Guanidinylated serinol polymeric nucleic acid transporters

Abstract

Provided are compounds in the form of copolymers derived from lipid-functionalized carbonate monomers and guanidinylated serinol monomers, which are useful for the delivery of nucleic acids to cells and tissues. Also provided are related compositions and methods.

Description

Docket No. 6045.0589WO | S23-281 GUANIDINYLATED SERINOL POLYMERIC NUCLEIC ACID TRANSPORTERS GOVERNMENT SUPPORT STATEMENT

[0001] This invention was made with Government support under contracts CA031845 and CA245533 awarded by the National Institutes of Health. The Government has certain rights in the invention. FIELD

[0002] The present invention relates to compositions and methods for nucleic acid delivery to cells and tissues and related biomedical applications. BACKGROUND

[0003] Nucleic acid based therapeutics have emerged as a new class of medicine thanks to the rapid development of technology in recent years. Among the different types of RNA medicines, mRNA has received significant attention, evident by the FDA approval of COVID-19 mRNA vaccines and multiple therapeutic assets in active clinical trials, spanning across various biomedical applications including genome editing, cancer, vaccines, and infectious diseases. Despite the enormous progress, naked mRNA is large and polyanionic, unable to efficiently cross non-polar biological barriers such as plasma membrane and reach cytosol to elicit its function in vivo. Moreover, disease phenotypes in which mRNA therapeutics take effect typically happen in specific cell types in selective organs. As a result, to realize the full potential of mRNA therapeutics in vivo requires efficient mRNA transporters with precise organ- and cell-selectivity. The most studied mRNA delivery system so far is lipid nanoparticles (LNPs), but current clinically utilized LNP formulations are restricted largely to liver delivery (when administered intravenously) and to local delivery (when administered intramuscularly). Efficient and selective delivery of therapeutic nucleic acids, including mRNA, to other organs such as lungs and lymphatic organs remains challenging. Accordingly, there is a need for delivery systems that are able to deliver their nucleic acid cargo, including mRNA, to other organs and tissues. BRIEF SUMMARY

[0004] The present invention provides polymer-based nucleic acid delivery vehicles derived from lipid-functionalized carbonate monomers and guanidinylated serinol monomers, andDocket No. 6045.0589WO | S23-281 related compositions and methods. The polymeric compounds are polycationic at low pH (~5.5) and effectively form complexes with polyanionic nucleic acids. Under physiological conditions (pH ~7.4), the once positively-charged guanidinium moieties of the polymer compounds undergo an irreversible and unique O-to-N acyl shift that neutralizes the cationic charges thereby releasing the polyanionic cargo. The chemical structures of the compounds are generally depicted herein with a guanidine (neutral) moiety; however it is understood that the guanidine moiety is positively-charged (protonated) at low pH and is converted to a neutral moiety at higher pH. The compounds disclosed herein can be used to deliver nucleic acids to cells and tissues in vitro, ex vivo, or in vivo. The compounds may also be used to deliver nucleic acids to specific cells and tissues, in part by adjusting the charge ratio of the complexes as described herein. Also provided are related compositions, including pharmaceutical compositions, and methods, including methods of delivering nucleic acids into cells and tissues, and related therapeutic methods.

[0005] Provided are compounds of Formula I: R1-X-[(L1)x1-(L2)x2-(A1)y1]-R2 (Formula I) where R1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; X is a bond, O, S, N or a linking group as defined herein; L1 and L2 are each independently a lipid carbonate monomer unit; A1 is a guanidinylated serinol monomer unit; x1 and y1 are each independently greater than zero and less than 100; x2 and y2 are each independently zero or less than 100; and R2 is H, C2-C24 alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl,Docket No. 6045.0589WO | S23-281 optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-6 alkyl, and carboxylato.

[0006] In aspects, L1 and L2 are each independently a lipid-functionalized methyl- trimethylene carbonate (MTC) unit having a structure ofa structure ofwhich may be fully saturated, mono- or polyunsaturated. In some aspects, RL is independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

[0007] In aspects, A1 is a guanidinylated serinol monomer unit having a structure of

[0008] In an aspect, X is O; L1 and L2 are each an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ia:Docket No. 6045.0589WO | S23-281Ia where R1 is substituted or unsubstituted C1-C24alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1and RL2are each independently a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated; x1 and x2 are each independently 1-100, 1-50, or 1-25; and y1 is 1-100, 1-50, or 1-25.

[0009] In aspects of a Formula Ia compound, R1 is benzyl and RL1and RL2are each independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

[0010] In aspects, a Formula Ia compound is selected from a compound designated GS-1a, GS-1b, or GS-1c, as described herein.

[0011] In an aspect, X is O; L1 is an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ib: IbDocket No. 6045.0589WO | S23-281 where R1 is substituted or unsubstituted C1-C24alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1is a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25; x2 is zero; and y1 is 1-100, 1-50, or 1-25.

[0012] In aspects of a Formula Ib compound, RL1 is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

[0013] In aspects, a Formula Ib compound is selected from a compound designated GS-2a, GS-2b, or GS-2c, as described herein.

[0014] In an aspect, X is O; L1 is a bAC unit; A1 is a GSer unit and the compound is represented by Formula Ic: Icwhere R1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H;Docket No. 6045.0589WO | S23-281 RL1 is a branched or unbranched C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25; x2 is zero; and y1 is 1-100, 1-50, or 1-25.

[0015] In aspects of a Formula Ic compound, RL1is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

[0016] In aspects, a Formula Ic compound is selected from a compound designated GS-3a, GS-3b, or GS-3c, as described herein.

[0017] Also provided are compositions and pharmaceutical compositions including complexes of a compound of Formula I, or a subformula thereof, non-covalently associated with a nucleic acid or a plurality of different nucleic acids. In aspects, the nucleic acid is RNA or DNA. In aspects, the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

[0018] The composition or pharmaceutical composition may also include where the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent.

[0019] The composition or pharmaceutical composition may also include where the composition is a vaccine.

[0020] The composition or pharmaceutical composition may also include where the nucleic acid is mRNA.

[0021] Also provided are methods of transfecting a nucleic acid into a cell in vitro or ex vivo, the methods including contacting the cell in vitro or ex vivo with a composition or pharmaceutical composition including complexes of a compound of Formula I, or a subformula thereof, non-covalently associated with a nucleic acid or a plurality of different nucleic acids. The methods may also include delivering a nucleic acid to a cell of a subject. In some aspects, the methods may include where the cell is a pulmonary endothelial cell, a pulmonary immuneDocket No. 6045.0589WO | S23-281 cell, or a splenic macrophage. The methods may also include where the complexes comprise a compound of Formula 1a, Formula 1b, or Formula 1c.

[0022] In an aspect, provided is a method of delivering a nucleic acid to the lungs of a subject, where the method includes administering to the subject a composition or pharmaceutical composition including complexes of a compound of Formula I, or a subformula thereof, non- covalently associated with a nucleic acid or a plurality of different nucleic acids. The method may also include where the complexes have a 25:1 (cation: anion) charge ratio. The method may also include where the complexes comprise a compound selected from the group consisting of GS-1a, GS-1b, GS-2a, and GS-3a.

[0023] In an aspect, provided is a method of delivering a nucleic acid to the lungs or spleen of a subject, where the method includes administering to the subject a composition or pharmaceutical composition including complexes of a compound of Formula I, or a subformula thereof, non-covalently associated with a nucleic acid or a plurality of different nucleic acids. The method may also include where the complexes of the composition have a 10:1 (cation: anion) charge ratio. The method may also include where the complexes comprise a compound selected from the group consisting of GS-2a, GS-2b, and GS-3a.

[0024] In an aspect, provided is a method of delivering a nucleic acid to the spleen of a subject, where the method includes administering to the subject a composition or pharmaceutical composition including complexes of a compound of Formula I, or a subformula thereof, non-covalently associated with a nucleic acid or a plurality of different nucleic acids. The method may also include where the complexes of the composition have a 4:1 (cation: anion) charge ratio. The method may also include where the complexes comprise a compound selected from the group consisting of GS-1a, GS-2a, GS-2b, and GS-3a.

[0025] Also provided are methods for treating a respiratory disease in a subject, where the methods include delivering a therapeutic nucleic acid to the lungs of a subject according to the methods described herein.

[0026] Also provided are methods for treating an inflammatory disease in a subject, where the methods include delivering a therapeutic nucleic acid to the spleen of a subject according to the methods described herein. In some aspects, the therapeutic nucleic acid may encode an anti- inflammatory protein, optionally interleukin 10 (IL-10) or apolipoprotein E (ApoE).Docket No. 6045.0589WO | S23-281

[0027] Also provided are methods for treating cancer in a subject, where the methods include delivering a therapeutic nucleic acid to the spleen of a subject according to the methods described herein.

[0028] Also provided are methods for treating cancer in a subject, where the methods include delivering a therapeutic nucleic acid to the lungs of a subject according to the methods described herein.

[0029] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims. BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG.1 illustrates a reaction scheme for synthesis of a small library of GSer copolymers.

[0031] FIG.2 illustrates the synthesis of model compounds 4, 5, and 6 utilized to investigate the mechanism of degradation.

[0032] FIG.3 illustrates the degradation scheme and kinetics of mono-carbonate 5 at neutral pH and characterization of the resulting products as monitored with1H NMR by the shift of benzylic hydrogens on alcohol 7 using dimethyl sulfone (MSM) as an internal standard.

[0033] FIG.4 illustrates NMR spectra showing peaks corresponding to compounds 5 (a) and 7 (a') over time in D2O (pH 7.6).

[0034] FIG.5 illustrates the degradation of di-carbonate 6 at neutral pH via a 2-step cyclization cascade to form 8, which occurs with the formation of intermediate 10. To minimize carbonate hydrolysis, the degradation of 6 was carried out under anhydrous conditions (methylene chloride solvent) with triethylamine as the base.

[0035] FIG.6 is a line graph showing change in size (nm) over time (min) of two representative polymer / mRNA complexes, GS-3a (circles) and GS-3b (squares), prepared by simple mixing of the polymers and mRNA at a 10:1 charge ratio for 20 seconds in a phosphate buffered saline solution at acidic pH (PBS pH 5.5).

[0036] FIG.7 is a line graph showing change in surface charge (Zeta potential, mV) over time (min) of two representative polymer / mRNA complexes GS-3a (circles) and GS-3b (squares) prepared by simple mixing of the polymers and mRNA at a 10:1 charge ratio for 20 seconds in a phosphate buffered saline (PBS) solution at either acidic pH (pH 5, filled circles / squares) or physiological pH (pH 7.5, open circles / squares).Docket No. 6045.0589WO | S23-281

[0037] FIG.8 is a scatter plot illustrating eGFP expression as measured by flow cytometry following incubation of representative polymer / mRNA complexes with Chinese Hamster Ovary cells in vitro (left to right: GS-1a, GS-1b, GS-1c, GS-2a, GS-2b, GS-2c, GS-3a, GS-3b, GS-3c).

[0038] FIG.9 is a bar graph illustrating performance (percentage (%) eGFP gene expression) of compound GS-3b compared to a commercial transfection reagent, Lipofectamine 2000, and a reference polymeric compound, ONA CART.

[0039] FIG.10 is a schematic showing the experimental protocol for evaluating the ability of the compounds to deliver mRNA in vivo using a mouse system.

[0040] FIG.11 is a scatter plot illustrating change in surface charge (Zeta potential, mV) over time (min) of lung tropic (circles) and spleen tropic (squares) polymer / mRNA complexes under physiological conditions (PBS pH 7.5).

