Poly (amine-co-ester) polymers with modified end groups and enhanced pulmonary delivery

Abstract

To provide poly (amine-co-ester) polymers, methods of forming polyplexes and particles loaded with active agents therefrom, and methods of using them for delivery of nucleic acid agents with optimal uptake.SOLUTION: The examples demonstrate important molecular weights in combination with exposed carboxylic acid and / or hydroxyl groups, and methods of making. Typically, the composition is less toxic, more efficient in drug delivery, or a combination thereof, as compared to other transfection reagents in the control. In some embodiments, the composition is suitable for invivo delivery and can be administered systemically to a subject to treat a disease or condition. In vivo delivery to the lung by inhalation has been demonstrated for poly (amine-co-ester) polymers having certain amine or hydroxyl group-containing end groups during mixing with PEGylated poly (amine-co-ester) polymers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Application No. 63 / 041,739, filed June 19, 2020, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under EB000487 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The field of the invention relates generally to polymeric compositions and methods for improved delivery of diagnostic, prophylactic and / or therapeutic agents, particularly nucleic acid-based agents, particularly to the lung. [Background technology]

[0004] Background of the Invention Non-viral vectors for gene delivery have attracted much attention over the past few decades due to their potential for limited immunogenicity, their ability to accommodate and deliver large-sized genetic material, and the possibility of modifying their surface structure. Major categories of non-viral vectors include cationic lipids and cationic polymers. Cationic lipid-derived vectors, pioneered by Felgner and colleagues, represent some of the most widely explored systems for non-viral gene delivery (Felgner, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. PNAS, 84, 7413-7417 (1987)) (Templeton, et al. Improved DNA: liposome complexes for increased systemic delivery and gene transfer). expression. Nat. Biotechnol. 15, 647-652 (1997)) (Chen, et al. Targeted nanoparticles deliver siRNA to melanoma. J. Invest. Dermatol. 130, 2790-2798 (2010)).

[0005] Cationic polymer non-viral vectors have attracted increasing attention due to their flexibility in synthesis and structural modification for specific biomedical applications. Both cationic lipid and cationic polymer systems deliver genes by forming condensed complexes with negatively charged DNA through electrostatic interactions; the complex formation protects the DNA from degradation and promotes its cellular uptake and intracellular transport to the nucleus.

[0006] Although polyplexes formed between cationic polymers and DNA are generally more stable than lipoplexes formed between cationic lipids and DNA, both are often unstable in physiological fluids containing serum components and salts, which tend to cause the complexes to disintegrate or aggregate (Al-Dosari, et al. Nonviral gene delivery: principles, limitations, and recent progress. AAPS J. 11, 671-681 (2009)) (Tros de Ilarduya, et al. Gene delivery by lipoplexes and polyplexes. Eur. J. Pharm. Sci. 40, 159-170 (2010)). In addition, although some studies have shown that anionic polymers, or even naked DNA, can provide some level of transfection under certain conditions, transfection with both lipids and polymers usually requires materials with excess charge, resulting in polyplexes or lipoplexes with a net positive charge on the surface (Nicol, et al. Gene. Ther. 9, 1351-1358 (2002))(Schlegel, et al.. J. Contr. Rel. 152, 393-401 (2011))(Liu, et al, AAPS J. 9, E92-E104 (2007) )(Liu, et al. Gene Ther. 6, 1258-1266 (1999)). Circulatory system in vivo When injected into the blood, the positive surface charge initiates the rapid formation of complex aggregates with the membranes of negatively charged serum molecules or cellular components, which are then removed by the reticuloendothelial system (RES).

[0007] More importantly, many cationic vectors developed so far exhibit substantial toxicity, which limits their clinical applicability (Tros de Ilarduya, et al. Eur. J. Pharm. Sci. 40, 159-170 (2010)) (Gao, et al. Biomaterials 32, 8613-8625 (2011)) (Felgner, et al. J. Biol. Chem. 269, 2550-2561 (1994)) (Kafil, et al. BioImpacts 1, 23-30 (2011)) (Lv, et al. J. Contr. Rel. 114, 100-109 (2006)). This also appears to be charge dependent, with the Excessive positive charges on surfaces can interact with cellular components such as cell membranes and inhibit normal cellular processes such as clathrin-mediated endocytosis, the activity of ion channels, membrane receptors, and enzymes, or cell survival signaling (Gao, et al. Biomaterials 32, 8613-8625 (2011)) (Felgner, et al. J. Biol. Chem. 269, 2550-2561 (1994)) (Kafil, et al. BioImpacts 1, 23-30 (2011)).

[0008] As a result, cationic lipids often trigger acute inflammatory responses in animals and humans, while cationic polymers such as PEI destabilize the plasma membrane of red blood cells and induce cell necrosis, apoptosis, and autophagy (Tros de Ilarduya, et al. Eur. J. Pharm. Sci. 40, 159-170 (2010)) (Gao, et al. Biomaterials 32, 8613-8625 (2011)) (Lv, et al. J. Contr. Rel. 114, 100-109 (2006)). Because of these undesirable effects, high-efficiency non-Williams with lower charge densities have been developed. There is a need for virus vectors.

[0009] The synthesis of a family of biodegradable poly(amine-co-ester)s formed via enzymatic copolymerization of diesters with amino-substituted diols has been reported by Liu, et al. J. Biomed. Mater. Res. A 96A, 456-465 (2011) and Jiang, Z. Biomacromolecules 11, 1089-1093. (2010). Diesters with various chain lengths (e.g., succinate to dodecanedioate) were copolymerized with diethanolamine bearing either alkyl (methyl, ethyl, n-butyl, t-butyl) or aryl (phenyl) substituents on the nitrogen. The high tolerance of the lipase catalyst allowed the copolymerization reaction to be completed in one step without the need for protection and deprotection of the amino functional group. Upon protonation under mildly acidic conditions, these poly(amine-co-esters) readily condense DNA to form nanosized polyplexes. Screening studies revealed that one of these materials, poly(N-methyldiethyleneamine sebacate) (PMSC), transfected various cell types, including HEK293, U87-MG, and 9L, with efficiencies comparable to those of major commercial products such as Lipofectamine 2000 and PEI14. PMSCs have previously been used for gene delivery, but the delivery efficiency of the enzymatically synthesized material was approximately five orders of magnitude higher than any previously reported one (Wang, et al.). al. Biomacromolecules 8, 1028-1037 (2007))(Wang, et al. Biomaterials 28, 5358-5368 (2007). However, these poly(amine-co-ester)s have not been effective for systemic delivery of nucleic acids in vivo. This may be due to the fact that the polyplexes formed by these polymers and genetic material (1) are not efficient enough for in vivo applications and / or (2) are not stable enough in blood, disintegrating or aggregating during circulation.

[0010] Thus, there remains a need for non-viral vectors suitable for efficient systemic in vivo or in vitro delivery of nucleic acids with high transfection efficiency and low toxicity.

[0011] There is also a need for polymeric nanocarriers whose molecular weight, polymer composition, and / or end groups can be easily controlled and modified.

[0012] It is therefore an object of the present invention to provide improved polymers capable of effectively delivering therapeutic, diagnostic, and / or prophylactic agents, particularly nucleic acids, in vivo or in vitro, as well as methods of making and using them.

[0013] It is an object of the present invention to provide improved polymers, and methods for making same, that are capable of effectively delivering genetic material to cells in vitro with high efficiency and are suitable for the in vivo delivery of nucleic acids.

[0014] It is also an object of the present invention to provide methods of using improved polymers for pulmonary delivery of nucleic acids in vivo. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Felgner, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. PNAS, 84, 7413-7417 (1987) [Non-patent document 2] Templeton, et al. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat. Biotechnol. 15, 647-652 (1997) [Non-patent document 3] Chen, et al. Targeted nanoparticles deliver siRNA to melanoma. J. Invest. Dermatol. 130, 2790-2798 (2010)

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[0016] Summary of the Invention Poly(amine-co-ester) polymers with modified end groups are used to deliver therapeutic, diagnostic, and / or prophylactic agents, particularly nucleic acid-based drugs, by forming polyplexes and particles therefrom. These exhibit improved loading of nucleic acid cargo, such as RNA, more specifically mRNA, improved cellular transfection, improved intracellular endosomal release, or a combination thereof. Examples demonstrate key molecular weights combined with exposed carboxylic acid and / or hydroxyl groups, as well as methods of preparation. The modified polymers also exhibit preferential uptake in specific tissues when administered by injection and high levels of uptake when administered to the pulmonary system. Typically, the compositions exhibit reduced toxicity, more efficient drug delivery, or a combination thereof, compared to comparable transfection reagents.

[0017] A polymer having the structure of Formula I is disclosed. [ka] [In the formula, n is an integer of 1 to 30, m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; R x is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof.

[0018] In some forms, Z is the same as Z'.

[0019] In some forms, Z is O and Z' is O. In some forms, Z is NR' and Z' is NR'. In some forms, Z is O and Z' is NR'. In some forms, Z is NR' and Z' is O.

[0020] In some embodiments, Z' is O and n is an integer from 1 to 24, for example, 4, 10, 13, or 14. In some embodiments, Z is also O.

[0021] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, and m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8. In some embodiments, Z is also O.

[0022] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and o and p are the same integer from 1 to 6, e.g., 2, 3, or 4. In some embodiments, Z is also O.

[0023] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and R is alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl, or aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, or xylyl. In some embodiments, Z is also O.

[0024] In some forms, n is 14 (e.g., pentadecalactone, PDL), m is 7 (e.g., sebacic acid), and o and p are 2 (e.g., N-methyldiethanolamine, MDEA).

[0025] In some embodiments, the polyplexes or particles are formed from polymers in which R1 and / or R2 do not consist of or include:

[0026] In some forms, the polymer has a structure of Formula II: [ka] wherein J1 and J2 are independently a linking moiety or absent; R3 and R4 are substituted alkyls containing hydroxyl groups, primary amine groups, secondary amine groups, tertiary amine groups, or combinations thereof.

[0027] In some embodiments, J1 is -O- or -NH-.

[0028] In some forms, J2 is -C(O)NH- or -C(O)O-.

[0029] In some forms, R3 is the same as R4.

[0030] Preferably, R3 and / or R4 are linear.

[0031] In some embodiments, R3, R4, or both contain a hydroxyl group. In some embodiments, R3, R4, or both contain a hydroxyl group and one or more amine groups, preferably secondary amine groups or tertiary amine groups. In some embodiments, R3, R4, or both contain a hydroxyl group and no amine groups.

[0032] In some forms, at least one of R3 and R4 does not contain a hydroxyl group.

[0033] In some forms, the polymer has the structure of Formula III: [ka]

[0034] In certain embodiments, the values ​​of x, y, and / or q are such that the weight average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, or greater than 2,000 daltons. In some forms, the weight average molecular weight of the polymer is from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 10,000 daltons.

