Nucleic acid complex and its use

Abstract

The present disclosure provides complexes and methods of use. In some embodiments, the complexes described herein comprise a cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer. In some embodiments, the complexes comprise a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT Application No. PCT / IB22 / 59136, filed September 26, 2022, and U.S. Provisional Patent Application No. 63 / 517338, filed August 2, 2023, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] The use of nucleic acids, e.g., RNA, has emerged in recent years as a means for delivering therapeutics. Nucleic acid therapeutics have shown particular success in the areas of infectious disease vaccines and show promise as cancer immunotherapy, therapeutic protein replacement therapy, and the treatment of genetic diseases. Delivering nucleic acid therapeutics to cellular targets faces some challenges because RNA is unstable and susceptible to degradation by nucleases. See Wadhwa, et al., Pharmaceutics, 12(2):102 (2020). Summary of the Invention

[0003] There remains a need for a delivery vehicle for nucleic acids, such as RNA, that can effectively and efficiently deliver nucleic acids to target cells while withstanding conditions in the body and that does not require large amounts of nucleic acid to achieve efficacy. Cationic polymers have been recognized as useful in developing delivery vehicles, such as those reported in PCT Application Publication No. WO 2021 / 001417, the entire contents of which are incorporated herein by reference. However, it is desirable to provide a delivery vehicle that provides improved functionality over previous formulations, such as improved stealth properties, improved or modified targeting of cells or tissues, includes tracking functionality (e.g., radionuclides, fluorophores, or chelators), and / or can beneficially respond to specific physiological conditions, such as pH, temperature, or redox potential.

[0004] The present disclosure provides, among other things, complexes for delivering nucleic acids, e.g., RNA, that exhibit certain improvements over previous complex formulations. In some embodiments, the present disclosure provides complexes comprising a cationic polymer, an anionic polymer, and RNA, wherein the cationic polymer and the RNA form a core complex encapsulated by the anionic polymer. Such complexes, exhibiting the improved properties described above, can be used in applications not previously realized with conventional formulations, such as systemic, subcutaneous, or intranasal administration. Furthermore, in some embodiments, the provided complexes allow for specific tuning of the surface charge, preferably from about -25 mV to about +25 mV, and exhibit particular stability when exposed to multiple freeze-thaw cycles while maintaining an acceptable PDI and consistent size distribution.

[0005] It has also been surprisingly found that certain cationic polymers, such as cationic polymers with a linear structure, confer improved properties compared to those with a branched structure. For example, as shown in Example 7, complexes comprising a linear cationic polymer were found to result in improved transfection in various cell lines compared to complexes comprising a branched cationic polymer. Thus, in some embodiments, the present disclosure provides complexes comprising a linear cationic polymer, an anionic polymer, and RNA, wherein the linear cationic polymer and the RNA form a core complex encapsulated by the anionic polymer.

[0006] In some embodiments, the present disclosure provides a method of increasing or causing an increase in expression of RNA in a target in a subject, comprising administering to the subject a complex described herein.

[0007] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition in a subject, comprising administering to the subject a conjugate described herein.

[0008] In some embodiments, the present disclosure provides a conjugate described herein for use as a medicament.

[0009] In some embodiments, the present disclosure provides a conjugate described herein for use in the treatment and / or prevention of a disease or disorder, wherein the disease or disorder is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a bar graph showing the mean hydrodynamic diameter of formulations containing saRNA after incubation in 10% v / v human serum at 37° C. for 30 minutes. [Figure 2] A-C show the physicochemical characterization of Alfa-tagged ternary modRNA PEI-PLX with PGA(50)-b-pSar(50)-Alfa, PGA(50)-Alfa, PGA(100)-Alfa, and PGA(50)-b-Ac-AEEA(14)-Alfa at Glu / N ratios of 0-3. A is a plot measuring the particle size distribution (Zavg). B is a plot measuring the polydispersity index (PDI). C is a plot measuring the zeta potential. [Figure 3] A–D show (A) PGA(50)-b-pSar(50)-Alfa (Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, and 1.5 followed by naked saRNA control), (B) PGA(50)-Alfa (Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, and 3 followed by naked saRNA control), (C) PGA(100)-Alfa (Glu / N ratios of 0, 0.2, 0.4, and 0.6 followed by naked saRNA control), and (D) PGA(50)b-Ac-AEEA(14)-Alfa (Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, and 1.5 followed by naked saRNA control). Each well represents a sample with increasing Glu / N ratio, followed by naked modRNA control. [Figure 4A]Freezing stability of ternary modRNA PEI-PLX exhibiting Zavg and PDI. PGA(50)-b-pSar(50)-Alfa at -80°C. [Figure 4B] Freezing stability of ternary modRNA PEI-PLX showing Zavg and PDI. PGA(50)-Alfa at -80°C. [Figure 4C] Freezing stability of ternary modRNA PEI-PLX showing Zavg and PDI. PGA(50)-Alfa at -20°C. [Figure 4D] Freezing stability of ternary modRNA PEI-PLX exhibiting Zavg and PDI. PGA(100)-Alfa at -80°C. [Figure 4E] Freezing stability of ternary modRNA PEI-PLX, PGA(50)-b-Ac-AEEA(14)-Alfa at -80°C, showing Zavg and PDI. [Figure 4F] Freezing stability of ternary modRNA PEI-PLX, PGA(50)-b-Ac-AEEA(14)-Alfa, showing Zavg and PDI at -20°C. [Figure 5] A-C are a set of plots showing the physicochemical characterization of alfa-tagged PGA(X) ternary PEI polyplexes (X = 50, 100), where A shows the particle size distribution, B shows the polydispersity index (PDI) distribution, and C shows the zeta potential distribution. [Figure 6] Agarose gel electrophoresis of ternary PEI-PLX with PGA(X)-Alfa at X = 50 and 100. Each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, and 1.5 (PGA(50)-Alfa) and 0, 0.2, 0.4, and 0.6 (PGA(100)-Alfa), followed by a naked saRNA control. [Figure 7] A–D show the freezing stability (Zavg and PDI) of ternary saRNA PEI-PLX using bar graphs; A and B report PGA(50)-Alfa at -20°C and -80°C, and C and D report PGA(100)-Alfa at -20°C and -80°C. [Figure 8] A–C are a series of plots showing the physicochemical characterization of PEI-PLX-coated saRNA polyplexes with PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-pAEEA(14)-Alfa. A shows the particle size distribution, B shows the polydispersity index (PDI) distribution, and C shows the zeta potential distribution. [Figure 9] A-B show agarose gel electrophoresis of ternary PEI-PLX, where A shows PGA(50)-b-pSar(50)-Alfa and B shows PGA(50)-b-pAEEA(14)-Alfa. (Each well shows increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, and 1 (PGA(50)-Alfa) followed by naked saRNA control.) [Figure 10] A–D show the freezing stability (Z-avg and PDI) of ternary saRNA PEI-PLX. A and B show PGA(50)-b-pSar(50)-Alfa at −20°C and −80°C, and C and D show PGA(50)-b-pAEEA(14)-Alfa at −20°C and −80°C. [Figure 11] The particle size and PDI of functionalized polyplexes at N / P = 15 are shown. Core particles. Size and PDI measurements were performed at 23 °C using a DynaPro Plate Reader III (Wyatt Technology) at a concentration of 5 ng / μL (5 μL of sample mixed with 95 μL of water). Before measurement, the sample was mixed in the well by pipetting. For each well, 10 acquisitions were used with an acquisition time of 5 seconds. [Figure 12] Zeta potential of functionalized polyplexes is shown. Zeta potential measurements were performed at 23 °C at a concentration of 0.002 μg / μL (16 μL sample mixed with 784 μL of 10 mM HEPES, pH = 6) using a Malvern Zetasizer Ultra Nano Series Instrument cuvette with a DTS1070 cuvette. Each sample was measured in 12 runs (attenuator setting 11). [Figure 13]Agarose gel electrophoresis analysis of functionalized polyplexes is shown. Agarose gels were run at 1% using TAE buffer at pH 7 and Gel-Red at the recommended dilution. For all samples, 10 μL (total nucleic acid (NA) concentration = 0.1 μg / μL) was loaded onto the gel, resulting in a total NA mass of 1 μg per well. Two controls for each NA were pipetted (0.1 μg, 0.5 μg). Of the 6x loading dye, 2 μL was added to all samples and controls. Separation was performed at 80 V and 500 mA for 40 min. Images were acquired with a BioRad ChemiDOC imager (automatic optimal exposure time). [Figure 14] PBMC transfection studies using functionalized polyplexes are shown. 10 μl of each nanoparticle formulation (total cargo concentration = 0.1 μg / μL, c(Thy1.1) = 0.025 g / L) was prediluted with 50 μl of X-Vivo15 in an ultra-low attachment 96-well plate. 0.3e6 thawed human PBMCs were diluted with 50 μl of human serum from male AB clotted whole blood and added to the nanoparticle dilution. After 30 minutes of incubation (37°C, 5% CO), 100 μl of X-Vivo15 containing human IL-2 [200 U / ml] was added per well. Cells were further cultured for 96 hours (37°C, 5% CO). A shows the viable cell counts of total cells (total viability, right y-axis) and cell subpopulations (CD4+, CD8+, CD19+, and CD14+, left y-axis) analyzed by flow cytometry. B. Cell type-specific RNA transfection (Thy1.1-RNA expression) analyzed by flow cytometry. Shown is the percentage of Thy1.1-expressing cells among all single cells and viable cells. [Figure 15]Transfection studies using functionalized polyplexes in Jurkat cells are shown. 5 μl of each nanoparticle formulation was prediluted in 50 μl of RPMI + 10% FBS and 1% Pen / Strep in an ultra-low attachment 96-well plate. 0.5e6 Jurkat cells were diluted in 50 μl of RPMI + 10% FBS and 1% Pen / Strep and added to the nanoparticle dilution. After 30 minutes of incubation (37°C, 5% CO2), 10 μl of Jurkat cell and nanoparticle solution was transferred to a new well, and 190 μl of RPMI + 10% FBS and 1% Pen / Strep was added. Cells were cultured for an additional 96 hours (37°C, 5% CO2). Figure 15 shows transfections expressed as the number of viable cells based on flow cytometry (Alive; right Y-axis) and the percentage of cells expressing Thy1.1-RNA and Venus-DNA among all single and viable cells (Thy1.1 / Venus; left Y-axis). [Figure 16] Figures A-B show the physicochemical characterization of binary polyplexes prepared using linear (l-PEI) and branched (b-PEI) PEI and modRNA and saRNA. Figure A shows the particle size distribution and monomeric RNA. Figure B shows an image of agarose gel electrophoresis (each well represents a sample followed by a naked modRNA or saRNA control). [Figure 17A] Figure 17 shows the freeze stability of binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) structures. Figure 17A reports the complex with modRNA. [Figure 17B] Figure 17B shows the freezing stability of binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) structures. Figure 17B reports the complexes with saRNA at -20°C and -80°C. [Figure 18] A–C show the in vitro transfection activity of modRNA binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) PEI in HepG2 (A), RAW (B), and C2C12 (C) cell lines. [Figure 19]A-C show the in vitro cellular activity of modRNA binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) PEI in HepG2 (A), RAW (B), and C2C12 (C) cell lines. [Figure 20] A–C show the in vitro transfection activity of saRNA binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) PEI in HepG2 (A), RAW (B), and C2C12 (C) cell lines. [Figure 21] A–C show the in vitro cellular activity of saRNA binary polyplexes prepared with linear (L-PEI) and branched (b-PEI) PEI in HepG2 (A), RAW (B), and C2C12 (C) cell lines. [Figure 22] Physicochemical characterization of the modRNA ternary system PEI-PLX with PGA(X) at X = 20, 50. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 23] Agarose gel electrophoresis of ternary PEI-PLX with PGA(X) at X=20, 50 is shown. Each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.8, 1, 1.5, and 3 for PGA(20), followed by a naked modRNA control. For PGA(50), each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.8, 1, and 1.5, followed by a naked modRNA control. [Figure 24] Serum stability of ternary PEI-PLX with PGA(X) at X = 20 and 50 is shown. For each set of ternary complexes, the top lane corresponds to a control sample without serum incubation, and the bottom lane corresponds to a sample that received serum incubation. For PGA(20), each well represents samples with increasing Glu / N ratios (0, 0.2, 0.4, 0.8, 1, 1.5, and 3), followed by a naked modRNA control. For PGA(50), each well represents samples with increasing Glu / N ratios (0, 0.2, 0.4, and 0.8), followed by a naked modRNA control. [Figure 25]Figure 1 shows the freezing stability of ternary modRNA PEI-PLX containing PGA(20) and PGA(50) at -20°C and -80°C, respectively. [Figure 26] Figure 1 shows the physicochemical characterization of ternary PEI-PLX containing PAsp(50). The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 27] Agarose gel electrophoresis of ternary PEI-PLX containing PAsp(50) is shown. Each well represents samples with increasing Asp / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, and 3, followed by a naked modRNA control. [Figure 28] Physicochemical properties of the ternary PEI-PLX system containing PGA(50)-b-pSar(X) with X = 10, 25, and 50. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 29] Image of agarose gel electrophoresis of ternary PEI-PLX containing PGA(50)-b-pSar(X) at X = 10, 25, and 50. Each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, and 0.6, followed by a naked modRNA control. [Figure 30] Serum stability of ternary PEI-PLX prepared with PGA(50)-b-pSar(50) at Glu / N of 0.6. The top lane corresponds to the control sample without serum incubation, while the bottom lane corresponds to the sample that underwent serum incubation. The order of the wells represents increasing incubation times: 30 min, 1 h, and 2 h, followed by the naked modRNA control. [Figure 31] Figure 1 shows the cell viability of modRNA binary and ternary PLX containing PGA(50)-b-pSar(50) in HepG2, RAW, and C2C12 cell lines at a Glu / N ratio of 0.6. [Figure 32]Figure 1 shows the in vitro transfection activity of modRNA binary and ternary PLX containing PGA(50)-b-pSar(50) in HepG2, RAW, and C2C12 cell lines at a Glu / N ratio of 0.6. [Figure 33] Physicochemical characterization of Alfa-tagged ternary modRNA PEI-PLX containing PGA(50)-b-pSar(50)-Alfa, PGA(50)-Alfa, PGA(100)-Alfa, and PGA(50)-b-Ac-AEEA(14)-Alfa at Glu / N ratios of 0 to 3. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 34] Agarose gel electrophoresis of ternary modRNA PEI-PLX with A) PGA(50)-b-pSar(50)-Alfa (Glu / N 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5)-x), B) PGA(50)-Alfa (Glu / N 0, 0.2, 0.4, 1, 1.5, 3), C) PGA(100)-Alfa (Glu / N 0, 0.2, 0.4), and D) PGA(50)b-Ac-AEEA(14)-Alfa (Glu / N 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5). Each well represents samples with increasing Glu / N ratios, followed by a naked modRNA control. [Figure 35] Figure 1 shows the freezing stability of ternary modRNA PEI-PLX containing PGA(50)-b-pSar(50)-Alfa, PGA(50)-Alfa, PGA(100)-Alfa, and PGA(50)-b-Ac-AEEA(14)-Alfa at −20°C and −80°C, respectively. [Figure 36] Figure 1 shows the physicochemical characterization of the saRNA ternary system PEI-PLX containing PGA(X) at X = 10, 20, and 50. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 37]Image of agarose gel electrophoresis of ternary PEI-PLX containing PGA(X) at X = 10, 20, and 50. Each well represents samples with increasing Glu / N ratios from 0, 0.2, 0.4, 1.5, and 3, followed by naked saRNA control for PGA(10), increasing Glu / N ratios from 0, 0.2, 0.4, 0.6, 0.8, 1.5, and 3, followed by naked saRNA control for PGA(20), followed by naked saRNA control for PGA(50), Glu / N ratios from 0, 0.2, 0.4, 0.8, 1. Each well contains samples with increasing Glu / N ratios of 0, 0.2, 0.4, 1.5, and 3 for PGA(10), followed by a naked saRNA control; samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, and 3 for PGA(20), followed by a naked saRNA control; and samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.8, and 1 for PGA(50), followed by a naked saRNA control. [Figure 38] Freezing stability of ternary saRNA PEI-PLX containing A) PGA(10), B) PGA(20), and C) PGA(50) at selected Glu / N ratios at -20°C and -80°C, respectively. [Figure 39] Physicochemical properties of the ternary PEI-PLX system containing PGA(50)-b-pSar(X) with X = 10, 25, and 50. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 40] Image of agarose gel electrophoresis of ternary PEI-PLX containing PGA(50)-b-pSar(X) at X = 10, 25, and 50. Each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, and 0.6, followed by a naked modRNA control. [Figure 41] Freezing stability of ternary saRNA PEI-PLX with A) PGA(50)-b-pSar(10), B) PGA(50)-b-pSar(25), and PGA(50)-b-pSar(50) at -80 °C. [Figure 42]Figure 1 shows the physicochemical characterization of alfa-tagged PGA(X) ternary PEI polyplexes (at X = 50, 100). The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 43] Image of agarose gel electrophoresis of ternary PEI-PLX with PGA(X)-Alfa at X=50, 100. Each well represents samples with increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, and 1.5 for PGA(50)-Alfa, followed by naked modRNA control; and samples with increasing Glu / N ratios of 0, 0.2, 0.4, and 0.6 for PGA(100)-Alfa, followed by naked modRNA control. [Figure 44] Figure 1 shows the freezing stability of ternary saRNA PEI-PLX containing PGA(50)-Alfa and PGA(100)-Alfa at -20°C and -80°C. [Figure 45] Physicochemical characterization of saRNA polyplexes coated with PEI-PLX containing PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-pAEEA(14)-Alfa. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 46] Image of agarose gel electrophoresis of ternary PEI-PLX containing PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-pAEEA(14)-Alfa. Each well represents increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, and 1, followed by a naked saRNA control. [Figure 47] Figure 1 shows the freezing stability of ternary saRNA PEI-PLX containing PGA(50)-b-pSar(50)-Alfa (A) and PGA(50)-b-pAEEA(14)-Alfa (B) at -20°C and -80°C. [Figure 48]Figure 1 shows the physicochemical characterization of the saRNA ternary system PGA(50)-b-Ac-AEEA(X) at X = 4, 8, and 14. The top graph shows the particle size distribution, the middle graph shows the polydispersity index, and the bottom graph shows the zeta potential. [Figure 49] Agarose gel electrophoresis images of ternary PEI-PLX systems containing PGA(50)-b-Ac-AEEA(X) at X = 4, 8, and 14. Each well represents increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, and 1 for PGA(50)-b-pAEEA(4) and PGA(50)-b-pAEEA(8), followed by naked saRNA control. For PGA(50)-b-pAEEA(14), each well represents increasing Glu / N ratios of 0, 0.2, 0.4, 0.6, 0.8, 1, and 1.5, followed by naked saRNA control. [Figure 50] Figure 1 shows the freezing stability of the ternary saRNA PEI-PLX containing PGA(50)-b-Ac-AEEA(X) with X = 4, 8, and 14 at -20°C and -80°C. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure provides, inter alia, complexes useful for the delivery of nucleic acids, e.g., RNA, and uses of such complexes. In some embodiments, such complexes exhibit improved properties over previous formulations, including one or more of the following: enhanced stealth properties, improved or altered cell or tissue targeting, the addition of tracking functionality (e.g., imageable moieties such as radionuclides), and / or favorable response to specific physiological conditions, such as pH, temperature, or redox potential. Furthermore, the inclusion of linear cationic polymers in the complexes reported herein has also been found to confer improved properties over complexes containing branched cationic polymers. For example, as exemplified in Example 7, the use of linear cationic polymers improved transfection efficiency of target cells (e.g., 2-3 log improvement) at various concentrations compared to branched polymers.

[0012] In some embodiments, the conjugates described herein are useful for treating various diseases. In some embodiments, such conjugates can be administered via systemic, intravenous, or intranasal means. Furthermore, in some embodiments, provided conjugates allow for specific tuning of the charge on their surface, preferably from about -25 mV to about +25 mV, and exhibit particular stability when exposed to multiple freeze-thaw cycles while maintaining an acceptable PDI and consistent size distribution.

[0013] definition The complexes of the present disclosure include those outlined above and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the contents of which are incorporated herein by reference in their entireties.

[0014] Unless otherwise specified, structures depicted herein are intended to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of this disclosure. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the provided compounds are within the scope of this disclosure. Unless otherwise specified, all tautomeric forms of the provided compounds are within the scope of this disclosure.

[0015] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of a hydrogen by deuterium or tritium, or 13 C or 14 Compounds having this structure including the replacement of a carbon with a C-enriched carbon are within the scope of this disclosure.

[0016] About or Approximately: As used herein, the term "about" or "approximately" refers to a value similar to a stated reference value, as applied to one or more subject values. Generally, a person skilled in the art who is familiar with the context will fully understand the relevant degree of difference encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" can encompass a range of values ​​within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0017] Administering: As used herein, the term "administering" or "administration" typically refers to administration of a composition to a subject to achieve delivery of the composition or an agent contained therein to a target site or site to be treated. Those skilled in the art will recognize various routes available for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration can be, e.g., ophthalmic, oral, injection, topical, etc. In some embodiments, administration can be transbronchial (e.g., by intrabronchial infusion), buccal, transdermal (e.g., can be or can include one or more of topical, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraorgan (e.g., intrahepatic), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (e.g., by intratracheal infusion), vaginal, intravitreal, etc. In some embodiments, administration can be parenteral. In some embodiments, administration may be oral. In some specific embodiments, administration may be intravenous. In some specific embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a set number of doses. In some embodiments, administration may involve dosing that is intermittent dosing (e.g., multiple doses separated in time) and / or cyclical dosing (e.g., individual doses separated by a common period of time). In some embodiments, administration may involve continuous administration (e.g., perfusion) over at least a selected period of time. In some embodiments, administration may involve a prime-and-boost protocol. A prime-and-boost protocol may include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine), followed, after a set interval of time, by administration of a second or subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol may result in an increased immune response in a patient.

