Fast-shedding polyethylene glycol lipids

Abstract

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

Description

FAST-SHEDDING POLYETHYLENE GLYCOL LIPIDSBACKGROUND

[0001] Polyethylene glycol (PEG) lipids have been incorporated into lipid nanoparticles for decades due to their ability to help control nanoparticle size and reduce immune cell interaction and clearance. It is known, however, that therapeutics including polyethylene glycol (PEGylated drugs) can elicit immune responses, including the production of anti-PEG antibodies, which can lead to accelerated blood clearance (ABC) and reduced or ablated efficacy upon repeated dosing. PEG lipids traditionally incorporated into lipid nanoparticles (LNPs) for gene delivery are thought to mainly elicit a thymusindependent (TI) immune response which does not lead to the formation of PEG-specific antibodies. However, with sufficient interaction of stably anchored PEG lipids with existing B cells, these B cells can produce anti-PEG IgM antibodies which can bind to LNPs and cause ABC upon subsequent repeat dosing.SUMMARY

[0002] The present disclosure provides PEG lipids and transfer vehicle compositions including the same. In some embodiments, incorporation of the PEG lipids of this disclosure into a transfer vehicle provides for one or more desirable properties, including low' immune response and / or immunogenicity (e.g., reduced antibody binding capacity and therefore reduced accelerated blood clearance (ABC)), fast-shedding of the PEG lipid from the transfer vehicle, and / or degradability of the PEG lipid (e.g., via ester hydrolysis or cleavage), as compared to a control PEG lipid (e.g., as described herein) or composition including the same.

[0003] The subject PEG lipid compounds can include at least one lipid, and a core moiety having a linking moiety that connects to a PEG moiety. In some embodiments, the subject compounds include a linker and an additional linking group connecting the core moiety to the PEG moiety and the one or more lipids. In some embodiments, the subject compounds include a branched core moiety.

[0004] In some embodiments, the transfer vehicles are lipid nanoparticles (LNPs). The present application also provides LNPs including a subject PEG lipid and an ionizable lipid. In some embodiments, the LNP includes a subject PEG lipid and a subject ionizable lipid (as described herein). Also provided herein are methods of preparing the subject PEG lipid compounds, and their use in various transfer vehicles, such as lipid nanoparticle formulations.

[0005] This disclosure provides PEG lipids, and transfer vehicle compositions including the same, where the PEG lipid is a compound of Formula (I):(A - ZA)n- B - YA- L - YB- P(I) wherein:YAIS selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH~, -NHC(O)O-, and - NHC(O)NH;P is a PEG moiety;L is an optional linker;Ydis an optional linking moiety selected from -OC(O)-, -C(O)O~, -OC(O)O~, -OC(O)NH-, - X'HCi ())()-. and -NHC(O)NH;A is a lipid;ZAis an optional linking moiety; and n is 1 or 2, wherein: when n is 2, B is a branched core moiety, and when n is 1, B is an optional linker.100061 In some embodiments of the compounds and compositions of this disclosure, the PEG lipid is a compound of Formula II:wherein:X1is O or NH;X2is a bond, O, or NH; andP is a PEG moiety;Ydis an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, ~OC(O)NH-, - X'HCi ())()-. and -NHC(O)NH;L is an optional linker; m is an integer selected from 1 to 10 (e.g., m is an integer selected from 1 to 8, 1 to 6, or 2 to 6).ZAis an optional linking moiety’; andA is a lipid.100071 In some embodiments of the compounds and compositions of this disclosure, the PEG lipid is a compound of Formula VA:wherein:P is a PEG moiety;YBIS 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;X2is a bond, O, or NH;X1is O or NH;B is a branched core moiety; each ZAis independently an optional linking moiety; and each A is independently a lipid.

[0008] In some embodiments of the compositions of this disclosure, the transfer vehicle further includes an ionizable lipid of Formula LIV:Formula (LIV) or a pharmaceutically acceptable salt thereof, wherein: n* is an integer selected from 1 to 7;Rais hydrogen or hydroxyl;Rhis hydrogen or Ci-Cs alkyl;R!is Ci-Cso alkyl or R!*;R:is Ci-Cso alkyl or R2*;R:and R2’ are independently selected from: ~(CH?)qC(O)O(CH2)rC(R8)(R9)(Ri0), -(CI12)qOC(O)(ad2.)rC(R8)(R9)(R!0), and -(CH2)qOC(O)O(CH2)rC(R8)(R9)(R,°); wherein: q is an integer selected from 0 to 12,r is an integer selected from 0 to 6, wherein at least one occurrence of r is not 0;R is I I or R .R9, R10, and R11are each independently G -CM alkyl or Cj-Czo-alkenyl; and wherein (i) R1is R!*. (ii) R2is R2*, or (iii) R1is R1* and R2is R2*.

[0009] In some embodiments of the compositions of this disclosure, the transfer vehicle further includes an ionizable lipid, and an RNA polynucleotide, such as a circular RNA polynucleotide.

[0010] In some embodiments of the compositions, the transfer vehicle is a lipid nanoparticle (LNP).BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depscts zeta potential measurements in millivolts (mV) for LNP formulations including ionizable lipid 204 (Table 3) and different PEG lipids from Table 1 or 2A.

[0012] FIG. 2 depicts 2-(p-toluidino)naphthalene-6-sulfonic acid TNS pKa assay curves for LNP formulations containing different PEG lipids from Tables 1 and 2A.

[0013] FIGs. 3A-3B show whole body luminescence expression in Balb / C mice 5 days post-dosing for 3 or 4 weeks of dosing with LNP encapsulating circular RNA that expresses firefly luciferase formulated with different combinations of ioni zable lipid from Table 3 and PEG lipid from Table 1 or2. In FIGs. 3A-3B, "‘Lipid A”, “Lipid B” and Lipid C” correspond to ionizable lipids 204, 138, and 160 of Table 3, respectively. FIG. 3A provides LNPs formulated with Lipid A. FIG. 3B provides LNPs formulated with Lipids B and C.

[0014] FIGs. 4A-4B shows change in body weight as a percentage of bodyweight for Balb / C mice dosed at 0.5 mg / kg of RNA for each of 4 weeks with LNP encapsulating circular RNA that expresses firefly luciferase formulated with different combinations of ionizable lipid from Table 3 and PEG lipid from Table 2A. In FIGs. 4A-4B, “Lipid B” and “Lipid C” correspond to ionizable lipids 138 and 160 of Table 3, respectively. FIG. 4B is continuation of FIG. 4A.

[0015] FIGs. 5A-5B shows measured anti-PEG IgM titers in Balb / C mice 5 days post-dosing for 3 or 4 weeks of dosing with LNP encapsulating circular RNA and formulated with different combinations of ionizable lipids from Table 3 and PEG lipids from Table 2A. PBS was used as a control. In FIGs. 5A-5B, “Lipid A'’, “Lipid B'’ and Lipid C” correspond to ionizable lipids 204, 138, and 160 of Table3, respectively. FIG. 5A provides LNPs formulated with Lipid A. FIG. 5B provides LNPs formulated with Lipids B and C.

[0016] FIG. 6 shows measured anti-PEG IgG titers in Balb / C mice 5 days post-dosing after 4 weekly doses of LNP containing circular RNA and formulated with different PEG lipids from Table 2A. PBS was used as a control.

[0017] FIGs. 7A-7B illustrate B cell depletion in the peripheral blood within Balb / C mice at 24 hours (FIG. 7A) and 72 hours (FIG. 7B) after treatment with LNP encapsulating circular RNA encoding aCD- 19 chimeric antigen receptor (CAR) protein formulated with different combinations of ionizable lipid and PEG lipid. PBS was used as control. In FIGs. 7A-7B, "‘Lipid B” and “Lipid C” correspond to ionizable lipids 138 and 160 of Table 3, respectively.|0018| FIGs. 8A-8E illustrate expression in various splenic cell types within Balb / C mice after treatment with LNP encapsulating circular RNA encoding a Wasabi fluorescent reporter protein formulated with different combinations of ionizable lipid from Table 3 and PEG lipid from Table 2A. The cell types shown in each of these figures include: CD45+ leukocytes (FIG. 8A), B220+ B cells (FIG. 8B), CD3+ T cells (FIG. 8C), CD1 lb+- myeloid cells (FIG. 8D), and NK-t natural killer cells (FIG. 8E). For FIGs. 8A-8E, PBS was used as a control. In FIGs. 8A-8E, “Lipid B” and “Lipid C” correspond to ionizable lipids 138 and 160 of Table 3, respectively.

[0019] FIGs. 9A-9E are graphs illustrating the results of dosing of BL / 6 mice with LNPs of the present disclosure comprising an hEPO oRNA payload. FIGs. 9A and 9C are bar graphs showing anti- PEG IgM expression m animals dosed with LN Ps comprising PEG lipids of the present disclosure as compared to LNPs comprising DMG-PEG2k and PBS controls, FIGs. 9B and 9D are bar graphs showing serum hEPO expression in animals dosed with LNPs comprising PEG lipids of the present disclosure as compared to LNPs comprising DMG-PEG2k and PBS controls. FIG. 9E is a bar graph showing the data of FIG. 9D, normalizing tire hEPO expression concentration of the LNPs comprising PEG lipids of the present disclosure against the LNP comprising DMG-PEG2k. For each of FIGs. 9A- 9E, in a given mouse group, the five bars represent measurements at week 1 , 2, 3, 4, and 5 from left to right.

[0020] FIGs. 10A-10C are bar graphs reporting the % of T cells expressing the CD19 CAR protein payload of the tested LNPs in peripheral blood, spleen and bone marrow samples, respectively, in humanized mice.|0021 | FIGs. 11A-11C are bar graphs reporting the B cell depletion in humanized mice after administration of LNPs comprising a CD 19 CAR protein payload m peripheral blood, spleen and bone marrow samples, respectively.DETAILED DESCRIPTION

[0022] The present application provides, among other things, PEG lipid compounds, and transfer vehicle formulations including the same. In some embodiments, the transfer vehicle is a lipid nanoparticle (LNP) that is useful for delivery of a polynucleotide. Provided are LNPs including a subject PEG and an ionizable lipid (e.g., as described herein). Hie subject lipid nanoparticles can include RNA polynucleotides, particularly circular RN A polynucleotides (aka circRNA or oRNA®).

[0023] In some embodiments, the subject PEG lipid compounds are fast-shedding, in that the entire PEG lipid is shed from the LNP without hydrolysis at the ester, as compared to one or more of the control lipids of Table 1.

[0024] In some embodiments, the subject PEG lipid compounds are fast -degrading, in that the PEG lipid is hydrolyzed faster at the ester, as compared to one or more of the control lipids of Table 1 .

[0025] In some embodiments, the subject PEG lipid compounds have better immune response and / or immunogenicity as compared to one or more of the control lipids of Table 1. In some embodiments, the subject PEG lipids exhibit one or more of lower anti-PEG response, lower anti-PEG immune response, and / or lower anti-PEG antibodies (e.g., lower anti-PEG IgM, and lower anti-PEG IgG in serum) as compared to one or more of the control lipids of Table 1 .

[0026] In some embodiments, the subject PEG lipid compounds provide for desirable or improved functional characteristics in a LNP formulation, as compared to one or more of the PEG lipids disclosed in one or more of the following International Pat. Pub, Nos. WO2022 / 173531, WO2018 / 232120, WO2018 / 232120, WO2022 / 169787, and WO2020 / 227510.

[0027] In some embodiments, the subject PEG lipid compounds can provide for desirable or improved functional characteristics in a LNP formulation, as compared to one or more of control PEG lipids, such as a PEG -lipid of Table 1.

[0028] Also disclosed herein is RNA therapy, along with associated compositions and methods. In some embodiments, the RNA therapy allows for increased RNA stability, expression, and prolonged half-life, among other things.

[0029] In some embodiments, provided herein are methods including administration of circular RNA polynucleotides provided herein into cells for therapy or production of useful proteins. In some embodiments, the method is advantageous in providing the production of a desired polypeptide inside eukaryotic cells with a longer half-life than linear RNA, due to the resistance of the circular RNA to ribonucleases.

[0030] Various aspects of the disclosure are described in detail in the following sections. The use of sections is not meant to limit the disclosure. Each section can apply to any aspect of the disclosure. In this application, the use of “or” means “and / or” unless stated otherwise.1. PEG LIPIDS

[0031] The present disclosure provides polyethylene glycol (PEG) lipid compounds.100321 In some embodiments, the PEG lipid compounds include at least one lipid attached to a core moiety having a linking moiety that connects to a PEG moiety. In some embodiments, the subject compounds include a core moiety that is a linear linker connecting the core moiety to the PEG moiety and the lipid moiety. In some embodiments, the subject compounds include a branched lipid connected to the core moiety. In some embodiments, the subject compounds include a branched core moiety to which two or more lipid moieties can be attached. The PEG lipid compounds can include one, two or more linking groups that are cleavable, e.g., susceptible to hydrolysis or enzymatic cleavage in vivo.

[0033] In some embodiments, the PEG lipid is a compound of Formula I:(A - ZA)n- B - YA- L - YB- P(I) wherein:YAis selected from ~OC(O)~, -C(O)O-, -OC(O)O~, ~OC(O)NH~, -NHC(O)O~, and - NHC(O)NH;P is a PEG moiety;L is an optional linker;YBis an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, - \l K (())()-. and -NHC(O)NH;A is a lipid;ZAis an optional linking moiety; and n is 1 or 2, wherein: when n is 2, B is a branched core moiety, andwhen n is 1, B is an optional linker.

[0034] In some embodiments of Formula I, n is 2 and B is a branched core moiety. In some embodiment, the core moiety is an optionally substituted branched C1 -C6 alkylene. In some embodiments, B includes an optionally substituted branched C3-C6 alkylene. In some embodiments, B includes an optionally substituted C3-alkylene. In some embodiments, B includes an optionally substituted C4-alkylene. In some embodiments, B includes an optionally substituted C5-alkylene. In some embodiments, B includes an optionally substituted C6-alkylene.100351 In some embodiments of Formula I, the branched core moiety B is of Formula B-I:(B-l) wherein W'-W3are each independently absent or a C1-C20 alkylene.

[0036] In some embodiments of Formula B-l, W!-W3are each independently absent or a C1-C6 alkylene. In some embodiments of Formula B-l , W‘-WJare each independently absent or a CI-C3 alkylene. In some embodiments of Formula B-l , W'-W5are each independently absent, or a C1-C2 alkylene. In some embodiments of Formula B-l, W’-W3are each independently absent or methylene.

[0037] In some embodiments of formula B-l, W'-W3are each absent, such that B is:

[0038] In some embodiments of formula B-l , two of W’-W3are absent, and the other is a C1-C20 alkylene, e.g., a. C1 -C6 alkylene, such as a C1 -alkylene, C2-alkylene, C3-alkylene, C4-alkylene, C5- alkylene, or C6-alkylene.

[0039] In some embodiments, B is selected from one of the following branched core moieties:

[0040] In some embodiments of formula B-l, one of W'-W’’ is absent, and the other two are C1-C20 alkylene, such as a Cl-alkylene, C2-alkylene, C3-alkylene, C4-alkylene, or a C5 -alkylene.

[0041] In some embodiments, B is selected from one of the following branched core moieties:

[0042] In some embodiments of formula B-l, W!-W5are each independently C1-C20 alkylene, e.g., a C1-C6 alkylene, such as a Cl-alkylene, C2 -alkylene, C3-alkylene, C4-alkylene, or a C-5 -alkylene.

[0043] In some embodiments, B is selected from one of the following branched core moieties:

[0044] 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 includes an optionally substituted linear C1-C6 alkylene. In some embodiments, B includes an optionally substituted linear C3-C6 alkylene. In some embodiments, B includes an optionally substituted linear C3-C6 alkylene. In some embodiments, B includes an optionally substituted linear C3-alkylene. In some embodiments, B includes an optionally substituted linear C4-alkylene. In some embodiments, B includes an optionally substituted linear C5 -alkylene. In some embodiments, B includes an optionally substituted linear Chalky lene.

[0045] In some embodiments of Formula I, YAis -OC(O)-. In some embodiments, ¥Ais -C(O)O~. In some embodiments, YAis -OC(O)O-. In some embodiments, YAis -OC(O)NH-. In some embodiments, YAis -NHC(O)O-. In some embodiments, YAis -NHC(O)NH-.

[0046] In some embodiments of Formula I, A, ZA, L, YBand P are as defined herein below,

[0047] In some embodiments of Formula I, n is 1 and the PEG lipid is a compound of Formula II:wherein:X is a bond, O orNH;X2is a bond, O, or NH; andP is a PEG moiety;YBis an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, - XH( (())()-. and -NHC(O)NH-;L is an optional linker; m is an integer selected from 1 to 10 (e.g., in is an integer selected from 1 to 8, 1 to 6, or 2 to 6);ZAis an optional linking moiety; andA is a lipid.

[0048] In some embodiments of Formula II, L or L-YBis present. In some embodiments, the linker (L) includes 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, L-Ybincludes a linking moiety that is -OC(O)-, -C(O)O-, or -OC(O)O-. In some embodiments, L-YBincludes a linking moiety that is - OC(O)NH~, -NHC(O)O-, or -NHC(O)NH-.

[0049] In some embodiments of Formula II, YBis absent. In some embodiments of Formula II, L or L-YBis absent.

[0050] In some embodiments of Formula II, L is selected from optionally substituted Cl -CIO alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-CI0 alkenylene, and optionally substituted C2-C10 alkynylene. In some embodiments of Formula II, L is optionally substituted Cl -CIO alkylene. In some embodiments of Formula II, L is optionally substituted C1-C6 alkylene. In some embodiments of Formula II, L is optionally substituted CI-C3 alkylene. In some embodiments of Formula II, L is methylene. In some embodiments of Formula II, L is ethylene.

[0051] In some embodiments of Formula II, X1is O. In some embodiments of Formula II, X!is NH.

[0052] In some embodiments of Formula II, X"1is (). In some embodiments of Formula II, X2is NH. In some embodiments of Formula II, X2is a bend.

[0053] In some embodiments of Formula II, X1is O, and X2is a bond. In some embodiments of Formula II, X1is O, and X2is NH. In some embodiments, X1and X2are each O. In some embodiments of Formula II, X1is a bond, and X2is O. In some embodiments of Formula II, X1is NH, and X"1is abond. In some embodiments of Formula II, X!is MH, and X2is O. In some embodiments, both X1, and X2are NH.

[0054] In some embodiments of Formula II, the compound is of Formula IIIA:wherein s is an integer from I to 10.100551 In some embodiments of Formula IIIA, s is from 1 to 8, such as from I to 6. In some embodiments of Formula IIIA, s is from 2 to 8. In some embodiments of Formula IIIA, s is from 3 to7. 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.

[0056] In some embodiments of Formula IIIA , A and ZAare as defined herein below.

[0057] In some embodiments of Formula IIIA, the compound is of Formula IIIA-1 :(IIIA- 1) wherein R2and IV are each independently CI-C15 alkyl or C2-C 15 -alkenyl.

[0058] In some embodiments of Formula IIIA or IIIA-1 , s is from 2 to 8, such as from 3 to 7. In some cases of Formula IIIA or IIIB, s is 2, 3, 4, 5, 6, 7, or 8.

[0059] In some embodiments of Formula IIIA- 1, R and R3are each independently Cl -Cl 5 alkyl, such as C1-C12 alkyl, or C4-C8 alkyl. In some embodiments, IV and R3are each independently a C4- alkyl chain. In some embodiments, R2and RJare each independently a C5 -alkyl chain. In some embodiments, R2and RJare each independently a C6-alkyl chain. In some embodiments, R2and R3are each independently a C7-alkyl chain. In some embodiments, Rzand R3are each independently a C8- alkyl chain. In some embodiments, R2and R3are each independently a C9-alkyl chain. In some embodiments, R2and R3are each independently a ClO-alkyl chain. In some embodiments, R2and R3are each independently a Cl I -alkyl chain. In some embodiments, R2and R5are each independently a C12-alkyl chain. In some embodiments, R:and R’ are each independently a CI3-alkyl chain. In someembodiments, R2and R3are each independently a C14-alkyl chain. In some embodiments, R2and R’ are each independently a Cl 5-alkyi chain.

[0060] In some embodiments of Formula IIIA-1 , R ' and R3are each independently C2-C15 alkenyl, such as C2-C12 alkenyl, or C4-C8 alkenyl. In some embodiments, R2and R’ are each independently a C4-alkenyl chain. In some embodiments, R2and R3are each independently a CS-alkenyl chain. In some embodiments, R2and R are each independently a C6-alkenyl chain. In some embodiments, R2and R3are each independently a C7-alkenyl chain. In some embodiments, R2and R’ are each independently a C8-alkenyl chain. In some embodiments, Rzand R’ are each independently a C9-alkenyl chain. In some embodiments, R2and R3are each independently a ClO-alkenyl chain. In some embodiments, R2and R3are each independently a Cl 1 -alkenyl chain. In some embodiments, R2and R3are each independently a CI2-aIkenyl chain. In some embodiments, R2and R3are each independently a CI3-alkenyl chain. In some embodiments, R2and R’ are each independently a C14-alkenyl chain. In some embodiments, R ’ and R3are each independently a C15-aikenyl chain.

[0061] In some embodiments of Formula IIIA-1, R2and R3are the same. In some embodiments of Formula IIIA-1, R2and R3are different.

[0062] In some embodiments of Formula II, the compound is of Formula IIIB:wherein r is an integer selected from 1 to 10 (e.g., r is an integer selected from 2 to 8, or from 1 to 6, or from 3 to 7, e.g., r is 1, 2, 3, 4, 5, 6 or 7).

[0063] In some embodiments of Formula II, the compound is of formula IIIC:wherein r is an integer selected from 1 to 10 (e.g., from 2 to 8, or from 1 to 6, or from 3 to 7, e.g., r is 1, 2, 3, 4, 5, 6 or 7).

[0064] In some embodiments of Formula IIIB and IIIC, r is an integer selected from 2 to 8, such as from 3 to 7. In some embodiments of Formula IIIB and IIIC, r is an integer selected from 1 to 6. In some embodiments of Formula IIIB and IIIC, r is I. In some embodiments of Formula IIIB and IIIC, ris 2. in some embodiments of Formula IIIB and IIIC, r is 3. in some embodiments of Formula IIIB and IIIC, r is 4. In some embodiments of Formula IIIB and IIIC, r is 5. In some embodiments of Formula IIIB and IIIC, r is 6. In some embodiments of Formula IIIB and IIIC, r is 7. In some embodiments of Formula IIIB and IIIC, r is 8.

[0065] In some embodiments of Formula II, the compound is of Formula HID:wherein q is an integer selected from 2 to 10.100661 In some embodiments of Formula HID, q is an integer selected from 2 to 8, such as from 2 to 6. In some embodiments of Formula HID, q is 2. In some embodiments of Formula HID, q is 3. In some embodiments of Formula HID, q is 4, In some embodiments of Formula HID, q is 5. In some embodiments of Formula HID, q is 6. In some embodiments of Formula HID, q is 7. In some embodiments of Formula HID, q is 8.

[0067] In some embodiments of Formula IIIA-IHD, ZAis absent; A is -CH(R2)(R3); and R2and R3are each independently C1-C15 alkyl, or C2 -Cl 5 -alkenyl,

[0068] In some embodiments of Formula HIA-IIID, A is -CH(R2)(R3), and R:and R3are each independently C1-C15 alkyl, such as C1-C12 alkyl, or C4-C8 alkyl. In some embodiments, R2and R’ are each independently a C4-alkyl chain. In some embodiments, R2and R3are each independently a C5-alkyl chain. In some embodiments, R3and R3are each independently a C6-alkyl chain. In some embodiments, R2and R3are each independently a C7-alkyl chain. In some embodiments, R2and R3are each independently a C8-alkyl chain. In some embodiments, R2and R3are each independently a C9- alkyl chain. In some embodiments, R2and R3are each independently a ClO-alkyl chain. In some embodiments, R2and R3are each independently a Cl 1 -alkyl chain. In some embodiments, R3and R3are each independently a C12-alkyl chain. In some embodiments, R3and R3are each independently a C13-alkyl chain. In some embodiments, R2and R3are each independently a C14-alkyl chain. In some embodiments, R2and R3are each independently a C15-alkyl chain.

[0069] In some embodiments of Formula IIIA-IHD, A is -CH(R2)(R3), and R3and R3are each independently C2-C15 alkenyl, such as C2-C12 alkenyl, or C4-C8 alkenyl. In some embodiments, R2and R3are each independently a C4-alkenyl chain. In some embodiments, R3and R’ are each independently a C5 -alkenyl chain. In some embodiments, R2and R3are each independently a C6- alkenyl chain. In some embodiments, R2and R3are each independently a C7~alkenyl chain. In some embodiments, R2and R3are each independently a C8-alkenyl chain. In some embodiments, R2and R3are each independently a C9-alkenyl chain. In some embodiments, R2and R3are each independently aClO-alkenyl chain. In some embodiments, R2and R3are each independently a Cl l-alkenyl chain. In some embodiments, R2and R.2are each independently a C12-alkenyl chain. In some embodiments, R2and R2are each independently a C13-alkenyl chain. In some embodiments, R2and R3are each independently a C14-alkenyl chain. In some embodiments, R2and R3are each independently a Cl 5- alkenyl chain.

[0070] In some embodiments of Formula III A-IIID, A is -CH(R2)(R2), and R2and R3are the same.In some embodiments of Formula IIIA-IIID, A is -CH(R2)(R3), and R2and R3are different.100711 In some embodiments of Formula II, L-YBis absent.

[0072] In some embodiments of Formula II, the compound is of Formula IVA:wherein t is an integer selected from 0 to 10 (e.g., t is an integer selected from 2 to 8 or from 2 to 6).