[0035] The polymers can be prepared from one or more lactones, one or more amine-diols (Z and Z' = O) or triamines (Z and Z' = NR'), and one or more diacids or diesters. In embodiments where two or more different lactones, diacids or diesters, and / or triamine or amine-diol monomers are used, the values ​​of n, o, p, and / or m can be the same or different.

[0036] In some embodiments, the polymer is between about 2 kDa and 20 kDa, or between about 2 kDa and about 10 kDa, or between about 2 kDa and about 5 kDa.

[0037] The polymers can be used to form microparticles and / or nanoparticles in which one or more therapeutic, diagnostic, and / or prophylactic agents are encapsulated. The encapsulated and delivered agents can be small molecule agents (e.g., non-polymeric agents having a molecular weight of less than 2,000, 1500, 1,000, 750, or 500 daltons) or macromolecules (e.g., oligomers or polymers), such as proteins, enzymes, peptides, nucleic acids, etc. The particles can be used for in vivo and / or in vitro delivery of agents.

[0038] In some embodiments, polymers can be used to form polymeric nanoparticle polynucleotide carriers, referred to herein as polyplexes, which are effective for delivering polynucleotides to cells in vitro and in vivo.

[0039] Poly(amine-co-ester)s or poly(amine-co-amide)s have been developed that have improved properties for delivery, including pulmonary delivery.

[0040] Poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] [In the formula, n is an integer of 1 to 30, m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; R x is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. Contains:

[0041] In some forms, Z is the same as Z'.

[0042] In some forms, Z is O and Z' is O. In some forms, Z is NR' and Z' is NR'. In some forms, Z is O and Z' is NR'. In some forms, Z is NR' and Z' is O.

[0043] In some embodiments, Z' is O and n is an integer from 1 to 24, for example, 4, 10, 13, or 14. In some embodiments, Z is also O.

[0044] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, and m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8. In some embodiments, Z is also O.

[0045] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and o and p are the same integer from 1 to 6, e.g., 2, 3, or 4. In some embodiments, Z is also O.

[0046] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and R is alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl, or aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, or xylyl. In some embodiments, Z is also O.

[0047] In some forms, n is 14 (e.g., pentadecalactone, PDL), m is 7 (e.g., sebacic acid), and o and p are 2 (e.g., N-methyldiethanolamine, MDEA).

[0048] In some embodiments, the polymer has Formula II: [ka] wherein Rx, Z, Z', m, n, o, p, q, x, and y are as described above for Formula I; J1 and J2 are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R3 and R4 in Formula II are independently a substituted alkyl containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. It contains the structure:

[0049] In some embodiments, J1 is -O- or -NH-.

[0050] In some forms, J2 is -C(O)-, -C(O)NH-, or -C(O)O-.

[0051] In some forms, R3 is the same as R4.

[0052] Preferably, R3 and / or R4 are linear.

[0053] In some embodiments, R3, R4, or both contain a hydroxyl group. In some embodiments, R3, R4, or both contain a hydroxyl group and one or more amine groups, preferably secondary amine groups or tertiary amine groups. In some embodiments, R3, R4, or both contain a hydroxyl group and no amine groups.

[0054] In some forms, at least one of R3 and R4 does not contain a hydroxyl group.

[0055] In some embodiments, the polymer has Formula III: [ka] wherein R, R, R, Z, Z', m, n, o, p, q, x, and y are as described above for Formula II. It contains the structure:

[0056] In certain embodiments, the values ​​of x, y, and / or q are such that the weight average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, or greater than 2,000 daltons. In some forms, the weight average molecular weight of the polymer is from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 10,000 daltons.

[0057] The polymers can be prepared from one or more lactones, one or more amine-diols (Z and Z' = O) or triamines (Z and Z' = NR'), and one or more diacids or diesters. In embodiments where two or more different lactones, diacids or diesters, and / or triamine or amine-diol monomers are used, the values ​​of n, o, p, and / or m can be the same or different.

[0058] In some embodiments, the polymer is between about 2 kDa and 20 kDa, or between about 2 kDa and about 10 kDa, or between about 2 kDa and about 5 kDa.

[0059] The poly(amine-co-ester) or poly(amine-co-amide) can be used to form polyplexes, microparticles, and / or nanoparticles in which one or more messenger ribonucleic acids are encapsulated. In some forms, the polyplexes or particles are formed by combining R1 and / or R2 with one or more of the following: [ka] The polymer is formed from a polymer that does not consist of or contain

[0060] In some forms, the poly(amine-co-ester) or poly(amine-co-amide) is in a mixture that contains a poly(amine-co-ester) or poly(amine-co-amide) conjugated to polyethylene glycol (PEG), i.e., a PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide).

[0061] In some forms, the PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] wherein m′ and m″ are independently 0 or 1, provided that m′+m″ is 1 or 2; J1 and J2 in Formula XI are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—. Contains:

[0062] In some forms of Formula XI, J1 is -O- or -NH-. In some forms of Formula XI, J2 is -C(O)-, -C(O)NH-, or -C(O)O-.

[0063] Delivery can be optimized by controlling the choice of polymer, blending or conjugating the polymer with hydrophilic polymers such as polyalkylene glycols, end groups and mixtures thereof, and molecular weight.

[0064] The examples demonstrate enhanced delivery and high levels of transfection after direct administration to cells, after systemic administration such as by injection, or after administration to the lungs (pulmonary delivery). PACE of defined molecular weights mixed or conjugated with PEG produced polyplexes containing mRNA with high levels of transfection in lung cells and little production in other tissues after pulmonary administration. [Brief explanation of the drawings]