[0041] FIG.12 illustrates three line graphs showing change in surface charge (Zeta potential, mV) over time (min) of polymer / mRNA complexes in buffered solution at physiological pH (PBS pH 7.5) for two charge ratios. From left to right: GS-1a (10:1 solid line, 4:1 dashed line), GS-2a (25:1 solid line, 4:1 dashed line), and GS-3a (25:1 solid line, 4:1 dashed line).

[0042] FIG.13 illustrates three line graphs showing change in surface charge (Zeta potential, mV) over time (min) of polymer / mRNA complexes in buffered solution at physiological pH (PBS pH 7.5) for two charge ratios. From left to right: GS-1b (25:1 solid line, 10:1 dashed line), GS-2b (10:1 solid line, 4:1 dashed line), and GS-3b (25:1 solid line, 10:1 dashed line).

[0043] FIG.14 is a bar graph illustrating particle size (nm) of polymer / mRNA complexes after incubation with mouse serum albumin (MSA). Charge ratios shown in parentheses. From left to right: RNA alone, GS-1a (10:1), GS-1a (4:1), GS-1b (25:1), GS-1b (10:1), GS-2a (25:1), GS-2a (4:1), GS-2b (10:1), GS-2b (4:1), GS-3a (25:1), GS-3a (4:1), GS-3b (25:1), GS-3b (10:1).

[0044] FIG.15 a picture of representative SDS-PAGE gel showing protein associated with polymer / mRNA complexes. From left to right: molecular weight markers, empty lane, 1.3 ug GS-3a (10:1), 15.5 ug GS-3b (25:1), and 690 ng RNA alone.

[0045] FIG.16 is a pari of bar graphs showing nanoparticle size of GS-CART / mRNA complexes with and without mouse plasma. Top panel shows lung-tropic GS-CARTs and bottom panel shows spleen-tropic GS-CARTs, n=3, bars represent SD.Docket No. 6045.0589WO | S23-281

[0046] FIG.17 is a scatter plot illustrating change in surface charge (Zeta potential, mV) over time (min) of lung tropic and spleen tropic polymer / mRNA complexes incubated with mouse serum albumin (MSA).

[0047] FIG.18 illustrates the results of a principal component analysis (PCA) by integrating particle size and surface charge to identify the most significant attributes of the complexes leading to distinct organ tropisms.

[0048] FIG.19 shows the in vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes for representative lung tropic compounds. Charge ratios are also indicated.

[0049] FIG.20 shows the in vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes for representative spleen tropic compounds. Charge ratios are also indicated.

[0050] FIG.21 shows dose-dependence of in vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes of GS-3a complexed with luciferase mRNA administered at 2 ug, 5 ug, or 7.5 ug luciferase RNA, corresponding to doses of about 0.1 mg / kg, or 0.25 mg / kg or 0.375 mg / kg.

[0051] FIG.22 shows dose-dependence of in vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes of GS-3b complexed with luciferase mRNA administered at 2 ug, 5 ug, or 7.5 ug luciferase RNA, corresponding to doses of about 0.1 mg / kg, or 0.25 mg / kg or 0.375 mg / kg.

[0052] FIG.23 shows protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes of GS-3a complexed with 0.25 mg / kg luciferase mRNA, or a reference compound complexed with the same amount of luciferase mRNA. The reference compounds were a commercially available mRNA delivery reagent (JetRNA) and a reference polymeric transporter, ONA CART.

[0053] FIG.24 shows protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes of GS-3b complexed with 0.25 mg / kg luciferase mRNA, or a reference compound complexed with the same amount of luciferase mRNA. The reference compounds were a commercially available mRNA delivery reagent (JetRNA) and a reference polymeric transporter, ONA CART.Docket No. 6045.0589WO | S23-281

[0054] FIG.25 is a graphical representation of experiments performed to investigate the types of cells being transfected within the lungs and spleens of the Ai14 mice.

[0055] FIG.26 shows expression (measured as % TdTomato positive cells) in spleen and lung following delivery of polymer / mRNA complexes of GS-3a formulated in a 25:1 charge ratio with mRNA-Cre.

[0056] FIG.27 shows expression (measured as % TdTomato positive cells) in spleen and lung cells following delivery of polymer / mRNA complexes of GS-3a formulated in a 10:1 charge ratio with mRNA-Cre.

[0057] FIG.28 is a stacked bar graph quantifying the types of cells transfected with GS-3a formulated in a 25:1 charge ratio with mRNA-Cre showing the percentage of each cell type out of the total TdTomato+ population.

[0058] FIG.29 shows the quantification of the types of cells transfected with GS-3a formulated in a 25:1 charge ratio with mRNA-Cre showing the percentage of TdTomato+ cells out of the total for each cell type.

[0059] FIG.30 is a stacked bar graph quantifying the types of cells transfected with GS-3b formulated in a 10:1 charge ratio with mRNA-Cre showing the percentage of each cell type out of the total TdTomato+ population.

[0060] FIG.31 shows the quantification of the types of cells transfected with GS-3b formulated in a 10:1 charge ratio with mRNA-Cre showing the percentage of TdTomato+ cells out of the total for each cell type.

[0061] FIG.32 is a pair of bar graphs illustrating viability of isolated lung cells and splenocytes after treatment with GS-CART / mRNA complexes. n=3, bars represent SD.

[0062] FIG.33 is a pair of bar graphs illustrating the cell composition of lung and spleen after GS-CART transfection, left bar graph shows untreated / GSer-3a; right bar graph shows untreated / Gser-3b, n=3, error bars represent SD.

[0063] FIG.34A is a bar graph illustrating the results of pre-clinical toxicology following the administration of representative complexes formed with mfluc mRNA (GS-3a, GS-3b) and a reference LNP formulation. AST, ALT, and alkaline phosphatase are illustrated.

[0064] FIG.34B is a bar graph illustrating the results of pre-clinical toxicology following the administration of representative complexes formed with mfluc mRNA (GS-3a, GS-3b) and a reference LNP formulation. BUN, Calcium, and phosphorus are illustrated.Docket No. 6045.0589WO | S23-281

[0065] FIG.35A is a bar graph illustrating in vitro stability of the GS-3b / mRNA complex over time for complexes formulated on ice.

[0066] FIG.35B is a bar graph illustrating in vitro stability of the GS-3b / mRNA complex over time for complexes formulated on ice after 3 days of storage at -80 °C, compared to freshly prepared complexes.

[0067] FIG.36 illustrates three bar graphs showing quantification of OVA-specific T cells in blood (top), lungs (middle) and spleens (bottom), at day 10 post-boost, n = 4, bars represent SD. Statistical significance was calculated using 2-way ANOVA.

[0068] FIG.37 is a line graph showing tumor volume monitoring over 21 days, n = 5, bars represent SEM. Statistical significance was calculated using One-way ANOVA of area under the curve.

[0069] FIG.38 is a line graph showing bioluminescence tumor quantification over 21 days (n = 5, bars represent SEM). Statistical significance was calculated using One-way ANOVA of area under the curve.

[0070] FIG.39 is a bar graph showing quantification of anti-OVA IgG antibodies in serum measured by ELISA, n = 4, bars represent SEM.

[0071] FIG.40 is a bar graph illustrating transfection efficiency of a leading GS-CART formulation incubated for 30 min on ice, n=3, bars represent SD. Statistical significance was calculated using 2-way ANOVA.

[0072] FIG.41 is a bar graph illustrating transfection efficiency of a leading GS-CART formulation stored at -20°C. n=3, bars represent SD. Statistical significance was calculated using Welch’s t-test.

[0073] FIG.42 is a bar graph illustrating transfection efficiency of a leading GS-CART formulation stored at -80°C. n=3, bars represent SD. Statistical significance was calculated using Welch’s t-test.

[0074] FIG.43 is a bar graph illustrating transfection efficiency of a leading GS-CART formulation after lyophilization and stored at -20°C. n=3, bars represent SD. Statistical significance was calculated using Welch’s t-test.

[0075] FIG.44 is a pair of bar graphs illustrating inflammatory cytokines 24 hours before and after delivery of luciferase mRNA with GS-3a (top) or GS-3b (bottom), n=3, bars represent SD.Docket No. 6045.0589WO | S23-281 DETAILED DESCRIPTION

[0076] The disclosure provides polymer-based nucleic acid delivery vehicles derived from lipid-functionalized carbonate monomers and guanidinylated serinol monomers, and related compositions and methods. The compounds disclosed herein are effective nucleic acid transporters as demonstrated by their ability to complex with mRNA and deliver the mRNA into cells where it is efficiently translated to produce high levels of protein expression in vitro and in vivo. In addition, certain compounds provide specific tropism to certain organs, such as lung and spleen. Also provided are related compositions, including pharmaceutical compositions, and methods of delivering nucleic acids into cells.

[0077] Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0078] The term “alkyl,” by itself or as part of another substituent, refers to an acyclic branched or unbranched hydrocarbon group containing from about 1 to 24 carbon atoms (C1‑C24) or from 1 to 18 carbon atoms (C1‑C18), which may be fully saturated, mono‑ or polyunsaturated and can include mono‑, di‑ and multivalent radicals. Examples of saturated alkyl groups include methyl, ethyl, n‑propyl, isopropyl, n‑butyl, t‑butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2‑propenyl, crotyl, 2‑isopentenyl, 2‑(butadienyl), 2,4‑pentadienyl, 3‑(1,4‑pentadienyl), ethynyl, 1‑ and 3‑propynyl, 3‑butynyl, and the higher homologs and isomers. In some aspects, an unsaturated alkyl group may be a linolenyl or oleyl. “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, including where two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group. For example, a substituted alkyl group may include a carbonyl (- C(=O)-) moiety. The term “heteroalkyl” refers to an alkyl substituent in which at least oneDocket No. 6045.0589WO | S23-281 carbon atom is replaced with a heteroatom, as described in more detail below. If not otherwise indicated, the term “alkyl” includes unsubstituted, substituted, and / or heteroatom-containing alkyl substituents.

[0079] The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage which may be represented as -O-alkyl, where alkyl is as defined above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. The term “alkylthio” refers to a group -S-alkyl.

[0080] The term “heteroalkyl”, which is shorthand for “heteroatom-containing alkyl”, refers to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom. Similarly, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, and the term “heteroaryl” refers to “aryl” substituents that are heteroatom-containing. In this context, “heteroatom-containing” refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, such as nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) or silicon (Si), and more typically in the context of the present disclosure with N, O, or S. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl groups are provided below.

[0081] In some aspects, the heteroatom is O, N, or S. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2- S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, -CH=CH- N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3,R-S-S-R’, and RO-S(O)x-OR’. In some aspects, a heteroalkyl moiety may include one, two, three, four, or five heteroatom (e.g., O, N, S). In some aspects, a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S).

[0082] The term “alkenyl” refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, octadecenyl, eicosenyl, and the like. In some aspects, alkenyl groups may contain 2 to about 18 carbon atoms (C2‑C18) or 2 to 12 carbon atoms (C2‑C12). The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the term “heteroalkenyl” refers to alkenyl in which at least one carbon atom is replaced with a heteroatom. A heteroalkenylDocket No. 6045.0589WO | S23-281 may optionally include more than one double bond and / or one or more triple bonds in additional to the one or more double bonds.

