, 800 μm, 825 μm, 850 μm, 875 μm, 900 μm, 925 μm, 950 μm, and 975 μm.

[0253] In another embodiment, the LNP may have the following structures: about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 10 to about 50 nm, about 20 to about 50 nm, about 30 to about 50 nm, about 40 to about 50 nm, about 20 to about 60 nm, about 30 to about 60 nm, about 40 to about 60 nm, about 20 to about 70 nm, about 30 to about 70 nm. Diameters of approximately 40 to 70 nm, approximately 50 to 70 nm, approximately 60 to 70 nm, approximately 20 to 80 nm, approximately 30 to 80 nm, approximately 40 to 80 nm, approximately 50 to 80 nm, approximately 60 to 80 nm, approximately 20 to 90 nm, approximately 30 to 90 nm, approximately 40 to 90 nm, approximately 50 to 90 nm, approximately 60 to 90 nm, and / or approximately 70 to 90 nm. Each possibility represents a separate embodiment of this disclosure.

[0254] The nanoparticle composition can be relatively homogeneous. A polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as its particle size distribution. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The nanoparticle composition may have a polydispersity index of about 0 to about 0.25 (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25). In some embodiments, the polydispersity index of the nanoparticle composition may be about 0.10 to about 0.20. Each possibility represents a separate embodiment of this disclosure.

[0255] The zeta potential of a nanoparticle composition can be used to indicate the electromotive force of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with relatively low charges (positive or negative) are generally desirable because higher charge types may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -20 mV to about +20 mV, about -20 mV to about +15 mV, about -20 mV to about +10 mV, about -20 mV to about +5 mV, about -20 mV to about 0 mV, about -20 mV to about -5 mV, about -20 mV to about -10 mV, about -20 mV to about -15 mV, about -20 mV to about +20 mV, about -20 mV to about +15 mV, about -20 mV to about +10 mV, about -20 mV to about +5 mV, about -20 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV. mV to approximately +10 mV. Each possibility represents a separate embodiment of this disclosure.

[0256] The encapsulation efficiency of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. A high encapsulation efficiency is desirable (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic agent in a solution containing the nanoparticle composition before and after decomposition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., nucleic acid) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic agent can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. Each possibility represents a separate embodiment of this disclosure. In some embodiments, the lipid nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH. In some embodiments, the lipid nanoparticles have an average diameter of 50-200 nm.

[0257] The properties of lipid nanoparticle formulations can be influenced by factors including, but not limited to: the selection of cationic lipid components, the degree of cationic lipid saturation, the selection of non-cationic lipid components, the degree of non-cationic lipid saturation, the selection of structural lipid components, the nature of PEGylation, the ratio of all components, and biophysical parameters (e.g., size). As described herein, the purity of the subject PEG lipid components can also be important to the properties and performance of LNPs.

[0258] In one embodiment, the lipid nanoparticle formulation can be prepared by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is incorporated herein by reference in its entirety. In some embodiments, the lipid nanoparticle formulation can be as described in International Publication No. WO2019131770, which is incorporated herein by reference in its entirety.

[0259] In some embodiments, the circular RNA is formulated according to the method described in U.S. Patent Application 15 / 809,680 (published as US20180153822A1). In some embodiments, this disclosure provides a process for encapsulating circular RNA in a transfer medium, the process comprising the steps of: forming a pre-formed transfer medium with lipids (i.e., forming it in the absence of RNA), and then combining the pre-formed transfer medium with RNA. In some embodiments, the novel formulation process produces RNA formulations with higher potency (peptide or protein expression) and higher efficacy (improved biologically relevant endpoints) both in vitro and in vivo, and potentially better tolerability, compared to the same RNA formulation prepared without the step of pre-forming lipid nanoparticles (e.g., combining lipids directly with RNA).

[0260] For certain cationic lipid nanoparticle formulations of RNA, the RNA in a buffer solution (e.g., citrate buffer) must be heated to achieve high RNA encapsulation. In those processes or methods, heating needs to be performed before the formulation process (i.e., heating a separate component) because post-formulation heating (after nanoparticle formation) does not increase the encapsulation efficiency of RNA in the lipid nanoparticles. In contrast, in some embodiments of the novel process disclosed herein, the heating sequence of RNA does not appear to affect the percentage of RNA encapsulation. In some embodiments, heating (i.e., maintaining at ambient temperature) of one or more of the following—a solution comprising pre-formed lipid nanoparticles, a solution comprising RNA, and a mixed solution comprising RNA encapsulated by lipid nanoparticles—is not required before or after the formulation process.

[0261] RNA can be provided in solution to mix with a lipid solution, allowing the RNA to be encapsulated within lipid nanoparticles. A suitable RNA solution can be any aqueous solution containing various concentrations of the RNA to be encapsulated. For example, a suitable RNA solution may contain RNA at concentrations of about 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml. In some embodiments, suitable RNA solutions may contain concentrations ranging from about 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0.05-0.8 mg / ml, 0.05-0.7 mg / ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4 mg / ml, 0.05-0.3 mg / ml, and 0.05-0.2 mg / ml. mg / ml, 0.05-0.1 mg / ml, 0.1-1.0 mg / ml, 0.2-0.9 mg / ml, 0.3-0.8 mg / ml, 0.4-0.7mg / ml or 0.5-0.6 mg / ml of RNA.

[0262] Typically, suitable RNA solutions may also contain buffers and / or salts. Buffers may typically include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, or sodium phosphate. In some embodiments, suitable buffer concentrations may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM.

[0263] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable salt concentrations in the RNA solution may be in the range of about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.

[0264] In some implementations, a suitable RNA solution may have a pH in the range of about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.

[0265] Various methods can be used to prepare RNA solutions suitable for this disclosure. In some embodiments, the RNA can be directly dissolved in the buffer solution described herein. In some embodiments, the RNA stock solution can be prepared by mixing the RNA stock solution with the buffer solution prior to mixing it with the lipid solution for encapsulation. In some embodiments, the RNA stock solution can be prepared by mixing the RNA stock solution with the buffer solution immediately prior to mixing it with the lipid solution for encapsulation.

[0266] According to this disclosure, the lipid solution comprises a mixture of lipids suitable for forming a transfer medium for encapsulating RNA. In some embodiments, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution may comprise a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropanol-based. In another embodiment, a suitable lipid solution is dimethyl sulfoxide-based. In yet another embodiment, a suitable lipid solution is a mixture of suitable solvents, including but not limited to ethanol, isopropanol, and dimethyl sulfoxide.

[0267] Suitable lipid solutions may contain mixtures of desired lipids at various concentrations. In some embodiments, suitable lipid solutions may contain mixtures of desired lipids at total concentrations ranging from about 0.1-100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml.

[0268] In some embodiments, the LNP of this disclosure further comprises one or more additional PEG lipids disclosed and described in PCT application disclosure WO 2024 / 044728 A1 (which is incorporated herein by reference in its entirety). In some embodiments, the PEGylated lipid is a lipid having any one of the following: formula PL-I', PL-I'', PL-I, PL-Ia, PL-Ib, PL-Iaa, PL-Iab, PL-Iac, PL-Iad, PL-Iae, PL-Iaf, PL-Iag, PL-Iah, PL-Iba, PL-Ibb, PL-Ibc, PL-Ibd, PL-Ibe, PL-Ibf, PL-Ibg, PL-Ibh, PL-Ica, PL-Icb, PL-Icc, PL-Icd, PL-Id PL-Ie, PL-If, PL-Ig, PL-Ih, PL-Ii, PL-Iha, PL-Ihb, PL-Ihc, PL-Ihd, PL-Iia, PL-Iib, PL-Iic, PL-Iid, PL-Ij, PL-Ik, L-Il, PL-Im, PL-In, PL-Io, PL-Ip, PL -Iq, PL-Ioa, PL-Iob, PL-Ioc, PL-Iod, PL-Ioe, PL-Iof, PL-Iog, PL-Ioh, PL-Ipa, PL-Ipb, PL-Ipc, PL-Ipd, PL-Ipe, PL-Ipf, PL-Ipg, PL-Iph, PL-Iqa, PL-Iqb, PL-Iqc, PL-Iqd, PL-Ir, PL-Is, PL-It, PL-Iu, PL-Iv, PL-Iw, PL-Iva, PL-Ivb, PL-Ivc, PL-Ivd, PL-Iwa, PL-Iwb, PL-Iwc, PL-Iwd, PL-Ix, PL-Ixx, PL-Iy, PL-I yy, PL-Iyyy, PL-Iz, PL-Izz, PL-Izzz, PL-II', PL-II'', PL-II, PL-IIc, PL-IId, PL-IIe, PL-IIf, PL-IIg, PL-IIh, PL-IIa, PL-IIb, PL-IIk, PL-IIm, or PL-IIn.

[0269] A. Ionizable lipids

[0270] In some embodiments, the lipid solutions and lipid nanoparticles disclosed herein comprise ionizable lipids. The subject matter of these ionizable lipids can be used as components of compositions to facilitate the encapsulation and release of nucleic acid cargo (e.g., circular RNA) into one or more target cells. In some embodiments, the ionizable lipids comprise one or more cleavable functional groups (e.g., disulfides) that allow, for example, hydrophilic functional head groups to dissociate from lipophilic functional tail groups of the compound (e.g., upon exposure to oxidizing, reducing, or acidic conditions).

[0271] In some embodiments, the ionizable lipid has a pKa of 6 to 12. In some embodiments, the ionizable lipid has a pKa of 7 to 9. In some embodiments, the ionizable lipid has a pKa of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0, or any range of pKa created from these values.

[0272] In some implementations, the ionizable lipids include amino groups.

[0273] In some embodiments, the ionizable lipid includes a divalent head group and one or more straight-chain hydrocarbon lipid tails. In some embodiments, the length of the straight-chain hydrocarbon lipid tail is 3-25 carbon atoms, for example, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 15, 10 to 20, or 10 to 25 carbon atoms.

[0274] In some embodiments, the ionizable lipid includes a divalent head group and one or more branched hydrocarbon lipid tails. In some embodiments, the length of the branched hydrocarbon lipid tail is 3-25 carbon atoms, for example, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 15, 10 to 20, or 10 to 25 carbon atoms.

[0275] In some implementations, the divalent head group is selected from guanidine and squaramide.

[0276] In some embodiments, the squaramide head group has the following formula: , Wherein RA and RB are each independently a C1-C6 alkyl group or H; and This represents the attachment point between the head group and the straight-chain or branched hydrocarbon lipid tail.

[0277] In some embodiments, the ionizable lipid includes a head group selected from the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , and , in This represents the attachment point between the head group and the straight-chain or branched hydrocarbon lipid tail.

[0278] In some embodiments, the ionizable lipid includes a head group having the following formula: Where n is an integer selected from 1 to 7 (for example, n is 1, 2, 3, 4, 5, 6 or 7).

[0279] In some embodiments, the ionizable lipid includes a head group selected from the following:

[0280] in This represents the attachment point between the head group and the straight-chain or branched hydrocarbon lipid tail.

[0281] In some embodiments, the ionizable lipid includes a hydrophilic head group as disclosed in Jayaraman et al. Angew. Chem. Int. Ed. (2012), 51, 8529-8533.

[0282] In some embodiments, the ionizable lipid is ethyl lauroyl arginine ester (ELA). In some embodiments, the ionizable lipid is ionizable lipid 1, wherein ionizable lipid 1 comprises: .

[0283] In some embodiments, one or more of the cationic or ionizable lipids are represented by formula (LI):

[0284] Formula (LI)

[0285] in: n is an integer chosen from 1 and 4; R a It is hydrogen or hydroxyl; and R1 and R2 are each independently linear or branched C6-C chains optionally substituted with one or more substituents selected from the following. 30 Alkyl, C6-C 30 alkenyl or C6-C 30Heteroalkyl: oxo, halogen, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic)(alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyl sulfoxide, alkyl sulfoxide alkyl, alkyl sulfonyl and alkyl sulfonyl alkyl.

[0286] In some embodiments, Ra is hydrogen. In some embodiments, Ra is a hydroxyl group.

[0287] In some implementations, the ionizable lipid is represented by formula (LIa-1), formula (LIa-2), or formula (LIa-3):

[0288] In some implementations, the ionizable lipid is represented by formula (LIb-1), formula (LIb-2), or formula (LIb-3):

[0289] In some embodiments, the ionizable lipids are represented by formula (LIb-4), (LIb-5), (LIb-6), (LIb-7), (LIb-8), or (LIb-9):

[0290] In some embodiments, one or more of the cationic or ionizable lipids are represented by formula (LI), wherein R1 and R2 are each independently selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0291] In some implementations, R1 and R2 are the same. In other implementations, R1 and R2 are different.

[0292] In various embodiments, one or more of the cationic or ionizable lipids are derived from formula (LI) )express:

[0293] Formula (LI) )

[0294] in: n It is an integer selected from 1 to 7. R a It is hydrogen or hydroxyl. R b It is hydrogen or C1-C6 alkyl. R1 and R2 are each independently linear or branched C1-C chains optionally substituted with one or more substituents selected from the following. 30 Alkyl, C2-C 30 alkenyl or C1-C 30Heteroalkyl: oxo, halogenated, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic) (alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl) (alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy Alkyl, alkyl carbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenyl carbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynyl carbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyl sulfoxide, alkyl sulfoxide alkyl, alkyl sulfonyl and alkyl sulfonyl alkyl.

[0295] In some embodiments, one or more of the cationic or ionizable lipids are represented by formula (LII):

[0296] Formula (LII)

[0297] in: Each n is an integer selected independently from 2 to 15; L1 and L3 are each independently –OC(O)– Or –C(O)O– ,in" "Indicates the attachment point with R1 or R3; R1 and R3 are each independently straight-chain or branched C9-C chains optionally substituted with one or more substituents selected from the following. 20 Alkyl or C9-C 20 Alkenyl: oxoyl, halogen, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic) (alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl) (alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl) (alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkynylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylsulfonyl and alkylsulfonylalkyl; and R2 is selected from: , , , , , , , , , , , , , , , , , , , , and .

[0298] In some embodiments, the ionizable lipid is selected from ionizable lipids having formula LII, wherein R1 and R3 are each independently selected from: , , , , , , , , , and .

[0299] In some implementations, R1 and R3 are the same. In other implementations, R1 and R3 are different.

[0300] In some embodiments, one or more of the cationic or ionizable lipids are represented by formula (LII-1) or formula (LII-2):

[0301] Formula (LII-1),

[0302] Formula (LII-2).

[0303] In some embodiments, the ionizable lipid is selected from the ionizable lipids of WO2015 / 095340. In some embodiments, the ionizable lipid is selected from the ionizable lipids of WO2021 / 021634, WO2020 / 237227, or WO2019 / 236673. In some embodiments, the ionizable lipid is selected from the ionizable lipids of WO2021226597 and WO2021113777. In some embodiments, the ionizable lipid is selected from the ionizable lipid of WO2023056033. In some embodiments, the ionizable lipid is selected from the ionizable lipid of WO2023081526.

[0304] In some embodiments, one or more of the cationic or ionizable lipids are represented by formula (LIII):

[0305] Formula (LIII)

[0306] Or its pharmaceutically acceptable salt, wherein

[0307] L 1 It is C2-C 11 Alkylene, C4-C 10 - Idemenyl or C4-C 10 - Hydrinyl group; X 1 OR 1 SR 1 or N(R) 1 )2, where R 1 Independently, it is H or an unsubstituted C1-C6 alkyl group; and R 2 and R 3 Each is independently C6-C 30 -alkyl, C6-C 30 -Alkenyl or C6-C 30 -Alkyne group.

[0308] In some embodiments, one or more of the cationic or ionizable lipids are derived from formula (LIII) )express:

[0309] Formula (LIII) )

[0310] Or its pharmaceutically acceptable salt, wherein: L 1 It is C2-C 11 Alkylene, C4-C 10 - Idemenyl or C4-C 10 - Hydrinyl group; X 1 OR 1 SR 1 or N(R) 1 )2, where R 1 Independently, it is H or an unsubstituted C1-C6 alkyl group; and R2 and R3 are each independently linear or branched C1-C chains optionally substituted with one or more substituents selected from the following. 30 Alkyl, C2-C 30 alkenyl or C1-C 30 Heteroalkyl: oxoyl, halogen, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic) (alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl) (alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonyloxy Alkyl carbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenyl carbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynyl carbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyl sulfoxide, alkyl sulfoxide alkyl, alkyl sulfonyl and alkyl sulfonyl alkyl.

[0311] In some implementations, the ionizable lipid is a compound of formula (LIV): , Formula (LIV) Or its pharmaceutically acceptable salts, of which: It is an integer selected from 1 to 7; R a It is hydrogen or hydroxyl; R h It is hydrogen or C1-C6 alkyl; R 1 It is C1-C 30 Alkyl or R 1 ；

[0312] R 2 It is C1-C 30 Alkyl or R 2 ; R 1 and R2 Selected independently from: –(CH2) q C(O)O(CH2) r C(R 8 (R) 9 (R) 10 ), –(CH2) q OC(O)(CH2) r C(R 8 (R) 9 (R) 10 )as well as –(CH2) q OC(O)O(CH2) r C(R 8 (R) 9 (R) 10 ); in: q is an integer selected from 0 to 12. r is an integer selected from 0 to 6, where r appears at least once and is not 0; R 8 Is it H or R? 11 ; R 9 R 10 and R 11 Each is independently C1-C 20 Alkyl or C2-C 20 -Alkenyl; and Where (i) R 1 It is R 1 (ii) R 2 It is R 2 , or (iii) R 1 It is R 1 And R 2 It is R 2 .

[0313] In some embodiments of formula (LIV), the ionizable lipid has formula (LIV-A):

[0314] Formula (LIV-A)

[0315] Or its pharmaceutically acceptable salt, wherein: n is an integer selected from 1 to 7; q and q' are each an integer selected independently from 0 to 12; r and r' are each independently an integer selected from 0 to 6, where at least one of r or r' is not 0; Z A and Z B Each is independently selected from ^-C(O)O-, ^-OC(O) and -OC(O)O-; where ^ represents -(CH2). q -or-(CH2) q’ - Attachment point; and R 9A R 9B R 10A and R 10B Each is independently C1-C 20 Alkyl or C2-C 20 Alkenyl group.

[0316] In some implementations of formula (LIV-A), Z A and Z B Both are ^-C(O)O-, where ^ represents -(CH2). q -or-(CH2) q’ - Attachment point.

[0317] In some implementations, the ionizable lipid is any of the following structures: (204); (138) or (160)

[0318] In some embodiments, ionizable lipid 138 is combined with PEG lipids P1, P2, P3, or P9, and exhibits a lower anti-PEG response compared to a combination of ionizable lipid 138 and DMG PEG. See, for example, Figure 5.

[0319] In some embodiments, ionizable lipid 138 is combined with PEG lipids P1, P2 or P9, and exhibits a lower anti-PEG response compared to ionizable lipid 160 combined with the same PEG lipids (e.g., P1, P2 or P9).

[0320] In some embodiments, ionizable lipid 160 is combined with PEG lipid P3 and exhibits an anti-PEG response comparable to that of ionizable lipid 138 combined with PEG lipid P3.

[0321] In some embodiments, ionizable lipid 138 is combined with PEG lipid C18-PEG-OH and exhibits an anti-PEG response comparable to that of ionizable lipid 160 combined with PEG lipid C18-PEG-OH.