[0065] [Figure 1-1] FIG. 1 shows the R3 and R4 groups that can be incorporated into Formula I. [Figure 1-2] FIG. 1 shows the R3 and R4 groups that can be incorporated into Formula I. [Figure 2] 2 is a bar graph showing the transfection efficiency of PACE-COOH and PACE-MAE with MW of either 5 kDa or 10 kDa, synthesized by the method described in Example 2. Statistical significance was determined by Student's t-test (denoted as follows: *, P<0.05; ***, P<0.001). [Figure 3-1] Figure 3A is a bar graph showing the effect of MW (average LUC per mg of protein) using classical PACE with 10% PDL content and molecular weights of 2 kDa, 5 kDa, 10 kDa, and 20 kDa. Figure 3B is a bar graph showing the effect of PDL content (average LUC per mg of protein) using a 10 kDa classical PACE polymer. Figure 3C is a bar graph showing the transfection efficiency (average LUC per mg of protein) of acidic and classical PACE with 5 kDa MW at 10% PDL content. Results are expressed as the mean ± SD of three independent experiments performed in duplicate. [Figure 3-2] Figure 3A is a bar graph showing the effect of MW (average LUC per mg of protein) using classical PACE with 10% PDL content and molecular weights of 2 kDa, 5 kDa, 10 kDa, and 20 kDa. Figure 3B is a bar graph showing the effect of PDL content (average LUC per mg of protein) using a 10 kDa classical PACE polymer. Figure 3C is a bar graph showing the transfection efficiency (average LUC per mg of protein) of acidic and classical PACE with 5 kDa MW at 10% PDL content. Results are expressed as the mean ± SD of three independent experiments performed in duplicate. [Figure 4] FIG. 4 is a line graph showing the evolution of the weight average MW (Da) of the aPACE polymer during the activation process (activation time in days) (5 kDa—lower line, 10 kDa—middle line, 20 kDa—upper line). [Figure 5-1]Figure 5A is a line graph showing luciferase mRNA transfection efficiency (average LUC per mg of protein) as a function of prime time (days), demonstrating the preferred prime time for each MW (lines identified as 20 kDa - 0 days, lower data point; 10 kDa - 0 days, middle data point; 5 kDa - 0 days, upper data point). Figure 5B is a bar graph showing transfection efficiency (average LUC per mg of protein) using non-priming PACE and primed PACE of different initial MW with their preferred prime times (5 days for 5 kDa polymers, 10 days for 10 kDa polymers, and 30 days for 20 kDa polymers, **p<0.005). Figure 5C is a line graph showing the cytotoxicity profile (cell viability (%)) of mRNA:aPACE polyplexes (5 kDa 5 days, 10 kDa 10 days, 20 kDa 30 days (cluster of upper lines)) compared to mRNA:TransIT complexes (lower lines). Figure 5D is a bar graph of the transfection efficiency (average LUC per mg of protein) of aPACE using different activation temperatures (**p<0.005). All results are expressed as the mean ± SD of three independent experiments performed in duplicate. [Figure 5-2]Figure 5A is a line graph showing luciferase mRNA transfection efficiency (average LUC per mg of protein) as a function of prime time (days), demonstrating the preferred prime time for each MW (lines identified as 20 kDa - 0 days, lower data point; 10 kDa - 0 days, middle data point; 5 kDa - 0 days, upper data point). Figure 5B is a bar graph showing transfection efficiency (average LUC per mg of protein) using non-priming PACE and primed PACE of different initial MW with their preferred prime times (5 days for 5 kDa polymers, 10 days for 10 kDa polymers, and 30 days for 20 kDa polymers, **p<0.005). Figure 5C is a line graph showing the cytotoxicity profile (cell viability (%)) of mRNA:aPACE polyplexes (5 kDa 5 days, 10 kDa 10 days, 20 kDa 30 days (cluster of upper lines)) compared to mRNA:TransIT complexes (lower lines). Figure 5D is a bar graph of the transfection efficiency (average LUC per mg of protein) of aPACE using different activation temperatures (**p<0.005). All results are expressed as the mean ± SD of three independent experiments performed in duplicate. [Figure 5-3]Figure 5A is a line graph showing luciferase mRNA transfection efficiency (average LUC per mg of protein) as a function of prime time (days), demonstrating the preferred prime time for each MW (lines identified as 20 kDa - 0 days, lower data point; 10 kDa - 0 days, middle data point; 5 kDa - 0 days, upper data point). Figure 5B is a bar graph showing transfection efficiency (average LUC per mg of protein) using non-priming PACE and primed PACE of different initial MW with their preferred prime times (5 days for 5 kDa polymers, 10 days for 10 kDa polymers, and 30 days for 20 kDa polymers, **p<0.005). Figure 5C is a line graph showing the cytotoxicity profile (cell viability (%)) of mRNA:aPACE polyplexes (5 kDa 5 days, 10 kDa 10 days, 20 kDa 30 days (cluster of upper lines)) compared to mRNA:TransIT complexes (lower lines). Figure 5D is a bar graph of the transfection efficiency (average LUC per mg of protein) of aPACE using different activation temperatures (**p<0.005). All results are expressed as the mean ± SD of three independent experiments performed in duplicate. [Figure 6-1]Figure 6A is a bar graph showing EPO blood concentrations 6 hours after IV administration of mRNA (20 mg total) using TransIT, 5 kDa non-activated PACE, 5-day activated 5 kDa aPACE, 10 kDa non-activated PACE, or 10-day activated 10 kDa aPACE. Results are presented as mean ± SD for N = 3 animals (****p < 0.0001). Figure 6B is a line graph showing the time course of EPO production after IV administration of mRNA (20 mg total) using vehicle (lower line), TransIT (middle line), or 10-day activated 10 kDa aPACE (upper line). Results are presented as mean ± SD for N = 3 animals (***p < 0.001 and *p < 0.05). Figures 6C-6F are bar graphs showing blood chemistry (AST (Figure 6C), ALT (Figure 6D), urea (Figure 6D), creatinine (Figure 6F)) analysis 24 h and 7 days after IV administration of acetate buffer, free mRNA, or mRNA:aPACE polyplexes. Results are presented as mean ± SEM for N = 3 animals. [Figure 6-2] Figure 6A is a bar graph showing EPO blood concentrations 6 hours after IV administration of mRNA (20 mg total) using TransIT, 5 kDa non-activated PACE, 5-day activated 5 kDa aPACE, 10 kDa non-activated PACE, or 10-day activated 10 kDa aPACE. Results are presented as mean ± SD for N = 3 animals (****p < 0.0001). Figure 6B is a line graph showing the time course of EPO production after IV administration of mRNA (20 mg total) using vehicle (lower line), TransIT (middle line), or 10-day activated 10 kDa aPACE (upper line). Results are presented as mean ± SD for N = 3 animals (***p < 0.001 and *p < 0.05). Figures 6C-6F are bar graphs showing blood chemistry (AST (Figure 6C), ALT (Figure 6D), urea (Figure 6D), creatinine (Figure 6F)) analysis 24 h and 7 days after IV administration of acetate buffer, free mRNA, or mRNA:aPACE polyplexes. Results are presented as mean ± SEM for N = 3 animals. [Figure 6-3]Figure 6A is a bar graph showing EPO blood concentrations 6 hours after IV administration of mRNA (20 mg total) using TransIT, 5 kDa non-activated PACE, 5-day activated 5 kDa aPACE, 10 kDa non-activated PACE, or 10-day activated 10 kDa aPACE. Results are presented as mean ± SD for N = 3 animals (****p < 0.0001). Figure 6B is a line graph showing the time course of EPO production after IV administration of mRNA (20 mg total) using vehicle (lower line), TransIT (middle line), or 10-day activated 10 kDa aPACE (upper line). Results are presented as mean ± SD for N = 3 animals (***p < 0.001 and *p < 0.05). Figures 6C-6F are bar graphs showing blood chemistry (AST (Figure 6C), ALT (Figure 6D), urea (Figure 6D), creatinine (Figure 6F)) analysis 24 h and 7 days after IV administration of acetate buffer, free mRNA, or mRNA:aPACE polyplexes. Results are presented as mean ± SEM for N = 3 animals. [Figure 6-4] Figure 6A is a bar graph showing EPO blood concentrations 6 hours after IV administration of mRNA (20 mg total) using TransIT, 5 kDa non-activated PACE, 5-day activated 5 kDa aPACE, 10 kDa non-activated PACE, or 10-day activated 10 kDa aPACE. Results are presented as mean ± SD for N = 3 animals (****p < 0.0001). Figure 6B is a line graph showing the time course of EPO production after IV administration of mRNA (20 mg total) using vehicle (lower line), TransIT (middle line), or 10-day activated 10 kDa aPACE (upper line). Results are presented as mean ± SD for N = 3 animals (***p < 0.001 and *p < 0.05). Figures 6C-6F are bar graphs showing blood chemistry (AST (Figure 6C), ALT (Figure 6D), urea (Figure 6D), creatinine (Figure 6F)) analysis 24 h and 7 days after IV administration of acetate buffer, free mRNA, or mRNA:aPACE polyplexes. Results are presented as mean ± SEM for N = 3 animals. [Figure 7]FIG. 7 is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Example 5) on mRNA loading on polyplexes. [Figure 8-1] Figure 8A is a schematic diagram of the mechanism that produces the results shown in Figures 8B-8D. Figure 8B is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on cellular uptake of polyplexes. Figure 8C is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on endosomal escape. Figure 8D is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on transfection efficiency. [Figure 8-2] Figure 8A is a schematic diagram of the mechanism that produces the results shown in Figures 8B-8D. Figure 8B is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on cellular uptake of polyplexes. Figure 8C is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on endosomal escape. Figure 8D is a bar graph showing the effect of PACE end groups (numbers correspond to the end groups identified in Table 1) on transfection efficiency. [Figure 9-1] Figures 9A-9C are plots showing the linear correlation between transfection efficiency (expressed as flux activity) and the different steps of transfection, including mRNA loading (Figure 9A), uptake (Figure 9B), and endosomal escape (Figure 9C). [Figure 9-2] Figures 9A-9C are plots showing the linear correlation between transfection efficiency (expressed as flux activity) and the different steps of transfection, including mRNA loading (Figure 9A), uptake (Figure 9B), and endosomal escape (Figure 9C). [Figure 10]Figures 10A-10B are graphs demonstrating that the in vitro translation rate of mRNA polyplexes does not correlate with transfection efficiency. Polyplexes containing mRNA, ddRLuc-Fc, and PACE are endocytosed by cells, and the mRNA encoding the luciferase gene is then released and translated. Figure 10A is a graph of the amount (log) of luciferase expression (expire) versus the translation rate (AU). Figure 10B is a graph of the translation rate (AU) versus the encapsulation efficiency. [Figure 11] Figure 11 is a graph of the transfection efficiency (percent) at pH 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, and 6.0 for polymers 2, 4, 14, 15, 16, 17, 20, 25, 27, and 31 in Table 1. The results demonstrate the optimal sodium acetate buffer pH for PACE polyplexes with different end groups. [Figure 12-1] Figure 12A is a graph of the effect of PEG on polyplex size compared to no PEG (showing aggregation over time (min) compared to no change in size over time with either 0.25% PACE-PEG or 1% PACE-PEG). Figure 12B is a graph of transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 from Table 1 as a function of PACE-PEG content (%) of 0, 0.05, 0.1.0, 0.25, 0.50, and 1. Figure 12C is a graph of EGFP expression for the same PACE-PEG mixtures, showing that PEG increased EGFP expression. [Figure 12-2]Figure 12A is a graph of the effect of PEG on polyplex size compared to no PEG (showing aggregation over time (min) compared to no change in size over time with either 0.25% PACE-PEG or 1% PACE-PEG). Figure 12B is a graph of transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 from Table 1 as a function of PACE-PEG content (%) of 0, 0.05, 0.1.0, 0.25, 0.50, and 1. Figure 12C is a graph of EGFP expression for the same PACE-PEG mixtures, showing that PEG increased EGFP expression. [Figure 13-1] Figure 13A is a graph of the effectiveness of 0%, 0.01%, 0.1%, 1%, or 10% DSPE-PEG on size (nm) and polydispersity. Figure 13B is a graph of the effective DSPE-PEG content on transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 in Table 1. Figure 13C is a graph of the effect of DSPE-PEG concentration (0, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, and 5%) on EGFP expression. [Figure 13-2] Figure 13A is a graph of the effectiveness of 0%, 0.01%, 0.1%, 1%, or 10% DSPE-PEG on size (nm) and polydispersity. Figure 13B is a graph of the effective DSPE-PEG content on transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 in Table 1. Figure 13C is a graph of the effect of DSPE-PEG concentration (0, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, and 5%) on EGFP expression. [Figure 14] Figures 14A and 14B are plots of data from IVIS images showing the biodistribution of polyplexes as measured by bioluminescence distribution in the heart, lungs, liver, kidneys, spleen, and intestine 6 hours after IP injection (Figure 12A) and 6 hours after IV injection (Figure 12B). [Figure 15]FIG. 15 is a graph of polyplex biodistribution as measured by bioluminescence distribution after pulmonary administration. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description of the Invention I. Definition The term "polyplex," as used herein, typically refers to polymeric microparticles and / or nanoparticles or micelles having one or more polynucleotides encapsulated therein, dispersed therein, and / or associated with their surface.

[0067] The term particle includes microspheres, microcapsules, microparticles, nanospheres, nanocapsules, and nanoparticles. The term "microparticle" includes microspheres and microcapsules, all of which have an average dimension of less than about 1000 microns. Microparticles may be spherical or non-spherical and may have any regular or irregular shape. When structures have a diameter of less than about 1 micron (1000 nm), the corresponding art-recognized terms "nanosphere," "nanocapsule," and "nanoparticle" may be utilized. In certain embodiments, populations of nanospheres, nanocapsules, and nanoparticles have an average diameter of about 500 nm, 200 nm, 100 nm, 50 nm, 10 nm, or 1 nm. In some embodiments, the average diameter of the particles is about 200 nm to about 600 nm, preferably about 200 to about 500 nm. The term "diameter" is used herein to refer to either the physical diameter or the hydrodynamic diameter. The diameter of an essentially spherical particle may refer to either the physical diameter or the hydrodynamic diameter. The diameter of non-spherical particle can refer primarily to hydrodynamic diameter.As used herein, the diameter of non-spherical particle can refer to the maximum linear distance between two points on the surface of particle.When referring to multiple particles, the diameter of particle typically refers to the average diameter of particle.Particle diameter can be measured using various techniques in the art, including but not limited to dynamic light scattering.

[0068] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0069] The term "biocompatible," as used herein, refers to one or more materials that are not themselves toxic to a host (e.g., an animal or human) or that do not degrade (if they degrade) at a rate that produces toxic concentrations of monomeric or oligomeric subunits or other by-products in the host.

[0070] The term "biodegradable," as used herein, means that a material is capable of being broken down or broken down into its component subunits, or, for example, by digestion of the material by biochemical processes into smaller (e.g., non-polymeric) subunits.

[0071] "Sustained release," as used herein, refers to the release of a substance over an extended period of time, as opposed to bolus-type administration, in which the entire amount of the substance is made bioavailable at once.

[0072] The phrases "parenteral administration" and "parenterally administered" are art-recognized terms and include modes of administration other than enteral and topical administration, such as injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0073] The term "surfactant," as used herein, refers to an agent that reduces the surface tension of a liquid.

[0074] As used herein, "transient" refers to expression of a non-integrated transgene over a period of hours, days, or weeks, where the period of expression is shorter than the period for expression of a gene when integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0075] As used herein, a "promoter site" refers to a region of a gene encoding a promoter that is capable of expressing a DNA-dependent RNA polymerase, such as the DNA-dependent RNA polymerase originally isolated from a bacteriophage described by Davanloo, et al., Proc. Natl. Acad. Sci. USA, 81:2035-39 (1984), or from another source, as described by Chamberlin, et al., Nature, 228:227-231 (1970). As described above, it is a sequence of nucleotides that binds with high specificity.

[0076] As used herein, "poly(A)" refers to a series of adenosines attached to an mRNA by polyadenylation. In preferred embodiments of constructs for transient expression, the poly(A) is 50 to 5000, preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functionality, such as localization, stability, or translation efficiency.