[0083] The term “alkylene” refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. In aspects, an alkylene substituent group may be methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)6-), octylene (-(CH2)8-) and the like. Similarly, the terms “alkenylene”, “alkynylene”, “arylene”, “aralkylene”, and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively. In some aspects, the aralkylene group is substituted on the alkylene moiety or the arylene moiety (e.g. at carbons 2, 3, 4, or 6) with a functional group. In the context of the present disclosure, alkylene groups are utilized as linking groups. Accordingly, these and other di-radical groups may be referred to herein as “linkers”, “linker groups”, “linker substituents”, “linking groups” or “linking substituents”. The alkylene, alkenylene, alkynylene, arylene, aralkylene, and alkarylene groups may also contain one or more functional groups. The term “functional group” in this context refers to di-radical moieties that contain one or more functional groups such as an oxo (-O-, such as in an ether linkage), amine (-NR-), carbonyl (-C(=O)-), carbonate, and the like. In some aspects, the functional group of a functional linker may be selected from oxo, ‑N3, ‑CN, ‑CHO, ‑OH, ‑NH2, ‑COOH, ‑CONH2, ‑NO2, ‑SH, ‑SO2CH3 ‑SO3H, , ‑OSO3H, ‑SO2NH2, ‑NHNH2, ‑ONH2, ‑NHC(O)NHNH2, substituted or unsubstituted C1‑C5alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl. In embodiments, the alkylarylene is unsubstituted.

[0084] The term "amino" is used herein to refer to the group -NZ1Z2 wherein Z1 and Z2 are hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0085] The terms “cycloalkyl” and “heterocycloalkyl” refer to cyclic versions of the “alkyl” and “heteroalkyl” groups defined above. Cycloalkyl and heterocycloalkyl are not aromatic. In aspects, a cycloalkyl group includes monocyclic hydrocarbon ring systems containing from 3 to 9 carbon atoms (C3-C9), one or more of which may be replaced with a heteroatom, and where such groups can be saturated or unsaturated, but not aromatic. The term “3 to 6 membered” in reference to a cycloalkyl or heterocycloalkyl refers to the number of atoms, i.e. carbon atoms or carbon and one or more heteroatoms, in the monocyclic ring. For example, the term “3 to 6Docket No. 6045.0589WO | S23-281 membered cycloalkyl or heterocycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, heterocyclopropyl, heterocyclobutyl, heterocyclopentyl, and heterocyclohexyl. In aspects, a cycloalkyl group includes a bicyclic or multicyclic cycloalkyl ring system where multiple rings are fused together, where at least one of the fused rings is a cycloalkyl ring and the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkyl ring of the multiple rings. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. In embodiments, the cycloalkyl is fully saturated. In aspects, the cycloalkyl is monounsaturated. In embodiments, the cycloalkyl is polyunsaturated. In aspects, the heterocycloalkyl is fully saturated. In embodiments, the heterocycloalkyl is monounsaturated. In aspects, the heterocycloalkyl is polyunsaturated.

[0086] The term “acyl” means, unless otherwise stated, ‑C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0087] The term “aryl” refers to cyclic groups that contain at least one aromatic ring, for example a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl). In some aspects of the present disclosure, the aryl group contains 6, 9 or 10 atoms such as phenyl, biphenyl, naphthyl, diphenylether, diphenylamine, benzophenone, indanyl, anthracenyl, 1,2- dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the term “heteroaryl” refers to an aryl substituent in which at least one carbon atom is replaced with a heteroatom, asDocket No. 6045.0589WO | S23-281 described in more detail below. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0088] The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “alkaryl” refers to an aryl group with an alkyl substituent, where “alkyl” and “aryl” are as defined above. In general, aralkyl and alkaryl groups may contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms or from 6 to 12 carbon atoms.

[0089] The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents as described herein.

[0090] An “arylene” and a “heteroarylene,” alone or as part of another substituent, means a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0091] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkylDocket No. 6045.0589WO | S23-281 group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0092] The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.

[0093] The term “substituted” as in “substituted alkyl”, “substituted aryl”, and the like, refers to at least one hydrogen atom bound to a carbon or other atom that is replaced with one or more non-hydrogen substituents in the alkyl, aryl, or other moiety. The term “substituted or unsubstituted” preceding a list, as in “substituted or unsubstituted C1-C24alkyl, C1- C24 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl” is intended to modify each member of the list, as in "“substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C1-C24heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkylaryl”. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C24alkoxy, C2-C24alkenyloxy, C5-C20aryloxy, acyl (including C2-C24alkylcarbonyl (-CO-alkyl) and C6-C20arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24alkylcarbonato (-O-(CO)-O-alkyl), C6-C20arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), mono-substituted C1-C24 alkylcarbamoyl (- (CO)-NH(C1-C24alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24alkyl)2), mono- substituted arylcarbamoyl (--(CO)--NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH- (CO)-NH2), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (- NH-(CO)-alkyl), C5-C20arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1- C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), sulfonamide (-SO2NH), C1-C24alkylsulfanyl (-S-alkyl or “alkylthio”), arylsulfanyl (-S-aryl or “arylthiol”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20arylsulfonyl (-SO2-aryl); and the hydrocarbyl moieties C1- C24alkyl (including C1-C18alkyl, C1-C12alkyl, C1-C8alkyl and C1-C6alkyl), C2-C24alkenylDocket No. 6045.0589WO | S23-281 (including C2-C18 alkenyl, C1-C12 alkenyl, C1-C8 alkenyl, and C1-C6 alkenyl), C5-C30 aryl (including C5-C20aryl, C5-C12aryl), and C6-C30aralkyl (including C6-C20aralkyl, and C6-C12aralkyl). In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above- mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. In addition, the hydrocarbyl moieties may contain one or more heteroatoms, optionally N, O, or both.

[0094] It is understood that due to resonance a charge may be distributed across the molecule. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts, and as such one of skill in the art would recognize the equivalency of the moieties possessing resonance structures. For example, - NHC(NH2+)NH2 refers to .

[0095] is divalent, i.e. inserted between two other groups.

[0096] The symbol “ ” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0097] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosureDocket No. 6045.0589WO | S23-281 Compounds

[0098] Provided are compounds in the form of copolymers derived from two different types of monomers, lipid-functionalized carbonate monomers and guanidinylated serinol monomers. The corresponding units of the copolymers derived from these monomers are referred to as “constitutional units” or “monomer units” or simply “units”.

[0099] The copolymers described here may be synthesized in a block architecture, where the units are distributed as blocks of identical monomer units, rather than statistically, or they may be synthesized in a statistical architecture. The nomenclature poly(A-stat-B) or polyA-block- polyB is adopted herein to differentiate statistical versus block copolymers, in accordance with Hodge et al., Pure Appl. Chem.202092(5):797-813, where such differentiation is warranted.

[0100] The polymer formulas described herein are presented as graphical representations of an “ideal” form of the polymer. For example, while the graphical representations may present the repeating monomer units as connected to each other in the same orientation, e.g., “head-to- tail”, it is understood that in the actual polymer there may also be some “head-to-head” and / or “tail-to-tail” dyads.

[0101] Provided are compounds of Formula I: R1-X-[(L1)x1-(L2)x2-(A1)y1]-R2 (Formula I) where R1 is substituted or unsubstituted C1-C24alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; X is a bond, O, S, N or a linking group as defined herein; L1 and L2 are each independently a lipid carbonate monomer unit; A1 is a guanidinylated serinol monomer unit; x1 and y1 are each independently greater than zero and less than 100; x2 and y2 are each independently zero or less than 100; and R2 is H, C2-C24 alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl, optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-6 alkyl, and carboxylato.

[0102] In accordance with aspects of any of the foregoing, a compound of Formula I may also include where R1 is a molecule useful for in vivo imaging or a ligand moiety that binds to aDocket No. 6045.0589WO | S23-281 cell-surface receptor, where R1 is joined to the copolymer via a linking group X. In some aspects, the compound may include where R1 is a fluorophore, optionally difluoroboron-β- diketonate (BDK), a “Black Hole Quencher” dye, or a naphthalene derivative, including dansyl derivatives such as 5-(dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-sulfonamide. In some aspects, the compound may also include where R1 is a ligand moiety, optionally a saccharide, a disaccharide, an oligosaccharide, a liposaccharide, a lipid, a peptide, an antibody, biotin, a biotin derivative, or fingolimod.

[0103] In aspects of Formula I, L1 and L2 are each independently a lipid-functionalized methyl-trimethylene carbonate (MTC) unit having a structure ofhaving a structure ofa or which may be fully saturated, mono- or polyunsaturated. In some aspects, RLis independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl. In aspects of Formula I, A1 is a guanidinylated serinol monomer unit having a structure ofDocket No. 6045.0589WO | S23-281X is O; L1 and L2 are each an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ia:Ia where R1 is substituted or unsubstituted C1-C24alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1 and RL2 are each independently a branched or unbranched C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated; x1 and x2 are each independently 1-100, 1-50, or 1-25; and y1 is 1-100, 1-50, or 1-25. In aspects, X is O; L1 is an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ib:Docket No. 6045.0589WO | S23-281 Ibor cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1is a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25; x2 is zero; and y1 is 1-100, 1-50, or 1-25. In aspects, X is O; L1 is a bAC unit; A1 is a GSer unit and the compound is represented by Formula Ic: IcR1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1 is a branched or unbranched C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25;Docket No. 6045.0589WO | S23-281 x2 is zero; and y1 is 1-100, 1-50, or 1-25. In aspects of Formula Ia, R1 is benzyl, RL1 and RL2 are each independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl. Representative compounds of Formula Ia are provided in Table 1.

[0104] Table 1: Representative compounds of Formula Ia: Compound R1 RL1 x1 RL2 x2 y1 R2 GS-1a benzyl oleyl 8 nonenyl 7 9 H GS-1b benzyl oleyl 4 nonenyl 5 12 H GS-1c benzyl oleyl 4 nonenyl 4 19 H

[0105] In aspects of Formula Ib, RL1 is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl. Representative compounds of Formula Ib are provided in Table 2.

[0106] Table 2: Representative compounds of Formula Ib: Compound R1 RL1 x1 y1 R2 GS-2a benzyl farnesyl 15 8 H GS-2b benzyl farnesyl 10 9 H GS-2c benzyl farnesyl 10 16 H

[0107] In aspects of Formula Ic, RL1is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl. Representative compounds of Formula Ic are provided in Table 3.

[0108] Table 3: Representative compounds of Formula Ic: Compound R1 RL1 x1 y1 R2 GS-3a benzyl farnesyl 18 10 HDocket No. 6045.0589WO | S23-281 GS-3b benzyl farnesyl 11 9 H GS-3c benzyl farnesyl 10 15 H

[0109] Structures of representative compounds of Formulas Ia, Ib, and Ic are shown in Table 4.

[0110] Table 4: Structures of representative compounds Compoun Structure dDocket No. 6045.0589WO | S23-281herein in its neutral , the guanidine moiety will be positively-charged (protonated) at around pH 5.5, which form can be represented as aguanidinium cation, -NHC(NH2+)NH2, and including resonance structures thereof, as defined herein. Synthetic Methods

[0112] Reference ONA CART compounds are copolymers of cationic poly(alpha-amino ester) monomers and lipid functionalized monomers having a cyclic methyl trimethylene carbonate (MTC) polymer backbone and were synthesized as described in WO 2018 / 022930 and WO 2020 / 097614.

[0113] Synthesis of bAC lipid monomers are described in US Provisional application no. 63 / 423,541.