[0018] Aliphatic: The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, with single or multiple points of attachment to the rest of the molecule, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic (also referred to herein as "cycloaliphatic"). Unless otherwise specified, an aliphatic group contains 1 to 12 aliphatic carbon atoms. As used herein, it is understood that aliphatic groups can also be divalent (e.g., encompassing divalent hydrocarbon chains that are saturated or contain one or more unsaturated units, e.g., -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, etc.). In some embodiments, an aliphatic group contains 1 to 6 aliphatic carbon atoms (e.g., C 1-6 In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms (e.g., C 1-5 In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C 1-4 In still other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., C 1-3 ), and in still other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C 1-2 In some embodiments, "cycloaliphatic" refers to a monocyclic C group that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has single or multiple points of attachment to the rest of the molecule. 3-8 Hydrocarbon or bicyclic C 7-10 "Aliphatic groups" refers to hydrocarbons. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, and hybrids thereof. Preferred aliphatic groups include C 1-6 It is alkyl.

[0019] Alkyl: The term “alkyl” used alone or as part of a larger moiety (unless otherwise specified) refers to an alkyl group having 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms (e.g., C 1-12 , C1-10 , C 1-8 , C 1-6 , C 1-4 , C 1-3 , or C 1-2 ) refers to a saturated, optionally substituted, straight or branched chain hydrocarbon group having a carbon atom. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0020] Alkenyl: The term “alkenyl,” used alone or as part of a larger moiety, refers to an alkenyl group having at least one double bond and (unless otherwise specified) 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C, C, C). 2-8 , C 2-6 , C 2-4 , or C 2-3 "cycloalkenyl" refers to an optionally substituted straight-chain, branched-chain, or cyclic hydrocarbon group having at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0021] Alkynyl: The term “alkynyl,” used alone or as part of a larger moiety, refers to an alkynyl group having at least one triple bond and (unless otherwise specified) 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched chain hydrocarbon group having an alkynyl group. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0022] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity to the reference substance, e.g., sharing a core structure or consensus structure, but differs in certain individual ways. In some embodiments, an analog is a substance that can be generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process that is substantially similar (e.g., sharing multiple steps) to that which generates the reference substance. In some embodiments, an analog is produced, or can be produced, by performing a synthetic process that is different from the synthetic process used to generate the reference substance.

[0023] Aryl: The term "aryl" refers to an alkyl group having a total of 5 to 14 ring members (e.g., C5-C 14 ) refers to monocyclic and bicyclic ring systems in which at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. In some embodiments, an "aryl" group refers to a ring system containing 6 to 12 total ring members (e.g., C6-C 12 ). The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to an aromatic ring system, including phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Unless otherwise specified, an "aryl" group is a hydrocarbon. In some embodiments, an "aryl" ring system is an aromatic ring (e.g., phenyl) fused to a non-aromatic ring (e.g., cycloalkyl). Examples of fused aryl rings include: [ka] Examples include:

[0024] Associated: As used herein, two events or entities are "associated" with one another when the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its presence, level, and / or state correlates with the occurrence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they interact directly or indirectly to be in physical proximity and / or remain in close proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0025] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture) as described herein. In some embodiments, the source of interest comprises an organism such as an animal or a human. In some embodiments, the biological sample is or comprises a biological tissue or fluid. In some embodiments, the biological sample can be or comprise bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, suspended nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavage fluids such as ductal lavage or bronchoalveolar lavage, aspirates, scrapings, bone marrow specimens, tissue biopsy specimens, surgical specimens, feces, other bodily fluids (e.g., sperm, sweat, tears), secretions, and / or excretions, and / or cells therefrom, etc. In some embodiments, the biological sample is or consists of cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.). In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more agents), e.g., filtration using a semi-permeable membrane. Such a "processed sample" can include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components, etc.

[0026] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in administering a composition. In some exemplary embodiments, a carrier can comprise sterile liquids such as, for example, water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, a carrier is or comprises one or more solid ingredients.

[0027] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents or modalities) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously. In some embodiments, the regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen). In some embodiments, the agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administering one or more agent(s) or modality(s) in combination to a subject receiving the other agent(s) or modality(s). For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combined composition or even a combined compound (e.g., as part of a single chemical complex or covalent conjugate).

[0028] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, etc. that are not necessarily identical to one another, but are sufficiently similar to permit a comparison between them, such that a person of ordinary skill in the art would understand that reasonable conclusions can be drawn based on the perceived differences or similarities. In some embodiments, an equivalent set of conditions, circumstances, individuals, or populations is characterized by multiple substantially identical characteristics and one or a few different characteristics. A person of ordinary skill in the art will understand from the context what level of identity is necessary for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable in any given situation. For example, a person of ordinary skill in the art will understand that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by, or indicate, variations in those characteristics.

[0029] Composition: Those skilled in the art will understand that the term "composition" can be used to refer to a separate physical entity that includes one or more defined components. Generally, unless otherwise specified, a composition can be in any form, e.g., gas, gel, liquid, solid, etc.

[0030] Cycloaliphatic: As used herein, "cycloaliphatic" refers to a monocyclic C ring that is fully saturated or contains one or more units of unsaturation, but is not aromatic, having a single point of attachment or more than one point of attachment to the rest of the molecule. 3-8 Hydrocarbon or bicyclic C 7-10 Refers to hydrocarbons.

[0031] Cycloalkyl: As used herein, the term "cycloalkyl" refers to an optionally substituted saturated monocyclic or multicyclic ring system having about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0032] Deoxyribonucleic Acid (DNA): As used herein, the term "DNA" refers to a polymer of nucleotides that is typically double-stranded and contains adenine, cytosine, guanine, and thymine, as well as the deoxyribose sugar backbone structure defined in the definition of "nucleic acid / polynucleotide." In some embodiments, the DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggiebone DNA, or transposon.

[0033] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to unmodified and modified deoxyribonucleotides. For example, unmodified deoxyribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and thymine (T). Modified deoxyribonucleotides include, but are not limited to, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse conjugation, etc.), 3'-terminal modifications (e.g., conjugation, reverse conjugation, etc.), (b) base modifications (e.g., modified bases, stabilizing bases, destabilizing bases, or substitutions with bases that base-pair with expanded repertoire partners, or substitutions with conjugate bases), (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages.

[0034] Dosage Form or Unit Dosage Form: Those of skill in the art will understand that the term "dosage form" may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is a unit dosage (or a whole fraction thereof) suitable for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) determined to correlate with a desired or beneficial outcome when administered to a relevant population. Those of skill in the art will understand that the overall amount of therapeutic composition or agent to be administered to a particular subject will be determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0035] Dosing regimen or treatment regimen: Those skilled in the art will appreciate that the terms "dosing regimen" and "treatment regimen" can be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can include one or more doses. In some embodiments, the dosing regimen includes multiple administrations, each separated in time from the other administrations. In some embodiments, the individual doses are separated from each other by periods of equal length. In some embodiments, the dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, the dosing regimen includes a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen comprises a first dosing at a first dosage amount, followed by one or more additional dosings at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0036] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0037] Heteroaliphatic: The terms "heteroaliphatic" or "heteroaliphatic group," as used herein, refer to an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, 1 to 5 heteroatoms, which may be linear (i.e., unbranched), branched, or cyclic ("heterocyclic"), and which may be fully saturated or contain one or more units of unsaturation, but is not aromatic. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, including any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1 to 10 carbon atoms, in which 1 to 3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1 to 4 carbon atoms, in which 1 to 2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, in which 1 carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, heteroaliphatic groups of 1-10 atoms include exemplary groups such as -O-CH, -CH-O-CH, and -O-CH-CH-O-CH-CH-O-CH.

[0038] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to a monocyclic or bicyclic ring group having 5 to 12 ring atoms (e.g., a 5- or 6-membered monocyclic heteroaryl or a 9- to 12-membered bicyclic heteroaryl), having 6, 10, or 14 pi electrons shared in a cyclic arrangement, and having 1 to 5 heteroatoms in addition to the carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzisoxazolyl. The terms "heteroaryl" and "heteroara-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms).Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzisoxazolyl. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently are optionally substituted.

[0039] Heteroatom: As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0040] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably one to four, heteroatoms as defined above. The term "nitrogen," when used in reference to a ring atom of a heterocycle, includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR +(as in the case of N-substituted pyrrolidinyl). A heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions are independently optionally substituted. Bicyclic heterocyclic rings also include groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, tetrahydroquinolinyl, [ka] Bicyclic heterocyclic rings can also be spirocyclic ring systems (e.g., 7- to 11-membered spirocyclic fused heterocyclic rings having, in addition to carbon atoms, one or more heteroatoms (e.g., 1, 2, 3, or 4 heteroatoms) as defined above). Bicyclic heterocyclic rings can also be bridged ring systems (e.g., 7- to 11-membered bridged heterocyclic rings having 1, 2, or 3 bridging atoms).

[0041] Nanoparticle: As used herein, the term "nanoparticle" refers to a discrete entity of small size, e.g., typically having a longest dimension less than about 1000 nanometers (nm), often less than 500 nm, or even less than 100 nm. In many embodiments, nanoparticles can be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 μm and about 500 nm, or between about 1 nm and 1000 nm. In many embodiments, a population of microparticles is characterized by an average size (e.g., longest dimension) less than about 1000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm, and often greater than about 1 nm. In many embodiments, the microparticles are substantially spherical (e.g., such that their longest dimension can be their diameter). In some embodiments, nanoparticles have a diameter less than 100 nm, as defined by the National Institutes of Health. In some embodiments, the nanoparticles are micelles in that they contain an encapsulated compartment separated from the bulk solution by a micellar membrane, typically composed of amphiphilic entities that surround and encapsulate a space or compartment (e.g., to define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, e.g., a biocompatible and / or biodegradable polymer.

[0042] Nucleic Acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid can comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof).In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template), replication in a recombinant cell or system in vivo or in vitro, or chemical synthesis. In some embodiments, the nucleic acid comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, and 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0043] Nucleic acid particles: "Nucleic acid particles" can be used to deliver nucleic acids to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid particles can be formed from at least one cationic lipid or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0044] Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When a number of nucleotides is used, for example, as an indicator of the size of a polynucleotide, a specific number of nucleotides refers to the number of nucleotides on a single strand, for example, a polynucleotide.

[0045] Oral: As used herein, the phrases "oral administration" and "orally administered" have their art-understood meanings, meaning administration of a compound or composition by mouth.

[0046] Parenteral: As used herein, the terms "parenteral administration" and "parenterally administered" have their art-understood meanings and refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0047] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties as defined herein.

[0048] Patient or Subject: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition is or can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient or subject suffers from or is prone to one or more disorders or conditions. In some embodiments, the patient or subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, the patient or subject is undergoing or has undergone a particular therapy to diagnose and / or treat a disease, disorder, or condition.

[0049] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment or dosing regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual; ophthalmic; transdermal; or compatible with the nose, lungs, and other mucosal surfaces.

[0050] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0051] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or organ filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0052] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" means a salt of a compound that is suitable for use in a pharmaceutical context, i.e., a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxybenzoate, benzoic acid, benzoates ... Examples of suitable salts include dimethylsulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.

[0053] Physiological conditions: As used herein, this term has its technically understood meaning and refers to the conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to the external or internal environmental conditions that may occur in nature for an organism or cellular system. In some embodiments, physiological conditions are conditions present inside the body of a human or non-human animal, particularly at and / or within a surgical site. Physiological conditions typically include, for example, a temperature range of 20-40°C, 1 atmosphere of pressure, a pH of 6-8, a glucose concentration of 1-20 mM, atmospheric levels of oxygen, and gravity as encountered on Earth. In some embodiments, conditions in a laboratory are manipulated and / or maintained at physiological conditions. In some embodiments, physiological conditions are encountered in an organism.

[0054] Polycyclic: As used herein, the term "polycyclic" refers to a saturated or unsaturated ring system having two or more rings (e.g., heterocyclyl, heteroaryl, cycloalkyl, or aryl rings) with 7 to 20 atoms, in which one or more carbon atoms are common to two adjacent rings. For example, in some embodiments, a polycyclic ring system refers to a saturated or unsaturated ring system having three or more rings (e.g., heterocyclyl, heteroaryl, cycloalkyl, or aryl rings) with 14 to 20 atoms, in which one or more carbon atoms are common to two adjacent rings. The rings in a polycyclic ring system may be fused (i.e., bicyclic or tricyclic), spirocyclic, or a combination thereof.

[0055] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. Those of skill in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both, and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, polypeptides contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof (e.g., peptidomimetics).

[0056] Polymer: As used herein, the term "polymer" refers to a composition comprising one or more molecules containing repeating units of one or more monomers. As used herein, "polymer" and "polymer composition" are used interchangeably and, unless otherwise specified, refer to a composition of polymer molecules. As will be understood by one of ordinary skill in the art, a polymer composition includes polymer molecules having different lengths (e.g., containing varying amounts of monomers). The polymer compositions described herein are characterized by one or more of the number average molecular weight (Mn), weight average molecular weight (Mw), and / or polydispersity index (PDI). The polymers described herein can also be characterized by the degree of polymerization (DP), which refers to the number of monomer units in the polymer. The polymers described herein can be homopolymers, heteropolymers, or block copolymers. As used herein, a "homopolymer" refers to a polymer having a single type of monomer repeating throughout the polymer chain, e.g., -AAAA-. As used herein, a "heteropolymer" refers to a polymer having more than one (e.g., two or more) type of monomer present throughout the polymer chain, e.g., -ABABA-. As used herein, "block copolymer" refers to a polymer having a block arrangement of polymerized monomers, e.g., -AAAABBBB- (diblock polymer) or -AAAABBBBAAA- (triblock polymer). The polymers described herein can be linear or branched. As used herein, "linear polymer" refers to a polymer whose molecules form long chains without branching or crosslinking. As used herein, "branched polymer" refers to a polymer that includes one or more additional monomers, or chains of monomers, extending from the polymer backbone, which can, for example, form branched or crosslinked structures.

[0057] Prevent or Prevention: As used herein, the terms "prevent" or "prevention," when used in connection with the occurrence of a disease, disorder, and / or condition, refer to a reduction in the risk of the disease, disorder, and / or condition occurring and / or a delay in the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition has been delayed for a predefined period of time.

[0058] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by one of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. It will be apparent to one of skill in the art when sufficient similarity exists to justify reliance on and / or comparison to a particular possible reference or control.

[0059] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides include, but are not limited to, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse conjugation, etc.), 3'-terminal modifications (e.g., conjugation, reverse conjugation, etc.), (b) base modifications (e.g., modified bases, stabilizing bases, destabilizing bases, or substitutions with bases that base-pair with expanded repertoire partners, or substitutions with conjugate bases), (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0060] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA includes both single-stranded and double-stranded portions. In some embodiments, the RNA may include a backbone structure as described in the definition of "nucleic acid / polynucleotide" above. The RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, the RNA is an mRNA. In some embodiments, the RNA is an mRNA, the RNA typically includes a polyA region at its 3' end. In some embodiments, the RNA is an mRNA, the RNA typically includes a cap structure at its 5' end, e.g., known in the art for recognition and binding of mRNA to ribosomes to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis and / or chemical synthesis). As used herein, "monomeric RNA" refers to an individual RNA molecule that is not an RNA aggregate, dimer, trimer, or oligomer.

[0061] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest can be or include a cell, tissue, or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the source of interest can be or include a preparation generated in a production process. In some embodiments, a sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or adding one or more agents).

[0062] Substituted or Optionally Substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens either explicitly or implicitly from the structure (e.g., [ka] At least [ka] means, [ka] At least [ka] (meaning "substituted" or "substituted"). Unless otherwise specified, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when multiple positions in a given structure can be substituted with multiple substituents selected from a specified group, the substituents can be the same or different at all positions. Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that remains substantially unchanged when subjected to conditions that permit the compound's production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes provided herein. Groups described as "substituted" preferably have 1 to 4 substituents, more preferably 1 or 2 substituents. Groups described as "optionally substituted" can be unsubstituted or "substituted" as described above.

[0063] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0-4 R°;-(CH2) 0-4 OR°;-O(CH2) 0-4 R o ,-O-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 CH(OR°)2;-(CH2) 0-4 SR°; optionally substituted with R° -(CH2) 0-4 Ph; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 Ph; optionally substituted with R° -CH=CHPh; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;-(CH2) 0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0-4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0-4N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0-4 C(O)R°;C(S)R°;-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 C(O)SR°;-(CH2) 0-4 C(O)OSiR°3;-(CH2) 0-4 OC(O)R°;-OC(O)(CH2) 0-4 SR°;-(CH2) 0-4 SC(O)R°;-(CH2) 0-4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-SC(S)SR°,-(CH2) 0-4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0-4 SSR°;-(CH2) 0-4 S(O)2R°;-(CH2) 0-4 S(O)2OR°;-(CH2) 0-4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0-4 S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O)(OR°)2;SiR°3;-(C 1-4 Linear or branched alkylene)ON(R°)2; or -(C 1-4 linear or branched alkylene)C(O)ON(R°)2, where each R° may be optionally substituted as defined below and independently represents hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1Ph, -CH2- (a 5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, notwithstanding the above definitions, two independent occurrences of R° together with the intervening atom(s) therebetween can form a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0064] Suitable monovalent substituents on R° (or the ring formed by combining two independent occurrences of R° and the intervening atom(s) therebetween) are independently halogen, —(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR · , -(CH2) 0-2 CH(OR · )2, -O(HaloR · ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● 、 -(C 1-4 Linear or branched alkylene)C(O)OR ● , or -SSR ● where each R ●is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and independently: C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 or a 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include ═O and ═S.

[0065] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: ═O ("oxo"), ═S, ═NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S-, and each R occurring independently in the formula * is hydrogen, optionally substituted as defined below 1-6 A 5- to 6-membered, saturated, partially unsaturated, or aryl ring is an aliphatic or unsubstituted group having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Preferred divalent substituents attached to vicinal substitutable carbon atoms of an "optionally substituted" group include -O(CR * 2) 2-3 O-, and each R occurring independently in the formula * is hydrogen, optionally substituted as defined below 1-6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0066] R * Suitable substituents on the aliphatic group include halogen, -R ● , (Halo R ● ), OH, -OR● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, and each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0067] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † wherein R † are independently hydrogen, optionally substituted as defined below, C 1-6 an aliphatic, unsubstituted -OPh, or an unsubstituted 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, two independent occurrences of R † together with the intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0068] R †Suitable substituents on the aliphatic groups are independently halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● ,-NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and independently: C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0069] Small molecule: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer.

[0070] Those of skill in the art will understand, upon reading this disclosure, that, for example, certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms, such as, for example, crystalline forms (e.g., polymorphs, solvates, etc.), salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0071] Those skilled in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers; in some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of a racemic mixture.

[0072] Those skilled in the art will recognize that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual tautomers or in forms that interconvert between tautomers.

[0073] Those skilled in the art will appreciate that a particular small molecule compound may have a structure that allows for isotopic substitution (e.g., 2 H or 3 H, 12 In the case of C 11 C. 13 C, or 14 C. 14 In the case of N 13 N or 15 N, 16 In the case of O 17 O or 18 O. 35 Cl or 37 In the case of Cl 36 Cl, 19 In the case of F 18 F, 127 In the case of I 131 It will be understood that such small molecules have one or more isotopically modified forms, or mixtures thereof, in accordance with the present disclosure.

[0074] In some embodiments, a reference to a particular small molecule compound may relate to a particular form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in the form of a salt (e.g., an acid addition or base addition salt, depending on the compound), and in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0075] In some embodiments, if a small molecule compound is a naturally occurring or naturally found compound, the compound may be provided and / or utilized in accordance with the present disclosure in a form that is different from that in which it naturally occurs or is found in nature. One of skill in the art will understand that in some embodiments, the absolute or relative amount of a compound, or a preparation of a particular small molecule compound containing that particular form, will differ from the compound present in the reference preparation or source (e.g., with respect to other components of the preparation that contain other forms of the compound), which differs from the absolute or relative amount of the compound or form present in a reference preparation (e.g., a primary sample from a source of interest, such as a biological or environmental source). Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a distinct form from a racemic mixture of the compound, a particular salt of a small molecule compound may be considered to be a distinct form from another salt form of the compound, a preparation that includes only forms of the compound that contain one stereoisomer of the double bond ((Z) or (E)) may be considered to be a distinct form from forms of the compound that contain the other stereoisomer of the double bond (E) or (Z), and a preparation in which one or more atoms are isotopes different from those present in a reference preparation may be considered to be a distinct form.

[0076] Those skilled in the art will recognize that in small molecule structures, the symbols [ka] It will further be understood to refer to the point of attachment between two atoms.

[0077] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect in an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria, such as a particular age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is a drug that has been, or needs to be, approved by a government agency before it can be commercially available for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

[0078] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of the disease, disorder, and / or illness. In some embodiments, treatment may be administered to subjects who exhibit only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing conditions associated with the disease, disorder, and / or condition.

[0079] Complex The present disclosure provides, inter alia, complexes for delivery of nucleic acids, such as RNA. In some embodiments, the present disclosure provides complexes comprising a cationic polymer, an anionic polymer, and RNA. In some embodiments, the present disclosure provides complexes comprising a cationic polymer, an anionic polymer, and RNA, wherein the cationic polymer and the RNA form a core complex encapsulated by the anionic polymer. In some embodiments, the complexes described herein comprise a monomeric RNA molecule.