[0322] In some embodiments, the ionizable lipids of this disclosure are represented by formula (LV): , Style (LV) Or its pharmaceutically acceptable salts, of which: R a It is hydrogen or hydroxyl; R 1 It is C1-C 30 Alkyl or R 1 ；

[0323] R 2 It is C1-C 30 Alkyl or R 2 ; R 1 and R 2 Selected independently from: –(CH2) q C(O)O(CH2) r C(R 4 (R) 5 (R) 6 ), –(CH2) q OC(O)(CH2) r C(R 4 (R) 5 (R) 6 )and –(CH2) q OC(O)O(CH2) r C(R 4 (R) 5 (R) 6 ); in: q is an integer selected from 0 to 12. r is an integer selected from 0 to 6, where r appears at least once and is not 0; R 4 Is it hydrogen or R? 7 ; R 5 R 6 and R 7 Each is independently C1-C 20 Alkyl or C2-C 20 -Alkenyl; Where (i) R 1 For R 1 (ii) R 2 For R 2 , or (iii) R 1 For R 1 And R 2 For R 2 ;and R 3 Let L-R' be a linear or branched chain C1-C. 10 alkylene, and R' is (i) a monocyclic or bicyclic heterocyclic or heteroaryl group, such as imidazolyl, pyrazolyl, 1,2,4-triazolyl or benzimidazolyl, each optionally substituted with a C1-C6 alkyl group at one or more available carbon and nitrogen sites, or (ii) R A R B or R C ,in R A Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ; R B Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ;and R C Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0324] In some embodiments, the LNP of this disclosure includes the ionizable lipid disclosed in PCT patent application publication WO2023044343A1 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is one of formula (VII-A), (VIII-A), (X), or (XA) of PCT application publication WO2023044343A1.

[0325] In some embodiments, the LNP of this disclosure includes the ionizable lipid disclosed in PCT application publication WO2023044333A1, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is one of formula (CY), (CY-I), (CY-IV), (CY-IV'), or (CY-VI') of PCT application publication WO2023044333A1.

[0326] In some embodiments, the LNPs of this disclosure include ionizable lipids disclosed in PCT Publication WO2023122752A1, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is one of the formulas II, III, VI, VI', VI'', VI''', VII, VII', VII'', VII''', VIII, VIII', VIII'', VIII''', IX, IX', IX'', IX''', X, X', X'', X''', XI, XI', XI'', XII, XII', XII'', XII''', XIII, XIII', XIII'', XIII''', XIV, XIV', XIV'', XV, XV', XV'', XV'', XVI, XVI', XVI'', XVI''', XVII, XVIII, XVIII', XIX, XX, or XXI of PCT application publication WO2023122752A1.

[0327] In some embodiments, the LNP of this disclosure comprises the ionizable lipid disclosed in PCT publication WO2023196931A1, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is one of formula (CX-I), (CX-i), or (CZ) of PCT application publication WO2023196931A1.

[0328] In some embodiments, the LNP of this disclosure comprises the ionizable lipid disclosed in PCT patent application publication WO2024192277A1 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is one of the formulas (SI), (SM), (AT), (AT-E'), (AT-F'''), (AT-M), (AT-N'), (AT-O'), (AT-P'''), (AC), (CO), (CC), (CC-A), (CC-C), (CC-E), (CC-F'), (CC-H), (CC-J), or (CC-L) of PCT application publication WO2024192277A1.

[0329] In some embodiments, this disclosure provides compounds having the formula (AX'''').

[0330] (AX''''), Or its pharmaceutically acceptable salt, wherein: A is selected from optionally substituted bridging bicyclic carbon rings, bridging polycyclic carbon rings, bridging heterocyclic bicyclic carbon rings, or bridging heterocyclic polycyclic carbon rings; n is an integer selected from 0, 1, and 2; m is an integer selected from 1 and 2, such that m plus n is less than or equal to 3; R 1 Selected from -OH, -OAc, -NR2, -N(R)R H , , , , , , , , , , , and ; Each R is independently -H or C1-C6 aliphatic; Each R H It is a C1-C6 aliphatic -OH; Each X 1 and X A C1-C6 aliphatic molecules that are independently bonded or optionally substituted; Each Y 1 Selected independently , , , , , , , and key; where marked " The key is connected to X. 1 ; Each X 2 and X 3 C1-C is independently a bond or optionally substituted. 12 aliphatic; Each Y 2 and Y 3 Selected independently , , , , , , and ; marked with " The key is connected to X. 2 or X 3 ; Each X 4 and X 5C1-C6 aliphatic molecules that are independently bonded or optionally substituted; Each Y 4 and Y 5 Independently selected from keys, , , , , , , and ; marked with " The key is connected to X. 4 or X 5 ; Each X 6 and X 7 C1-C6 aliphatic molecules that are independently bonded or optionally substituted; R 2 It is -CH(OR) 6 (OR) 7 -CH(SR) 6 (SR) 7 ), -CH(R 6 (R) 7 ), -CF(R 6 (R) 7 -R 10 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 10 , where R 2 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Each R 3 Independently is -CH(OR) 8 (OR) 9 -CH(SR) 8 (SR) 9 ), -CH(R 8 (R) 9 ), -CF(R 8 (R) 9 -R 11 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 11 , where R 3One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 6 and R 7 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 10 Or optionally replace -C1-C 14 Aliphatic-R 10 ;where R 6 and R 7 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 8 and R 9 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 11 Or optionally replace -C1-C 14 Aliphatic-R 11 ;where R 8 and R 9 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkylene group, phenyl group, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; and Each R 10 and R 11 Independently or optionally, cyclic, double-cyclic, bridged double-cyclic, multi-cyclic, or bridged multi-cyclic C4-C 14 Cycloalkyl, or optionally substituted cyclic, bicyclic, bridged bicyclic, polycyclic, or bridged polycyclic 4-14 membered heterocyclic groups, or two R groups. 10 Or two Rs 11 Together they form optional bridging double or multi-ring C4-C 14 Cycloalkyl, or optionally substituted bridging bicyclic or polycyclic 4-14 membered heterocyclic groups; Where X 1 X A X 2 X 3 X 4 X 5 X 6 X 7R 2 and R 3 One or more of them may be optionally and independently substituted by one or more substituents selected from -F, -Cl, -Br and -I.

[0331] In some implementations, A is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0332] In some embodiments, the LNP of this disclosure comprises an ionizable lipid having the formula (AX).

[0333] (AX), Or its pharmaceutically acceptable salt, wherein: A is selected from optionally substituted bridging carbocyclic or heterocyclic cores, and is chosen from the group consisting of: , , , , , and ; n is an integer selected from 1 or 2; R 1 Choose from the following groups: -OH, -OAc, -NR2, , , , , , , , , , , and ; Each R is independently -H or C1-C6 aliphatic; X 1 and X A Each is an independent C1-C6 aliphatic compound with either a bond or optional substitution; Y 1 Choose from the following groups: , , , , , , , and key; where marked " The key is connected to X. 1 ; Each X 2 and X 3 C1-C is independently a bond or optionally substituted. 12 aliphatic; Each Y 2 and Y 3 Choose independently from the following groups: , , , , , , and ; marked with " The key is connected to X. 2 or X 3 ; Each X 4 and X 5 Independently, the C1-C6 aliphatic molecules are optionally substituted; R 2 It is -CH(OR) 6 (OR) 7 -CH(SR) 6 (SR) 7 ), -CH(R 6 (R) 7 -R 10 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 10 , where R 2 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Each R 3 Independently is -CH(OR) 8 (OR) 9 -CH(SR) 8 (SR) 9 ), -CH(R 8 (R) 9 -R 11 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 11 , where R 3 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 6 and R 7 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 10 Or optionally replace -C1-C 14 Aliphatic-R 10 ;where R 6 and R7 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 8 and R 9 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 11 Or optionally replace -C1-C 14 Aliphatic-R 11 ;where R 8 and R 9 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Each R 10 and R 11 Independently or optionally, cyclic, double-cyclic, bridged double-cyclic, multi-cyclic, or bridged multi-cyclic C4-C 14 Cycloalkyl, or optionally substituted cyclic, bicyclic, bridged bicyclic, polycyclic, or bridged polycyclic 4-14 membered heterocyclic groups, or two R groups. 10 Or two Rs 11 Together they form optional bridging double or multi-ring C4-C 14 Cycloalkyl, or optionally substituted bridging bicyclic or polycyclic 4-14-membered heterocyclic groups.

[0334] In some embodiments, this disclosure provides compounds having any one of the following formulas:

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350] Or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 X A X 2 X 3 X 4 X 5 Y 1 Y 2 Y 3 R 3 R 6 R 7 R 8 R 9 R 10 and R 11 As described in formula (AX) or as further described in any of the embodiments below.

[0351] In some implementations, the ionizable lipids are selected from the lipids in Table 3: Table 3: Exemplary ionizable lipids

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481] B. assist lipids

[0482] In some embodiments, the lipid solutions and lipid nanoparticles described herein comprise one or more non-cationic assist lipids. In some embodiments, the assist lipid is a phospholipid. In some embodiments, the assist lipid is a phospholipid substitute or alternative. In some embodiments, the phospholipid or phospholipid substitute may be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or combinations thereof. Typically, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0483] The phospholipid portion may be selected from, for example, the following non-restrictive group: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin.

[0484] The fatty acid portion may be selected from, for example, the non-restrictive group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0485] Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include sphingolipids, such as sphingomyelin.

[0486] In some implementations, the auxiliary lipid is a 1,2-distearyl-177-glycerol-3-phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0487] In some implementations, the auxiliary lipid is a nonphosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a DSPC substitute.

[0488] In some embodiments, the auxiliary lipids are described in PCT / US2018 / 053569. Auxiliary lipids suitable for the lipid compositions of this disclosure include, for example, various neutral, uncharged, or zwitterionic lipids. Such auxiliary lipids are preferably used in combination with one or more of the compounds and lipids disclosed herein. Examples of co-acting lipids include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1 Palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-disarachido-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-biseicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearate phosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the adjuvant lipid may be distearate phosphatidylcholine (DSPC) or dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the assisting lipid may be distearylphosphatidylcholine (DSPC). The assisting lipid serves to stabilize and improve the processing of lipid solutions and lipid nanoparticles. Such assisting lipids are preferably used in combination with other excipients, such as one or more ionizable lipids disclosed herein.

[0489] In some embodiments, the LNP comprises a phospholipid selected from the non-limiting group consisting of: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DI ... Acyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), (S)-2-ammonium-3-((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxy)phosphoyl)oxy)propionate sodium salt (L-α-phosphatidylserine);Brain PS), myristoyl phosphatidylcholine (DMPC), myristoyl phosphatidyl ethanolamine (DMPE), myristoyl phosphatidylglycerol (DMPG), dioleoyl-phosphatidyl ethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-(phospho-L-serine) (DOPS), Cell Fusion Phospholipids (DPhPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Ditransoleoyl-sn-phosphatidylethanolamine (DEPE), Dipalmitoylphosphatidylglycerol (DPPG), Dipalmitoylphosphatidylserine (DPPS), Distearylphosphatidylcholine (DSPC), Distearyl-phosphatidyl-ethanolamine (DSPE), Distearylphosphatidylethanolamine imidazole (DSPEI), 1,2-Diundecanoyl-sn-glycerol-phosphocholine (DUPC), Lecithinylcholine (EPC), 1,2-Dioleoyl-sn-glycerol-3-phosphate (18:1 PA; DOPA), Bis((S)-2-hydroxy-3-(oleoyloxy)propyl)ammonium phosphate (18:1 DMP; LBPA), 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol) (DOPI; 18:1) PI), 1,2-distearyl-sn-glycerol-3-phosphate-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:0 Lyso PS) and sphingomyelin. In some embodiments, the lipid nanoparticles contain DSPC. In some embodiments, the LNP contains DOPE. In some embodiments, the lipid nanoparticles contain both DSPC and DOPE.

[0490] In some embodiments, the LNP comprises a phospholipid selected from the following: 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-myristoyl-2-stearoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-myristoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-glycerol-3-phosphate choline, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-arachidonic acid... 1-Steayl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1-oleoyl- 2-Myristoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-acetyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-3',4'-bisphosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-3',5'-bisphosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-4',5'-bisphosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-4',5'-bisphosphate), 1,2-dioleoyl -sn-glycerol-3-phosphate-(1'-inositol-3',4',5'-triphosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-3'-phosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-4'-phosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol-5'-phosphate), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine and 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycerol-3-phosphate choline.

[0491] In some embodiments, the LNP comprises a phospholipid selected from the following: DSPS (distearylphosphatidylserine), DSPG (1,2-distearyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)), DSPA (1,2-distearyl-sn-glycerol-3-phosphate), diPhyPC (1,2-diphytanyl-sn-glycerol-3-phosphate choline), diPhy-diether-PC (1,2-di-O-phytyl-sn-glycerol-3-phosphate choline), diPhyPE (1,2-diphytanyl-sn-glycerol-3-phosphate ethanolamine), diPhy-diether-PE (1,2-di-O-phytyl-sn-glycerol-3-phosphate ethanolamine), diPhyPS (1,2-diphytanyl-sn-glycerol-3-phosphate-L-serine), diPhyPG (1,2-Diphytyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)), diPhyPA (1,2-Diphytyl-sn-glycerol-3-phosphate), egg PA (L-α-phosphatidic acid) and soybean PA (L-α-phosphatidic acid).

[0492] In some embodiments, the LNP comprises phospholipids selected from the following: 18:1 (Δ9-cis) PE (DOPE), 18:0-18:1 PE (SOPE), C16-18:1 PE, 16:0-18:1 PE (POPE), 18:1 BMP (S,R), 18:0-18:1 PC (SOPC), 16:0-18:1 PC (POPC), 4ME 16:0 diether PE (4Me), 18:1 (Δ9-trans) PE (DEPE), 16:1 PE (DPPE), and CL. In some embodiments, the LNP comprises phospholipids described or disclosed in the following: Alvarez-Benedicto et al. (Biomater. Sci., 2022, 10, 549) and Li et al. (Asian Journal of Pharmaceutical Sciences, 2015, 10, 81-98).

[0493] In some embodiments, the LNP comprises sphingosine lipids or sphingolipids, such as, but not limited to, sphingomyelin. As used herein, the terms “sphingosine lipid” and “sphingolipid” are intended to refer to a class of lipids containing a backbone comprising a sphingosine base. An exemplary sphingosine base is sphingosine. In some embodiments, the LNP comprises a sphingolipid selected from the following: egg sphingomyelin (egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitoyl-octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), brain or porcine sphingomyelin (brain SM / (2S,3R,E)-3-hydroxy-2-stearoyl-octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), milk or bovine sphingomyelin (milk SM / (2S,3R,E)-3-hydroxy-2-tetradecanoamide-octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), 28:0 SM (N-octadecanoyl-D-erythrosine dihydrosphocholine), 14:0 SM (N-myristoyl-D-erythrosine dihydrosphocholine), 16:1 SM (N-Palmyl-D-erythrodihydrosphingosine phosphocholine), 12:0 dihydroSM (N-Lauroyl-D-erythrodihydrosphingosine phosphocholine), Lyso SM (Sphingosine phosphocholine), Lyso SM (Sphingosine phosphocholine), Lyso SM (dihydro) (Dihydrosphingosine phosphocholine), 24:1 SM (N-Ceramide-D-erythrodihydrosphingosine phosphocholine), 24:0 SM (N-Teicosyl-D-erythrodihydrosphingosine phosphocholine), 18:1 SM (N-Oleoyl-D-erythrodihydrosphingosine phosphocholine), 18:0 SM (N-Stearyl-D-erythrodihydrosphingosine phosphocholine), 17:0 SM (N-Heptadecyl-D-erythrodihydrosphingosine phosphocholine), 16:0 SM (N-palmitoyl-D-erythrodihydrosphocholine), 12:0 SM (N-lauroyl-D-erythrodihydrosphocholine), 06:0 SM (N-hexanoyl-D-erythrodihydrosphocholine), 02:0 SM (N-acetyl-D-erythrodihydrosphocholine), 3-O-methylLyso SM (3-O-methyl-sphocholine), 3-O-methyl-N-methylLyso SM (3-O-methyl-N-methyl-sphocholine) and 3-N-methylLyso SM (3-N-methyl-sphocholine).

[0494] In some embodiments, the LNP comprises a phospholipid containing at least one constrained tail, such as those described by Gan (Bioeng Transl Med. 2020 Sep; 5(3): e10161.). In some embodiments, the phospholipid is selected from one of the following: .

[0495] In some embodiments, the LNP contains phospholipids containing ceramide analogs with triazole linkages, such as those described by Kim et al., Bioorg. Med. Chem. Lett., 17(16), 2007, 4584-4587.

[0496] In some embodiments, the LNP comprises the phospholipids disclosed in WO 2023 / 141470, which is incorporated herein by reference in its entirety. In some embodiments, the phospholipids are (a15:0-i15:0PE).

[0497] In some implementations, LNP includes the phospholipids disclosed in WO 2022 / 040641 (which is incorporated herein by reference in its entirety).

[0498] In some implementations, the phospholipid tail may be modified to facilitate endosome escape, as described in U.S. Patent Application Publication 2021 / 0121411, which is incorporated herein by reference.

[0499] In some implementations, the LNP includes US 2019 / 0240354; US 2010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 2021 / 0128488; US 2020 / 0121809; US 2017 / 0119904; US2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US 2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095A1; WO 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO Phospholipids disclosed in one of the documents 2019 / 089828A1, each of which is incorporated herein by reference in its entirety.

[0500] In some embodiments, the LNP comprises the phospholipids disclosed in PCT disclosure WO2023141470A2 (which is incorporated herein by reference in its entirety). In some embodiments, the LNP comprises phospholipids having formula (I) of PCT disclosure WO2023141470A2, including but not limited to 2-ammonium ethyl ((S)-3-(((S)-12-methyltetradecanoyl)oxy)-2-((13-methyltetradecanoyl)oxy)propyl) phosphate.

[0501] In some embodiments, the phospholipids disclosed in US 2020 / 0121809 have the following structure:

[0502] R1 and R2 are each independently branched or straight-chain, saturated or unsaturated carbon chains (e.g., alkyl, alkenyl, alkynyl).

[0503] C. Structural lipids

[0504] In some embodiments, the lipid solutions and lipid nanoparticles described herein comprise one or more structured lipids. Incorporating one or more structured lipids into the lipid nanoparticles can help reduce the aggregation of other lipids in the particles. Structured lipids may include, but are not limited to, cholesterol, coccidosterol, ergosterol, campesterol, tomatine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structured lipid may be selected from cholesterol, coccidosterol, fucosterol, β-sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, campesterol, tomatine, cholic acid, sitosterol, lithocholic acid, tomatine, ursolic acid, α-tocopherol, vitamin D3, vitamin D2, calcipotriol, botulinum toxin, lupeol, oleanolic acid, β-sitosterol acetate, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof. In some embodiments, structural lipids are described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

[0505] In some embodiments, the structural lipids are described in international patent application PCT / US2019 / 015913, which is incorporated herein by reference in its entirety.

[0506] In some embodiments, the structured lipid is a sterol. In some embodiments, the structured lipid is a steroid. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is a cholesterol analogue. In some embodiments, the structured lipid is α-tocopherol.

[0507] The lipid solutions and lipid nanoparticles described herein include one or more structured lipids. Incorporation of structured lipids into transfer media (e.g., lipid nanoparticles) can help mitigate the aggregation of other lipids within the particles. In some embodiments, the structured lipids include cholesterol, corticosteroids (such as, for example, prednisolone, dexamethasone, and hydrocortisone), or combinations thereof.

[0508] In some embodiments, the structural lipid is a sterol. The structural lipid may include, but is not limited to, sterols (e.g., phytosterols or animal sterols).

[0509] In some implementations, the structural lipids are steroids. For example, steroids may include, but are not limited to, cholesterol, β-sitosterol, coccosterol, ergosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatine, tomatine, ursolic acid, or α-tocopherol.

[0510] In some embodiments, the lipid solution and lipid nanoparticles comprise an effective amount of immune cell delivery-enhancing lipids, such as cholesterol analogs or amino lipids or combinations thereof, which, when present in the transfer medium (e.g., lipid nanoparticles), can act by enhancing cell association and / or uptake, internalization, intracellular transport and / or processing, and / or endosome escape, and / or enhance recognition and / or binding to immune cells, relative to transfer media lacking immune cell delivery-enhancing lipids. Therefore, while not intended to be bound by any particular mechanism or theory, in one embodiment, the structural lipids or other immune cell delivery-enhancing lipids of this disclosure bind to C1q, or promote the binding of transfer media including such lipids to C1q. Therefore, for the in vitro use of the transfer media of this disclosure for the delivery of nucleic acid molecules to immune cells, culture conditions including C1q are used (e.g., using a culture medium including serum or adding exogenous C1q to a serum-free culture medium). For the in vivo use of the transfer media of this disclosure, the demand for C1q is provided by endogenous C1q.