[0114] Boc protected GS cationic monomer is synthesized in 2 steps by first coupling 2- amino-1,3-propanediol with N,N’-Di-Boc-1H-pyrazole-1-carboxamidine in DMF for 16 hr at room temperature. The reaction mixture was then extracted with EtOAc and 1N HCl and solvent evaporated to yield the diol intermediate 1. The intermediate was subsequently cyclized with triphosgene in dry DCM at -20C for 2hr. The reaction mixture was washed with saturated ammonium sulfate and the solvent evaporated to yield a slightly yellow crude oil / solid mixture. The crude product was purified by silica gel chromatography to yield the GS cationic monomer.Docket No. 6045.0589WO | S23-281

[0115] All GS compounds were prepared without a glove box using standard Schlenk techniques. A representative procedure to synthesize Boc-protected GS-3b starts by adding bAC-F monomer (32.88 mg, 10 equiv, 92.47 umol) and thiourea catalyst (7.00 mg, 19.76 umol, TU, TU = 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea)) into a glass GPC vial with a stir bar. The vial was flushed with nitrogen, and solids were then dissolved in 75 ul DCM. 30 mg / mL benzyl alcohol solution was prepared in a separate vial, and 34.3 ul was added to the monomer solution (1.00 mg, 1 equiv, 9.25 umol). 3.01 mg of DBU (2 equiv, 19.76 umol, DBU = 1,8-diazabicyclo [5.4.0]undec-7-ene) was added to start the polymerization. 3 hours later, 33.23mg GS monomer (10 equiv, 92.47 umol) was added as a solid. After 3 hours, the reaction was quenched with 6 drops of AcOH and transferred to a 2 kDa dialysis bag and dialyzed against MeOH for 4 hours. The dialyzed solution was concentrated to yield a clear oil as the product. The block length (DP) and number average molecular weight (Mn) was determined by 1H-NMR end group analysis. Dispersity (Ð) was determined by gel permeation chromatography (GPC).

[0116] With reference to FIG. 1, Boc groups were removed to yield the deprotected oligomer as the active vehicle as follows. In a 1-dram vial, Boc-protected oligomers (0.5 µmol) were deprotected in a trifluoracetic acid / distilled DCM solution (1:4 v / v, 500 µL) under slow stirring and ambient atmosphere for 16 hour. Solvent was removed in vacuo and the samples were stored under high vacuum for 18 hours. The deprotected oligomer, as a thin film, was dissolved in DMSO to achieve 30 mM cation concentrations and stored at -20°C ready to use.

[0117] Proton NMR characterization of representative GS-CARTs (Boc-protected) is provided below.

[0118] GSer-1a: O8N7GSer9 1H NMR (400 MHz, CDCl3) δ 11.48 (d, J = 7.1 Hz, 9H), 8.84 – 8.48 (m, 9H), 7.42 – 7.31 , 5.35 (pd, J = 10.0, 4.0 Hz, 29H), 5.16 (s, 2H), 4.78 (dt, J = 9.5, 5.2 Hz, 6H), 4.48Docket No. 6045.0589WO | S23-281 – 4.20 (m, 91H), 4.14 (dt, J = 10.5, 5.5 Hz, 35H), 3.88 – 3.58 (m, 15H), 2.03 (dt, J = 9.9, 7.0 Hz, 61H), 1.65 (p, J = 6.4 Hz, 33H), 1.55 – 1.44 (m, 181H), 1.42 – 1.18 (m, 259H), 0.97 (t, J = 7.5 Hz, 22H), 0.89 (t, J = 6.7 Hz, 25H).

[0119] GSer-1b: O4N5GSer12 1H NMR (400 MHz, CDCl3) δ 11.48 (s, 12H), 8.85 – 8.43 (m, 12H), 7.39 (d, J = 4.5 Hz, 5H), 5.47 – 5.26 (m, J = 5.7 Hz, 17H), 5.18 (d, J = 9.3 Hz, 2H), 4.79 (d, J = 8.1 Hz, 8H), 4.32 (p, J = 11.1 Hz, 84H), 4.14 (s, 24H), 3.94 – 3.54 (m, 18H), 2.39 (d, J = 7.2 Hz, 2H), 2.03 (dt, J = 14.2, 9.2 Hz, 38H), 1.50 (d, J = 5.7 Hz, 258H), 1.44 – 1.15 (m, 153H), 0.97 (t, J = 7.5 Hz, 15H), 0.90 (t, J = 6.7 Hz, 13H).

[0120] GSer-1c: O4N4GSer19 1H NMR (400 MHz, CDCl3) δ 11.46 (d, J = 7.1 Hz, 19H), 8.76 – 8.56 (m, 19H), 7.36 (s, 5H), 5.47 – 5.25 (m, 19H), 5.14 (s, 2H), 4.77 (s, 17H), 4.29 (d, J = 11.0 Hz, 116H), 4.11 (s, 21H), 3.97 – 3.66 (m, 17H), 2.02 (dd, J = 10.3, 6.2 Hz, 35H), 1.62 (d, J = 9.4 Hz, 40H), 1.57 – 1.40 (m, 421H), 1.40 – 1.12 (m, 145H), 0.95 (t, J = 7.6 Hz, 13H), 0.88 (t, J = 6.6 Hz, 12H).

[0121] GSer-2a: MTCF15GSer8 1H NMR (400 MHz, CDCl3) δ 11.46 (d, J = 7.8 Hz, 8H), 8.85 – 8.46 (m, 8H), 7.40 – 7.30 (m, 5H), 5.14 (s, 2H), 4.76 (s, 6H), 4.29 (d, J = 9.9 Hz, 82H), 4.15 (t, J = 6.3 Hz, 32H), 3.87 – 3.50 (m, 15H), 1.75 – 1.55 (m, 39H), 1.51 – 1.46 (m, 173H), 1.34 – 1.00 (m, 241H), 0.92 – 0.81 (m, 184H).

[0122] GSer-2b: MTCF10GSer9 1H NMR (400 MHz, CDCl3) δ 11.44 (d, J = 8.0 Hz, 8H), 8.92 – 8.37 (m, 8H), 7.40 – 7.28 (m, 5H), 5.12 (s, 2H), 4.74 (s, 7H), 4.27 (d, J = 10.1 Hz, 70H), 4.13 (d, J = 7.7 Hz, 23H), 3.97 – 3.64 (m, 9H), 1.57 – 1.41 (m, 190H), 1.41 – 0.95 (m, 185H), 0.85 (td, J = 9.8, 6.3 Hz, 128H).

[0123] GSer-2c: MTCF10GSer16 1H NMR (400 MHz, CDCl3) δ 11.45 (d, J = 7.2 Hz, 16H), 8.81 – 8.49 (m, 16H), 7.36 (s, 5H), 5.14 (s, 2H), 4.77 (s, 11H), 4.29 (d, J = 10.8 Hz, 98H), 4.16 (s, 25H), 3.88 – 3.53 (m, 19H), 1.51 – 1.45 (m, 322H), 1.41 – 0.98 (m, 180H), 0.86 (td, J = 9.9, 6.4 Hz, 125H).

[0124] GSer-3a: bACF18GSer10Docket No. 6045.0589WO | S23-281 1H NMR (400 MHz, CDCl3) δ 11.44 (s, 10H), 8.78 – 8.49 (m, 10H), 7.34 (s, 5H), 4.75 (s, 5H), 4.42 – 4.04 (m, 128H), 3.61 (d, J = 6.7 Hz, 101H), 2.37 – 2.04 (m, 47H), 2.01 (d, J = 11.3 Hz, 25H), 1.57 – 1.41 (m, 238H), 1.41 – 0.97 (m, 363H), 0.89 (d, J = 6.6 Hz, 69H), 0.83 (dd, J = 9.5, 6.4 Hz, 213H).

[0125] GSer-3b: bACF11GSer9 1H NMR (400 MHz, CDCl3) δ 11.47 (s, 9H), 8.58 (s, 9H), 7.36 (s, 5H), 5.15 (s, 2H), 4.78 – 4.12 3.75 – 3.51 (m, 65H), 2.37 – 2.10 (m, 32H), 1.48 (t, J =3.1 , – , 0.91 (d, J = 6.4 Hz, 47H), 0.88 – 0.83 (m, 130H).

[0126] GSer-3c: bACF10GSer15 1H NMR (400 MHz, CDCl3) δ 11.48 (t, J = 4.9 Hz, 15H), 8.85 – 8.50 (m, 14H), 7.39 (d, J = 3.7 Hz, 5H), 5.17 (d, J = 2.3 Hz, 2H), 4.79 (d, J = 7.3 Hz, 8H), 4.54 – 4.07 (m, 107H), 3.90 – 3.40 (m, 70H), 2.40 – 2.11 (m, 25H), 2.04 (s, 14H), 1.61 – 1.44 (m, 313H), 1.45 – 1.01 (m, 177H), 0.87 (dd, J = 9.6, 6.6 Hz, 112H). Compositions

[0127] Also provided are compositions, including pharmaceutical compositions, comprising a compound as described herein and a carrier or excipient, including a pharmaceutically acceptable carrier or excipient.

[0128] In aspects, the composition or pharmaceutical composition comprises a compound of Formula I or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the complexes condense to form nanoparticles ranging in size from about 100-400 nanometers (nm) in diameter or preferably from about 150-250 nm. In this context, size refers to the mean particle size (Z). Thus, the disclosure also provides compositions or pharmaceutical compositions comprising nanoparticles of a compound of Formula I, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids.

[0129] In aspects, the nucleic acid is an RNA or DNA. In aspects, the nucleic acid is messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA),Docket No. 6045.0589WO | S23-281 minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

[0130] In aspects, the nucleic acid is a therapeutic agent. In aspects, the nucleic acid encodes a therapeutic agent.

[0131] In aspects, the nucleic acid includes one or more vectors, which may be eukaryotic expression vectors, bacterial plasmid vectors or viral vectors. In some aspects where the vector is a eukaryotic expression vector, the vector may include (a) a first polynucleotide encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence in the genome of eukaryotic cell, and (b) a second polynucleotide encoding a Cas9 protein, optionally wherein the Cas9 protein is codon optimized for expression in the cell. In aspects, the first (a) and second (b) polynucleotides are located in the same or different vectors. In aspects, the nucleic acid comprises a CRISPR RNA (crRNA), optionally wherein the crRNA is in the same plasmid vector as the first nucleotide sequence. In aspects, the nucleic acid comprises a transactivating RNA (tracrRNA). In aspects, the tracrRNA is optionally in the same vector as the second nucleotide sequence.

[0132] In aspects, the nucleic acid includes (a) a first polynucleotide encoding a transposase; and (b) a second polynucleotide comprising a nucleic acid sequence of a gene of interest flanked by a transposase recognition site. In aspects, the first (a) and second (b) polynucleotides are located in the same or different vectors. In accordance with this aspect, the transposase recognizes and excises a genomic sequence of interest.

[0133] In general, a compound of Formula I, or subformula thereof, is complexed with nucleic acid in an amount effective to produce a theoretical charge ratio of cationic compound to anionic nucleic acid of from about 4:1 to about 25:1 (cation:anion). In aspects, the theoretical charge ratio is 4:1, 5:1, 10:1, 15:1, 20:1, or 25:1. Theoretical (+ / -) charge ratios are calculated as moles of guanidinium cations to moles of phosphate anions, assuming full amine protonation and phosphate deprotonation. In some aspects, the charge ratio (+ / -) is 4:1, 10:1, or 25:1. Complexation of nucleic acid with a compound described herein may take place in the presence of a coordinating metal such as Zn+2, Mg+2, Ca+2; a dynamic non-covalent cross linker such as a carbohydrate; a counterion such as Cl-, AcO-, succinate, or citrate; or a solubility modulator such as a lipid or a polyethyleneglycol (PEG), or any combination thereof.

[0134] In other aspects, the composition or pharmaceutical composition comprises a compound of Formula I, or a subformula thereof, without a nucleic acid cargo, for exampleDocket No. 6045.0589WO | S23-281 where the nucleic acid is to be complexed at a later time such as before administration of the pharmaceutical composition to a subject.

[0135] In some aspects, the composition is a vaccine. In aspects where the composition is a vaccine, the composition may further include an immunological adjuvant. The immunological adjuvant can include, but is not limited to, agonists of Toll-like Receptors (TLRs), agonists of the STING pathway, agonistic antibodies against CD40, OX40, CTLA4, PD1, or PD1-L, Freund’s adjuvant, bryostatins, PKC modulators, and ligands for CD40, OX40, CD137, PD1, CTLA4 and any combinations thereof.