[0073] In some embodiments of Formula IVA, A and ZAare as defined herein below.

[0074] In some embodiments of Formula IVA, the compound is of Formula IVB:wherein:R2and R3are independently C1-C15 alkyl or C2-C 15 -alkenyl (e.g., C1-C12 alkyl, or C4-C8 alkyl).

[0075] In some embodiments of Formula IVB, R2and R3are each independently C1-C15 alkyl, such as C 1 -C 12 aakyl, or C4-C8 alkyl. In some embodiments, R2and R3are each independently a C4-alkyl chain. In some embodiments, R2and R’ are each independently a C5 -alkyl chain. In some embodiments, R2and R3are each independently a C6-alkyl chain. In some embodiments, R2and R3are each independently a C7-alkyl chain. In some embodiments, R2and R3are each independently a C8- alkyl chain. In some embodiments, R2and R3are each independently a C9-alkyl chain. In some embodiments, R2and R5are each independently a ClO-alkyl chain. In some embodiments, R2and R’ are each independently a C11-alkyl chain. In some embodiments, R2and R2are each independently a C12-alkyl chain. In some embodiments, R2and R2are each independently a C13-alkyl chain. In someembodiments, R2and R3are each independently a C14-alkyl chain. In some embodiments, R2and R’ are each independently a Cl 5-alkyl chain.

[0076] In some embodiments of Formula IVB, R2and R ' are each independently C2-C15 alkenyl, such as C2-C12 alkenyl, or C4-C8 alkenyl. In some embodiments, R2and R’ are each independently a C4-alkenyl chain. In some embodiments, R2and R3are each independently a C5-alkenyl chain. In some embodiments, R2and R are each independently a C6-alkenyl chain. In some embodiments, R2and R3are each independently a C7-alkenyl chain. In some embodiments, R2and R’ are each independently a C8-alkenyl chain. In some embodiments, R and R’ are each independently a C9-alkenyl chain. In some embodiments, R2and R3are each independently a ClO-alkenyl chain. In some embodiments, R2and R3are each independently a Cl 1 -alkenyl chain. In some embodiments, R2and R3are each independently a C12-alkenyl chain. In some embodiments, R2and R3are each independently a C13-alkenyl chain. In some embodiments, R2and R’ are each independently a C14-alkenyl chain. In some embodiments, R and R3are each independently a CI5-alkenyl chain.

[0077] In some embodiments of Formula IVA-IVB, R2and R3are the same. In some embodiments of Formula IVA-IVB, R2and R3are different.

[0078] In some embodiments of Formula IVA or IVB, t is an integer selected from 2 to 8, such as from 2 to 6, or from 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.

[0079] In some embodiments of any one of Formulae II, IIIA, IIIB, IVA, or IVB, m is an integer selected from I to 8, such as from 1 to 6, or from 2 to 6. In some embodiments of any one of Formulae 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.

[0080] In another aspect, provided herein is a compound of Formula VA:wherein:P is a PEG moiety;Y'3is an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -M K i O)O-. and -NHC(O)NH-;L is an optional linker;X2is a bond, O, MH, or CH2;X1is O or NH;B is a branched core moiety; each ZAis independently an optional linking moiety; and each A is independently a lipid.

[0081] In some embodiments of Formula VA, the compound is not:where r is an integer selected from 40 to 50;|0082| In some embodiments of Formula VA, B includes an optionally substituted branched Cl -C6 alkylene. In some embodiments, B includes an optionally substituted branched C3-C6 alkylene. In some embodiments, B includes an optionally substituted C3-a.lkylene. In some embodiments, B includes an optionally substituted C4-alkylene. In some embodiments, B includes an optionally substituted C5-alkylene. In some embodiments, B includes an optionally substituted C6-alkylene.

[0083] In some embodiments of Formula VA, the branched core moiety B is of Formula B-l:(B-l ) wherein W'-W3are each independently absent or a C1-C20 alkylene.

[0084] In some embodiments of Formula B-l, W!~W3are each independently absent or a C1-C6 alkylene. In some embodiments of Formula B-l , W‘-WJare each independently absent or a C1-C3 alkylene. In some embodiments of Formula B-l , W'-W5are each independently absent or a C1-C2 alkylene. In some embodiments of Formula B-l, W’-W3are each independently absent or methylene.

[0085] In some embodiments of B-l, two of W’-W3are absent, and the other is a C1-C20 alkylene, e.g., a C1-C6 alkylene, such as a C 1 -alkylene, C2 -alkylene, C3-alkylene, C4-alkylene, or a C5-alkylene.

[0086] In some embodiments, B includes the structure B-2:wherein a and c are independently 1, 2 or 3, and b is 0, 1, 2 or 3.

[0087] In some embodiments of formula 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.

[0088] In some embodiments, B-2 is selected from one of the following branched core moieties:

[0089] 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 .

[0090] In some embodiments of Formula B-2, a, b and c are 0 and B is:|00911 In some embodiments, B is selected from the following branched core moieties:

[0092] In some embodiments, B is selected from one of the following branched core moieties:

[0093] In some embodiments, the compound of Formula VA is of Formula VB:wherein: a and c are independently I, 2 or 3, and b is 0, I, 2 or 3; andL is selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene.

[0094] In some embodiments, the compound of Formula V A or VB includes a linking moiety ZAthat covalently ataches the lipid to the branch core moiety (B). As described herein, ZAis an optional moiety, and thus in some cases ZAis absent such that the one or more lipids (A) are covalently attached directly to the branched core moiety (B).

[0095] In some embodiments of Formula VA or VB, linking moiety ZAis an ether, ester, carbonate, amide, carbamate, urea, amine, anhydride or a disulfide group. In some embodiments, ZAis selected from -SS-, -C(O)OC(O)-, -NH-, -NHCONH-, -OCONH-, -NHCO-, -OC(O)O-, -OCO-, and -O-. In some embodiments, ZAis -SS-. In some embodiments, ZAis -C(O)OC(O)~. In some embodiments, ZAis -NH-. In some embodiments, ZAis -NHCONH-. In some embodiments, ZAis -OCONH-. In someembodiments, ZAis -NHCO-. In some embodiments, ZAis -OC(O)O-. In some embodiments, ZAis - OCO-. In some embodiments, ZAis -O-.

[0096] In some embodiments of Formula VA or VB, linking moiety ZAis selected from -CONH-, - NHCO-, -CO2-, -OCO-, -O-, OCO2-, -OCONH-, -NHCOO-, -OPO(O)O-, a maleimide-thiol conjugation, and a triazole. In some embodiments, ZAis -O-. In some embodiments, ZAis -CO2-. In some embodiments, ZAis -OCO-. In some embodiments, ZAis -OPO(O)O-.

[0097] In some embodiments of Formula VA or VB, L or L-Ybis present. In some embodiments, the linker (L) includes a linear or branched alkylene group. In some embodiments, the alkylene group includes from 1 to 10 carbon atoms. In some embodiments, the alkyl chain includes from 2 to 8 carbon atoms. In some embodiments, the alkyl chain includes from 3 to 7 carbon atoms. In some embodiments, L is selected from optionally substituted Cl -CIO alkylene, optionally substituted Cl -CIO heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene. In some embodiments, L-Ydincludes a linking moiety that is -OC(O)-, -C(O)O-, or - OC(O)O-. In some embodiments, L-Ybincludes a linking moiety that is -OC(O)NH~, -NHC(O)O-, or -NHC(O)NH.

[0098] In some embodiments of Formula VA or VB, L is absent. In some embodiments of Formula VA or VB, L-YBIS absent.

[0099] In some embodiments of Formula VB, the compound is of Formula VC:wherein:Xsis O orNH;Xzis a bond, NH, O, or CH?;R2, and R3are each independently C1-C15 alkyl, or C2.-C 15 -alkenyl (e.g., C8-C15 alkyl, or C10-C15 alkyl); s is an integer from 2 to 100 (e.g., from 20 to 50, from 30 to 50, or from 40 to 50); andR° is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a protecting group.

[0100] In some embodiments of Formula VA, VB, or VC, X!is O. In some embodiments of FormulaVA, VB. or VC, X1is NH.

[0101] In some embodiments of Formula VA, VB, or VC, X2is O. In some embodiments of Formula VA, VB, or VC, X2is NH. In some embodiments of Formula VA, VB, or VC, X2is a bond.

[0102] In some embodiments of Formula VC, R2and R3are each independently Cl-Cl 5 alkyl, such as C4-C15 alkyl, or C4-C8 alkyl. In some embodiments, R2and R3are each independently a C4-alkyl chain. In some embodiments, R2and R3are each independently a C5 -alkyl chain. In some embodiments, R2and R3are each independently a C6-aikyl chain. In some embodiments, R2and R3are each independently a C7-alkyl chain. In some embodiments, R2and R3are each independently a C8- alkyl chain. In some embodiments, R and R3are each independently a C9-alkyl chain. In some embodiments, R2and R3are each independently a ClO-alkyl chain. In some embodiments, R2and R3are each independently a Cl 1 -alkyl chain. In some embodiments, R2and R3are each independently a C12-alkyl chain. In some embodiments, R:and R3are each independently a C13-alkyl chain. In some embodiments, R2and R3are each independently a C14-alkyl chain. In some embodiments, R2and R3are each independently a C15-alkyl chain.

[0103] In some embodiments of Formula VC, R2and R3are each independently C2-C15 alkenyl, such as C4-C12 alkenyl, or C4-C8 alkenyl. In some embodiments, R2and R3are each independently a C4-alkenyl chain. In some embodiments, R2and R3are each independently a C5-alkenyl chain. In some embodiments, R2and R3are each independently a C6-alkenyl chain. In some embodiments, R2and R3are each independently a C7-alkenyl chain. In some embodiments, R2and R.3are each independently a C8-alkenyl chain. In some embodiments, R2and R3are each independently a C9-alkenyl chain. In some embodiments, R2and R3are each independently a ClO-alkenyl chain. In some embodiments, R2and R’ are each independently a C 11 -alkenyl chain. In some embodiments, R2and R3are each independently a C12-alkenyl chain. In some embodiments, R2and R3are each independently a C13-alkenyl chain. In some embodiments, R2and R3are each independently a C14-alkenyl chain. In some embodiments, R2and R3are each independently a C15-alkenyl chain.

[0104] In some embodiments of Formula VC, R2and R3are the same. In some embodiments of Formula VC, R2and R3are different.

[0105] In some embodiments of Formula VC, s is an integer selected from 2 to 80, such as from 10 to 80, from 20 to 80, from 20 to 70, from 20 to 60, from 20 to 50, from 30 to 50, or from 40 to 50. In some embodiments s is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0106] In some embodiments of Formula VC, Ruis hydrogen, optionally substituted alkyl, optionally substituted acyl, or a protecting group. In some embodiments, R° is hydrogen. In some embodiments, R° is optionally substituted C1-C6 alkyl. In some embodiments, R° is acyl. In some embodiments, R° is a protecting group. In some embodiments, Ruis a trialkylsilyl protecting group. In some embodiments, R° is a diarylalkylsilyl protecting group. In some embodiments, Ruis a tetrahydropyranyl protecting group. In some embodiments, R'Jis a benzyl protecting group.

[0107] As described herein, the compounds of Formulae VA-VC can have two lipid moieties (A).

[0108] In some embodiments of Formula VA or VB, A and ZAare as defined herein below.

[0109] In some embodiments, the PEG lipid is a compound of Formula (VI):wherein:P is a PEG moiety;YBis 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 optionally substituted Cl -CIO alkylene, optionally substituted Cl -CIO heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene;YAis 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; andR2and R3are each independently optionally substituted Cl -C l 5 alkyl or optionally substituted C2-C 15-alkenyl.

[0110] In some embodiments, the PEG lipid is a compound of Formula (VI):wherein:P is a PEG moiety of formulaz is an integer selected from 1 to 200;R is hydrogen or methyl;Ybis an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH~, - \H( (())()-. and -NHC(O)NH-;L is an optional linker selected from optionally substituted Cl -CIO alkylene, optionally substituted Cl -CIO heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C 2 -C 10 alkynylene ;YAis 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; andRf and R3are each independently optionally substituted C 1-C 15 aliphatic.

[0111] In some embodiments of Formula VI, YBis present and 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, YBis absent. In some embodiments of Formula VI, Ybis -OC(O)-. In some embodiments of Formula VI, YBis -C(O)O-. In some embodiments of Formula VI, YBis -OC(O)O-. In some embodiments of Formula VI, YBis -OC(O)NH~. In some embodiments of Formula VI, YBis -NHC(O)O-. In some embodiments of Formula VI, YBis -NHC(O)NH-.

[0112] In some embodiments of Formula VI, L is present and selected from optionally substituted Cl -CIO alkylene, optionally substituted Cl -CIO heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene. In some embodiments of Formula VI, L is absent. In some embodiments of Formula VI, L. is optionally substituted Cl-Cl 0 alkylene. In some embodiments of Formula VI, Lis optionally substituted C1 -C10 heteroalkylene. In some embodiments of Formula VI, L is optionally substituted C2-C10 alkenylene. In some embodiments of Formula VI, L is optionally substituted C2-C10 alkynylene. In some embodiments of Formula VI, L is optionally substituted C1-C9 alkylene. In some embodiments of Formula VI, L is optionally substituted C3-C9 alkylene. In some embodiments of Formula VI, Lis C1-C9 alkylene. In some embodiments ofFormula VI, L is C3-C9 alkylene. In some embodiments of Formula VI, L is --(CH2)-. In some embodiments of Formula VI, L is -(CFfY-. In some embodiments ofFormula VI, L is -(CB)?-. In some embodiments of Formula VI, L is -(CH?.) / !-. In some embodiments of Formula VI, L is -(CHz);-. In some embodiments of Formula VI, L is -(CILJe-. In some embodiments of Formula VI, L is -(CFL)?-. In some embodiments of Formula VI, L is ~(CH2)s-. In some embodiments of Formula VI, L is --(CFLIs-

[0113] In some embodiments of Formula VI, YAis selected from -OC(O)-, -C(O)O~, -OC(O)O-, - OC(O)NH-, -NIIC(O)O-, and -NHC(O)NH-. In some embodiments of Formula VI, YAis -OC(O)-. In some embodiments of Formula VI, YAis -C(O)O-. In some embodiments of Formula VI, YAis - OC(O)O-. In some embodiments of Formula VI, YAis -OC(O)NH-. In some embodiments of Formula VI, YAis -NHC(O)O-. In some embodiments of Formula VI, YAis -NHC(O)NH-.

[0114] 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 FormulaVI, 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.

[0115] In some embodiments of Formula VI, R2and R3are each independently optionally substituted C1-C15 aliphatic. In some embodiments of Formula VI, R ' and R’ are the same. In some embodiments of Formula VI, R2and R3are different.

[0116] In some embodiments of Formula VI, R2is optionally substituted Cl -Cl 5 aliphatic. In some embodiments of Formula VI, R2is optionally substituted Cl -CIS alkyl. In some embodiments of Formula VI, R2is optionally substituted C1-C15 branched alkyl. In some embodiments of Formula VI, R2is optionally substituted C1-C15 straight chain alkyl. In some embodiments of Formula VI, Rzis optionally substituted C2-C15 alkenyl. In some embodiments of Formula VI, R2is optionally substituted C2-C 15 branched alkenyl. In some embodiments of Formula VI, R2is optionally substituted C2-C15 straight chain alkenyl. In some embodiments of Formula VI, R2is optionally substituted C4- Cl l alkyl. In some embodiments of Formula VI, R2is optionally substituted straight chain C4-C11 alkyl. In some embodiments of Formula VI , R2is unsubstituted C4-CH alkyl. In some embodiments of Formula VI, R2is unsubstituted straight chain C4-C11 alkyl. In some embodiments of Formula VI, R2is optionally substituted C6-C11 alkyl. In some embodiments of Formula VI, R2' is optionally substituted straight chain C6-C11 alkyl. In some embodiments of Formula VI, R2is unsubstituted C6- C11 alkyl. In some embodiments of Formula VI, R2is unsubstituted straight chain C6-C11 alkyl. In some embodiments of Formula VI, R2is unsubstituted C5-C7 alkyl. In some embodiments of Formula VI, R2is unsubstituted straight chain C5-C7 alkyl. In some embodiments of Formula VI, R2is optionally substituted -(Q-fc^CHs. In some embodiments of Formula VI, R2is optionally substituted “(CH2)4CH3. In some embodiments of Formula VI, R2is optionally substituted --(CHzJsCHs. In some embodiments of Formula VI, R2is optionally substituted -(CHj^CHb. In some embodiments of Formula VI, R2is optionally substituted -(CHjJyCHs. In some embodiments of Formula VI, R2is optionally substituted --(CEhlsCHj. In some embodiments of Formula VI, R2is optionally substituted -(CH2)9CH3. In some embodiments of Formula VI, R2is optionally substituted -(CH2)!oCH3.

[0117] In some embodiments of Formula VI, R3is optionally substituted C1-CI5 aliphatic. In some embodiments of Formula VI, R3is optionally substituted CI-C-15 alkyl. In some embodiments of Formula VI, R3is optionally substituted CI-CI5 branched alkyl. In some embodiments of Formula VI, R3is optionally substituted C1-CI5 straight chain alkyl. In some embodiments of Formula VI, R2is optionally substituted C2-C15 alkenyl. In some embodiments of Formula VI, R2is optionally substituted C2-C15 branched alkenyl. In some embodiments of Formula VI, R2is optionally substitutedC2-C15 straight chain alkenyl. In some embodiments of Formula VI, R3is optionally substituted C4- Cl 1 alkyl. In some embodiments of Formula VI, R3is optionally substituted straight chain C4-C11 alkyl. In some embodiments of Formula VI, R3is unsubstituted C4-C1 1 alkyl. In some embodiments of Formula VI, R3is unsubstituted straight chain C4-C11 alkyl. In some embodiments of Formula VI, R3is optionally substituted C6-C11 alkyl. In some embodiments of Formula VI, IV is optionally substituted straight chain C6-C11 alkyl. In some embodiments of Formula VI, R3IS unsubstituted C6- Cl l alkyl. In some embodiments of Formula VI, R3is unsubstituted straight chain C6-C11 alkyl. In some embodiments of Formula VI, R’ is unsubstituted C5-C7 alkyl. In some embodiments of Formula VI, R3is unsubstituted straight chain C5-C7 alkyl. In some embodiments of Formula VI, R3is optionally substituted -(CFFVCHj. In some embodiments of Formula VI, RJis optionally substituted -(CHs^CHs. In some embodiments of Formula VI, R3is optionally substituted -(CI-L^CH?,. In some embodiments of Formula VI, R’ is optionally substituted --(CI-VFCHs. In some embodiments of Formula VI, R3is optionally substituted -(CHO / CHs. In some embodiments of Formula VI, IV is optionally substituted -(CH2)8CH3. In some embodiments of Formula VI, R3is optionally substituted -(CJHb^CHj. In some embodiments of Formula VI, Rzis optionally substituted “(CIVJioCH?.

[0118] In some embodiments of Formula VI, R2and R’ are each independently selected from

[0119] In certain embodiments, R2and R are each independently selected from

[0120] In certain embodiments.is selected fromR is hydrogen or methyl . In certain embodiments, P isIn certain embodiments, P

[0122] 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 of Formula VI is part of a composition comprising a plurality of compounds of Formula VI, wherein z is an integer from 30 to 75, and wherein the compounds of Formula VI in the composition have an approximately normal distribution of z within said range. In some embodiments, the compound of Formula VI is part of a composition comprising a plurality of compounds of Formula VI, wherein z is an integer from 35 to 50, and wherein the compounds of Formula VI in the composition have an approximately normal distribution of z within said range. In some embodiments, the compound of Formula VI is part of a composition comprising a plurality of compounds of Formula VI, wherein z is an integer from 40 to 50, and wherein the compounds of Formula VI in the composition have an approximately normal distribution of z around z = 45.

[0123] In some embodiments of Formula VI, P is a PEG moiety of formulawherein z is from 40 to 50 and R is hydrogen; YBand L are absent; YAis -OC(O)-; m is from 1 to 9; and R2and R3are each independently Cl -Cl 5 alkyl.

[0124] In some embodiments of Formula VI, P is a PEG moiety' of formulawherein z is from 40 to 50 and R is methyl; YBand L are absent; Yrtis -OC(O)-; m is from 1 to 9; and R2and R are each independently Cl -Cl 5 alkyl.[0125[ In some embodiments of Formula VI, P is a PEG moiety of formulawherein z is from 40 to 50 and R is hydrogen; YBis -C(O)O-; L is C1-C10 alkylene; YAis -OC(O)-; m is from 1 to 4; and R2and R~ are each independently Cl-Cl 5 alkyl.

[0126] In some embodiments of Formula VI, P is a PEG moiety of formulawherein z is from 40 to 50 and R is methyl; YBis -C(O)O~; L is Cl-Cl 0 alkylene; YAis -OC(O)-; m is from 1 to 4; and R2and R3are each independently Cl -Cl 5 alkyl.A. Lipid (A)

[0127] The PEG lipid compounds of Formula I, II, IIIA-IIID, I VA , VA-VB, can have A and ZAlinked lipid moieties as defined herein below.

[0128] Each lipid (A) can independently be selected from optionally substituted C6-C30 alkyl, optionally substituted C6-C30 alkenyl, and optionally substituted C6-C30 heteroalkyl. The lipid can be linear or branched, and unsubstituted or substituted.

[0129] In some embodiments, each lipid (A) independently includes a linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalky] aminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino,(aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl , alkyloxycarbonyl , aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,(alkylaminoalkyl)(alkyl)anrinocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, alkylsulfonealkyl, and phosphonate.

[0130] In some embodiments, at least one lipid (A) includes a C8-C30 linear or branched alkyl. In some embodiments, at least one lipid (A) includes a linear C8-C30 alkyl. In some embodiments of Formulae V A or VB, at least one lipid (A) includes decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, or icosane. In some embodiments, at least one lipid (A) includes a branched C8-C30 alkyl. In some embodiments, the branched C8-C30alkyl includes two lipid tails, wherein each lipid tail includes a C7-C12 alkyl chain. In some embodiments, each lipid tail includes a C 7 -alkyl chain. In some embodiments, each lipid tail includes a C8-alkyl chain. In some embodiments, each lipid tail includes a C9-alkyl chain. In some embodiments, each lipid tail includes a ClO-alkyl chain. In some embodiments, each lipid tail includes a Cl 1 -alkyl chain. In some embodiments, each lipid tail includes a C12-alkyl chain.

[0131] In some embodiments, at least one lipid (A) includes a C8-C30 linear or branched alkenyl. In some embodiments, at least one lipid (A) includes a linear C8-C30 alkenyl. In some embodiments of Formulae VA or VB, at least one lipid (A) includes myristoleane, palmitoleane, oleane, or iinoleane.

[0132] In some embodiments, at least one lipid (A or A-ZA-) is selected from:-Cl 0-C30 linear or branched alkyl;-Cl 0-C30 linear or branched alkenyl;-(CH2)qC(O)O(CH2)rCH(R1)(R2);-(CH2)qOC(O)(CH2)rCH(R1)(R2) ;-(CH2)qOC(O)O(CH2)rCH(R5)(R2) ;-(CH2)qOCH(R!)(R2);--(CH2)qOC(O)CH(Rl)(R2);-(CH2)qC(O)OCH(R,)(R2); and-(CH2)qOC(O)OCH(R5)(R2), wherein: q is an integer selected from 0 to 12, each r is independently an integer selected from 0 to 6;R1is hydrogen or R3; andR2, and R3are each independently Cl -Cl 2 alkyl, or C2-C12-alkenyl.

[0133] In some embodiments, at least one lipid (A) includes di-myristoyl-glycerol (DMG):

[0134] In some embodiments, at least one lipid (A) includes di-palmitoyl-glycerol (DPG):

[0135] In some embodiments, at least one lipid (A) includes di-stearoyl-glycerol (DSG):

[0136] In some embodiments, at least one lipid (A) includes a phospholipid. In some embodiments, at least one lipid (A) includes di-myristoyl-phosphatidyl-ethanolamine (DMPE):

[0137] In some embodiments, at least one lipid (A) includes di-palmitoyl-phosphatidyl-etlianolamine(DPPE):

[0138] In some embodiments, at least one lipid (A) includes di-stearoyl-phosphatidyl-ethanolamine(DSPE):

[0139] In some embodiments, at least one lipid (A) includes di-oleoyl-phosphatidyl-ethanolamine(DOPE):

[0140] In some embodiments, at least one lipid (A or A-ZA-) is selected from: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.

[0141] In some embodiments, at least one lipid (A or A-ZA-) is selected from:

[0142] In some embodiments, at least one lipid (A) is a sterol or cholesterol

[0143] In some embodiments, at least one lipid (A) is cholesterol:101441 In some embodiments, at least one lipid (A) is P-sitosterol:

[0145] In some embodiments, at least one lipid (A) is stigmasterol:

[0146] In some embodiments, at least one lipid (A) is lanosterol:

[0147] In some embodiments, at least one lipid (A) is 7-dehydrocholesterol:

[0148] In some embodiments, at least one lipid (A) is zymosterol:|0149| In some embodiments, at least one lipid (A) is lanosterol:

[0150] In some embodiments, at least one lipid (A) is brassicasterol:101511 In some embodiments, at least one lipid (A) is campesterol:B. Optional linking moiety ZA

[0152] In some embodiments, the PEG-lipid compound of Formula I, II, IIIA-IIID, IVA, VA-VB includes a linking moiety ZAthat covalently attaches a lipid to the compound. As described herein, ZAis an optional moiety, and thus in some cases ZAis absent such that the lipid (A) is covalently attached directly to the compound. It is understood that the exemplary linked lipid moieties described herein are depicted as A groups in some embodiments, and as A-ZA- groups in certain other embodiments.