[0511] In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is a cholesterol analogue. In some embodiments, the structural lipid is one of the lipids listed in Table 4 below: Table 4: Exemplary structural lipids

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531] D. Other lipid components

[0532] In some embodiments, the LNP compositions of this disclosure further comprise one or more additional lipid components capable of influencing the tropism of LNPs. In some embodiments, the LNPs further comprise at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200 (see Cheng, et al. Nat Nanotechnol. April 2020; 15(4): 313–320; Dillard, et al. PNAS 2021 Vol. 118 No. 52).

[0533] In some embodiments, the LNP of this disclosure further comprises one or more additional ionizable lipids, such as, but not limited to, those disclosed in one of the following: US 2023 / 0053437; US 2019 / 0240354; US 2010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 2021 / 0128488; US 2020 / 0121809; US 2017 / 0119904; US 2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US 2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095A1; WO WO 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO 2019 / 089828A1, each of which is incorporated herein by reference in its entirety. In some embodiments, the LNP of this disclosure further comprises one or more additional ionizable lipids selected from those disclosed in WO2023044343A1 or WO2023044333A1 (both of which are incorporated herein by reference in their entirety).

[0534] In some embodiments, the LNP compositions of this disclosure comprise or further comprise one or more lipids selected from the group consisting of: 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1,2-dilinoyl-sn-glycerol-3-phosphocholine (18:3 PC), acylcarnosine (AC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 LysoPC), N-oleoyl-sphingomyelin (SPM) (C18:1), and N-tetracosyl-SPM. (C24:0), N-sphingomyelin (C24:l), cardiolipin (CL), 1,2-bis(tetrazol-10,12-diynyl)-sn-glycerol-3-phosphocholine (DC8-9PC), diceryl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoylphosphatidylcholine (DHPC), dihexadecanoyl-phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycerol-3-PE (DLPE), Dimyroxyglycerol hemisuccinate (DMGS), Dimyroxyphosphatidylcholine (DMPC), Dimyroxyphosphatidylethanolamine (DMPE), Dimyroxyphosphatidylglycerol (DMPG), Dioleoylbenzyl alcohol (DOBA), 1,2-Dioleoylglycerol-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-bis-octadecane-9-enoxy-propoxy)-ethoxy]-ethoxy}-ethoxy)- [3,4,5-Dihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylthioalkyl]-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-(phospho-L-serine) (DOPS), Cell Fusion Phospholipids (DPhPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dipalmitoylphosphatidylglycerol (DPPG), Dipalmitoylphosphatidylserine (DPPS), Distearylphosphatidylcholine (DSPC), Distearyl-phosphatidyl-ethanolamine (DSPE), Distearylphosphatidylethanolamine imidazole (DSPEI), 1,2-Diundecanoyl-sn-glycerol-phosphocholine (DUPC), Lecithinylcholine (EPC), Histamine distearylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-histidine-hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-tetrapentacarbidediyneamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyromethesphophyllate-a (HPPH), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dipalmitoylglycerol-O-α-histidine-Nα-hemisuccinate (IsohistsuccDG), mannosylated dipalmitoylphosphatidylethanolamine (ManDOG), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimidemethyl)cyclohexane-formamide] (MCC-PE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16:0) PE), 1-myristoyl-2-hydroxy-sn-glycerol-choline-phosphocholine (MHPC), thiol-reactive maleimide head group lipids (e.g., 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[4-(p-maleimide phenyl)butyramide (MPB-PE)), nervonic acid (NA), sodium cholate (NaChol), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycerol-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipids (PE), and PE lipids conjugated with polyethylene glycol (PEG). (Examples include polyethylene glycol-distearate phosphatidylethanolamine lipids (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soybean phosphatidylcholine (PHSPC), phosphatidylinositol lipids (PI), phosphatidylinositol-4-phosphate (PIP), palmitoyloleoyl phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleoyl phosphatidylglycerol (POPG), phosphatidylserine (PS), rhodamine β-phosphatidylethanolamine lipids (Rh-PE), purified soybean-derived phospholipid mixtures (SIOO), phosphatidylcholine (SM), and 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).) Soybean phosphatidylcholine (SPC), sphingomyelin (SPM), α,α-trehalose-6,6'-dibenzenesulfonate (TDB), 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine (trans-DOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methylmethyl phosphate, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-Dicosahedroyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycerol-3-phosphate choline, 1,2-dilinoleyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearateyl-sn-glycerol-3-phosphate ethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleoylphosphatidylethanolamine.

[0535] E. Targeted portion

[0536] In some embodiments, the lipid nanoparticles further include a targeting portion. The targeting portion may be an antibody or a fragment thereof. The targeting portion may be capable of binding to a target antigen. In some embodiments, the lipid nanoparticles include more than one targeting portion. In some embodiments, the lipid nanoparticles include more than one targeting portion, wherein the targeting portion targets at least two different receptors, and in some embodiments, the at least two different receptors are ubiquitous in different types of cells or tissues.

[0537] In some embodiments, the pharmaceutical composition includes a targeting portion operatively linked to the lipid nanoparticles. In some embodiments, the targeting portion is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed in a greater extent in the target organ than in the liver.

[0538] In some embodiments, the targeting portion is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeting particle is conjugated to a specific anti-CD38 monoclonal antibody (mAb), which enables the siRNA encapsulated within the particle to be specifically delivered to B-cell lymphocytic malignancies (such as MCL) at a greater percentage than to other subtypes of leukocytes.

[0539] In some embodiments, the targeting portion targets receptors selected from the following: CD20, CCR7, CD3, CD4, CD5, CD8, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD35, CD40, CD45RA, CD45RO, CD52, CD62L, CD80, CD95, CD127, and CD137. In some embodiments, the targeting portion targets receptors selected from the following: CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153. The target receptors are CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7. In some embodiments, the target portion targets receptors selected from CD2, CD3, CD5, and CD7. In some embodiments, the target portion targets receptors selected from CD2, CD3, CD5, CD7, CD8, CD4, β7 integrin, β2 integrin, and C1q. In some embodiments, the target portion targets CD117. In some embodiments, the target portion targets CD90. In some implementations, the targeting portion targets receptors selected from mannose receptors, CD206, and C1q.In some implementations, the targeting portion is selected from T-cell receptor motif antibodies, T-cell α-chain antibodies, T-cell β-chain antibodies, T-cell γ-chain antibodies, T-cell δ-chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25 antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40 antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56 antibodies, CD62L antibodies, CD68 antibodies, CD80 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, and CD137 antibodies. (4-1BB) antibody, CD163 antibody, F4 / 80 antibody, IL-4Rα antibody, Sca-1 antibody, CTLA-4 antibody, GITR antibody, GARP antibody, LAP antibody, granzyme B antibody, LFA-1 antibody, transferrin receptor antibody, and fragments thereof. In some embodiments, the target portion is any of those described or considered in US20230312713A1, US20230203538A1, US20230320995A1, US20160145348, and US20110038941, each of which is incorporated herein by reference in its entirety.

[0540] In some implementations, lipid nanoparticles can be targeted when conjugated / attached / associated with a target motif (e.g., an antibody or a fragment thereof).

[0541] F. Exemplary LNP Compositions

[0542] In some embodiments, the present invention provides an LNP comprising: (a) at least one ionizable lipid; (b) at least one PEG lipid; (c) at least one structural lipid; and (d) at least one non-ionizable lipid and / or zwitterionic lipid. In some embodiments, the LNP further comprises additional ionizable lipids besides those disclosed herein. In some embodiments, the LNP further comprises any additional lipid components of any class other than those disclosed herein.

[0543] In some embodiments, the PEG-lipid is the PEG-lipid disclosed and described herein. In some embodiments, the LNP further comprises a PEG-lipid selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0544] In some implementations, the structural lipids are selected from the group consisting of: cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, ursolic acid, and α-tocopherol.

[0545] In some embodiments, the non-ionizable lipids are phospholipids selected from the group consisting of: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl 1,2-Diundecanoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-Diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:O diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 LysoPC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-diphyranoyl-sn-glycerol-3-phosphate ethanolamine (ME 16).0 PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), (S)-2-ammonium-3-((((R)-2 Sodium α-phosphatidylserine (L-α-phosphatidylserine; brain PS), myristoyl phosphatidylcholine (DMPC), myristoyl phosphatidylethanolamine (DMPE), myristoyl phosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-(phospho-L-serine) (DOPS), Cell Fusion Phospholipids (DPhPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dipalmitoylphosphatidylglycerol (DPPG), Dipalmitoylphosphatidylserine (DPPS), Distearylphosphatidylcholine (DSPC), Distearyl-phosphatidyl-ethanolamine (DSPE), Distearylphosphatidylethanolamine imidazole (DSPEI), 1,2-Diundecanoyl-sn-glycerol-phosphocholine (DUPC), Lecithinylcholine (EPC), 1,2-Dioleoyl-sn-glycerol-3-phosphate (18:1 PA; DOPA), Bis((S)-2-hydroxy-3-(oleoyloxy)propyl)ammonium phosphate (18:1 DMP; LBPA), 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol) (DOPI; 18:1 PI), 1,2-distearyl-sn-glycerol-3-phosphate-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:0 Lyso PS) and sphingomyelin.

[0546] In some embodiments, the non-ionizable lipids are phospholipids selected from the group consisting of: egg sphingomyelin (egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitoamide octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), brain or porcine sphingomyelin (brain SM / (2S,3R,E)-3-hydroxy-2-stearoamide octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), milk or bovine sphingomyelin (milk SM / (2S,3R,E)-3-hydroxy-2-tetracosamide octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), 28:0 SM (N-octadecanoyl-D-erythrosine dihydrosphocholine), 14:0 SM (N-myristoyl-D-erythrosine dihydrosphocholine), 16:1 SM (N-Palmyl-D-erythrodihydrosphingosine phosphocholine), 12:0 dihydroSM (N-Lauroyl-D-erythrodihydrosphingosine phosphocholine), Lyso SM (Sphingosine phosphocholine), Lyso SM (Sphingosine phosphocholine), Lyso SM (dihydro) (Dihydrosphingosine phosphocholine), 24:1 SM (N-Ceramide-D-erythrodihydrosphingosine phosphocholine), 24:0 SM (N-Teicosyl-D-erythrodihydrosphingosine phosphocholine), 18:1 SM (N-Oleoyl-D-erythrodihydrosphingosine phosphocholine), 18:0 SM (N-Stearyl-D-erythrodihydrosphingosine phosphocholine), 17:0 SM (N-Heptadecyl-D-erythrodihydrosphingosine phosphocholine), 16:0 SM (N-palmitoyl-D-erythrodihydrosphocholine), 12:0 SM (N-lauroyl-D-erythrodihydrosphocholine), 06:0 SM (N-hexanoyl-D-erythrodihydrosphocholine), 02:0 SM (N-acetyl-D-erythrodihydrosphocholine), 3-O-methylLyso SM (3-O-methyl-sphocholine), 3-O-methyl-N-methylLyso SM (3-O-methyl-N-methyl-sphocholine) and 3-N-methylLyso SM (3-N-methyl-sphocholine).

[0547] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 0 mol% to about 10 mol% of PEG lipids; (b) about 0 mol% to about 30 mol% of structural lipids; (c) about 20 mol% to about 45 mol% of phospholipids, non-ionizable lipids or zwitterionic lipids; and (d) about 30 mol% to about 60 mol% of ionizable lipids.

[0548] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 1 mol% to about 2 mol% of PEG lipids; (b) about 25 mol% to about 40 mol% of structural lipids; (c) about 20 mol% to about 45 mol% of phospholipids, non-ionizable lipids or zwitterionic lipids; and (d) about 30 mol% to about 60 mol% of ionizable lipids.

[0549] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 2 mol% PEG lipids; (b) about 25 mol% structural lipids; (c) about 40 mol% phospholipids, non-ionizable lipids, or zwitterionic lipids; and (d) about 33 mol% ionizable lipids.

[0550] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 0.5 mol% to about 10 mol% of PEG lipids; (b) about 0.5 mol% to about 50 mol% of structural lipids; (c) about 5 mol% to about 45 mol% of non-ionizable lipids (e.g., phospholipids), non-ionizable lipids, or zwitterionic lipids; and (d) about 30 mol% to about 60 mol% of ionizable lipids.

[0551] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipids; (b) about 20 mol% to about 45 mol% of structural lipids; (c) about 35 mol% to about 45 mol% of non-ionizable lipids (e.g., phospholipids), non-ionizable lipids, or zwitterionic lipids; and (d) about 30 mol% to about 40 mol% of ionizable lipids.

[0552] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 1.5 mol% to about 3.5 mol% of PEG lipids; (b) about 20 mol% to about 45 mol% of structural lipids; (c) about 35 mol% to about 45 mol% of non-ionizable lipids (e.g., phospholipids), non-ionizable lipids, or zwitterionic lipids; and (d) about 30 mol% to about 40 mol% of ionizable lipids.

[0553] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipids; (b) about 20 mol% to about 45 mol% of structural lipids; (c) about 5 mol% to about 15 mol% of non-ionizable lipids (e.g., phospholipids), non-ionizable lipids, or zwitterionic lipids; and (d) about 45 mol% to about 55 mol% of ionizable lipids.

[0554] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 1.5 mol% to about 3.5 mol% of PEG lipids; (b) about 20 mol% to about 45 mol% of structural lipids; (c) about 5 mol% to about 15 mol% of non-ionizable lipids (e.g., phospholipids), non-ionizable lipids, or zwitterionic lipids; and (d) about 45 mol% to about 55 mol% of ionizable lipids.

[0555] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 2.5 mol% PEG lipids; (b) about 39 mol% structural lipids; (c) about 10 mol% phospholipids, non-ionizable lipids, or zwitterionic lipids; and (d) about 48.5 mol% ionizable lipids.

[0556] In some embodiments, the lipid component of the nanoparticles comprises: (a) about 1.5 mol% PEG lipids; (b) about 40 mol% structural lipids; (c) about 10 mol% phospholipids, non-ionizable lipids, or zwitterionic lipids; and (d) about 48.5 mol% ionizable lipids.

[0557] In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol% to about 60 mol% of ionizable lipids, about 0 mol% to about 30 mol% of phospholipids, about 18.5 mol% to about 48.5 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 20 mol% to about 45 mol% of ionizable lipids, about 30 mol% to about 60 mol% of phospholipids, about 10 mol% to about 30 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol% to about 55 mol% of ionizable lipids, about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 40 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol% to about 40 mol% of ionizable lipids, about 35 mol% to about 45 mol% of phospholipids, about 20 mol% to about 30 mol% of structural lipids, and about 0.5 mol% to about 5 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 25 mol% to about 45 mol% of ionizable lipids, about 35 mol% to about 50 mol% of phospholipids, about 10 mol% to about 25 mol% of structural lipids, and about 1 mol% to about 5 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In a particular embodiment, the lipid component comprises about 50 mol% of ionizable lipids, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another particular embodiment, the lipid component comprises about 40 mol% of ionizable lipids, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In yet another particular embodiment, the lipid component comprises about 48.5 mol% of ionizable lipids, about 10 mol% of phospholipids, about 40 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another specific embodiment, the lipid component comprises about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 39 mol% structural lipids and about 2.5 mol% PEG lipids.In another specific embodiment, the lipid component comprises about 33 mol% ionizable lipids, about 40 mol% phospholipids, about 25 mol% structural lipids, and about 2 mol% PEG lipids. In some embodiments, the phospholipids are DOPE or DSPC. In some embodiments, the phospholipids are DSPC. In some embodiments, the phospholipids are sphingolipids. In some embodiments, the sphingolipids are ESM (egg sphingomyelin). In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 20 mol% sphingolipids, about 20 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 10 mol% sphingolipids, about 30 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 30 mol% sphingolipids, about 10 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 20 mol% sphingolipids, about 20 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 10 mol% sphingolipids, about 30 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 30 mol% sphingolipids, about 10 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 25 mol% cholesterol, about 2 mol% PEG lipids, and about 40% a mixture of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingosine lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 25 mol% cholesterol, about 2 mol% PEG lipids, and about 40% a mixture of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingosine lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingosine lipids is present in an amount less than 30 mol% of the total lipid component of the LNP.In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 25 mol% cholesterol, about 2 mol% PEG lipids, and about 40% a mixture of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingosine lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingosine lipids is present in an amount less than 25 mol% of the total lipid component of the LNP.

[0558] In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 39.5 mol% DSPC, about 25 mol% cholesterol, and about 2.5 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% sphingomyelin, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% ESM, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% DOPE, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% DOPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% DLPC, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% DOPS, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 40 mol% phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In another specific embodiment, the LNP comprises about 33 mol% ionizable lipids, about 20 mol% sphingomyelin, about 20 mol% DSPC, about 25 mol% cholesterol and about 2 mol% PEG lipids.

[0559] In some embodiments, the LNP comprises about 43 mol% ionizable lipids, about 15 mol% sphingolipids, about 15 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 25 mol% sphingolipids, about 15 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 33 mol% ionizable lipids, about 15 mol% sphingolipids, about 25 mol% non-sphingolipid phospholipids, about 25 mol% cholesterol, and about 2 mol% PEG lipids. In some embodiments, the LNP comprises about 50 mol% ionizable lipids, about 10 mol% phospholipids (e.g., DSPC), about 38.5 mol% cholesterol, and about 1.5 mol% PEG lipids.

[0560] In another specific embodiment, the lipid component comprises about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 38.5 mol% structural lipids, and about 3 mol% PEG lipids. In another specific embodiment, the lipid component comprises about 48.5 mol% ionizable lipids, about 10 mol% phospholipids, about 38 mol% structural lipids, and about 3.5 mol% PEG lipids.

[0561] In some embodiments, the LNP further includes a targeting portion. In some embodiments, the targeting portion is an antibody or a fragment thereof.

[0562] The amount of active agent in a nanoparticle composition can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the active agent. For example, the amount of active agent available in a nanoparticle composition can be determined by the size, sequence, and other characteristics of the active agent. The relative amounts of active agents and other elements (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of the lipid component to the payload in the nanoparticle composition is from about 5:1 to about 60:1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. The amount of effective load in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0563] In some embodiments, the nanoparticle compositions of this disclosure are formulated to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA activator (e.g., a linear or circular mRNA payload). Generally, a lower N:P ratio is preferred. One or more polynucleotides, lipids, and their amounts are selected to provide an N:P ratio of about 2:1 to about 30:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1). In some embodiments, the N:P ratio is about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio is about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. In some embodiments, the nanoparticle compositions of this disclosure are formulated to provide a specific IL:P ratio, which refers to the molar ratio of the number of phosphate groups in the ionizable lipid (as a whole compound) to the number of phosphate groups in the RNA-active reagent. One or more polynucleotides, lipids, and their amounts are selected to provide an IL:P ratio of about 2:1 to about 30:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1). In some embodiments, the IL:P ratio is about 2:1 to about 8:1. In other embodiments, the IL:P ratio is about 5:1 to about 8:1. For example, the IL:P ratio is approximately 5.0:1, approximately 5.5:1, approximately 5.67:1, approximately 6.0:1, approximately 6.5:1, approximately 7.0:1, or approximately 7.5:1.

[0564] The dosage of the pharmaceutical composition provided herein can be measured in mg / kg, where mg / kg refers to the total nucleic acid used to prepare the LNP in mg / kg of the subject's body weight (total mRNA in mg / kg). In some embodiments, the pharmaceutical composition is present in a dosage form at a dose of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001 mg / kg (mg / kg or mpk) of body weight, or within a range of any two of the foregoing values ​​(inclusive). In some embodiments, the pharmaceutical composition is present in a dosage form at a dose not exceeding about 10 mg / kg (mg / kg or mpk) of body weight. In some embodiments, the pharmaceutical composition is present in a dosage form at a dose not exceeding about 9 mg / kg, not exceeding about 8 mg / kg, not exceeding about 7 mg / kg, not exceeding about 6 mg / kg, not exceeding about 5 mg / kg, not exceeding about 4 mg / kg, not exceeding about 3 mg / kg, not exceeding about 2 mg / kg, not exceeding about 1 mg / kg, not exceeding about 0.5 mg / kg, not exceeding about 0.2 mg / kg, not exceeding about 0.1 mg / kg, not exceeding about 0.05 mg / kg, or not exceeding about 0.01 mg / kg.