[0080] As described herein, reference to a "binary formulation" or "binary particle" is also intended to refer to a "core complex" as described throughout this application. That is, a binary formulation comprises a cationic polymer and a nucleic acid. A "ternary formulation" or "ternary particle" is also intended to refer to a complex comprising a cationic polymer, a nucleic acid, and an anionic polymer, where the cationic polymer and nucleic acid form a core complex encapsulated by the anionic polymer.

[0081] The complexes described herein exhibit numerous advantages over prior formulations due, at least in part, to the presence of an anionic polymer that acts as a shell to encapsulate the cationic polymer-RNA core complex. Without being bound by theory, it is understood that the anionic polymer induces release of the RNA from the core complex by displacement of negatively charged phosphates.

[0082] In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and RNA. In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule. In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and RNA, where the linear cationic polymer and the RNA form a core complex encapsulated by the anionic polymer. In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule, where the linear cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer. In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and RNA, where the linear cationic polymer and the RNA form a core complex encapsulated by the anionic polymer, where the cationic polymer is linear poly(ethyleneimine). In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the linear cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer, and the ionic polymer is linear poly(ethyleneimine). In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and RNA, wherein the linear cationic polymer and the RNA form a core complex encapsulated by the anionic polymer, and the cationic polymer is linear poly(ethyleneimine), and the anionic polymer is a block copolymer comprising blocks of polymers selected from poly-L-glutamic acid, poly-L-aspartic acid, and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid.In some embodiments, the complex comprises a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the linear cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer, wherein the cationic polymer is linear poly(ethyleneimine), and the anionic polymer is a block copolymer comprising blocks of polymers selected from poly-L-glutamic acid, poly-L-aspartic acid, and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0083] Core complex As generally described herein, in some embodiments, the complex comprises a core complex (also referred to as a binary particle) comprising a cationic polymer and RNA (e.g., monomeric RNA as described herein). In some embodiments, the complex comprises a core complex comprising a cationic polymer, RNA, and additional nucleic acid (e.g., DNA). Examples of cationic polymers and conditions suitable for forming core complexes are described in PCT Application Publication No. WO 2021 / 001417, which is incorporated herein by reference in its entirety. Such complexes are characterized by the "N / P ratio," which is the molar ratio of cationic (nitrogen) groups in the cationic polymer (the "N" in N / P) to anionic (phosphate) groups in the RNA (the "P" in N / P). The cationic groups are in cationic form (e.g., N + ) or that can be ionized to become cationic. The use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number relative to 1, e.g., an N / P ratio of about 5 will be washed at about 5:1. The use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) means that the number is relative to 1, e.g., an N / P ratio of about 5 means a ratio of about 5:1.

[0084] In some embodiments, the N / P ratio for a core complex described herein is about 5 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 10 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 12 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 20 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 40 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 70 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 100 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 200 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 300 or greater. In some embodiments, the N / P ratio for a core complex described herein is about 5 to about 20 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20). In some embodiments, the N / P ratio for a complex described herein is about 10 to about 50. In some embodiments, the N / P ratio for a complex described herein is about 10 to about 70. In some embodiments, the N / P ratio for a complex described herein is about 10 to about 120. In some embodiments, the N / P ratio for a core complex described herein is about 40 to about 70. In some embodiments, the N / P ratio for a core complex described herein is about 40 to about 120 (e.g., about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, or about 120). In some embodiments, the N / P ratio of the complexes described herein is about 70 to about 120. In some embodiments, the N / P ratio is 48 or greater. In some embodiments, the N / P ratio is about 48 to about 300. In some embodiments, the N / P ratio is about 60 to about 200. In some embodiments, the N / P ratio is about 80 to about 150.

[0085] In some embodiments, the core complexes described herein are characterized by RNA concentration. In some embodiments, the RNA concentration in the core complex is about 0.05 mg / mL or greater. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL or greater. In some embodiments, the RNA concentration in the core complex is about 0.25 or greater. In some embodiments, the RNA concentration in the core complex is about 0.5 or greater. In some embodiments, the RNA concentration in the core complex is about 0.01 mg / mL to about 0.5 mg / mL. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL to about 0.25 mg / mL. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.35 mg / mL, about 0.4 mg / mL, about 0.45 mg / mL, or about 0.5 mg / mL. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL.

[0086] In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL, and the N / P ratio is about 45 or greater. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL, and the N / P ratio is about 48 to about 300. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL, and the N / P ratio is about 60 to about 200. In some embodiments, the RNA concentration in the core complex is about 0.1 mg / mL, and the N / P ratio is about 80 to about 150.

[0087] In some embodiments, the RNA concentration in the core complex is about 0.05 mg / mL and the N / P ratio is about 5 or greater. In some embodiments, the RNA concentration in the core complex is about 0.05 mg / mL and the N / P ratio is about 12 or greater. In some embodiments, the RNA concentration in the core complex is about 0.05 mg / mL and the N / P ratio is about 45 or greater. In some embodiments, the RNA concentration in the core complex is about 0.05 mg / mL and the N / P ratio is about 60 or greater.

[0088] In some embodiments, the core complexes described herein are prepared according to a process comprising contacting a solution comprising RNA with a solution comprising a cationic polymer at a ratio of greater than about 3:1 (v:v). In some embodiments, the core complexes described herein are prepared according to a process comprising contacting a solution comprising RNA with a solution comprising a cationic polymer at a ratio of greater than about 20:1 (v:v). In some embodiments, the core complexes described herein are prepared according to a process comprising contacting a solution comprising RNA with a solution comprising a cationic polymer at a ratio of greater than about 50:1 (v:v). In some embodiments, the core complexes described herein are prepared according to a process comprising contacting a solution comprising RNA with a solution comprising a cationic polymer at a ratio of greater than about 99:1 (v:v).

[0089] In some embodiments, the complexes described herein comprise monomeric RNA. As used herein, "monomeric RNA" refers to RNA aggregates, or individual RNA molecules that are not dimers, trimers, or oligomers. Thus, reference to a complex that comprises monomeric RNA is understood to refer to a complex that comprises a single RNA molecule that is not an aggregate, dimer, trimer, or oligomer.

[0090] In some embodiments, the complex further comprises DNA.

[0091] It is understood that the complexes described herein are included as part of a solution or formulation.As such, many complexes constitute a solution or formulation.The characterization of any complex is intended to represent the average of all complexes in a given solution or formulation, or to represent an individual complex characterized according to methods known in the art.

[0092] cationic polymers As generally described above, the conjugates described herein comprise a cationic polymer. In some embodiments, the cationic polymer is a polycationic polymer, e.g., a polymer having one or more cationic or ionizable groups. As described herein, a "cationic" group is a group that has a net positive charge. As used herein, an "ionizable" group is a group that may have a neutral charge at a particular pH, but may become charged (e.g., cationic) at a different pH. For example, in some embodiments, the ionizable group becomes cationic (i.e., positively charged) at physiological pH (e.g., a pH of about 7.4). Thus, reference to a cationic polymer is understood to refer to both the neutral and charged (i.e., ionic) forms. Those of skill in the art will also understand that the cationic groups described herein can also exist as salts (e.g., pharmaceutically acceptable salts) comprising the cationic group and one or more suitable counterions. For example, in some embodiments, the cationic groups described herein comprise ammonium chloride. Suitable counterions include halogens (e.g., Br - , Cl - , I - , F - ), acetate (e.g., C(O)O - For additional examples, see the definition of pharmaceutically acceptable salts herein.

[0093] In some embodiments, one or more of the cationic or ionizable groups comprises a nitrogen atom. Cationic polymers useful for preparing the conjugates described herein can be homopolymers, heteropolymers, or block copolymers.

[0094] In some embodiments, the cationic polymer is a homopolymer selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0095] As will be apparent, the cationic polymers described herein can be linear or branched. In some embodiments, the cationic polymer is linear. In some embodiments, the cationic polymer is a linear polymer selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0096] In some embodiments, the cationic polymer is a branched polymer selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic polymer is linear poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0097] In some embodiments, the cationic polymer is poly(ethyleneimine). In some embodiments, the cationic polymer is poly(propyleneimine). In some embodiments, the cationic polymer is polybrene. In some embodiments, the cationic polymer is polyallylamine. In some embodiments, the cationic polymer is polyvinylamine. In some embodiments, the cationic polymer is polyamidoamine. In some embodiments, the cationic polymer is poly-L-lysine. In some embodiments, the cationic polymer is poly-L-arginine. In some embodiments, the cationic polymer is poly-L-histidine. In some embodiments, the cationic polymer is poly(2-aminoethyl methacrylate).

[0098] In some embodiments, the cationic polymer is linear poly(ethyleneimine). In some embodiments, the cationic polymer is linear poly(propyleneimine). In some embodiments, the cationic polymer is linear polybrene. In some embodiments, the cationic polymer is linear polyallylamine. In some embodiments, the cationic polymer is linear polyvinylamine. In some embodiments, the cationic polymer is linear polyamidoamine. In some embodiments, the cationic polymer is linear poly-L-lysine. In some embodiments, the cationic polymer is linear poly-L-arginine. In some embodiments, the cationic polymer is linear poly-L-histidine. In some embodiments, the cationic polymer is linear poly(2-aminoethyl methacrylate).

[0099] In some embodiments, the cationic polymer is a heteropolymer comprising a copolymer of one or more of poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic polymer is a heteropolymer comprising poly(ethyleneimine) and poly(propyleneimine). In some embodiments, the cationic polymer is a linear heteropolymer comprising a copolymer of one or more of linear poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic polymer is a heteropolymer comprising poly(ethyleneimine) and poly(propyleneimine).

[0100] In some embodiments, the cationic polymer is a block copolymer comprising blocks of polymers selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic polymer is a block copolymer of poly(ethyleneimine) and poly(propyleneimine) (i.e., poly(ethyleneimine)-block-poly(propyleneimine)). In some embodiments, the cationic polymer is a linear block copolymer comprising blocks of polymers selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate). In some embodiments, the cationic polymer is a linear block copolymer of poly(ethyleneimine) and poly(propyleneimine) (ie, poly(ethyleneimine)-block-poly(propyleneimine)).

[0101] In some embodiments, the cationic polymer has 2 to 2000 repeating monomer units. In some embodiments, the cationic polymer has 100 to 2000 repeating monomer units. In some embodiments, the cationic polymer has 500 to 2000 repeating monomer units. In some embodiments, the cationic polymer has 1000 to 2000 repeating monomer units. In some embodiments, the cationic polymer has 1500 to 2000 repeating monomer units. In some embodiments, the cationic polymer comprises about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 repeating monomer units.

[0102] In some embodiments, the cationic polymer is a polymer or copolymer comprising Formula I and / or II: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic, or G; Each R 2 is independently H, an optionally substituted C1-C6 aliphatic, or G; X 1 and X 3 are each independently C1-C6 aliphatic; X 2 is a bond or an optionally substituted C1-C6 aliphatic; m is an integer from 2 to 2000, n is an integer from 2 to 2000, Each G is independent, [ka] and R 1’ is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic, or G′; Each R 2’ is independently H, an optionally substituted C1-C6 aliphatic, or G'; X 1’ and X 3’ are each independently an optionally substituted C1-C6 aliphatic; X 2’ is a bond or an optionally substituted C1-C6 aliphatic; n' is an integer from 2 to 2000, m' is an integer from 2 to 2000; Each G' is independently [ka] and R1’’ is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic; Each R 2’’ are independently H or C1-C6 aliphatic; X 1’’ and X 3’’ are each independently an optionally substituted C1-C6 aliphatic; X 2’’ is a bond or an optionally substituted C1-C6 aliphatic; n'' is an integer from 2 to 2000, m'' is an integer of 2 to 2000.

[0103] In some embodiments, the cationic polymer has a number average molecular weight (M) of about 600 Daltons (Da) to about 400,000 Da. n In some embodiments, the cationic polymer is a polymer described herein having an M of about 1,000 Da to about 300,000 Da. n In some embodiments, the cationic polymer has an M of about 10,000 Da to about 250,000 Da. n In some embodiments, the cationic polymer has an M of about 10,000 Da to about 120,000 Da. n In some embodiments, the cationic polymer has an M of about 20,000 Da to about 120,000 Da. n It has.

[0104] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to a salt thereof, unless otherwise specified.

[0105] Polyamine derivatives (Viromer) In some embodiments, a complex comprises a cationic polymer, an anionic polymer, and RNA, wherein the cationic polymer is or comprises a polyamine derivative. As described herein, polyamine derivatives are also referred to as "viromers." In some embodiments, a complex comprises a cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the cationic polymer is or comprises a polyamine derivative. In some embodiments, a complex comprises a cationic polymer, an anionic polymer, RNA, and DNA, wherein the cationic polymer is or comprises a polyamine derivative.

[0106] In some embodiments, the complex comprises a cationic polymer, an anionic polymer, and RNA, wherein the cationic polymer is or comprises a polyamine derivative, and the polyamine derivative and RNA form a core complex encapsulated by the anionic polymer. In some embodiments, the complex comprises a cationic polymer, an anionic polymer, and monomeric RNA, wherein the cationic polymer is a polyamine derivative, and the polyamine derivative and monomeric RNA form a core complex encapsulated by the anionic polymer.

[0107] In some embodiments, the complexes described herein include polyamine derivatives, e.g., carboxylated polyamine derivatives. Without being bound by theory, it is understood that polyamines form polycations in solution, which facilitate complexation with polyanions, such as nucleic acids.

[0108] In some embodiments, the polyamine derivative is a polyamine moiety containing multiple amino groups; a plurality of carboxylated substituents comprising carboxyl groups attached to the amino groups of the polyamine moiety via hydrophobic linkers; and and a plurality of hydrophobic substituents attached to the amino groups of the polyamine moiety.

[0109] In some embodiments, the polyamine derivative is a polyamine moiety containing multiple amino groups; a plurality of carboxylated substituents comprising carboxyl groups attached to amino groups of the polyamine moiety via hydrophobic linkers, each of the carboxylated substituents containing 6 to 40 carbon atoms, preferably 6 to 20 carbon atoms, more preferably 8 to 16 carbon atoms, and each of the hydrophobic linkers may contain 1 to 3 heteroatoms selected from O, N, and S; The polyamine moiety includes a plurality of hydrophobic substituents attached to the amino groups, each of which contains at least 2 carbon atoms, preferably 6 to 40 carbon atoms, and may contain 1 to 3 heteroatoms selected from O, N, and S if the hydrophobic substituent has at least 6 carbon atoms.

[0110] In some embodiments, each of the carboxylated substituents of the polyamine derivative comprises any one or more of the following moieties as the hydrophobic linker: alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and combinations thereof; and / or each of the hydrophobic substituents of the polyamine derivative comprises any one or more of the following moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, and combinations thereof.

[0111] In some embodiments, polyamine derivatives useful as polyanion delivery vehicles in the present disclosure are polyalkyleneimine derivatives having one or more carboxyalkyl substituents containing 6 to 40 carbon atoms and one or more hydrophobic substituents selected from hydrocarbon substituents having at least 2 carbon atoms, preferably 6 to 40 carbon atoms, each of said hydrophobic substituents may be or may include an alkyl group and / or each of said hydrophobic substituents may be or may include an aryl group.

[0112] In some embodiments, the polyalkyleneimine is selected from the group consisting of polyethyleneimine, polypropyleneimine, and polybutyleneimine.

[0113] In some embodiments, the polyamine portion of the polyamine derivative may contain 4 to 20,000 nitrogen atoms, more preferably 6 to 10,000 nitrogen atoms, such as 6 to 1,000 nitrogen atoms or 6 to 100 nitrogen atoms per polyamine molecule.

[0114] In some embodiments, the polyamine portion of the polyamine derivative may be a branched polyamine, preferably a branched polyalkyleneimine.

[0115] In some embodiments, the carboxylated substituents contain one or two carboxyl groups, preferably one carboxyl group. In some embodiments, each carboxylated substituent contains 6 to 40 carbon atoms, preferably 6 to 20 carbon atoms, and more preferably 8 to 16 carbon atoms. The hydrophobic linker of the carboxylated substituents can contain one to three, preferably one or two, heteroatoms selected from O, N, and S. Preferably, the heteroatoms are selected from O and S. In some embodiments, one or two heteroatoms selected from O, N, and S, preferably O and S, are contained in the hydrophobic linker. In some embodiments, the carboxylated substituents can be carboxyhydrocarbyl groups. In some embodiments, the carboxylated substituents are carboxyheterohydrocarbyl groups containing one to three heteroatoms selected from O, N, and S, preferably O and S.

[0116] In some embodiments, among the multiple carboxylated substituents of the polyamine derivative molecule, only carboxyhydrocarbyl groups, only carboxyheterohydrocarbyl groups, or carboxyhydrocarbyl groups and carboxyheterohydrocarbyl groups may be present. In some embodiments, all of the multiple carboxylated substituents are carboxyhydrocarbyl groups. In some embodiments, all of the multiple carboxylated substituents are carboxyheterohydrocarbyl groups.

[0117] In some embodiments, the carboxylated substituent is a carboxyhydrocarbyl group, the hydrocarbyl portion of which can be a saturated aliphatic moiety, an alicyclic moiety, an aromatic moiety, or a moiety comprising two or more from the foregoing list.

[0118] Examples of carboxyhydrocarbyl groups are carboxyalkyl groups, carboxyalkenyl groups, carboxyalkynyl groups, carboxycycloalkyl groups, carboxycycloalkenyl groups, carboxyalkylcycloalkyl groups, carboxycycloalkylalkyl groups, carboxyalkylcycloalkylalkyl groups, carboxyaryl groups, carboxyalkylaryl groups, carboxyarylalkyl groups, and carboxyalkylarylalkyl groups. One, two, or three, preferably one or two, carbon atoms in the hydrocarbyl portion of the carboxylated substituent can be replaced with oxygen, nitrogen, or sulfur, thereby forming a carboxyheterohydrocarbyl moiety. It should be understood that such formal substitution with a heteroatom includes adjustment of the hydrogen atoms bonded to adjust to the valence of the replaced heteroatom. In preferred embodiments, such carboxyheterohydrocarbyl moieties comprise one or more functional groups in the hydrophobic linker selected from -O-, -S-, -N(H)C(O)-, -C(O)O-, -OC(O)N(H)-, -C(O)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-, or -SS-.

[0119] In some embodiments, the hydrophobic linker is or includes an alkylene group, such as a linear or branched alkylene group, or the linker is or includes a cycloalkylene group. The alkylene group can be an n-alkylene group or an isoalkylene group. Examples of alkylene groups include propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tetradecylene, or hexadecylene. Examples of cycloalkylene groups include cyclopentylene, cyclohexylene, and cycloheptylene. Examples of alkylcycloalkyl groups are methylcyclopentylene, ethylcyclopentylene, propylcyclopentylene, butylcyclopentylene, pentylcyclopentylene, hexylcyclopentylene, methylcyclohexylene, ethylcyclohexylene, propylcyclohexylene, butylcyclohexylene, pentylcyclohexylene, and hexylcyclohexylene, one or more of which may be combined in the hydrophobic linker.

[0120] In some embodiments, the carboxylated substituent is or includes a carboxyalkyl or carboxycycloalkyl group containing 6 to 20 carbon atoms. Such carboxylated substituents can be selected from the group consisting of carboxy-n-alkyl, branched, or cyclic carboxyalkyl groups and structural or conformational isomers thereof. In preferred embodiments, the carboxyalkyl group is the radical of an acid selected from hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 2-cyclohexylacetic acid, 4-cyclohexylbutyric acid, 6-cyclohexylhexanoic acid, 2-(2',3', or 4' ethylcyclohexyl)-acetic acid, 4-(2',3', or 4' ethylcyclohexyl)-butyric acid, or 6-(2',3', or 4' ethylcyclohexyl)-hexanoic acid.

[0121] In some embodiments, the hydrophobic linker is or contains an arylene group and has 6 to 20 carbon atoms. The aryl groups forming the arylene group include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, anthracenyl, and phenanthryl. In some embodiments, the nitrogen-containing heteroaryl group has a pK value of <5 to avoid additional cationic charge at neutral pH. Examples of such nitrogen-containing heteroaryl groups include indolyl, pyrazinyl, pyridazinyl, pyrimidinyl, cinnolinyl, phthalazinyl, and purinyl groups. In some embodiments, the oxygen-containing heterohydrocarbyl group forming the hydroxy group has a pK value >12 to avoid negative charge at neutral pH.

[0122] Examples of alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, and xylyl groups. Examples of arylalkyl (aralkyl) groups are benzyl, phenylethyl, and trityl groups. Examples of alkylarylalkyl groups are methylbenzyl and 4-isopropylbenzyl groups. The carboxyarylalkyl moiety can be, for example, a radical derived from o-, m-, or p-methylbenzoate, or o-, m-, or p-ethylbenzoic acid. The carboxyalkylarylalkyl moiety can be, for example, o-, m-, or p-methylphenylacetic acid. The carboxyalkenylarylalkyl moiety can be, for example, o-, m-, or p-methylcinnamic acid, or o-, m-, or p-methylcinnamic acid.