[0153] In some embodiments, linking moiety ZAis an ether, ester, carbonate, amide, carbamate, urea, amine, anhydride or a disulfide group. In some embodiments, ZAis selected from -SS-, -C(O)OC(O)~, -NH-, -NHCONH-, -OC(O)NH-, -NHCO-, -OC(O)O~, -OC(O)-, and -O-. In some embodiments, ZAis -SS-. In some embodiments, ZAis -C(O)OC(O)-. In some embodiments, ZAis -NH-. In some embodiments, ZAis -NHCONH-. In some embodiments, ZAis -OCONH-. In some embodiments, ZAis -NHCO-. In some embodiments, ZAis -OC(O)O-. In some embodiments, ZAis -OCO-, In some embodiments, ZAis -O-.

[0154] In some embodiments, linking moiety ZAis selected from -CONH-, -NHCO-, -CO?.-, -OC(O)- , -O-, OCO2-, -OCONH-, -NHC(O)O-, -OPO(O)O-, a maleimide-thiol conjugation, and a triazole. In some embodiments, ZAis -O-. In some embodiments, ZAis -CO?-. In some embodiments, ZAis - OC(O)-. In some embodiments, ZAis -OPO(O)O-.C. PEG moiety P

[0155] In some embodiments, the PEG moiety (P) includes 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 includes 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, 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 22.0 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 embodiments, P includes 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, %- 10%.101561 In some embodiments, P includes a PEG block of 500 Da to 20 kDa, such as 5 OODa to 1 OkDa, 500 Da to 7kDa, 500Da to 5kDa, or 500Da to 2kDa. In some embodiments, P includes a PEG block of IkDa to 20kDa, such as IkDa to 17.5kDa, IkDa to l5kDa, IkDa to lOkDa, 5kDa to 20KDa, SkDa to 17.5ka, 5kDa to 15kDa, 5kDa to l OkDa, or 5kDa to 7.5kDa. In some embodiments, P includes a PEG block of 1.5kDa to 3.5kDa, such as 1.5kDa to 3kDa, 1.5kDa to 2.5kDa, or 1.5 to 2kDa. In some embodiments, P includes a PEG block of 500Da, IkDa, 1.5kDa, 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 7.5kDa, SkDa, 8.5kDa, 9kDa, 9.5kDa, l OkDa, 10.5kDa, HkDa, 11 . SkDa, 12kDa, 12.5kDa, 13kDa, 13. SkDa, 14kDa, 14. SkDa, 15kDa, 15.5kDa, 16kDa, I6.5kDa, 17kDa, 17.5kDa, 18kDa, I8.5kDa, 19kDa, 19.5kDa, or 20kDa + / - 10%.

[0157] In some embodiments, P includes one PEG block.

[0158] In some embodiments, P includes two or more PEG blocks.

[0159] In some embodiments, P includes the Formula Pl :P -T PE)U(Pl) wherein:PAand PBare each independently the two or more PEG blocks;T is an optional linking group; and u is an integer selected from 1 to 6.

[0160] In some embodiments of Formula Pl, 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.|0161 | In some embodiments of Formula Pl, PAand P3are the same PEG blocks. In some embodiments of Formula PI, PAand P3are different PEG blocks.

[0162] In some embodiments, Formula Pl does not include a linking group (T).

[0163] In some embodiments, Formula P l includes a linking group (T). Any suitable linking group can find use in the compound of FormulaPl . In some embodiments, T includes, atriazole, amaleimide, -S-, -NH-, -CO-, -CONH-, -NHCO-, -CO2-, -OCO-. -O-. OCO2-, -OCONH-. -NHCOO-, -OPO(O)O-, or any combination thereof.

[0164] In some embodiments. Formula PI includes a linking group (T) that is a cleavable linker. The cleavable linker can render the compound biodegradable. Any convenient cleavable linker can find use m the subject macrocycles including Formula Pl. In some embodiments, the cleavable linker can be cleaved by exposure to a stimulus. A non-exhaustive list of stimulus includes pH, temperature, light, redox change, over-expressed enzymes, hypoxia, sound, magnetic force, electrical energy, and anycombination thereof. In some embodiments, Formula Pl includes a linking group (T) is the cleavable linker including a group selected from disulfide, hydrazone, vinyl ether, imine, ortho ester, borate ester. amide, a peptide, an azo, and any combination thereof.[01651 In some embodiments, the PEG lipid compound is selected from:

[0166] In some embodiments, the PEG lipid compound is selected from:

[0168] In some embodiments, the PEG lipid compound is

[0169] In some embodiments, the PEG lipid compound is

[0170] In some embodiments, the PEG lipid compound is

[0171] In some embodiments, the PEG lipid compound is|0172| In some embodiments, the PEG lipid compound is

[0173] Exemplary' PEG lipid compounds of the present disclosure are shown in Table 2A.

[0175] In some embodiments of the compounds of 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 individual species of a compound of Table 2B having any above described value of “z” are to be understood as specifically contemplated herein as an exemplary compound of the present disclosure (E.g. Compound P131, wherein z = 45; P131 , wherein z = 44; P 131 , wherein z = 43;. ..etc.).2. TRANSFER VEHICLES

[0176] The present disclosure provides transfer vehicle formulations including a subject PEG lipid. A transfer vehicle is a particulate vehicle or carrier that can be used for encapsulation and / or delivery of a variety of therapeutic agents. A transfer vehicle can be a nanoparticle, micelle, liposome, emulsion and / or dendrimer. In general terras a PEG lipid can be useful as a component of such transfer vehicles, e.g., as a coating to protect the particles during drug deli very. In some embodiments, the transfer vehicle is lipid nanoparticle, solid lipid nanoparticle, nanostructured lipid carrier, polymeric nanoparticle, metal nanoparticle, nanogel.

[0177] In some embodiments, the transfer vehicle encapsulates a therapeutic agent selected from contrast agents, chemotherapy drugs, polynucleotides, and genes.|0178| Exemplary transfer vehicles are now described in greater detail.3. LIPOSOMAL FORMULATIONS

[0179] In some embodiments, the formulation includes a subject PEG-lipid in a liposomal formulation. In these embodiments, the molar ratio of the PEG-lipid is from about 0.1% to 100% of the total lipid. In some embodiments, the molar ratio of the 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 lipid. In some embodiments, the PEG-lipid is of Formula (I) (as described herein). In some embodiments, the PEG-lipid is of Formula (11) (as described herein). In some embodiments, the PEG- lipid is of Formula (III A-IIID) (as described herein). In some embodiments, the PEG-lipid is of Formula(1VA-IVB) (as described herein). In some embodiments, the PEG-lipid is of Formula (VA-VC) (as described herein). In some embodiments, the PEG-lipid is of Formula (VI) (as described herein).

[0180] In some embodiments, the liposomal formulation includes a helper lipid, a structural lipid, and a subject PEG-lipid. In some embodiments, the molar ratio of helper lipid: structural lipid: PEG- lipid in the liposomal formulation is about 56:38:5. In some embodiments, the molar ratio of helper lipid: structural lipid: PEG-lipid in the liposomal formulation is about 50:45:5. In some embodiments, the molar ratio of helper lipid: structural lipid: PECs -lipid in the liposomal formulation is about 45:45: 10. In some embodiments, the molar ratio of helper hpid:structural lipid: PEG-lipid in the liposomal formulation is about 60:38:2. In some embodiments, the molar ratio of helper lipid: structural lipid:PEG- lipid in the liposomal formulation is about 60:35:5. In some embodiments, the molar ratio of each of the helper lipid, structural lipid, and PEG-lipid is within 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% of the stated value.

[0181] In some embodiments of the liposomal formulation, the helper lipid is a phospholipid (e.g., as described herein). In some embodiments, the phospholipid is hydrogenated soy phosphatidylcholine (HSPC). In some embodiments of the liposomal formulation, the structure lipid is cholesterol. In some embodiments, the liposomal formulation includes a molar ratio of HSPC:cholesterol:PEG-lipid of about 56:38:5. In some embodiments, the molar ratio of each of HSPC, cholesterol, and the PEG-lipid is within 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 % of the stated value.4. LIPID NANOPARTICLE FORMULATIONS

[0182] In some embodiments, the present disclosure provides iipid nanoparticles (LNPs) including a subject PEG lipid, lire subject LNP formulation can also include one or more ionizable lipids, helper lipids, structural lipids, and polynucleotides (as described herein below). In some embodiments, an LNP further comprises a fifth lipid, or more additional lipids, in addition to the aforementioned lipid components. In some embodiments, an LNP delivery vehicle further comprises one or more targeting moieties covalently or non-covalently bound to the outer surface of the LNP delivery vehicle. In some embodiments, a targeting moiety is a moiety that binds to, or otherwise facilitates uptake by, cells of one or more specific cell populations.

[0183] The formation of a lipid nanoparticle (LNP) described herein may be accomplished by any methods known in the art. For example, as described in U.S. Pat. Pub. No. US2012 / 0178702 Al, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172-176; Jayaraman et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier etal. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0184] In one embodiment, the LNP formulation may be prepared by, e.g., the methods described in International Pat. Pub. No. WO 2011 / 127255 or WO 2008 / 103276, the contents of each of which are herein incorporated by reference in their entirety.

[0185] In one embodiment, LNP formulations described herein may include a polycationic composition. As a non-limiting example, the polycationic composition may be a composition selected from Formulae 1-60 of U.S. Pat. Pub. No. US2005 / 0222064 Al, the content of which is herein incorporated by reference in its entirety.

[0186] In one embodiment, the lipid nanoparticle may be formulated by the methods described in U.S. Pat. Pub. No. IJS2013 / 0156845 Al , and International Pat. Pub. No. WO2013 / 093648 A2 or WO2012 / 024526 A2, each of which is herein incorporated by reference in its entirety.

[0187] In one embodiment, the lipid nanoparticles described herein may be made m a sterile environment by the system and / or methods described in U.S. Pat. Pub. No. US 2013 / 0164400 Al , which is incorporated herein by reference in its entirely.

[0188] In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in U.S. Pat. No. 8,492,359, which is incorporated herein by reference in its entirety'.

[0189] A nanoparticle composition may optionally include one or more coatings. For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0190] In some embodiments, the lipid nanoparticles described herein may be synthesized using methods including microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to, a slit interdigital micromixer including, but not limited to, those manufactured by Precision Nanosystems (Vancouver, BC, Canada), Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (Zhigaltsev, l.V. et al. (2012) Langmuir. 28:3633-40; Belliveau, N.M. et al. Mol. Ther. Nucleic. Acids. (2012) l:e37; Chen, D. et al. J. Am. Chem. Soc. (2012) 134(22):6948-51; each of which is herein incorporated by reference in its entirety).

[0191] In some embodiments, methods of LNP generation including SHM, further include the mixing of at least two input streams wherein mixing occurs by microstructure -induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. Th is method may also include a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Pat. Pub. Nos.US2004 / 0262223 Al and US2012 / 0276209 Al, each of which is incorporated herein by reference in their entirety.

[0192] In one embodiment, the lipid nanoparticles may be formulated using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging -jet (IJMM)from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany). In one embodiment, the lipid nanoparticles are created using microfluidic technology (see, Whitesides (2006) Nature. 442: 368-373; and Abraham et al. (2002) Science. 295: 647-651; each of which is herein incorporated by reference in its entirety). As a nonlimiting example, controlled microti uidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. (2002) Science. 295: 647651 ; which is herein incorporated by reference in its entirety).

[0193] In one embodiment, the polynucleotide (e.g., circRNA) of the present disclosure may 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). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0194] In some embodiments, the LNP of tire present disclosure includes a molar ratio of between about 40% and about 60 % ionizable lipid, a molar ratio of between about 3.5% and about 14% helper lipid, a molar ratio of between about 28% and about 50% structural lipid, and a molar ratio of between about 0.5% and about 5% subject PEG-lipid, inclusive of all endpoints. In some embodiments, the total molar percentage of tire ionizable lipid, the helper lipid, the structural lipid, and the PEG-lipid is 100% in the LNP.

[0195] In some embodiments, the molar ratio of the ionizable lipid in the LNP is from about 40 to about 60% of the total lipid present in the LNP. In some embodiments, the molar ratio of the ionizable lipid 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 lipid present in the LNP. All values are inclusive of all endpoints.

[0196] In some embodiments, the molar ratio of the helper lipid in the LNP is from about 3.5% to about 14% of the total lipid present in the LNP. In some embodiments, the molar ratio of the helper lipid in the LNP is 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 lipid present in the LNP. In some embodiments, tire helper lipid is DSPC. In some embodiments, the helper lipid is DOPE. All values are inclusive of all endpoints.

[0197] In some embodiments, the molar ratio of the structural lipid in the LNP is from about 28% to about 50% of the total lipid present in the LNP. In some embodiments, the molar ratio of the structural lipid 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 tire total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol. All values are inclusive of all endpoints.|0198| In some embodiments, the molar ratio of the subject PEG-lipid in the LNP is from about 0. 1 % to about 5% of the total lipid present in tire 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 lipid present in the LNP. In some embodiments, the PEG-lipid is of Formula (I) (as described herein). In some embodiments, the PEG-lipid is of Formula (II) (as described herein). In some embodiments, the PEG-lipid is of Formula (III) (as described herein). In some embodiments, the PEG- lipid is of Formula (IV) (as described herein). In some embodiments, the PEG-lipid is of Formula (V) (as described herein). In some embodiments, the PEG-lipid is of Formula (VI) (as described herein). In some embodiments, the PEG-lipid is of Formula (VII) (as described herein). In some embodiments, the PEG-lipid is of Formula (VIII) (as described herein). In some embodiments, the PEG-lipid is of Formula (VI) (as described herein), . All values are inclusive of all endpoints.

[0199] In some embodiments, the molar ratio of ionizable lipid:helper lipid: structural lipid: PEG- lipid in the LNP is about 45:9:44:2. In some embodiments, the molar ratio of ionizable lipid:helper lipid structural lipid: PEG-lipid in the LNP is about 50: 10:38.5: 1.5. In some embodiments, the molar ratio of ionizable lipid:helper lipid structural lipid:PEG-lipid in the LNP is about 41 : 12:45:2. In some embodiments, the molar ratio of ionizable lipid:helper lipid: structural lipid:PEG-lipid in the LNP is about 62:4:33: 1. In some embodiments, the molar ratio of ionizable lipid:helper lipidstructural lipid: PEG-lipid in the LNP is about 53:5:41: 1. In some embodiments, the molar ratio of each of the ionizable lipid, helper lipid, structural lipid, and PEG-lipid is within 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% of the stated value.

[0200] In one embodiment, the lipid nanoparticles may have a diameter from 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 80nm, 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 30 to about 60 nm, about 30 to about 70 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 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm . In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm. In one embodiment, the lipid nanoparticle 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 the present disclosure.

[0201] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10- 500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.[0202[ In some embodiments, the lipid nanoparticles described herein can have a diameter from below 0 .1 pm to up to 1 mm such as, but not limited to, less than 0 .1 um, 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 um, 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, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 um, less than 700 um, less than 725 um, less than 750 um, less than 775 um, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, less than 975 pm.[0203[ In another embodiment, LNPs may have a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about I nm to about 60 nm, from about I nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and / or from about 70 to about 90 nm. Each possibility represents a separate embodiment of the present disclosure.102041 A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 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 a nanoparticle composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment of the present disclosure.[0205[ Hie zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition . Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably wi th cells, tissues, and oilier elements in the body. In some embodiments, the zeta potential of a nanoparticle composition maybe from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 m V to about +5 m V, from about -20 m V to about 0 m V, from about -20 mV to about -5 mV, from about -20 mV to about -10 mV, from about -20 mV to about -15 mV from about -20 mV to about -+20 mV, from about -20 mV to about + 15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 m V, from about -20 mV to about 0 mV, from about 0 mV to about +2.0 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +■5 mV to about +10 mV. Each possibility represents a separate embodiment of the present disclosure.

[0206] "Die efficiency of encapsulation of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. Hie encapsulation efficiency is desirably high (e.g., close to100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic agent in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic agent (e.g., nucleic acids) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent may be at least 50%, for example 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 may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. Each possibility represents a separate embodiment of the present disclosure. In some embodiments, the lipid nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200nm. 102071 The properties of a lipid nanoparticle formulation may be influenced by factors including, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the selection of the non-cationic lipid component, the degree of noncationic lipid saturation, the selection of the structural lipid component, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. As described herein, the purity of a subject PEG lipid component can also be important to an LNP’s properties and performance.

[0208] In one embodiment, a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO201 1 127255 or W02008103276, each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, which is herein incorporated by reference in its entirety'.

[0209] In some embodiments, circular RNA is formulated according to a process described in US patent application 15 / 809,680 (Published as US20180153822A1). In some embodiments, the present disclosure provides a process of encapsulating circular RNA in transfer vehicles including tire steps of forming lipids into pre-formed transfer vehicles (i.e., formed in the absence of RNA) and then combining the pre-formed transfer vehicles with RNA. In some embodiments, the novel formulation process results in an RNA formulation with higher potency (peptide or protein expression) and higher efficacy (improvement of a biologically relevant endpoint) both in vitro and in vivo with potentially better tolerability as compared to the same RNA formulation prepared without the step of preforming the lipid nanoparticles (e.g., combining the lipids directly wdth the RNA).|0210| For certain cationic lipid nanoparticle formulations of RNA, in order to achieve high encapsulation of RNA, the RNA in buffer (e.g., citrate buffer) has to be heated. In those processes or methods, the heating is required to occur before the formulation process (i.e., heating the separate components) as heating post-formulation (post-formation of nanoparticles) does not increase theencapsulation efficiency of the RNA in the lipid nanoparticles. In contrast, in some embodiments of the novel processes of the present disclosure, the order of heating of RNA does not appear to affect the RNA encapsulation percentage. In some embodiments, no heating (i.e., maintaining at ambient temperature) of one or more of the solutions including the pre-formed lipid nanoparticles, the solution including the RNA and the mixed solution including the lipid nanoparticle encapsulated RNA is required to occur before or after the formulation process,

[0211] RNA may be provided in a solution to be mixed with a lipid solution such that the RNA may be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain an RNA at a concentration of or greater than 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, a suitable RNA solution may contain an RNA at a concentration in a range 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. S3 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, 0.05-0.2 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, 0.4-0.7 mg / ml, or 0.5-0.6 mg / ml.

[0212] Typically, a suitable RNA solution may also contain a buffering agent and / or salt. Generally, buffering agents can include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate or sodium phosphate. In some embodiments, suitable concentration of the buffering agent may be in a 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.

[0213] Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an RNA solution may be in a range from 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.

[0214] In some embodiments, a suitable RN A solution may have a pH in a range from 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.

[0215] Various methods may be used to prepare an RNA solution suitable for the present disclosure. In some embodiments, RNA may be directly dissolved in a buffer solution described herein. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an RNA solutionmay be generated by mixing an RNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation.

[0216] According to the present disclosure, a lipid solution contains a mixture of lipids suitable to form transfer vehicles for encapsulation of RNA. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

[0217] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration in a range 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.

[0218] In some embodiments, an LNP of the present disclosure further comprises one or more additional PEG lipids disclosed and described in PCT Application Publication WO 2024 / 044728 Al, which is incorporated by reference herein, in its entirety. In some embodiments, the PEGylated lipid is a lipid of any one of formulas PL-T, PL-I”, PL. -I, PL-Ia, PL-Ib, PL-Iaa, PL-Iab, Pl .-lac. PL, -lad, PL- lae, PL-Iaf, PL -lag, 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-lib, PL-Iic, PL-Iid, PL-lj, PL-lk, L-Il, PL-Im, PL-In, PL-Io, PL-Ip, PL-Iq, PL-Ioa, PL-Iob, PL- loc, Pl ,-lod. Pl.-loe. PL-Iof, Pl. -log. PL-Ioh, PL-Ipa, Pl.-lpb. PL-Ipc, PL-Ipd, PL -Ipe, Pl ,-lpf Pl.-lpg. 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-Iyy, PL-Iyyy, PL-Iz, PL- Izz, PL-Izzz, PL-II’, PL-II”, PL-II. PL-IIc, PL-lId, PL-IIe. PL-IIf, PL-Ilg, PL-IIh, PL-IIa, PL-IIb, PL- Ilk, PL-IIm or PL-IIn.A. Ionizable lipids

[0219] In some embodiments, the lipid solutions and lipid nanoparticles disclosed herein include ionizable lipids. The subject ionizable lipids may be used as a component of a composition to facilitate encapsulation and release of nucleic acid cargo (e.g., circular RNA) to one or more target cells. In some embodiments, an ionizable lipid includes one or more cleavable functional groups (e.g., a disulfide) that allow, for example, a hydrophilic functional head-group to dissociate from a lipophilic functional tail- group of the compound (e.g., upon exposure to oxidative, reducing or acidic conditions).

[0220] In some embodiments, the ionizable lipid has a pKa from 6 to 12. In some embodiments, the ionizable lipid has a pKa from 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 ranges created by these.

[0221] In some embodiments, the ionizable lipid includes an amino group.

[0222] In some embodiments, the ionizable lipid includes a divalent headgroup and one or more straight hydrocarbon lipid tails. In some embodiments, the straight hydrocarbon lipid tails are from 3- 25 carbon atoms in length, such as 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 15, 10 to 20, or 10 to 25 carbon atoms in length.

[0223] In some embodiments, the ionizable lipid includes a divalent headgroup and one or more branched hydrocarbon lipid tails. In some embodiments, the branched hydrocarbon lipid tails are from 3-25 carbon atoms in length, such as 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 15, 10 to 20, or 10 to 25 carbon atoms in length.

[0224] In some embodiments, the divalent headgroup is selected from guanidine and squaramids

[0225] In some embodiments, the squaramide headgroup is of the following formula:Wherein R A and RB are each independently a C1-C6 alkyl group or H; and represents the point of attachment of the headgroup to a straight or branched hydrocarbon lipid tail.

[0226] In some embodiments, the ionizable lipid includes a head group selected from:wherein represents the point of attachment of the headgroup to a straight or branched hydrocarbon lipid tail.

[0227] In some embodiments, the ionizable lipid includes a head group of formula:integer selected from 1 to 7 (e.g., n is 1, 2, 3, 4, 5, 6 or 7).

[0228] In some embodiments, the ionizable lipid includes a head group selected from:wherein represents the point of attachment of the headgroup to a straight or branched hydrocarbon lipid tail.

[0229] In some embodiments, the ionizable lipid includes a hydrophilic headgroup as disclosed in Jayaraman et al. Angew. Chem. Int. Ed. (2012), 51, 8529-8533.

[0230] In some embodiments, the ionizable lipid is ethyl lauryl arginate (ELA). In some embodiments, the ionizable lipid is ionizable lipid I, wherein ionizable lipid 1 includes:

[0231] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (LI):Formula (LI) wherein: n is an integer selected from 1 and 4;Ra is hydrogen or hydroxyl; andRi and R?_ are each independently a linear or branched Cs-Cho alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkyl aminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,(alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl.

[0232] In some embodiments, Ra is hydrogen. In some embodiments, Ra is hydroxyl.

[0233] In some embodiments, the ionizable lipid is represented by Formula (Lla -1), Formula (Lla-2), or Formula (LIa-3):Formula (LIa-1) Formula (Lla -2) Formula (LIa-3).|0234| In some embodiments, the ionizable lipid is represented by Formula (LIb-1), Formula (Lib-2), or Formula (LIb-3):Formula (LIb-1 ) Formula (LIb-2) Formula (LIb-3).

[0235] In some embodiments, the ionizable lipid is represented by Formula (LIb-4), Formula (Lib-5), formula (LIb-6), Formula (LIb-7), Formula (LIb-8), or Formula (LIb-9):Formula (LIb-4) Formula (LIb-5) Formula (LIb-6)Formula (LIb-7) Formula (LIb-8) Formula (LIb-9).

[0236] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (LI), wherein Ri and R2 are each independently selected from:102371 In some embodiments, R1 and R2 are the same. In some embodiments, R-;and R2 are different.

[0238] In various embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (LI*):Formula (LI*) wherein: n* is an integer selected from 1 to 7,Rais hydrogen or hydroxyl,Rbis hydrogen or Ci-Cs alkyl,Ri and R2are each independently a linear or branched C1-C30 alkyl, C2-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl , alkylaminoalkyl, dialky laminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkyl carbonyl oxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxy carbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,(alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl.

[0239] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (LII):each n is independently an integer selected from 2 to 15;Li and L3 are each independently -OC(O)---* or -C(O)O--*, wherein indicates the attachment point to Ri or R3;Ri and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl.alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)ammoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl. aminoalkylaminocarbonyl. alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocaibonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; andR2 is selected from:

[0240] In some embodiments, the ionizable lipid is selected from an ionizable lipid of Formula LIT, wherein Ri and R3are each independently selected from:|0241| In some embodiments, Ri and Rs are the same. In some embodiments, RI and R3 are different.

[0242] In some embodiments, the one or more of the cationic or ionizable lipids are represented byFormula (LII-1) or Fonnula (LII-2):Formula (L.II-2).

[0243] In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2015 / 095340. In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2021 / 021634, WO2020 / 237227, or WO2019 / 236673. In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2021226597 and WO2021 1 13777. In some embodiments, the ionizable lipid is selected from an ionizable lipid of W02023056033. In some embodiments, the ionizable lipid is selected from an ionizable lipid ofWO2023081526.

[0244] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (LIII):or a pharmaceutically acceptable salt thereof, whereinL* is C2-C11 alkylene, C4-C10-alkenylene, or C1-C10-alkynylene;X’ is OR1, SR!, orN(R’)2, where R1is independently H or unsubstituted C1-C6 alkyl; andR2and R3are each independently C6-C30-alkyl, C6-C30-alkenyl, or C6-C30-alkynyl.