[0077] As used herein, an "open reading frame" or "ORF" is a series of nucleotides containing a sequence of bases that can potentially encode a polypeptide or protein. An open reading frame is located between an initiation coding sequence (initiation codon or start codon) and a termination codon sequence (stop codon).

[0078] The term "construct" refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences.

[0079] The term "expression control sequence" refers to a nucleic acid sequence that controls and regulates the transcription and / or translation of another nucleic acid sequence. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome binding site, etc. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0080] The term "gene" refers to a DNA sequence that, through its template or messenger RNA, codes for a sequence of amino acids characteristic of a particular peptide, polypeptide, or protein. The term "gene" also refers to a DNA sequence that codes for an RNA product. When used herein with reference to genomic DNA, the term gene includes intervening non-coding and regulatory regions and can include a 5' end and a 3' end.

[0081] The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell in which they are utilized, such as human polypeptides produced by bacterial cells. Polypeptides contain as amino acids. Amino acid sequences are written from left to right in the direction from amino to carboxy terminus and are represented by either a three-letter or one-letter code, as indicated below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0082] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. A variant and a reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitution, addition, and / or deletion). The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be a naturally occurring one, such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0083] Modifications and changes can be made in the structure of a polypeptide that do not significantly alter its characteristics (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without appreciable loss of activity. Because it is the interactive ability and characteristics of a polypeptide that define its biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence to still obtain a polypeptide with similar properties. When making such changes, the hydropathic index of the amino acid can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function in a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indexes or scores and still result in polypeptides with similar biological activity. Substitutions of similar amino acids can also be made based on hydrophilicity, particularly when the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, and size.

[0084] The terms "lactone" and "lactone unit" are used to describe compounds containing cyclic esters or the open-chain chemical structure resulting from the cleavage of the ester bond in a cyclic ester. For example, lactones can be derived from the cyclic esters shown below, and the corresponding lactone-derived open-chain structures: [ka] [n is an integer] The term "chain structure" is used to describe the chain structure formed via methods known in the art, including but not limited to solvolysis, e.g., hydrolysis, and enzymatic cleavage.

[0085] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0086] In preferred embodiments, a straight or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1 to C6 for a straight chain). 30 , for branched chains C3 to C 30 ), preferably having 20 or fewer, more preferably 15 or fewer, and most preferably 10 or fewer carbon atoms. All integer values ​​for the number of skeletal carbon atoms between 1 and 30 are contemplated and disclosed for straight or branched chain alkyls. Likewise, preferred cycloalkyls have from 3 to 10 carbon atoms in their ring structure, and more preferably have 5, 6, or 7 carbons in the ring structure. All integer values ​​for the number of ring carbon atoms between 3 and 10 are contemplated and disclosed for cycloalkyls.

[0087] The term "alkyl" (or "lower alkyl"), as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties.

[0088] Unless the number of carbons is otherwise specified, "lower alkyl," as used herein, refers to an alkyl group, as defined above, but having from 1 to 10 carbons, more preferably 1 to 6 carbon atoms, in its backbone structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this application, preferred alkyl groups are lower alkyls. In preferred embodiments, substituents designated herein as alkyl are lower alkyls.

[0089] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, substituents on substituted alkyls can include halogen, hydroxy, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Cycloalkyls can also be substituted in the same manner.

[0090] "Aryl," as used herein, refers to a C5-C 10 refers to a 1-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring system. In some embodiments, the ring system has 3 to 50 carbon atoms. The broad definition of "aryl" as used herein includes 5-, 6-, 7-, 8-, 9-, 10-, and 24-membered monocyclic aromatic groups that may contain 0 to 4 heteroatoms, such as benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. These aryl groups with heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring may be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, -CF3, -CN; and combinations thereof.

[0091] The term "aryl" also includes polycyclic ring systems (i.e., "fused rings") having two or more cyclic rings that have two or more carbons in common with two adjacent rings, where at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic.Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, Furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl , 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindryl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl Examples include thiadiazinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolidinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.One or more of the rings may be substituted as defined above for "aryl."

[0092] "Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, n-pentoxy, s-pentoxy, and derivatives thereof.

[0093] Primary amines occur when one of the three hydrogen atoms in ammonia is replaced by a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group. Secondary amines have two organic substituents (substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or a combination thereof) attached to the nitrogen, along with one hydrogen. In tertiary amines, the nitrogen has three organic substituents.

[0094] As used herein, "substituted" means that one or more atoms or groups of atoms on a monomer are replaced with one or more atoms or groups of atoms that are different from the atoms or groups of atoms being replaced. In some embodiments, one or more hydrogen atoms on a monomer are replaced with one or more atoms or groups of atoms. Examples of functional groups that can replace hydrogen atoms are listed above in the definitions. In some embodiments, one or more functional groups can be added to change the chemical and / or physical properties of the resulting monomer / polymer, such as charge or hydrophilicity / hydrophobicity. Exemplary substituents include, but are not limited to, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, nitro, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0095] II. Polymers Polymers comprising poly(amine-co-esters), poly(amine-co-amides), or combinations thereof, and polyplexes and solid core particles formed therefrom. Poly(amine-co-esters) are discussed in WO 2013 / 082529, WO 2017 / 151623, WO 2017 / 197128, U.S. Published Application No. 2016 / 0251477, U.S. Published Application No. 2015 / 0073041, and U.S. Patent No. 9,272,043.

[0096] When used to deliver genetic material, the transfection efficiency of polymers is strongly dependent on the end groups on the polymer. Replacing the diester monomers in a polymer with a diacid, such as sebacic acid, can yield a polymer with a mixture of hydroxyl and carboxyl end groups. Both of these end groups can be activated with 1,1'-carbodiimidazole. The activated product can then be reacted with an amine-containing molecule to yield a polymer with the new end groups.

[0097] The polymer can be further hydrolyzed to release more reactive end groups such as -OH and -COOH, both of which can typically result from hydrolysis of ester bonds in the polymer (also referred to herein as "activation"), for example, by incubating the polymer at a controlled temperature (e.g., 37°C or 100°C) for days or weeks. In some embodiments, the polymer is not hydrolyzed and thus can be referred to as "non-activation."

[0098] In some embodiments, the content of hydrophobic monomers in a polymer is increased relative to the content of the same hydrophobic monomers when used to form polyplexes. Increasing the content of hydrophobic monomers in a polymer results in a polymer that is capable of forming solid-core nanoparticles in the presence of nucleic acids, including RNA.

[0099] Unlike polyplexes, these particles are stable for extended periods of time during incubation in buffered water or serum, or upon administration (e.g., injection) to animals. They also provide sustained release of nucleic acids (e.g., siRNA) resulting in long-term activity (e.g., siRNA-mediated knockdown).

[0100] A. Polymer Structure The poly(amine-co-ester) or poly(amine-co-amide) can be represented by Formula I: [ka] [In the formula, n is an integer of 1 to 30, m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; R x is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. It has the structure shown as:

[0101] R x Examples of R' groups include, but are not limited to, hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, xylyl, and the like.

[0102] In certain embodiments, the values ​​of x, y, and / or q are such that the weight average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, or greater than 2,000 daltons. In some forms, the weight average molecular weight of the polymer is from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 10,000 daltons.

[0103] The polymers can be prepared from one or more lactones, one or more amine-diols (Z and Z'=O), triamines (Z and Z'=NR'), or hydroxy-diamines (Z=O and Z'=NR', or Z=NR' and Z'=O), and one or more diacids or diesters. In embodiments in which two or more different lactones, diacids or diesters, and / or triamines, amine-diols, or hydroxy-diamine monomers are used, the values ​​of n, o, p, and / or m can be the same or different.

[0104] In some embodiments, the percent composition of lactone units is about 10% to about 100%, calculated as lactone units to (lactone units + diester / diacid). Expressed in terms of molar ratio, the lactone unit to (lactone units + diester / diacid) content is about 0.1 to about 1, i.e., x / (x+q) is about 0.1 to about 1. Preferably, the number of carbon atoms in the lactone units is about 10 to about 24, more preferably about 12 to about 16. Most preferably, the number of carbon atoms in the lactone units is 12 (dodecalactone), 15 (pentadecalactone), or 16 (hexadecalactone). In some embodiments, Z is the same as Z'.

[0105] In some forms, Z is O and Z' is O. In some forms, Z is NR' and Z' is NR'. In some forms, Z is O and Z' is NR'. In some forms, Z is NR' and Z' is O.

[0106] In some embodiments, Z' is O and n is an integer from 1 to 24, for example, 4, 10, 13, or 14. In some embodiments, Z is also O.

[0107] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, and m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8. In some embodiments, Z is also O.

[0108] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and o and p are the same integer from 1 to 6, e.g., 2, 3, or 4. In some embodiments, Z is also O.

[0109] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and R is alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl, or aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, or xylyl. In some embodiments, Z is also O.

[0110] In some forms, n is 14 (e.g., pentadecalactone, PDL), m is 7 (e.g., sebacic acid), and o and p are 2 (e.g., N-methyldiethanolamine, MDEA).

[0111] In some embodiments, the polyplexes or particles are formed from polymers where R1 and / or R2 are not associated with the corresponding polyplex, and where R1 and / or R2 are: [ka] consisting of or including.

[0112] In some embodiments, the polyplexes or particles formed from the polymers are those in which R1 and / or R2 are: [ka] and (iii) exhibit improved loading, improved cellular transfection, improved intracellular endosomal release, or a combination thereof, of nucleic acid cargo, e.g., RNA, more particularly, mRNA, compared to a corresponding polyplex consisting of or comprising:

[0113] In some forms, the polymer has a structure of Formula II: [ka] wherein J1 and J2 are independently a linking moiety or absent; R3 and R4 are independently substituted alkyls containing hydroxyl groups, primary amine groups, secondary amine groups, tertiary amine groups, or combinations thereof. In some forms, the molecular weight of R3, R4, or both is 500 daltons or less, 200 daltons or less, or 100 daltons or less.

[0114] In some embodiments, J1 is -O- or -NH-.

[0115] In some forms, J2 is -C(O)NH- or -C(O)O-.

[0116] In some forms, R3 is the same as R4.

[0117] Preferably, R3 and / or R4 are linear.

[0118] In some forms, R3, R4, or both contain a primary amine group. In some forms, R3, R4, or both contain a primary amine group and one or more secondary or tertiary amine groups.

[0119] In some embodiments, R3, R4, or both contain a hydroxyl group. In some embodiments, R3, R4, or both contain a hydroxyl group and one or more amine groups, preferably secondary amine groups or tertiary amine groups. In some embodiments, R3, R4, or both contain a hydroxyl group and no amine groups.

[0120] In some forms, at least one of R3 and R4 does not contain a hydroxyl group.