[0123] Multiple carboxylated substituents, such as those that are or include carboxyalkyl groups, present on a polyamine derivative may be the same or different. For simplicity, they may be the same. The carboxyl group of the carboxylated substituent may be attached to any carbon atom of the hydrophobic linker. Preferably, the carboxyl group is attached to a carbon atom as follows: if z is the number of carbon atoms in the longest carbon chain in the carboxylated substituent (e.g., carboxyalkyl group) relative to the carbon atom attached to the polyamine nitrogen atom, and the carbon atom attached to the polyamine nitrogen atom is counted as position 1, then the carboxyl group is attached to a carbon atom that is more than z / 2 atom positions away from the polyamine nitrogen. If the value of z / 2 is not an integer, the above definition translates to the position defined by the next integer > z / 2. In one embodiment, the carboxyl group is attached to the carbon atom of the hydrophobic linker that is furthest (in terms of the number of carbon atoms) from the polyamine nitrogen atom to which the hydrophobic linker (the alkylene chain in the case of a carboxyalkyl group) is attached. The carboxyl group may be attached to the carbon atom in the carboxylated substituent (or carboxyl alkyl group) that is farthest from the polyamine nitrogen, for example, in the structure of a linear carboxylated substituent. The carboxyl group may be attached to the carbon atom in the carboxylated substituent (or carboxyl alkyl group) that is farthest from the polyamine nitrogen, for example, to the terminal (omega) carbon atom of the linear carboxylated substituent (or carboxyl alkyl group).

[0124] In some embodiments, the hydrophobic substituent contains 2 to 40 carbon atoms, in some embodiments, 3 to 40 carbon atoms, in some embodiments, 6 to 40 carbon atoms, and in some embodiments, 6 to 20 carbon atoms. The hydrophobic substituent may contain 1 to 3, preferably 1 or 2, heteroatoms selected from O, N, and S, provided that the hydrophobic substituent contains 6 or more carbon atoms. Preferably, the heteroatoms are selected from O and S. Thus, the hydrophobic substituent may be a hydrocarbyl group or a heterohydrocarbyl group, the latter containing 1 to 3 heteroatoms. Among the multiple hydrophobic substituents on the molecule of the polyamine derivative, only hydrocarbyl groups, only heterohydrocarbyl groups, or both hydrocarbyl and heterohydrocarbyl groups may be present. In some embodiments, all of the multiple hydrophobic substituents are hydrocarbyl groups. In some embodiments, all of the multiple hydrophobic substituents are heterohydrocarbyl groups.

[0125] When the hydrophobic substituent is a hydrocarbyl group, it can be selected from alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, alkylcycloalkyl groups, alkylcycloalkylalkyl groups, aryl groups, alkylaryl groups, arylalkyl groups, and alkylarylalkyl groups, and groups containing two or more groups from the above list. When the hydrophobic substituent contains six or more carbon atoms, one, two, or three of the carbon atoms of the hydrocarbyl group can be replaced with oxygen, nitrogen, or sulfur, preferably oxygen or sulfur, thereby forming a heterohydrocarbyl substituent. Such heterohydrocarbyl substituents can contain functional groups selected from -O-, -S-, -N(H)C(O)-, -C(O)O-, -OC(O)N(H)-, -C(O)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-, or -SS-.

[0126] In some embodiments, the hydrophobic substituent is or includes an alkyl group, such as a linear or branched alkyl group, or a cycloalkyl group. The alkyl group can be an n-alkyl group or an isoalkyl group. Examples of alkyl groups are propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, or hexadecyl groups. Examples of cycloalkyl groups are cyclopentyl, cyclohexyl, and cycloheptyl groups.

[0127] Examples of alkenyl groups are propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl, and hexadecenyl groups. Examples of alkynyl groups are propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, and hexadecenyl groups.

[0128] Examples of cycloalkenyl groups are cyclopentenyl, cyclohexenyl, and cycloheptenyl. Cycloalkylalkyl groups are groups in which a cycloalkyl group is linked to an alkylene group corresponding to the alkyl group. Examples include cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, etc.

[0129] Alkylcycloalkyl groups are groups in which an alkyl group is linked to a cycloalkylene group corresponding to the cycloalkyl group. Examples of alkylcycloalkyl groups are methylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, butylcyclopentyl, pentylcyclopentyl, hexylcyclopentyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, butylcyclohexyl, pentylcyclohexyl, and hexylcyclohexyl.

[0130] An alkylcycloalkylalkyl group is an alkyl group linked to a cycloalkylalkylene group.

[0131] In some embodiments, the hydrophobic substituent comprises an aryl group having 6 to 20, preferably 7 to 15, carbon atoms. Aryl groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, and phenanthryl. In some embodiments, the nitrogen-containing heteroaryl group has a pK value of <5 to avoid additional cationic charge at neutral pH. Examples of such nitrogen-containing heteroaryl groups include indolyl, pyrazinyl, pyridazinyl, pyrimidinyl, cinnolinyl, phthalazinyl, and purinyl groups. In some embodiments, the oxygen-containing heterohydrocarbyl group forming the hydroxy group has a pK value >12 to avoid negative charge at neutral pH.

[0132] Examples of alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, methylindole, and xylyl groups. Examples of arylalkyl (aralkyl) groups are benzyl, phenylethyl, indolylmethyl, and trityl groups. Examples of alkylarylalkyl groups are methylbenzyl and 4-isopropylbenzyl groups.

[0133] The various hydrophobic substituents on the molecule of the polyamine derivative may be the same or different. For simplicity, they may be the same.

[0134] In some embodiments, the polyamine derivative has a linear polyethyleneimine moiety (as number average molecular weight) of 2 to 500 kDa (as number average molecular weight), the carboxylated substituent has 10 to 16 carbon atoms and is an n-alkyl carboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl, and / or alkylarylalkyl.

[0135] In some embodiments, the polyamine derivative has a branched polyethyleneimine moiety of 0.5 to 200 kDa (number average molecular weight), the carboxylated substituent has 10 to 16 carbon atoms and is an n-alkylcarboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl, and / or alkylarylalkyl.

[0136] In some embodiments, the polyamine derivative is [ka] or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the complex comprises a cationic polymer, an anionic polymer, RNA, and a polyamine, wherein the polyamine is [ka] or a pharmaceutically acceptable salt thereof.

[0138] RNA In some embodiments, the complexes described herein comprise a cationic polymer, an anionic polymer, and a nucleic acid. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA comprises a monomeric RNA molecule (i.e., the complexes described herein comprise a monomeric RNA molecule). As used herein, "monomeric RNA" refers to an RNA aggregate, or an individual RNA molecule, not as a dimer, trimer, or oligomer. Thus, a reference to a complex comprising monomeric RNA (e.g., a monomeric RNA molecule) is understood to refer to a complex comprising a single RNA molecule, not as an aggregate, dimer, trimer, or oligomer. Those skilled in the art will understand how to assess the presence of monomeric RNA in a sample, for example, by centrifugation and quantification methods. That is, in some embodiments, the presence and quantification of monomeric RNA in a sample is determined by centrifuging the sample containing RNA, extracting the supernatant, and then measuring the concentration of RNA in the supernatant relative to the total amount of RNA in the sample. In some embodiments, the presence and assessment of monomeric RNA in a sample is performed by an ultracentrifugation assay, in which the sample is measured in an analytical ultracentrifuge and the absorbance of light is measured at two different wavelengths, 255 nm and 650 nm. In some embodiments, such samples are centrifuged at 80,000 x g at room temperature.

[0139] In some embodiments, the RNA acceptable to the techniques described herein is single-stranded RNA. In some embodiments, the RNA disclosed herein is linear RNA. In some embodiments, the single-stranded RNA is non-coding RNA in that its nucleotide sequence does not contain an open reading frame (or its complementary strand). In some embodiments, the single-stranded RNA has a nucleotide sequence that encodes (or is the complementary strand of) a polypeptide or multiple polypeptides (e.g., epitopes) of the present disclosure.

[0140] In some embodiments, the RNA is or comprises siRNA, miRNA, or other non-coding RNA.

[0141] In many embodiments, the related RNAs comprise at least one open reading frame (ORF) (e.g., are mRNAs), in some embodiments, the related RNAs comprise a single ORF, and in some embodiments, the related RNAs comprise more than one ORF.

[0142] In some embodiments, the RNA comprises, for example, an ORF encoding a polypeptide of interest, or encoding multiple polypeptides of interest. In some embodiments, the RNA produced in accordance with the techniques provided herein comprises multiple ORFs (e.g., encoding multiple polypeptides). In some embodiments, the RNA produced in accordance with the techniques provided herein comprises a single ORF encoding multiple polypeptides. In some such embodiments, the polypeptide is or comprises an antigen or epitope (e.g., an associated antigen) thereof.

[0143] In some embodiments, an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence that is functional in mammalian cells, e.g., an endogenous signal sequence or a heterologous signal sequence. In some embodiments, the signal sequence directs secretion of the encoded polypeptide, and in some embodiments, the signal sequence directs transport of the encoded polypeptide to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment.

[0144] In some embodiments, the ORF encodes a polypeptide that includes a multimerization element (e.g., a native or heterologous multimerization element). In some embodiments, an ORF that encodes a surface polypeptide (e.g., includes a signal sequence that directs surface localization) includes a multimerization element.

[0145] In some embodiments, the ORF encodes a polypeptide that includes a transmembrane element or domain.

[0146] In some embodiments, the ORF is codon-optimized for expression in a particular host, eg, a mammalian host, eg, a human cell.

[0147] In some embodiments, the RNA comprises unmodified uridine residues, and RNA comprising only unmodified uridine residues may be referred to as "uRNA." In some embodiments, the RNA comprises one or more modified uridine residues, and in some embodiments, such RNA (e.g., RNA comprising fully modified uridine residues) is referred to as "modRNA." In some embodiments, the RNA may be self-amplifying RNA (saRNA). In some embodiments, the RNA may be trans-amplifying RNA (see, e.g., WO2017 / 162461).

[0148] In some embodiments, the relevant RNA comprises a portion encoding a polypeptide or a portion encoding multiple polypeptides. In certain embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or includes a biologically active polypeptide or portion thereof (e.g., an enzyme or cytokine, or a therapeutic protein such as a replacement protein or antibody or portion thereof). In certain embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or includes an antigen (or epitope thereof), cytokine, enzyme, etc. In some embodiments, the encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with tumors. In some embodiments, the encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.). In certain embodiments, the encoded polypeptide may be a variant of a wild-type polypeptide.

[0149] In some embodiments, the single-stranded RNA (e.g., mRNA) may include a region encoding a secretion signal (e.g., a region encoding a secretion signal that enables the encoded target entity or entities to be secreted upon translation by the cell). In some embodiments, such a secretion signal coding region may be or include a non-human secretion signal. In some embodiments, such a secretion signal coding region may be or include a human secretion signal.

[0150] In some embodiments, a single-stranded RNA (e.g., an mRNA) can include at least one non-coding element (e.g., to improve RNA stability and / or translation efficiency). Examples of non-coding elements include, but are not limited to, a 3' untranslated region (UTR), a 5' UTR, a cap structure (e.g., in some embodiments, an enzymatically added cap; in some embodiments, a co-transcriptional cap), a polyadenine (polyA) tail (e.g., in some embodiments, can be or include 100 or more A residues, and / or in some embodiments can include one or more "interrupt" [i.e., non-A] sequence elements), and any combination thereof. Exemplary embodiments of such non-coding elements can be found, for example, in WO2011015347, WO2017053297, US10519189, US10494399, WO2007024708, WO2007036366, WO2017060314, WO2016005324, WO2005038030, WO2017036889, WO2017162266, and WO2017162461, each of which is incorporated herein by reference in its entirety.

[0151] format At least four formats useful for RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines) have been developed: unmodified uridine containing mRNA (uRNA), nucleoside-depleted mRNA (modRNA), self-amplifying mRNA (saRNA), and trans-amplifying RNA.

[0152] Characteristics of the unmodified uridine platform may include, for example, one or more of inherent adjuvant effect, good tolerability and safety, and potent antibody and T cell responses.

[0153] The characteristics of the modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune sensor activation capacity, and thus increased antigen expression, good tolerability and safety, and strong antibody and CD4+ T cell responses. As described herein, the present disclosure provides insight that such strong antibody and CD4+ T cell responses may be particularly useful for vaccination.

[0154] Characteristics of a self-amplifying platform may include, for example, long-term polypeptide (eg, protein) expression, good tolerability and safety, and high potential for efficacy at very low vaccine doses.

[0155] In some embodiments, a self-amplifying platform (e.g., RNA) comprises two nucleic acid molecules, one nucleic acid molecule encoding a replicase (e.g., a viral replicase) and another nucleic acid molecule capable of being replicated by the replicase in trans (a trans-replication system) (e.g., a repcon). In some embodiments, a self-amplifying platform (e.g., RNA) comprises multiple nucleic acid molecules, the nucleic acids encoding multiple replicases and / or replicas.

[0156] In some embodiments, the trans-replication system comprises the presence of both nucleic acid molecules within a single host cell.

[0157] In some such embodiments, the nucleic acid encoding the replicase (e.g., a viral replicase) is unable to autonomously replicate in a target cell and / or target organism, hi some such embodiments, the nucleic acid encoding the replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (+)-strand template-based (-)-strand synthesis and / or (+)-strand template-based (-)-strand synthesis.

[0158] In some embodiments, the self-amplifying RNA comprises a 5'-cap, and in some trans-replication systems, at least the RNA encoding the replicase is capped. Without wishing to be bound by any one theory, it has been found that the 5'-cap can be important for high-level expression of the gene of interest in trans.

[0159] In some embodiments, the self-amplifying platform does not require viral particle propagation (e.g., is not associated with undesired viral particle formation). In some embodiments, the self-amplifying platform is incapable of forming viral particles.

[0160] In some embodiments, the RNA may contain an internal ribosome entry site (IRES) element. In some embodiments, the RNA does not contain an IRES site, and in particular, in some embodiments, the saRNA does not contain an IRES site. In some such embodiments, translation of the gene of interest and / or replicase is not driven by an IRES element. In some embodiments, the IRES element is replaced by a 5'-cap. In some such embodiments, the replacement with a 5'-cap does not affect the sequence of the polypeptide encoded by the RNA.

[0161] In some embodiments, the complexes described herein comprise mRNA, modRNA, and saRNA. In some embodiments, the complexes comprise mRNA. In some embodiments, the complexes comprise modRNA. In some embodiments, the complexes comprise saRNA.

[0162] Anionic Polymers As generally described herein, the complexes of the present disclosure also include anionic polymers capable of encapsulating the core complexes described herein. The anionic polymers described herein can be linear or branched and contain one or more anionic moieties or groups. As used herein, an "anionic" moiety or group is a group that is negatively charged at neutral pH (e.g., about pH 7) or that can otherwise become negatively charged upon a change in physiological pH (e.g., pH 7.4). In some embodiments, the anionic polymer is a polyanionic polymer, e.g., a polymer with one or more anionic groups. In some embodiments, the anionic group is -CO2 - , -OSO3 - , or -OPO3 2- It is the base.

[0163] In some embodiments, the anionic polymer is selected from poly-L-glutamic acid, poly-L-aspartic acid, polysaccharides, derivatives of poly(2-oxazoline), derivatives of poly(2-oxazine), poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), and polyphosphoric acid.

[0164] In some embodiments, the anionic polymer is selected from poly-L-glutamic acid, poly-L-aspartic acid, polysaccharides, poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), and polyphosphoric acid.

[0165] In some embodiments, the anionic polymer is polyglutamic acid. In some embodiments, the anionic polymer is poly-L-glutamic acid. In some embodiments, the anionic polymer is fondaparinux, represented by the following structure: [ka] In some embodiments, the anionic polymer is a derivative of poly(2-oxazoline). In some embodiments, the anionic polymer is a derivative of poly(2-oxazine). In some embodiments, the anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) (pC2MestOx). In some embodiments, the anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazoline) (pC3MestOx). In some embodiments, the anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) (pC2MestOz). In some embodiments, the anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazine) (pC3MestOz). In some embodiments, the anionic polymer is polyphosphoric acid (PolyP).

[0166] In some embodiments, the anionic polymer is a homopolymer. In some embodiments, the anionic polymer is a homopolymer comprising about 10 to about 150 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 10 to about 100 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 20 to about 100 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 50 to about 150 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 50 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 100 repeating monomer units.

[0167] In some embodiments, the homopolymeric anionic polymer is poly-L-glutamic acid having about 10 to about 150 glutamic acid repeat units. In some embodiments, the homopolymeric anionic polymer is poly-L-glutamic acid having about 20 to about 100 glutamic acid repeat units. In some embodiments, the homopolymeric anionic polymer is poly-L-glutamic acid having about 50 to about 150 glutamic acid repeat units. In some embodiments, the homopolymeric anionic polymer is poly-L-glutamic acid having about 50 glutamic acid repeat units. In some embodiments, the homopolymeric anionic polymer is poly-L-glutamic acid having about 100 glutamic acid repeat units.

[0168] In some embodiments, the homopolymeric anionic polymer is poly-L-aspartic acid having about 10 to about 150 repeating aspartic acid units. In some embodiments, the homopolymeric anionic polymer is poly-L-aspartic acid having about 20 to about 100 repeating glutamic aspartic acid units. In some embodiments, the homopolymeric anionic polymer is poly-L-aspartic acid having about 50 to about 150 repeating aspartic acid units. In some embodiments, the homopolymeric anionic polymer is poly-L-aspartic acid having about 50 repeating aspartic acid units. In some embodiments, the homopolymeric anionic polymer is poly-L-aspartic acid having about 100 repeating aspartic acid units.

[0169] In some embodiments, the homopolymeric anionic polymer is a polyphosphate having about 10 to about 150 repeating phosphate units. In some embodiments, the homopolymeric anionic polymer is a polyphosphate having about 20 to about 100 repeating phosphate units. In some embodiments, the homopolymeric anionic polymer is a polyphosphate having about 50 to about 150 repeating phosphate units. In some embodiments, the homopolymeric anionic polymer is a polyphosphate having about 50 repeating glutamic acid units.

[0170] In some embodiments, the homopolymeric cationic polymer is a derivative of poly(2-oxazoline) having from about 10 to about 150 repeating units of a derivative of 2-oxazoline. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazoline) having from about 20 to about 100 repeating units of a derivative of 2-oxazoline. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazoline) having from about 50 to about 150 repeating units of a derivative of 2-oxazoline. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazoline) having from about 50 repeating units of a derivative of 2-oxazoline. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazoline) having from about 100 repeating units of a derivative of 2-oxazoline.

[0171] In some embodiments, the homopolymeric cationic polymer is a derivative of poly(2-oxazine) having from about 10 to about 150 repeating units of a derivative of 2-oxazine. In some embodiments, the homopolymeric cationic polymer is a derivative of poly(2-oxazine) having from about 20 to about 100 repeating units of a derivative of 2-oxazine. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazine) having from about 50 to about 150 repeating units of a derivative of 2-oxazine. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazine) having from about 50 repeating units of a derivative of 2-oxazine. In some embodiments, the homopolymeric anionic polymer is a derivative of poly(2-oxazine) having from about 100 repeating units of a derivative of 2-oxazine.

[0172] In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) having from about 10 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) having from about 20 to about 100 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) having from about 50 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazoline) having from about 50 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the anionic polymer that is a homopolymer is poly(2-methoxycarbonylethyl-2-oxazoline) having about 100 repeating units of 2-methoxycarbonylethyl-2-oxazoline.

[0173] In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazoline) having from about 10 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazoline) having from about 20 to about 100 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazoline) having from about 50 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazoline) having from about 50 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the anionic polymer that is a homopolymer is poly(2-methoxycarbonylpropyl-2-oxazoline) having about 100 repeating units of 2-methoxycarbonylethyl-2-oxazoline.

[0174] In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 10 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 20 to about 100 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 50 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazoline. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 50 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the anionic polymer that is a homopolymer is poly(2-methoxycarbonylethyl-2-oxazine) having about 100 repeating units of 2-methoxycarbonylethyl-2-oxazine.

[0175] In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 10 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazine) having from about 20 to about 100 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylethyl-2-oxazine) having from about 50 to about 150 repeating units of 2-methoxycarbonylethyl-2-oxazine. In some embodiments, the homopolymeric anionic polymer is poly(2-methoxycarbonylpropyl-2-oxazine) having from about 50 repeating units of 2-methoxycarbonylpropyl-2-oxazine. In some embodiments, the anionic polymer that is a homopolymer is poly(2-methoxycarbonylpropyl-2-oxazine) having about 100 repeating units of 2-methoxycarbonylpropyl-2-oxazine.

[0176] In some embodiments, the anionic polymer is a heteropolymer. In some embodiments, the anionic polymer is a heteropolymer comprising monomers of glutamic acid, ethylene glycol, propylene glycol, sarcosine, phosphate, 2-methyl-2-oxazoline, 2-methoxycarbonylethyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-methyl-2-oxazine, 2-methoxycarbonylethyl-2-oxazine, 2-methoxycarbonylpropyl-2-oxazoline, 2-methoxycarbonylpropyl-2-oxazine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and / or 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic polymer is a heteropolymer comprising monomers of glutamic acid and ethylene glycol. In some embodiments, the anionic polymer is a heteropolymer comprising monomers of glutamic acid and propylene glycol. In some embodiments, the anionic polymer is a heteropolymer comprising monomers of glutamic acid and sarcosine. In some embodiments, the anionic polymer is a heteropolymer comprising monomers of glutamic acid and 2-methoxycarbonylethyl-2-oxazoline.

[0177] In some embodiments, the anionic polymer is a block copolymer, ie, a block copolymer comprising blocks of polymers selected from the group consisting of poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, polyphosphate, poly(N-methyl-sarcosine), poly(ethylene glycol), poly(propylene glycol), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0178] In some embodiments, the block copolymer comprises about 10 to about 100 monomer repeat units in each block. In some embodiments, the block copolymer comprises about 20 to about 100 monomer repeat units in each block. In some embodiments, the block copolymer comprises about 50 monomer repeat units in each block.