[0245] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (I.III*):or a pharmaceutically acceptable salt thereof, wherein:L1is C2-Cn alkylene, Q-Cio-alkenylene, or Ca-Cio-alkynylene;X‘ is OR1, SR1, or NCR1)?:, where R1is independently H or un substituted Cs-Ce alkyl; andR2and R?, are each independently a linear or branched C1-C30 alkyl, (V-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl , alkylammoalkyl , dialkylaminoalkyl, (heterocyclyi)(aikyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, ammo, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxy carbonyl , aikynylcarbonyloxy , alkynyl carbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,(alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. p246^ In some embodiments, an ionizable lipid is a compound of Formula (LIV):Formula (LIV) or is a pharmaceutically acceptable salt thereof wherein: n* is an integer selected from 1 to 7;Rais hydrogen or hydroxyl;R“ is hydrogen or Ci-Cg alkyl;R!is G-Go alkyl or R1*,R2is C1-C30 alkyl or R2*;R‘” and R2* are independently selected from:(( :H2)qC(O)O(CH )(Ri0),-(CH2)G.()C(O)(CH2)rC(R8)(R9)(R!0), and ~(CH2)qOC(O)O(CH2)rC(R8)(R9)(R,°); wherein: q is an integer selected from 0 to 12, r is an integer selected from 0 to 6, wherein at least one occurrence of r is not 0;Rsis H or R11;R9, R10, and Rnare each independently C1-C20 alkyl or C2-C2o-alkenyl; and wherein (i) R1is R‘\ (ii) R2is R2*, or (iii) R!is R‘* and R2is R2*.

[0247] In some embodiments of Formula (LTV), the ionizable lipid is of Formula (LIV-A):Formula (LIV-A) or a pharmaceutically acceptable salt thereof, wherein: n is an integer selected from 1 to 7; q and q’ are each independently integers selected from 0 to 12; r and r’ are each independently integers selected from 0 to 6, wherein at least one of r or r’ is not 0;ZAand ZBare each independently selected fromA-C(O)O~,A-OC(O), and -OC(O)O-; whereAdenotes the atachment point to -(CH?)q- orandR9A, R9B, R,OA. and R,0Bare each independently C1-C20 alkyl or C2-C20 alkenyl.

[0248] In some embodiments of Formula (LIV-A), ZAand ZBare bothA-C(O)O- whereAdenotes the attachment point to -(CHjjq- or -(CTfeX-.

[0249] In some embodiments, the ionizable lipid is any one of structures:

[0250] In some embodiments, ionizable lipid 138 is combined with PEG lipid Pl, P2, P3, or P9 and exhibits lower anti-PEG response as compared to ionizable lipid 138 combined with DMG PEG. See, e.g., Fig. 5.

[0251] In some embodiments, ionizable lipid 138 is combined with PEG lipid Pl, P2, or P9 and exhibits lower anti-PEG response as compared to ionizable lipid 160 combined with the same PEG lipid (e.g,, P1, P2, or PS)).

[0252] In some embodiments, ionizable lipid 160 is combined with PEG lipid P3 and exhibits comparative anti-PEG response as ionizable lipid 138 combined with PEG lipid P3.

[0253] In some embodiments, ionizable lipid 138 is combined with PEG lipid C18-PEG-OH and exhibits comparative anti-PEG response as ionizable lipid 160 combined with PEG lipid C 18-PEG-OH.

[0254] In some embodiments, an ionizable lipid of the present, disclosure is represented by Formula (LV):Formula (LV) or is a pharmaceutically acceptable salt thereof, wherein:Rais hydrogen or hydroxyl;R1is C1-C30 alkyl or R1’R2is C1-C30 alkyl or R2’;R‘* and R2’ are independently selected from:~(CH2)qC(O)O(CH2)rC(R4)(R5)(R6),-(CH2)qOC ;(O)(CH2)rC(R4)(R5)(R6), and“(CH2)qOC(O)O(CH2),.C(R4)(R5)(R6); wherein: q is an integer selected from 0 to 12, r is an integer selected from 0 to 6, wherein at least one occurrence of r is not 0;R4is hydrogen or R ';R5, R6, and R' are each independently C1-C20 alkyl or C2-C2o-alkenyl; wherein (i) R1is R4*, (ii) R2is R2*, or (iii) R1is R4* and R2is R2*; andR2is L-R’, wherein L is linear or branched CI-CJO alkylene, and R’ is (i) mono- or bicyclic heterocyclyl or heteroaryl, such as imidazolyl, pyrazolyl, 1,2,4-triazolyl, or benzimidazolyl, each optionally substituted at one or more available carbon and nitrogen by C1-C6 alkyl, or (ii) RA, RE, or RcwhereinRAis selected from:

[0255] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044343A1, which is incorporated by reference herein, in its entirety. In certain embodiments, the ionizable lipid is one of Formulae (VII-A), (VIII-A), (X), or (X- A) of PCT Application Publication WO2023044343A1.

[0256] In some embodiments, an LNP ofthe present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044333A1, which is incorporated by reference herein, in its entirety. In certain embodiments, the ionizable lipid is one of Formulae (CY), (CY-I), (CY-IV), (CY- IV’), or (CY-VT) of PCT Application Publication WO2023044333A1 .

[0257] In some embodiments, an LNP ofthe present disclosure comprises an ionizable i i pi d disclosed in PCT Publication WO2023122752A1, which is incorporated by reference herein, in its entirety. In certain embodiments, the ionizable iipid is one of Formulae II, III, VI, VI’, VI”, VI VII, VIP, VII”, VIP”, VIII, VTIF, VIII”, VIIF”, IX, IX’, IX”, IX’”, X, X’, X”, X”’, XI, XI’, XI”, XI’”, XII, XIF, XII”, XIF”, XIII, XIII’, XIII”, XIII”’, XIV, XIV’, XIV”, XIV’”, XV, XV’, XV”, XV’”, XVI, XVI’, XVI”, XVI’”, XVII, XVIII, XVIII’, XIX, XX, or XXI of PCT Application Publication WO2023122752A1.

[0258] In some embodiments, an LNP ofthe present disclosure comprises an ionizable lipid disclosed in PCT Publication WO2023196931 Al, which is incorporated by reference herein, in its entirety. In certain embodiments, the ionizable lipid is one of Formulae (CX-I), (CX-i), or (CZ) of PCT Application Publication WO2023196931A 1 .

[0259] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. In some embodiments, the ionizable lipid is one of Formulae (S-I), (S-M), (AT), (AT-E’), (AT-F’”), (AT- M), (AT-bF), (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 WO2024192277A 1 .

[0260] In some embodiments, the present disclosure, provides compounds of Formula (AX"”)or a pharmaceutically acceptable salt thereof, wherein:A is selected from an optionally substituted bridged carbocyclic bicycle, bridged carbocyclic multicycle, bridged heterocyclic bicycle, or bridged heterocyclic multicycle; 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;R1is selected from -OH, -OAc, -NRs, -N(R)R:\each R is independently -H or C)-C& aliphatic; ach R:iis Cj-Cc aliphatic-OH; each X' and XAis independently a bond or optionally substituted Ci-Cs aliphatic; each Y4is independently selected fromR. and a bond; wherein the bond marked with an is attached to X1; each Xzand XJis independently a bond or optionally substituted CI-CJS aliphatic; each Yzand YJis independently selected fromR-U A / ~r f : wherein die bond marked with an is attached to X" or XJ; each X4and X’ is independently a bond or optionally substituted Cj-Cg aliphatic; each Y4and Y5is independ ently selected from a bond.RX. XX / z ; wherein the bond marked with an is attached to X" or X\ each X6and X7is indepen dently a bond or optionally substituted Ci-Cs aliphatic;R2is -CH(OR6)(()R7), -CH(SR6)(SR7), -CH(Re)(R7), -CF(R6)(R7), -R‘°, optionally substituted C5-CS8aliphatic, or optionally substituted CI-CM aliphatic-R1'', wherein one or more methylene linkages of Rzare each optionally and independently replaced with an optionally substituted Ch-Cg cycloalkylenyl, phenyl, -O-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each R3is independently ~CH(ORS)(ORS), -CH(SRs)(SRs), ~CH(RSXR9), -CF(R8)(R3), -R; ;, optionally substituted C5-C1? aliphatic, or optionally substituted Cj-Cw aliphatic-R", wherein one or more methylene linkages of R’ are each optionally and independently replaced with an optionally substituted C3-C& cycloalkylenyl, phenyl, -O-. -S-, -SS-, -C(O)-, -OC(O)O-. -OC(O)-, -NHC(O)-. or -0'0)0-;R° and R; are each independently optionally substituted -Cj -Cu aliphatic, -R!u, or optionally substituted -CI-CM aliphatic-R'1'; wherein one or more methylene linkages of R° and R' are each optionally and independently replaced with an optionally substituted C?t~Cs cycloalkylenyl, phenyl, - O-, -NH-, -S-. -SS-. -C(O)-, -OC(O)O~, -OC(())-, -NHC(O)-. or -C(O)O-;Rsand R9are each independently optionally substituted -C1-C14 aliphatic, -R1or optionally- substituted -C;-Ci4 aliphatic-R1wherein one or more methylene linkages of R* and R' are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-. -SS-. -C(O)-, -OC(O)O -, -OCR)}.. -NHC(O)-, or -t (OK)-. and each R’uand R“ is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicychc, or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic, or bridged multicyclic 4-14 membered beterocyclyl, or two R,uor two R" taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally- substituted bridged bicyclic or multicyclic 4-14 membered beterocyclyl; wherein one or more of X’ , XA, X2, X\ X4, XJ, X°, X', Rz, and R-’ is optionally and independently substituted with one or more substituents selected from -F, -Cl, -Br and -I iments, A is selected from:

[0262] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid of Formula (AX)(AX), or a pharmaceutically acceptable salt thereof, wherein:A is selected from an optionally substituted bridged carbocyclic or heterocyclic core selected from the group consisting of:n is and integer selected from 1 or 2;R1is selected from the group consisting of -OH, -OAc, -NR?,each R is independently -H or C1-C6 aliphatic;X1and XAare each independently a bond or optionally substituted C1-C6 aliphatic;Y1is selected from the group consisting ofR, and bond; wherein the bond marked with an is attached to X!;:ach X2and X2is independently a bond or optionally substituted C1-C12 aliphatic;:ach Y2and Y2is independently selected from the group consisting ofR; wherein the bond marked with an is attached to X‘ or X ; each X4and X5is independently optionally substituted C1-C6 aliphatic;R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R°)(R7), -R’°, optionally substituted Cs-Cis aliphatic, or optionally substituted Ci-Cu aliphatic-R.10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted Cg-Cg cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each R3is independently -CH(OR8)(ORy), -CH(SR8)(SRy), -CH(RS)(R9), -Rn, optionally substituted Cs-Cig aliphatic, or optionally substituted Ci-C« aliphatic-R11, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted Cg-Cg cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-;R° and R7are each independently optionally substituted -Ci-Cu aliphatic, -R’°, or optionally substituted -C1-C14 aliphatic-Rlu; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted Cg-Cg cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-;R8and Ryare each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R6and R9are each optionally and independently replaced with an optionally substituted Cg-Cg cycloalkylenyl, phenyl, - O-, -Nil-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each R10and R" is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two Rluor two R11taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.

[0263] The present disclosure, m some embodiments, provides compounds of any one of theFormulae below:(AX-F),(AX-K’), (AX-K”),(AX-M),(AX-R),(AX-Y),(AX-AA’), (AX-AA”),(AX-AD),(AX-AF’), (AX-AF”),(AX-AL), (AX-AU),(AX-AO), (AX-AO’) or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, XA, X2, X’. X4, X5, Y;, Y2, YJ, R3, R6, R7, Rs, R9, R!0, and R11are as described in Formula (AX) or as otherwise described in any embodiments below.

[0264] In some embodiments, the ionizable lipid is selected from a lipid of Table 3:Table 3: Exemplary Ionizable Lipids001911201C£3B. Helper lipids

[0265] In some embodiments, the lipid solutions and lipid nanoparticles described herein includes one or more non-cationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or replacement. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids include a phospholipid moiety and one or more fatty acid moieties.

[0266] A phospholipid moiety' can be selected, for example, from the non-limiting group of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0267] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behemc acid, docosapentaenoic acid, and docosahexaenoic acid.

[0268] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0269] In some embodiments, the helper lipid is a l,2-distearoyl-177-glycero-3-phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0270] In some embodiments, a helper lipid is a non -phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a DSPC substitute.

[0271] In some embodiments, a helper lipid is described in PCT / US2018 / 053569. Helper lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwiterionic lipids. Such helper lipids are preferably used in combination with one or more of the compounds and lipids disclosed herein. Examples of helper lipids include, but are not limited to, 5-heptadecylbenzene-l,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoylsn-glycero-3- phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1 -myristoyl-2- palmitoyl phosphatidylcholine (MPPC), 1 -paimitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1 -stearoyl -2 -palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), paimitoyioieoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanol amine (DOPE) dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC) or dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC). Helper lipids function to stabilize and improve processing of the lipid solutions and lipid nanoparticles. Such helper lipids are preferably used in combination with other excipients, for example, one or more of the ionizable lipids disclosed herein.

[0272] In some embodiments, the LNP comprises a phospholipid selected from the non-limiting group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl- sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl~sn~glycero~3-phosphocholine (DOPC), 1 ,2- dipalmitoyl -sn-gly cero-3 -phosphochol ine (DPPC), 1 ,2 -di undecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoy 1-2 -oleoyl-sn-gly cero-3 -phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholme (18:0 Diether PC), 1 -oleoyl -2 -cholesterylhemisuc cinoy 1-sn-gly cero-3 - phosphocholine (OChemsPC), 1 ~hexadecyl~sn~glycero~3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, l ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoiyl)oxy)propanoate (L-a-phosphatidylserine;Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanol amine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG),1.2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1 ,2-Dielaidoyl-sn-phospbatidylefhanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2 -diol eoyl-sn-glycero-3 -phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), l,2-dioleoyl-sn-glycero-3- phospho-(l ' -myo-inositol) (DOPI; 18: 1 PI), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS),1.2-dilinoleoyl -sn-glycero-3-phospho-L-serine (18:2 PS), 1 -palmitoyl -2-oleoyl -sn-glycero-3 -phospho- L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), 1- stearoyl-2-linoleoyl-sn-glycero-3 -phospho-L-serine ( 18 : 0- 18 : 2 PS), 1 -oleoyl-2 -hydroxy-sn-glycero-3- phospho-L-serine (18: 1 Lyso PS), l-stearoyl-2-hydroxy-sn-gly cero-3 -phospho-L-serine (18:0 Lyso PS), and sphingomyelin. In some embodiments, an LNP comprises DSPC. In certain embodiments, an LNP comprises DOPE. In some embodiments, an LNP comprises both DSPC and DOPE.102731 In some embodiments, an LNP comprises a phospholipid selected from 1 -pentadecanoyl-2- oleoyl-sn-glycero-3 -phosphocholine, 1 -myristoyl-2-palmitoyl-sn-gly cero-3 -phosphocholine, 1 - myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1 -palmitoyl -2 -oleoyl-glycero-3 -phosphocholine, l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, I -palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine, 1 -palmitoyl -2 -docosahexaenoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, l-stearoyl-2-palmitoyl-sn-glycero-3- phosphocholine, l-stearoyI-2-oleoyI-sn-glycero-3-phosphocho!ine, l-stearoyl-2-linoleoyl-sn-glycero- 3 -phosphocholine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, l-stearoyl-2- docosahexaenoyl-sn-glycero-3-phosphocholine, 1 -oleoyl -2 -myristoyl-sn-gly cero-3 -phosphocholine, 1 -oleoyl-2 -palmitoyl-sn-glycero-3-phosphocholine, l-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo- inositol-31,4' -bisphosphate), l,2-dioleoyl-sn-glycero-3-phospho-( 1 ' -myo-inositol-3s,5 ' - bisphosphate), l,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol-41,5 -' -bisphosphate), 1,2- dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol-3f,4' ,51-trisphosphate), l,2-dioleoyl-sn-glycero-3~ phospho-(l ' -myo-inositol-31-phosphate), 1 ,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol-4f-phosphate), l,2-dioleoyl-sn-glycero-3-phospho-(l ’ -myo-inositol-5 ' -phosphate), 1,2-dioleoyl-sn- g!ycero-3-phospho-(l ’ -myo-inositol), l,2-dioleoy]-sn-glycero-3-phospho-L-serine, and 1-(8Z- octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine.|0274| In some embodiments, the LNP comprises a phospholipid selected from DSPS (Distearoylphosphatidylserine), DSPG (l,2-distearoyl-sn-glycero-3-phospho-(l ■' -rac -glycerol)), DSPA (1 ,2-Distearoyl-sn-glycero-3-phosphate), diPhyPC (l,2-diphytanoyl-sn-glycero-3- phosphocholine), diPhy -diether-PC (l,2-di-O-phytanyl-sn-glycero-3-phosphocholine), diPhyPE (1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine), diPhy -diether-PE (l,2-di-O-phytanyl-sn-glycero-3- phosphoethanolamine), diPhyPS (1 ,2~diphytanoyl-sn-glycero-3~phospho-L-serine), diPhyPG (1,2- diphytanoy1-sn-glycero-3-phospho-(l ' -rac-glycerol)), diPhyPA (1 ,2-diphytanoyl-sn-glycero-3~ phosphate), Egg PA (L-a-phosphatidic acid), and Soy PA (L-a-phosphatidic acid).

[0275] In some embodiments, the LNP comprises a phospholipid selected from 18: 1 (A9-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 (A9-Trans) PE (DEPE), 16: 1 PE (DPPE), and CL. In certain embodiments, the LNP comprises a phospholipid described or disclosed in Alvarez-Benedicto, et al. (Biomater. Sci., 202.2, 10, 549) and Li, et al. (Asian Journal of Pharmaceutical Sciences, 2015, 10, 81-98).

[0276] In certain embodiments, the LNP comprises a sphingoid lipid or sphingolipid, such as, but not limited to sphingomyelin. As used herein, the terms ‘‘sphingoid lipid” and “sphingolipid” are meant to refer to a class of lipids containing a backbone comprising a sphingoid base. An exemplary' sphingoid base is sphingosine. In certain embodiments, the LNP comprises a sphingolipid selected from Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamidooctadec-4-en-l-yl (2- (trimethylammonio)ethyl) phosphate), Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3- hydroxy~2-stearamidooctadec-4~en-l~yl (2-(trimethylammonio)ethyl) phosphate). Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3-hydroxy-2-t.ricosanamidooctadec-4-en-l-yl (2- (trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro- sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D-erythro-sphingosylphosphorylcholine), 16: 1 SM (N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D- erylhro-sphinganylphosphoryicholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24: 1 SM (N- nervonoyl-D-erythro-sphingosylphosphorylcholine), 24:0 SM (N-hgnoceroyl-D-erythro- sphingosylphosphorylcholme), 18: 1 SM (N-oleoyl-D-erythro-sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palnritoyl-D-erythro-sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erythro-sphingosylphosphorylcholine), 06:0 SM (N-hexanoyl-D-erythro-sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D-erythro-sphingosylphosphorylcholme), 3-O- methyl Lyso SM (3-O-methyl-spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O- methyl-N-methyl-spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphorylcholine).

[0277] In some embodiments, the LNP comprises a phospholipid comprising at least one constrained tail, such as those described by Gan, et al. (Bioeng Trans! Med. 2020 Sep; 5(3): e 10161 .). In certain embodiments, the phospholipid is one selected from:

[0278] In some embodiments, the LNP comprises a phospholipid comprising a ceramide analogue having a triazole linkage, such as those described by Kim et al., Bioorg. Med. Chem. Lett., 17(16), 2007, 4584-4587.

[0279] In some embodiments, the LNP comprises a phospholipid disclosed in WO 2023 / 141470, which is incorporated by reference herein, in its entirety. In certain embodiments, the phospholipid is

[0280] In some embodiments, the LNP comprises a phospholipid disclosed in WO 2022 / 040641, which is incorporated by reference herein, in its entirety.|0281 | In some embodiments, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S. Application Publication 2021 / 0121411, which is incorporated herein by reference.

[0282] In some embodiments, the LNP comprises a phospholipid disclosed in one of US 2019 / 0240354; US 2.010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 202.1 / 012.8488; US 2020 / 0121809; US 2017 / 0119904; US 2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US 2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095AI; WO 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO 2019 / 089828A1, each of which is incorporated by reference herein in their entirety.

[0283] In some embodiments, the LNP comprises a phospholipid disclosed in PCT Publication WO2023141470A2, which is incorporated by reference herein in its entirety. In certain embodiments, the LNP comprises a phospholipid of Formula (I) of PCT Publication WO2023141470 A2, including but not limited to 2-ammonioethyl ((S)-3-(((S)-12-methyltetradecanoyl)oxy)-2-((13- methy Itetradecanoy l)oxy )propyl) phosphate .

[0284] In some embodiments, phospholipids disclosed in US 2020 / 0121809 have the following structure:

[0285] wherein R1 and R2 are each independently a branched or straight, saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl).C. Structural lipids

[0286] In some embodiments, the lipid solutions and lipid nanoparticles described herein includes one or more structural lipids. Incorporation of structural lipid(s) in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof. In certain embodiments, structural lipids can be selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol. Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In some embodiments, a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

[0287] In some embodiments, a structural lipid is described in international patent application PCT / US2019 / 015913, which is incorporated by reference herein in its entirety.

[0288] In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid . In certain embodiments, the structural lipid is cholesterol. In certain embodiments, thestructural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alphatocopherol.

[0289] The lipid solutions and lipid nanoparticles described herein include one or more structural lipids. Incorporation of structural lipids in a transfer vehicle, e.g., a lipid nanoparticle, may help mitigate aggregation of other lipids in the particle. In some embodiments, tire structural lipid includes cholesterol, a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.|0290| In some embodiments, the structural lipid is a sterol. Structural lipids can include, but are not limited to, sterols (e.g., phytosterols or zoosterols).

[0291] In some embodiments, the structural lipid is a steroid. For example, sterols can include, but are not limited to, cholesterol, 0-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol.

[0292] In some embodiments, the lipid solutions and lipid nanoparticles include an effective amount of an immune cell delivery’ potentiating lipid, e.g., a cholesterol analog or an amino lipid or combination thereof, that, when present in a transfer vehicle, e.g., an lipid nanoparticle, may function by enhancing cellular association and / or uptake, internalization, intracellular trafficking and / or processing, and / or endosomal escape and / or may enhance recognition by and / or binding to immune cells, relative to a transfer vehicle lacking the immune cell delivery potentiating lipid. Accordingly, while not intending to be bound by any particular mechanism or theory'-, in one embodiment, a structural lipid or other immune cell delivery' potentiating lipid of the disclosure binds to Clq or promotes the binding of a transfer vehicle including such lipid to Clq. Thus, for in vitro use of the transfer vehicles of the disclosure for delivery' of a nucleic acid molecule to an immune cell, culture conditions that include Clq are used (e.g., use of culture media that includes serum or addition of exogenous Clq to serum- free media). For in vivo use of the transfer vehicles of the disclosure, the requirement for Clq is supplied by endogenous Clq.

[0293] In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is a lipid in Table 4 below:Table 4: Example Structural lipidsD. Additional lipid components|0294| In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional iipid components capable of influencing the tropism of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200 (see Cheng, et al. Nat Nanotechnol. 2020 April; 15(4): 313—320; Dillard, et al. PNAS 2021 Vol. 118 No. 52).

[0295] In some embodiments, an LNP of the present disclosure further comprises one or more additional ionizable lipids, such as, but not limited to those disclosed in one of US 2.023 / 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 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO 2019 / 089828A1 , each of which is incorporated by reference herein in theirentirety. In certain embodiments, an LNP of the present disclosure further comprises one or more additional ionizable lipids selected from those disclosed m WO2023044343A1 or WO2023044333A1, both of which are incorporated by reference herein in their entirety.102961 In some embodiments, the LNP compositions of the present disclosure comprise, or further comprise one or more lipids selected from l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcamosine (AC), 1- hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), N-oleoyl-sphingomyelin (SPM) (Cl 8:1), N- lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12- diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate ( DC P 1), 1 ,2-Dipalmitoylglycerol -3 -hemi succinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1 ,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2- dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)- ethoxy J -ethoxy } -ethoxy)-ethyl ] -3 -(3 ,4,5 -dihy droxy-6-hydroxymethyI- 1 etrahy dro-pyran-2-y isulfanyl)- propionamide (DOGP4o:Man), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidyl serine (DPPS), distearoylphosphatidylcholine (DSPC), di stearoyl - phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol~hemisuccinate-Na-Histidinyl- Hemisuccinate (HistSuccDG), N~(5?-hydroxy-3 ’ -oxypentyl)-! 0-12-pentacosadiynamide (h-Pegi- PCDA), 2-[l-hexyloxyethyl] -2 -de vinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-ce-histidinyl-Nce- hemisuccmate (IsohistsuccDG), mamiosialized dipalmitoylphosphatidylethanolamine (ManDOG), 1,2- Dioleoyl~sn~Glycero-3~Phosphoethanolamine-N-[4-(p~maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), l,2-diphytanoyl-sn-g]ycero-3-phosphoethanolamine (ME 16:0 PE), 1 -myristoyl -2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol -reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3~phosphoethanolamine~N~[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC 12-DOPE), 1 -oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoyiphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidyl serine (PS), lissamine rhodamine B- phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1 -trans-PE, 1 -stearoyl -2 -oleoyl -phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha~trehalose-6, 6f-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3~phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)tetrahydrofi.iran- 2-yl)methylmethylphosphate, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, I,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphocholine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-dilmoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleyl-sn-glycero-3-phosphoethanolamine, l,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.E. Targeting moieties

[0297] In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen. In certain embodiments, the lipid mtnoparticle comprises more than one targeting moiety. In certain embodiments, the lipid nanoparticle comprises more than one targeting moiety, wherein the targeting moieties target at least two different receptors, and in some embodiments, the at least two different receptors are prevalent on different types of cells or tissues.