[0136] Pharmaceutically acceptable carriers or excipients include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additional acceptable carriers, excipients, or stabilizers may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0137] In some aspects, the composition or pharmaceutical composition may include a cryoprotectant agent. Non-limiting examples of cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof. In an aspect, the composition or pharmaceutical composition may comprise 10-30% sucrose formulated at pH 5.5.Docket No. 6045.0589WO | S23-281 Methods of Use

[0138] The disclosure also provides methods for delivery of nucleic acids to cells and / or tissues in vitro, ex vivo, or in vivo using a compound as described herein. In aspects, the methods comprising contacting a target cell or tissue with a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I, or subformula thereof. In aspects, the methods comprise administering to a subject a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I, or subformula thereof, including a pharmaceutical composition or vaccine. Administration may be according to any suitable route, for example, parenteral, including e.g., intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intracranial, etc., or transmucosal, including e.g., buccal, nasal, sublingual, transdermal, etc. In aspects, the subject is a mammal, for example a human, a non-human primate, a murine (i.e., mouse and rat), a canine, a feline, or an equine. In embodiments, the subject is a human.

[0139] Also provided are methods for delivery of nucleic acids to a specific target site in vivo, such as the lung or spleen; or to specific cells, such as pulmonary endothelial cells, pulmonary immune cells, or splenic macrophages. The methods comprise delivering a composition comprising a nucleic acid complexed with a compound of Formula I, or subformula thereof, in a specific charge ratio (cation: anion), as discussed herein. In general, a charge ratio greater than 10:1, such as about 25:1, can be used to target the nucleic acid for delivery to the lungs while a charge ratio of less than 10:1 , such as about 4:1, can be used to target the nucleic acid for delivery to the spleen. Generally, intermediate charge ratios of about 10:1 can be used to target both lung and spleen.

[0140] In this context, the term “charge ratio” refers to a theoretical charge ratio of cationic co-oligomer to anionic nucleic acid (cation:anion). In aspects, the theoretical charge ratio is about 4:1, 10:1, or 25:1. In aspects, where it is desired to target nucleic acid delivery to both the spleen and lung, the theoretical charge ratio may be about 8:1, 9:1, 10:1, 11:1, 12:1, 13:1 14:1, or 15:1. In aspects, where it is desired to target nucleic acid delivery primarily to the lung, the theoretical charge ratio may be about 16:1, 18:1, 20:1, 22:1, 25:1, 26:1, 28:1 or 30:1. In aspects, where it is desired to target nucleic acid delivery primarily to the spleen, the theoretical charge ratio may be about 2:1, 3:1, 4:1, 5:1, or 6:1. Theoretical (+ / -) charge ratios are calculated as moles of ammonium cations to moles of phosphate anions, assuming full amine protonation and phosphate deprotonation.Docket No. 6045.0589WO | S23-281

[0141] In aspects, the nucleic acid may be RNA or DNA. In aspects, the RNA is messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), or transactivating RNA (tracrRNA). In aspects, the DNA is plasmid DNA (pDNA), minicircle DNA, or genomic DNA (gNDA).

[0142] In aspects, the nucleic acid may be a therapeutic nucleic acid or the nucleic acid may encode one or therapeutic agents, for example a cytokine, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0143] In some aspects, a method of transfecting a nucleic acid into a cell as described herein may be part of a method for gene editing or genetic engineering. For example, one or more nucleic acids may be transfected using the methods described herein in a CRISPR-based system or a transposon-based system for gene editing or genetic engineering. Accordingly, one or more nucleic acids may be transfected according to the methods described here, which nucleic acids may be located on one or more vectors. For example, the one or more nucleic acids may comprise a vector having a first nucleotide sequence encoding a CRISPR-Cas system guide RNA (gRNA) that hybridizes with a target sequence in the genome of a target cell and a second nucleotide sequence encoding a Cas9 protein. Alternatively, the gRNA and Cas9 protein can be located on different vectors, or either the gRNA or Cas9 protein may be produced in the target cell. The one or more nucleic acids may also comprise a CRISPR RNA (crRNA) and / or transactivating RNA (tracrRNA), each of which may be located on the same or a different vector as the gRNA and / or Cas9 encoding sequence.

[0144] In another aspect, the one or more nucleic acids may comprise a sequence encoding a transposase and a nucleic acid sequence of a gene of interest flanked by a transposase recognition site, which may be located on the same or different vectors.

[0145] In some aspects, a compound of Formula I, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptides which may form a vaccine composition. In some aspects, a mixture of two or more different compounds of Formula I, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptides. Examples and Biological Assays

[0146] The compounds described here represent a new class of charge-altering releasable transporters (CARTs) having cationic guanidinylated serinol moieties, abbreviated herein asDocket No. 6045.0589WO | S23-281 either “GSer”-CARTs, or for conciseness in the text simply “GS”-CARTs. The terms “GSer- CARTS” and “GS-CARTS” are used interchangeably herein. GS-CARTs undergo a guanidinium-to-carbonate cyclization process at physiological pH, resulting in neutralization of cationic moieties and efficient release of mRNA cargo. Certain compounds were also found to be highly selective in delivery of mRNA to the lung (up to 96%) and spleen (up to 98%) following intravenous administration. The synthesis and characterization of a series of GS- CARTs is described below. Certain biophysical properties of GS-CART / mRNA complexes, particularly in the context of the plasma environment, were found to differentiate lung-tropic from spleen-tropic formulations.

[0147] The compounds described here are particularly advantageous for selective RNA delivery which is required for the broad implementation of RNA clinical applications including prophylactic and therapeutic vaccinations, immunotherapies for cancer, and genome editing. Current polyanion delivery relies heavily on cationic amines while cationic guanidinium systems have received limited attention due in part to their strong polyanion association which impedes intracellular polyanion release. The GS-CARTs disclosed here provide a general solution to this problem in which cationic guanidinium groups are used to form stable RNA complexes upon formulation but at physiological pH undergo a charge-neutralization process resulting in RNA release. Significantly, systematic variations in structure and formulation resulted in GS-CARTs that exhibit highly selective mRNA delivery to the lung (~97%) and spleen (~98%) without targeting ligands. Illustrative of their breadth and translational potential, GS-CARTs also deliver circRNA, providing the basis for a cancer vaccination strategy, which in a murine model resulted in antigen-specific immune responses and effective suppression of established tumors. Synthesis and characterization of Guanidinylated Serinol (GSer) compounds

[0148] A series of compounds was synthesized to evaluate their ability to act as oligonucleotide transporters. FIG.1 illustrates the general synthetic scheme. For polymerization, benzyl alcohol was used as an initiator of the reaction with a DBU / TU catalyst system as described in McKinlay, C. J. et al. Proc. Natl. Acad. Sci. U.S.A.114, E448–E456 (2017). Each compound was synthesized as an oligomer containing 1 or 2 lipid functionalized carbonate monomers (“lipid units”) and cationic guanidinylated serinol monomers (“cationic units”). Lipid groups included oleyl / nonenyl or H6-farnesol (“farnesyl”). The carbonate monomers were either methyl-trimethylene carbonate (MTC) or beta-amido carbonate (bAC).Docket No. 6045.0589WO | S23-281 The total number of monomer units is referred to as the degree of polymerization (DP). Polymers were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) prior to deprotection.

[0149] FIG.2 illustrates the synthesis of model compounds 4, 5, and 6 utilized to investigate the mechanism of degradation. Serinol 1 was selected as the scaffold compound due to its commercial availability and biocompatibility. In particular, polymers of a serinol carbonate have previously been demonstrated to degrade via an O-to-N acyl shift of the amine which yielded the corresponding carbamate products (Qiu et al., ACS Macro Lett.2015, 4, 11, 1220– 1224). Here, guanidinium groups were added to the serinol amines in an effort to create a more highly cationic molecule. Serinol was first guanidinylated and the resulting diol 2 cyclized to provide the GSer monomer 3. Organocatalytic ring opening oligomerization (OROP) of 3 with a benzyl alcohol initiator and 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) / thiourea (TU) catalysts (followed by TFA deprotection yielded Guanidinylated Serinol (GSer) polymer 4 with an average block length of 53, which served as the model compound for GSer degradation studies.

[0150] Discrete compounds mono-carbonate 5 and di-carbonate 6 were also prepared by reacting diol 2 with the indicated equivalents of benzyl chloroformate (Cbz-Cl) followed by TFA deprotection. Compounds 5 and 6 served as the proxy substrates for GSer degradation behavior at the oligomer chain end (with a terminal hydroxy group) and for intra-chain degradation (with a guanidinium flanked by two carbonate groups), respectively.

[0151] Degradation of the mono-carbonate 5 was examined first due to its structural simplicity. 5 was stable for at least 3 days in unbuffered D2O (pH = 2.0). In contrast, when the solution pH was adjusted to 7.6 with 100 mM Na2CO3 solution, 5 degraded exponentially with a half-life of 11.3 min and reached 93.9% conversion in 2 hours (FIG. 3). Bicyclic acylated isourea 8, a previously unreported compound, and diol 9 resulting from hydrolysis were identified in 53% and 41% yield, respectively. The structures were established by NMR and mass spectrometric analysis after HPLC purification of the degradation mixtures. A representative NMR spectra showing peaks corresponding to compounds 5 (a) and 7 (a') over time in unbuffered D2O (pH 5.5) is shown in FIG.4.

[0152] The mechanism for the formation of the bicyclic acylated isourea 8 is believed to be unprecedented but finds analogy in a related amine-to-carbonate cyclization which results in oxazolidinone formation. We hypothesize that the novel formation of 8 occurred through a 2-Docket No. 6045.0589WO | S23-281 step cascade involving a guanidine-to-carbonate cyclization to initially give 10 (FIG.5), followed by carbonate hydrolysis and a further cyclization to afford 8. To isolate the proposed intermediate 10, di-carbonate 6 was selected for study as the oxygens required for cyclization are sequestered in the flanking carbonate groups as would be found in the oligomer. This enabled isolation and characterization of the guanidinylated-oxazolidinone intermediate 10 (64% yield), indicating that the guanidine-to-carbonate cyclization involving a 5-centered transition state is viable, as illustrated in FIG.5. When 10 was then subjected to the degradation conditions described above, the formation of 8 was observed, supporting our hypothesis about the cyclization cascade leading to charge cancellation.

[0153] Collectively, these results reveal a novel mechanism in which the guanidinium group, which strongly associates with phosphates at low pH, undergoes charge-cancellation at higher pH thereby eliminating its electrostatic phosphate association. Amongst other consequences, this charge-cancellation process opens the door for the potential use of guanidinium groups as surrogates for protonated amines in a range of LNP delivery technologies.

[0154] Importantly, bicyclic acylated isourea 8 and diol 9 were also isolated and identified when di-carbonate 6 and GSer polymer 4 were degraded under the same conditions, indicating that the degradation mechanism is similar for the three model compounds (4-6).

[0155] The ability of the polymers to form complexes with mRNA was tested using a luciferase mRNA assay as described in Kranz et al., Nature 534: 396–401 (2016). Simple mixing of the polymers and mRNA at a 10:1 charge ratio for 20 seconds in a phosphate buffered saline solution at acidic pH (PBS pH 5.5) gave greater than 92% RNA encapsulation. The change in size (nm) over time (min) of two representative complexes under these conditions is shown in FIG.6. The complexes were 201 nm and 227 nm in size and were stable for at least 30 min under acidic conditions.