[0121] In some forms, R, R, or both are -unsubstituted C-C 10 Alkylene-Aq-Unsubstituted C1-C 10 Alkylene-Bq, -unsubstituted C1-C 10 Alkylene-Aq-substituted C1-C 10 Alkylene-Bq, -substituted C1-C 10 Alkylene-Aq-Unsubstituted C1-C 10 Alkylene-Bq or -substituted C1-C 10 Alkylene-Aq-substituted C1-C 10 Alkylene-Bq (wherein Aq is absent or -NR5-, Bq is hydroxyl, primary amine, secondary amine, or tertiary amine, and R5 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl).

[0122] In some forms, R3, R4, or both are selected from the groups shown in Figure 1.

[0123] In some forms, the polymer has the structure of Formula III: [ka]

[0124] The monomer units can be substituted at one or more positions with one or more substituents. Exemplary substituents include, but are not limited to, alkyl groups, cyclic alkyl groups, alkene groups, cyclic alkene groups, alkynes, halogens, hydroxyl, carbonyls (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, nitro, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties.

[0125] The polymers are biocompatible. Readily available lactones of various ring sizes are known to have low toxicity; for example, polyesters prepared from small lactones, such as poly(caprolactone) and poly(p-dioxanone), are commercially available biomaterials used in clinical applications. Larger (e.g., C 16 ~C 24 ) Lactones and their polyester derivatives are natural products that have been identified in organisms such as bees. Lactones containing 16 to 24 ring carbon atoms are specifically contemplated and disclosed.

[0126] In some embodiments, the polymer can be further activated via temperature-controlled hydrolysis, thereby exposing one or more activated end groups. The one or more activated end groups can be, for example, hydroxyl or carboxylic acid end groups, both of which can arise via hydrolysis of ester bonds within the polymer. The activated polymer can have a weight-average molecular weight of about 5 to 25 kDa, preferably about 5 to 10 kDa. As used herein, the term "about" refers to slight variations within acceptable parameters. For clarity, "about" refers to ±10% of a given value. In some embodiments, the activated polymer contains R1 or R2 at one end and a hydroxyl or carboxylic acid end group at the other end, arising via hydrolysis.

[0127] In some forms, the polymer has the structure of Formula IV: [ka]

[0128] In some forms, the polymer has the structure of Formula V: [ka]

[0129] In some forms, the polymer has the structure of Formula VI: [ka] wherein X' is -OH or -NHR'.

[0130] Formulas VI, V, and VI are structures of intermediate products that can be used to synthesize a wide variety of polymers having the structures of Formulas I, II, or III.

[0131] Poly(amine-co-ester)s or poly(amine-co-amide)s have been developed that have improved properties for delivery, including pulmonary delivery.

[0132] Poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] [In the formula, n is an integer of 1 to 30, m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; R x is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. Contains:

[0133] In some forms, Z is the same as Z'.

[0134] In some forms, Z is O and Z' is O. In some forms, Z is NR' and Z' is NR'. In some forms, Z is O and Z' is NR'. In some forms, Z is NR' and Z' is O.

[0135] In some embodiments, Z' is O and n is an integer from 1 to 24, for example, 4, 10, 13, or 14. In some embodiments, Z is also O.

[0136] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, and m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8. In some embodiments, Z is also O.

[0137] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and o and p are the same integer from 1 to 6, e.g., 2, 3, or 4. In some embodiments, Z is also O.

[0138] In some embodiments, Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13, or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7, or 8, and R is alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl, or aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, or xylyl. In some embodiments, Z is also O.

[0139] In some forms, n is 14 (e.g., pentadecalactone, PDL), m is 7 (e.g., sebacic acid), and o and p are 2 (e.g., N-methyldiethanolamine, MDEA).

[0140] In some embodiments, the polymer has Formula II: [ka] wherein Rx, Z, Z', m, n, o, p, q, x, and y are as described above for Formula I; J1 and J2 are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R3 and R4 in Formula II are independently a substituted alkyl containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. It contains the structure:

[0141] In some embodiments, J1 is -O- or -NH-.

[0142] In some forms, J2 is -C(O)-, -C(O)NH-, or -C(O)O-.

[0143] In some forms, R3 is the same as R4.

[0144] Preferably, R3 and / or R4 are linear.

[0145] In some embodiments, R3, R4, or both contain a hydroxyl group. In some embodiments, R3, R4, or both contain a hydroxyl group and one or more amine groups, preferably secondary amine groups or tertiary amine groups. In some embodiments, R3, R4, or both contain a hydroxyl group and no amine groups.

[0146] In some forms, at least one of R3 and R4 does not contain a hydroxyl group.

[0147] In some embodiments, the polymer has Formula III: [ka] wherein R, R, R, Z, Z', m, n, o, p, q, x, and y are as described above for Formula II. It contains the structure:

[0148] In certain embodiments, the values ​​of x, y, and / or q are such that the weight average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, or greater than 2,000 daltons. In some forms, the weight average molecular weight of the polymer is from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 10,000 daltons.

[0149] The polymers can be prepared from one or more lactones, one or more amine-diols (Z and Z' = O) or triamines (Z and Z' = NR'), and one or more diacids or diesters. In embodiments where two or more different lactones, diacids or diesters, and / or triamine or amine-diol monomers are used, the values ​​of n, o, p, and / or m can be the same or different.

[0150] In some embodiments, the polymer is between about 2 kDa and 20 kDa, or between about 2 kDa and about 10 kDa, or between about 2 kDa and about 5 kDa.

[0151] The poly(amine-co-ester) or poly(amine-co-amide) can be used to form polyplexes, microparticles, and / or nanoparticles in which one or more messenger ribonucleic acids are encapsulated. In some forms, the polyplexes or particles are formed by combining R1 and / or R2 with one or more of the following: [ka] The polymer is formed from a polymer that does not consist of or contain

[0152] In some forms, the poly(amine-co-ester) or poly(amine-co-amide) is in a mixture that contains a poly(amine-co-ester) or poly(amine-co-amide) conjugated to polyethylene glycol (PEG), i.e., a PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide).

[0153] In some forms, the PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] wherein m′ and m″ are independently 0 or 1, provided that m′+m″ is 1 or 2; J1 and J2 in Formula XI are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—. Contains:

[0154] In some forms of Formula XI, J1 is -O- or -NH-. In some forms of Formula XI, J2 is -C(O)-, -C(O)NH-, or -C(O)O-.

[0155] B. Methods for Making Polymers The polymer is generally modified from a synthetic polymer. Exemplary synthetic polymers include poly(amine-co-esters) formed from lactones, dialkyl acids, and dialkylamines. Also provided is a method for synthesizing poly(amine-co-esters) from lactones, dialkyl acids, and dialkylamines using an enzyme catalyst such as lipase. Exemplary lactones are disclosed in U.S. Patent Publication No. 20170121454. In some embodiments, poly(amine-co-esters) are prepared as shown in Scheme 1. Scheme 1: Preparation of unmodified poly(amine-co-ester) [ka]

[0156] The molar ratio of monomers (e.g., lactone:aminodiol:diacid) can vary, for example, from about 10:90:90 to about 90:10:10. In some embodiments, the ratio is 10:90:90, 20:80:80, 40:60:60, 60:40:40, or 80:20:20. The weight average molecular weight, as determined by GPC using narrow polydispersity polystyrene standards, can vary, for example, from about 2,000 daltons to about 50,000 daltons, preferably from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 20,000 daltons, and most preferably from about 5,000 daltons to about 10,000 daltons.

[0157] The hydrophobicity of the polymer can be adjusted by varying the percentage of lactone, for example, from about 10% to about 100% (calculated as lactone units to (lactone units + diester / diacid)). The molecular weight of the polymer can be adjusted by adjusting the reaction time of the second stage, for example, from about 8 hours to about 72 hours.

[0158] Enzymatic methods allow for the synthesis of polymers with diverse chain architectures and tunable hydrophobicity. In some embodiments, hydrophobicity is varied by varying the ring size and / or molar amount of the lactone monomer. Lactones with a wide range of ring sizes (e.g., C4-C6) can be used. 24 , preferably C6 to C 24 , more preferably C6 to C 16 ) can be used as a comonomer. The reaction can be carried out in a single step without protection and deprotection of the amino group. Such amino-bearing copolyesters are extremely difficult to prepare using conventional organometallic catalysts because they are often sensitive to or inactivated by organic amines. These catalysts are also known to be inefficient in polymerizing large lactone ring monomers. Enzyme catalysts have distinct advantages for producing biomedical polymers due to the high activity and selectivity of the enzymes and the resulting high-purity, metal-free products.

[0159] Polymers having the structure of Formula IV, V, or VI can be synthesized by reacting the unmodified polymer of Formula VII with 1,1'-carbonyldiimidazole (CDI) in a molar ratio of about 1:10 to about 1:60, preferably about 1:40.

[0160] Polymers having the structure of Formula I or II can be obtained by modifying the end group of an unmodified polymer of Formula VII using coupling reactions known in the art. For example, a polymer having the structure of Formula III can be synthesized by (1) reacting an unmodified polymer of Formula VII with CDI to obtain a polymer of Formula IV, and (2) reacting the polymer of Formula IV with R3-NH2 and R4-NH2. In some embodiments, R3, R4, or both are selected from those shown in Figure 1. Preferably, R3 and R4 are the same.

[0161] Alternatively, a polymer having the structure of Formula III can be synthesized via the steps of: (1) reacting an unmodified polymer of Formula VII with CDI to obtain a polymer of Formula V or VI; (2) protecting the -COOH or -X' groups in the polymer from step (1); (3) reacting the protected polymer from step (2) with R4-NH2 or R3-NH2; (4) deprotecting the -COOH or -X' groups in the polymer from step (3); and (5) reacting the deprotected polymer from step (4) with R3-NH2 or R4-NH2.

[0162] Hydrolysis-mediated activation of polymers of Formula I, II, or III can be carried out in a temperature-controlled manner for up to 30 days or longer. The length of hydrolysis can vary depending on the molecular weight of the polymer being activated. Larger molecular weight polymers (e.g., about 20-25 kDa) are optimally hydrolyzed for longer periods, e.g., about 30-40 days. Smaller molecular weight polymers (e.g., about 5-7 kDa) are optimally hydrolyzed for shorter periods, e.g., about 4-10 days.

[0163] In some forms, the polymer is hydrolyzed at a temperature anywhere from about 30° C. to 42° C., or up to about 100° C. PACE polymers can be hydrolyzed at a temperature of about 35° C. to 40° C., for example, about 37° C.

[0164] In some forms, the polymer is hydrolyzed, for example, at about 1 atm. Higher pressures accelerate the process (e.g., pressures of about 1 to about 100 atm). The rate for the process will be determined by one of skill in the art for the particular formulation being made.

[0165] The weight average molecular weight of the resulting hydrolysis product can vary from about 5 kDa to about 25 kDa, preferably from about 5 to about 10 kDa.

[0166] Preferably, one or more of the ester bonds in the polymer are hydrolyzed. The hydrolysis product may have R1 or R2 at one end and a carboxyl or hydroxyl group generated through hydrolysis at the other end.