[0179] In some embodiments, the block copolymer comprises an anionic block and a stealth block. In some embodiments, the block copolymer comprises an anionic block, wherein the anionic block is selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine), and the stealth block is selected from poly(ethylene glycol), poly(propylene glycol), poly(2-methyl-2-oxazoline), poly(N-methylsarcosine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA), and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid. In some embodiments, the block copolymer comprises about 10 to about 100 repeating monomer units of the anionic block and about 10 to about 100 repeating monomer units of the stealth block.

[0180] In some embodiments, the block copolymer comprises a first anionic block and a second anionic block, hi some embodiments, the first anionic block and the second anionic block are not the same. In some embodiments, the block copolymer comprises a first anionic block selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, (2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine), and a second anionic block selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine). In some embodiments, the block copolymer comprises about 10 to about 100 repeating monomer units of a first anionic block and about 10 to about 100 repeating monomer units of a second anionic block.

[0181] In some embodiments, the block copolymer comprises three or more anionic blocks, each block comprising an anionic polymer independently selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine). In some embodiments, each anionic block of the block copolymer comprises from about 10 to about 100 monomer units of the anionic polymer.

[0182] In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly(2-methoxy-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic block copolymer polymer is poly-L-glutamic acid-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0183] In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly(2-methoxy-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic block copolymer polymer is poly-L-aspartic acid-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0184] In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly(2-methyl-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic polymer that is a block copolymer is poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0185] In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly(2-methyl-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic polymer that is a block copolymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0186] In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly(2-methyl-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic polymer that is a block copolymer is poly(2-methoxycarbonylethyl-2-oxazine)-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0187] In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazine)-block-poly(2-methyl-2-oxazoline). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazine)-block-poly(N-methyl-sarcosine). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazine)-block-poly(ethylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazine)-block-poly(propylene glycol). In some embodiments, the anionic block copolymer polymer is poly(2-methoxycarbonylpropyl-2-oxazoline)-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the anionic polymer that is a block copolymer is poly(2-methoxycarbonylpropyl-2-oxazine)-block-poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0188] In some embodiments, the anionic polymer is a homopolymer poly-α-L-glutamic acid having 10, 20, 50, or 100 repeating units (PGA(10), (20), (50), and (100), respectively). In some embodiments, the anionic polymer is a homopolymer poly-Lα-L-glutamic acid having 10, 20, 50, or 100 repeating units (PGA(10), (20), (50), and (100), respectively). In some embodiments, the anionic polymer is a homopolymer of n-butyl-poly-L-aspartic acid having 10, 20, 50, or 100 repeating units (PGA(10), (20), (50), and (100), respectively).

[0189] In some embodiments, the anionic polymer is a block copolymer of poly-α-L-glutamic acid (PGA)-block-poly-N-methylsarcosine (pSar) having 10, 20, 50, or 100 PGA units and about 10, 25, or 50 pSar units (PGA(10, 20, 50, or 100)-b-pSar(10, 25, or 50)). In some embodiments, the anionic polymer is a block copolymer of poly-α-L-glutamic acid (PGA)-block-2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA). In some embodiments, the PGA-b-AEEA block copolymer has 10, 20, 50, or 100 PGA units and 4, 8, or 14 AEEA units. In some embodiments, the PGA-b-AEEA block copolymer is PGA(50)-Ac-AEEA(14), where "Ac" refers to an acetate group.

[0190] In some embodiments, the anionic polymers described herein comprise a targeting moiety or an imaging moiety. For example, the anionic polymer may comprise a radionuclide (e.g., 18 F, 131 The antibody may comprise an imaging moiety such as 1H, 1H, 1H-I, 1H-II ...

[0191] In some embodiments, the targeting moiety is a derivative of an agent that has strong binding affinity for a specific cell marker on target cells, thereby enhancing targeting and internalization of the complex and / or cargo into the target cells. In some embodiments, the targeting moiety is or includes one or more of a small molecule, peptide, aptamer, or other nucleic acid sequence, or nanobody. In some embodiments, the small molecule targeting moiety is folic acid, phenylboronic acid, alendronate, telmisartan, glycyrrhetinic acid, adenosine, a vitamin, or a carbohydrate. In some embodiments, the vitamin is vitamin H (biotin) or vitamin B (flavin) mononucleotide. In some embodiments, the carbohydrate is a monosaccharide selected from glucose, mannose, galactose, fucose, sialic acid, mannose-6-phosphate, and lactobionic acid, or an oligosaccharide comprising one or more monosaccharides described herein. In some embodiments, the peptide is a cysteine-arginine-glutamic acid-lysine-alanine (CREKA) peptide, a K237 peptide, an F3 peptide, an A54 peptide, apamin, a hexapeptide ligand AE, an epidermal growth factor, an octreotide, an RGD (Arg-Gly-Asp) motif, an iRGD, an RGDF peptide, a cRGD, a U11 peptide, an HIV transactivating transcription factor (TAT) peptide, a Lyp-1 peptide, a rabies virus glycoprotein (RVG), a chlorotoxin (ClTx), an Aβ binding peptide (KLVFF), a TGNYKALHPHNG (TGN), a QSHYRHISPAQV (QSH), a Tet-1 peptide, a T7 peptide, an apolipoprotein, an E peptide (ApoE), a peptide motif B6, angiopep-2, a cell penetrating peptide (CPP), or a ferritin. In some embodiments, the peptide is SRLEEELRRRLTE and is referred to as an "ALfa" peptide. In some embodiments, compositions comprising an Alfa peptide are referred to as "Alfa-tagged."In some embodiments, the aptamer is the nucleotide-targeting ADN aptamer AS1411, an anti-epidermal growth factor receptor aptamer, a mucin-1 aptamer, a prostate-specific membrane antigen (PSMA) aptamer A9g, or PSMA A10. In some embodiments, the nanobody is an anti-HER2 nanobody 2Rb17c or an EGa1 nanobody. In some embodiments, the nanobody targets Alfa peptide (NbALFA). In some embodiments, the nanobody is NbALFA. In some embodiments, the targeting molecule is a multivalent dendron moiety comprising several identical or different targeting motifs.

[0192] In some embodiments, the anionic polymer described herein further comprises a stealth linker between the anionic polymer and the targeting moiety. Stealth moieties used herein are any suitable polymers, for example, comprising repeating units of ethylene glycol, sarcosine, or AEEA. For example, in some embodiments, the anionic polymer comprising the targeting moiety is a heteroblock copolymer of poly(glutamic acid)-block-poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid-Alfa (referred to as PGA-b-Ac-AEEA-Alfa). In some embodiments, the anionic polymer comprising the targeting moiety is PGA(50)-b-Ac-AEEA(14)-Alfa. In some embodiments, the anionic polymer comprising the targeting moiety is a block copolymer of poly-α-L-glutamic acid and polysarcosine, and the targeting moiety is an Alfa peptide. In some embodiments, the anionic polymer comprising the targeting moiety is PGA(50)-b-pSar(50)-Alfa.

[0193] In some embodiments, the anionic polymer comprising a targeting moiety is a homopolymer of poly-α-L-glutamic acid and the targeting moiety is an Alfa peptide.

[0194] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to a salt thereof, unless otherwise specified.

[0195] Characteristics of the complex As described herein, the complex comprises a core complex encapsulated in an anionic polymer. In some embodiments, the encapsulated complex is characterized by the charge ratio of the anionic groups in the anionic polymer to the cationic groups in the cationic polymer of the core complex. In some embodiments, the charge ratio of the anionic polymer to the cationic polymer is about 0.5:1 to about 3:1. In some embodiments, the charge ratio of the anionic polymer to the cationic polymer is about 0.5:1 to about 2:1. In some embodiments, the charge ratio of the anionic polymer to the cationic polymer is about 1:1 to about 2:1. In some embodiments, the charge ratio of the anionic polymer to the cationic polymer is about 1.25:1 to about 2:1. In some embodiments, the charge ratio of the anionic polymer to the cationic polymer is about 1.5:1.

[0196] In some embodiments, a complex described herein (e.g., an encapsulated complex) is characterized by a diameter of about 10 nm to about 200 nm. In some embodiments, a complex described herein (e.g., an encapsulated complex) is characterized by a diameter of about 10 nm to about 150 nm. In some embodiments, a complex described herein (i.e., an encapsulated complex) is characterized by a diameter of about 10 nm to about 100 nm. In some embodiments, a complex described herein (e.g., an encapsulated complex) is characterized by a diameter of about 10 nm to about 70 nm. In some embodiments, a complex described herein is characterized by a diameter of about 20 nm to about 50 nm. In some embodiments, a complex described herein is characterized by a diameter of about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. In some embodiments, a complex described herein is characterized by a diameter of less than 50 nm. Methods for characterizing the diameter of complexes are described in PCT Application Publication No. WO2021 / 001417.

[0197] In some embodiments, compositions comprising the conjugates described herein exhibit a uniform particle distribution (i.e., have a polydispersity index (PDI) indicative of a population of particles that are similarly sized). For example, in some embodiments, compositions of the conjugates described herein have a PDI of less than 0.5 (e.g., less than 0.5, less than 0.4, less than 0.3).

[0198] One advantage of the provided complexes is, among other things, that the surface charge can be adjusted to a desired value depending on the selection of the anionic polymer. For example, in some embodiments, the surface charge of the provided complexes can be from about +25 mV to about -25 mV. Without wishing to be bound by theory, it is hypothesized that increasing the amount of anionic polymer in the complex allows for a surface charge of up to about -25 mV.

[0199] Furthermore, the complexes described herein exhibit improved colloidal stability over multiple freeze-thaw cycles (e.g., over one, two, three or more freeze-thaw cycles). Such complexes exhibit the above stability over freeze-thaw cycles at both -20°C and -80°C. In some embodiments, the stability of certain complexes is improved using anionic polymers having fewer than 100 repeating units. In some embodiments, the anionic polymers described herein have a total of 50 or fewer monomer units. In some embodiments, the anionic polymers described herein have a total of 25 or fewer monomer units. In some embodiments, the anionic polymers described herein have a total of 10 or fewer monomer units.

[0200] In some embodiments, the complexes described herein comprise a core complex encapsulated by an anionic polymer shell. Determining that a complex comprises a core / shell structure can be performed by any method known to those skilled in the art. For example, in some embodiments, determining the core / shell structure can be performed by microscopy, such as transmission electron microscopy (TEM). In TEM, a sample is scanned with an electron beam; the electrons pass through the sample, are collected, and the signal is converted into an image. Identifying the core and shell can be achieved by applying a stain to alter the contrast between the components.

[0201] In some embodiments, the determination of the core / shell structure can be by small-angle scattering techniques, such as small-angle X-ray scattering (SAXS). In SAXS, a sample is treated with monochromatic X-rays, which are then scattered by the electrons of the atoms present in the formulation. SAXS measures the excess electron density, i.e., the density difference between the particle and its surrounding medium. However, sufficient differentiation between the core and shell can be achieved if the scattering intensities of the electrons from the core and shell are different. SAXS can be useful for providing quantitative information about the size and shape of the particle (which may change upon coating) as well as specific surface modifications (e.g., coating layer thickness). SAXS can be useful for providing quantitative information about the size and shape of the particle (which may change upon coating) as well as specific surface modifications (e.g., coating layer thickness).

[0202] In some embodiments, determining the core / shell structure can be by differential scanning calorimetry (DSC). DSC uses changes in temperature to measure the amount of heat emitted or absorbed by a sample. The change in heat capacity of each segment (core and shell) can be used to confirm the presence of the two components (if the components are not completely miscible, there will be two distinct signals) and to quantitatively determine the weight fraction.

[0203] In some embodiments, determining the core / shell structure can be performed by infrared spectroscopy (IR), including, for example, Fourier transform infrared (FTIR). FTIR is based on the principle that infrared radiation is passed through a sample and some of the radiation is absorbed (and recorded). This radiation is converted into energy based on specific interactions of the radiation with functional groups, producing a spectrum that is a molecular fingerprint of the sample. If new functional groups are introduced to the sample (as a result of a coating material), the FTIR spectrum will change as a result.

[0204] Delivery method The present disclosure provides, inter alia, a conjugate (e.g., a pharmaceutical composition or pharmaceutical formulation referred to herein) administered to a subject. For example, in some embodiments, the conjugate is administered as monotherapy. In some embodiments, the conjugate is administered as part of a combination therapy. In some embodiments, the concentration of RNA in the pharmaceutical compositions described herein is from about 0.01 mg / mL to about 0.5 mg / mL, or from about 0.05 mg / mL to about 0.1 mg / mL.

[0205] Pharmaceutical compositions, as used herein, may further comprise pharmaceutically acceptable excipients, including any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., suitable for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional excipient vehicle is incompatible with the substance or its derivatives, such as by producing some undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present disclosure.

[0206] In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeias.

[0207] Pharmaceutically acceptable additives used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may be optionally included in the pharmaceutical formulation. At the discretion of the formulator, excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents, and / or aromatic agents may be present in the composition.

[0208] In some embodiments, the pharmaceutical formulations described herein may further comprise a buffering agent, hi some embodiments, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), a Bis-tris buffer system, a carboxylic acid buffer system, a phosphate buffer system, or a citrate buffer system.

[0209] General considerations regarding the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated by reference in its entirety).

[0210] In some embodiments, the pharmaceutical compositions provided herein can be formulated using one or more pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and additives, according to conventional methods, e.g., as disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated by reference in its entirety).

[0211] The pharmaceutical conjugates and compositions described herein can be administered by any suitable method known in the art. As will be apparent to those skilled in the art, the route and / or mode of administration may depend on several factors, including, but not limited to, the stability and / or pharmacokinetics and / or pharmacodynamics of the pharmaceutical compositions described herein.

[0212] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, which typically includes modes of administration other than enteral and topical administration by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0213] In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions.

[0214] In some specific embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous (sc) administration. In some specific embodiments, the pharmaceutical compositions described herein are formulated for intramuscular (im) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intranasal administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous (iv) administration.

[0215] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, dispersions, powders (e.g., lyophilized powders), microemulsions, lipid nanoparticles, or other ordered structures suitable to high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0216] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by microfiltration sterilization.

[0217] In some embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0218] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0219] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the pharmaceutical compositions described herein. Furthermore, prolonged absorption of injectable pharmaceutical forms can be brought about by the addition of agents that delay absorption, such as aluminum monostearate and gelatin.

[0220] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with a diluent or other excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose unit.

[0221] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and / or methods described herein.

[0222] In some embodiments, the active agent that can be included in the pharmaceutical compositions described herein is or includes a therapeutic agent administered in a combination therapy described herein. The pharmaceutical compositions described herein can be administered in combination therapy, i.e., in conjunction with other agents. In some embodiments, such a therapeutic agent can include an agent that depletes or functionally inactivates regulatory T cells. For example, in some embodiments, the combination therapy can include a provided pharmaceutical composition and at least one immune checkpoint inhibitor.

[0223] In some embodiments, the pharmaceutical compositions described herein may be administered in conjunction with radiation therapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0224] In some embodiments, the pharmaceutical compositions described herein can be frozen to allow for long-term storage.

[0225] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any type of animal. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with no more than routine experimentation, if any.

[0226] It should also be understood that a variety of administration methods can be used to treat the diseases, disorders, or conditions described herein. For example, in some embodiments, a composition comprising a conjugate described herein is administered using the methods described herein in one or more doses (e.g., one or more doses by parenteral means, intranasal means, oral means, etc.). In some embodiments, a first dose is administered by a first means, and a second dose is administered by the same or different means. For example, in some embodiments, a first dose of a composition comprising a conjugate described herein is administered by intramuscular, subcutaneous, or intravenous means, and a second dose of a composition comprising a conjugate described herein is administered by intranasal means. In some embodiments, doses may be administered in the usual manner by any means described herein after the initial dose. For example, in some embodiments, a first dose is administered by intramuscular, subcutaneous, or intravenous means, and subsequent doses are administered by intranasal means.

[0227] In some embodiments, a first dose is administered and a second dose is administered at a period following the first dose. In some embodiments, the period between the first and second doses is 1 day to 1 year. In some embodiments, the period between the first and second doses is 1 week to 6 months. In some embodiments, the period between the first and second doses is 1 month to 3 months.

[0228] In some embodiments, subsequent doses after the first dose are administered at regular intervals, for example, in some embodiments, subsequent doses are administered annually after the administration of the first dose.

[0229] How to use The conjugates described herein are useful for the treatment and prevention in subjects of the diseases, disorders, and conditions described herein, hi some embodiments, the disease, disorder, or condition is an infectious disease, cancer, autoimmune disease, or rare disease.

[0230] In some embodiments, the infectious disease is caused by or associated with a viral pathogen. In some embodiments, the viral pathogen is from a family selected from the families Poxviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Hepadnaviridae, Coronaviridae, Caliciviridae, Picornaviridae, Reoviridae, Retroviridae, and Orthomyxoviridae. In some embodiments, the infectious disease is a virus selected from SARS-CoV-2, influenza, Crimean-Congo hemorrhagic fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

[0231] In some embodiments, the infectious disease is caused by or associated with a bacterial pathogen. In some embodiments, the bacterial pathogen is Actinomyces israelii, bacillus antracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campolobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium idphteriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia ricektssii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigelladysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0232] In some embodiments, the infectious disease is caused by or associated with a parasite, hi some embodiments, the parasite is from a family selected from Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomes, Ascaris, Echinococcus, and Taeniidae.

[0233] In some embodiments, the disease, disorder, or condition is cancer, hi some embodiments, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, renal cancer, lung cancer, lymphoma, melanoma, oral / oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0234] In some embodiments, the disease, disorder, or condition is a genetic disorder. In some embodiments, the genetic disorder is associated with a gain-of-function or loss-of-function mutation.

[0235] In some embodiments, the disease, disorder, or condition is an autoimmune disease, hi some embodiments, the autoimmune disease is selected from Addison's disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, pernicious anemia, Graves' disease, Hashimoto's disease, myasthenia gravis, and Sjogren's syndrome vasculitis.

[0236] In some embodiments, the disease, disorder, or condition is a rare disease. As described herein, a rare disease refers to a life-threatening or chronically debilitating disease that has a very low incidence rate (e.g., less than 1 / 2000 people) and requires a special combination of efforts to address it.

[0237] In some embodiments, the present disclosure provides conjugates that can selectively target specific systems in the body. As used herein, reference to "targeting" a specific system refers to causing increased expression of RNA derived from the cargo in the conjugate in the desired system. For example, in some embodiments, the conjugates described herein can selectively target the lung, liver, spleen, heart, brain, lymph nodes, bladder, kidney, and pancreas. As described herein, a conjugate "selectively targets" an organ if a single target expresses mRNA at 65% or more of the amount expressed in other organs after administration (e.g., 65% or more of the mRNA in the entire body is expressed from a single organ, with the remaining 35% distributed among one or more different organs). In some embodiments, the conjugates described herein selectively target the lung. In some embodiments, the conjugates described herein selectively target the liver. In some embodiments, the conjugates described herein selectively target the spleen. In some embodiments, the conjugates described herein selectively target the heart.

[0238] Illustrative Embodiments Embodiment 1. A complex comprising a cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer.

[0239] Embodiment 2. The conjugate of embodiment 1, wherein the cationic polymer is or comprises a copolymer of one or more of poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0240] Embodiment 3. The conjugate of embodiment 1 or 2, wherein the cationic polymer is a homopolymer selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0241] Embodiment 4. The conjugate of embodiment 2 or 3, wherein the cationic polymer is linear poly(ethyleneimine).

[0242] Embodiment 5. The linear poly(ethyleneimine) has an M of about 600 Da to about 400,000 Da. n 5. The conjugate of embodiment 4, having

[0243] Embodiment 6. The linear poly(ethyleneimine) has an M of about 10,000 Da to about 120,000 Da. n 6. The conjugate of embodiment 5, having

[0244] Embodiment 7. The conjugate of any one of embodiments 1 or 2, wherein the cationic polymer is a linear block copolymer comprising poly(ethyleneimine) and poly(propyleneimine).

[0245] Embodiment 8. The conjugate of embodiment 1, wherein the cationic polymer is a polymer or copolymer comprising blocks of Formula I and / or II: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic, or G; Each R 2 is independently H, an optionally substituted C1-C6 aliphatic, or G; X 1 and X 3 are each independently C1-C6 aliphatic; X 2 is a bond or an optionally substituted C1-C6 aliphatic; m is an integer from 2 to 2000; n is an integer from 2 to 2000, Each G is independent, [ka] R 1’ is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic, or G′; Each R 2’ is independently H, an optionally substituted C1-C6 aliphatic, or G'; X 1’ and X 3’ are each independently an optionally substituted C1-C6 aliphatic; X 2’ is a bond or an optionally substituted C1-C6 aliphatic; n' is an integer from 2 to 2000, m' is an integer from 2 to 2000, Each G' is independently [ka] and R 1’’ is H, —C(O)-optionally substituted C1-C6 aliphatic, optionally substituted C1-C6 aliphatic; Each R 2’’ are independently H or C1-C6 aliphatic; X 1’’ and X 3’’ are each independently an optionally substituted C1-C6 aliphatic; X 2’’ is a bond or an optionally substituted C1-C6 aliphatic; n'' is an integer from 2 to 2000, m'' is an integer of 2 to 2000.

[0246] Embodiment 9. The conjugate of any one of embodiments 1 to 8, wherein the cationic polymer is a homopolymer.

[0247] Embodiment 10. The conjugate of any one of embodiments 1 to 9, wherein the anionic polymer is a homopolymer selected from poly-L-glutamic acid, poly-L-aspartic acid, polysaccharides, poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), and polyphosphoric acid.