[0298] In some embodiments, the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle. In some embodiments, the targeting moiety 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 more in the target organ than it is in the liver.

[0299] In some embodiments, the targeting moiety is an antibody as described in WO2016I89532A1, which is incorporated herein by reference. For example, in some embodiments, the targeted particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows specific delivery of the siRNAs encapsulated within the particles at a greater percentage to B-cell lymphocytes malignancies (such as MCL) than to other subtypes of leukocytes.

[0300] In some embodiments, the targeting moiety targets a receptor selected from CD20, CCR7, CD3, CD4, CDS, CD8, CD 16, CD 19, CD20, CD21, CD22, CD25, CD28, CD35, CD40, CD45RA, CD45RO, CD52, CD62L, CD80, CD95, CD127, and CD137. In some embodiments, the targeting moiety targets a receptor selected from CD1, CD2, CD3, CD5, CD7, CD8, CD 16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, 0X40, 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 targeting moiety targets a receptor selected from CD2, CD3, CD5 and CD7. In some embodiments, the targeting moiety targets a receptor selected from CD2, CD3, CDS, CD7, CDS, CD4, beta 7 integrin, beta 2 integrin, and Clq. In some embodiments, the targeting moiety targets CD117. In some embodiments, the targeting moiety targets CD90. In some embodiments, the targeting moiety targets a receptor selected from a mannose receptor, CD206 and Clq. In some embodiments, the targeting moiety' is selected from T-cell receptor motif antibodies, T-cell a chain antibodies, T-cell B chain antibodies, T-cell y chain antibodies, T-cell o chain antibodies, CCR7 antibodies, CDS antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD l ib antibodies, CDl lc antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD2.2 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, CD137 (4-1BB) antibodies, CD163 antibodies, F4 / 80 antibodies, IL-4Ra antibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARP antibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies, transferrin receptor antibodies, and fragments thereof. In certain embodiments, the targeting moiety is any one described or contemplated in US20230312713A1, US20230203538A1, DS20230320995A1, US20160145348, and US20110038941, each of which is incorporated by reference herein in its entirety.

[0301] In some embodiments, the lipid nanoparticles may be targeted when conjugated / attached / associated with a targeting moiety such as an antibody, or a fragment thereof.F. Exemplar}- LNP compositions

[0302] In some embodiments, provided herein are LNPs 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 a zwitterionic lipid. In some embodiments, the LNPs further comprise an additional ionizable lipid, besides a compound disclosed herein. In some embodiments, the LNPs further comprise an additional lipid component, of any class, besides a compound disclosed herein.

[0303] In some embodiments, the PEG-lipid is a PEG-lipid disclosed and described herein. In some embodiments, an 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.

[0304] In some embodiments, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol ,

[0305] In some embodiments, the non-ionizable lipid is a phospholipid selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl- sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl -2 -cholesterylhemisuc cinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (Cl 6 Lyso PC), 1 ,2- dilinolenoyl-sn-glycero-3-phosphocholine, l ,2-dia.rachidonoyl-sn-glycero-3-phosphocholme, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn- gly cero-3 -phosphoethanolamine, 1 ,2 -didocosahexaenoyl-sn-gly cero-3 -phosphoethanolamine , 1,2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)- 2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-a-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-gIycero-3 -(phospho-L-serine) (DOPS), acell-fusogemcphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), di stearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(o1eoyloxy)propyl) phosphate (18: 1 DMP; LBPA), 1 , 2 -dioleoyl-sn-gly cero-3 -phospho-(l ’ -myo-inositol) (DOPI; 18: 1 PI), 1,2- distearoyl-sn-glycero-3 -phospho-L-serine (18:0 PS), 1 ,2-dilinoleoyl-sn-glycero-3 -phospho-L-serine (18:2 PS), 1 -palmitoyl -2 -oleoyl-sn-gly cero-3 -phospho-L-serine (16:0-18: 1 PS; POPS), 1 -stearoyl-2- oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2-linoleoyl-sn-g1ycero-3-phospho-L-serine (18:0-18:2 PS), l-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18: 1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

[0306] In some embodiments, the non -ionizable lipid is a phospholipid selected from the group consisting of Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamidooctadec-4-en- 1 -yl (2-(tnmetliylammonio)etliyl) phosphate), Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3-hydroxy-2-stearamidooctadec~4-en-l~yl (2-(trimethylammonio)ethyl) phosphate). Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3-hydroxy-2-tricosanamidooctadec-4-en-l-yl (2- (trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro- sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D-eiytliro-sphingosylphosphoiylcholine), 16: 1 SM (N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D- erythro-sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24: 1 SM (N- nervonoyl-D-erytfiro-sphingosylphosphorylcholine), 24:0 SM (N-lignoceroyi-D-erythro- sphingosylphosphorylcholine), 18: 1 SM (N-oleoyl-D-erythro-sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erytiiro-sphiiigosylphosphorylchoiine), 06:0 SM (N-hexanoyl-D-erythro- sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D-erythro-sphingosylpbosphorylcholine), 3-O- methyl Lyso SM (3-O-methyl-spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O- methyl-N-niethyl-spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphory Ichohne) .

[0307] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0 mol% to about 10 mol% of PECs lipid; (b) about 0 mol%to about 30 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of an ionizable lipid.

[0308] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol%to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of an ionizable lipid.

[0309] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid; (c) about 40 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 33 mol% of an ionizable lipid.

[0310] In some embodiments, the lipid component of tire nanoparticle comprises: (a) about 0.5 mol% to about 10 mol% of PEG lipid; (b) about 0.5 mol% to about 50 mol% structural lipid; (c) about 5 mol%to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of an ionizable lipid.

[0311] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 35 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 40 mol% of an ionizable lipid.

[0312] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 .5 mol% to about 3.5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 35 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 40 mol% of an ionizable lipid.

[0313] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 5 mol% to about 15 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 45 mol % to about 55 mol% of an ionizable lipid.

[0314] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 .5 mol% to about 3.5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 5 mol% to about 15 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 45 mol % to about 55 mol% of an ionizable lipid.

[0315] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid.

[0316] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of an ionizable lipid.

[0317] In certain embodiments, the lipid component of the nanoparticle composition comprises about 30 mol% to about 60 mol% ionizable lipid, about 0 mol% to about 30 mol% phospholipid, about 18.5 mol% to about 48.5 mol% structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol% does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 2.0 mol% to about 45 mol% ionizable lipid, about 30 mol% to about 60 mol% phospholipid, about 10 mol% to about 30 mol% structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, 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% ionizable lipid, about 5 mol% to about 25 mol% phospholipid, about 30 mol% to about 40 mol% structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle compositioncomprises about 30 mol% to about 40 mol% ionizable lipid, about 35 mol% to about 45 mol% phospholipid, about 20 mol% to about 30 mo1% structural lipid, and about 0.5 mol% to about 5 mol% of PEG lipid, provided that the total mol% does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 25 mol%to about 45 mol% ionizable lipid, about 35 mol% to about 50 mol% phospholipid, about 10 mol% to about 25 mol% structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol% does not exceed 100%. In a particular embodiment, the lipid component comprises about 50 mol% ionizable lipid, about 10 mol% phospholipid, about 38.5 mol% structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 40 mol% ionizable lipid, about 20 mol% phospholipid, about 38.5 mol% structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol% ionizable lipid, about 10 mol% phospholipid, about 40 rnol% structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol% ionizable lipid, about 10 mol% phospholipid, about 39 mol% structural lipid, and about 2.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 33 mol% ionizable lipid, about 40 mol% phospholipid, about 25 rnol% structural lipid, and about 2 rnol% of PEG lipid. In some embodiments, the phospholipid is DOPE or DSPC. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is a sphingolipid. In some embodiments, the sphingolipid is ESM (egg sphingomyelin). In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 20 mol% of a sphingolipid, about 20 rnol% of a non-sphingolipid phospholipid, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 10 mo1% of a sphingolipid, about 30 mol% of a non-sphingolipid phospholipid, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 30 mol% of a sphingolipid, about 10 mol% of a non-sphingolipid phospholipid, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 20 mol% sphingomyelin, about 20 mol% of a DSPC, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 10 mol% sphingomyelin, about 30 mol% of a DSPC, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 30 mol% sphingomyelin, about 10 mol% of a DSPC, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 25 mol% cholesterol, about 2 mol% of a PEG lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 25 mo1% cholesterol, about 2 mol% of a PEG lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of thephosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 25 mol% cholesterol, about 2 mol% of a PECs lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolaniine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP.

[0318] In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 39.5 mol% DSPC, about 25 mol% cholesterol, and about 2.5 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% sphingomyelin, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% ESM, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% DOPE, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% DOPC, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% DLPC, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% DOPS, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 40 mol% phospholipid, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. In another particular embodiment, LNP comprises about 33 mol% ionizable lipid, about 20 mol% sphingomyelin, about 20 mol% DSPC, about 25 mol% cholesterol, and about 2 mol% of PEG lipid. |0319| In certain embodiments, the LNP comprises about 43 mol% ionizable lipid, about 15 mol% of a sphingolipid, about 15 mol% of a non-sphingolipid phospholipid, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 25 mol% of a sphingolipid, about 15 mol% of a non-sphingolipid phospholipid, about 25 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 33 mol% ionizable lipid, about 15 mol% of a sphingolipid, about 25 mol% of a non-sphingolipid phospholipid, about 2.5 mol% cholesterol and about 2 mol% of a PEG lipid. In certain embodiments, the LNP comprises about 50 mol% ionizable lipid, about 10 mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol, and about 1.5 mol% PEG lipid.

[0320] In another particular embodiment, the lipid component includes about 48.5 mol% ionizable lipid, about 10 mol% phospholipid, about 38.5 mol% structural lipid, and about 3 mol% of PEG lipid.In another particular embodiment, the lipid component includes about 48.5 mol% ionizable lipid, about 10 mol% phospholipid, about 38 mol% structural lipid, and about 3.5 mol% of PEG lipid.

[0321] In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the targeting moiety is an antibody or a fragment thereof.

[0322] The amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the active agent. For example, the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt / wt ratio of the lipid component to payload in a nanoparticle composition is from about 5:1 to about 60: 1, such as 5: 1, 6: 1 , 7: 1, 8: 1, 9: 1 , 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. The amount of a payload in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0323] In some embodiments, a nanoparticle composition of the present disclosure is 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 m an RNA active agent (e.g., a linear or circular mRNA payload). In general, a lower N:P ratio is preferred. The one or more polynucleotides, lipids, and amounts thereof is selected to provide an N:P ratio from about 2: 1 to about 30: 1, such as 2: 1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio is from about 2: 1 to about 8: 1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio 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, a nanoparticle composition of the present disclosure is formulated to provide a specific IL:P ratio, referring to the molar ratio of ionizable lipid (as a whole compound) to the number of phosphate groups in an RNA active agent. The one or more polynucleotides, lipids, and amounts thereof is selected to provide an IL:P ratio from about 2: 1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the 1L:P ratio is from about 2:1 to about 8:1. In oilier embodiments, the IL:P ratio is from about 5:1 to about 8:1. For example, the IL:P ratio is about 5.0: 1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, about 7.0:1, or about 7.5:1.

[0324] The dose of a pharmaceutical composition provided herein can be measured in units of mg / kg, which refers to mg of total nucleic acid used to formulate the LNPs per kg of body weight of a subject (mg of total mRNA / kg). In some embodiments, the pharmaceutical composition is present in the 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, .005, 0.002, or 0.001 milligram per kilogram (mg / kg, or mpk) body weight, or of a range between (inclusive) any twoof the foregoing values. In some embodiments, the pharmaceutical composition is present in the dosage form at a dose of no more than about 10 milligram per kilogram (mg / kg, or mpk) body weight. In some embodiments, the pharmaceutical composition is present in the dosage form at a dose of no more than about 9 mg / kg, no more than about 8mg / kg , no more than about 7 mg / kg, no more than about 6 mg / kg, no more than about 5 mg / ka, no more than about 4 mg / kg, no more than about 3 mg / kg, no more than about2 mg / kg, no more than about 1 mg / kg, no more than about 0.5 mg / kg, no more than about 0.2 mg / kg, no more than about 0.1 mg / kg, no more than about 0.05 mg / kg, or no more than about 0.01 mg / kg.

[0325] In some embodiments, the pharmaceutical composition is present m the dosage fonn at a concentration of no more than about 5 milligram per milliliter (mg / mL). In some embodiments, the pharmaceutical composition is present in the dosage fonn at a concentration of about 5, 4-, 3, 2, 1, 0.5, 0.2, or 0.1 milligram per milliliter(mg / mL), or of a range between (inclusive) any two of the foregoing values.

[0326] In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of no more than about 5 milligram per milliliter (mg / mL). In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of no more than about 2 milligram per milliliter (mg / mL). In some embodiments, the pharmaceutical composition is present in the dosage fonn at a concentration of no more than about 1 milligram per milliliter (mg / mL, ). In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of no more than about 0.5 milligram per milliliter (mg / mL). In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration of no more than about 0.1 milligram per mi Hi liter (m g / m L) .

[0327] 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 microgram per milliliter (pg / mL), or of a range between (inclusive) any two of tire foregoing values. In some embodiments, the pharmaceutical composition is present in the dosage fonn at a concentration of no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about. 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1, no more than about 0.5, no more than about 0.2, no more than about 0. 1 microgram per milliliter (ug / mL).

[0328] The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different, characteristics than a nanoparticle composition that comprises a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different, characteristics than a nanoparticle composition including a lowermolar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure Zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, Such as particle size, polydispersity index, and Zeta potential.

[0329] In some embodiments, the mean size of a nanoparticle composition is between 10s of nm and 100s of nm, e.g., measured by dynamic light scatering (DLS). For example, the mean size may 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, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition is from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about. 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition is from about 70 nm to about 100 nm. In a particular embodiment, the mean size is about 80 nm. In other embodiments, the mean size is about 100 nm.

[0330] In some embodiments, the LNPs of the present disclosure can be characterized by their shape. In some embodiments, the LNPs are essentially spherical. In some embodiments, the LNPs are essentially rod-shaped (i.e., cylindrical). In some embodiments, the LNPs are essentially disk shaped.

[0331] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about O to about 0.2.5, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0,06, 0.07, 0.08, 0.09, 0.10, 0.1 1 , 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 0.25.103321 The efficiency of encapsulation of a payload describes the amount of payload that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%), The encapsulation efficiency may be measured, for example, by comparing the amount, of payload in asolution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free payload in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 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 certain embodiments, the encapsulation efficiency is at least 90%.

[0333] A nanoparticle composition may comprise any substance useful in pharmaceutical compositions. For example, the nanoparticle composition may comprise one or more pharmaceutically acceptable excipients or accessory' ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be comprised. Pharmaceutically acceptable excipients are well known in the art. (see for example Remington' s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006).5. POLYNUCLEOTIDES

[0334] In some embodiments, the present disclosure provides loaded lipid nanoparticles including a polynucleotide. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is linear RNA. In preferred embodiments, the polynucleotide is circular RNA.

[0335] Wesselhoeft et al. , (2.019) 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 tor Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629 are incorporated by reference in their entirety.

[0336] Transcription of a DNA template (e.g., including a 3’ intron element, 3’ exon element, a core functional element, a 5 ’ exon element, and a 5 ’ intron element) results in formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, this DNA template includes a vector, PCR product, plasmid, minicircle DNA, cosmid, artificial chromosome, complementary DNA (cDNA), extrachromosomal DNA (ecDNA), or a fragment therein. In certain embodiments, the minicircle DNA may be linearized or non -linearized. In certain embodiments, the plasmid may be linearized or non-lmearized. 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 includes in whole or m part from a viral, bacterial, or eukaryotic vector.

[0337] The present disclosure, as provided herein, includes a DMA template that shares the same sequence as the precursor linear RNA polynucleotide prior to splicing of the precursor linear RNA polynucleotide (e.g., a 3’ intron element, a 3’ exon element, a core functional element, and a 5’ exon element, a 5’ intron element). In some embodiments, said linear precursor RNA polynucleotide undergoes splicing leading to the removal of the 3 ’ intron element and 5 ' intron element during the process of circularization. In some embodiments, the resulting circular RNA polynucleotide lacks a 3’ intron fragment and a 5’ intron fragment, but maintains a 3’ exon fragment, a core functional element, and a 5’ exon element.

[0338] In some embodiments, the precursor linear RNA polynucleotide circularizes when incubated in the presence of one or more guanosine nucleotides or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg24). In some embodiments, the 3’ exon element, 5’ exon element, and / or core functional element in whole or in part promotes the circularization of the precursor linear RNA polynucleotide to form the circular RNA polynucleotide provided herein.

[0339] In certain embodiments circular RNA provided herein is produced inside a cell. In some embodiments, precursor RNA is transcribed using a DNA template (e.g., in some embodiments, using a vector provided herein) in the cytoplasm by a bacteriophage RNA polymerase, or in the nucleus by host RNA polymerase II and then circularized.

[0340] In certain embodiments, the circular RNA provided herein is injected into an animal (e.g., a human), such that a polypeptide encoded by the circular RNA molecule is expressed inside the animal. |0341 | In some embodiments, the DNA template (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotide provided herein is 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 length. In some embodiments, 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 length. In some embodiments, the polynucleotide is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length. In some embodiments, the length of a DNA, linear RNA, and / or circular RNA polynucleotide provided herein is 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,

[0342] In some embodiments, the circular RNA provided herein has higher functional stability than mRNA including the same expression sequence. In some embodiments, the circular RNA provided herein has higher functional stability than mRNA including the same expression sequence, 5moU modifications, an optimized UTR, a cap, and / or a poly A tail.

[0343] In some embodiments, the circular RNA polynucleotide provided herein has 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 polynucleotide provided herein has a functional half-life of 5-80, 10-70, 15-60, or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis. |0344| In some embodiments, the circular RNA polynucleotide, or pharmaceutical composition thereof, has a functional half-life in a human cell greater than or equal to that of a pre -determined threshold value. 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 the media of human cells (e.g. HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay, wherein levels of a protein encoded by the expression sequence of the circular RNA polynucleotide are measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the pre -determined threshold value is the functional half-life of a reference linear RNA polynucleotide including the same expression sequence as the circular RNA polynucleotide.103451 In some embodiments, the circular RNA provided herein may have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circular RNA provided herein has a higher magnitude of expression than mRNA including the same expression sequence, 5moU modifications, an optimized UTR, a cap, and / or a poly A tail.

[0346] In some embodiments, the circular RNA provided herein may be less immunogenic than an equivalent mRNA when exposed to an immune system of an organism or a certain type of immune cell . In some embodiments, the circular RNA provided herein is associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. For example, in some embodiments, the circular RNA provided herein is associated with reduced production of IFN-pl , RIG-I, IL-2, IL-6, IFNy, and / or TNFa when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA including the same expression sequence. In some embodiments, the circular RNA provided herein is associated with less IFN-pl, RIG-I, IL-2, IL-6, IFNy, and / or TNFa transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA including the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNAincluding the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA including the same expression sequence, 5moU modifications, an optimized UTR, a cap, and / or a poly A tail.103471 In certain embodiments, the circular RNA provided herein can be transfected into a cell as is or can be transfected in DNA vector form and transcribed in the cell. Transcription of circular RNA from a transfected DNA vector can be via added polymerases or polymerases encoded by nucleic acids transfected into the cell, or preferably via endogenous polymerases. i. Enhanced Intron Elements & Enhanced Exon Elements

[0348] In some embodiments, the DNA template (e.g., vector) or linear RNA (e.g., precursor RNA) includes an enhanced intron element and / or enhanced exon element. The enhanced intron elements and enhanced exon elements may include spacers, duplex regions, affinity sequences, intron fragments, exon fragments and various untranslated elements. These sequences within the enhanced intron elements or enhanced exon elements are arranged to optimize circularization or protein expression.

[0349] In certain embodiments, the DNA template, precursor linear RNA polynucleotide and circular RNA provided herein include a first (5’) and / or a second (3’) spacer. In some embodiments, the DNA template or precursor linear RNA polynucleotide includes one or more spacers in the enhanced intron elements. In some embodiments, the DNA template, precursor linear RNA polynucleotide includes one or more spacers in the enhanced exon elements. In certain embodiments, the DNA template or linear RNA polynucleotide includes a spacer in the 3’ enhanced intron fragment and a spacer in the 5’ enhanced intron fragment. In certain embodiments, DNA template, precursor linear RNA polynucleotide, or circular RNA includes a spacer in the 3 ’ enhanced exon fragment and another spacer in the 5’ enhanced exon fragment to aid with circularization or protein expression due to symmetry created in the overall sequence.103501 In some embodiments, including a spacer between the 3 ’ group I intron fragment and the core functional element may conserve secondary structures in those regions by preventing them from interacting, thus increasing splicing efficiency. In some embodiments, the first (between 3’ group I intron fragment and core functional element) and second (between the two expression sequences and core functional element) spacers include additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. In other embodiments, the first (between 3’ group I intron fragment and core functional element) and second (between the one of the core functional element and 5’ group I intron fragment) spacers include additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. In some embodiments, such spacer base pairing brings the group I intron fragments in close proximity to each other, further increasing splicing efficiency. Additionally, in some embodiments, the combination of base pairing between the first and second duplex regions, and separately, base pairing between the firstand second spacers, promotes the formation of a splicing bubble containing the group I intron fragments flanked by adjacent regions of base pairing. Typical spacers are contiguous sequences with one or more of the following qualities: 1) predicted to avoid interfering with proximal structures, for example, the IRES, expression sequence, aptamer, or intron; 2) is at least 7 nt long and no longer than 100 nt; 3) is located after and adjacent to the 3 ’ intron fragment and / or before and adjacent to the 5 ’ intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt long, b) a region of base pairing at least 5 nt long to a distal sequence, including another spacer, and c) a structured region at least 7 nt long limited in scope to the sequence of the spacer. Spacers may have several regions, including an unstructured region, a base pairing region, a hairpin / structured region, and combinations thereof In an embodiment, the spacer has a structured region with high GC content. In an embodiment, a region within a spacer base pairs with another region within the same spacer. In an embodiment, a region within a spacer base pairs with a region within another spacer. In an embodiment, a spacer includes one or more hairpin structures. In an embodiment, a spacer includes one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In an embodiment, there is an additional spacer between the 3’ group I intron fragment and the core functional element. In an embodiment, this additional spacer prevents the structured regions of the IRES or aptamer of a TIE from interfering with the folding of the 3’ group I 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 in length. In some embodiments, the 5’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5’ spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and / or 25 and 35 nucleotides in length. In certain embodiments, the 5’ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2.1, 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 length. In one embodiment, the 5’ spacer sequence is a polyA sequence. In another embodiment, the 5’ spacer sequence is apolyAC sequence. In one embodiment, a spacer includes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, a spacer includes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.|0351 | In some embodiments, the DNA template and precursor linear RNA polynucleotides and circular RNA polynucleotide provided herein include a first (5’) duplex region and a second (3’) duplex region. In certain embodiments, the DNA template and precursor linear RNA polynucleotide includes a 5’ external duplex region located within the 3’ enhanced intron fragment and a 3’ external duplex region located within the 5’ enhanced intron fragment. In some embodiments, the DNA template, precursor linear RNA polynucleotide and circular RNA polynucleotide include a 5’ internal duplex region located within the 3' enhanced exon fragment and a 3’ internal duplex region located within the5 ’ enhanced exon fragment. In some embodiments, the DNA polynucleotide and precursor linear RN A polynucleotide includes a 5’ external duplex region, 5’ internal duplex region, a 3’ internal duplex region, and a 3’ external duplex region.103521 In certain embodiments, the first and second duplex regions may form perfect or imperfect duplexes. Thus, in certain embodiments at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second duplex regions may be base paired with one another. In some embodiments, the duplex regions are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with unintended sequences in the RNA (e.g., non-duplex region sequences). In some embodiments, including such duplex regions on the ends of the precursor RNA strand, and adjacent or very' close to the group I intron fragment, bring the group I intron fragments in close proximity to each other, increasing splicing efficiency. In some embodiments, the duplex regions are 3 to 100 nucleotides in length (e.g., 3-75 nucleotides in length, 3-50 nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides in length, 5-25 nucleotides m length, 9- 19 nucleotides in length). In some embodiments, the duplex regions are 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 length. In some embodiments, the duplex regions have a length of 9 to 50 nucleotides. In one embodiment, the duplex regions have a length of 9 to 19 nucleotides. In some embodiments, the duplex regions have a length of 20 to 40 nucleotides. In certain embodiments, the duplex regions have a length of 30 nucleotides.

[0353] In other embodiments, the DNA template, precursor linear RNA polynucleotide, or circular RNA polynucleotide does not include of any duplex regions to optimize translation or circularization.

[0354] 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 the 3’ enhanced intron element. In some embodiments, the affinity tag is located in the 5 ’ enhanced intron element. In some embodiments, both (3’ and 5’) enhanced intron elements each include an affinity tag. In one embodiment, an affinity tag of the 3’ enhanced intron element is the length as an affinity tag in the 5’ enhanced intron element. In some embodiments, an affinity tag of the 3’ enhanced intron element is the same sequence as an affinity tag in the 5’ enhanced intron element. In some embodiments, the affinity sequence is placed to optimize oligo-dT purification.