[0167] PEG-conjugated poly(amine-co-ester)s or poly(amine-co-amide)s can be synthesized under conditions similar to those described in Scheme 1, except that PEG containing terminal carboxyl, hydroxyl, or amine groups can be added to the reaction in addition to (i) lactones, (ii) diacids / diesters, and (iii) amine diols and / or triamines.

[0168] III. Microparticles formed from polymers The polymers can be used to prepare microparticles and / or nanoparticles having one or more therapeutic, diagnostic, or prophylactic agents encapsulated therein. The agents can be encapsulated within the particle, dispersed within the polymer matrix forming the particle, covalently or noncovalently associated with the surface of the particle, or a combination thereof.

[0169] In other embodiments, the polymer is biocompatible and biodegradable. The nucleic acid encapsulated by and / or associated with the particles can be released through different mechanisms, including diffusion and degradation of the polymer matrix. The release rate can be controlled by changing the monomer composition of the polymer and therefore the rate of degradation. For example, if simple hydrolysis is the main mechanism of degradation, increasing the hydrophobicity of the polymer can slow the rate of degradation and therefore increase the duration of release. In all cases, the polymer composition is selected so that an effective amount of nucleic acid is released to achieve the desired purpose / result.

[0170] The polymers can be used to encapsulate, mix with, or ionically or covalently couple any of a variety of therapeutic, prophylactic, or diagnostic agents. A wide variety of biologically active materials can be encapsulated or incorporated into the polymer for delivery to a site or to impart properties to the polymer, such as bioadhesion, cell attachment, enhanced cell growth, inhibiting bacterial growth, and preventing blood clot formation.

[0171] In some forms, the agent to be encapsulated and delivered can be a small molecule agent (i.e., a non-polymeric agent having a molecular weight of less than 2,000, 1500, 1,000, 750, or 500 daltons), or a macromolecule (e.g., an oligomer or polymer), such as a protein, peptide, nucleic acid, etc. Suitable small molecule active agents include organic compounds, inorganic compounds, and / or organometallic compounds. The particles can be used for in vivo and / or in vitro delivery of agents.

[0172] Examples of suitable therapeutic and prophylactic agents include synthetic inorganic and organic compounds, proteins and peptides, polysaccharides and other sugars, lipids, and DNA and RNA nucleic acid sequences with therapeutic, prophylactic, or diagnostic activity. Nucleic acid sequences include genes, antisense molecules that bind to complementary DNA and inhibit transcription, and ribozymes. Compounds with a wide range of molecular weights, for example, from 100 to 500,000 grams per mole or more, can be encapsulated. Examples of suitable materials include proteins such as antibodies, receptor ligands, and enzymes, peptides such as adhesion peptides, sugars and polysaccharides, synthetic organic or inorganic drugs, and nucleic acids. Examples of materials that can be encapsulated include enzymes, blood clotting factors, inhibitors or clot-dissolving agents such as streptokinase and tissue plasminogen activator; antigens for immunization; hormones and growth factors; polysaccharides such as heparin; oligonucleotides such as antisense oligonucleotides and ribozymes; and retroviral vectors for use in gene therapy. Polymers can also be used to encapsulate cells and tissues.

[0173] Exemplary therapeutic agents that can be incorporated into the particles include, but are not limited to, tumor antigens, CD4+ T cell epitopes, cytokines, chemotherapeutic agents, radionuclides, small molecule signaling inhibitors, photothermal antennas, monoclonal antibodies, immunological danger signals, and the like. immunologic danger signaling molecules, other immunotherapeutics, enzymes, antibiotics, antivirals (particularly protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic agents (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized antibodies, single chain antibodies, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, immunomodulatory agents (ligands that bind to toll-like receptors that activate the innate immune system, and antibodies that stimulate the adaptive immune system) These include molecules that recruit and optimize immune responses, molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and molecules that inactivate or downregulate suppressor or regulatory T cells, agents that promote particle uptake into cells (including dendritic cells and other antigen-presenting cells), dietary supplements such as vitamins, and oligonucleotide drugs (including DNA, RNA, antisense, aptamers, small interfering RNA, ribozymes, external guide sequences for RNase P, and triplex formers).

[0174] Representative anti-cancer agents include, but are not limited to, alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), mitotic inhibitors (taxanes, e.g., paclitaxel and docetaxel, and vinca alkaloids, e.g., vincristine, vinblastine, anti-VEGF compounds; anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycins, e.g., actinomycin D); cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin); topoisomerase inhibitors (including camptothecins, e.g., camptothecin, irinotecan, and topotecan, and derivatives of epipodophyllotoxins, e.g., amsacrine, etoposide, etoposide phosphate, and teniposide); antibodies against vascular endothelial growth factor (VEGF), e.g., bevacizumab (AVASTIN®), other anti-VEGF compounds; thalid angiostatin; receptor tyrosine kinase (RTK) inhibitors, such as sunitinib (SUTENT®); tyrosine kinase inhibitors, such as sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib; transforming growth factor alpha or transforming growth factor beta inhibitors, and antibodies against the epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

[0175] Exemplary immunomodulatory agents include cytokines, xanthines, interleukins, interferons, oligodeoxynucleotides, glucans, growth factors (e.g., TNF, CSF, GM-CSF and G-CSF), hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA® (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

[0176] Examples of immunological adjuvants that can be associated with the particles include, but are not limited to, TLR ligands, C-type lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands.TLR ligands can include lipopolysaccharide (LPS) and its derivatives, and lipid A and its derivatives, such as, but not limited to, monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'-monophosphoryl lipid A.

[0177] Particles may also contain antigens and / or adjuvants (i.e., molecules that enhance the immune response). Peptide-, protein-, and DNA-based vaccines can be used to induce immunity against various diseases or conditions. Cellular immunity is necessary for detecting and destroying virus-infected cells. Most conventional vaccines (e.g., protein-based vaccines) can only induce humoral immunity. DNA-based vaccines can induce both humoral and cellular immunity, making them a unique means of vaccinating against viruses or parasites. In addition, DNA-based vaccines are potentially safer than conventional vaccines. DNA vaccines are relatively more stable and cost-effective for production and storage. DNA vaccines consist of two main components: a DNA carrier (or delivery vehicle) and DNA encoding the antigen. The DNA carrier can protect the DNA from degradation and facilitate DNA entry into specific tissues or cells and efficient expression.

[0178] Representative diagnostic agents are agents that can be detected by X-ray, fluorescence, magnetic resonance imaging, radioactivity, ultrasound, computed tomography (CT) and positron emission tomography (PET).Ultrasound contrast agents are typically gases, such as air, oxygen, or perfluorocarbons.Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, as well as X-ray imaging agents.

[0179] In some embodiments, the particles produced using the methods described herein contain less than 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% by weight of the drug. In some embodiments, the drug may be a mixture of pharmaceutically active drugs. The loading percentage depends on various factors, including the drug to be encapsulated, the polymer used to prepare the particles, and the method used to prepare the particles.

[0180] The particles can provide controlled release of drugs. For example, unmodified particles can provide effective drug release over time based on the diffusion rate of the drug forming the particles and / or the rate of degradation of the polymer. The polymer composition can be changed to manipulate the degradation behavior of the polymer and therefore the release rate / time of the delivered drug. Alternatively, the particles can be coated with one or more materials to provide controlled release, such as sustained or delayed release, of one or more delivered drugs.

[0181] Sustained-release and delayed-release materials are well known in the art. Solid esters of fatty acids hydrolyzed by lipase can be spray-coated onto microparticles or drug particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto drug-containing microparticles or drug particles by spray coating or wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with a cross-linking procedure, resulting in the formation of an insoluble network. Many methods for cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods for achieving cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized sugars and natural sugars have been used to cross-link gelatin. Cross-linking can also be achieved using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood. Finally, crosslinking can be initiated by physical means such as heat treatment, UV irradiation and gamma irradiation.

[0182] To produce a coating layer of crosslinked protein surrounding drug-containing microparticles or drug particles, a water-soluble protein can be spray-coated onto the microparticles and then crosslinked by one of the methods described above. Alternatively, the drug-containing microparticles can be microencapsulated in a protein by coacervation-phase separation (e.g., by adding salt) and then crosslinked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0183] Polysaccharides can also be cross-linked to form water-insoluble networks. For many polysaccharides, this can be achieved by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose, and guar gum undergo cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can complex through electrostatic interactions.

[0184] Controlled release polymers known in the art include acrylic and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymers, poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic anhydride), and glycidyl methacrylate copolymers.

[0185] In certain preferred embodiments, the acrylic polymer is composed of one or more ammonio methacrylate copolymers, which are well known in the art and described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[0186] In a preferred embodiment, the acrylic polymer is an acrylic resin lacquer, such as that commercially available from Rohm Pharma under the trade name EUDRAGIT®. In a more preferred embodiment, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the trade names EUDRAGIT® RL30D and EUDRAGIT® RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT® RS30D are copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups, with the molar ratio of ammonium groups to the remaining neutral (meth)acrylic acid esters being 1:20 for EUDRAGIT® RL30D and 1:40 for EUDRAGIT® RS30D. The average molecular weight is approximately 150,000. EUDRAGIT® S-100 and EUDRAGIT® L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these drugs. EUDRAGIT® RL / RS mixtures are insoluble in water and digestive fluids. However, multiparticulate systems formulated to contain them are swellable and permeable in aqueous solutions and digestive fluids.

[0187] Polymers such as EUDRAGIT® RL / RS can be mixed together in any desired ratio to ultimately obtain a sustained-release formulation with a desirable dissolution profile. Desirable sustained-release multiparticulate systems can be obtained, for example, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGIT® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. Those skilled in the art will recognize that other acrylic polymers, such as EUDRAGIT® L, can also be used.

[0188] Other controlled-release materials include methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulose polymers such as methyl and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose and Carbopol® 934, polyethylene oxide, and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate, and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.

[0189] Suitable coating materials for providing delayed release include, but are not limited to, cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethyl cellulose; acrylic acid polymers and copolymers, preferably those formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate, and those available under the trade name EUDRAGIT® (Rohm Other methacrylic resins commercially available from Pharma (Westerstadt, Germany) include EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above as a result of a higher degree of esterification), and EUDRAGIT® NE, RL, and RS (water-insoluble polymers with different degrees of permeability and extensibility); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonate copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azopolymers, pectin, chitosan, amylose, and guar gum; zein, and shellac.

[0190] A. Compositions for Transfection of Polynucleotides The gene delivery capabilities of polycationic polymers have been found to be due to several factors, including the polymer's molecular weight, hydrophobicity, and charge density. Many synthetic polycationic materials have been tested as vectors for non-viral gene delivery, but nearly all have been ineffective due to their low efficiency or high toxicity. Most previously described polycationic vectors exhibit high charge density, which is considered a key requirement for effective DNA condensation. As a result, although they are capable of highly efficient gene delivery in vitro, their in vivo applications are limited due to the toxicity associated with excessive charge density.