[0248] Embodiment 11. The conjugate of any one of embodiments 9 or 10, wherein the anionic polymer comprises from about 10 to about 100 repeating monomer units.

[0249] Embodiment 12. The conjugate of any one of embodiments 1 to 8, wherein the anionic polymer is a heteropolymer.

[0250] Embodiment 13. The conjugate of embodiment 12, wherein the heteropolymer comprises monomers of glutamic acid, aspartic acid, ethylene glycol, propylene glycol, sarcosine, phosphate, 2-methyl-2-oxazoline, 2-methoxycarbonylethyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-methyl-2-oxazine, 2-methoxycarbonylethyl-2-oxazine, 2-methoxycarbonylpropyl-2-oxazoline, 2-methoxycarbonylpropyl-2-oxazine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and / or 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0251] Embodiment 14. The conjugate of embodiment 12 or 13, wherein the anionic polymer comprises from about 10 to about 100 monomer units.

[0252] Embodiment 15. The conjugate of any one of embodiments 1 to 8, wherein the anionic polymer is a block copolymer.

[0253] Embodiment 16. The conjugate of embodiment 15, wherein the block copolymer comprises blocks of polymers selected from the group consisting of poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, polyphosphate, poly(N-methyl-sarcosine), poly(ethylene glycol), poly(propylene glycol), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0254] Embodiment 17. The conjugate of embodiment 16, wherein the block copolymer comprises an anionic block and a stealth block.

[0255] Embodiment 18. The conjugate of embodiment 17, wherein the anionic block is selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, polyphosphate, poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine).

[0256] Embodiment 19. The conjugate of embodiment 17 or 18, wherein the stealth block is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazine), poly(N-methyl-sarcosine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

[0257] Embodiment 20. The conjugate of any one of embodiments 15 to 19, wherein each block comprises from about 10 to about 100 repeating monomer units.

[0258] Embodiment 21. The conjugate of embodiment 1, wherein the cationic polymer is linear poly(ethyleneimine) and the anionic polymer is a block copolymer comprising blocks selected from poly-L-glutamic acid, poly-L-aspartic acid, and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0259] Embodiment 22. The conjugate of any one of embodiments 1 to 21, wherein the anionic polymer comprises a targeting moiety.

[0260] Embodiment 23. The conjugate of embodiment 22, wherein the targeting moiety is an Alfa peptide.

[0261] Embodiment 24. The conjugate of any one of embodiments 1-23, wherein the charge ratio of the anionic groups in the anionic polymer to the cationic groups in the cationic polymer is from about 0.25:1 to about 3:1.

[0262] Embodiment 25. The conjugate of embodiment 24, wherein the charge ratio of the anionic groups in the anionic polymer to the cationic groups in the cationic polymer is about 1.5:1.

[0263] Embodiment 26. The complex of any one of embodiments 1 to 25, further comprising a buffer.

[0264] Embodiment 27. The conjugate of embodiment 26, wherein the buffering agent is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), 2-amino-2(hydroxymethyl)propane-1,3-diol (TRIS), a Bis-tris buffer system, a carboxylic acid buffer system, a phosphate buffer system, and a citrate buffer system.

[0265] Embodiment 28. The conjugate of any one of embodiments 1, 9-20, or 22-27, wherein the cationic polymer is or comprises a polyamine derivative.

[0266] Embodiment 29. The conjugate of embodiment 28, wherein the polyamine derivative is: [ka]

[0267] Embodiment 30. The complex of any one of embodiments 1-29, wherein the RNA is mRNA, modRNA, saRNA, or taRNA.

[0268] Embodiment 31. The complex of any one of embodiments 1 to 30, wherein the complex has a diameter of about 10 nm to about 150 nm.

[0269] Embodiment 32. The complex of any one of embodiments 1 to 31, wherein the complex has a diameter of about 20 nm to about 50 nm.

[0270] Embodiment 33. A method of increasing or causing an increase in expression of RNA in a target in a subject, the method comprising administering to the subject a complex of any one of embodiments 1 to 32.

[0271] Embodiment 34. The method of embodiment 33, wherein the target is selected from lung, liver, spleen, heart, brain, lymph node, bladder, kidney, and pancreas.

[0272] Embodiment 35. A method of treating a disease, disorder, or condition in a subject, comprising administering to the subject a conjugate of any one of embodiments 1 to 32.

[0273] Embodiment 36. The method of embodiment 35, wherein the disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

[0274] Embodiment 37. The method of any one of embodiments 33-36, wherein the conjugate is administered parenterally.

[0275] Embodiment 38. The method of embodiment 37, wherein the conjugate is administered intramuscularly, subcutaneously, or intravenously.

[0276] Embodiment 39. The method of any one of embodiments 33-36, wherein the complex is administered intranasally.

[0277] Embodiment 40. The method of any one of embodiments 33-36, wherein a first dose of the conjugate is administered by a first route and a second dose of the conjugate is administered by a second route.

[0278] Embodiment 41. The method of embodiment 40, wherein the first route is parenteral.

[0279] Embodiment 42. The method of embodiment 40 or 41, wherein the second route is intranasal.

[0280] Embodiment 43. A conjugate according to any one of embodiments 1 to 32 for use as a medicament.

[0281] Embodiment 44. The conjugate of any one of embodiments 1 to 32 for use in the treatment and / or prevention of a disease, disorder, or condition, wherein said disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease. [Example]

[0282] As described in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures: While the general methods refer to the synthesis of specific compounds of the present disclosure, it will be understood that the following general methods, and other methods known to those of skill in the art, can be applied to all compounds and subclasses and species of each of these compounds as described herein. [Table 1]

[0283] Example 1 The saRNA-containing composition was complexed with linear polyethyleneimine (PEI; 500 DP or 22.5 kDa) and N / P12 at a final RNA concentration of 0.1 mg / mL in MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.1). The mixing ratio of the RNA-containing solution to the polymer-containing solution was 99:1 (v:v). Ternary formulations of saRNA were formed after the addition of a homopolymer of poly-α-L-glutamic acid (PGA50) or a block copolymer of poly-α-L-glutamic acid-block-poly-N-methylsarcosine (PGA(50)-b-pSar(50), with 50 repeat units in each block), or a homopolymer of poly(2-methoxycarbonylethyl-2-oxazoline) (pC2MestOx(50), with 50 repeat units), or a block copolymer of poly(2-methoxycarbonylethyl-2-oxazoline)-block-poly(2-methyl-2-oxazoline) (pC2MestOx(50)-b-pMeOx(50), with 50 repeat units in each block), resulting in an anion:PEI charge ratio of 1.5. The anion:PEI charge ratio is based on the ratio of negative charges in the anionic polymer to the positive charges present in PEI.

[0284] After adding the appropriate amount of each anionic polymer, the concentration was adjusted to 0.08 mg / mL RNA in all samples in MBG buffer (final concentration: 5% w / v glucose, 10 mM MES, pH 6.1). Control groups were also included in which samples were further diluted with buffer to match the 0.08 mg / mL RNA concentration found in the other samples. Compositions containing saRNA and buffer or buffer alone were used as controls and are simply referred to as "N / P12s" in Figure 1. The colloidal stability of the samples after serum incubation was investigated by adding human serum to the samples (to a final human serum concentration of 10% v / v) or buffer to a final RNA concentration of 0.05 mg / mL. All samples were diluted with human serum or buffer and incubated at 37°C for 30 minutes. After 30 minutes, the samples were cooled in a 4°C ice bath for 10 minutes, and the size of the complexes was measured using dynamic light scattering. FIG. 1 is a bar graph showing the mean hydrodynamic diameter of formulations containing saRNA after incubation in 10% v / v human serum at 37° C. for 30 minutes.

[0285] Example 2 In MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.1) at a final RNA concentration of 0.1 mg / ml, the saRNA-containing composition was complexed with linear PEI (500 DP or 22.5 kDa) and N / P12 at a mixing ratio of 99:1 (v:v) between the RNA-containing solution and the polymer-containing solution. Ternary formulations of saRNA are generated after the addition of different lengths of poly-α-L-glutamic acid (10, 20, 50, or 100 repeat units, referred to as PGA(10), PGA(20), PGA(50), or PGA(100)) or block copolymers of poly-α-L-glutamic acid (PGA)-poly-N-methylsarcosine (pSar) with varying pSar lengths (10, 25, or 50 repeat units, hereafter referred to as PGA(50)-b-pSar(10), PGA(50)-b-pSar(25), or PGA(50)-b-pSar(50)), within a concentration range that varies depending on the desired final charge ratio of the anion to PEI. The anion:PEI charge ratio is based on the negative charges in the anionic polymer relative to the positive charges present in the PEI.

[0286] After adding the appropriate amount of each anionic polymer, the concentration is adjusted to 0.08 mg / mL RNA in all samples in MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.1). A total of eight different anion:PEI charge ratios are tested for each type of PGA polymer. One control sample containing only RNA and buffer is also tested. The colloidal stability of the samples after serum incubation is investigated by adding human serum to the samples (to a final human serum concentration of 10% v / v) or buffer to a final concentration of 0.05 mg / mL. All samples are diluted with human serum or buffer and incubated at 37°C for 30 minutes. After 30 minutes, the samples are cooled in a 4°C ice bath for 10 minutes, and the size of the complexes is measured using dynamic light scattering to evaluate their biological performance in vitro.

[0287] Example 3 The saRNA-containing composition was complexed with PEI and then with one of five different anions to obtain a ternary formulation for testing in BALB / c mice. Twenty-one BALB / c mice were divided into seven different groups and administered an intramuscular dose of 1 μg of the RNA-containing composition in each leg. One group received only the control example, either Example 1 or 2. The saRNA-containing composition was complexed with PEI at an N / P ratio of 12 in MBG buffer (final concentration of 5% w / v glucose, 10 mM MES, pH 6.1) at a final RNA concentration of 0.125 mg / mL, where the mixing ratio of the RNA-containing solution to the polymer-containing solution was 99:1 (v:v).

[0288] The subsequent five ternary formulations were prepared by selective addition of appropriate amounts of either PGA(10), PGA(20), PGA(50), PGA(100), or PGA(50)-b-pSar(50) at a charge ratio of 1.5 anion:PEI in MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.1) to obtain a final RNA concentration of 0.1 mg / mL. The anion:PEI charge ratio is based on the negative charge in the anionic polymer relative to the positive charge present in PEI. BALB / c mice were administered the following formulations: [Table 2]

[0289] All groups received the same amount of formulated luciferase-encoding saRNA (1 μg) administered im into each leg. In vivo luciferase expression was measured in the treated tissues at six time points (6 h, 24 h, 72 h, and days 6, 9, and 20).

[0290] Example 4 The saRNA-containing composition was complexed with PEI and then with one of four different anionic polymers to obtain a ternary formulation. Thirty BALB / c mice were divided into five groups. The saRNA-containing composition was complexed with PEI and N / P12 in MBG buffer (final concentration of 5% w / v glucose, 10 mM MES, pH 6.1) at a final RNA concentration of 0.125 mg / mL, where the mixing ratio of the RNA-containing solution to the polymer-containing solution was 99:1 (v:v).

[0291] The subsequent four ternary formulations (formulations 2-5) were prepared by selectively adding appropriate amounts of either PGA(50), PGA(50)-b-pSar(10), PGA(50)-b-pSar(25), or PGA(50)-b-pSar(50) at a charge ratio of 1.5 anion:PEI in MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.1) to obtain a final RNA concentration of 0.1 mg / mL. The anion:PEI charge ratio is based on the negative charge in the anionic polymer relative to the positive charge present in PEI.

[0292] Groups of 5 mice receive the following formulations: [Table 3]

[0293] All groups receive the same amount of formulated luciferase-encoding saRNA (1 μg) administered im into the tail vein. 24 hours after injection, luciferase expression is measured in vivo and ex vivo.

[0294] Example 5 Preparation of binary complexes A binary formulation consisting of luciferase-saRNA was complexed with linear PEI500 (H[CHNH.HCl]OH, Mw 54.960 kDa) at N / P12. Fabrication was performed using a syringe pump-based fluidic pathway manufacturing process equipped with a Y-mixing element at a total flow rate of 250 mL / min, using a 3:1 mixing ratio (RNA to polymer phase). The binary formulation, or core polyplex (PLX), was produced at a final RNA concentration of 0.1 mg / mL in 1X MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0). These binary saRNA-PEI PLX are approximately 35 nm in diameter and have a positive zeta potential of 20–25 mV. No free RNA was detected using agarose gel electrophoresis (AGE). PLX is freezable, and no increase in size was observed over at least three freeze-thaw cycles at -80 °C. The binary saRNA complex exhibits a monomeric RNA content of 40% of the total RNA.

[0295] A binary formulation consisting of luciferase modRNA was complexed with linear PEI500 (H[C2H4NH.HCl]500OH, Mw 54.960 kDa) at N / P12. Fabrication was performed using a syringe pump-based fluidic pathway manufacturing process with a T-shaped mixing element. The final buffer composition consisted of MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0) and a final RNA concentration of 0.125 mg / ml. These binary modRNA-PEI PLXs were approximately 30 nm in diameter and had a positive zeta potential of 20-25 mV. No free RNA was detected using agarose gel electrophoresis (AGE). The PLXs were freeze-safe, and no increase in size was observed after thawing at -80°C. The binary modRNA complexes exhibited a high monomeric RNA content, with typically 80% of the total RNA in monomeric form.

[0296] Preparation of ternary complex The formation of an Alfa-tagged ternary formulation of saRNA was generated after the addition of a homopolymer of poly-α-L-glutamic acid with an Alfa-peptide sequence (PGA(X), X = 50 or 100 repeat units). The formation of the ternary complex is within the range of Glu / N charge ratios of 0 to 3. The final ternary formulation has an RNA concentration of 0.08 mg / ml and a storage matrix of 1X MBG buffer (5% w / v glucose, 10 mM MES, pH 6.0).

[0297] Ternary formulations of modRNA are generated after adding block copolymers of Alfa-tagged poly-α-L-glutamic acid block-poly-N-methyl-sarcosine (PGA(50)-b-pSar(50)-Alfa) or Alfa-tagged poly-α-L-glutamic acid block-poly-N-Ac-AEEA14 (PGA(50)-b-Ac-AEEA(14)-Alfa). Ternary complex formation occurs within a Glu / N charge ratio range of 0 to 3. The final ternary formulation has an RNA concentration of 0.1 mg / ml and a storage matrix of 1X MBG buffer (5% w / v glucose, 10 mM MES, pH 6.0).

[0298] Characterization of binary and ternary particles Physicochemical characterization of binary and ternary saRNA-PLX and modRNA-PLX particles included Z-average size and polydispersity index (PDI) using Wyatt dynamic light scattering (DLS). Surface charge zeta potential was measured using a Malvern zetasizer. Variations in zeta potential as a result of changes in the charge exposed by the particle surface further confirm the systematic coating of the core polyplexes with anionic polymers. Free RNA content was measured by agarose gel electrophoresis (AGE). Freezing stability testing was performed by measuring particle size distribution using three consecutive freeze-thaw cycles using Wyatt. During these cycles, particles were frozen overnight at either -20°C or -80°C, thawed at room temperature for 1 hour, measured by the standard Wyatt protocol, and then frozen again at the same temperature. This procedure was repeated three times unless a significant size increase was observed.

[0299] Monomeric RNA was assessed using a centrifugation and RNA quantification method. Briefly, 400 μl of binary PLX was loaded into a 1.5 mL Eppendorf® tube and centrifuged at 20,000 rcf for 1.5 hours at 4°C (acceleration gradient 9, deceleration gradient 5). 250 μl of supernatant was collected from each centrifuged formulation. The RNA concentration of the supernatant (where monomeric RNA is present) was measured and related to the overall RNA content, indicating the monomeric RNA of the formulation as a percentage of total RNA. This method has been proven to be comparable to ultracentrifugation assays, in which samples are measured in an analytical ultracentrifuge and the light absorption is measured at two different wavelengths, 255 nm and 650 nm. MBG sample buffer was used as the blank sample for the measurement. Samples were centrifuged at 80,000 × g at room temperature.

[0300] An example of a typical result for modRNA PEI-PLX is shown in Table 1. In this example, to derive the monomeric RNA content of the formulation, the ratio of RNA concentration from centrifuged PLX to non-centrifuged PLX is considered; this ratio results in a monomeric RNA content of 70%. [Table 4]

[0301] The RNA concentration is quantified using a NanoDrop spectrophotometer. A small volume option is used for RNA quantification, specifically 1.5 μl of formulated RNA. To calculate the RNA concentration, the 260 / 280 and 260 / 230 nm purity ratios are taken into account, and the formulation buffer is used as a blank solution for RNA quantification of each sample.

[0302] Evaluation of Alfa-tagged block copolymers of Poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine, Poly-α-L-glutamic acid, and Poly-α-L-glutamic acid-block-poly-N-Ac-AEEA for forming ternary complexes with modRNA-PEI polyplexes. Ternary formulations of modRNA were generated after the addition of either an Alfa-tagged block copolymer of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine (PGA(50)-b-pSar(50)-Alfa), an Alfa-tagged homopolymer of poly-α-L-glutamic acid (PGA(X)-Alfa, where X = 50 or 100 repeating monomer units), or an Alfa-tagged block copolymer of poly-α-L-glutamic acid-block-poly-N-Ac-AEEA (PGA(50)-b-Ac-AEEA(14)-Alfa).

[0303] Ternary complexes with PGA(50)-b-pSar(50)-Alfa, PGA(50)-b-Ac-AEEA(14)-Alfa, PGA(50)-Alfa, and PGA(100)-Alfa can be successfully formed, with sizes varying depending on the polymer used and the Glu / N ratio tested (Figure 2A). The presence of a stealth moiety as a linker between the PGA and Alfa peptides (pSar(50) and pAEEA(14)) allows for particles with a Z-average size of less than 100 nm.

[0304] Across the majority of Glu / N ratios tested, the ternary complexes exhibited an acceptable distribution with PDI values ​​≤ 0.4, with higher PDIs typically obtained at higher Glu / N ratios (Figure 2B). At the same time, as the amount of anionic polymer increased, the surface charge of the particles could be tuned, resulting in a complete shift in the zeta potential value from approximately +25 mV to -25 mV, as seen in Figure 2C.

[0305] Agarose gel electrophoresis (AGE) analysis shows that Alfa-tagged modRNA ternary complexes can be formulated within specific Glu / N ratios that achieve complete RNA encapsulation (Figure 3A-D).

[0306] Ternary complexes coated with Alfa-tagged polymers could be successfully stabilized at both -80°C and -20°C over up to three freeze-thaw cycles and a wide range of Glu / N ratios, thus ensuring the stability of positively, neutrally, and negatively charged particles (Figure 4A-F). Without wishing to be bound by theory, the frozen stability data suggest that the stealth moiety is useful for ensuring the stability of polymer complexes with a neutral zeta potential, as seen in the case of PGA(50)-b-pSar(50)-Alfa (compare PGA(50)-Alfa in Figure 4A-F).

[0307] Overall, the results show that modRNA-based complexes can be coated with Alfa-tagged polymers to enable active targeting of these delivery systems and to be modified with functionality to achieve organ affinity.

[0308] Evaluation of the length of homopolymeric Alfa-tagged poly-α-L-glutamic acid for forming ternary complexes with saRNA-PEI polyplexes. Physiochemical characterization showed that Alfa-tagged ternary complexes could be formed in the size range of 40–200 nm with both PGA(50)-Alfa and PGA(100)-Alfa, with larger particle sizes obtained at Glu / N ratios of 0.8–1 (Figure 5A–C). The particle size remained relatively small at either very small (0.2) or very large (3) Glu / N ratios. The PDI of the particles shown in Figure 5A–C is ≤0.4. Without wishing to be bound by theory, it is understood that the surface charge of the particles can be tuned by increasing the amount of ternary polymer, thereby shifting it from approximately +25 mV to -30 mV, as seen in Figure 5A–C.

[0309] Agarose gel electrophoresis (AGE) analysis showed that RNA could be successfully encapsulated when saRNA binary polyplexes were coated with both PGA(50)-Alfa and PGA(100)-Alfa (Figure 6).

[0310] Freezing stability studies show that PGA(50)-Alfa colloids are stable for up to three freeze-thaw cycles at low amounts of PGA (e.g., Glu / N ratio of 0.2), where the particles remain highly positively charged. For the same polymer length, stability at -80 °C can also be achieved at higher Glu / N ratios (0.8). Increasing the polymer length allows negatively charged complexes (Glu / N ratios of 1 and 1.5) to achieve colloidal stability at -20 °C and -80 °C (Figure 7A-C).

[0311] Evaluation of Alfa-tagged block copolymers of Poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine and Poly-α-L-glutamic acid-block-poly-N-Ac-AEEA14 for forming ternary complexes with saRNA-PEI polyplexes. Physicochemical characterization indicates that ternary complexes with PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-Ac-AEEA(14)-Alfa can be successfully formed, maintaining a size between 40 and 60 nm (Figure 8A). At most Glu / N ratios, no significant difference in size is observed between the two different stealth moieties used (pSar(50) and pAEEA(14)). PDI values ​​of ≤0.4 are obtained with both anionic polymers (Figure 8B). Without wishing to be bound by theory, we hypothesize that the size of the ternary complex can be maintained below 70 nm when a stealth moiety is present as a linker between the anionic polymer and the Alfa moiety (pSar(50) and pAEEA(14)).

[0312] Without wishing to be bound by theory, it is understood that the surface charge of the particles can be tuned by increasing the amount of anionic polymer, completely shifting the zeta potential value from approximately +25 mV to -25 mV, as seen in Figure 8C.

[0313] The AGE results show that saRNA can be successfully encapsulated when the binary polyplexes are coated with PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-Ac-AEEA(14)-Alfa (Figure 9A-B).