[0355] In some embodiments, an affinity tag includes a polyA region. In some embodiments the poly A region is at least 15, 30, or 60 nucleotides long. In some embodiments, one or both polyA regions is 15-50 nucleotides long. In some embodiments, one or both polyA regions is 20-25 nucleotides long. The polyA sequence is removed upon circularization. Thus, an oligonucleotide hybridizing with the polyA sequence, such as a deoxythymine oligonucleotide (oligo(dT)) conjugated to a solid surface (e.g. , a resin), can be used to separate circular RNA from its precursor RNA.

[0356] In certain embodiments, the 3 ’ enhanced intron element includes a leading untranslated sequence. In some embodiments, the leading untranslated sequence is a the 5 ’ end of the 3’ enhanced intron fragment. In some embodiments, the leading untranslated sequence includes of the last nucleotide of a transcription start site (TSS). In some embodiments, the TSS is chosen from a viral, bacterial, or eukaryotic DNA template. In one embodiment, the leading untranslated sequence include the last nucleotide of a TSS and 0 to 100 additional nucleotides. In some embodiments, the TSS is a terminal spacer. In one embodiment, the leading untranslated sequence contains a guanosine at the 5’ end upon translation of an RNA T7 polymerase.

[0357] In certain 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 partial restriction digest sequence. In one embodiment, the trailing untranslated sequence is in whole or in part a restriction digest site used to linearize the DNA template. In some embodiments, the restriction digest site is in whole or in part from a natural viral, bacterial or eukaryotic DNA template. In some embodiments, the trailing untranslated sequence is a terminal restriction site fragment. a. Enhanced Intron Fragments

[0358] In some embodiments, the 3‘ enhanced intron element and 5’ enhanced intron element each include an intron fragment. In certain embodiments, a 3’ intron fragment is a contiguous sequence at least 75% homologous (e.g„ at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 3’ proximal fragment of a natural group I or II intron including the 3’ splice site dinucleotide. Typically, a 5’ intron fragment is a contiguous sequence at least 75% homologous (e.g,, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 5’ proximal fragment of a natural group I or II intron including the 5’ splice site dinucleotide. In some embodiments, the 3’ intron fragment includes the first nucleotide of a 3’ group I or II splice site dinucleotide. In some embodiments, the 5 ’ intron fragment includes the first nucleotide of a 5’ group I or II splice site dinucleotide. In other embodiments, the 3’ intron fragment includes the first and second nucleotides of a 3’ group I or II intron fragment splice site dinucleotide; and the 5’ intron fragment includes the first and second nucleotides of a 3’ group I or II intron fragment dinucleotide. In some embodiments the 3’ enhanced intron element and 5’ enhanced intron element includes a synthetic intron fragment. b. Enhanced Exon Fragments103591 In certain embodiments, as provided herein, the DNA template, linear precursor RNA polynucleotide, and circular RNA polynucleotide each include an enhanced exon fragment. In some embodiments, following a 5’ to 3’ order, the 3’ enhanced exon element is located upstream to corefunctional element. In some embodiments, following a 5’ to 3’ order, the 5 ’ enhanced intron element is located downstream to the core functional element.

[0360] In some embodiments, the 3’ enhanced exon element and 5’ enhanced exon element each include an exon fragment. In some embodiments, the 3’ enhanced exon element includes a 3’ exon fragment. In some embodiments, the 5 ' enhanced exon element includes a 5 ’ exon fragment. In certain embodiments, as provided herein, the 3’ exon fragment and 5’ exon fragment each includes a group I or II intron fragment and I to 100 nucleotides of an exon sequence. In certain embodiments, a 3 ’ intron fragment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 3 ’ proximal fragment of a natural group I or II intron including the 3 ’ splice Site dinucleotide. Typically, a 5 ’ group I or II intron fragment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 5’ proximal fragment of a natural group I or II intron including the 5 ’ splice site dinucleotide. In some embodiments, the 3 ’ exon fragment includes a second nucleotide of a 3’ group I or II intron splice site dinucleotide and 1 to 100 nucleotides of an exon sequence. In some embodiments, the 5’ exon fragment includes the first nucleotide of a 5’ group I or II intron splice site dinucleotide and 1 to 100 nucleotides of an exon sequence. In some embodiments, the exon sequence includes in part or m whole from a naturally occurring exon sequence from a vims, bacterium or eukaryotic DNA vector. In other embodiments, the exon sequence further includes a synthetic, genetically modified (e.g., containing modified nucleotide), or other engineered exon sequence.

[0361] In one embodiment, where the 3’ intron fragment includes both nucleotides of a 3’ group I or II splice site dinucleotide and the 5 ’ intron fragment includes both nucleotides of a 5 ’ group I or II splice site dinucleotide, the exon fragments located within the 5’ enhanced exon element and 3’ enhanced exon element does not include of a group I or II splice site dinucleotide.

[0362] For means of example and not intended to be limiting, in some embodiment, a 3 ’ enhanced intron element includes in the following 5’ to 3’ order: a leading untranslated sequence, a 5’ affinity tag, an optional 5’ external duplex region, a 5’ external spacer, and a 3’ intron fragment. In same embodiments, the 3 ’ enhanced exon element includes in the following 5 ’ to 3 ’ order: a 3 ’ exon fragment, an optional 5 ’ internal duplex region, an optional 5 ’ internal duplex region, and a 5 ’ internal spacer. In the same embodiments, the 5’ enhanced exon element includes in the following 5’ to 3’ order: a 3’ internal spacer, an optional 3’ internal duplex region, and a 5’ exon fragment. In still the same embodiments, the 3’ enhanced intron element includes in the following 5’ to 3’ order: a 5’ intron fragment, a 3 ’ external spacer, an optional 3 ’ external duplex region, a 3 ’ affinity tag, and a trailing untran slated sequence .n. Core Functional Element

[0363] In some embodiments, the DN A template, linear precursor RNA polynucleotide, and circular RNA polynucleotide include a core functional element. In some embodiments, the core functional element includes a coding or noncoding element. In certain embodiments, the core functional element may contain both a coding and noncoding element. In some embodiments, the core functional element further includes translation initiation element (TIE) upstream to the coding or noncoding element. In some embodiments, the core functional element includes a termination element. In some embodiments, the termination element is located downstream to the TIE and coding element. In some embodiments, the termination element is located downstream to the coding element but upstream to the TIE. In certain embodiments, where the coding element includes a noncoding region, a core functional element lacks a TIE and / or a termination elemen t.? / / . Coding or Noncoding Element

[0364] In some embodiments, the polynucleotides herein include a coding element, a noncoding element, or a combination of both. In some embodiments, the coding element includes an expression sequence. In some embodiments, the coding element encodes at least one therapeutic protein.

[0365] In some embodiments, the circular RNA encodes two or more polypeptides. In some embodiments, tire circular RNA is a bicistronic RNA. The sequences encoding the two or more polypeptides can be separated by a ribosomal skipping element or a nucleotide sequence encoding a protease cleavage site. In certain embodiments, the ribosomal skipping element encodes thosea-asigna virus 2 A peptide (T2A), porcine teschovirus-1 2 A peptide (P2A), foot-and-mouth disease virus 2 A peptide (F2A), equine rhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2 A), or flachene vims of B. mori 2 A peptide (BmlF V 2A). iv. Translation Initiation Element (TIE)103661 As provided herein in some embodiments, the core functional element includes at least one translation initiation element (TIE). TIEs are designed to allow translation efficiency of an encoded protein. Thus, optimal core functional elements including only of noncoding elements lack any TIEs. In some embodiments, core functional elements including one or more coding element will further include one or more TIEs.

[0367] In some embodiments, a TIE includes an untranslated region (UTR). In certain embodiments, the TIE provided herein include an internal ribosome entry site (IRES), In some embodiments, IRES is a naturally occurring IRES. In some embodiments, the IRES is a non -naturally occurring or synthetic IRES. Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequences). The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem, Biophys. Res. Comm.(1996) 229:295-298; Rees el 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 entireties. v. Additional Accessory Elements (Sequence Elements)

[0368] As described in this disclosure, the circular RNA polynucleotide, linear RNA polynucleotide, and / or DNA template may further include of accessory elements. In certain embodiments, these accessory elements may be included within the sequences of the circular RNA, linear RNA polynucleotide and / or DNA template for enhancing circularization, translation or both. Accessory elements are sequences, in certain embodiments that are located with specificity between or within the enhanced intron elements, enhanced exon elements, or core functional element of the respective polynucleotide. As an example, but not intended to be limiting, an accessory element includes, a IRES transacting factor region, a miRNA binding site, a restriction site, an RNA editing region, a structural or sequence element, a granule site, a zip code element, an RNA trafficking element or another specialized sequence as found in the art that enhances promotes circularization and / or translation of the protein encoded within the circular RNA polynucleotide.

[0369] In certain embodiments, the accessory' element includes an IRES transacting factor (ITAF) region. In some embodiments, the IRES transacting factor region modulates the initiation of translation through binding to PCBP1 - PCBP4 (polyC binding protein), PABP1 (polyA binding protein), PTB (polyprimidine tract binding), Argonaute protein family, HNRNPK (Heterogeneous nuclear ribonucleoprotein K protein), or La protein. In some embodiments, tire IRES transacting factor region includes a polyA, polyC, polyAC, or polyprimidine track.

[0370] In some embodiments, the ITAF region is located within the core functional element, In some embodiments, the ITAF region is located within the TIE.

[0371] In certain embodiments, the accessory element includes a miRNA binding site. In some embodiments the miRNA binding site is located within the 5 ’ intron element, 5’ exon element, core functional element, 3’ exon element, and / or 3’ intron element.103721 In some embodiments, wherein the miRNA binding site is located within the spacer within the intron element or exon element. In certain embodiments, the miRNA binding site includes the entire spacer regions.

[0373] In some embodiments, the 5’ intron element and 3’ intron elements each include identical miRNA binding sites. In another embodiment, the miRNA binding site of the 5 ’ intron element includes a different, in length or nucleotides, miRNA binding site than the 3 ’ intron element. In one embodiment. the 5’ exon element and 3’ exon element include identical miRNA binding sites. In other embodiments,the 5 ' exon element and 3 ’ exon element includes different, in length or nucleotides, miRN A binding sites.

[0374] In some embodiments, the miRNA binding sites are located adjacent to each other within the circular RNA polynucleotide, linear RNA polynucleotide precursor, and / or DMA template. In certain embodiments, the first nucleotide of one of the miRNA binding sites follows the first nucleotide last nucleotide of the second miRNA binding site.

[0375] In some embodiments, the miRNA binding site is located within a translation initiation element (TIE) of a core functional element. In one embodiment, the miRNA binding site is located before, trailing or within an internal ribosome entry site (IRES). In another embodiment, the miRNA binding site is located before, trailing, or within an aptamer complex.

[0376] The unique sequences defined by the miRNA nomenclature are -widely known and accessible to those working in the microRNA field. For example, they can be found in the rniRDB public database. vi. Natural Ties: Viral & Eukaryotic / Cellular Internal Ribosome Entry Sites (IRES)

[0377] A multitude of IRES sequences are available and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang etal., J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al. , Proc. Natl. Acad. Sci. (2003) 100(25): 15125- 15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like.

[0378] For driving protein expression, the circular RNA includes an IRES operably linked to a protein coding sequence. Modifications of IRES and accessory sequences are disclosed herein to increase or reduce IRES activities, for example, by truncating the 5 ’ and / or 3 ’ ends of the IRES, adding a spacer 5’ to the IRES, modifying the 6 nucleotides 5’ to the translation initiation site (Kozak sequence), modification of 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 a native IRES.

[0379] In some embodiments, the IRES is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus. Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuioendotheliosis vims. Human poliovirus 1, Plautia stali intestine vims, Kashmir bee virus. Human rhinovirus 2, Homalodisca coagulata vims- 1 , Human Immunodeficiency Vims type I, Himetobi P vims. Hepatitis C vims, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus. Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis vims, Bovine viral diarrhea virus 1, Black Queen Cell Vims, Aphid lethal paralysisvirus, Avian encephalomyelitis virus. Acute bee paralysis virus, Hibiscus chlorotic ringspot virus. Classical swine fever virus. Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c- IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipi, 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, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobimavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV -A21, Salivirus A SHI, Sail vims FHB, Salivirus NG-J 1, Human Parechovirus 1 , Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Vitus HA 16, Phopi virus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepaci virus K, Hepaci virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picoma-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GET, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G.103801 In some embodiments, the IRES includes in whole or in part from a eukaryotic or cellular IRES. In certain embodiments, the IRES is from a human gene, where the human gene is ABCF1, ABCG1, ACAD10, ACOT7, ACSS3, ACTG2, ADCYAP1, ADK, AGTR1, AHCYL2, AHI1, AKAP8L, AKR1A1, ALDH3A1, ALDOA, ALG13, AMMECR1L, ANGPTL4, ANK3, AOC3, AP4BI, AP4EI, APAFI, APBB1, APC, APH1A, APOBEC3D, APOM, APP, AQP4, ARHGAP36, ARL13B, ARMC8, ARMCX6, ARPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURKA, B3GALNT1, B3GNTL1, B4GALT3, BAAT, BAGl, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, BTN3A2, CI2orf43, C14orf93, C17orf62, CTorf226, C21orf62, C2orfl5, C4BPB, C4or£22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP2, CAV1, CBX5, CCDC120, CCDC17, CCDC186, CCDC51, CCN1, CCND1, CCNT1, CD2BP2, CD9, CDC25C, CDC42, CDC7, CDCA7L, CDIP1, CDKI, CDKI 1A, CDKN1B, CEACAM7, CEP295NL, CFLAR, CHCHD7, CHIA, CHICI, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, COG4, COMMD1, C0MMD5, CPEB1, CPS1, CRACR2B, CRBN, CREM, CRYBGI, CSDEI, CSF2RA, CSNK2A1, CSTF3, CTCFL, CTH, CTNNA3, CTNNB1, CTNNBI, CTNND1, CTSL,CUTA, CXCR5, CYB5R3, CYP24A1, CYP3A5, DAG1, DAP3, DAP5, DAXX, DCAF4, DCAF7, DCLRE1A, DCP1A, DCTN1, DCTN2, DDX19B, DDX46, DEFBI 23, DGKA, DGKD, DHRS4, DHX15, DI03, DLG1, DLL4, DMD UTR, DMD exS, DMKN, DNAH6, DNAL4, DUSP13, DUSP19, DYNC1I2, DYNLRB2, DYRK1A, ECI2, ECT2, EIF1AD, EIF2B4, EIF4G1, EIF4G2, EIF4G3, ELANE. EL0VL6, ELP5. EMCN, EN01, EPB41, ERMN, ERVV-1, ESRRG, ETFB, ETFBKMT, ETV1, ETV4, EXD1, EXT1, EZH2, FAM11 IB, FAM157A, FAM213A, FBXO2.5, FBXO9, FBXW7, FCMR, FGF1, FGF1, FGF1A, FGF2, FGF2, FGF-9, FHL5, FMRI, FN1, FOXP1, FTH1, FUBPJ , G3BP1, GABBR1, GALC, GART, GAS7, gastrin, GATA1, GATA4, GFM2, GHR, GJB2, GLI1, GLP. A2. GMNN, GPAT3, GPATCH3, GPR137, GPR34, GPR55, GPR89A, GPRASP1, GRAP2. GSDMB, GSTO2, GTF2B, GTF2H4, GUCY1B2, HA.X1, HCST, HIGDIA, HIGD1B, HIPK1, HIST1H1C, HIST1H3H, HK1, HLA-DRB4, HMBS, HMGAJ , HNRNPC, HOPX, H0XA2, H0XA3, HPCAL1, HR, HSP90AB1, HSPA1A, HSPA4L, HSPA5, HYPK, IFFO1, IFT74, IFT81, IGF1, IGF1R, IGF1R, IGF2, ILH, IL17RE, IL1RL1, IL1RN, II .32. IL6, ILF2, ILVBL, INSR, INTS13, IP6K1, ITGA4, ITGAE, KCNE4, KERA, KIAA0355, KIAA0895L, KIAA1324, KIAA1522, KIAA1683, KIF2C, KIZ, KLHL31, KLK7, KRR1, KRT14, KRT17, KRT33A, KRT6A, KRTAP10-2, KRTAP13- 3, KRTAP13-4, KRTAP5-11, KRTCAP2, LACRT, LAMB1, LAMB3, LANCL1, LBX2, LCAT, LDHA, LDHAL6A, LEFT, L1NC-P1NT, LM03, LRRC4C, LRRC7, LRTOMT, LSM5, LTB4R, LYRM1, LYRM2, MAGEA11, MAGEA8, MAGEB1, MAGEB16, MAGEB3, MAPT, MARS, MC1R, MCCC1, METTL12, METTL7A, MGC 16025, MGC16025, MIA2, MIA2, MITF, MKLN1, MNT, MORF4L2, MPD6, MRFAP1, MRPL21, MRPS12, MSI2, MSLN, MSN, MT2A, MTFR1L, MTMR2, MTRR, MTUS1, MYB, MYC, MYCL, MYCN, MYL10, MYL3, MYLK, MYO1A, MYT2. MZB1, NAP1L1, NAVI, NBAS, NCF2, NDRG1, NDST2, NDUFA7, NDUFB11, NDUFC1, NDUFS1, NEDD4L, NF ATS, NFE2L2, NFE2L2, NFIA, NHEJ1, NHP2, NITI, NKRF, NME1-NME2, NPAT, NR3C1 , NRBF2, NRF1 , NTRK2, NUDCD1, NXF2, NXT2, ODC1, ODF2, OPTN, OR10R2, ORl 1L1, OR2M2, OR2M3, OR2M5, OR2T10, OR4C15, OR4F17, OR4F5, OR5H1, OR5K1, OR6C3, OR6C75, OR6N1, OR7G2, p53, P2RY4, PAN 2, PAQR6, PARP4, PARP9, PC, PCBP4, PCDHGC3, PCLAF, PDGFB, PDZRN4, PELO, PEMT, PEX2, PFKM, PGBD4, PGLYRP3, PHLDA2, PHTFl , PI4KB, PIGC, PIM1, PKD2L1, PKM, PLCB4, PLD3, PLEKHA1, PLEKHB1, PLS3, PML, PNMA5, PNN, POC1A, POC1B, POLD2, POLD4, POU5F1, PPIG, PQBP1, PR.4ME, PRPF4, PRR11, PRRT1, PRSS8, PSMA2, PSMA3, PSMA4, PSMD11, PSMD4, PSMD6, PSME3, PSMG3, PTBP3, PTC H I . PTHLH, PTPRD, PUS7L, PVRIG, QPRT, RAB27A, RAB7B, RABGGTB, RAET1E, RALGDS, RALYL, RARB, RCVRN, REG3G, RFCS, RGL4, RGS19, RGS3, RHD, RINL, RIPOR2, R1TA1, RMDN2, RNASE 1, RNASE4, RNF4, RPA2, RPL17, RPL21, RPL26L1, RI’L28, RPL29. RPL4L RI’L9, Rl’Sl l, RI’S13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RENXITI, RUNX1T1, RLTNX2. RUSC1, RXRG, S100A13, S100A4, SAT1, SCRIP 1, SCMH1, SEC14L1, SEMA4A, SERPINA1, SERPINB4, SERTAD3, SFTPD, SH3D19, SHC1, SHMT1, SHPRH, SIM1, SIRT5, SLC11A2,SLC12A4, SLC16A1. SLC25A3, SLC2.6A9, SLC5A11, SLC6A12, SLC6A19, SLC7A1, SLFN 11, SLIRP, SMAD5, SMARCAD1, SMN1, SNCA, SNRNP200, SNRPB2, SNX12, S0D1, SOX13, SOX5, SP8, SPARCL1, SPATA12, SPATA31C2, SPN, SPOP, SQSTM1, SRBD1, SRC, SREBF1 , SRPK2, SSB, SSB, SSBP1, ST3GAL6, STAB I, STAMBP, STAU1, STAU1, STAC I, STAU1, STAU1, STK16, STK24, STK38, STMN1, STX7, SULT2B1, SYK, SYNPR, TAF1C, TAGLN, TANK, TAS2R40, TBC1D15, TBXAS1 , TCF4, TDGF1, TDP2, TDRD3, TDRD5, TESK2, THAP6, THBD, THTPA, TIAM2, TKFC, TKTLI, TLR10, TM9SF2, TMC6, TMC02, TMED10, TMEM116, TMEM126A, TMEM159, TMEM208, TMEM230, TMEM67, TMPRSS13, TMUB2, TNFSF4, TNIP3, TP53, TP53, TP73, TRAFT, IRAKI, TR1M31, TRIM6, TRMT1, TRMT2B, TRPM7, TRPM8, TSPEAR . TTC39B, TTLL1 1 , TUBB6, TXLNB, TXNIP, TXNL1, TXNRD1, TYROBP, U2AF 1, UBA1, UBE2D3, UBE2I, UBE2L3, UBE2V1 , UBE2V2, UMPS, UNG, UPP2, USMG5, USP18, UTPI4A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS29, VSIG10L, WDHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY 1AP1, ZBTB32, ZNF146, ZNF250, ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF707, orZNRDl. vii. Synthetic Ties: Aptamer Complexes. Modified Nucleotides, IRES Variants & Other Engineered Ties

[0381] As contemplated herein, m some embodiments, a translation initiation element (TIE) includes a synthetic TIE. In some embodiments, a synthetic TIE includes aptamer complexes, synthetic IRES or other engineered TIES capable of initiating translation of a linear RNA or circular RNA polynucleotide.

[0382] In some embodiments, one or more aptamer sequences is capable of binding to a component of a eukaryotic initiation factor to either enhance or initiate translation. In some embodiments, aptamer may be used to enhance translation in vivo and in vitro by promoting specific eukaryotic initiation factors (elF) (e.g., aptamer in WO2019081383A1 is capable of binding to eukaryotic initiation factor 4F (eIF4F). In some embodiments, the aptamer or a complex of aptamers may be capable of binding to EIF4G, EIF4E, EIF4A, EIF4B, EIF3, EIF2, EIF5, EIF1, EIF1 A, 40S ribosome, PCBP1 (polyC binding protein), PCBP2, PCBP3, PCBP4, PABP1 (polyA binding protein), PTB, Argonaute protein family, HNRNPK (heterogeneous nuclear ribonucleoprotein K), or La protein. viii. Termination Sequence

[0383] 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 stop cassette. In some embodiments, the stop cassette includes at least 2 stop codons. In some embodiments, the stop cassette includes at least 2 frames of stop codons. In the same embodiment, the frames of the stop codons in a stop cassette each include 1, 2 or more stop codons. Insome embodiments, the stop cassette includes a LoxP or a RoxStopRox, or frt-flanked stop cassette. In the same embodiment, the stop cassette inchides a lox-stop-lox stop cassette. ix. Variants103841 In some embodiments, a circular RNA polynucleotide provided herein includes modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleoside is nr C (5-methylcytidme). In one embodiment, the modified nucleoside is m’U (5-methyhiridine). In another embodiment, the modified nucleoside is m°A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2-thiouridine). In another embodiment, the modified nucleoside is ‘P (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine). In other embodiments, the modified nucleoside is m‘A (1 -methyladenosine); m2A (2-methyladenosine); Am (2’-O-methyladenosine); ms2m°A (2-methylthio-N6-inetbyla.denosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (Nb-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N°- glycinylcarbamoyladenosine); t°A (N6-threonylcarbamoyladenosine); ms2t°A (2-methy1thio-N°- threonyl carbamoyladenosine); m°t6A (N°-methyl-N6-threonylcarbamoyladenosine); hn°A(N°- hydroxynorvatylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2’-O-ribosyladenosine (phosphate)); I (inosine); m'l (I -methylinosine); m‘Im (l ,2’-O-dimethylinosine); nr'C (3-methylcytidine); Cm (2’-O-methylcytidine); s2C (2- thiocytidine); aclC (N4-acetylcytidine); FC (5-formylcytidine); rn’Cm (5,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2’-O-methylcytidine); k2C (lysidine); m’G (1 -methylguanosine); m2G (N2- methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2- dimethyl guanosine); m2Gm (N2,2’-O-dimethylguanosine); m22Gm (N2,N2,2’-O-trimethylguanosine); Grip) (2’-O-ribosylguanosine(phosphate)); yW (wybutosine); O2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosme); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosme); preQs (7-aminomethyl~7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m’Um (5,2’-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5- methyl-2-thiouridine); s2lJm (2-thio-2’-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho’U (5-hydroxyuridme); mo5U (5-methoxyuridine); cmo>U (uridine 5- oxyacetic acid); mcmo3U (uridine 5 -oxyacetic acid methyl ester); chm'’U (5- (carboxyhydroxymethyl)uridine)); mcbm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylrnethyluridine); mcm’Um (5-methoxycarbonylmethyl-2’-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5S2U (5-aminomethyl-2-thiouridine); mnm5ll (5 -methylaminomethyluridine); mnm's2U (5 -methylaminomethyl -2 -thiouridine); mnm5se2U (5- methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridme); cmnm5U (5-carboxymethylaminomethyluridine); cmnnriUm (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U (5 -carboxymethylaminomethyl -2- thiouridine); m° ?.A (N\N°-dimethyladenosine); Im (2’-O-methylinosine); m4C- (N4-methylcytidine); m4Cm (N4,2’-O-dimethylcytidine); hnriC (5-hydroxymethyicytidine); mJU (3 -methyl uridine); cm5U (5-carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); m62Am (N6,N6,O-2’- trimethyladenosine); mA?G (N\7-dimetbylguanosine); m2’2, / G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m'D (5-methyldihydrouridine); FCm (S-fonnyl^’-O-methylc^idine); m‘Gm (l,2’-O-dimethylguanosine); m'Am (l,2’-O-dimethyladenosine); un5U (5- taurinometliyluridine): -nnVU (5 -taurinomethyl -2 -thiouridine)); imG-14 (4-demethyiwyosme); imG2 (isowyosine); or ac6A (N°-acetyladenosine).