[0565] In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 5 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 mg / mL, or within any two of the foregoing values ​​(inclusive).

[0566] In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 5 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 2 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 1 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 0.5 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 0.1 mg / mL.

[0567] In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 micrograms per milliliter (µg / mL), or within any two of the foregoing values ​​(inclusive). In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 10, not exceeding about 9, not exceeding about 8, not exceeding about 7, not exceeding about 6, not exceeding about 5, not exceeding about 4, not exceeding about 3, not exceeding about 2, not exceeding about 1, not exceeding about 0.5, not exceeding about 0.2, or not exceeding about 0.1 micrograms per milliliter (µg / mL).

[0568] The characteristics of nanoparticle compositions can depend on their components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different characteristics than a nanoparticle composition containing different structural lipids. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids may have different characteristics than a nanoparticle composition containing a lower molar fraction of phospholipids. Characteristics can also vary depending on the preparation method and conditions of the nanoparticle composition. Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure a variety of characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0569] In some embodiments, the average size of the nanoparticle composition is between tens of nanometers and hundreds of nanometers, for example, measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition is about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the nanoparticle composition is about 70 nm to about 100 nm. In a specific embodiment, the average size is about 80 nm. In other embodiments, the average size is about 100 nm.

[0570] In some embodiments, the LNP of this disclosure can be characterized by its shape. In some embodiments, the LNP is substantially spherical. In some embodiments, the LNP is substantially rod-shaped (i.e., cylindrical). In some embodiments, the LNP is substantially disk-shaped.

[0571] Nanoparticle compositions can be relatively homogeneous. A polydispersity index can be used to indicate the uniformity of a nanoparticle composition, such as its particle size distribution. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. Nanoparticle compositions can have a polydispersity index of about 0 to about 0.25 (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25).

[0572] Encapsulation efficiency describes the amount of payload that is encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. High encapsulation efficiency is desirable (e.g., close to 100%). For example, encapsulation efficiency can be measured by comparing the amount of payload in a solution containing the nanoparticle composition before and after decomposition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free payload in solution. For the nanoparticle compositions described herein, the encapsulation efficiency for treatment and / or prevention may be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency is at least 80%. In some embodiments, the encapsulation efficiency is at least 90%.

[0573] Nanoparticle compositions can contain any substance useful in a pharmaceutical composition. For example, a nanoparticle composition may contain one or more pharmaceutically acceptable excipients or adjuvants, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation aids, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. Excipients may also be included, such as waxes, butters, colorants, coating agents, flavoring agents, and aromatizers. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition, AR Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0574] 5. Polynucleotides

[0575] In some embodiments, this disclosure provides supported lipid nanoparticles comprising polynucleotides. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is linear RNA. In a preferred embodiment, the polynucleotide is circular RNA.

[0576] Wesselhoeft et al., (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In vivo. Molecular Cell. 74(3), 508-520 and Wesselhoeft et al., (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629 are incorporated in their entirety by reference.

[0577] Transcription of a DNA template (e.g., including 3' intron elements, 3' exon elements, core functional elements, 5' exon elements, and 5' intron elements) results in the formation of a circularizable precursor linear RNA polynucleotide. In some embodiments, the DNA template includes a vector, PCR product, plasmid, microcircular DNA, granules, artificial chromosome, complementary DNA (cDNA), extrachromosomal DNA (ecDNA), or fragments thereof. In some embodiments, the microcircular DNA may be linearized or non-linearized. In some embodiments, the plasmid may be linearized or non-linearized. In some embodiments, the DNA template may be single-stranded. In other embodiments, the DNA template may be double-stranded. In some embodiments, the DNA template comprises wholly or partially derived from a viral, bacterial, or eukaryotic vector.

[0578] As provided herein, this disclosure includes a DNA template that shares the same sequence as the precursor linear RNA polynucleotide (e.g., 3' intron element, 3' exon element, core functional element, and 5' exon element, 5' intron element) prior to splicing. In some embodiments, the linear precursor RNA polynucleotide undergoes splicing, resulting in the removal of the 3' intron element and 5' intron element during the circularization process. In some embodiments, the resulting circular RNA polynucleotide lacks the 3' intron fragment and 5' intron fragment, but retains the 3' exon fragment, core functional element, and 5' exon element.

[0579] In some embodiments, the precursor linear RNA polynucleotide is in one or more guanosine nucleotides or nucleosides (e.g., GTP) and divalent cations (e.g., Mg). 2+Circularization occurs during incubation in the presence of [a specific element]. In some embodiments, the 3' exon element, the 5' exon element, and / or the core functional element, wholly or partially, promote the circularization of the precursor linear RNA polynucleotide to form the circular RNA polynucleotide provided herein.

[0580] In some embodiments, the circular RNA provided herein is generated inside the cell. In some embodiments, the precursor RNA is transcribed in the cytoplasm by phage RNA polymerase using a DNA template (e.g., in some embodiments, using the vector provided herein), or in the cell nucleus by host RNA polymerase II, and then circularized.

[0581] In some embodiments, the circular RNA provided herein is injected into an animal (e.g., a human) such that the polypeptide encoded by the circular RNA molecule is expressed within the animal.

[0582] In some embodiments, the DNA template (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotide provided herein are between 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and / or 1500 and 5000 nucleotides (nt). In some embodiments, the length of the polynucleotide is at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt. In some embodiments, the length of the polynucleotide does not exceed 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt. In some implementations, the lengths of the DNA, linear RNA, and / or circular RNA polynucleotides provided herein are 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt.

[0583] In some embodiments, the circular RNAs provided herein exhibit higher functional stability than mRNAs comprising the same expression sequence. In some embodiments, the circular RNAs provided herein exhibit higher functional stability than mRNAs comprising the same expression sequence, 5 molU modification, optimized UTR, cap, and / or poly-A tail.

[0584] In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of 5-80, 10-70, 15-60, or 20-50 hours. In some embodiments, the functional half-life of the circular RNA polynucleotides provided herein is greater than the functional half-life of an equivalent linear RNA polynucleotide encoding the same protein (e.g., at least 1.5 times, at least 2 times). In some embodiments, the functional half-life can be assessed by detecting functional protein synthesis.

[0585] In some embodiments, the functional half-life of the circular RNA polynucleotide or its pharmaceutical composition in human cells is greater than or equal to a predetermined threshold functional half-life. In some embodiments, the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussia luciferase (GLuc) is measured in a culture medium of human cells (e.g., HepG2) expressing the circular RNA polynucleotide at 1, 2, 6, 7, or 14 days, every 1, 2, 6, 12, or 24 hours. In other embodiments, the functional half-life is determined by an in vivo assay, wherein the level of the protein encoded by the expression sequence of the circular RNA polynucleotide is measured in a patient serum or tissue sample at 1, 2, 6, 12, or 24 hours, at 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the predetermined threshold is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the circular RNA polynucleotide.

[0586] In some embodiments, the circular RNAs provided herein can have a higher expression level than equivalent linear mRNAs, for example, a higher expression level 24 hours after RNA administration to cells. In some embodiments, the circular RNAs provided herein have a higher expression level than mRNAs comprising the same expression sequence, 5 molU modification, optimized UTR, cap and / or poly-A tail.

[0587] In some embodiments, the circular RNAs provided herein may have lower immunogenicity than equivalent mRNAs when exposed to the immune system or a certain type of immune cells of an organism. In some embodiments, the circular RNAs provided herein are associated with the regulated production of cytokines when exposed to the immune system or a certain type of immune cells of an organism. For example, in some embodiments, the circular RNAs provided herein are associated with reduced production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to the immune system or a certain type of immune cells of an organism compared to mRNAs comprising the same expression sequence. In some embodiments, the circular RNAs provided herein are associated with less transcriptional induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to the immune system or a certain type of immune cells of an organism compared to mRNAs comprising the same expression sequence. In some embodiments, the circular RNAs provided herein have lower immunogenicity than mRNAs comprising the same expression sequence. In some implementations, the circular RNAs provided herein exhibit lower immunogenicity than mRNAs comprising the same expression sequence, 5 molU modification, optimized UTR, cap, and / or poly-A tail.

[0588] In some embodiments, the circular RNA provided herein can be transfected into cells as is, or it can be transfected into a DNA vector and transcribed in cells. The circular RNA can be transcribed from the transfected DNA vector via an added polymerase or a polymerase encoded by the nucleic acid transfected into the cells, or preferably via an endogenous polymerase.

[0589] i. Enhanced intron elements and enhanced exon elements

[0590] In some implementations, the DNA template (e.g., a vector) or linear RNA (e.g., precursor RNA) includes enhancing intronic elements and / or enhancing exon elements. These enhancing intronic and exon elements may include spacer regions, double-stranded regions, affinity sequences, intronic fragments, exon fragments, and various non-translational elements. These sequences within the enhancing intronic or exon elements are arranged to optimize circularization or protein expression.

[0591] In some embodiments, the DNA template, precursor linear RNA polynucleotide, and circular RNA provided herein include a first (5') and / or a second (3') spacer region. In some embodiments, the DNA template or precursor linear RNA polynucleotide includes one or more spacer regions within an enhanced intron element. In some embodiments, the DNA template or precursor linear RNA polynucleotide includes one or more spacer regions within an enhanced exon element. In some embodiments, the DNA template or linear RNA polynucleotide includes a spacer region within a 3' enhanced intron fragment and a spacer region within a 5' enhanced intron fragment. In some embodiments, the DNA template, precursor linear RNA polynucleotide, or circular RNA includes a spacer region within a 3' enhanced exon fragment and another spacer region within a 5' enhanced exon fragment to facilitate circularization or protein expression due to the symmetry generated in the overall sequence.

[0592] In some embodiments, the spacer regions included between the 3' I-group introns and the core functional element can protect the secondary structures in those regions by preventing them from interacting, thereby improving splicing efficiency. In some embodiments, the first spacer region (between the 3' I-group introns and the core functional element) and the second spacer region (between the two expressed sequences and the core functional element) include additional base-pairing regions predicted to pair with each other but not with the first and second double-stranded regions. In other embodiments, the first spacer region (between the 3' I-group introns and the core functional element) and the second spacer region (between one of the core functional elements and the 5' I-group intron) include additional base-pairing regions predicted to pair with each other but not with the first and second double-stranded regions. In some embodiments, such spacer region base pairing brings the I-group introns closer together, thereby further improving splicing efficiency. Furthermore, in some embodiments, the combination of base pairings between the first and second double-stranded regions, and the base pairings between the first and second spacer regions individually, promote the formation of splice bubbles of group I intron fragments containing adjacent regions with side-paired base pairings. A typical spacer region is a continuous sequence having one or more of the following characteristics: 1) expected to avoid interference with proximal structures, such as IRES, expression sequences, aptamers, or introns; 2) a length of at least 7 nt and not exceeding 100 nt; 3) located after and adjacent to a 3' intron fragment and / or before and adjacent to a 5' intron fragment; and 4) containing one or more of the following: a) an unstructured region at least 5 nt long, b) a region at least 5 nt long that pairs with the distal sequence (including another spacer region), and c) a structured region at least 7 nt long whose extent is confined to the spacer region sequence. The spacer region may have several regions, including unstructured regions, base-pairing regions, hairpin / structured regions, and combinations thereof. In one embodiment, the spacer region has a structured region with a high GC content. In one embodiment, a region within a spacer region pairs base-to-base with another region within the same spacer region. In another embodiment, a region within a spacer region pairs base-to-base with a region within another spacer region. In another embodiment, the spacer region comprises one or more hairpin structures. In another embodiment, the spacer region comprises one or more hairpin structures having a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In another embodiment, an additional spacer region exists between the 3' I group intron fragment and the core functional element. In one embodiment, this additional spacer region prevents the structured regions of the IRES or the aptamers of the TIE from interfering with the folding of the 3' I group intron fragment or reduces the extent to which this occurs.In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the 5' spacer sequence is 5 to 50, 10 to 50, 20 to 50, 20 to 40, and / or 25 to 35 nucleotides long. In some embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In one embodiment, the 5' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a poly-AC sequence. In one embodiment, the spacer includes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% poly-AC content. In one embodiment, the spacer region includes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0593] In some embodiments, the DNA template and precursor linear RNA polynucleotide and circular RNA polynucleotide provided herein include a first (5') double-stranded region and a second (3') double-stranded region. In some embodiments, the DNA template and precursor linear RNA polynucleotide include a 5' outer double-stranded region located within a 3' enhanced intron fragment and a 3' outer double-stranded region located within a 5' enhanced intron fragment. In some embodiments, the DNA template, precursor linear RNA polynucleotide, and circular RNA polynucleotide include a 5' inner double-stranded region located within a 3' enhanced exon fragment and a 3' inner double-stranded region located within a 5' enhanced exon fragment. In some embodiments, the DNA polynucleotide and precursor linear RNA polynucleotide include a 5' outer double-stranded region, a 5' inner double-stranded region, a 3' inner double-stranded region, and a 3' outer double-stranded region.

[0594] In some embodiments, the first and second double-stranded regions can form perfect or imperfect double strands. Therefore, in some embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second double-stranded regions are base-paired with each other. In some embodiments, the predicted base pairing between the double-stranded region and unexpected sequences (e.g., non-double-stranded sequences) in the RNA is less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%). In some embodiments, including such double-stranded regions at the ends of the precursor RNA strand and placing them adjacent to or very close to Group I intron fragments brings the Group I intron fragments close together, thereby increasing splicing efficiency. In some embodiments, the length of the double-stranded region is 3 to 100 nucleotides (e.g., 3-75 nucleotides, 3-50 nucleotides, 20-50 nucleotides, 35-50 nucleotides, 5-25 nucleotides, or 9-19 nucleotides). In some embodiments, the length of the double-stranded region is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the double-stranded region has a length of 9 to 50 nucleotides. In one embodiment, the double-stranded region has a length of 9 to 19 nucleotides. In some embodiments, the double-stranded region has a length of 20 to 40 nucleotides. In some embodiments, the double-stranded region has a length of 30 nucleotides.

[0595] In other implementations, the DNA template, precursor linear RNA polynucleotide, or circular RNA polynucleotide does not include any double-stranded regions to optimize translation or circularization.

[0596] As provided herein, the DNA template or precursor linear RNA polynucleotide may include an affinity tag. In some embodiments, the affinity tag is located in a 3' enhanced intron element. In some embodiments, the affinity tag is located in a 5' enhanced intron element. In some embodiments, each of the two (3' and 5') enhanced intron elements includes an affinity tag. In one embodiment, the affinity tag of the 3' enhanced intron element is the same length as the affinity tag in the 5' enhanced intron element. In some embodiments, the affinity tag of the 3' enhanced intron element has the same sequence as the affinity tag in the 5' enhanced intron element. In some embodiments, the affinity sequence is placed to optimize oligo-dT purification.

[0597] In some embodiments, the affinity tag includes a poly-A region. In some embodiments, the poly-A region is at least 15, 30, or 60 nucleotides long. In some embodiments, one or both poly-A regions are 15-50 nucleotides long. In some embodiments, one or both poly-A regions are 20-25 nucleotides long. The poly-A sequence is removed after circularization. Therefore, oligonucleotides that hybridize with the poly-A sequence (such as deoxythymidine oligonucleotides (oligo(dT)) conjugated to solid surfaces (e.g., resins)) can be used to separate circular RNA from its precursor RNA.

[0598] In some embodiments, the 3' enhancing intron element includes a leading untranslated sequence. In some embodiments, the leading untranslated sequence is the 5' end of the 3' enhancing intron fragment. In some embodiments, the leading untranslated sequence includes the last nucleotide of the transcription start site (TSS). In some embodiments, the TSS is selected from viral, bacterial, or eukaryotic DNA templates. In one embodiment, the leading untranslated sequence includes the last nucleotide of the TSS and 0 to 100 additional nucleotides. In some embodiments, the TSS is a terminal spacer region. In one embodiment, the leading untranslated sequence contains a guanosine at the 5' end during translation by RNA T7 polymerase.

[0599] In some embodiments, the 5' enhanced intron element includes a trailing untranslated sequence. In some embodiments, the 5' trailing untranslated sequence is located at the 3' end of the 5' enhanced intron element. In some embodiments, the trailing untranslated sequence is a partially restriction digestion sequence. In one embodiment, the trailing untranslated sequence is wholly or partially a restriction digestion site for linearizing the DNA template. In some embodiments, the restriction digestion site is wholly or partially derived from a natural viral, bacterial, or eukaryotic DNA template. In some embodiments, the trailing untranslated sequence is a terminal restriction site fragment.

[0600] a. Enhanced intron fragments

[0601] In some embodiments, the 3' enhanced intron element and the 5' enhanced intron element each comprise an intron fragment. In some embodiments, the 3' intron fragment is a continuous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 3' proximal fragment of a natural group I or II intron containing a 3' splice site dinucleotide. Typically, the 5' intron fragment is a continuous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5' proximal fragment of a natural group I or II intron containing a 5' splice site dinucleotide. In some embodiments, the 3' intron fragment comprises the first nucleotide of the 3' group I or II splice site dinucleotide. In some embodiments, the 5' intron fragment comprises the first nucleotide of a 5' group I or II splice site dinucleotide. In other embodiments, the 3' intron fragment comprises the first and second nucleotides of a 3' group I or II intron splice site dinucleotide; and the 5' intron fragment comprises the first and second nucleotides of a 3' group I or II intron dinucleotide. In some embodiments, the 3' enhancing intron element and the 5' enhancing intron element comprise synthetic intron fragments.

[0602] b. Enhanced exon fragments

[0603] In some embodiments, as provided herein, the DNA template, linear precursor RNA polynucleotide, and circular RNA polynucleotide each include an enhancing exon fragment. In some embodiments, the 3' enhancing exon element is located upstream of the core functional element in a 5' to 3' sequence. In some embodiments, the 5' enhancing intron element is located downstream of the core functional element in a 5' to 3' sequence.

[0604] In some embodiments, the 3' enhancing exon element and the 5' enhancing exon element each comprise an exon fragment. In some embodiments, the 3' enhancing exon element comprises a 3' exon fragment. In some embodiments, the 5' enhancing exon element comprises a 5' exon fragment. In some embodiments, as provided herein, the 3' exon fragment and the 5' exon fragment each comprise a group I or II intron fragment and an exon sequence of 1 to 100 nucleotides. In some embodiments, the 3' intron fragment is a continuous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 3' proximal fragment of a natural group I or II intron that includes a 3' splice site dinucleotide. Typically, the 5' group I or II intron fragment is a continuous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5' proximal segment of a natural group I or II intron including a 5' splice site dinucleotide. In some embodiments, the 3' exon fragment comprises the second nucleotide of the 3' group I or II intron splice site dinucleotide and an exon sequence of 1 to 100 nucleotides. In some embodiments, the 5' exon fragment comprises the first nucleotide of the 5' group I or II intron splice site dinucleotide and an exon sequence of 1 to 100 nucleotides. In some embodiments, the exon sequence comprises, partially or entirely, naturally occurring exon sequences derived from viruses, bacteria, or eukaryotic DNA vectors. In other embodiments, the exon sequence further comprises synthetic, genetically modified (e.g., containing modified nucleotides), or otherwise engineered exon sequences.

[0605] In one embodiment, when the 3' intron fragment includes two nucleotides of a 3' group I or II splice site dinucleotide and the 5' intron fragment includes two nucleotides of a 5' group I or II splice site dinucleotide, the exon fragments located within the 5' enhanced exon element and the 3' enhanced exon element do not include a group I or II splice site dinucleotide.