[0314] The ternary complex coated with PGA(50)-b-pSar(50)-Alfa could be successfully stabilized at both -80 °C and -20 °C for up to three freeze-thaw cycles and over a wide range of Glu / N ratios, thus ensuring the stability of positively, neutrally, and negatively charged particles. This result is particularly important as it broadens the application of drug delivery systems depending on specific administration routes. On the other hand, the ternary complex obtained with PGA(50)-b-Ac-AEEA(14)-Alfa exhibits good freeze stability at a low Glu / N ratio (0.2) (Figure 10A-D).

[0315] Furthermore, a stabilizing effect can be confirmed in the case of the PGA(50)-b-pSar(50)-Alfa polymer, where the presence of the alfa-tag on the anionic polymer improves the stability of negatively charged particles (high Glu / N ratio) (Figure 10A and B) compared to the anionic polymer without the alfa-tag.

[0316] Without wishing to be bound by theory, it is hypothesized that the stealth moiety ensures the freeze stability of polymer complexes with a neutral or negative zeta potential, and that pSar (50) is particularly effective in maintaining colloidal properties upon repeated freeze-thaw cycles.

[0317] Example 6 Preparation of mixed RNA- and DNA / Viromer core particles A nucleic acid mixture containing Thy1.1 RNA and nanoplasmid Venus DNA was mixed with an acidified cationic polymer (Viromer VL3 in 0.5% acetic acid) to form core polyplex particles at concentrations greater than 0.1 g / L with an N / P ratio (ratio of cationic or ionizable nitrogen in the polymer / phosphorus backbone to the nucleic acid backbone) of ≥ 7.5. Performing this mixing in a microfluidic device was observed to improve reproducibility and allow for particle size control compared to mixing with a handheld pipette; however, actuated particles were obtained with both methods. Buffer concentrate was then added to reach a final concentration of 10 mM HEPES and 10% trehalose ("1X HBT") at a pH of approximately 7.

[0318] Formation of PGA-AEEA(14)-Alfa-coated polyplex particles The PGA-AEEA(14)-Alfa shell was first added by mixing an appropriate amount of PGA-conjugate solution with 1x HBT in water, followed by the addition of the core-polyplex dispersion. The ratio of the negatively charged glutamic acid residues of the PGA to the positively charged nitrogen of the cationic polymer ("Glu / N ratio") was found to be an appropriate measure of PGA loading. As the Glu / N ratio increased, the zeta potential of the preparation decreased, approaching the neutral region (broadly defined as + / - 15 mV) at Glu / N ≥ 0.16. Concurrently, an increase in the amount of free cargo and free PGA conjugate could be observed. Therefore, PGA-AEEA(14)-Alfa polyplexes with Glu / N ratios of 0.01 to 0.04 were prepared.

[0319] Functionalization and testing of PGA-AEEA(14)-Alfa coated polyplexes. Ligand-functionalized particles were prepared by first mixing the appropriate amount of ligand (aCD3VHH × NbAlfa) with 1x HBT buffer, followed by the addition of the PGA-AEE(14)-Alfa-coated polyplex dispersion. To ensure that no unbound ligand remained in solution, the molar ratio of Alfa tag to ligand ("X:L ratio") was always maintained with Alfa tag in excess. In this example, X:L ratios from 1.5 to 6.0 were tested in addition to an unfunctionalized reference ("X:L = 0"). The volume of buffer added was chosen to achieve a final cargo (RNA + DNA) concentration of 0.1 g / L.

[0320] At low PGA loadings (Glu / N = 0.01-0.04) and across the ligand loadings tested, core particle size is maintained. Size generally increases only at the highest measured Glu / N ratios, indicating a greater dependence on the amount of bound ligand (Figure 11). Increasing PGA loading across the range tested decreases PdI and zeta potential (Figures 11-12). No free RNA or DNA was observed for any of the formulations tested (Figure 13).

[0321] In vitro, at low PGA loadings, core particles and alfa-AEEA(14)-PGA PLX showed reduced cell numbers compared to untreated controls, indicating toxicity. At higher PGA loadings, not only was this toxicity mitigated, but cell numbers increased compared to untreated controls, likely due to the induction of proliferation by CD3 ligands present on the particle surface (Figure 14A-B).

[0322] Ligand-mediated selective transfection of primary T cells was demonstrated by the difference in transfection between non-functionalized and functionalized particles in a PBMC assay (Figure 14A-B). Surprisingly, within the range measured, transfection efficiency was primarily determined by the amount of PGA-AEEA(14)-Alfa present, rather than the amount of bound ligand (Figure 14A-B).

[0323] Similar results were obtained for Thy1.1 and Venus in Jurkat cells, with the difference that transfection of non-functionalized and core formulations was generally higher (FIG. 15).

[0324] Example 7 This example evaluates linear and branched PEI to form binary complexes with modRNA and saRNA.

[0325] Preparation of dual-core particles with linear and branched PEI Binary formulations consisting of luciferase saRNA or luciferase modRNA were complexed with linear PEI500 (H[C2H4NH.HCl]500OH Mw 54.960 kDa) and branched PEI500 at N / P12 by mixing the two phases at a 24:1 mixing ratio (RNA to polymer). Binary formulations, or core polyplexes (PLX), were prepared at a final RNA concentration of 0.125 mg / mL in 1xMBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0).

[0326] Characterization of binary particles Physiochemical characterization of binary saRNA-PLX and modRNA-PLX included Zavg size and polydispersity index (PDI) using Wyatt DLS. Free RNA content was measured by agarose gel electrophoresis (AGE). Freezing stability testing was performed by measuring particle size distribution using three consecutive freeze-thaw cycles using Wyatt. In these cycles, particles were frozen overnight at either -20°C or -80°C, thawed at room temperature for 1 hour, measured by the standard Wyatt protocol, and frozen again at the same temperature. This procedure was repeated three times unless a significant size increase was observed.

[0327] Monomeric RNA was assessed using a centrifugation and RNA quantification method. Briefly, 400 μl of binary PLX was loaded into a 1.5 mL Eppendorf® tube and centrifuged at 20,000 rcf for 1.5 hours at 4°C (acceleration gradient 9, deceleration gradient 5). 250 μl of supernatant was collected from each centrifuged formulation. The RNA concentration of the supernatant (where monomeric RNA is present) was measured and related to the overall RNA content, expressing the monomeric RNA of the formulation as a percentage of total RNA. This method is comparable to an ultracentrifugation assay, in which samples are measured in an analytical ultracentrifuge and the light absorption is measured at two different wavelengths, 255 nm and 650 nm. MBG sample buffer was used as a blank for the measurements. Samples were centrifuged at 80,000 G at room temperature.

[0328] RNA concentration was quantified using NanoDrop. The microvolume option was used for RNA quantification, and 1.5 μl of formulated RNA was used. For RNA concentration calculation, the 260 / 280 and 260 / 230 nm purity ratios were considered, and formulation buffer was used as a blank solution for RNA quantification of each sample.

[0329] The luciferase assay is a reliable and sensitive method for detecting luciferase expression in live cells. In this assay, firefly luciferase catalyzes the monooxygenation of beetle luciferin via ATP and Mg2+-dependent pathways, resulting in light emission in the 550-620 nm range. For cell transfection, C2C12 and RAW 264.7 cells were seeded at 5,000 and 25,000 cells / well, respectively, into Nunc white flat-bottom 96-well plates (Merck KGaA, Darmstadt, Germany) and centrifuged at 500 x g for 5 minutes. After 18-24 hours, the medium was replaced with fresh medium at 90 μl / well. DPBS and medium alone served as negative and blank controls, respectively. Formulation samples were tested at mRNA assay concentrations of 12.5-100 ng per well. Samples and controls were added at 10 μl / well. After 24 hours, luciferase expression was measured by the Bright-Glo™ Luciferase Assay (Promega, Madion, WI, USA) according to the manufacturer's protocol. The reagent was added to the cells in the medium at a 1:1 (v / v) ratio, followed by incubation in the dark for 5 minutes to allow complete cell lysis.

[0330] Viability was measured using CellTiter-Glo® (Promega, Madison, WI, USA) according to the manufacturer's protocol. The reagent was added to cells in culture medium at a 1:1 (v / v) ratio, followed by 10 minutes of incubation on a shaker, followed by 20 minutes of incubation to stabilize the signal. Alternatively, luciferase and viability were measured using the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega GmbH, Madison, WI, USA) according to the manufacturer's instructions. 20 μl of 5X CellTiter-Fluor™ reagent was added to the wells, mixed on a shaker (300–500 rpm for approximately 30 seconds), and incubated at 37°C for 30 minutes. Fluorescence was then measured (viability) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Next, 100 μl of ONE-Glo™ reagent was added per well. After 3 minutes of incubation, bioluminescence was measured (luciferase expression). Bioluminescence signals (photons per second [p / s]) and fluorescence were measured using a microplate luminescence reader Infinite M200 (Tecan, Maneedorf, Switzerland). Relative luminescence was calculated by subtracting the signal of the DPBS control from the sample control. Relative viability was calculated as follows: Viability % = [RLU sample / RLU cells only] x 100

[0331] The binary saRNA-PEI PLX exhibits a size and monomeric RNA content of approximately 40 nm and 40% of the total RNA, respectively (Figure 16A). The binary modRNA-PEI PLX exhibits a size of 30 nm and a monomeric RNA content of approximately 75% (Figure 16A). No free RNA is detected using agarose gel electrophoresis (AGE) (Figure 16B). PLX is freezable, and no increase in size is observed after at least three freeze-thaw cycles at -80°C and -20°C (Figures 17A and 17B).

[0332] In vitro expression was evaluated in C2C12, RAW, and HEPG2 cell lines. ModRNA-PEI and saRNA-PEI polyplexes were evaluated at four different RNA concentrations: 100 ng, 50 ng, 25 ng, and 12.5 ng of RNA per well. The results show that for both modRNA and saRNA, polyplexes prepared with linear PEI (l-PEI) exhibit systematically higher luciferase signals than polyplexes prepared using branched PEI (b-PEI) (Figures 18A-C, 19A-C, 20A-C, and 21A-C), confirming the superior in vitro performance of linear cationic polymers compared with branched cationic polymers.

[0333] Example 8 This example describes the preparation and characterization of certain specific complexes, including at least those used in Examples 9-17, but these complexes can be applied to the various complexes described throughout this application. As described herein, references to a "binary formulation" or "binary particle" are also intended to refer to a "core complex" as described throughout this application. That is, a binary formulation includes a cationic polymer and a nucleic acid. A "ternary formulation" or "ternary particle" is also intended to refer to a complex including a cationic polymer, a nucleic acid, and an anionic polymer, where the cationic polymer and nucleic acid form a core complex encapsulated by the anionic polymer.

[0334] Preparation of binary core particles A binary formulation consisting of luciferase-saRNA was complexed with linear PEI500 (H[CHNH.HCl]OH, Mw 54.960 kDa) at N / P12. Fabrication was performed using a fluidic process by mixing the RNA and polymer phases. Binary formulations, or core polyplexes (PLX), were produced at a final RNA concentration of 0.1 mg / mL in 1xMBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0). These binary saRNA-PEI PLX exhibit a size of approximately 35 nm and a strong positive zeta potential of 20-25 mV. No free RNA was detected using agarose gel electrophoresis (AGE). PLX is freezable, and no increase in size was observed at -80°C for at least three freeze-thaw cycles. The binary saRNA complexes exhibit a monomeric RNA content of 40% of the total RNA.

[0335] A binary formulation consisting of luciferase modRNA was complexed with linear PEI500 (H[CHNH.HCl]OH, Mw 54.960 kDa) at N / P12. Fabrication was performed using a fluidic pathway process by mixing the RNA and polymer phases. The binary formulation (also referred to herein as core complex or core polyplex (PLX)) was fabricated in 1xMBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0) to a final RNA concentration of 0.125 mg / mL. These binary modRNA-PEI PLXs are approximately 30 nm in size as measured by Wyatt and exhibit a strong positive zeta potential of 20 to 25 mV. No free RNA was detected using agarose gel electrophoresis (AGE). PLXs can be frozen, and no increase in size was observed after thawing at -80 °C. Binary modRNA complexes exhibit high monomeric RNA content, with typically 80% of the total RNA in monomeric form.

[0336] Preparation of ternary complex Ternary formulations of modRNA were formed after the addition of an anionic PGA-based polymer onto the binary formulation (core polyplex). The ratio of negatively charged glutamic acid residues in PGA to positively charged nitrogen atoms in the cationic polymer ("Glu / N ratio") was found to be an appropriate measure of PGA loading. Ternary complex formation was tested within a range of Glu / N charge ratios from 0 to 3. After addition of the appropriate amount of each anionic polymer, the concentration was adjusted to 0.10 mg / ml RNA in all samples, and the final storage matrix was 1xMBG buffer (5% w / v glucose, 10 mM MES, pH 6.0).

[0337] Ternary formulations of modRNA were formed after the addition of a homopolymer of n-butyl-poly(L-aspartic acid sodium salt) 50 (PAsp). The ratio of the negatively charged aspartic acid residues of PAsp to the positively charged nitrogen atoms of the cationic polymer (the "Asp / N ratio") was found to be a suitable indicator for measuring the loading amount of PAsp. Ternary complex formation was tested within a range of Glu / N charge ratios from 0 to 3. After the addition of the appropriate amount of each anionic polymer, the concentration was adjusted to 0.10 mg / ml RNA in all samples, and the final storage matrix was 1xMBG buffer (5% w / v glucose, 10 mM MES, pH 6.0).

[0338] Ternary formulations of modRNA were generated by mixing RNA, cationic polymer, and anionic polymer to form core-shell complexes. The final buffer composition consisted of 1xMBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0).

[0339] Ternary formulations of saRNA were generated after the addition of an anionic PGA-based polymer onto the binary formulation (core polyplex). The ratio of negatively charged glutamic acid residues in PGA to positively charged nitrogen residues in the cationic polymer ("Glu / N ratio") was found to be an appropriate measure of PGA loading. Ternary complex formation was tested within a range of Glu / N charge ratios from 0 to 3. After the addition of the appropriate amount of each anion, the concentration was adjusted to 0.08 mg / ml RNA in all samples and MBG buffer (final concentration 5% w / v glucose, 10 mM MES, pH 6.0).

[0340] Characterization of binary and ternary particles Physiochemical characterization of binary and ternary saRNA-PLX and modRNA-PLX particles included Zavg size and polydispersity index (PDI) using Wyatt DLS. Surface charge zeta potential was measured using a Malvern zetasizer. Variations in zeta potential as a result of changes in the charge exposed by the particle surface further confirm the systematic coating of the core polyplexes with anionic polymers. Free RNA content was measured by agarose gel electrophoresis (AGE). Freezing stability testing was performed using a Wyatt system by measuring particle size distribution using three consecutive freeze-thaw cycles. For these cycles, particles were frozen overnight at either -20°C or -80°C, thawed at room temperature for 1 hour, measured by the standard Wyatt protocol, and then frozen again at the same temperature. This procedure was repeated three times.

[0341] Monomeric RNA was assessed using a centrifugation and RNA quantification method. Briefly, 400 μl of binary PLX was loaded into a 1.5 mL Eppendorf® tube and centrifuged at 20,000 rcf for 1.5 hours at 4°C (acceleration gradient 9, deceleration gradient 5). 250 μl of supernatant was collected from each centrifuged formulation. The RNA concentration of the supernatant (where monomeric RNA is present) was measured and related to the overall RNA content, expressing the monomeric RNA of the formulation as a percentage of total RNA. This method is comparable to an ultracentrifugation assay, in which samples are measured in an analytical ultracentrifuge and the light absorption is measured at two different wavelengths, 255 nm and 650 nm. MBG sample buffer was used as a blank for the measurements. Samples were centrifuged at 80,000 G at room temperature.

[0342] An example of a typical result for modRNA PEI-PLX is shown in the table below. In this example, to derive the monomeric RNA content of the formulation, the ratio of RNA concentration from centrifuged PLX to non-centrifuged PLX is considered, which results in a monomeric RNA content of 70%. [Table 5]

[0343] The RNA concentration was quantified using NanoDrop. A small volume option was used for RNA quantification, specifically 1.5 μl of formulated RNA. To calculate the RNA concentration, the 260 / 280 and 260 / 230 nm purity ratios were taken into account, and the formulation buffer was used as a blank solution for RNA quantification of each sample.

[0344] The protective effect of modRNA and saRNA against RNases can be assessed by incubation in serum, as these RNAs are degraded very rapidly in serum. Human serum solution was prepared by diluting samples with 5% MBG (5% w / v D-glucose, 10 mM MES, pH 6.0) to a final concentration of 0.05 mg / mL and 10% v / v human serum. A negative control was incubated in 5% MBG. Samples were incubated at 37°C for 30 minutes and then cooled in a 4°C bath for 10 minutes. Agarose gels were prepared using 100 ml of 1% agarose in TAE buffer. For RNA fluorescent labeling, 10 μl of GelRed was added before gel solidification. Each sample containing 1 μg of RNA was diluted with gel loading buffer to a final volume of 15 to 24 μl, which was loaded into each well of the agarose gel. Samples were scanned at 80 V and 50 mA for 40 minutes. UV fluorescence was measured after a 0.5 second exposure time to improve the visibility of faint bands.

[0345] The luciferase assay is a reliable and sensitive method for detecting luciferase expression in live cells. In this assay, firefly luciferase catalyzes the monooxygenation of beetle luciferin via ATP and Mg2+-dependent pathways, resulting in light emission in the 550-620 nm range. For cell transfection, C2C12 and RAW 264.7 cells were seeded at 5,000 and 25,000 cells / well, respectively, into Nunc white flat-bottom 96-well plates (Merck KGaA, Darmstadt, Germany) and centrifuged at 500 x g for 5 minutes. After 18-24 hours, the medium was replaced with fresh medium at 90 μl / well. DPBS and medium alone served as negative and blank controls, respectively. Formulation samples were tested at mRNA assay concentrations of 12.5-100 ng per well. Samples and controls were added at 10 μl / well. After 24 hours, luciferase expression was measured by the Bright-Glo™ Luciferase Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol: the reagent was added to cells in culture medium at a 1:1 (v / L) ratio, followed by 5 minutes of incubation in the dark to allow complete cell lysis. Viability was measured by the CellTiter-Glo® (Promega, Madison, WI, USA) according to the manufacturer's protocol: the reagent was added to cells in culture medium at a 1:1 (v / v) ratio, followed by 10 minutes of incubation on a shaker, followed by 20 minutes of incubation to stabilize the signal. Alternatively, luciferase and viability were determined by the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega GmbH, Madison, WI, USA) according to the manufacturer's instructions. Briefly, 20 μl of 5X CellTiter-Fluor™ Reagent was added to the wells and mixed on a shaker (300-500 rpm, approximately 30 seconds). After a 30-minute incubation at 37°C, fluorescence was measured (viability) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Next, 100 μl / well of ONE-Glo™ Reagent was added.After 3 minutes of incubation, bioluminescence was measured (luciferase expression). Bioluminescence signals (photons per second [p / s]) and fluorescence were measured using a microplate luminescence reader Infinite M200 (Tecan, Maneedorf, Switzerland). Relative luminescence was calculated by subtracting the signal of the DPBS control from the sample control. Relative viability was calculated as follows: Viability % = [RLU sample / RLU cells only] x 100

[0346] Example 9 This example provides an evaluation of the length of homopolymer poly-α-L-glutamic acid to form a ternary complex with modRNA-PEI.

[0347] Physicochemical characterization demonstrated that ternary polyplexes could be formulated when PGAs with different monomer units were used, with sizes ranging from 50 nm to 150 nm. The size of the ternary complexes depended on the Glu / N ratio and PGA length. As a general trend, ternary PLXs became larger at Glu / N ratios close to neutral surface charge (ratios 0.4–0.8), whereas particle size remained relatively small at either very small (0.2) or very large (3) Glu / N ratios (Zavg ≤ 100 nm) (Figure 22). The length of the PGA affected both size and PDI. PGA(50) and PGA(20) yielded the lowest complex sizes (Zavg ≤ 150 nm) among all Glu / N ratios tested. At the same time, the shortest PGA, PGA(20), yielded the most uniform particle size distribution (PDI ≤ 0.3). The surface charge of the particles can be tuned by increasing the amount of anionic polymer added to the core polyplex, which completely shifts the zeta potential from approximately +25 mV to -25 mV, as can be seen from the zeta potential values ​​in Figure 22. The variation in zeta potential as a result of the change in charge exposed by the particle surface further confirms the systematic coating of the core polyplex with the anionic polymer.

[0348] Free RNA content was measured by AGE and is shown in Figure 23. For PGA(20) and PGA(50), no free RNA was detected in any of the Glu / N tested, suggesting successful encapsulation of the cargo.

[0349] The ternary complexes were tested for cargo release and RNAse degradation upon serum incubation. Figure 24 shows the corresponding AGE images of all tertiary complexes with different PGA lengths and ratios, including the control (untreated sample) and serum-incubated samples. Compaction of modRNA induced by interaction with PEI successfully protects it against RNase degradation. Furthermore, with shorter PGA chains (PGA(20)), no increase in free RNA content is observed upon serum incubation, while longer chains (PGA(50)) show the presence of free RNA starting at a Glu / N ratio of 0.8.

[0350] FIG. 25 shows that the PGA coating confers colloidal stability upon freeze-thaw at both −20° C. and −80° C., and that the length of the polymer influences the Glu / N range over which stability is achieved.

[0351] Example 10 This example describes the evaluation of a homopolymer of n-butyl-poly-(L-aspartic acid) to form a ternary complex with modRNA-PEI.