[0385] In some embodiments, the modified nucleoside may include a compound selected from: pyridin -4-one ribonucleoside, 5 -aza-uridine, 2 -thio-5 -aza-uridine, 2 -thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyiuridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5 -propynyl -uridine, 1 -propynyl -pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl -pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridme, 5- methyl -uridine, 1 -methyl-pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio- 1 -methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridme, 4-m ethoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4 -acetylcytidine, 5- formyicytidine, N4-methylcytidine, 5 -hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio-l-methyl-l -deaza-pseudoisocytidine, 1- methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7- deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyiadenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2~metliylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glyciny1carbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2- methylthio-adenine, 2 -methoxy -adenine, inosine, 1 -methyl -inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8- aza-guanosine, 7-methyl-guanosine, 6-thio-7~m ethyl -guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2 -methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosme, 7-me±yl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2^2- dimethyl-6-thio-guanosme. In another embodiment, the modifications are independently selected from 5-methylcytosine, pseudouridine and 1 -methylpseudouridine.103861 In some embodiments, the modified ribonucleosides include 5 -methylcytidine, 5- methoxyuridine, 1-methyi-pseudouridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.|0387| In particular embodiments, polynucleotides may be codon -optimized. A codon optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted in order to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRN As transcribed from the DNA sequence, (ix) prior knowledge the function of the DNA sequences upon which design of the codon substitution set is to be based, and / or (x) systematic variation of codon sets for each amino acid. In some embodiments, a codon optimized polynucleotide may minimize ribozyme collisions and / or limit structural interference between the expression sequence and the core functional element. x. Payloads

[0388] 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 the following Table 5.

[0389] In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the expression sequence encodes a cytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21,IFM-a, IFN- P, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. In some embodiments, the expression sequence encodes an immune checkpoint inhibitor. In some embodiments. the expression sequence encodes an agonist (e.g., a TNFR family member such as CD137L, 0X401,, ICOSL, LIGHT, or CD70). In some embodiments, the expression sequence encodes a chimeric antigen receptor. In some embodiments, the expression sequence encodes an inhibitory receptor agonist (e.g.,PDL1, PDL2, Galectin-9, VISTA, B7H4, or MHCII) or inhibitory receptor (e.g., PD1, CTLA4, TIGIT,LAG3, or TIM3). In some embodiments, the expression sequence encodes an inhibitory receptor antagonist. In some embodiments, the expression sequence encodes one or more TCR chains (alpha andbeta chains or gamma and delta chains). In some embodiments, the expression sequence encodes a secreted T cell or immune cell engager (e.g,, a bispecific antibody such as BiTE, targeting, e.g,, CD3, CD137, or CD28 and a tumor-expressed protein e.g., CD 19, CD20, or BCMA etc.). In some embodiments, the expression sequence encodes for a BCMA chimeric antigen receptor as disclosed in US Patent Application US 2021 / 0128618A1, the contents of which are hereby incorporated in its entirety by reference. In some embodiments, the expression sequence encodes a transcription factor (e.g., FOXP3, HELIOS. TOX1, or TOX2). In some embodiments, the expression sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39 / CD73). In some embodiments, the expression sequence encodes a GvHD (e.g., anti-HLA-A2 CAR-Tregs).

[0390] In some embodiments, the precursor RNA polynucleotide and circular RNA constructs include at least one expression sequence encoding an antigen, adjuvant, or adjuvant-like protein, e.g., from an infectious agent. In these embodiments, the circular RNA construct may be used as a vaccine. In some embodiments, the one or more circular RNA polynucleotide encodes 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 a virus, bacterium, fungus, protozoan, and / or parasite. In some embodiments, the antigen is a viral antigen or viral antigenic polypeptide.

[0391] In an embodiment, the antigen is selected from or derived from the group consisting of rotavirus, foot and mouth disease virus, influenza A vims, influenza B virus, influenza C vims, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza type 2, herpes simplex virus, Epstein-Barr virus, varicella virus, porcine herpesvirus 1, cytomegalovirus, lyssavirus. Bacillus anthracis, anthrax PA and derivatives, poliovirus. Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, distemper virus, Venezuelan equine encephalomyelitis, feline leukemia vims, reovirus, respiratory syncytial virus, Lassa fever vims, polyoma tumor virus, canine parvovirus, papilloma virus, tick bome encephalitis virus, rinderpest virus, human rhinovirus species. Enterovirus species, Mengovirus, paramyxovirus, avian infectious bronchitis vims, human T-cell leukemia-lymphoma virus 1, human immunodeficiency virus-1, human immunodeficiency virus-2, lymphocytic choriomeningitis virus, parvovirus Bl 9, adenovirus, rubella virus, yellow fever vims, dengue virus, bovine respirator}' syncitial virus, corona virus, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis. Brucella abortis. Brucella melitensis. Brucella suis. Brucella ovis, Brucella species, Escherichia coli. Salmonella species. Salmonella typhi, Streptococci, Vibrio cholera. Vibrio parahaemolyticus, Shigella, Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin, Mycobacterium leprae. Pneumococci, Staphlylococci, Enterobacter species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella multocida. Chlamydia trachomatis. Chlamydia psittaci, Lymphogranuloma venereum. Treponema pallidum, Haemophilus species, Mycoplasma bovigenitalium. Mycoplasma pulmonis. Mycoplasma species, Borrelia burgdorferi, Legionalla pneumophila, Colstridium botulinum, Corynebacteriumdiphtheriae, Yersinia entercolitica, Rickettsia ricketsii, Rickettsia typhi, Rickettsia prowsaekii, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes, trypanosomes, Leishmania species. Filarial nematodes, trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albican, Aspergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae, malaria circumsporozoite protein, malaria merozoite protein, trypanosome surface antigen protein, pertussis, alphaviruses, adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, Influenza hemagglutinin, cancer antigen, tumor antigens, toxins, Clostridium perfringens epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins, neurotoxins, cytokines, cytokine receptors, monokines, monokine receptors, plant pollens, animal dander, and dust mites.103921 In some embodiments, the antigenic polypeptide is a viral polypeptide from an adenovirus; Herpes simplex, type 1; Herpes simplex, type 2; encephalitis virus, papillomavirus, Varicella-zoster virus; Epstein -barr virus; Human cytomegalovirus; Human herpes virus, type 8; Human papillomavirus; BK virus; JC vims; Smallpox; polio virus; Hepatitis B virus; Human bocavirus; Parvovirus B 19; Human astrovirus; Norwalk virus; 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); Influenza virus; Guanarito virus; Junin virus; Lassa vims; Machupo vims; Sabia vims; Crimean-Congo hemorrhagic fever vims; Ebola virus; Marburg virus; Measles vims; Mumps virus; Parainfluenza virus; Respiratory syncytial virus; Human metapneumo virus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Coltivirus; Banna vims; Human Enterovirus; Hanta virus; West Nile virus; Middle East Respiratory Syndrome Corona Virus; Japanese encephalitis virus; Vesicular exanthemavirus; SARS- CoV-2; Eastern equine encephalitis, or a combination of any two or more of the foregoing.

[0393] 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, Lumazine synthase, 0mpl6, 0mpl9, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, Falgellin, IFN- aipha, IFN-gamma, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-lb, IL-6, TNF-a, IL-7, IL-17, IL- IBeta, anti-CTLA4, anti-PDl, anti-41BB, PD-L1, Tim-3, Lag-3, T1G1T, GITR, and andti-CD3.

[0394] In some embodiments, a polynucleotide encodes a protein that is made up of subunits that 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 that more than one circRNA molecule is delivered in the transfer vehicle and each circRN A encodes a separate subunit of the protein. Alternatively, a single circRNA may- be engineered to encode more than one subunit. In certainembodiments, separate circRNA molecules encoding the individual subunits may be administered in separate transfer vehicles.

[0395] Additional polynucleotides, including but not limited to intron elements, exon elements, translation initiation elements, expression sequences, and lipids are in WO2019236673; WO2020237227; WO2021113777; WO2021226597; WO2021189059; WO2021236855; WO2022261490; W02023056033; WO2023081526; WO2023250375; the contents of which are hereby incorporated by reference in their entireties.(I) Chimeric Antigen Receptors (CARs)

[0396] Chimeric antigen receptors (CARs or CAR-Ts) are genetically-engineered receptors. These engineered receptors may be inserted into and expressed by immune cells, including T cells via circular RNA as described herein. With a CAR, a single receptor may be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor ceils, an immune cell that expresses the CAR may target and kill the tumor cell. In some embodiments, the CAR encoded by the polynucleotide includes (i) an antigen-binding molecule that specifically binds to a target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activating domain.

[0397] In some embodiments, an orientation of tire CARs in accordance with the disclosure includes an antigen binding domain (such as an scFv) m tandem with a costimulatory’ domain and an activating domain. The costimulatory' domain may include one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains may be utilized in tandem.

[0398] CARs may be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, e.g., one or more single chain antibody fragment (scFv). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody’ linked together. See U.S. Patent Nos, 7,741 ,465, and 6,319,494 as well as Eshhar et al.. Cancer Immunol Immunotherapy (1997) 45: 131 -136, An scFv retains the parent antibody’s ability to specifically interact with target antigen. scFvs are useful in chimeric antigen receptors because they may be engineered to be expressed as part of a single chain along with the other CAR components. Id. See also Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et at., Journal of Immunology, 1998, 161: 2791-2797. It will be appreciated that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the present disclosure, with specificity’ to more than one target of interest.

[0399] In some embodiments, the antigen binding molecule includes a single chain, wherein the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N terminus of the linker and the VL is located at the C terminus of the linker. In other embodiments, the VL is located at the N terminus of the linker and the VH is located at the C terminus of the linker. In some embodiments, the linker includes at least 5, at least 8, at least 10, at least 13, at least 15, at least 18, at least 20, at least 2.5, 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.104001 In some embodiments, the antigen binding molecule includes a nanobody. In some embodiments, the antigen binding molecule includes a DARPin. In some embodiments, the antigen binding molecule includes an anticalin or other synthetic protein capable of specific binding to target protein .104011 In some embodiments, the CAR includes an antigen binding domain specific for an antigen selected from CD 19, 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 ((Tn Ag) or (GalNAca-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 (EPC AM), B7H3 (CD276), KIT (CD117), Interleukin- 13 receptor subunit alpha-2, mesothelm. Interleukin 11 receptor alpha (IL-1 IRa), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAI.X), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gplOO), oncogene fission protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type- A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o~ acetyl-GD2 ganglioside (OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 -related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD 179a, anaplastic lymphoma kinase (ALK), Polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH),mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), Hepatitis A vims cellular receptor 1 (HA VCR 1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), Olfactory' receptor 51E2 (OR51E2), TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1), Cancer / testis antigen 1 (NY-ESO-1), Cancer / testis antigen 2 (LAGE-la), MAGE family members (including MAGE-A1, MAGE-A3 and MAGE-A4), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA 17), 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-related antigen 1, tumor protein p53 (p53), p53 mutant, prostem, surviving, telomerase, prostate carcinoma tumor antigen- 1, melanoma antigen recognized by T cells 1 , Rat sarcoma (Ras) mutant, human Telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N -Acetyl glucosaminyl -transferase V (NA 17), paired box protein Pax-3 (PAX3), Androgen receptor, Cyclin Bl, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2 (TRP-2), Cytochrome P450 1B1 (CYP1B1), CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TESl), lymphocyte -specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), Receptor for Advanced Glycation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papilloma vims E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinal carboxyl esterase, heat shock protein 70-2 mutated (mut hsp70-2), CD79a, CD79b, CD72, Leukocyte-associated immunoglobulin -like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), Leukocyte immunoglobulin -like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptorlike 2 (EMR2), lymphocyte antigen 75 (LY75), Glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8119. av'PO integrin, avp6 integrin, alphafetoprotein (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 insert domain receptor (KDR), k-light chain, LI cell adhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4), tumor / testis-antigen IB, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT 10, MART-1, immunoglobulin lambda-like polypeptide 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, haemagglutinin (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 polyoma virus small T antigen, Merkel cell polyoma vims large T antigen, Kaposi sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and KSHV latent nuclear antigen. In some embodiments, an antigen binding domain includes SEQ ID NO: 321 and / or 322.

[0402] In some embodiments, a CAR includes a hinge or spacer domain. In some embodiments, the hinge / spacer domain may include a truncated hinge / spacer domain (THD) the THD domain is a truncated version of a complete hinge / spacer domain (“CHD”). In some embodiments, an extracellular domain is from or derived from (e.g., includes all or a fragment of) ErbB2, glycophorin A (GpA), CD2, CD 3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T CD1 la (IT GAL), CD1 lb (IT GAM), CD1 lc (ITGAX), CDS Id (IT GAD), CD 18 (ITGB2), CD 19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA 1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex- associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD 100 (SEMA4D), CD 103 (ITGAE), CD134 (0X40), 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), CD2.47 (CD3-zeta), 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-stimulator (ICOS), LFA-1 (CD1 la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2.R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG 1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll ligand receptor, and fragments or combinations thereof. A hinge or spacer domain may be derived either from a natural or from a synthetic source.

[0403] In some embodiments, a hinge or spacer domain is positioned between an antigen binding molecule (e.g., an scFv) and a transmembrane domain. In this orientation, the hinge / spacer domain provides distance between the antigen binding molecule and the surface of a cell membrane on which the CAR is expressed. In some embodiments, a hinge or spacer domain is from or derived from an immunoglobulin. In some embodiments, a hinge or spacer domain is selected from the hinge / spacerregions of IgGl, IgG2, lgG3, lgG4, IgA, IgD, IgE, IgM, or a fragment thereof. In some embodiments, a hinge or spacer domain includes, is from, or is derived from the hinge / spacer region of CDS alpha. In some embodiments, a hinge or spacer domain includes, is from, or is derived from the hinge / spacer region of CD28. In some embodiments, a hinge or spacer domain includes a fragment of the hinge / spacer region of CD8 alpha or a fragment of the hinge / spacer region of CD28, wherein tire fragment is anything less than the whole hinge / spacer region. In some embodiments, the fragment of the CD8 alpha hinge / spacer region or the fragment of the CD28 hinge / spacer region includes an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 ammo acids at the N-tenninus or C-Termmus, or both, of the CDS alpha hinge / spacer region, or of the CD28 hinge / spacer region.|0404| The CAR may further include a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. It may similarly be fused to the intracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in a CAR is used. In some instances, the transmembrane domain may be selected or modified (e.g., by an amino acid substitution) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein.

[0405] Transmembrane regions may be derived from (i.e., include) a receptor tyrosine kinase (e.g., ErbB2), glycophorin A (GpA), 4-1BB / CD137, activating NK cell receptors, an immunoglobulin protein, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CDI00 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a. CD49D. CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, GDI 1c, CD1 Id, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1 , ICAM-1, Ig alpha (CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cell costimulator (ICOS), integnns, 1TGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, a ligand that specifically binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function -associated antigen- 1 (LFA-1 ; CDl-la / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death- 1 (PD-1), PSGL1, SELPLG (CD 162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SL.AMF6(NTB-A; LylO8), SLAMF7, SLP-76, INF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VL.A1 , or VLA-6, or a fragment, truncation, or a combination thereof.

[0406] In some embodiments, suitable intracellular signaling domain include, but are not limited to, activating Macrophage / Myeloid cell receptors CSFR1, MYD88, CD 14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN 1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4 / 80, CD91, C1 QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, Lactoferrin, or a fragment, truncation, or combination thereof.|0407| In some embodiments, a receptor tyrosine kinase may be derived from (e.g., include) Insulin receptor (InsR), Insulin-like growth factor I receptor (IGF1R), Insulin receptor-related receptor (IRR), platelet derived growth factor receptor alpha (PDGFRa), platelet derived growth factor receptor beta (PDGFRfi). KIT proto-oncogene receptor tyrosine kinase (Kit), colony stimulating factor 1 receptor (CSFR), fins related tyrosine kinase 3 (FLT3), fins related tyrosine kinase 1 (VEGFR-1), kinase insert domain receptor (VEGFR-2), fins related 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 (R0R1), receptor tyrosine kinase like orphan receptor 2 (R0R2), muscle associated receptor tyrosine kinase (MuSK), MET protooncogene, receptor tyrosine kinase (MET), macrophage stimulating 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto -oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin like and EGF like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor Al (EphAl), EPH receptor A 2 (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 (EphAlO), EPH receptor Bl (EphBl), 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), discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin 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.iyrosine kinase 1 (STYK1).

[0408] In some embodiments, the CAR includes a costimulatory domain. In some embodiments, the costimulatory domain includes 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In a preferred embodiment, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (Q. 4- IBB, CD28, CD3 zeta may include less than the whole 4-1BB, CD28 or CDS zeta, respectively. Chimeric antigen receptors mayincorporate costimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465, and 6,319,494, as well as Krause etal. and Finney etal. (supra}. Song etal., Blood 119:696- 706 (2012); Kalos etal.. Sei Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (201 1), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0409] In some embodiments, a costimulatory domain includes the amino acid sequence of SEQ ID NO: 318 or 320.

[0410] The intracellular (signaling) domain of the engineered T cells disclosed herein may provide signaling to an activating domain, which then activates at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0411] In some embodiments, suitable intracellular signaling domain include (e.g., include), but are not limited to 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 100 (SEMA4D), CD 103, CD 160 (BY55), CD 18, CD19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CDSalpha, CDSbeta, CD96 (Tactile), CD1 la, CD1 lb, GDI 1c, CD1 Id, CDS, CEACAM 1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (L1GHTR), IA4, ICAM-1, Ig alpha (CD79a). IL- 2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1 , ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function- associated antigen- 1 (LFA-1; CDl-la / CD18), MHC class 1 molecule, NKG2.C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX -40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1 ; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

[0412] CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs. In some embodiments, the CD3 is CD3 zeta. In some embodiments, the activating domain includes an amino acid sequence 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.

[0413] In some embodiments, the sequence encoding the CAR includes a sequence from Table 6.(2) T-Cell Receptors (TCR)

[0414] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly; each chain may include variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region may include three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable ( Va) regions and several types of beta chain variable (Vp) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Va types are referred to in IMGT nomenclature by a unique TRAV number. Thus “TRAV21” defines a TCR Va region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an ammo acid sequence which is preserved from TCR to TCR but which also includes an ammo acid sequence which varies from TCR to TCR. In the same way, “TRBV5-1” defines a TCR Vp region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence.

[0415] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature.

[0416] The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD / TRBJ regions are often considered together as the joining region.

[0417] The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The “T cell Receptor Factsbook”, (2001 ) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database.

[0418] Native TCRs exist in heterodimeric ap or y8 forms. However, recombinant TCRs consisting of aa or pp homodimers have previously been shown to bind to peptide MHC molecules. Therefore, the TCR of the present disclosure may be a heterodimeric ap TCR or may be an aa or pp homodimeric TCR,

[0419] For use in adoptive therapy, an ap heterodimeric TCR may, for example, be transfected as full length chains having both cytoplasmic and transmembrane domains. In certain embodiments TCRs of the present disclosure may have an introduced disulfide bond between residues of the respective constant domains, as described, for example, in WO 2006 / 000830.

[0420] TCRs of the present, disclosure, particularly alpha-beta heterodimeric TCRs, may include an alpha chain TRAC constant domain sequence and / or a beta chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chain constant domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. Tire alpha and / or beta, chain constant domain sequence(s) may also be modified by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulfide bond between the alpha and beta constant domains of the TCR.

[0421] Binding affinity (inversely proportional to the equilibrium constant KD) and binding half-life (expressed as TN) can be determined by any appropriate method . It will be appreciated that doubling the affinity of a TCR results in halving the KD. T'A is calculated as In 2 divided by the off-rate (koff). So doubling of TN results in a halving in koff. KD and koff values for TCRs are usually measured for soluble forms of the TCR, i.e., those forms which are truncated to remove cytoplasmic and transmembrane domain residues. Therefore, it is to be understood that a given TCR has an improved binding affinity for, and / or a binding half-life for the parental TCR if a soluble form of that TCR has the said characteristics. Preferably the binding affinity or binding half-life of a given TCR is measured several times, for example 3 or more times, using the same assay protocol, and an average of the results is taken.|0422| Since the TCRs of the present disclosure have utility in adoptive therapy, the present disclosure includes a non-naturally occurring and / or purified and / or or engineered cell, especially a T- cell, presenting a TCR of the present disclosure. There are a number of methods suitable for the transfection of T cells with nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of thepresent disclosure (see for example Robbins et al. , (2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of the present disclosure will be suitable for use in adoptive therapy-based treatment of cancers such as those of the pancreas and liver. As will be known to those skilled in the art, there are a number of suitable methods by which adoptive therapy can be carried out (see for example Rosenberg et al. , (2008) Nat Rev Cancer 8(4): 299-308).

[0423] As is well-known in the art TCRs of the present disclosure may be subject to post-translational modifications when expressed by transfected cells. Glycosylation is one such modification, which may include the covalent attachment of oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine residues, or serine / threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e., oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Glycosylation of transfected TCRs may be controlled by mu tations of the transfected gene (Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations are also encompassed in this disclosure.

[0424] A TCR may be specific for an antigen in the group MAGE -Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-Al l, 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, brain glycogen phosphorylase, Melan-A, MAGE-CI, MAGFAC2, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT- 7, alpha-actmin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA- A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, Lage-1, Mage- C2, NA-88, Lage-2, SP17, and TRP2-Int2, (MART-I), gpl OO (Pmel 17), TRP-1 , TRP-2, MAGE-1 , MAGE-3, p!5(58), CEA, NY-ESO (LAGE), SCP-I, Hom / Mel-40, p53, H-Ras, HER-2 / neu, BCR- ABL, E2A-PRL, H4-RET, IGH-1GK, MYL-RAR, Epstein Barr vims antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p!85erbB2, p!80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1 , NuMa, K-ras, beta- catenin, CDK4, Mum-1, p!6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, a- fetoprotem, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 2.7.29\BCAA), CA 195, CA 242, CASO, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, M0VI8, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding proteinkycloplnlin C-associated protein), TAAL6, TAG72, TLP, and TPS.(3) B-Cell Receptors (BCR)104251 B-cell receptors (BCRs) or B-cell antigen receptors are immunoglobulin molecules that form a type I transmembrane protein on the surface of a B cell. A BCR is capable of transmitting activatory signal into a B cell following recognition of a specific antigen. Prior to binding of a B cell to an antigen, the BCR wall remain in an unstimulated or “resting” stage. Binding of an antigen to a BCR leads to signaling that initiates a humoral immune response.

[0426] A BCR is expressed by mature B cells. These B cells work with immunoglobulins (Igs) in recognizing and tagging pathogens. The typical BCR includes a membrane -bound immunoglobulin (e.g., mlgA, mlgD, mlgE, mlgG, and mlgM), along with associated and Iga / Igp (CD79a / CD79b) heterodimers (a / P). These membrane -bound immunoglobulins are tetramers consisting of two identical heavy and two light chains. Within the BCR, the membrane bound immunoglobulins is capable of responding to antigen binding by signal transmission across the plasma membrane leading to B cell activation and consequently clonal expansion and specific antibody production (Friess M et ai. (2018), Front. Immunol. 2947(9)). The Iga / IgP heterodimers is responsible for transducing signals to the cell interior.

[0427] A Iga / IgP heterodimer signaling relies on the presence of immunoreceptor tyrosine -based activation motifs (ITAMs) located on each of the cytosolic tails of the heterodimers. ITAMs include two tyrosine residues separated by 9-12 amino acids (e.g., tyrosine, leucine, and / or valine). Upon binding of an antigen, the tyrosine of the BCR’s ITAMs become phosphorylated by Src -family tyrosine kinases Blk, Fyn, or Lyn (Janeway C etal., Immunobiology: The Immune System in Health and Disease (Garland Science, 5th ed. 2001 )).(4) Other Chimeric Proteins

[0428] In addition to tire chimeric proteins provided above, the circular RNA polynucleotide may encode for a various number of other chimeric proteins available in the art. The chimeric proteins may include recombinant fusion proteins, chimeric mutant protein, or other fusion proteins.104291 In some embodiments, the circular RNA polynucleotide encodes for an immune modulatory ligand. In certain embodiments, the immune modulatory ligand may be immunostimulatory; while in other embodiments, the immune modulatory' ligand may be immunosuppressive.

[0430] In some embodiments, the circular RNA polynucleotide encodes for a cytokine. In some embodiments, the cytokine includes a chemokine, interferon, interleukin, lyrnphokine, and tumor necrosis factor. Chemokines are chemotactic cytokine produced by a variety of cell types in acute and chronic inflammation that mobilizes and activates white blood cells. An interferon includes a family of secreted a-helical cytokines induced in response to specific extracellular molecules throughstimulation of TLRs (Borden, Molecular Basis of Cancer (Fourth Edition) 2015). Interleukins are cytokines expressed by leukocytes.

[0431] Descriptions and / or amino acid sequences of IL -2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-270, IFNy, and / or TGF01 are provided herein and at the www.uniprot.org database at accession numbers: P60568 (IL-2), P29459 (IL-I2A). P29460 (IL-I2B), P13232 (IL-7), P22301 (IL-10), P40933 (IL-15), Q141 16 (IL-18), Q14213 (11.-270), P01579 (IFNy), and / or P01137 (TGFpi).