[0606] By way of example and not intended to be limiting, in some embodiments, the 3' enhanced intron element comprises, in the following 5' to 3' order: a leading untranslated sequence, a 5' affinity tag, an optional 5' outer double-stranded region, a 5' outer spacer region, and a 3' intron fragment. In the same embodiment, the 3' enhanced exon element comprises, in the following 5' to 3' order: a 3' exon fragment, an optional 5' inner double-stranded region, an optional 5' inner double-stranded region, and a 5' inner spacer region. In the same embodiment, the 5' enhanced exon element comprises, in the following 5' to 3' order: a 3' inner spacer region, an optional 3' inner double-stranded region, and a 5' exon fragment. Still in the same embodiment, the 3' enhanced intron element comprises, in the following 5' to 3' order: a 5' intron fragment, a 3' outer spacer region, an optional 3' outer double-stranded region, a 3' affinity tag, and a trailing untranslated sequence.

[0607] ii. Core functional components

[0608] In some embodiments, the DNA template, linear precursor RNA polynucleotide, and circular RNA polynucleotide comprise a core functional element. In some embodiments, the core functional element comprises a coding element or a non-coding element. In some embodiments, the core functional element may contain both coding and non-coding elements. In some embodiments, the core functional element further comprises a translation initiation element (TIE) upstream of the coding or non-coding element. In some embodiments, the core functional element comprises a termination element. In some embodiments, the termination element is located downstream of the TIE and the coding element. In some embodiments, the termination element is located downstream of the coding element but upstream of the TIE. In some embodiments, when the coding element comprises a non-coding region, the core functional element lacks a TIE and / or a termination element.

[0609] iii. Encoded elements or non-encoded elements

[0610] In some embodiments, the polynucleotides herein include coding elements, non-coding elements, or a combination of both. In some embodiments, the coding element includes an expressed sequence. In some embodiments, the coding element encodes at least one therapeutic protein.

[0611] In some embodiments, the circular RNA encodes two or more polypeptides. In some embodiments, the circular RNA is bicistronic RNA. The sequences encoding two or more polypeptides can be separated by ribosomal jumping elements or nucleotide sequences encoding protease cleavage sites. In some embodiments, the ribosomal jumping elements encode 2A peptides of *Melilothorax muscularis* virus (T2A), *P. spp.* cyclosporine virus-1 (P2A), foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), or silkworm fibrosis virus (BmIFV 2A).

[0612] iv. Translation Initiation Element (TIE)

[0613] As provided in some embodiments herein, the core functional element includes at least one translation initiation element (TIE). The TIE is designed to allow for efficient translation of the encoded protein. Therefore, an optimal core functional element includes only non-coding elements and lacks any TIE. In some embodiments, a core functional element that includes one or more coding elements will further include one or more TIEs.

[0614] In some embodiments, the TIE includes an untranslated region (UTR). In some embodiments, the TIE provided herein includes an internal ribosome entry site (IRES). In some embodiments, the IRES is a naturally occurring IRES. In some embodiments, the IRES is a non-natural or synthetic IRES. The inclusion of an IRES allows for the translation of one or more open reading frames (e.g., open reading frames forming an expression sequence) from circular RNA. The IRES element attracts the eukaryotic ribosomal translation initiation complex and facilitates translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161. In some embodiments, the IRES element is selected from those disclosed in International Publication WO / 2022 / 261490, the contents of which are hereby incorporated in their entirety.

[0615] v. Additional auxiliary elements (sequential elements)

[0616] As described in this disclosure, the circular RNA polynucleotide, linear RNA polynucleotide, and / or DNA template may further include auxiliary elements. In some embodiments, these auxiliary elements may be included within the sequence of the circular RNA, linear RNA polynucleotide, and / or DNA template for enhancing circularization, translation, or both. In some embodiments, the auxiliary element is a sequence specifically located between or within an enhancing intron element, enhancing exon element, or core functional element of the respective polynucleotide. As an example, but not intended to be limiting, auxiliary elements include IRES trans-acting factor regions, miRNA binding sites, restriction sites, RNA editing regions, structural or sequence elements, particulate sites, postcode elements, RNA transport elements, or other specialized sequences found in the art that enhance and promote the circularization and / or translation of proteins encoded within the circular RNA polynucleotide.

[0617] In some embodiments, the auxiliary element includes the IRES trans-acting factor (ITAF) region. In some embodiments, the IRES trans-acting factor region regulates translation initiation by binding to PCBP1-PCBP4 (poly-C binding protein), PABP1 (poly-A binding protein), PTB (polypyrimidine strand binding protein), the Argonaute protein family, HNRNPK (heterogeneous nucleoribonucleoprotein K protein), or La protein. In some embodiments, the IRES trans-acting factor region includes poly-A, poly-C, poly-AC, or polypyrimidine strands.

[0618] In some implementations, the ITAF region is located within the core functional element. In some implementations, the ITAF region is located within the TIE.

[0619] In some embodiments, the auxiliary element includes a miRNA binding site. In some embodiments, the miRNA binding site is located within a 5' intron element, a 5' exon element, a core functional element, a 3' exon element, and / or a 3' intron element.

[0620] In some embodiments, the miRNA binding site is located within a spacer region within an intron or exon element. In some embodiments, the miRNA binding site encompasses the entire spacer region.

[0621] In some embodiments, the 5' intron element and the 3' intron element each include the same miRNA binding site. In another embodiment, the miRNA binding site of the 5' intron element includes a miRNA binding site that differs from that of the 3' intron element in length or nucleotides. In one embodiment, the 5' exon element and the 3' exon element include the same miRNA binding site. In other embodiments, the 5' exon element and the 3' exon element include miRNA binding sites that differ in length or nucleotides.

[0622] In some embodiments, miRNA binding sites are located adjacent to each other within circular RNA polynucleotides, linear RNA polynucleotide precursors, and / or DNA templates. In some embodiments, the first nucleotide of one miRNA binding site follows the last nucleotide of the second miRNA binding site.

[0623] In some embodiments, the miRNA binding site is located within the translation initiation element (TIE) of the core functional element. In one embodiment, the miRNA binding site is located before, following, or within the internal ribosome entry site (IRES). In another embodiment, the miRNA binding site is located before, following, or within the aptamer complex.

[0624] The unique sequences defined by miRNA nomenclature are widely known and available to those working in the field of microRNA. For example, they can be found in the miRDB public database.

[0625] vi. Natural Tie: Virus-eukaryotic / intracellular ribosome entry site (IRES)

[0626] Many IRES sequences are available and include sequences derived from a variety of viruses, such as leader sequences derived from piconemaviruses, such as encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63:1651-1660), poliovirus leader sequences, hepatitis A virus leader sequences, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), Giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc.

[0627] To drive protein expression, circular RNAs include IRES operatively linked to a protein-coding sequence. This document discloses modifications to IRES and accessory sequences to increase or decrease IRES activity, for example, by truncating the 5' and / or 3' ends of the IRES, adding a spacer region at the 5' end of the IRES, modifying the 6 nucleotides at the 5' end of the translation initiation site (Kozak sequence), modifying alternative translation initiation sites, and creating chimeric / hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNA disclosed herein includes one or more of these modifications relative to the native IRES.

[0628] In some implementations, IRES are the following IRES sequences: Taura syndrome virus, blood-sucking stink bug virus, Tyler's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, grain constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, skeletal bug enterovirus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus- 1. Human immunodeficiency virus type 1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid moth microRNA-like virus, encephalomyocarditis virus, fruit fly C virus, human Coxsackievirus B3, cruciferous tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black bee queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, fruit fly antennae and legs, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, hairless Drosophila, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, tobacco etch virus, turnip shrunken virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobina virus, HCV QC64, human Coxsackievirus E / D, human Coxsackievirus F, human Coxsackievirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sally virus A SH1, Sally virus FHB, Sally virus NG-J1, Human paraenteritis virus 1, Croshi virus B, Yc-3, Rosa virus M-7, Sanba virus A, Pasi virus A, Pasi virus A 2, Echovirus E14, Human paraenteritis virus 5, Aichi virus, Hepatitis A virus HA16, Vonpivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human hepatic tropism virus 2, GBV-CGT110, GBV-C K1737, GBV-C Iowa, Hepatic tropism virus A 1220, Pasi virus A 3. Aptamers of Sapellovirus, Rosavirus B, Bakunsavirus, Tremor Virus A, Porcine Parsis Virus 1, PLV-CHN, Parsis Virus A, Scinni Virus, Hepatitis C Virus K, Hepatitis C Virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Bicistronic Virus, Hubei MicroRNA-like Virus, CRPV, Sally Virus A BN5, Sally Virus A BN2, Sally Virus A 02394, Sally Virus A GUT, Sally Virus A CH, Sally Virus A SZ1, Sally Virus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0629] In some embodiments, the IRES includes all or part of a eukaryotic or cellular IRES. In some embodiments, the IRES are derived from human genes, wherein the human genes are ABCF1, ABCG1, ACAD10, ACOT7, ACSS3, ACTG2, ADCYAP1, ADK, AGTR1, AHCYL2, AHI1, AKAP8L, AKR1A1, ALDH3A1, ALDOA, ALG13, AMMECR1L, ANGPTL4, ANK3, AOC3, AP4B1, AP4E1, APAF1, APBB1, APC, APH1A, APOBEC3D, APOM, APP, AQP4, ARHGAP36, ARL13B, ARMC8, ARMCX6, A RPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURKA, B3GALNT1, B3GN TL1, B4GALT3, BAAT, BAG1, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, BTN3A2, C12orf43, C1 4orf93, C17orf62, C1orf226, C21orf62, C2orf15, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP2, CAV1, CBX5, CCDC120 , CCDC17, CCDC186, CCDC51, CCN1, CCND1, CCNT1, CD2BP2, CD9, CDC25C, CDC42, CDC7, CDCA7L, CDIP1, CDK1, CDK11A, CDKN1B, CEACAM7, CEP295NL, CFL AR, CHCHD7, CHIA, CHIC1, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, COG4, COMMD1, COMMD5, CPEB1, CPS1 , CRACR2B, CRBN, CREM, CRYBG1, CSDE1, CSF2RA, CSNK2A1, CSTF3, CTCFL, CTH, CTNNA3, CTNNB1, CTNNB1, CTNND1, CTSL, CUTA, CXCR5, CYB5R3, CYP24A1,CYP3A5、DAG1、DAP3、DAP5、DAXX、DCAF4、DCAF7、DCLRE1A、DCP1A、DCTN1、DCTN2、D DX19B、DDX46、DEFB123、DGKA、DGKD、DHRS4、DHX15、DIO3、DLG1、DLL4、DMDUTR、DMD ex5、DMKN、DNAH6、DNAL4、DUSP13、DUSP19、DYNC1I2、DYNLRB2、DYRK1A、ECI2、ECT2、EIF1AD、EIF2B4、EIF4G1、EIF4G2、EIF4G3、ELAN E、ELOVL6、ELP5、EMCN、ENO1、EPB41、ERMN、ERVV-1、ESRRG、ETFB、E TFBKMT、ETV1、ETV4、EXD1、EXT1、EZH2、FAM111B、FAM157A、FAM213 A、FBXO25、FBXO9、FBXW7、FCMR、FGF1、FGF1、FGF1A、FGF2、FGF2、FG F-9、FHL5、FMR1、FN1、FOXP1、FTH1、FUBP1、G3BP1、GABBR1、GALC、G ART、GAS7、gastrin、GATA1、GATA4、GFM2、GHR、GJB2、GLI1、GLRA2、GMNN、GPAT3、GPATCH3、GPR137、GPR34、GPR55、GPR89A、GPRASP1、G RAP2、GSDMB、GSTO2、GTF2B、GTF2H4、GUCY1B2、HAX1、HCST、HIGD1A 、HIGD1B、HIPK1、HIST1H1C、HIST1H3H、HK1、HLA-DRB4、HMBS、HMGA 1、HNRNPC、HOPX、HOXA2、HOXA3、HPCAL1、HR、HSP90AB1、HSPA1A、HS PA4L、HSPA5、HYPK、IFFO1、IFT74、IFT81、IGF1、IGF1R、IGF1R、IGF 2、IL11、IL17RE、IL1RL1、IL1RN、IL32、IL6、ILF2、ILVBL、INSR、IN TS13、IP6K1、ITGA4、ITGAE、KCNE4、KERA、KIAA0355、KIAA0895L、K IAA1324、KIAA1522、KIAA1683、KIF2C、KIZ、KLHL31、KLK7、KRR1、K RT14、KRT17、KRT33A、KRT6A、KRTAP10-2、KRTAP13-3、KRTAP13-4、KRTAP5-11、KRTCAP2、LACRT、LAMB1、LAMB3、LANCL1、LBX2、LCAT、LDHA、LDHA L6A、LEF1、LINC-PINT、LMO3、LRRC4C、LRRC7、LRTOMT、LSM5、LTB4R、LYRM1、LY RM2、MAGEA11、MAGEA8、MAGEB1、MAGEB16、MAGEB3、MAPT、MARS、MC1R、MCCC1、 METTL12、METTL7A、MGC16025、MGC16025、MIA2、MIA2、MITF、MKLN1、MNT、MORF 4L2、MPD6、MRFAP1、MRPL21、MRPS12、MSI2、MSLN、MSN、MT2A、MTFR1L、MTMR2、 MTRR、MTUS1、MYB、MYC、MYCL、MYCN、MYL10、MYL3、MYLK、MYO1A、MYT2、MZB1、NA P1L1、NAV1、NBAS、NCF2、NDRG1、NDST2、NDUFA7、NDUFB11、NDUFC1、NDUFS1、N EDD4L、NFAT5、NFE2L2、NFE2L2、NFIA、NHEJ1、NHP2、NIT1、NKRF、NME1-NME2、N PAT、NR3C1、NRBF2、NRF1、NTRK2、NUDCD1、NXF2、NXT2、ODC1、ODF2、OPTN、OR1 0R2、OR11L1、OR2M2、OR2M3、OR2M5、OR2T10、OR4C15、OR4F17、OR4F5、OR5H1、O R5K1、OR6C3、OR6C75、OR6N1、OR7G2、p53、P2RY4、PAN2、PAQR6、PARP4、PARP9 PC、PCBP4、PCDHGC3、PCLAF、PDGFB、PDZRN4、HEAD、PEMT、PEX2、PFKM、PGBD4、 PGLYRP3、PHLDA2、PHTF1、PI4KB、PIGC、PIM1、PKD2L1、PKM、PLCB4、PLD3、PLE KHA1、PLEKHB1、PLS3、PML、PNMA5、PNN、POC1A、POC1B、POLD2、POLD4、POU5F1、 PPIG、PQBP1、PRAME、PRPF4、PRR11、PRRT1、PRSS8、PSMA2、PSMA3、PSMA4、PSMD 11、PSMD4、PSMD6、PSME3、PSMG3、PTBP3、PTCH1、PTHLH、PTPRD、PUS7L、PVRIG、QPRT, RAB27A, RAB7B, RABGGTB, RAET1E, RALGDS, RALYL, RARB, RCVRN, REG3G, RFC5, RGL4, RGS19, RGS3, RHD, RINL, RIPOR2, RITA1, RMDN2, RNASE1, RNASE 4.RNF4, RPA2, RPL17, RPL21, RPL26L1, RPL28, RPL29, RPL41, RPL9, RPS11, RPS13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RUNX1T1, RUNX1T1, RUNX2, RUSC1, RX RG, S100A13, S100A4, SAT1, SCHIP1, SCMH1, SEC14L1, SEMA4A, SERPINA1, SERPINB4, SERTAD3, SFTPD, SH3D19, SHC1, SHMT1, SHPRH, SIM1, SIRT5, SLC11A2, SLC12A4, SLC16A1, SLC25A3, SLC26A9, SLC5A11, SLC6A12, SLC6A19, SLC7A1, SLFN11, SLIRP, SMAD5, SMARCAD1, SMN1, SNCA, SNRNP200, SNRPB2, SNX12, SOD1, SOX13, SOX5, SP8, SPARCL1, SPATA12, SPATA31C2, SPN, SPOP, SQSTM1, SRBD1, SRC, SREBF1, SRPK2, SSB, SSB, SSBP1, ST3GAL6, STAB1, STAMBP, STAU1, STAU1, STAU1, STAU1, STK16, STK24, STK38, STMN1, STX7, SULT2B1, SYK, SYNPR, TAF1C, TAGLN, TANK, TAS2R40, TBC1D15, TBXAS1, TCF4, TDGF1, T DP2, TDRD3, TDRD5, TESK2, THAP6, THBD, THTPA, TIAM2, TKFC, TKTL1, TLR10, TM9SF2, TMC6, TMCO2, TMED10, TMEM116, TMEM126A, TMEM159, TMEM208, TMEM 230、TMEM67、TMPRSS13、TMUB2、TNFSF4、TNIP3、TP53、TP53、TP73、TRAF1、TRAK1、TRIM31、TRIM6、TRMT1、TRMT2B、TRPM7、TRPM8、TPEAR、TTC39B、TTLL11TUBB6, TXLNB, TXNIP, TXNL1, TXNRD1, TYROBP, U2AF1, UBA1, UBE2D3, UBE2I, UBE2L3, UBE2V1, UBE2V2, UM PS, UNG, UPP2, USMG5, USP18, UTP14A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS29, VSIG10L, W DHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY1AP1, ZBTB32, ZNF146, ZNF2 50. ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF707 or ZNRD1. ,

[0630] vii. Synthetic Ties: Aptamer Complexes, Modified Nucleotides, IRES Variant and Other Engineered Ties

[0631] As considered herein, in some embodiments, translation initiation elements (TIEs) include synthetic TIEs. In some embodiments, synthetic TIEs include aptamer complexes, synthetic IRES, or other engineered TIEs capable of initiating the translation of linear RNA or circular RNA polynucleotides.

[0632] In some embodiments, one or more aptamer sequences are capable of binding to components of eukaryotic initiation factors to enhance or initiate translation. In some embodiments, the aptamer can be used to enhance in vivo and in vitro translation by promoting a specific eukaryotic initiation factor (eIF) (e.g., the aptamer in WO2019081383A1 is capable of binding to eukaryotic initiation factor 4F (eIF4F)). In some embodiments, the aptamer or a complex of the aptamer may be capable of binding to: EIF4G, EIF4E, EIF4A, EIF4B, EIF3, EIF2, EIF5, EIF1, EIF1A, 40S ribosome, PCBP1 (poly-C binding protein), PCBP2, PCBP3, PCBP4, PABP1 (poly-A binding protein), PTB, the Argonaute protein family, HNRNPK (heteronucleoribonucleoprotein K), or La protein.

[0633] viii. Terminating sequence

[0634] In some embodiments, the core functional element includes a termination sequence. In some embodiments, the termination sequence includes a stop codon. In one embodiment, the termination sequence includes a termination box. In some embodiments, the termination box includes at least two stop codons. In some embodiments, the termination box includes at least two stop codon subframes. In the same embodiment, each stop codon subframe in the termination box includes one, two, or more stop codons. In some embodiments, the termination box includes a LoxP or RoxStopRox, or a frt-flanked termination box. In the same embodiment, the termination box includes a lox-stop-lox termination box.