[0156] In terms of surface charge, both complexes exhibited positive zeta potentials upon mRNA complexation. Charge remained stable over 30 min under acidic conditions (also referred to as “low pH” conditions, PBS pH 5.5), as shown in FIG.7. Under physiological conditions (pH 7.5) the zeta potential became negative between 10-20 minutes as the complexes dissociated. These size and surface charge data together indicated that the polymers formed stable complexes with mRNA under acidic conditions while under physiological conditions cyclization of GSer units neutralized the cationic guanidinium species resulting in release of mRNA.Docket No. 6045.0589WO | S23-281

[0157] To evaluate the ability of the compounds to deliver mRNA into cells, compounds were complexed with 210 ng of eGFP mRNA under acidic conditions (PBS pH 5.5) in a 4:1 charge ratio (determined empirically). Compounds and mRNA were mixed for 20 seconds then transferred (2ul) into wells containing Chinese Hamster Ovary (CHO) cells (50 ng per well, n=4). After 2-4 hours, 150 ul complete media was added to each well. eGFP expression was measured by flow cytometry. Results are shown in FIG. 8. Compounds GS-1a, GS-1b, GS-3a, and GS-3b performed considerably better than compounds GS-2a and GS-2b, while compounds GS-1c, GS-2C, and GS-3c did not result in expressed protein at all (<1% eGFP+). In general, these compounds had longer cationic block length compared to the compounds that effectively delivered and released the mRNA as measured by cellular protein expression. Long block lengths (>15) tended to reduce cellular uptake of transporters, possibly due to strong complexation of guanidinium groups with cell membrane preventing efficient dissociation required for subsequent entry.

[0158] As shown in FIG. 9, the best performing compound, GS-3b outperformed a commercial transfection reagent, Lipofectamine 2000, and was slightly better than the reference compound, ONA CART, which is described in McKinlay, et al., Proc. Natl. Acad. Sci. U.S.A. 115, E5859–E5866 (2018). Percentage is proportion of eGFP positive cells by flow cytometry. The structure of the reference compound is:Docket No. 6045.0589WO | S23-281

[0159] The ability of the compounds to deliver mRNA in vivo was evaluated in a mouse system by intravenous injection (retro-orbital) of complexes of compounds and luciferase mRNA (5 ug, 1.9 kb). The charge ratio was 10:1 for this initial evaluation. Luciferase protein expression was evaluated 6 hours post-injection by whole body imaging and then in individual organs, specifically liver, lung, and spleen. FIG.10 shows a schematic representation of the experiments. All compounds resulted in robust protein expression with various organ tropisms.

[0160] GS-1a and GS-2b induced highly selective (96.6%, 90.8%) lung protein expression while GS-1b and GS-3b induced highly selective (96.2%, 97.3%) expression in the spleen, indicating that different lipid substituents could target complexes to the spleen versus the lung. Lipid block length had mixed effects on organ tropism. Compounds with a balanced lipid:GSer block length (GS-1b, 2b, 3b) tended to induce more organ specific delivery. The composition of the polymeric backbone, MTC or bAC, did not seem to significantly impact organ tropism in compounds with lipid block lengths ~15 (compare GS-2a and GS-3a). However, different organ tropisms were observed among compounds having lipid block lengths ~10 (compare GS-2b and GS-3b). Notably, minimal liver expression was observed for all compounds. This is in contrast to FDA approved LNP formulations, including Dlin-MC3-DMA, DSPC, and DMG-PEG2000, which resulted primarily in delivery to the liver, about 89% (data not shown).

[0161] Together, these data suggested that the chemical structures of the compounds play a role in determining organ selectivity, but no particular chemical feature could be identified to predict organ selectivity. After considering various parameters of the compounds and / or complexes that may impact organ tropism, it was decided to evaluate the charge ratio of the compound:RNA complexes. Charge ratio had been suggested as one factor impacting organ tropism in the context of liposomal formulations. See Kranz et al., Nature 534:396–401 (2016)).

[0162] To test the effects of charge ratio on organ tropism, compounds were formulated with luciferase mRNA at 25:1, 10:1, or 4:1 charge ratios and administered to mice via intravenous injection. The results are summarized in Table 5. Strikingly, while 10:1 formulations tended to deliver mRNA to both lung and spleen, the 25:1 formulations tended to deliver mRNA selectively to the lung, as indicated by strong protein expression in the lung. Further unexpectedly, the 4:1 formulations tended to deliver mRNA selectively to the spleen. Remarkably, most of the compounds tested exhibited drastic tropism shifts with higher and lower charge ratios as compared to the 10:1 charge ratio. Moreover, a consistent trend wasDocket No. 6045.0589WO | S23-281 observed, indicating that higher charge ratio favors lung delivery and lower charge ratio favors spleen delivery.

[0163] The organ selectivity of the lung tropic (GS-3a 25:1) and spleen tropic (GS-3b 10:1) nanoparticle (NP) formulations was verified by the absence in detectable protein expression in off-target organs, including liver, heart, kidney, and stomach (data not shown).

[0164] Table 5: Protein expression following administration of complexes to mice. The percentage represents the allocation of the luciferase signal across all three organs, lung, liver, and spleen. “nd” indicates not determined. Compound charge ratio lung % liver % spleen % GS-1a 25:1 nd nd nd GS-1a 10:1 96.6 1.7 1.7 GS-1a 4:1 4.8 2.1 93.1 GS-1b 25:1 78.8 4.5 16.7 GS-1b 10:1 96.2 2.4 1.4 GS-1b 4:1 nd nd nd GS-2a 25:1 81.4 4.1 14.5 GS-2a 10:1 63.2 3.7 33.1 GS-2a 4:1 3.8 3.9 92.3 GS-2b 25:1 nd nd nd GS-2b 10:1 90.8 1.3 7.9 GS-2b 4:1 16.9 6.9 77.8 GS-3a 25:1 90.6 4.6 3.6 GS-3a 10:1 59.4 2.5 38.1Docket No. 6045.0589WO | S23-281 GS-3a 4:1 0.5 1.4 98.1 GS-3b 25:1 9.2 6.3 84.5 GS-3b 10:1 0.7 2.1 97.3 GS-3b 4:1 nd nd nd

[0165] Identifying the characteristics of drug delivery systems that impact organ tropism is crucial to rational design for targeted delivery. Physicochemical properties of lipid nanoparticles such as size and surface charge have been shown to play important roles in mRNA delivery. See e.g., Sato et al., Mol Ther 24:788-795 (2016); Kumar et al., Mol Ther Nucleic Acids 3:e210 (2014). In order to understand whether these properties played a role with the present compounds, the size and surface charge of compound:mRNA complexes was examined under acidic (PBS pH 5.5) and physiological (PBS pH 7.5) conditions. Under acidic conditions, there was no significant variation in either nanoparticle size or surface charge (not shown). However, under physiological conditions (PBS pH 7.5) lung-tropic nanoparticles exhibited higher surface charge over time compared to spleen-tropic compounds, as shown in FIG.11.

[0166] The change in surface charge (zeta potential) over time (min) of polymer / mRNA complexes under physiological conditions (PBS pH 7.5) with different charge ratios is shown in FIG.12. The figure illustrates, from left to right: GS-1a (10:1 solid line, 4:1 dashed line), GS-2a (25:1 solid line, 4:1 dashed line), and GS-3a (25:1 solid line, 4:1 dashed line). For complexes formed with each of GS-1a, GS-2a, and GS-3a, increasing the charge ratio resulted in an elevation of surface charge.

[0167] However for other compounds, such as GS-3b, which did not show lung tropism, increasing the charge ratio from 10:1 to 25:1 did not result in increased surface charge, as illustrated in FIG.13. The figure illustrates, from left to right: GS-1b (25:1 solid line, 10:1 dashed line), GS-2b (10:1 solid line, 4:1 dashed line), and GS-3b (25:1 solid line, 10:1 dashed line).

[0168] These results indicated that surface charge of the complexes at physiological pH is highly correlated with organ tropism, and that surface charge could be altered at least in someDocket No. 6045.0589WO | S23-281 cases by altering the charge ratio of compound to mRNA used to form the complexes. This observation is in contrast to LNP systems, where the surface charge has been reported to be not correlated with organ tropism (Dilliard et al., Proc. Natl. Acad. Sci.118(52) e2109256118 (December 21, 2021); Qiu et al., Proc. Natl. Acad. Sci.119(8) e2116271119 (February 16, 2022)).

[0169] It is known that nanoparticles rapidly associate with serum proteins to form protein corona upon intravenous delivery. This process alters the physicochemical properties of lipid nanoparticles and there is emerging evidence suggesting that the protein corona may play a role in organ selectivity, but a link between nanoparticle properties and protein corona formation is not well established. We speculated that surface charge of the complexes may impact protein corona formation. To test this, complexes were incubated with mouse plasma, and their size was monitored over time (time points 0, 10, 20, and 30 min). FIG.14 shows size (nm) 1 minute after complexes were mixed with plasma. Remarkably, lung-tropic nanoparticles GS-1a, GS- 1b, GS-2a, GS-2b, and GS-3a exhibited a significant increase in size, from approximately 200 nanometers (nm) to over 2 micrometers (um). On the other hand, the spleen-tropic nanoparticles of GS-3b displayed more moderate increases in size, ranging from around 200 nm to less than 350 nm. See FIG.16. Complexes associated with protein corona maintained their sizes over 30 min. In addition, protein corona formation seemed to be reversible, as mild vortexing of complexes led to reduced nanoparticle size. This indicates that the observed change in size could also be reversible following administration.

[0170] To further characterize the protein corona associated with the complexes, protein associated with lung-tropic and spleen-tropic complexes was isolated and quantified following the protocol of Dilliard et al., Proc. Natl. Acad. Sci.118:e2109256118 (2021). In general, the lung-tropic complexes yielded more protein than spleen-tropic compounds, as determined by SDS-PAGE gel chromatography. FIG.15 shows a picture of a representative gel comparing GS-3a (10:1) with GS-3b (25:1). This result was consistent with the larger nanoparticle size observed for the lung-tropic GS-3a (10:1) complexes incubated with mouse plasma, as shown in FIG.14 and FIG.16. Since most serum proteins have an isoelectric point below 7, nanoparticles with positive surface charges may be attracting more protein resulting in larger nanoparticle sizes.

[0171] In addition to particle size, surface charge of the complexes in plasma was also evaluated. As shown in FIG.17, all complexes exhibited negative surface charge upon proteinDocket No. 6045.0589WO | S23-281 corona formation, in contrast with the complexes formulated in buffer in the absence of MSA (compare FIG.17 to FIG.11). Moreover, the magnitude of the change in surface charge was much smaller, less than 5 mV over 30 minutes, suggesting protein corona may impact the kinetics of charge alteration of the compounds. Lung-tropic (circles) and spleen-tropic (squares) complexes displayed a small but discernible surface charge difference in plasma, which suggests surface charge may still play a role in organ selectivity after protein corona formation.

[0172] To identify the most significant attributes of the complexes leading to distinct organ tropisms, we conducted a principal component analysis (PCA) by integrating particle size and surface charge. The results are depicted in FIG.18. The results revealed a distinct clustering of complexes exhibiting lung versus spleen tropisms. PC1 accounted for 59% of the variation observed between lung / spleen complexes, with the size of the complex in the presence of serum being the primary determining factor, closely followed by the change in surface charge of the complex at physiological pH. These data suggest the surface charge of the complexes greatly impacted protein corona formation and composition, which together re-shape the biophysical properties of nanoparticles in circulation and lead to distinct organ tropism.

[0173] In vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes formed using representative lung-tropic complexes is illustrated in FIG.19 using the assay described above. Charge ratios are also indicated.

[0174] In vivo protein production (measured as bioluminescence) following delivery of polymer / mRNA complexes formed using representative spleen-tropic complexes is illustrated in FIG.20 using the assay described above.