[0191] We disclose high molecular weight polymers, particularly terpolymers, and methods for their preparation using enzyme-catalyzed copolymerization of lactones with dialkyl diesters and aminodiols. These poly(amine-co-ester) terpolymers have low charge density. In addition, their hydrophobicity can be varied by selecting lactone comonomers with specific ring sizes and by adjusting the lactone content in the polymer. The high molecular weight and increased hydrophobicity of lactone-diester-aminodiol terpolymers, at the expense of low charge density, provide efficient gene delivery with minimal toxicity.

[0192] In preferred embodiments, the terpolymer exhibits efficient gene delivery with reduced toxicity. The terpolymer may be significantly more efficient than commercially available non-viral vectors. For example, the terpolymer may be over 100 times more efficient than commercially available non-viral vectors such as PEI and LIPOFECTAMINE® 2000 based on luciferase expression assays, yet exhibit minimal toxicity at toxic doses of up to 0.5 mg / ml compared to these commercially available non-viral vectors. Preferably, the terpolymer is non-toxic at concentrations suitable for both in vitro and in vivo transfection of nucleic acids. For example, in some embodiments, the terpolymer causes less non-specific cell death compared to other approaches to cell transfection.

[0193] As described in more detail below, in some embodiments, the terpolymer is an ω-pentadecalactone-diethyl sebacate-N-methyldiethanolamine terpolymer containing 20% ​​PDL (also referred to as Terpolymer III-20% PDL).

[0194] IV. Micelles formed from polymers A. Properties of Micelles 1. Micelle size Polymers such as PEG block-containing polymers can be used to prepare micelles. The average micelle size typically ranges from about 100 to about 500 nm, preferably from about 100 to about 400 nm, more preferably from about 100 to about 300 nm, more preferably from about 150 to about 200 nm, and most preferably from about 160 to about 190 nm. These copolymers are stable at a physiological pH of 7.4 in the presence of serum proteins. The copolymers are highly hemocompatible and exhibit minimal hemolysis and aggregation-inducing activity.

[0195] 2.Surface charge The size and zeta potential of micelles were found to change significantly as the pH of the aqueous medium containing the micelles was changed. For example, the trends in the size-pH and zeta-pH curves were remarkably similar for micelles of three PEG2K-PPMS copolymers with different PDL contents (11%, 30%, and 51%). It is clear that the average size of the micellar samples gradually increases as the medium pH is lowered from 7.4 to 5.0, and then remains nearly constant at pH values ​​below 5.0. This pH-responsive behavior observed for micelles is expected as the pH is lowered from 7.4 to 5.0; the PPMS core of the micelles becomes protonated and more hydrophilic, thus absorbing more water molecules from the aqueous medium and causing the micelles to swell. The micelle core is already fully protonated at pH 5.0, and as a result, the size of the micelles remains fairly constant as the pH is further reduced from 5.0. The effect of PDL content in PEG2K-PPMS copolymers on the magnitude of micelle size change at pH values ​​from 7.4 to 5.0 is also noteworthy. A decrease in PDL content and an increase in tertiary amino group content in the copolymer are expected to increase the ability of the micelle core to absorb protons and water molecules. Therefore, as the pH decreased from 7.4 to 5.0, the change in average micelle size was more pronounced for PEG2K-PPMS-11%PDL (200 nm to 234 nm) compared to PEG2K-PPMS-30%PDL (184 nm to 214 nm) and PEG2K-PPMS-51%PDL (163 nm to 182 nm) (Figure 5A).

[0196] The zeta potential of micelles in aqueous media also exhibits significant pH dependence. At physiological and alkaline pH (7.4-8.5), the surface charge of blank PEG2K-PPMS copolymer micelles was negative, which changed to positive as the pH of the medium decreased into the acidic range (4.0-6.0). For example, micelles of PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL had zeta potential values ​​of -5.8, -7.1, and -5.1 mV, respectively, at pH 7.4, which correspondingly changed to +7.6, +5.8, and +4.0 mV at a lower pH of 5.0. Based on the above discussion, this surface charge dependence on pH is attributed to the protonation or deprotonation of the PPMS core of the micelles at different medium pHs. At alkaline pH (7.4-8.5), most of the amino groups in the micelles are likely not protonated, and the micellar particles remain negatively charged, likely due to the absorption of HPO4 2- and / or H2PO4 - anions in PBS by the micelles. In particular, at pH 8.5, the zeta potential values ​​were -8.1 mV, -7.9 mV, and -9.0 mV for PEG2K-PPMS-11%PDL, PEG2K-PPMS-30%PDL, and PEG2K-PPMS-51%PDL, respectively. Lowering the pH from 7.4 to 5.0, the tertiary amino moieties in the micellar PPMS core are mostly protonated, transforming the micelles into positively charged particles. Consistently, among the three micellar samples, PEG2K-PPMS-11% PDL micelles, which have the greatest ability to absorb protons, exhibited the highest zeta potential values ​​at pH 4.0–5.0, whereas PEG2K-PPMS-51% PDL micelles, which have the smallest protonation capacity, exhibited the lowest zeta potential. The observed micelle surface charge response to medium pH is highly desirable because the negative surface charge of micelles at physiological pH can mitigate interactions between micelles and serum proteins in the blood and extend their in vivo circulation time. On the other hand, the reversal of the surface charge to a positive one at an extracellular pH of approximately 6.5 may enhance the uptake of these micelles by target tumor cells.

[0197] The surface charge of the particles / micelles is slightly negative in PBS solution (0.01 M, pH = 7.4), which is beneficial for in vivo drug delivery applications of micelles. It is known that nanoparticles with a nearly neutral surface charge (zeta potential of -10 to +10 mV) can reduce their uptake by the reticuloendothelial system (RES) and extend their circulation time in the blood. The negative surface charge of the micelles is induced by the ionic charge of the micelles in PBS via hydrogen bonding interactions between the anions and the ether groups of the PEG shell or the amino groups of the PPMS core. 2- and / or H2PO4 - This may result from the absorption of anions. For amphiphilic block copolymer micelles, the hydrophilic chain segments (e.g., PEG) in the outer shell of the micelles can shield the charge in the micelle core, and it is expected that the long chain blocks are more effective at reducing the zeta potential than the short chain blocks. Therefore, significantly lower zeta potential values ​​were observed for PEG5K-PPMS copolymer micelles compared to PEG2K-PPMS copolymer micelles.

[0198] The copolymer micelles are pH-responsive: lowering the medium pH from 7.4 to 5.0 significantly increased micelle size, while the micelle surface charge reversed from negative to positive. Correspondingly, DTX-encapsulated copolymer micelles exhibited gradual and sustained drug release at pH 7.4, but significantly accelerated DTX release at acidic pH 5.0. Because the tumor microenvironment is known to be typically slightly acidic (e.g., 5.7–7.0) as a result of lactic acid accumulation due to insufficient oxygen perfusion, this phenomenon can be exploited to improve drug release at tumor sites. In contrast, the extracellular pH of normal tissues and blood is slightly basic (pH 7.2–7.4). Therefore, enhanced drug delivery efficiency is expected for anticancer drug-loaded micelles that are pH-responsive and can be triggered by acidic pH to accelerate drug release. Furthermore, even more acidic conditions (pH = 4.0-6.0) are encountered in endosomes and lysosomes after uptake of micelles by tumor cells via the endocytic pathway, which may further increase the cytotoxicity of drug-encapsulated micelles.

[0199] V. Therapeutic, Preventive, and Diagnostic Agents The polymers can form a variety of polymer compositions, which are useful for preparing various biodegradable medical devices and for drug delivery. Devices prepared from PHA copolymers can be used for a wide range of different medical applications. Examples of such applications include controlled release of therapeutic, preventive, or diagnostic agents; drug delivery; tissue engineering scaffolds; cell encapsulation; targeted delivery; biocompatible coatings; biocompatible implants; guided tissue regeneration; wound dressings; orthopedic devices; prosthetics and bone cements (including adhesives and / or structural fillers); and diagnostics.

[0200] The polymers can be used to encapsulate, mix with, or ionically or covalently couple any of a variety of therapeutic, prophylactic, or diagnostic agents. A wide variety of biologically active materials can be encapsulated or incorporated into the polymer for delivery to a site or to impart properties to the polymer, such as bioadhesion, cell attachment, enhancing cell growth, inhibiting bacterial growth, and preventing blood clot formation.

[0201] Examples of suitable therapeutic and prophylactic agents include synthetic inorganic and organic compounds, proteins and peptides, polysaccharides and other sugars, lipids, and DNA and RNA nucleic acid sequences with therapeutic, prophylactic, or diagnostic activity. Nucleic acid sequences include genes, antisense molecules that bind to complementary DNA and inhibit transcription, and ribozymes. Compounds with a wide range of molecular weights can be encapsulated, for example, from 100 to 500,000 grams per mole or more. Examples of suitable materials include proteins such as antibodies, receptor ligands, and enzymes, peptides such as adhesion peptides, sugars and polysaccharides, synthetic organic or inorganic drugs, and nucleic acids. Examples of materials that can be encapsulated include enzymes, blood clotting factors, inhibitors or clot-dissolving agents such as streptokinase and tissue plasminogen activator; antigens for immunization; hormones and growth factors; polysaccharides such as heparin; oligonucleotides such as antisense oligonucleotides and ribozymes; and retroviral vectors for use in gene therapy. Polymers can also be used to encapsulate cells and tissues. Representative diagnostic agents are agents that can be detected by X-ray, fluorescence, magnetic resonance imaging, radioactivity, ultrasound, computed tomography (CT) and positron emission tomography (PET).Ultrasound diagnostic agents are typically gases, such as air, oxygen, or perfluorocarbons.In a preferred embodiment, polymers are used for the delivery of nucleic acids.

[0202] A. Polynucleotides As discussed in more detail below, the terpolymers can be used to transfect cells with nucleic acids. Accordingly, also disclosed are polyplexes comprising the terpolymer and one or more polynucleotides.

[0203] The polynucleotide can encode one or more proteins, functional nucleic acids, or a combination thereof. The polynucleotide can be monocistronic or polycistronic. In some embodiments, the polynucleotide is multigenic.

[0204] In some embodiments, the polynucleotide is transfected into a cell and remains extrachromosomal. In some embodiments, the polynucleotide is introduced into a host cell and integrated into the host cell's genome. As discussed in more detail below, the composition can be used in gene therapy methods. Gene therapy methods can include the introduction of a polynucleotide into a cell that changes the genotype of the cell. The introduction of the polynucleotide can correct, replace, or otherwise alter an endogenous gene via genetic recombination. The method can include the introduction of an entire replacement copy of a defective gene, a heterologous gene, or a small nucleic acid molecule such as an oligonucleotide. For example, the corrective gene can be introduced into a non-specific location within the host's genome.