[0635] ix. Variants

[0636] In some embodiments, the circular RNA polynucleotides provided herein comprise modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleosides are m 5 C (5-methylcytidine). In one embodiment, the modified nucleoside is m 5 U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A (N 6 -methyladenosine). In another embodiment, the modified nucleoside is S-methyladenosine. 2 U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2′-O-methyluridine). In other embodiments, the modified nucleoside is m. 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A (2-methylthio-N) 6 -methyladenosine); i 6 A (N 6 -Isopentenyl adenosine); ms 2 i6A (2-methylthio-N) 6 (Isopentenyl adenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A (2-methylthio-N) 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A (N 6 -glycylcarbamoyladenosine); t 6 A (N 6 -Threonylcarbamoyladenosine); ms2 t 6 A (2-methylthio-N) 6 -Threonylcarbamoyladenosine); m 6 t 6 A (N 6 -Methyl-N 6 -Threonylcarbamoyladenosine); hn 6 A(N 6 -hydroxyn-valinecarbamoyladenosine); ms 2 hn 6 A (2-methylthio-N) 6 -hydroxyvaline carbamoyl adenosine); Ar(p) (2'-O-ribosyl adenosine (phosphate)); I (inosine); m 1 I (1-methylinosine); m 1 Im (1,2'-O-dimethylinosine); m 3 C (3-methylcytidine); Cm (2'-O-methylcytidine); s 2 C (2-thiocytidine); ac 4 C (N 4 -acetylcytidine); f 5 C (5-formylcytidine); m 5 Cm (5,2′-O-dimethylcytidine); ac 4 Cm (N 4 -acetyl-2'-O-methylcytidine); k 2 C (lysine); m 1 G (1-methylguanosine); m 2 G (N 2 -methylguanosine); m 7 G (7-methylguanosine); Gm (2′-O-methylguanosine); m 2 2G (N 2 N 2 -dimethylguanosine); m 2 Gm (N 2 ,2'-O-dimethylguanosine); m 2 2Gm (N 2 N 2 ,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (huaitin); o2yW (peroxyhuaitin); OHyW (hydroxyhuaitin); OHyW (Undermodified hydroxyhuaitoside); imG (huaitoside); mimG (methylhuaitoside); Q (huaitoside); oQ (epoxyhuaitoside); galQ (galactosylhuaitoside); manQ (mannosylhuaitoside); preQ0 (7-cyano-7-deazoguanosine); preQ1 (7-aminomethyl-7-deazoguanosine); G + (Ancient purine); D (dihydrouridine); m 5 Um (5,2'-O-dimethyluridine); s 4 U (4-thiouridine); m 5 s 2 U (5-methyl-2-thiouridine); s 2 Um (2-thio-2'-O-methyluridine); acp 3 U (3-(3-amino-3-carboxypropyl)uridine); ho 5 U (5-hydroxyuridine); mo 5 U (5-methoxyuridine); cmo 5 U (uridine 5-hydroxyacetic acid); mcmo 5 U (uridine 5-hydroxyacetic acid methyl ester); chm 5 U (5-(carboxyhydroxymethyl)uridine)); mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U (5-methoxycarbonylmethyluridine); mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U (5-methoxycarbonylmethyl-2-thiouridine); nm 5 S 2 U (5-aminomethyl-2-thiouridine); mnm 5 U (5-methylaminomethyluridine); mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U (5-methylaminomethyl-2-selenouridine); ncm 5 U (5-carbamoylmethyluridine); ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm 5 U (5-Carboxymethylaminomethyluridine); cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2U (5-carboxymethylaminomethyl-2-thiouridine); m 6 2A (N 6 N 6 -dimethyladenosine); Im (2'-O-methylinosine); m 4 C (N 4 -methylcytidine); m 4 Cm (N 4 ,2'-O-dimethylcytidine); hm 5 C (5-hydroxymethylcytidine); m 3 U (3-methyluridine); cm 5 U (5-carboxymethyluridine); m 6 Am (N 6 ,2'-O-dimethyladenosine); m 6 2Am(N 6 N 6 (,O-2'-trimethyladenosine); m 2,7 G (N 2 ,7-Dimethylguanosine); m 2,2,7 G (N 2 N 2 ,7-Trimethylguanosine); m 3 Um(3,2'-O-dimethyluridine); m 5 D (5-methyldihydrouridine); f 5 Cm (5-formyl-2'-O-methylcytidine); m 1 Gm(1,2'-O-dimethylguanosine); m 1 Am (1,2'-O-dimethyladenosine); τm 5 U (5-Taurine methyluridine); τm 5 s 2 U (5-Tauratemethyl-2-thiouridine); imG-14 (4-Demethylwoyoside); imG2 (Isowoyside); or ac 6 A (N 6 -acetyl adenosine.

[0637] In some embodiments, the modified nucleoside may include compounds selected from: pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurate methyluridine, 1-taurate methylpseudouridine, 5-taurate methyl-2-thiouridine, 1-taurate methyl-4-thiouridine, 5-methyluridine, 1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazo-pseudouridine, 2- Thio-1-methyl-1-deazo-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deazo-pseudoisocytidine, 1-methyl-1-deazo-pseudoisocytidine Nitrogen-pseudoisocytidine, zabran, 5-aza-zabran, 5-methyl-zabran, 5-aza-2-thio-zabran, 2-thio-zabran, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-aminopurine, 7-deadenine-8-aza-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl) Adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, inosine, 1-methyl-inosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-Dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In another embodiment, the modification is independently selected from 5-methylcytosine, pseudouridine, and 1-methylpseudouridine.

[0638] In some embodiments, the modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseuuridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified ribonucleosides provide additional stability and resistance to immune activation.

[0639] In specific implementations, the polynucleotide may be codon-optimized. A codon-optimized sequence may be a sequence in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or synthetic constructs of bias tables; (ii) variations in the degree of codon bias within an organism, gene, or gene set; (iii) systematic variations in codons (including background); (iv) codon variations based on the decoding of tRNA; (v) codon variations based on the GC% of a position in the whole or triad; (vi) variations in the degree of similarity to a reference sequence (e.g., a naturally occurring sequence); (vii) variations in codon frequency cutoff values; (viii) structural characteristics of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; and / or (x) systematic variations in the codon set for each amino acid. In some implementations, codon-optimized polynucleotides can minimize ribozyme collisions and / or structural interference between the restricted expression sequence and the core functional element.

[0640] x. Payload

[0641] In some embodiments, the polynucleotide (e.g., circRNA) expression sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the proteins listed in Table 5 below.

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652] In some embodiments, a sequence encoding a therapeutic protein is expressed. In some embodiments, a sequence encoding a cytokine is expressed, such as IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN-β, IL-10, TGF-β, IL-4, or IL-35, or a functional fragment thereof. In some embodiments, a sequence encoding an immune checkpoint inhibitor is expressed. In some embodiments, a sequence encoding an agonist (e.g., a member of the TNFR family, such as CD137L, OX40L, ICOSL, LIGHT, or CD70) is expressed. In some embodiments, a sequence encoding a chimeric antigen receptor is expressed. In some embodiments, a sequence encoding an inhibitory receptor agonist (e.g., PDL1, PDL2, galactolectin-9, VISTA, B7H4, or MHCII) or an inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3, or TIM3) is expressed. In some embodiments, a sequence encoding an inhibitory receptor antagonist is expressed. In some embodiments, the expressed sequence encodes one or more TCR chains (α and β chains or γ and δ chains). In some embodiments, the expressed sequence encodes a secretory T cell or immune cell adaptor (e.g., a bispecific antibody (such as BiTE) targeting, for example, CD3, CD137, or CD28, and a tumor-expressed protein, such as CD19, CD20, or BCMA). In some embodiments, the expressed sequence encodes a BCMA chimeric antigen receptor as disclosed in U.S. Patent Application US 2021 / 0128618A1, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the expressed sequence encodes a transcription factor (e.g., FOXP3 , HELIOS, TOX1 or TOX2 In some embodiments, the expressed sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39 / CD73). In some embodiments, the expressed sequence encodes GvHD (e.g., anti-HLA-A2 CAR-Treg).

[0653] In some embodiments, the precursor RNA polynucleotide and circular RNA construct includes at least one expression sequence encoding an antigen, adjuvant, or adjuvant-like protein (e.g., derived from an infectious agent). In these embodiments, the circular RNA construct can be used as a vaccine. In some embodiments, one or more circular RNA polynucleotides encode an antigen or adjuvant derived from an infectious agent. In some embodiments, the infectious agent from which the antigen or adjuvant is derived or engineered includes, but is not limited to, viruses, bacteria, fungi, protozoa, and / or parasites. In some embodiments, the antigen is a viral antigen or a viral antigenic peptide.

[0654] In one implementation scheme, the antigen is selected from or derived from the group consisting of: rotavirus, foot-and-mouth disease virus, influenza A virus, influenza B virus, influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza virus type 2, herpes simplex virus, Epstein-Barr virus, varicella virus, swine herpesvirus 1, cytomegalovirus, rabies virus, Bacillus anthracis, anthrax PA and its derivatives, poliovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis E, canine distemper virus, Venezuelan equine encephalitis virus, feline leukemia virus, reovirus, respiratory syncytial virus, Lassa fever virus. Polyomavirus, Canine Parvovirus, Papillomavirus, Tick-borne Encephalitis Virus, Rinderpest Virus, Human Rhinovirus Species, Enterovirus Species, Mengovirus, Paramyxovirus, Avian Infectious Bronchitis Virus, Human T-cell Leukemia-Lymphoma Virus 1, Human Immunodeficiency Virus-1, Human Immunodeficiency Virus-2, Lymphocytic Choroid Meningitis Virus, Parvovirus B19, Adenovirus, Rubella Virus, Yellow Fever Virus, Dengue Virus, Bovine Respiratory Syncytial Virus, Coronavirus, Bordetella pertussis, Bordetella bronchitidis, Bordetella parapertussis, Brucella abortus, Brucella ovis, Brucella swine, Brucella ovis, Brucella species, Escherichia coli, Salmonella species, Salmonella typhi, Streptococcus typhi Cocci, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, Pseudomonas, tuberculosis, birds, BCG, Mycobacterium leprae, Streptococcus pneumoniae, Staphylococcus, Enterobacter species, Rocalimabril henle, hemolytic Pasteurella, Pasteurella multocida, Chlamydia trachomatis, Chlamydia psittaci, Lymphogranuloma venereum, Treponema pallidum, Haemophilus species, Bovine genital mycoplasma, Mycoplasma pneumoniae, Mycoplasma species, Borrelia burgdorferi, Legionella pneumophila, Clostridium botulinum, Corynebacterium diphtheriae, Yersinia enterocolitica, Rickettsia rickettsiae, Rickettsia typhi, Rickettsia prowleri, Ehrlich aspergillus chafing, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosoma, Trypanosoma, Leishmania species Filarial nematodes, trichomoniasis, myosocystis infection, beef tapeworm, pork tapeworm, Leishmania, Toxoplasma gondii, trichinella, coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae, malaria circospore protein, malaria merozoonin, trypanosome surface antigen protein, pertussis, alpha virus, adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, influenza hemagglutinin, cancer antigen, tumor antigen, toxins, Clostridium perfringens ε toxin, ricin, Pseudomonas exotoxin, exotoxins, neurotoxins, cytokines, cytokine receptors, mononuclear factors, mononuclear factor receptors, plant pollen, animal dander, and dust mites.

[0655] In some implementations, the antigenic polypeptide is derived from the following viral polypeptides: adenovirus; herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus; hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; norovirus; Coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV). HIV; influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; circovirus; Colorado tick fever virus (Coltivirus); Banna virus; human enterovirus; Hantavirus; West Nile virus; Middle East respiratory syndrome coronavirus; Japanese encephalitis virus; vesicular herpesvirus; SARS-CoV-2; Eastern equine encephalitis; or any two or more combinations thereof.

[0656] In some embodiments, the adjuvant is selected from or derived from the group consisting of: BCSP31, MOMP, FomA, MymA, ESAT6, PorB, PVL, porin, OmpA, PepO, OmpU, dioxetine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, pneumococcal hemolysin, falgellin, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-α, IL-7, IL-17, IL-1β, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and anti-CD3.

[0657] In some embodiments, the polynucleotide encodes a protein composed of subunits, which are encoded by more than one gene. For example, the protein may be a heterodimer, wherein each chain or subunit of the protein is encoded by a separate gene. It is possible to deliver more than one circRNA molecule in a transfer medium, and each circRNA encodes a separate subunit of the protein. Alternatively, a single circRNA may be engineered to encode more than one subunit. In some embodiments, separate circRNA molecules encoding the individual subunits may be administered in separate transfer media.

[0658] Other polynucleotides (including but not limited to intronic elements, exonic elements, translation initiation elements, expression sequences, and lipids) are contained in WO2019236673; WO2020237227; WO2021113777; WO2021226597; WO2021189059; WO2021236855; WO2022261490; WO2023056033; WO2023081526; WO2023250375; the contents of these documents are hereby incorporated herein by reference in their entirety.

[0659] (1) Chimeric antigen receptor (CAR)

[0660] Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be inserted into and expressed by immune cells, including T cells, via circular RNA as described herein. Using CARs, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate the immune cell to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells. In some embodiments, the CAR encoded by a polynucleotide includes (i) an antigen-binding molecule that specifically binds to the target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activation domain.

[0661] In some embodiments, the orientation of the CAR according to this disclosure includes an antigen-binding domain (e.g., scFv) in tandem with a co-stimulatory domain and an activation domain. The co-stimulatory domain may include one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple co-stimulatory domains may be utilized in tandem.

[0662] CARs can be engineered to bind to antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. In some embodiments, the antigen-binding molecule is its antibody fragment, such as one or more single-chain antibody fragments (scFvs). scFvs are single-chain antibody fragments having variable regions of the heavy and light chains of an antibody linked together. See U.S. Patents 7,741,465 and 6,319,494 and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. scFvs retain the ability of the parent antibody to specifically interact with the target antigen. scFvs are useful in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain along with other CAR components. Ibid. See also Krause et al., J. Exp. Med., Vol. 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It should be understood that antigen-binding molecules are typically contained within the extracellular portion of the CAR, enabling it to recognize and bind to antigens of interest. Bispecific and multispecific CARs, which possess specificity for more than one target, are considered within the scope of this disclosure.

[0663] In some embodiments, the antigen-binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are linked by a linker. In some embodiments, VH is located at the N-terminus of the linker, and VL is located at the C-terminus of the linker. In other embodiments, VL is located at the N-terminus of the linker, and VH is located at the C-terminus of the linker. In some embodiments, the linker comprises at least 5, at least 8, at least 10, at least 13, at least 15, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

[0664] In some embodiments, the antigen-binding molecule includes a nanobody. In some embodiments, the antigen-binding molecule includes DARPin. In some embodiments, the antigen-binding molecule includes an anticarrier protein or other synthetic protein capable of specifically binding to a target protein.

[0665] In some embodiments, the CAR includes an antigen-binding domain specific to an antigen selected from CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((TnAg) or (GaINAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit α-2, mesothelin, interleukin-11 receptor α (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor β (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor α, HER2, HER3, mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostate enzyme, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), liver ligand B2, fibroblast activating protein α (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macroprotein factor) subunit β-type 9 (LMP2), glycoprotein 100 (gp100), homolog of breakpoint cluster region (BCR) and Apson mouse leukemia virus oncogene 1 The following are listed: oncogene fusion protein (bcr-abl) composed of (Abl), tyrosinase, liver ligand A receptor 2 (EphA2), fucose GM1, sialic acid Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), O-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-associated (TEM7R), sealing protein 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), and chromosome X open reading frame 61.(CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), plaque protein 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pan-connector 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR γ-substituted reading frame protein (TARP), Wilms' tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3, and MAGE-A4), ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, spermin 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-associated antigen 1, tumor protein p53 (p53), p53 mutant, prostaglandin, surviving protein, telomerase, prostate cancer tumor antigen-1, T cell-recognized melanoma antigen 1, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma cell apoptosis inhibitor (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosamine transferase V (NA17), Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-associated protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like, T cell-recognized squamous cell carcinoma antigen 3 (SART3), Pax-5 (PAX5), pro-acromial protein-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), pod protein, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E6)E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA receptor Fc fragment (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecular-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2), lymphocyte antigen 75 (LY75), phosphatidylinositol proteoglycan-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, ανβθ integrin, αvβ6 integrin, alpha-fetoprotein (AFP), B7-H6, ca-125, CA9, CD44, CD44v7 / 8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate-binding protein (FBP), kinase insertion domain receptor (KDR), κ light chain, L1 cell adhesion molecule, MUC18, NKG2D, carcinoembryonic antigen (h5T4), tumor / testis antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin λ-like peptide 1 (IGLL1), Hepatitis B surface antigen binding protein (HBsAg), viral capsid antigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, hemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, human metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lymphotropic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, Kaposi's sarcoma-associated herpesvirus (KSHV) lysate nuclear antigen, and KSHV latent nuclear antigen. In some implementations, the antigen-binding domain includes SEQ ID NO: 321 and / or 322.

[0666] In some embodiments, the CAR includes a hinge or spacer region structural domain. In some embodiments, the hinge / spacer region structural domain may include a truncated hinge / spacer region structural domain (THD), which is a truncated version of the full hinge / spacer region structural domain (“CHD”). In some embodiments, the extracellular domains are derived from or comprise all or all of the following fragments: ErbB2, blood group glycoprotein A (GpA), CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8[T], CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (tactile protein), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229(SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270(TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulatory molecules (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activated NK cell receptors, Toll ligand receptors, and fragments or combinations thereof. The hinge or spacer domain can be derived from natural or synthetic sources.

[0667] In some embodiments, the hinge or spacer region domain is located between the antigen-binding molecule (e.g., scFv) and the transmembrane domain. In this orientation, the hinge / spacer region provides the distance between the antigen-binding molecule and the cell membrane surface on which the CAR is expressed. In some embodiments, the hinge or spacer region domain is derived from or originates from immunoglobulins. In some embodiments, the hinge or spacer region domain is selected from hinge / spacer regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof. In some embodiments, the hinge or spacer region domain includes, is derived from, or is derived from: the hinge / spacer region of CD8α. In some embodiments, the hinge or spacer region domain includes, is derived from, or is derived from: the hinge / spacer region of CD28. In some embodiments, the hinge or spacer region domain includes a fragment of the hinge / spacer region of CD8α or a fragment of the hinge / spacer region of CD28, wherein said fragment is any portion less than the entire hinge / spacer region. In some embodiments, the fragments of the CD8α hinge / spacer region or the CD28 hinge / spacer region comprise an amino acid sequence excluding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least five, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least nineteen, at least eighteen, at least nineteen, or at least twenty amino acids at the N-terminus or C-terminus of the CD8α hinge / spacer region or the CD28 hinge / spacer region.

[0668] The CAR may further include a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be programmed to fuse with the extracellular domain of the CAR. It may similarly fuse with the intracellular domain of the CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain may be selected or modified (e.g., by amino acid substitution) to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain may be derived from a natural or synthetic source. In the case of a natural source, the domain may be derived from any membrane-binding or transmembrane protein.

[0669] Transmembrane regions can be derived from (i.e., include) receptor tyrosine kinases (e.g., ErbB2), blood group glycoprotein A (GpA), 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (tactile protein), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IE-2Rβ, IE-2Rγ, IE-7Rα, inducible T cell co-stimulatory molecules (ICOS), integrins, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligands that specifically bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms or combinations thereof.

[0670] In some implementations, suitable intracellular signal transduction domains include, but are not limited to, activated macrophage / myeloid cell receptors CSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4 / 80, CD91, C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, lactoferrin, or fragments, truncated forms, or combinations thereof.

[0671] In some implementations, the receptor tyrosine kinase may be derived from (e.g., including) the insulin receptor (InsR), insulin-like growth factor I receptor (IGF1R), insulin receptor-associated receptor (IRR), platelet-derived growth factor receptor α (PDGFRa), and platelet-derived growth factor receptor β (PDGFRfi).KIT proto-oncogene receptor tyrosine kinase (Kit), colony-stimulating factor 1 receptor (CSFR), fms-associated tyrosine kinase 3 (FLT3), fms-associated tyrosine kinase 1 (VEGFR-1), kinase insertion domain receptor (VEGFR-2), fms-associated tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), muscle-associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage-stimulated receptor 1 (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase 1 with immunoglobulin-like and EGF-like domains (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), (EPH receptor A3) EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphA10), EPH receptor B1 (EphB1), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret proto-oncogene (Ret), receptor-like tyrosine kinase (RYK), discoid domain receptor tyrosine kinase 1 (DDR1), discoid domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis-associated tyrosine kinase (Lmrl), lemur tyrosine kinase 2 (Lmr2), lemur tyrosine kinase 3 (Lmr3), leukocyte receptor tyrosine kinase (LTK), ALK receptor tyrosine kinase (ALK), or serine / threonine / tyrosine kinase 1 (STYK1).