[0175] The best performing compounds were further evaluated for dose-dependence. GS- 3a / luciferase mRNA (FIG. 21) and GS-3b / luciferase mRNA (FIG.22) complexes were administered at doses 2, 5, or 7.5 ug RNA. Charge ratios were 25:1 for GS-3a and 10:1 for GS- 3b. Robust luciferase expression was detected and selective organ tropism was maintained.

[0176] Interestingly, addition of 20 % sucrose (w / v) to PBS 5.5 buffer significantly improved protein expression in the spleen for complexes formed with GS-3a. While addition of sucrose to RNA formulations as a cryoprotectant for long term storage is known, to our knowledge sucrose has not previously been demonstrated to improve delivery efficiency.

[0177] The best performing formulations, GS-3a (25:1) and GS-3b (10:1) were tested against a commercially available mRNA delivery reagent (JetRNA) and the reference polymericDocket No. 6045.0589WO | S23-281 compound, ONA CART. Both substantially outperformed these two reference reagents in terms of luciferase protein expression in the lung (GS-3a, FIG.23) and the spleen (GS-3b, FIG. 24).

[0178] Additional experiments were performed to investigate the types of cells being transfected within the lungs and spleens of the animals. The protocol is depicted graphically in FIG.25. A murine Cre recombinase model (Ai14 mice) was utilized, as described in Kauffman, K. J. et al. Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse. Mol Ther Nucleic Acids 10, 55–63 (2018). With this mouse model, successful delivery of Cre recombinase mRNA to cells leads to tdTomato fluorescent protein expression detectable by flow cytometry.15 ug Cre mRNA was formulated with GS-3a (25:1, lung-tropic) or GS-3b (10:1, spleen-tropic) and administered intravenously. Lungs and spleens were isolated 48 hr after injection and single cell suspensions of the main cell populations were analyzed by flow cytometry for tdTomato expression.

[0179] As shown in FIG. 26, GS-3a (lung tropic) provided 37 % lung cell transfection with less than 0.4 % splenocyte transfection. In contrast, as shown in FIG.27, GS-3b (spleen tropic) provided 6 % splenocyte transfection with less than 0.3 % lung cell transfection, confirming the excellent organ selectivity of the mRNA complexes formed with these compounds.

[0180] With respect to the specific types of cells being transfected, the data indicated that in the lungs complexes formed with GS-3a (25:1) transfected a significant population of non- immune cells (CD45-), and in particular, endothelial cells (CD45-, CD31+), as illustrated in FIG.28 and FIG. 29. The endothelium is the first cell layer encountered from the circulation and it represents the largest cell population (> 60 %) in murine lungs. Among lymphocytes (CD45+), macrophages (CD45+CD11B-CD11C+CD64+) were the most prominently transfected (>60%), followed by B cells (CD45+CD19+) at 20%. Successful delivery was confirmed by confocal imaging of lung sections, where tdTomato was detected in the lung parenchyma but not in the bronchioles.

[0181] In the spleen, complexes formed with GS-3b (10:1) resulted in splenic macrophages being transfected with high selectivity, as illustrated in FIG.30 and FIG.31. Individual analysis of lymphocyte populations showed that over 65% of splenic macrophages (CD45+F4 / 80+) and over 30% of dendritic cells (CD45+CD11C+) were transfected. Confocal imaging of spleen sections indicated that transfection primarily occurred in the red pulp, aDocket No. 6045.0589WO | S23-281 region predominantly composed of lymphocytes and macrophages with a primary function of blood filtration.

[0182] Importantly, treatment with GS-CART showed minimal impact on the transfected organs as indicated by the viability of isolated lung cells and splenocytes (FIG.32) after treatment and the similarity in cell composition of the lung and spleen in untreated versus treated animals (FIG.33). Delivery of circRNA with GS-CARTs elicit strong anti-tumor response

[0183] Circular RNA (circRNA) represents a new class of RNA with increased stability and more durable protein expression when compared to mRNA. In addition, circRNA possesses unique adjuvant activity which induces potent T cell immunity and leads to tumor clearance upon intraperitoneal (i.p.) administration with CARTs29. Given the promising results of GS-CART delivery with reporter mRNAs, the ability of GS-CARTs to deliver a circRNA vaccine was tested using a lower dose administered via intravenous injection, a route of administration relevant for clinical applications. We first complexed GS-3a (lung-tropic) and GS-3b (spleen-tropic) with a previously designed circRNA encoding chick Ovalbumin (circOVA) (Amaya, L. et al. Proc. Natl. Acad. Sci. 120, e2302191120 (2023). The resulting complexes had nanoparticle sizes similar to mRNA complexes. GS-CART / circOVA complexes were administered by intravenous (retro- orbital) injection and evaluated for activation of adaptive immune responses. Specifically, mice were immunized with circOVA complexed with ONA CART, GS-3a, and GS-3b at days 1 and 21. The antigen specific CD8 T cell responses were assessed by MHC class I tetramer staining (SIINFEKL epitope) of blood, spleen, and lung T cells at Day 10 post-boost (FIG. 36). Additionally, antibody responses were measured by bleeding mice at day 9 post-boost. We observed significantly higher anti-Ova IgG in the GS-3b / circOVA (spleen-tropic) group (FIG. 38).

[0184] Remarkably, compared to mice treated with an irrelevant circRNA control, GS-3b induced robust T cell responses. These findings underscore that employing GS-CARTs complexed with circRNA encoding an antigenic sequence (circOVA) can effectively elicit antigen-specific CD8 T cell responses following immunization. Notably, the spleen-specific GS- 3b formulation yielded the most potent antibody and T cell responses, surpassing the ONA CART formulation, and potentially attributable to the spleen's ideal environment for activating dendritic cells and priming antigen specific T cell responses.Docket No. 6045.0589WO | S23-281

[0185] The potent cytotoxic T cell response observed in these experiments indicated potential utility of GS-CART / circOVA as a cancer vaccine. To test this, B16F10-OVA-Luc cancer cells were subcutaneously inoculated into C57BL / 6J mice and circOVA formulations were intravenously administered 6, 10, and 14 days after tumor injection, with tumor growth monitored over time. Encouragingly, both GS-3a / circOVA and GS-3b / circOVA formulations demonstrated significant tumor growth inhibition compared to the control (ONA-circFOR, which is an irrelevant circRNA) (FIG.37 and FIG.39).

[0186] Assessing in vivo tolerability is a critical aspect in determining the clinical viability of drug delivery systems. Accordingly, we conducted a comprehensive pre-clinical toxicology blood analysis following the administration of representative complexes form with mfluc mRNA. For comparison, FDA-approved MC3 LNP was formulated with mfluc mRNA according to Wang et al., Nature Protocols 18:265–291 (2023) and used as a benchmark. As shown in FIG.34A and FIG.34B, both lung-tropic (GS-3a, 25:1) and spleen-tropic (GS-3b, 10:1) formulations exhibited similar levels of blood metabolites, which serve as indicators of liver (ALT, AST, ALP) or kidney (blood urea nitrogen, calcium, and phosphorus) damage, compared to MC3 formulations. These findings suggest the absence of clinical signs of toxicity or alterations in clinical pathology. In addition, the levels of inflammatory cytokines, including IL-1b, IL-6, and TNFa, remained similar 24 hours before and after GSer CART delivery (FIG. 44).

[0187] The in vitro stability of the GS-3b / mRNA complexes was evaluated over time for complexes formulated on ice (FIG.35A) and after storage at -20 °C for three days (FIG.35B). The complexes maintained transfection efficiency for at least 30 min after formulation on ice and could be stored at -20 °C for long term use. Expanding on these data, FIG.40 shows transfection efficiency of leading GS-CART formulations incubated for 30 min on ice and following storage at -20°C (FIG.41), -80°C (FIG.42) or after lyophilization and stored at - 20°C (FIG.43).

[0188] These results collectively demonstrate the delivery efficacy, selectivity, versatility, tolerability, and stability of GS-CARTs for mRNA delivery.

[0189] Guanidinium motifs have been used in drug delivery systems due to their excellent ability to interact with negatively charged lipids on cellular membranes and enhance cellular uptake. However, persistent guanidinium groups proved to be inefficient for mRNA delivery, likely due to their strong association with and thus poor release of polyanionic cargoes. Here,Docket No. 6045.0589WO | S23-281 we solved this problem by designing guanidinium containing compounds that effectively complex polyanions at low pH (~pH 5.5) but but undergo an irreversible and unique O-N acyl shift that neutralizes cationic charges under physiological conditions (~pH 7.5) thereby releasing the polyanionic cargo. These compounds show remarkably efficient mRNA delivery both in vitro and in vivo, surpassing the performance of commercially available transfection reagents as well as a reference polymeric compound, ONA CART. The compounds also show organ-specific (lung, spleen) and cell-selective (splenic macrophage) delivery upon intravenous administration in vivo as well as good tolerability.

[0190] The results here also demonstrate that nanoparticle surface charge impacts the formation and composition of protein corona, which reciprocally alter the size and surface charge of the original mRNA complexes. Whether this is reversible or leads to other complexes and aggregates that enter cells is not known. Both biophysical properties and protein corona display significant differences for formulations targeting different organs, suggesting they likely play a role in determining organ selectivity. The detailed mechanism of how size / surface charge, protein corona, and organ tropism are connected remains unknown. Nevertheless, the data presented here illustrate the paramount importance of nanoparticle biophysical properties, particularly in the plasma environment on selective in vivo delivery.

[0191] Upon in vivo administration, mRNA complexes with compounds described here demonstrated efficient transfection of various lung cells and splenocytes. Notably, both lung and spleen delivery led to significant transfection of immune cells, without the use of targeting ligands. This is highly relevant for applications involving prophylactic and therapeutic vaccinations relevant to therapy for diseases such as cancer and the treatment of infectious diseases. The preferential transfection of lung endothelial cells and splenic macrophages is also relevant for therapy of respiratory diseases and inflammation, respectively. Endothelial cells are the primary cells affected in the pathogenesis of various respiratory diseases while macrophages, as essential components of the innate immune system, play pivotal roles in inflammatory diseases and cancer. Targeting these specific macrophages could enable focused depletion during chronic inflammation or facilitate the generation of CAR-macrophages for tumor immune cell therapy.