[0432] In some embodiments, the circular RNA polynucleotide may encode for a transcription factor. Regulatory T cells (Treg) are important in maintaining homeostasis, controlling the magnitude and duration of the inflammatory response, and in preventing autoimmune and allergic responses.

[0433] In general, Tregs are thought to be mainly involved in suppressing immune responses, functioning in part as a “self-check” for the immune system to prevent excessive reactions. In particular, Tregs are involved in maintaining tolerance to self-antigens, harmless agents such as pollen or food, and abrogating autoimmune disease.

[0434] Tregs are found throughout the body including, without limitation, the gut, skin, lung, and liver. Additionally, Treg cells may also be found 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 have one or more unique features and additional information may 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 disclosure of which is hereby incorporated in its entirety.

[0435] Typically , Tregs are known to require TGF-0 and IL-2 for proper activation and development. Tregs, expressing abundant, amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-0, both potent immune suppressive cytokines. Additionally, Tregs are known to inhibit the ability of antigen presenting cells (APCs) to stimulate T cells. One proposed mechanism for APC inhibition is via CTLA-4, which is expressed by Foxp3+ Tregs. It is thought that CTLA-4 may bind to B7 molecules on APCs and either block these molecules or remove them by causing internalization resulting in reduced availability of B7 and an inability to provide adequate co -stimulation for immune responses. Additional discussion regarding the origin, differentiation and function of Tregs may be found in Dhamne el al. , Peripheral and thymic Foxp3+ regulatory' T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4 (253): 1-11 , the disclosure of which is hereby incorporated in its entirety.104361 As provided herein, in certain embodiments, the coding element of the circular RNA polynucleotide encodes for one or more checkpoint inhibitors or agonists.

[0437] In some embodiments, the immune checkpoint inhibitor is an inhibitor of 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, B7HL B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30. CD40, CD70, CD80, CD86, CD! 37, CD 160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagenous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1, CTL.A4, PD-1, LAG3, PD-L1, TIM3, or combinations 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 IDO L104381 As described herein, at least in one aspect, the present disclosure encompasses 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 rnucin domain 3 (TIM-3). An antagonist of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 interferes with CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 function, respectively. Such antagonists of CTLA-4, PD-1, PDL-1, LAG-3, and TIM-3 can include antibodies which specifically bind to CTLA-4, PD-1, PDL-1 , LAG-3, and TIM-3, respectively and inhibit and / or block biological activity and function.

[0439] In some embodiments, the pay load encoded within one or more of the coding elements is a hormone, FC fusion protein, anticoagulant, blood clotting factor, protein associated with deficiencies and genetic disease, a chaperone protein, an antimicrobial protein, an enzyme (e.g., metabolic enzyme), a structural protein (e.g., a channel or nuclear pore protein), protein variant, small molecule, antibody, nano body, an engineered non-body antibody, or a combination thereof. xi. Production of polynucleotides

[0440] DNA templates can be made using standard techniques of molecular biology. For example, the 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 the polynucleotides from a DMA template known to include the same.

[0441] The various elements of the DNA template can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.. Science (1984) 223: 1299; and Jay et al,, J. Biol. Chem, (1984) 259:631 1.

[0442] Thus, particular nucleotide sequences can be obtained from DNA template harboring the desired sequences or synthesized completely, or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. One method of obtaining nucleotide sequences encoding the desired DNA template elements is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al.. Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally, oligonucleotide- directed synthesis (Jones et al.. Nature (1986) 54:75-82), oligonucleotide directed mutagenesis of preexisting nucleotide regions (Riechmann et al,, Nature (1988) 332:323-327 and Verhoeyen et al.. Science (1988) 239: 1534-1536), and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al.. Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used.

[0443] The precursor RNA can be generated by incubating a DNA template under conditions permissive of transcription of the precursor RNA encoded by the DNA template. For example, in some embodiments a precursor RNA is synthesized by incubating a DNA template provided herein that includes an RNA polymerase promoter upstream of its 5 ’ duplex sequence and / or expression sequences with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the DNA template is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II. xii. Linear mRNA payloads

[0444] In various embodiments, the LNP compositions described herein can be used to deliver an RNA payload that is a linear mRNA molecule.

[0445] In various embodiments, the LNP -based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems, may include one or more linear mRNA molecules or linear mRNA payloads. In various embodiments, the mRN A payloads may encode one or more components of the herein described gene editing systems. For example, an 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 Casl2a, CRISPR Casl2f, CRISPR Cas 13a, CRISPR C las 13b, or TnpB).

[0446] mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and / or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.

[0447] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars atached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., IncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., IncRNA) sequence.104481 In various embodiments, the LNP compositions described herein can be used to deliver a mRNA payload that is a linear mRNA molecule. In embodiments, the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a vaccine antigen, a component, of a gene editing system (e.g., an endonuclease, a prime editor, etc.) and / or a therapeutic protein.

[0449] In some embodiments, the RNA payload may be a linear mRNA. As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo.

[0450] Generally, a mRNA molecule comprises at least a coding region, a 5’ untranslated region (UTR.), a 3’ UTR, a 5’ cap and a poly-A tail. In some aspects, one or more structural and / or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized m a nucleic acid without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.

[0451] Generally, a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the mRNA may encode a peptide of 2.-30 amino acids, e.g. 5-30, 10-30, 2.-25, 5-25, 10-25, or 10-2.0 amino acids. The mRNA may encode a peptide of at least 10, 1 1 , 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ammo acids, or a peptide that is no longer 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.

[0452] Generally, the length of the region of the mRNA encoding a product of interest is greater than about 30 nucleotides in length (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, 1,400, 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).

[0453] In some embodiments, the mRNA has a total length that spans from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1 ,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1 ,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides),

[0454] In some embodiments, the region or regions flanking the region encoding the product of interest may range independently from 15-1,000 nucleotides in length (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, 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).

[0455] In some embodiments, the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90. 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined m units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional .

[0456] In some embodiments, the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap. The capping sequence may be from 1 to 10, e.g. 2-9, 3- 8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the caping sequence is absent.

[0457] In some embodiments, the mRNA comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0458] In some embodiments, the mRNA comprises a region comprising a stop codon. The region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0459] In some embodiments, the mRNA comprises a region comprising a restriction sequence. The region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.Untranslated Regions (UTRs)

[0460] In various embodiments, the mRNA payloads of the LNP compositions described herein, may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and / or is incorporated within the mRNA molecule. UTRs are transcribed by not translated. The mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs.

[0461] The RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where nucleic acids are designed to encode at least one polypeptide of interest, tire nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles play ed by the UTRs in terms of stability’ of the nucleic acid molecule and translation. The regulator}' features of a UTR can be incorporated into the RNA payload molecules (e.g., linear and circular mRNA molecules) of the present disclosure to, among oilier things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down -regulation of the transcript in case they are misdirected to undesired organs sites, A variety of 5 'UTR and 3 'UTR sequences are known and available in the art.

[0462] In various embodiments, the mRNA payloads of the LNP compositions described herein, may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S, Patent No. 10,709,779, which is incorporated herein by reference.5 ’ UTR regions

[0463] In various embodiments, the mRN A payloads of the LNP compositions described herein, may comprise at least one 5' UTR.

[0464] A 5' UTR. is region of an mRNA that is directly upstream (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5' UTR does not encode a protein (is non-coding). Natural 5'UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved m the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5 'UTR also have been known to form secondary structures which are involved in elongation factor binding. 5’ UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6). In addition, 5’ UTR sequences may confer increased half-life, increased expression and / or increased activity of a polypeptide encoded by the RNA pay load described herein.

[0465] In various embodiments, the RNA payload constructs contemplated herein may include 5 ’UTRs that are found in nature and those that are not. For example, the 5 ’UTRs can be synthetic and / or can be altered in sequence with respect to a naturally occurring 5 ’UTR. Such altered 5 ’UTRs can include one or more modifications relative to a naturally occurring 5 ’UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide.104661 The 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 ‘ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulator}' role in terms of translation and stability of the nucleic acid.104671 Natural 5’ UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5 ’UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0468] In some embodiments of the disclosure, a 5' UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different mRNA. In another embodiment, a 5' UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5' UTRs include Xenopus or human derived alpha-globin or beta-globin (e.g., US8.278.063 and US9,012,219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus. CMV immediate -early 1 (IE!) gene (see US20140206753 and WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 29) (WO2014144196) may also be used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO / 2015101414, W02015101415, WO / 2015 / 062738,WO2015024667, WO2015024667; 5’ UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015101414, W02015101415, WO / 2015 / 062738)), 5' UTR element derived from the 5 'UTR of an hydroxysteroid (17-P) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from die 5' UTR of ATP5A1 (WO2015024667) can be used. In one embodiment, an internal ribosome entry site (IRES) is used as a substitute for a 5' UTR.3 ’ UTR regions

[0469] In various embodiments, the mRNA payloads of the LNP compositions described herein, may comprise at least one 3' UTR. 3' UTRs may be heterologous or synthetic.

[0470] A 3’ UTR is region of an mRN A that is directly downstream (3') from die stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3' UTR does not encode a protein (is non-coding). Natural or wild type 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all tire three classes. Engineering tire HuR specific binding sites into tire 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0471] 3’ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3 ’ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0472] Introduction, removal or modification of 3’ UTR AU rich elements (AREs) can be used to modulate the stability of the mRNA payloads described herein. For example, one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein. Alternatively, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of tire resultant protein.

[0473] In some embodiments, the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and / or tissue. As a non-limiting example, the feature can be a UTR. As another example, the feature can be introns or portions of introns sequences.

[0474] Those of ordinary skill in the art will understand that 5' UTRs that are heterologous or synthetic may be used with any desired 3' UTR sequence. For example, a heterologous 5' UTR may be used with a synthetic 3' UTR with a heterologous 3' UTR.

[0475] Non-UTR sequences may also be used as regions or subregions within an RNA payload construct. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

[0476] Combinations of features may be included in flanking regions and may be contained within other features. For example, the polypeptide coding region of interest in an mRNA payload may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and / or a 3' UTR which may include an oligo(dT) sequence for ternplated addition of a poly-A tail. 5' UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes such as the 5’ UTRs described in US Patent Application Publication No. 20100293625 and PCT / US2014 / 069155, herein incorporated by reference in its entirety104771 It should be understood that any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA). Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3’ UTR may be inverted, shortened, lengthened, made with one or more other 5’ UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' UTR or 5' UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

[0478] In some embodiments, a double, triple or quadruple UTR such as a 5’ UTR or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety .

[0479] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter. A, B, or C represent a different UTR at tire nucleotide level.

[0480] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0481] The untranslated region may also include translation enhancer elements (TEE). As a nonlimiting example, the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.5 ’ Capping

[0482] In various embodiments, the mRNA pay loads of the LN P compositions described herein, may comprise a 5’ cap structure.

[0483] The 5’ cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5 ’ proximal introns removal during mRNA splicing.104841 Endogenous mRNA molecules may be 5 ’-end capped generating a 5 ’-ppp-5 ’-triphosphate linkage between a terminal guanosine cap residue and the 5 ’-terminal transcribed sense nucleotide of the mRNA molecule. This 5’-guanylate cap may then be methylated to generate an N7-methyl- guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5’ end of the mRNA may optionally also be 2’-0-methylated. 5 ’-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0485] Modifications to mRNA may generate a non -hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 ’-ppp-5’ phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio- guanosine nucleotides according to the manufacturer’s instructions to create a phosphorotluoate linkage in the 5 ’-ppp-5’ cap.

[0486] Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides .

[0487] Additional modifications include, but are not limited to, 2’-0-methylation of the ribose sugars of 5 ‘-terminal and / or 5’-anteterminal nucleotides of the mRNA (as mentioned above) on the Azhydroxyl group of the sugar ring. Multiple distinct 5 ‘-cap structures can be used to generate the 5 ‘-cap of a nucleic acid molecule, such as an mRNA molecule.

[0488] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wildtype or physiological) 5 ’-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non -enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule.

[0489] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 ‘-5 ‘-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3 ’-0-methyl group (i.e., N7,3’-0-dimethyl-guanosine-5’-triphosphate-5 ‘-guanosine (m'G-3’mppp-G; which may equivalently be designated 3’ O-Me-m7G(5’)ppp(5’)G). The 3’-0 atom of the other, unmodified, guanine becomes linked to the 5 ’-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA). The N7- and 3’-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA).

[0490] Another exemplary cap is mCAP, which is similar to ARCA but has a 2’-0-methyl group on guanosine (i .e ., N7,2 ’ -0-dimethyl -guanosine-5 ’ -triphosphate-5 ’ -guanosine , m?Gm -ppp-G) .

[0491] While cap analogs allow' tor the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 ’-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.104921 mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5 ’-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiologicalcellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 "cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’ endonucleases and / or reduced 5 ’decapping, as compared to synthetic 5 ‘cap structures known in the art (or to a wild-type, natural or physiological 5 ‘cap structure). For example, recombinant Vaccinia Vims Capping Enzyme and recombinant 2’-0-methyltransferase enzyme can create a canonical 5 ‘-5 ‘~ triphosphate linkage between the 5 ‘-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 ‘-terminal nucleotide of the mRNA contains a 2’-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational -competency and cellular stability and a reduced activation of cellular pro-inflammatory' cytokines, as compared, e.g., to other 5 ‘cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5”)- ppp(5’)NlmpN2mp (cap 2).

[0493] In some embodiments, the 5’ terminal caps may include endogenous caps or cap analogs.|0494| In some embodiments, a 5’ terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2 ’fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA -guanosine, and 2-azido-guanosine.IRES Sequences104951 In various embodiments, the mRNA payloads of the LNP compositions described herein, may comprise one or more IRES sequences.Poly-A tails and 3 ’stabilizing region

[0496] In various embodiments, the mRNA payloads of the LNP compositions described herein, may comprise a poly-A tail.

[0497] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately' after transcription, the 3 ’ end of the transcript may be cleaved to free a 3 ’ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the free 3’ hydroxyl end. The process, called polyadenylation, adds a poly-A tail of a certain length.

[0498] In some embodiments, the length of a poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (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. 1,000, 1,100, 1,200, 1,300, 1,400, 1.500. 1,600, 1,700, 1,800, 1,900, 2,000, 2.500. and 3,000 nucleotides) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length. In some embodiments, the mRNA includes a poly-A tail fromabout 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1 ,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1 ,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1 ,000 to 3,000, from 1,500 to 2,000, from 1 ,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0499] In some embodiments, the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA.

[0500] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of mRN A for poly-A binding protein may enhance expression.

[0501] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3 ’-end using modified nucleotides at the 3 '-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.

[0502] In some embodiments, the mRNA are designed to include a polyA-G Quartet, The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. Stop Codons

[0503] In various embodiments...

Claims

WHAT IS CLAIMED IS1 . A PEG lipid of Formula VI:wherein:P is a PEG moiety;YBis an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, - \H( (())()-. and -NHC(O)NH-;L is an optional linker selected from optionally substituted Cl -CIO alkylene, optionally substituted Cl -CIO heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene;YAis selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -XHC (())()-. and - NHC(O)NH-; m is an integer selected from 1 to 10; andR2and R3are each independently optionally substituted Cl -C l 5 alkyl or optionally substituted C2-C15 alkenyl.The PEG lipid of claim 1, wherein:z is an integer selected from 1 to 2.00;R is hydrogen or methyl ; andR2and R3are each independently optionally substituted Cl -Cl 5 aliphatic.

3. The PEG lipid of claim 1 or claim 2, wherein:YAis -OC(O)-; andYBis absent.

4. The PEG lipid of claim 1 or claim 2, wherein:YAis -OC(O)-; andYBis -( (())()-.

5. The PEG lipid of any one of claims 1-4, wherein L is optionally substituted Cl -CIO alkylene.

6. The PEG lipid of claim 1 or claim 2, wherein:YAis -OC(O)-; andL-YBIS absent.

7. The PEG lipid of any one of claims 1 -6, wherein R2and R3are each independently optionally substituted C1-C15 alkyl (e.g., optionally substituted C4-C11 alkyl or optionally substituted C6-C11 alkyl).

8. The PEG lipid of any one of claims 1-6, wherein R2and R are each independently unsubstituted C1-C15 alkyl (e.g., unsubstituted C4-C11 alkyl or unsubstituted C6-C11 alkyl).

9. The PEG lipid of any one of claims 1 -8, wherein R2and R3are each independently selected1 1 . 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 of any one of claims 1-11, wherein ni is an integer selected from 1 to 9.

13. The PEG of claim 1 , wherein the PEG lipid is selected from:wheresn z is selected from 1 to 200, e.g., from 30 to 75.

13. A transfer vehicle (e.g., lipid nanoparticle (LNP)) comprising a PEG lipid of any one of claims 1-12.

14. The transfer vehicle of claim 13, further comprising an ionizable lipid of Formula AX””:Formula (AX””) or a pharmaceutically acceptable salt thereof, wherein:A is selected from an optionally substituted bridged carbocyclic bicycle, bridged carbocyclic multicycle, bridged heterocyclic bicycle, or bridged heterocyclic multicycle; n is an integer selected from 0, 1, and 2; m is ail integer selected from 1 and 2, such that m plus n is less than or equal to 3;R1is selected from -OH, -OAc, -NR2, -N(R)RH,each R is independently -H or Ci-CT aliphatic; each RHis C1-C6 aliphatic-OH; each X!and XAis independently a bond or optionally substituted Ci-Cs aliphatic; eachY’ is independently selected fromR, and a bond; wherein the bond marked with anc’*” is attached to X1; each X2and X3is independently a bond or optionally substituted Ci-CY aliphatic; each Y2and Y is independently selected fromandwherein the bond marked with an is atached to X3or X3; each X4and X3is independently a bond or optionally substituted C1-C5 aliphatic; each Y4and Y5is independently selected from a bond.R and; wherein the bond marked with an ’ is attached to X’ or X : each X6and X7is independently a bond or optionally substituted Ci -Ce aliphatic;R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -CF(R6)(R7), -R10, optionally substituted Cs-Csg aliphatic, or optionally substituted Ci-C-;4 aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C?-Cs cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)~, -OC(O)O-, -OC(O)~, -NHC(O)-, or -C(O)O-; each R3is independently -CH(ORS)(ORS), -CH(SRs)(SRs), -CH(RS)(R9), -CF(RS)(R9), -Rn, optionally substituted Cs-Cis aliphatic, or optionally substituted C1-C14 aliphatic-R1’1, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C?-Cs cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, - OC(O)O-, -OC(O)-, -M R (())-. or -C(O)C)-;R6and R7are each independently optionally substituted -Ci-CY aliphatic, -Rlu, or optionally substituted -CrCY aliphatic-R10; wherein one or more methylene linkages of R° and R7are each optionally and independently replaced with an optionally substituted Cs-Ck cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or - C(O)O-;Rsand R9are each independently optionally substituted -C1-C14 aliphatic, -R”, or optionally substituted -C)-Ci4 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted Cj-Cs cycloalkylenyl, phenyl, -()-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or - C(O)O-; and each Rluand R11is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic, or bridged multicyclic C4-C;4 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic, or bridged multicyclic 4-14 membered heterocyclyl, or two Rluor two R1 !taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl; wherein one or more of X1, XA, X2, X3, X4, X3, X°, X7, R2, and R3is optionally and independently substituted with one or more substituents selected from -F, -Cl, -Br and -I. , The transfer vehicle of claim 14, wherein the ionizable lipid is of Formula AX-T’ :Formula (AX-T’) or a pharmaceutically acceptable sal t thereof, wherein:R1is selected from -OH, -OAc, -NR2,each R is independently -H or Ci-Cg aliphatic;X1and XAare each independently a bond or optionally substituted C1-C6 aliphatic;X2and X3are each independently a bond or optionally substituted C1-C12 aliphatic;X4and X5are each independently optionally substituted Ci-Cg aliphatic;R° and R7are each independently optionally substituted -CI-CM aliphatic, -Rlu, or optionally substituted -Ci-Cu aliphatic-R10; wherein one or more methylene linkages of R° and R / are each optionally and independently replaced with an optionally substituted Cs-Ck cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or - C(O)O-;R8and R9are each independently optionally substituted -CI-CM aliphatic, -Rl!, or optionally substituted -CI-CM aliphatic-R1wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted Cs-Cs cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)~, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; and each R10and R1!is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R!l!or two R!1taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4- 14 membered heterocyclyl.

16. The transfer vehicle of claim 15, wherein the ionizable lipid is selected from:

17. The transfer vehicle of any one of claims 13-16, further comprising a RNA polynucleotide.

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

19. The transfer vehicle of any one of claims 13-18, further comprising a further PEG lipid, a structural lipid, a non-ionizable lipid, or a combination thereof20. The transfer vehicle of claim 19, wherein the transfer vehicle further comprises a further PEG lipid.21 . Tire transfer vehicle of claim 20, wherein the further PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

22. The transfer vehicle of claim 19, wherein the transfer vehicle further comprises a structural lipid.

23. The transfer vehicle of claim 22, wherein tire structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tom ati dine, ursolic acid, and alpha-tocopherol.

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

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

26. The transfer vehicle of claim 25, wherein the phospholipid is selected from 1,2-di stearoyl -sn- glycero-3 -phosphocholine (DSPC), l ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocho line (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3- phosphocholine, l,2-diarachidonoyi-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, l,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyi-sn-glycero-3-phosphoethanoiamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyi-sn-glycero-3 -phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn- glycero-3~phospho-rac-(l -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2- (o1eoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-a-phosphatidylserine; BrainPS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl -phosphatidylethanolamme4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG),1.2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), di stearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), di stearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3 -phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3 -(oleoyloxy jpropyl) phosphate (18: 1 DMP; LBPA), l,2-dioleoyl-sn-glycero-3-phospho-(l ’-myo-inositol) (DOPI; 18: 1 PI),1.2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), l,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18: 1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L~serine (18:0-18: 1 PS), 1 -stearoyl -2 -linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), l-oleoyl~2-hydroxy-sn-glycero~3-phospho~L-serine (18:1 Lyso PS), l-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate). Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3~hydroxy-2-stearamidooctadec- 4-en-l-yl (2-(trimethylammonio)ethyl) phosphate), Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3-hydroxy-2-tricosananridooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro-sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D- erythro-sphingosylphosphorylcholine), 16: 1 SM (N-pahnitoleoyl-D-erythro- sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D-erythro- sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24: 1 SM (N- nervonoyl-D-erythro-sphingosylphosphorylcholine), 24:0 SM (N-lignoceroyl-D-erythro- sphingosylphosphorylcholine), 18: 1 SM (N-oleoyl-D-erythro-sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erythro-sphingosylphosphorylcholine), 06:0 SM (N-hexanoyl-D-erythro- sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D-erythro-sphingosylphosphorylcholme), 3-O- methyl Lyso SM (3-O-methyl-spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O- methyl-N-methyl-spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphorylchohne)sphingomyelin.

27. The transfer vehicle of claim 19, comprising:(a) about 0.5 mol% to about 10 mol% of PEG lipid;(b) about 0.5 mol%to about 50 mol% structural lipid;(c) about 5 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid); and(d) about 30 mol% to about 60 mol% ionizable lipid.

28. The transfer vehicle of claim 27, comprising:(a) about 0.5 mol% to about 5 mol% of PEG lipid;(b) about 20 mol% to about 45 mol% structural lipid(c) about 5 mol% to about 15 mol% or about 35 mol% to about 45 mol% non -ionizable lipid (e.g., phospholipid);(d) about 30 mol% to about 40 mol% or about 45 mol% to about 55 mol% ionizable lipid.

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

30. Tire transfer vehicle of any one of claims 17-29, wherein the molar ratio of ionizable lipid to the number of phosphate groups in the RNA (IL:P ratio) is from about 2: 1 to about 30: 1.31 . The transfer vehicle of claim 30, wherein the IL.:P ratio is from about 2: 1 to about 8: 1 .

32. A method of treating and / or preventing a condition in a subject in need thereof comprising administering to the subject an effective amount of pharmaceutical composition comprising a transfer vehicle of 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 carcinoma); bone cancer; brain cancer (e.g., medulloblastoma and glioblastoma multiforme); breast cancer; cancer of the anus, anal canal, or anorectum; cancer of the eye; cancer of the intrahepatic bile duct; cancer of the joints; cancer of the neck; gallbladder cancer; cancer of the pleura; cancer of the nose, nasal cavity, or middle ear; cancer of the oral cavity; cancer of the vulva; chronic lymphocytic leukemia; chronic myeloid cancer; colon cancer; esophageal cancer, cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumor; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin lymphoma; hypopharynx cancer; kidney cancer; larynx cancer; leukemia; liquid tumors; lipoma; liver cancer; lung cancer (e.g., non-small cell lung carcinoma, lung adenocarcinoma, and small cell lung carcinoma); lymphoma; mesothelioma; mastocytoma; melanoma; multiplemyeloma; nasopharynx cancer; non-Hodgkin lymphoma; B-chronic lymphocytic leukemia; hairy cell leukemia; Burkit’s lymphoma; ovarian cancer; pancreatic cancer; cancer of the peritoneum; cancer of the omentum; mesentery cancer; pharynx cancer; prostate cancer; rectal cancer; renal cancer; skin cancer; small intestine cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; teratoma; testicular cancer; thyroid cancer; and ureter cancer.

35. Tire 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,Grave’s disease, Crohn’s disease, Sjogren’s disease, multiple sclerosis, Hashimoto’s disease, psoriasis, myasthenia gravis, autoimmune polyendocrinopathy syndromes. Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and the generalized autoimmune diseases typified by human Lupus.

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

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

39. The method of claim 38, wherein the hemoglobinopathy is selected from sickle cell disease and beta-thalassemia.

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