[0672] In some embodiments, the CAR includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain includes 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In a preferred embodiment, the co-stimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3ζ (ζ). 4-1BB, CD28, and CD3ζ may each include less than the entire 4-1BB, CD28, or CD3ζ. Chimeric antigen receptors may incorporate the co-stimulatory (signaling) domain to increase their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (ibid.), Song et al., Blood 119:696-706 (2012); Kalos et al., SciTransl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0673] In some implementations, the co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 318 or 320.

[0674] The intracellular (signal transduction) domain of the engineered T cells disclosed herein can provide signal transduction to an activation domain, which in turn activates at least one of the normal effector functions of the immune cell. The effector functions of the T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion.

[0675] In some implementations, suitable intracellular signal transduction domains include (e.g., include), but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (tactile protein), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, and DNAM1. (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, Inducible T cell co-stimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Lyl08, Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms or combinations thereof.

[0676] CD3 is a component of the T cell receptor on natural T cells and has been shown to be an important intracellular activating element in CARs. In some embodiments, CD3 is CD3ζ. In some embodiments, the activation domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 319.

[0677] In some implementations, the sequence encoding the CAR includes sequences from Table 6.

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689] (2) T cell receptor (TCR)

[0690] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. Natural αβ heterodimer TCRs have α and β chains. Broadly, each chain may include variable, linker, and constant regions, and the β chain typically also contains a short region of diversity between the variable and linker regions, but this diverse region is usually considered part of the linker region. Each variable region may include three CDRs (complementarity-determining regions) embedded in the frame sequence, one of which is a hypervariable region named CDR3. Several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions exist, distinguished by their frame, CDR1 and CDR2 sequences, and by a partially defined CDR3 sequence. The Vα type is designated in the IMGT nomenclature by a unique TRAV number. Thus, “TRAV21” defines a TCRVα region with a unique frame and CDR1 and CDR2 sequences, and a CDR3 sequence defined partly by amino acid sequences that remain constant between TCRs but also including amino acid sequences that vary between TCRs. In the same manner, “TRBV5-1” defines a TCR Vβ region with a unique frame and CDR1 and CDR2 sequences but only a partially defined CDR3 sequence.

[0691] The connection region of the TCR is similarly defined by the unique IMGT TRAJ and TRBJ nomenclatures, and the constant region is defined by the IMGTTRAC and TRBC nomenclatures.

[0692] In the IMGT nomenclature, the β-chain diversity region is referred to by the abbreviation TRBD, and as mentioned above, tandem TRBD / TRBJ regions are usually considered together as connection regions.

[0693] The unique sequences defined by the IMGT nomenclature are widely known and available to those skilled in the art of TCR. For example, they can be found in the IMGT public database. Sequences defined by the IMGT nomenclature are also disclosed in “T cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, but due to their publication date and the resulting time lag, the information therein sometimes needs to be confirmed by referring to the IMGT database.

[0694] Natural TCRs exist as heterodimers in the form of αβ or γδ. However, recombinant TCRs composed of αα or ββ homodimers have previously been shown to bind to peptide MHC molecules. Therefore, the TCRs disclosed herein can be heterodimer αβ TCRs, or they can be αα or ββ homodimer TCRs.

[0695] For use in adoptive therapy, αβ heterodimeric TCRs can be transfected, for example, as a full-length chain having both cytoplasmic and transmembrane domains. In some embodiments, the TCRs of this disclosure may have introduced disulfide bonds between residues of their respective constant domains, as described, for example, in WO 2006 / 000830.

[0696] The TCRs disclosed herein (particularly α-β heterodimer TCRs) may include an α-chain TRAC constant domain sequence and / or a β-chain TRBC1 or TRBC2 constant domain sequence. The α-chain and β-chain constant domain sequences may be modified by truncation or substitution to remove the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α- and / or β-chain constant domain sequences may also be modified by substituting Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 with cysteine ​​residues that form a disulfide bond between the α- and β-constant domains of the TCR.

[0697] Combining affinity (with equilibrium constant K) D The inversely proportional affinity (Tc) and binding half-life (denoted as T½) can be determined by any suitable method. It should be understood that the doubling of affinity of the TCR leads to Kc... D Halved. T½ is calculated as ln 2 divided by the dissociation rate (koff). Therefore, doubling T½ results in koff being halved. K of TCR D Koff values ​​are typically measured for soluble forms of TCRs (i.e., those truncated to remove cytoplasmic and transmembrane domain residues). Therefore, it should be understood that if a soluble form of a given TCR possesses the aforementioned characteristics, then that TCR has improved binding affinity and / or binding half-life to its parental TCR. Preferably, the binding affinity or binding half-life of a given TCR is measured several times using the same assay protocol, e.g., three or more times, and the results are averaged.

[0698] Because the TCRs of this disclosure are effective in adoptive therapy, this disclosure includes non-naturally occurring and / or purified and / or engineered cells, particularly T cells, that present the TCRs of this disclosure. Numerous methods are suitable for transfecting T cells with nucleic acids encoding the TCRs of this disclosure (such as DNA, cDNA, or RNA) (see, for example, Robbins et al., (2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of this disclosure will be suitable for use in adoptive therapy-based treatment of cancers (e.g., pancreatic and liver cancers). As will be known to those skilled in the art, many suitable methods exist by which adoptive therapy can be performed (see, for example, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

[0699] As is well known in the art, the TCRs of this disclosure may undergo post-translational modifications when expressed by transfected cells. Glycosylation is one such modification that may include covalently linking an oligosaccharide moiety to a specific amino acid in the TCR chain. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on many factors, including protein sequence, protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation state (i.e., oligosaccharide type, covalent bonds, and total number of attachments) can affect protein function. Therefore, controlling glycosylation is generally desirable when producing recombinant proteins. Glycosylation of transfected TCRs can be controlled by mutations in the transfected gene (Kuball J et al. (2009), JExp Med 206(2):463-475). Such mutations are also covered in this disclosure.

[0700] TCRs can be specific for antigens in the following groups: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE- A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), Tyrosinase, Glycogen Phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, α-actin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein Synthesis protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2 and 3, neo-PAP, class I myosin, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, Lage-2, SP17 and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom / Mel-40, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19--9, CA 72--4, CAM 17.1. NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\Cyclophilic C-related protein), TAAL6, TAG72, TLP, and TPS. .

[0701] (3) B-cell receptor (BCR)

[0702] B cell receptors (BCRs), or B cell antigen receptors, are immunoglobulin molecules that form type I transmembrane proteins on the surface of B cells. BCRs transmit activation signals to B cells upon recognition of specific antigens. Before binding to the antigen, the BCR remains in an unstimulated or "resting" phase. The binding of the antigen to the BCR triggers the signaling that initiates the humoral immune response.

[0703] BCRs are expressed by mature B cells. These B cells, along with immunoglobulins (Ig), play a role in recognizing and labeling pathogens. Typical BCRs include membrane-bound immunoglobulins (e.g., mIgA, mIgD, mIgE, mIgG, and mIgM), along with the associated Igα / Igβ (CD79a / CD79b) heterodimer (α / β). These membrane-bound immunoglobulins are tetramers composed of two identical heavy chains and two light chains. Within the BCR, membrane-bound immunoglobulins can respond to antigen binding via transmembrane signaling, leading to B cell activation and consequently clonal expansion and specific antibody production (Friess M et al. (2018), Front. Immunol. 2947(9)). The Igα / Igβ heterodimer is responsible for transducing signals into the cellular interior.

[0704] Igα / Igβ heterodimer signaling depends on the presence of an immune receptor tyrosine-based activation motif (ITAM) located on each cytoplasmic tail of the heterodimer. An ITAM consists of two tyrosine residues separated by 9–12 amino acids (e.g., tyrosine, leucine, and / or valine). Upon antigen binding, the tyrosine residues of the BCR's ITAM are phosphorylated by Src family tyrosine kinases Blk, Fyn, or Lyn (Janeway C et al., Immunobiology: The Immune System in Health and Disease (Garland Science, 5th ed., 2001)).

[0705] (4) Other chimeric proteins

[0706] In addition to the chimeric proteins described above, circular RNA polynucleotides can encode a wide variety of other chimeric proteins available in the art. Chimeric proteins may include recombinant fusion proteins, chimeric mutant proteins, or other fusion proteins.

[0707] In some embodiments, the circular RNA polynucleotide encodes an immunomodulatory ligand. In some embodiments, the immunomodulatory ligand may be immunostimulatory; while in other embodiments, the immunomodulatory ligand may be immunosuppressive.

[0708] In some embodiments, circular RNA polynucleotides encode cytokines. In some embodiments, cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Chemokines are chemokines produced by various cell types in acute and chronic inflammation, which mobilize and activate leukocytes. Interferons include the family of secreted α-helical cytokines induced by stimulating TLR responses to specific extracellular molecules (Borden, Molecular Basis of Cancer (4th Edition) 2015). Interleukins are cytokines expressed by leukocytes.

[0709] Descriptions and / or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-27β, IFNγ, and / or TGFβ1 are provided in this paper and in the www.uniprot.org database under the following accession numbers: P60568 (IL-2), P29459 (IL-12A), P29460 (IL-12B), P13232 (IL-7), P22301 (IL-10), P40933 (IL-15), Q14116 (IL-18), Q14213 (IL-27β), P01579 (IFNγ), and / or P01137 (TGFβ1).

[0710] In some implementations, circular RNA polynucleotides may encode transcription factors. Regulatory T cells (Tregs) are important in maintaining homeostasis, controlling the magnitude and duration of inflammatory responses, and preventing autoimmune and allergic reactions.

[0711] Generally, Tregs are thought to primarily participate in suppressing immune responses, partially acting as a "self-check" mechanism of the immune system to prevent overreaction. In particular, Tregs are involved in maintaining tolerance to self-antigens, harmless agents such as pollen or food, and eliminating autoimmune diseases.

[0712] Treg cells are distributed throughout the body, including but not limited to the intestines, skin, lungs, and liver. Furthermore, Treg cells may also be present in certain compartments of the body that are not directly exposed to the external environment, such as the spleen, lymph nodes, and even adipose tissue. Each of these Treg cell populations is known or suspected to possess one or more unique characteristics, and further information can be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294): 1-10, the contents of which are hereby incorporated in their entirety.

[0713] Typically, Tregs are known to require TGF-β and IL-2 for proper activation and development. Tregs expressing high levels of the IL-2 receptor (IL-2R) depend on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β, both of which are potent immunosuppressive cytokines. Furthermore, Tregs are known to suppress the ability of antigen-presenting cells (APCs) to stimulate T cells. One proposed mechanism of APC suppression is through CTLA-4, which is expressed by Foxp3+ Tregs. It is believed that CTLA-4 can bind to B7 molecules on APCs and block or remove these molecules by inducing internalization, thereby reducing B7 availability and failing to provide adequate co-stimulation for the immune response. Further discussion on the origin, differentiation, and function of Tregs can be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4 (253): 1-11, the contents of which are hereby incorporated in their entirety.

[0714] As provided herein, in some embodiments, the coding element of the circular RNA polynucleotide encodes one or more checkpoint inhibitors or agonists.

[0715] In some implementations, immune checkpoint inhibitors are the following inhibitors: programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell co-stimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1, CTLA4, PD-1, LAG3, PD-L1, TIM3, or a combination thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1.

[0716] As described herein, at least in one aspect, this disclosure includes the use of immune checkpoint antagonists. Such immune checkpoint antagonists include antagonists of immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (Pd-1), programmed death ligand 1 (PDL-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain 3 (TIM-3). Antagonists of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 interfere with the function of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3, respectively. Such antagonists of CTLA-4, PD-1, PDL-1, LAG-3, and TIM-3 may comprise antibodies that specifically bind to CTLA-4, PD-1, PDL-1, LAG-3, and TIM-3 and inhibit and / or block their biological activity and function, respectively.

[0717] In some embodiments, the payload encoded within one or more coding elements is a hormone, FC fusion protein, anticoagulant, coagulation factor, protein associated with defects and genetic diseases, chaperone protein, antimicrobial protein, enzyme (e.g., metabolic enzyme), structural protein (e.g., channel or nucleoporin), protein variant, small molecule, antibody, nanobody, engineered non-body antibody, or a combination thereof.

[0718] xi. Production of polynucleotides

[0719] DNA templates can be prepared using standard molecular biology techniques. For example, various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving polynucleotides from DNA templates known to contain polynucleotides.

[0720] Various elements of a DNA template can also be synthesized based on known sequences, rather than cloned. The complete sequence can be assembled from overlapping oligonucleotides prepared and assembled into a complete sequence using standard methods. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311.

[0721] Therefore, specific nucleotide sequences can be obtained from a DNA template having the desired sequence, or synthesized, fully or partially, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR), where appropriate. One method of obtaining a nucleotide sequence encoding a desired DNA template element is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional automated polynucleotide synthesizer, followed by ligation with a suitable DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Alternatively, oligonucleotide-guided synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-guided mutagenesis of pre-existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of nicked oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0722] Precursor RNA can be generated by incubating a DNA template under conditions that allow transcription of the precursor RNA encoded by the DNA template. For example, in some embodiments, the precursor RNA is synthesized by incubating a DNA template, provided herein, which includes an RNA polymerase promoter upstream of its 5' double-stranded sequence and / or expression sequence, with a compatible RNA polymerase under conditions that allow in vitro transcription. In some embodiments, the DNA template is incubated intracellularly by phage RNA polymerase or in the cell nucleus by host RNA polymerase II.

[0723] xii. Linear mRNA payload

[0724] In various embodiments, the LNP compositions described herein can be used to deliver RNA payloads, which are linear mRNA molecules.

[0725] In various embodiments, the LNP-based pharmaceutical compositions described herein, such as LNP-based gene editing systems, may include one or more linear mRNA molecules or linear mRNA payloads. In various embodiments, the mRNA payload may encode one or more components of the gene editing system described herein. For example, the mRNA payload may encode an amino acid sequence-programmable DNA-binding domain (e.g., TALENS and zinc finger binding domains) or a nucleic acid sequence-programmable DNA-binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).

[0726] Depending on the nature of the gene editing system, the mRNA payload may also encode one or more functional effector domains that provide to facilitate alterations in nucleotide sequence and / or gene expression, such as, but not limited to, single-stranded DNA-binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminase or adenosine deaminase), polymerases (e.g., reverse transcriptase), integrases, recombinases, and fusion proteins containing one or more functional domains linked together.

[0727] Ribonucleic acid (RNA) is a molecule composed of nucleotides, which are ribose sugars linked to a nitrogenous base and a phosphate group. Nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). RNA is typically single-stranded, but can exist in double strands in some cases. The length, form, and structure of RNA vary depending on its purpose. For example, RNA length can vary from short sequences (e.g., siRNA) to long sequences (e.g., lncRNA), it can be linear (e.g., mRNA) or circular (e.g., oRNA), and it can be coding (e.g., mRNA) or non-coding (e.g., lncRNA) sequences.

[0728] In various embodiments, the LNP compositions described herein can be used to deliver mRNA payloads, which are linear mRNA molecules. In embodiments, the mRNA payload may contain one or more nucleotide sequences encoding a target product, such as, but not limited to, vaccine antigens, components of gene editing systems (e.g., endonucleases, leader editors, etc.), and / or therapeutic proteins.

[0729] In some implementations, the RNA payload may be linear mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a target protein and can be translated in vitro, in vivo, in situ, or ex vivo to produce the target protein.

[0730] Typically, mRNA molecules contain at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. In some respects, one or more structural and / or chemical modifications or alterations can be included in RNA that can reduce the innate immune response of the cell in which the mRNA is introduced. As used herein, a "structural" feature or modification is a feature or modification that inserts, deletes, repeats, inverts, or randomizes two or more linked nucleotides in a nucleic acid without requiring significant chemical modification of the nucleotides themselves. Because chemical bonds must necessarily break and reform to achieve structural modification, structural modification is chemical in nature and therefore a chemical modification. However, structural modifications will produce different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G".

[0731] Typically, the target coding region within the mRNA used herein can encode dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, or decapeptides. In another embodiment, the mRNA can encode peptides of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide of no more than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0732] Typically, the region of mRNA encoding the product of interest is longer than about 30 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides). 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides.

[0733] In some implementations, the total length of the mRNA spans from about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 10...). 0 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 50 0 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides.

[0734] In some implementations, the length of one or more regions lateralized to encode the product of interest can independently range from 15 to 1,000 nucleotides (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, etc.). 600, 700, 800, and 900 nucleotides, or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides.

[0735] In some embodiments, the mRNA includes a tailing sequence whose length can range from absent to 500 nucleotides (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). When the tailing region is a poly-A tail, the length can be determined based on the number of poly-A binding protein units or the relationship to the poly-A binding protein. In this embodiment, the poly-A tail is long enough to bind at least four poly-A binding protein monomers. The poly-A binding protein monomers bind to an extension of approximately 38 nucleotides. Therefore, poly-A tails of approximately 80 and 160 nucleotides have been observed to be functional.

[0736] In some embodiments, the mRNA includes a capping sequence comprising a single cap or a series of nucleotides forming a cap. The length of the capping sequence can be from 1 to 10, for example 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2 or 10 or fewer nucleotides. In some embodiments, there is no capping sequence.

[0737] In some implementations, the mRNA contains a region containing a start codon. The length of the region containing the start codon can range from 3 to 40 nucleotides, for example, 5-30, 10-20, 15, or at least 4, 30, or fewer nucleotides.

[0738] In some implementations, the mRNA contains a region containing a stop codon. The length of the region containing the stop codon can range from 3 to 40 nucleotides, for example, 5-30, 10-20, 15, or at least 4, 30, or fewer nucleotides.

[0739] In some implementations, the mRNA contains a region containing a restriction sequence. The length of the region containing the restriction sequence can range from 3 to 40 nucleotides, for example, 5-30, 10-20, 15, or at least 4, 30, or fewer nucleotides.

[0740] Untranslated Region (UTR)

[0741] In various embodiments, the mRNA payload of the LNP composition described herein may include at least one untranslated region (UTR) flanking a region encoding the target product and / or incorporated into the mRNA molecule. The UTR is transcribed but not translated. The mRNA payload may include a 5' UTR sequence and a 3' UTR sequence, as well as an internal UTR.

[0742] The RNA payloads of this disclosure may include one or more regions or portions that function as untranslated regions or perform untranslated regions. When the nucleic acid is designed to encode at least one polypeptide of interest, the nucleic acid may include one or more of these untranslated regions (UTRs). The wild-type untranslated region of the nucleic acid is transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start site and extends to the start codon, but does not include the start codon; while the 3' UTR immediately follows the stop codon and extends until the transcription termination signal. Increasing evidence suggests that UTRs play a regulatory role in the stability and translation of nucleic acid molecules. Regulatory features of UTRs may be incorporated into the RNA payload molecules of this disclosure (e.g., linear and circular mRNA molecules) to enhance molecular stability, among other things. Specific features may also be incorporated to ensure controlled downregulation of transcripts to prevent them from being misdirected to undesired organ sites. Various 5'UTR and 3'UTR sequences are known and available in the art.

[0743] In various embodiments, the mRNA payload of the LNP composition described herein may include at least one UTR, which may be selected from any UTR sequence listed in Table 19 or Table 20 of U.S. Patent No. 10,709,779 (which is incorporated herein by reference).

[0744] 5' UTR zone

[0745] In various embodiments, the mRNA payload of the LNP composition described herein may include at least one 5'UTR.

[0746] The 5′ UTR is a region in mRNA located directly upstream (5′) of the start codon (the first codon in the mRNA transcript translated by the ribosome). The 5′ UTR does not encode proteins (it is non-coding). Native 5′ UTRs are characterized by their role in translation initiation. They are characterized by sequences such as the Kozak sequence, which is known to be involved in the ribosome-initiated translation of many genes. The Kozak sequence has a consistent CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. 5′ UTRs are also known to form secondary structures involved in elongation factor binding. 5′ UTR sequences are also known to be important for ribosome recruitment to mRNA and have been reported to play a role in translation (Hinnebusch A et al., (2016) Science, 352:6292:1413-6). In addition, the 5' UTR sequence can confer increased half-life, increased expression, and / or increased activity to the polypeptide encoded by the RNA payload described herein.