[0352] Poly(L-aspartic acid) (PAsp) was tested as a polymer coating for core polyplexes because of its desirable properties for the development of novel drug delivery carriers. PAsp is indeed biocompatible, biodegradable, and exhibits low cytotoxicity. See Nie, et al., ACS Appl. Mater. Interfaces 2015, 7, 1, 553-562.

[0353] Physiochemical characterization is shown in Figure 26. Ternary complexes can be prepared using PAsp, yielding particle sizes ranging from 50-150 nm, depending on the Asp:N ratio used. Ternary PLX exhibits a large particle size distribution at ratios close to neutral surface charge (ratio 0.6), but particle size remains relatively small at either very low (0.2) or very high (3) Asp:N ratios (Figure 26). Very low and very high Asp:N ratios (Asp:N ≥ 0.2 and ≥ 1.5) result in the least uniform particle size distribution (PDI > 0.3), while Asp:N ratios in the range of 0.4-0.6 result in more uniform complex distributions. The particle surface charge can be tuned by increasing the amount of anionic polymer added to the core polyplex, which completely shifts the zeta potential from approximately +25 mV to -25 mV, as seen from the zeta potential values ​​in Figure 26. The variation in zeta potential as a result of changes in the charge exposed by the particle surface further confirms the systematic coating of the core polyplexes with the anionic polymer.

[0354] The free RNA content was measured by AGE (Figure 27), and the images show that it is possible to obtain ternary complexes coating PEI-RNA with a layer of biodegradable polymer such as PAsp with acceptable size and PDI (size ≤ 150 nm and PDI ≤ 0.3), and that the amount of biodegradable polymer can be adjusted on the core polyplex surface to obtain complexes with different surface charges (Asp: No free RNA up to N 0.6) without changing the polyplex structure.

[0355] Example 11 This example describes the evaluation of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine long block copolymers to form ternary complexes with modRNA-PEI, including investigation of their stability in serum and in vitro transfection activity.

[0356] Ternary formulations of modRNA were generated after adding block copolymers of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine (PGA(50)-b-pSar(X), where X = 10, 25, or 50 repeating units).

[0357] Physiochemical characterization indicates that a decrease in size was observed in all cases with increasing pSar length (Figure 28). Complexes prepared with PGA(50)-b-pSar(50) showed a minimum size of approximately 50 nm for all Glu / N tested.

[0358] Furthermore, as the length of the pSar increases, the PDI generally decreases, i.e., the particle size distribution becomes more uniform (Figure 28). As the length of the pSar increases, the particles become more stable. Without wishing to be bound by theory, it is hypothesized that stability improves as the steric repulsive interactions between particles become stronger. With the addition of increasing amounts of PGA(50)-b-pSar(50), the size distribution of the ternary formulation remains nearly constant at 50–60 nm, but the surface charge systematically changes from +25 to -25 mV.

[0359] Free RNA content was measured by AGE and is shown in Figure 29. In this case, increasing the length of pSar did not result in significant differences in free RNA detection, as good encapsulation of cargo was achieved in all cases up to a Glu / N of 0.6.

[0360] Ternary complexes formed with PGA(50)-b-pSar(50) at Glu:N 0.6 were tested for cargo release upon serum incubation for 30 min, 1 h, and 2 h (Figure 30). No increase in free RNA content was observed upon serum incubation for up to 2 h, indicating that PGA(50)-b-pSar(50) can successfully protect polyplexes from serum protein destabilization.

[0361] In vitro expression was evaluated in C2C12, RAW, and HEPG2 cell lines. Ternary modRNA-PEI polyplexes complexed with PGA(50)-bpSar(50) at a Glu / N ratio of 0.6 were evaluated at four different concentrations: 100 ng, 50 ng, 25 ng, and 12.5 ng of RNA per well. A control sample of uncoated core polyplexes was included. The results show that cell viability is maintained when core polyplexes are coated with PGA(50)-bpSar(50) (Figure 31). Most importantly, the data show that while luciferase expression of core polyplexes is reduced upon serum incubation, the coated complexes can successfully maintain luciferase expression in all cell lines tested, especially at low doses (Figure 32).

[0362] Example 12 This example describes the evaluation of Alfa-tagged block copolymers of Poly-α-L-glutamic acid-block-Poly-N-methyl-sarcosine, Poly-α-L-glutamic acid, and Poly-α-L-glutamic acid-block-Poly-N-Ac-AEEA to form ternary complexes with modRNA-PEI polyplexes.

[0363] Ternary formulations of modRNA were generated after the addition of either an Alfa-tagged block copolymer of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine (PGA(50)-b-pSar(50)-Alfa), an Alfa-tagged homopolymer of poly-α-L-glutamic acid (PGA(X)-Alfa, where X = 50 or 100 repeating monomer units), or an Alfa-tagged block copolymer of poly-α-L-glutamic acid-block-poly-N-Ac-AEEA (PGA(50)-b-Ac-AEEA(14)-Alfa).

[0364] Ternary complexes with PGA(50)-b-pSar(50)-Alfa, PGA(50)-b-Ac-AEEA(14)-Alfa, PGA(50)-Alfa, and PGA(100)-Alfa can be successfully formed, with sizes varying depending on the polymer used and the Glu / N ratio tested (Figure 33, top graph). The presence of a stealth moiety as a linker between the PGA and Alfa peptides (pSar(50) and pAEEA(14)) allows the Z-average size to be maintained below 100 nm. Across the majority of Glu / N ratios tested, the ternary complexes show an acceptable distribution with PDI values ​​≤ 0.4, with higher PDIs typically obtained at higher Glu / N ratios (Figure 33, middle graph). At the same time, as the amount of anionic polymer increases, the surface charge of the particles can be tuned, resulting in a complete shift in the zeta potential value from approximately +25 mV to -25 mV, as seen in Figure 33, bottom graph.

[0365] Agarose gel electrophoresis (AGE) analysis shows that Alfa-tagged modRNA ternary complexes can be formulated within specific Glu / N ratios that achieve complete RNA encapsulation (Figure 34).

[0366] The ternary complexes coated with Alfa-tagged polymers could be successfully stabilized at both -80°C and -20°C for up to three freeze-thaw cycles and a wide range of Glu / N ratios, thus ensuring the stability of positively, neutrally, and negatively charged particles (Figure 35).

[0367] Example 13 This example describes the evaluation of the length of homopolymer poly-α-L-glutamic acid to form a ternary complex with saRNA-PEI.

[0368] Physiochemical characterization indicates that ternary complexes were obtained even when larger RNA constructs were used, as in the case of saRNA, with sizes ranging from 50 nm to 150 nm. The size of the ternary complexes was affected by the Glu / N ratio and PGA length tested. Ternary PLX exhibited larger particle sizes at Glu / N ratios close to neutral surface charge (ratios 0.6–0.8), but particle sizes remained small at either very small (0.2) or very large (3) Glu / N ratios (Zavg ≤ 100 nm) (Figure 36, top graph). The PGA length primarily affected the uniformity of the size distribution, with the longest PGAs (PGA(50) and PGA(100)) leading to a highly uniform population of complexes (PDI ≤ 0.3) (Figure 36, middle graph). The surface charge of the particles can be tuned by increasing the amount of anionic polymer added to the core polyplex, which completely shifts the zeta potential from approximately +25 mV to -25 mV, as can be seen from the zeta potential values ​​in Figure 36, bottom graph.

[0369] The AGE results show that ternary complexes can be prepared that achieve complete cargo encapsulation across the Glu / N ratios tested (Figure 37).

[0370] Figure 38 shows that the PGA coating ensures colloidal stability upon freeze-thaw at both -20°C and -80°C, and that the length of the polymer can influence the Glu / N range over which stability is achieved. In this regard, PGA(50) is particularly efficient at maintaining colloidal properties after repeated freeze-thaw cycles over the widest range of Glu / N tested.

[0371] Example 14 This example describes the evaluation of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine long block copolymers to form ternary complexes with saRNA-PEI, including investigations into in vitro transfection activity.

[0372] The formation of ternary formulations of saRNA was generated after adding block copolymers of poly-α-L-glutamic acid-block-poly-N-methyl-sarcosine (PGA(50)-b-pSar(X), where X is 10, 25, or 50 repeating units).

[0373] Physicochemical characterization showed that a decrease in size was observed in all cases with increasing pSar length, which was particularly significant for particles with a near-neutral charge (0.6) (Figure 39, top graph). Complexes prepared with PGA(50)-b-pSar(50) showed a minimum size of approximately 50 nm for all Glu / N ratios tested.

[0374] The ternary complexes exhibited a narrow size distribution with a PDI ≤ 0.3 at low Glu:N charge ratios (up to 0.8) (Figure 39, middle graph). With the addition of increasing amounts of PGA(50)-b-pSar(50), the size distribution of the ternary formulations remained nearly constant at 50–60 nm, but the surface charge systematically shifted from +25 to −25 mV.

[0375] Free RNA content was measured by AGE and is shown in Figure 40. In this case, increasing the length of pSar did not result in significant differences in the detection of free RNA, since good encapsulation of cargo was obtained in all cases up to a Glu / N of 0.6.

[0376] Figure 41 shows that the PGA coating ensures colloidal stability upon freeze-thaw at -80 °C, and that the length of the stealth moiety can influence the Glu / N range over which stability is achieved. In this context, PGA(50)-b-pSar(50) is particularly efficient at maintaining colloidal properties after repeated freeze-thaw cycles over the widest range of Glu / N tested.

[0377] Example 15 This example describes the evaluation of the length of homopolymeric Alfa-tagged poly-α-L-glutamic acid to form ternary complexes with saRNA-PEI polyplexes.

[0378] Physiochemical characterization showed that alfa-tagged ternary complexes could be formed in the size range of 40–150 nm with both PGA50-Alfa and PGA100-Alfa, with larger particle sizes obtained at Glu / N ratios of 0.4–1 (Figure 42, top graph). The particle size remained relatively small at either very small (0.2) or very large (3) Glu / N ratios. The PDI, shown in the middle graph of Figure 42, was approximately ≤0.4. The surface charge of the particles could be tuned with increasing amounts of ternary polymer, resulting in a complete shift from approximately +25 mV to -30 mV, as seen in the bottom graph of Figure 42.

[0379] Agarose gel electrophoresis (AGE) analysis shows that RNA can be successfully encapsulated when saRNA binary polyplexes are coated with both PGA(50)-Alfa and PGA(100)-Alfa (Figure 43).

[0380] Freezing stability studies show that PGA(50)-Alfa colloids are stable to up to three freeze-thaw cycles at low amounts of PGA (e.g., Glu / N ratio of 0.2), while the particles remain highly positively charged. For the same polymer length, stability at -80°C can also be achieved at higher Glu / N ratios (0.8). Increasing the polymer length allows colloidal stability to be achieved at -20°C and -80°C, even for negatively charged complexes (Glu / N ratios of 1 and 1.5) (Figure 44).

[0381] Example 16 This example describes the evaluation of Alfa-tagged block copolymers of Poly-α-L-glutamic acid-block-Poly-N-methyl-sarcosine and Poly-α-L-glutamic acid-block-Poly-N-Ac-AEEA14 to form ternary complexes with saRNA-PEI polyplexes.

[0382] Physicochemical characterization indicates that ternary complexes with PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-Ac-AEEA(14)-Alfa can be successfully formed, maintaining a size between 40 and 60 nm (Figure 45, top graph). At most Glu / N ratios, no significant differences in size are observed between the two different stealth polymers used (pSar(50) and pAEEA(14)). PDI values ​​of ≤0.4 are obtained with both anionic polymers (Figure 45, middle graph). These results indicate that the size of the ternary complex can be maintained below 70 nm when the stealth moiety is present as a linker between the cationic polymer and alfa (pSar(50) and pAEEA(14)).

[0383] At the same time, as the amount of anionic polymer increases, the surface charge of the particles can be tuned, resulting in a complete shift in the zeta potential value from approximately +25 mV to -25 mV, as seen in Figure 45, bottom graph.

[0384] The AGE results indicate that saRNA can be successfully encapsulated when the binary polyplexes are coated with PGA(50)-b-pSar(50)-Alfa and PGA(50)-b-Ac-AEEA(14)-Alfa (Figure 46).

[0385] Ternary complexes coated with PGA(50)-b-pSar(50)-Alfa could be successfully stabilized at both -80 °C and -20 °C over a wide range of freeze-thaw cycles and Glu / N ratios, thus ensuring the stability of positively, neutrally, and negatively charged particles. The ternary complex obtained with PGA(50)-b-Ac-AEEA(14)-Alfa exhibits good freeze stability at a low Glu / N ratio (0.2) (Figure 47). Furthermore, the stabilizing effect can be confirmed in the case of the PGA(50)-b-pSar(50)-Alfa polymer. The presence of the Alfa tag on the anionic polymer improves the stability of negatively charged particles (high Glu / N ratio) (Figure 47) compared to anionic polymers without the Alfa tag (Figure 41).

[0386] Example 17 Evaluation of poly-α-L-glutamic acid-block-poly-N-Ac-AEEA-length block copolymers for forming ternary complexes with saRNA-PEI The formation of ternary formulations of saRNA was generated after adding block copolymers of poly-α-L-glutamic acid-block-poly-N- Ac-AEEA (PGA(50)-Ac-AEEA(X), where X = 4, 8, or 14 repeating units).

[0387] Physiochemical characterization showed that ternary complexes were formed with all tested AEAAs and PGA(50)-Ac-AEEA(X), with the longer AEA (PGA(50)-Ac-AEEA(14)) exhibiting the smallest complex size, Zavg ≤ 100 nm (Figure 48, top graph). The complex distribution was uniform (PDI ≤ 0.4), and the particle surface charge varied with the amount of added coating polymer (the zeta potential shifted from +25 to -25 mV) (Figure 48, middle and bottom graphs).

[0388] AGE revealed good encapsulation of saRNA up to a Glu / N ratio of 1, with no significant effect due to the length of the stealth moiety (Figure 49).

[0389] Figure 50 shows that PGA(50)-Ac-AEEA(X) coatings can ensure colloidal stability during freeze-thaw cycles at -20°C and -80°C within a specific range of Glu / N ratios, and that PGA(50)-b-Ac-AEEA(14) is particularly effective in maintaining colloidal properties after repeated freeze-thaw cycles over the widest range of Glu / N ratios tested.

[0390] The embodiments of the present disclosure described above are intended to be merely illustrative, and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Claims

1. A complex comprising a linear cationic polymer, an anionic polymer, and a monomeric RNA molecule, wherein the cationic polymer and the monomeric RNA molecule form a core complex encapsulated by the anionic polymer.

2. 2. The conjugate of claim 1, wherein the cationic polymer is or comprises one or more copolymers of poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

3. 3. The conjugate of claim 1 or 2, wherein the cationic polymer is a homopolymer selected from poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

4. 4. The conjugate of claim 2 or 3, wherein the cationic polymer is poly(ethyleneimine).

5. The linear poly(ethyleneimine) has an M of about 600 Da to about 400,000 Da. ｎ 5. The complex of claim 4, having the formula:

6. The linear poly(ethyleneimine) has an M of about 10,000 Da to about 120,000 Da. ｎ 6. The complex of claim 5, having the formula:

7. 3. The composite of claim 1, wherein the cationic polymer is a block copolymer comprising poly(ethyleneimine) and poly(propyleneimine).

8. 10. The conjugate of claim 1, wherein the cationic polymer is a polymer or copolymer comprising blocks of Formula I and / or II: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: R １ is H, —C(O)— optionally substituted C １ -C ６ Aliphatic, optionally substituted C １ -C ６ aliphatic, or G; Each R ２ are independently H, optionally substituted C １ -C ６ aliphatic, or G; X １ and X ３ However, each independently, C １ -C ６ is aliphatic, X ２ is a bond or an optionally substituted C １ -C ６ is aliphatic, m is an integer from 2 to 2000; n is an integer from 2 to 2000, Each G is independent, 【Chemistry 2】 and R １’ is H, —C(O)— optionally substituted C １ -C ６ Aliphatic, optionally substituted C １ -C ６ aliphatic, or G'; Each R ２’ are independently H, optionally substituted C １ -C ６ aliphatic, or G'; X １’ and X ３’ each independently represents an optionally substituted C １ -C ６ is aliphatic, X ２’ is a bond or an optionally substituted C １ -C ６ is aliphatic, n' is an integer from 2 to 2000; m' is an integer from 2 to 2000; Each G' is independently 【Chemistry 3】 and R １’’ is H, —C(O)— optionally substituted C １ -C ６ Aliphatic, optionally substituted C １ -C ６ is aliphatic, Each R ２’’ are independently H, or C １ -C ６ is aliphatic, X １’’ and X ３’’ are each independently optionally substituted C １ -C ６ is aliphatic, X ２’’ is a bond or an optionally substituted C １ -C ６ is aliphatic, n″ is an integer from 2 to 2000; The above complex, wherein m″ is an integer of 2 to 2000.

9. The complex of any one of claims 1 to 8, wherein the anionic polymer is a homopolymer.

10. 10. The complex according to claim 1, wherein the anionic polymer is a homopolymer selected from poly-L-glutamic acid, poly-L-aspartic acid, polysaccharides, poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), and polyphosphoric acid.

11. 11. The conjugate of claim 9, wherein the anionic polymer comprises from about 10 to about 100 repeating monomer units.

12. The complex of any one of claims 1 to 8, wherein the anionic polymer is a heteropolymer.

13. 13. The conjugate of claim 12, wherein the heteropolymer comprises monomers of glutamic acid, aspartic acid, ethylene glycol, propylene glycol, sarcosine, phosphate, 2-methyl-2-oxazoline, 2-methoxycarbonylethyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-methyl-2-oxazine, 2-methoxycarbonylethyl-2-oxazine, 2-methoxycarbonylpropyl-2-oxazoline, 2-methoxycarbonylpropyl-2-oxazine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and / or 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

14. 14. The composite of claim 12 or 13, wherein the anionic polymer comprises from about 10 to about 100 monomer units.

15. The complex of any one of claims 1 to 8, wherein the anionic polymer is a block copolymer.

16. 16. The conjugate of claim 15, wherein the block copolymer comprises blocks of polymers selected from the group consisting of poly(2-methoxycarbonylethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, polyphosphate, poly(N-methyl-sarcosine), poly(ethylene glycol), poly(propylene glycol), poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), poly(2-methoxycarbonylpropyl-2-oxazine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

17. 17. The composite of claim 16, wherein the block copolymer comprises an anionic block and a stealth block.

18. 18. The composite of claim 17, wherein the anionic block is selected from poly(2-methoxycarbonylethyl-2-oxazoline), poly-L-glutamic acid, poly-L-aspartic acid, polyphosphoric acid, poly(2-methoxycarbonylpropyl-2-oxazoline), poly(2-methoxycarbonylethyl-2-oxazine), and poly(2-methoxycarbonylpropyl-2-oxazine).

19. 19. The composite of claim 17 or 18, wherein the stealth block is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazine), poly(N-methyl-sarcosine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid.

20. 20. The conjugate of any one of claims 15 to 19, wherein each block comprises from about 10 to about 100 repeating monomer units.

21. 2. The conjugate of claim 1, wherein the cationic polymer is linear poly(ethyleneimine) and the anionic polymer is a block copolymer comprising blocks selected from poly-L-glutamic acid, poly-L-aspartic acid, and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid.

22. The conjugate of any one of claims 1 to 21, wherein the anionic polymer comprises a targeting moiety.

23. 23. The conjugate of claim 22, wherein the targeting moiety is an Alfa peptide.

24. 24. The complex of any one of claims 1 to 23, wherein the charge ratio of the anionic groups in the anionic polymer to the cationic groups in the cationic polymer is from about 0.25:1 to about 3:

1.

25. 25. The complex of claim 24, wherein the charge ratio of the anionic groups in the anionic polymer to the cationic groups in the cationic polymer is about 1.5:

1.

26. The complex of any one of claims 1 to 25, further comprising a buffer.

27. 27. The complex of claim 26, wherein the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), 2-amino-2(hydroxymethyl)propane-1,3-diol (TRIS), a Bis-tris buffer system, a carboxylic acid buffer system, a phosphate buffer system, and a citrate buffer system.

28. The complex of any one of claims 1 to 27, wherein the RNA is mRNA, modRNA, saRNA, or taRNA.

29. 29. The complex of any one of claims 1 to 28, wherein the complex has a diameter of about 10 nm to about 150 nm.

30. 30. The complex of any one of claims 1 to 29, wherein the complex has a diameter of about 20 nm to about 50 nm.

31. 31. A method of increasing or causing an increase in expression of RNA in a target in a subject, the method comprising administering to the subject a complex according to any one of claims 1 to 30.

32. 32. The method of claim 31 , wherein the target is selected from the lung, liver, spleen, heart, brain, lymph nodes, bladder, kidney, and pancreas.

33. 31. A method of treating a disease, disorder, or condition in a subject, comprising administering to the subject a conjugate of any one of claims 1 to 30.

34. 34. The method of claim 33, wherein the disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

35. The method of any one of claims 31 to 34, wherein the complex is administered parenterally.

36. 36. The method of claim 35, wherein the conjugate is administered intramuscularly, subcutaneously, or intravenously.

37. 35. The method of any one of claims 31-34, wherein the complex is administered intranasally.

38. 35. The method of any one of claims 31 to 34, wherein a first dose of the complex is administered by a first route and a second dose of the complex is administered by a second route.

39. 39. The method of claim 38, wherein the first route is parenteral.

40. 40. The method of claim 38 or 39, wherein the second route is intranasal.

41. A conjugate according to any one of claims 1 to 30 for use in medicine.

42. 31. The conjugate of any one of claims 1 to 30, for use in the treatment and / or prevention of a disease, disorder, or condition, wherein the disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

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