[0192] While the vast majority of RNA delivery systems are based on protonatable amines (ammonium cations), guanidinium cations have featured significantly in the cell-penetrating performance of peptidic and non-peptidic molecular transporters and are known to facilitateDocket No. 6045.0589WO | S23-281 cellular uptake. However, their use for polyanion delivery has been limited due in part to their strong polyanion association which inhibits release. Here this limitation is addressed with a unique guanidinium-based delivery system in which the cationic guanidinium group is dynamically converted at physiological pH into neutral byproducts thereby enabling release of its polyanionic cargo. This new charge-cancellation process opens the door for the broader use of guanidinium cations as delivery components in place of ammonium ion counterparts. Significantly, GS-CARTs exhibit efficient and selective mRNA delivery to extrahepatic organs (lung up to 97 % and spleen up to 98 %) in a tunable and predictable manner, functioning as a facile strategy for future CART and potentially other delivery systems. The translational potential of GS-CARTs is illustrated by its successful use in cancer vaccination via the delivery of circOVA, which showed robust generation of cytotoxic, antigen specific T cells and tumor growth inhibition. The proof-of-concept development of GS-CARTs / circOVA cancer vaccines underscores the clinical potential of GS-CARTs, suggesting broader applications in various biomedical research and therapeutic contexts. Methods Dynamic light scattering (DLS) for size analysis and Zeta-potential for surface charge analysis

[0193] For size and zeta potential measurement in acidic formulation buffer, CARTs were added to PBS pH 5.5 containing 420 ng of luciferase mRNA (Trilink L-7211) at various charge ratios indicated above (cation: anion). The formulations were mixed for 20 seconds using a micropipette (drawing and dispensing 8 μl about 3 times / second) and subsequently diluted with 1 mL DI water. The dilutions were then immediately transferred to a disposable clear plastic cuvette, and the sizes measured by NanoBrook Omni (Serial No: 280097). Surface charge was monitored at 0, 10, 20, 30 min after formulation using the same instruments. For size and zeta potential measurements in physiological pH, GS-CART / mRNA formulations were prepared as described above, 100 μl 100 mM Na2CO3M was added and mixed for 10 sec and subsequently diluted with DI water for measurement. For size and zeta potential measurements in mouse serum albumin (MSA) solution (conc) and mouse plasma, GS-CART / mRNA formulations were prepared as described above, 8.4 μl protein solution or plasma was then added and mixed for 10 sec and subsequently diluted with DI water for measurement. In vivo mRNA deliveryDocket No. 6045.0589WO | S23-281

[0194] All experiments were approved by the Stanford Administrative Panel on Laboratory Animal Care and were conducted in accordance with Stanford University Animal Facility and NIH (National Institutes of Health) guidelines. Luciferase mRNA biodistribution

[0195] Female BALB / c mice were purchased from Jackson Laboratory and housed in the Laboratory Animal Facility of the Stanford University Medical Center. Luciferase mRNA was purchased from Trilink Biotechnologies (Trilink L-7202) and nanoluciferase mRNA was prepared in house as previously reported. CARTs were complexed with 2, 5, or 7.5 ug mRNA in PBS pH 5.5 of PBS 5.5 containing 20% sucrose to a total volume of 100 μl at 4:1, 10:1, or 25:1 N:P ratio with the same formulation procedure described above. The formulations were mixed for 20 seconds using a micropipette (drawing and dispensing 100 μl twice / second) before retro-orbital injections.6 hours after injection, D-luciferin solution (100 μl, 30 mg / mL) was injected intraperitoneally, and whole body luminescence was measured using AMI Imaging system CCD camera and analyzed with Aura. Mice were subsequently sacrificed in CO2 for 8 min and their lungs, livers, spleens isolated to quantify organ specific luciferase expression. Ai14 Cre-mediated recombination (from bAC)

[0196] Female Ai14 mice were purchased from Jackson Laboratory and housed in the Laboratory Animal Facility of the Stanford University Medical Center. CARTs were complexed with 15 μg Cre mRNA (Trilink L-7211) in PBS pH 5.5 to a total volume of 100 μl at 10:1 N:P ratio, with the same formulation procedure described above. Spleens were isolated 48 h after transfection to measure Cre-mediated recombination. Spleens were smashed with a 100 μm strainer to make a single-cell suspension. Red blood cells were lysed before staining. Single-cell samples were then stained with Zombie NIR (BUV570, BioLegend 423103), anti- CD45 (clone 145-2C11, BioLegend), anti-CD8α (clone 53-6.7, BioLegend), anti-CD4 (clone RM4-5, BioLegend), anti-CD11c (clone IM7, BioLegend), anti-CD19 (clone 30-F11, BioLegend), anti-F4 / 80 (clone H1.2F3, BioLegend). Cells were then washed twice and analyzed by flow cytometry. Data were collected on a Attune Nxt Flow Cytometer. Statistical analysis (from bAC)

[0197] All other statistical analysis was performed with Prism (GraphPad Software v9.2.0). For comparing two groups, P values were determined using Student’s t-tests (two-tailed). ForDocket No. 6045.0589WO | S23-281 comparing more than two groups, 2-way ANOVAs were applied. Differences between groups were considered significant for P values < 0.05. No statistical methods were used to predetermine sample sizes. Mice were assigned to the various experimental groups randomly. Data collection and analysis were not performed blind to the conditions of the experiments.

[0198] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0199] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical sciences.

[0200] The term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0201] The term “initiator” refers to a compound that initiates a polymerization reaction. Thus, the initiator is typically incorporated at the end of the synthesized polymer. For example, a plurality of monomer molecules can be reacted with an initiator to provide a polymer or copolymer. The initiator can be present on at least one end of the resulting polymer or copolymer and not constitute a repeating monomer unit of the polymer.

[0202] The terms “oligomer” and “polymer” are used interchangeably herein to refer to a compound that has a plurality of repeating subunits, which may be referred to as blocks or monomer units, or simply as monomers. The terms “co-oligomer” or “copolymer” are used interchangeably herein to refer to an oligomer or polymer that includes two or more different types of monomers.

[0203] The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” are used interchangeably herein to refer to polymers of deoxyribonucleotides or ribonucleotides in either single-, double- or multiple-stranded form, or complements thereof. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer.Docket No. 6045.0589WO | S23-281 Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. RNA may include messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), and transactivating RNA (tracrRNA). DNA may include plasmid DNA (pDNA), minicircle DNA, genomic DNA (gNDA), and fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids has one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0204] Polynucleotides may comprise known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids andDocket No. 6045.0589WO | S23-281 analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0205] The term “small organic molecule” refers to a small organic compound, including heteroorganic and organometallic compounds, having a molecular weight less than about 1,000 grams per mole, or less than about 500 grams per mole and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0206] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.

[0207] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details that are not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0208] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. The transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the recited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

Claims

Docket No. 6045.0589WO | S23-281 CLAIMS What is claimed is:

1. A compound of Formula I: R1-X-[(L1)x1-(L2)x2-(A1)y1]-R2 (Formula I) where R1 is substituted or unsubstituted C1-C24alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; X is a bond, O, S, N or a linking group as defined herein; L1 and L2 are each independently a lipid carbonate monomer unit; A1 is a guanidinylated serinol monomer unit; x1 and y1 are each independently greater than zero and less than 100; x2 and y2 are each independently zero or less than 100; and R2 is H, C2-C24 alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl, optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-6 alkyl, and carboxylato.

2. The compound of claim 1, wherein L1 and L2 are each independently a lipid-functionalized methyl-trimethylene carbonate (MTC) unit having a structure ofa structure ofDocket No. 6045.0589WO | S23-281 where RL is a branched or unbranched C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated. In some aspects, RLis independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

3. The compound of claim 1 or 2, wherein A1 is a guanidinylated serinol monomer unit having a structure of 4. Thewherein X is O; L1 and L2 are each an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ia:Ia where R1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1 and RL2 are each independently a branched or unbranched C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated; x1 and x2 are each independently 1-100, 1-50, or 1-25; and y1 is 1-100, 1-50, or 1-25.

5. The compound of claim 4, wherein R1 is benzyl andDocket No. 6045.0589WO | S23-281 RL1 and RL2 are each independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

6. The compound of claim 4 or 5, wherein the compound of Formula Ia is GS-1a, GS-1b, or GS-1c.

7. The compound of any one of claims 1 to 3, wherein X is O; L1 is an MTC unit; A1 is a GSer unit and the compound is represented by Formula Ib: IbR1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1is a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25; x2 is zero; and y1 is 1-100, 1-50, or 1-25.

8. The compound of claim 7, wherein RL1 is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

9. The compound of claim 7 or 8, wherein the compound of Formula Ib is GS-2a, GS-2b, or GS-2c.

10. The compound of any one of claims 1 to 3, wherein X is O; L1 is a bAC unit;Docket No. 6045.0589WO | S23-281 A1 is a GSer unit and the compound is represented by Formula Ic: Icor cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; R2 is H; RL1is a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated; x1 is 1-100, 1-50, or 1-25; x2 is zero; and y1 is 1-100, 1-50, or 1-25.

11. The compound of claim 10, wherein RL1 is stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, or farnesyl.

12. The compound of claim 10 or 11, wherein the compound of Formula 1c is GS-3a, GS-3b, or GS-3c.

13. The compound of claim 12, wherein the compound is GS-3a or GS-3b.

14. A composition or pharmaceutical composition comprising complexes of the compound of any one of claims 1 to 13, non-covalently associated with a nucleic acid or a plurality of different nucleic acids.

15. The composition or pharmaceutical composition of claim 14, wherein the complexes form nanoparticles having a net positive or a net negative surface charge in aqueous buffer.

16. The composition or pharmaceutical composition of claim 14 or 15, wherein the nucleic acid is RNA or DNA.Docket No. 6045.0589WO | S23-281 17. The composition or pharmaceutical composition of any one of claims 14 to 16, wherein the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA, plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

18. The composition or pharmaceutical composition of any one of claims 14 to 17, wherein the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent.

19. The composition or pharmaceutical composition of any one of claims 14 to 18, wherein the composition is a vaccine.

20. The composition or pharmaceutical composition of any one of claims 14 to 19, wherein the nucleic acid is mRNA or circRNA.

21. A method of transfecting a nucleic acid into a cell in vitro or ex vivo, the method comprising contacting the cell in vitro or ex vivo with the composition or pharmaceutical composition of any one of claims 14 to 20.

22. A method of delivering a nucleic acid to a cell of a subject, the method comprising administering to the subject the composition or pharmaceutical composition of any one of claims 14 to 20.

23. The method of claim 22, wherein the cell is a pulmonary endothelial cell, a pulmonary immune cell, or a splenic macrophage.

24. The method of claim 22 or 23, wherein the complexes of the composition comprise a compound of Formula 1c.

25. A method of delivering a nucleic acid to the lungs of a subject, the method comprising administering to the subject the composition or pharmaceutical composition of any one of claims 14 to 20.

26. The method of claim 25, wherein the complexes of the composition form nanoparticles having a surface charge above zero millivolts (mV) zeta potential in aqueous buffer.Docket No. 6045.0589WO | S23-281 27. The method of claim 25 or 26, wherein the complexes of the composition have a 25:1 (cation: anion) charge ratio.

28. The method of any one of claims 25 to 27, wherein the complexes comprise a compound selected from the group consisting of GS-1a, GS-1b, GS-2a, and GS-3a.

29. A method of delivering a nucleic acid to the lungs or spleen of a subject, the method comprising administering to the subject the composition or pharmaceutical composition of any one of claims 14 to 20.

30. The method of claim 29, wherein the complexes of the composition have a 10:1 (cation: anion) charge ratio.

31. The method of claim 29 or 30, wherein the complexes comprise a compound selected from the group consisting of GS-2a, GS-2b, and GS-3a.

32. A method of delivering a nucleic acid to a spleen of a subject, the method comprising administering to the subject a composition or pharmaceutical composition comprising of any one of claims 14 to 20.

33. The method of claim 32, wherein the complexes of the composition form nanoparticles having a surface charge below zero millivolts (mV) zeta potential in aqueous buffer.

34. The method of claim 32 or 33, wherein the complexes of the composition have a 4:1 (cation: anion) charge ratio.

35. The method of any one of claims 32 to 34, wherein the complexes comprise a compound selected from the group consisting of GS-1a, GS-2a, GS-2b, and GS-3a.

36. A method for treating a respiratory disease in a subject, the method comprising delivering a therapeutic nucleic acid to the lungs of a subject according to the method of any one of claims 22 to 31.

37. A method for treating an inflammatory disease in a subject, the method comprising delivering a therapeutic nucleic acid to the spleen of a subject according to the method of any one of claims 22-24 or 29-35.Docket No. 6045.0589WO | S23-281 38. The method of claim 37, wherein the therapeutic nucleic acid encodes an anti-inflammatory protein, optionally interleukin 10 (IL-10) or apolipoprotein E (ApoE).

39. A method for treating cancer in a subject, the method comprising delivering a therapeutic nucleic acid to the spleen of a subject according to the method of any one of claims 22-24 or 29-35.

40. A method for treating cancer in a subject, the method comprising delivering a therapeutic nucleic acid to the lungs of a subject according to the method of any one of claims 22 to 31.

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