[0747] In various implementations, the RNA payload constructs considered herein may include 5'UTRs found in nature and 5'UTRs not found in nature. For example, the 5'UTR may be synthetic and / or may be altered in sequence relative to the naturally occurring 5'UTR. Such altered 5'UTRs may include one or more modifications relative to the naturally occurring 5'UTR, such as insertions, deletions, or altered sequences, or substitutions of one or more nucleotide analogs for the naturally occurring nucleotides.

[0748] The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. While we do not wish to be bound by theory, the UTR may play a regulatory role in nucleic acid translation and stability.

[0749] Natural 5'UTRs typically include features that play a role in translation initiation because they tend to include Kozak sequences, which are generally known to be involved in the process by which ribosomes initiate the translation of many genes. Kozak sequences have a common sequence CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0750] In some embodiments of this disclosure, the 5' UTR is a heterologous UTR, i.e., a UTR found in nature that is associated with a different mRNA. In another embodiment, the 5' UTR is a synthetic UTR, i.e., one that does not exist in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties (e.g., increase gene expression) as well as completely synthetic UTRs. Exemplary 5' UTRs include Xenopus laevis or human-derived α-globin or β-globin (e.g., US8,278,063 and US9,012,219), human cytochrome b-245 polypeptide, hydroxysteroid (17b) dehydrogenase, and tobacco etch virus. The CMV Immediate Early 1 (IE1) gene (see US20140206753 and WO2013 / 185069), sequence GGGAUCCUACC (SEQ ID NO: 29)(WO2014144196), may also be used. In another embodiment, the 5′ UTR of the TOP gene is the 5′ UTR of the TOP gene lacking the 5′ TOP motif (oligopyrimidine string) (e.g., WO / 2015101414, WO2015101415, WO / 2015 / 062738, WO2015024667, WO2015024667; 5′ UTR elements derived from the ribosomal protein large 32 (L32) gene (WO / 2015101414, WO2015101415, WO / 2015 / 062738)), and may use a 5′ UTR element derived from the 5′ UTR of the hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5′ UTR element derived from the ATP5A1 5′ UTR (WO2015024667). In one implementation, the internal ribosome entry site (IRES) is used as a substitute for the 5′ UTR.

[0751] 3' UTR zone

[0752] In various embodiments, the mRNA payload of the LNP composition described herein may include at least one 3′ UTR. The 3′ UTR may be heterologous or synthetic.

[0753] 3′ UTRs are regions in mRNA located directly downstream (3′) of a stop codon (the codon that signals the termination of translation in the mRNA transcript). 3′ UTRs do not encode proteins (they are non-coding). Natural or wild-type 3′ UTRs are known to contain embedded adenosine and uridine extensions. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonmers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are not well-defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myopoietin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize the messenger, while members of the ELAV family (most notably HuR) have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3′ UTR of a nucleic acid molecule will result in HuR binding, thereby stabilizing the messenger in vivo.

[0754] 3' UTRs are known to contain extensions of adenosine and uridine. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are not well-defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myopoietin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize messengers, while members of the ELAV family (most notably HuR) have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3' UTR of a nucleic acid molecule will result in HuR binding, thereby stabilizing the messenger in vivo.

[0755] The introduction, removal, or modification of AU-rich elements (AREs) in the 3' UTR can be used to modulate the stability of the mRNA payload described herein. For example, one or more copies of ARE can be introduced to make the mRNA less stable, thereby inhibiting translation and reducing the production of the resulting protein. Alternatively, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein.

[0756] In some embodiments, introducing a feature typically expressed in genes of a target organ can enhance mRNA stability and protein production in that organ and / or tissue. As a non-limiting example, the feature may be a UTR. As another example, the feature may be an intron or a portion of an intron sequence.

[0757] Those skilled in the art will understand that heterologous or synthetic 5' UTRs can be used with any desired 3' UTR sequence. For example, a heterologous 5' UTR can be used with a synthetic 3' UTR having a heterologous 3' UTR.

[0758] Non-UTR sequences can also be used as regions or subregions within the RNA payload construct. For example, introns or portions of intron sequences can be incorporated into the nucleic acid regions of this disclosure. Incorporation of intron sequences can increase protein production and nucleic acid levels.

[0759] Combinations of features may be included in flanking regions and may be contained within other features. For example, a polypeptide coding region of interest in an mRNA payload may be flanked by a 5' UTR (which may contain a strong Kozak translation initiation signal) and / or a 3' UTR (which may contain an oligonucleotide (dT) sequence for template addition of a polyA tail). The 5' UTR may contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes, such as the 5' UTR described in U.S. Patent Application Publication No. 20100293625 and PCT / US2014 / 069155, which are incorporated herein by reference in their entirety.

[0760] It should be understood that any UTR from any gene can be incorporated into a region of an RNA payload molecule (e.g., linear mRNA). Furthermore, multiple wild-type UTRs of any known gene can be utilized. Artificial UTRs that provide variants that are not wild-type regions are also within the scope of this disclosure. These UTRs, or portions thereof, may be positioned in the same orientation as the transcript from which they were selected, or may be altered in orientation or position. Thus, a 5' UTR or a 3' UTR can be inverted, shortened, lengthe...

Claims

1. A PEG lipid having formula VI: (VI) in: P is the PEG portion; Y B is an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-; L is an optional linker selected from the following: optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenyl, and optionally substituted C2-C10 ynylene. Y A is selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-; m is an integer selected from 1 to 10; and R 2 and R 3 each independently is optionally substituted C1-C15 alkyl or optionally substituted C2-C15 alkenyl.

2. The PEG lipid of claim 1, wherein: P is a formula The PEG portion; z is an integer selected from 1 to 200; R is either hydrogen or methyl; and R 2 and R 3 each independently is optionally substituted C1-C15 aliphatic.

3. The PEG lipid according to claim 1 or claim 2, wherein: Y A is -OC(O)-; and Y B Not present.

4. The PEG lipid according to claim 1 or claim 2, wherein: Y A is -OC(O)-; and Y B is -C(O)O-.

5. The PEG lipid according to any one of claims 1-4, wherein L is optionally a substituted C1-C10 alkylene group.

6. The PEG lipid of claim 1 or claim 2, wherein: Y A It is -OC(O)-; and LY B It does not exist.

7. The PEG lipid according to any one of claims 1-6, wherein R 2 and R 3 Each is independently a optionally substituted C1-C15 alkyl group (e.g., optionally substituted C4-C11 alkyl or optionally substituted C6-C11 alkyl).

8. The PEG lipid according to any one of claims 1-6, wherein R 2 and R 3 Each is independently an unsubstituted C1-C15 alkyl group (e.g., an unsubstituted C4-C11 alkyl group or an unsubstituted C6-C11 alkyl group).

9. The PEG lipid according to any one of claims 1-8, wherein R 2 and R 3 Each is selected independently from: , , , , , , , , , , , and .

10. The PEG lipid according to any one of claims 1-9, wherein Selected from: , , , , , , , , and .

11. The PEG lipid of any one of claims 1-10, wherein z is an integer selected from 40 to 50 (e.g., z is 41, 42, 43, 44, 45, 46, 47, 48 or 49).

12. The PEG lipid according to any one of claims 1-11, wherein m is an integer selected from 1 to 9.

13. The PEG of claim 1, wherein the PEG lipid is selected from: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and ， Where z is selected from 1 to 200, for example, selected from 30 to 75. A transfer medium (e.g., lipid nanoparticles (LNP)) comprising PEG lipids as described in any one of claims 1-12.

14. The transfer medium of claim 13, further comprising an ionizable lipid having the formula AX'''': Formula (AX'''') Or its pharmaceutically acceptable salt, wherein: A is selected from optionally substituted bridging bicyclic carbon rings, bridging polycyclic carbon rings, bridging heterocyclic bicyclic carbon rings, or bridging heterocyclic polycyclic carbon rings; n is an integer selected from 0, 1, and 2; m is an integer selected from 1 and 2, such that m plus n is less than or equal to 3; R 1 Selected from -OH, -OAc, -NR2, -N(R)R H , , , , , , , , , , , and ; Each R is independently -H or C1-C6 aliphatic; Each R H It is a C1-C6 aliphatic -OH; Each X 1 and X A C1-C6 aliphatic molecules that are independently bonded or optionally substituted; Each Y 1 Selected independently , , , , , , , and key; where marked " The key is connected to X. 1 ; Each X 2 and X 3 C1-C is independently a bond or optionally substituted. 12 aliphatic; Each Y 2 and Y 3 Selected independently , , , , , , and ; marked with " The key is connected to X. 2 or X 3 ; Each X 4 and X 5 C1-C6 aliphatic molecules that are independently bonded or optionally substituted; Each Y 4 and Y 5 Independently selected from keys, , , , , , , and ; marked with " The key is connected to X. 4 or X 5 ; Each X 6 and X 7 C1-C6 aliphatic molecules that are independently bonded or optionally substituted; R 2 It is -CH(OR) 6 (OR) 7 -CH(SR) 6 (SR) 7 ), -CH(R 6 (R) 7 ), -CF(R 6 (R) 7 -R 10 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 10 , where R 2 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Each R 3 Independently is -CH(OR) 8 (OR) 9 -CH(SR) 8 (SR) 9 ), -CH(R 8 (R) 9 ), -CF(R 8 (R) 9 -R 11 Optional replacement of C5-C 18 Aliphatic or optionally substituted C1-C 14 Aliphatic-R 11 , where R 3 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 6 and R 7 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 10 Or optionally replace -C1-C 14 Aliphatic-R 10 ;where R 6 and R 7 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 8 and R 9 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 11 Or optionally replace -C1-C 14 Aliphatic-R 11 ;where R 8 and R 9 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkylene group, phenyl group, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; and Each R 10 and R 11 Independently or optionally, cyclic, double-cyclic, bridged double-cyclic, multi-cyclic, or bridged multi-cyclic C4-C 14 Cycloalkyl, or optionally substituted cyclic, bicyclic, bridged bicyclic, polycyclic, or bridged polycyclic 4-14 membered heterocyclic groups, or two R groups. 10 Or two Rs 11 Together they form optional bridging double or multi-ring C4-C 14 Cycloalkyl, or optionally substituted bridging bicyclic or polycyclic 4-14 membered heterocyclic groups; Where X 1 X A X 2 X 3 X 4 X 5 X 6 X 7 R 2 and R 3 One or more of them may be optionally and independently substituted by one or more substituents selected from -F, -Cl, -Br and -I.

15. The transfer medium of claim 14, wherein the ionizable lipid has the formula AX-T': ， Equation (AX-T') Or its pharmaceutically acceptable salt, wherein: R 1 Selected from -OH, -OAc, -NR2, , , , , , , , , , , and ; Each R is independently -H or C1-C6 aliphatic; X 1 and X A Each is an independent C1-C6 aliphatic compound with either a bond or optional substitution; X 2 and X 3 Each is an independent bond or optionally a C1-C substituted bond. 12 aliphatic; X 4 and X 5 Each is an optional substitution of C1-C6 aliphatic molecules; R 6 and R 7 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 10 Or optionally replace -C1-C 14 Aliphatic-R 10 ;where R 6 and R 7 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. R 8 and R 9 Each is independently and arbitrarily replaced -C1-C 14 Aliphatic, -R 11 Or optionally replace -C1-C 14 Aliphatic-R 11 ;where R 8 and R 9 One or more methylene bonds are each optionally and independently replaced by a C3-C8 cycloalkylene group, phenyl group, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; and Each R 10 and R 11 Independently or optionally, cyclic, double-cyclic, bridged double-cyclic, multi-cyclic, or bridged multi-cyclic C4-C 14 Cycloalkyl, or optionally substituted cyclic, bicyclic, bridged bicyclic, polycyclic, or bridged polycyclic 4-14 membered heterocyclic groups, or two R groups. 10 Or two Rs 11 Together they form optional bridging double or multi-ring C4-C 14 Cycloalkyl, or optionally substituted bridging bicyclic or polycyclic 4-14-membered heterocyclic groups.

16. The transfer medium of claim 15, wherein the ionizable lipid is selected from: 、 、 、 、 、 、 、 、 、 、 、 and 。 17. The transfer medium according to any one of claims 13-16, further comprising RNA polynucleotides.

18. The transfer medium of claim 17, wherein the RNA polynucleotide is a circular RNA polynucleotide.

19. The transfer medium of any one of claims 13-18, further comprising additional PEG lipids, structural lipids, non-ionizable lipids, or combinations thereof.

20. The transfer medium of claim 19, wherein the transfer medium further comprises additional PEG lipids.

21. The transfer medium of claim 20, wherein the additional PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

22. The transfer medium of claim 19, wherein the transfer medium further comprises structural lipids.

23. The transfer medium of claim 22, wherein the structural lipid is selected from the group consisting of: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, campesterol, tomatine, ursolic acid and α-tocopherol.

24. The transfer medium of claim 19, wherein the transfer medium further comprises a non-ionizable lipid.

25. The transfer medium of claim 24, wherein the non-ionizable lipid is a phospholipid.

26. The transfer medium of claim 25, wherein the phospholipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline. 1,2-Diundecanoyl-sn-glycerol-3-phosphate choline (DPPC), 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-Di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-Oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-Hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-Diarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-di-phosphatidyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), (S)-2-ammonium-3-((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxy)oxy)propionate sodium salt (L-α-phosphatidylserine);Brain PS), myristoyl phosphatidylcholine (DMPC), myristoyl phosphatidyl ethanolamine (DMPE), myristoyl phosphatidylglycerol (DMPG), dioleoyl-phosphatidyl ethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-(phospho-L-serine) (DOPS), Cell Fusion Phospholipids (DPhPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dipalmitoylphosphatidylglycerol (DPPG), Dipalmitoylphosphatidylserine (DPPS), Distearylphosphatidylcholine (DSPC), Distearyl-phosphatidyl-ethanolamine (DSPE), Distearylphosphatidylethanolamine imidazole (DSPEI), 1,2-Diundecanoyl-sn-glycerol-phosphocholine (DUPC), Lecithinylcholine (EPC), 1,2-Dioleoyl-sn-glycerol-3-phosphate (18:1 PA; DOPA), Bis((S)-2-hydroxy-3-(oleoyloxy)propyl)ammonium phosphate (18:1 DMP; LBPA), 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1'-inositol) (DOPI; 18:1 PI), 1,2-distearyl-sn-glycerol-3-phosphate-L-serine (18:0 PS), 1,2-dilinoleyl-sn-glycerol-3-phosphate-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (16:0-18:1 PS);POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine (18:0 Lyso PS), sphingomyelin (egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamide octadecane-4-en-1-yl(2-(trimethylammonium)ethyl) phosphate), brain or porcine sphingomyelin (brain SM / (2S,3R,E)-3-hydroxy-2-stearoamide octadecane-4-en-1-yl (2-(trimethylammonium)ethyl) phosphate, milk or bovine sphingomyelin (milk SM / (2S,3R,E)-3-hydroxy-2-tetracosamidooctadecane-4-en-1-yl (2-(trimethylammonium)ethyl) phosphate), 28:0 SM (N-octadecanoyl-D-erythrodihydrosphingomyelin), 14:0 SM (N-myristoyl-D-erythrodihydrosphingomyelin), 16:1 SM (N-palmitoyl-D-erythrodihydrosphingomyelin), 12:0 dihydro SM (N-lauroyl-D-erythrodihydrosphingomyelin), Lyso SM (sphingomyelin), Lyso SM (sphingomyelin), Lyso SM (dihydro) (N-Ceramide dihydrosphingosine phosphate choline), 24:1 SM (N-Ceramide-D-erythrodihydrosphingosine phosphate choline), 24:0 SM (N-Teicosacyl-D-erythrodihydrosphingosine phosphate choline), 18:1 SM (N-Oleoyl-D-erythrodihydrosphingosine phosphate choline), 18:0 SM (N-Stearyl-D-erythrodihydrosphingosine phosphate choline), 17:0 SM (N-Heptadecanyl-D-erythrodihydrosphingosine phosphate choline), 16:0 SM (N-Palmitoyl-D-erythrodihydrosphingosine phosphate choline), 12:0 SM (N-Lauroyl-D-erythrodihydrosphingosine phosphate choline), 06:0 SM (N-Hexanoyl-D-erythrodihydrosphingosine phosphate choline), 02:0 SM (N-acetyl-D-erythrodihydrosphingosine phosphocholine), 3-O-methylLyso SM (3-O-methyl-Sphingosine phosphocholine), 3-O-methyl-N-methylLyso SM (3-O-methyl-N-methyl-Sphingosine phosphocholine), and 3-N-methylLyso SM (3-N-methyl-Sphingosine phosphocholine) sphingomyelin.

27. The transfer medium of claim 19, comprising: (a) Approximately 0.5 mol% to approximately 10 mol% of PEG lipids; (b) Structural lipids of approximately 0.5 mol% to approximately 50 mol%; (c) About 5 mol% to about 45 mol% of non-ionizable lipids (e.g., phospholipids); and (d) Approximately 30 mol% to approximately 60 mol% of ionizable lipids.

28. The transfer medium of claim 27, comprising: (a) Approximately 0.5 mol% to approximately 5 mol% of PEG lipids; (b) Structural lipids of approximately 20 mol% to approximately 45 mol% (c) About 5 mol% to about 15 mol% or about 35 mol% to about 45 mol% of non-ionizable lipids (e.g., phospholipids); (d) Ionizable lipids of about 30 mol% to about 40 mol% or about 45 mol% to about 55 mol%.

29. The transfer medium of claim 27 or claim 28, wherein the structural lipid is cholesterol, and the non-ionizable lipid is selected from DSPC and ESM.

30. The transfer medium according to any one of claims 17-29, wherein the molar ratio (IL:P ratio) of the ionizable lipid to the number of phosphate groups in the RNA is about 2:1 to about 30:

1.

31. The transfer medium of claim 30, wherein the IL:P ratio is about 2:1 to about 8:

1.

32. A method for treating and / or preventing a condition in a subject in need, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a transfer medium as described in any one of claims 17-31.

33. The method of claim 32, wherein the condition is cancer.

34. The method of claim 33, wherein the cancer is selected from acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B-cell malignancies; bladder cancer (e.g., bladder epithelial carcinoma); bone cancer; brain cancer (e.g., medulloblastoma and glioblastoma multiforme); breast cancer; anal cancer, anal canal cancer, or anorectal cancer; eye cancer; intrahepatic bile duct cancer; joint cancer; neck cancer; gallbladder cancer; pleural cancer; nasal cancer, nasal cavity cancer, or middle ear cancer; oral cancer; vulvar cancer; chronic lymphocytic leukemia; chronic myeloid carcinoma; colon cancer; esophageal cancer, cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumors; head and neck cancer (e.g., squamous cell carcinoma of the head and neck). Cancer cells (including carcinoma of the ovarian cell line); Hodgkin's lymphoma; hypopharyngeal cancer; kidney cancer; laryngeal cancer; leukemia; fluid-filled tumors; lipomas; liver cancer; lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, and small cell lung cancer); lymphoma; mesothelioma; mast cell tumor; melanoma; multiple myeloma; nasopharyngeal carcinoma; non-Hodgkin's lymphoma; B-cell chronic lymphocytic leukemia; hairy cell leukemia; Burkitt lymphoma; ovarian cancer; pancreatic cancer; peritoneal cancer; omental cancer; mesenteric cancer; pharyngeal cancer; prostate cancer; rectal cancer; kidney cancer; skin cancer; small bowel cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; teratoma; testicular cancer; thyroid cancer; and ureteral cancer.

35. The method of claim 32, wherein the condition is an autoimmune disorder.

36. The method of claim 35, wherein the autoimmune disorder is selected from scleroderma, Graves' disease, Crohn's disease, Sjögren's syndrome, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrine syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveitis, polymyositis, colitis, thyroiditis, and systemic autoimmune diseases represented by lupus erythematosus.

37. The method of claim 36, wherein the autoimmune disorder is B cell mediated.

38. The method of claim 32, wherein the condition is hemoglobinopathies.

39. The method of claim 38, wherein the hemoglobinopathies are selected from sickle cell disease and β-thalassemia.

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