[0205] In some embodiments, the polynucleotide is incorporated into or part of a vector. Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression vectors generally contain regulatory sequences and elements necessary for the translation and / or transcription of the inserted coding sequence, which may be, for example, a polynucleotide of interest. The coding sequence may be operably linked to a promoter and / or enhancer that helps control the expression of the desired gene product. Promoters used in biotechnology are of different types according to the intended type of gene expression regulation. They can generally be divided into constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0206] For example, in some embodiments, the polynucleotide of interest is operably linked to a promoter or other regulatory elements known in the art. Thus, the polynucleotide may be a vector, such as an expression vector. Manipulation of polynucleotides for expression in prokaryotic or eukaryotic systems can be performed by techniques commonly known to those skilled in the art of recombinant expression. Expression vectors typically contain one of the compositions under the control of one or more promoters. To place a coding sequence "under the control" of a promoter, the 5' end of the translation initiation site of the reading frame is generally positioned approximately 1-50 nucleotides "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the inserted DNA and promotes expression of the encoded recombinant protein. This is the meaning of "recombinant expression" in the context used herein.

[0207] Many standard techniques are available for constructing expression vectors containing appropriate nucleic acids and transcription / translation control sequences to achieve protein or peptide expression in various host expression systems. Cell types that can be used for expression include, but are not limited to, bacteria such as E. coli and B. subtilis transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors. It will be recognized that any of these vectors can be packaged and delivered using polymers.

[0208] Expression vectors for use in mammalian cells usually contain a replication origin (if necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding site, RNA splice site, polyadenylation site, and transcription terminator sequence. The replication origin can be provided by constructing the vector to contain an exogenous origin, such as from SV40 or other viruses (e.g., polyoma, adeno, VSV, BPV) sources, or can be provided by the host cell chromosome replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0209] Promoters may be derived from the genomes of mammalian cells (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). Furthermore, it may be possible and desirable to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell system.

[0210] Several viral-based expression systems can be utilized; for example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40 (SV40). The early and late promoters of the SV40 virus are useful because both are easily obtained from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication is included.

[0211] When adenovirus is used as an expression vector, the coding sequence can be ligated to the adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the protein in infected hosts.

[0212] Specific initiation signals may also be required for efficient translation of the composition. These signals include the ATG initiation codon and adjacent sequences. It may be necessary to provide additional exogenous translational control signals, including the ATG initiation codon. Those skilled in the art will be readily able to determine this requirement and provide the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of sources, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators.

[0213] For eukaryotic expression, it will also typically be desirable to incorporate an appropriate polyadenylation site into the transcription unit if one was not contained within the original cloned segment. Typically, a poly A addition site is placed approximately 30-2000 nucleotides "downstream" of the protein's termination site, prior to the termination of transcription.

[0214] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express a protein-encoding construct can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with a vector controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells may be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. do.

[0215] In a preferred embodiment, the polynucleotide cargo is RNA, such as mRNA, which is capable of encoding a polypeptide of interest.

[0216] In some embodiments, the mRNA has a cap at the 5' end and / or a 3' poly(A) tail, which can modulate ribosome binding, translation initiation, and mRNA stability in the cell.

[0217] B. Polypeptide of Interest The polynucleotide can encode one or more polypeptides of interest. The polypeptide can be any polypeptide. For example, the polypeptide encoded by the polynucleotide can be a polypeptide that provides a therapeutic or preventive effect to an organism or a polypeptide that can be used to diagnose a disease or disorder in an organism. For example, for the treatment of cancer, autoimmune disorders, parasitic, viral, bacterial, fungal, or other infections, the expressed polynucleotide can encode a polypeptide that functions as a ligand or receptor for cells of the immune system or can function to stimulate or inhibit the organism's immune system. As discussed in the Examples below, a polynucleotide encoding TNF-related apoptosis-inducing ligand (TRAIL) can be delivered to tumor cells using polyplexes in methods for treating cancer.

[0218] In some embodiments, the polynucleotide supplements or replaces a defective polynucleotide in an organism.

[0219] In some embodiments, the polynucleotide comprises a selectable marker that is effective in eukaryotic cells, such as a drug resistance selectable marker. The selectable marker gene can encode a factor necessary for the survival or growth of transformed host cells grown in a selective culture medium. Typical selectable genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, kanamycin, gentamicin, zeocin, or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients that are subtracted from the culture medium.

[0220] In some embodiments, the polynucleotide comprises a reporter gene. A reporter gene is typically a gene that is not present or expressed in the host cell. A reporter gene typically encodes a protein that provides some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include glucuronidazole, thiazolinone ... Examples of genes that may be used include the gene encoding GUS and the gene encoding GFP.

[0221] C. Functional nucleic acid The polynucleotide can be or encode a functional nucleic acid. A functional nucleic acid is a nucleic acid molecule that has a specific function, such as binding to a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex-forming molecules, RNAi, and external guide sequences. Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional nucleic acid molecules can have de novo activity independent of any other molecules.

[0222] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide, or carbohydrate chain.Therefore, functional nucleic acids can interact with the mRNA or genomic DNA of target polypeptide, or they can interact with polypeptide itself.In many cases, functional nucleic acids are designed to interact with other nucleic acids based on the sequence homology between target molecule and functional nucleic acid molecule.In other situations, the specific recognition between functional nucleic acid molecules and target molecule is not based on the sequence homology between functional nucleic acid molecules and target molecule, but rather based on the formation of tertiary structure, which allows specific recognition to occur.

[0223] Antisense molecules are designed to interact with target nucleic acid molecules through either standard or non-standard base pairing. The interaction between antisense molecules and target molecules is designed to promote the destruction of target molecules, for example, by RNAseH-mediated RNA-DNA hybrid degradation. Alternatively, antisense molecules are designed to interrupt the processing functions that would normally occur in target molecules, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many methods for optimizing antisense efficiency by finding the most accessible region of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. Antisense molecules can be designed based on the sequence of the target molecule. -6 , 10 -8 , 10 -10 , or 10 -12 Less than or equal to the dissociation constant (K d ) to bind to the target molecule.

[0224] Aptamers are molecules that interact with target molecules, preferably in a specific manner. Typically, aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophylline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind to 10 -12 K from target molecules less than M d The aptamer can bind very tightly at 10 -6 , 10 -8 , 10 -10 , or 10 -12 Less than K d Aptamers can bind to target molecules with extremely high specificity. For example, aptamers have been isolated that have a binding affinity between a target molecule and another molecule that differs at only a single position on the molecule that is more than 10,000-fold different. Aptamers have a K dK with the target molecule that is at most 10, 100, 1000, 10,000, or 100,000 times lower d When the comparison is made for a molecule such as a polypeptide, the background molecule is preferably a different polypeptide.

[0225] Ribozymes are nucleic acid molecules that can catalyze either intramolecular or intermolecular chemical reactions. Ribozymes preferably catalyze intermolecular reactions. There are several different types of ribozymes that catalyze nuclease or nucleic acid polymerase-type reactions based on ribozymes found in natural systems, such as hammerhead ribozymes. Some ribozymes are not found in natural systems but have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, more preferably RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate followed by cleavage. This recognition is often based primarily on standard or non-standard base pair interactions. Because target substrate recognition is based on the target substrate sequence, this property makes ribozymes particularly good candidates for target-specific cleavage of nucleic acids.

[0226] Triplex-forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acids.When triplex molecules interact with a target region, a structure called a triplex is formed, in which three strands of DNA form a complex that relies on both Watson-Crick and Hoogsteen base pairing.Triplex molecules are preferred because they can bind to target regions with high affinity and specificity.Triplex-forming molecules are preferred because they can bind to target regions with high affinity and specificity. -6 , 10 -8 , 10 -10 , or 10 -12 Less than K d It is preferred that the target molecule be bound by the

[0227] An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule, forming a complex that is recognized by RNase P, which then cleaves the target molecule. EGSs can be designed to specifically target a selected RNA molecule. RNAse P assists in the processing of transfer RNA (tRNA) within cells. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, the directed cleavage of RNA by eukaryotic EGS / RNAse P can be utilized to cleave desired targets within eukaryotic cells. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules are known in the art.

[0228] Gene expression can also be effectively silenced in a highly specific manner by RNA interference (RNAi), which was originally observed by the addition of double-stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). Once dsRNA enters the cell, it is cleaved by the RNase III-like enzyme Dicer into 21-23 nucleotide long double-stranded small interfering RNAs (siRNAs) containing two nucleotide overhangs at the 3' end (Elbashir, et al. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATP-dependent step, the siRNAs are incorporated into a multisubunit protein complex commonly known as the RNAi-induced silencing complex (RISC), which guides the siRNA to the target RNA sequence (Nykanen, At some point, the siRNA duplex unwinds, and the antisense strand remains bound to RISC, where it appears to direct the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). However, the siRNA duplex unwinds and the antisense strand remains bound to RISC, where it directs the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). The effects or their use are not limited to any type of mechanism.

[0229] Short interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression.In one example, siRNA triggers the specific degradation of homologous RNA molecules such as mRNA within the region of sequence identity between both siRNA and target RNA.For example, WO02 / 44321 discloses siRNA that can sequence-specifically degrade target mRNA when base-paired with 3' overhang end, and the method for producing these siRNAs is incorporated herein by reference.Sequence-specific gene silencing can be achieved in mammalian cells by using synthetic short double-stranded RNA that mimics the siRNA produced by the enzyme Dicer (Elbashir, et al. (2001) Nature, 411:494 498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can be chemically or in vitro synthesized, or can be the result of short double-stranded hairpin-like RNA (shRNA) that is processed into siRNA inside cells.Synthetic siRNA is generally designed using algorithms and conventional DNA / RNA synthesizers.Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands).siRNA can be synthesized in vitro using kits such as Ambion's SILENCER® siRNA construction kit. It can also be synthesized in vitro.

[0230] The production of siRNA from vectors is more commonly carried out by transcription of short hairpin RNAse (shRNA). For example, kits for generating vectors containing shRNA are available, such as Imgenex's GENESUPPRESSOR™ construction kit and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentiviral vector.

[0231] Polynucleotide Compositions Polynucleotides can be DNA or RNA nucleotides that typically contain a heterocyclic base (nucleobase), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl function of the sugar moiety. The majority of naturally occurring nucleotides contain uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases and ribose or deoxyribose sugars linked by phosphodiester bonds.

[0232] Polynucleotides may be composed of chemically modified nucleotide analogs to improve stability, half-life, or specificity or affinity for target sequences compared to their DNA or RNA counterparts. Chemical modifications include chemical modifications of the nucleobase, sugar moiety, nucleotide linkage, or a combination thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide with one or more chemical modifications of the heterocyclic base, sugar moiety, or phosphate moiety components. In some embodiments, the charge of the modified nucleotide is reduced compared to a DNA or RNA oligonucleotide of the same nucleobase sequence. For example, the oligonucleotide can have a low negative charge, no charge, or a positive charge. The modification should not interfere with, and preferably enhance, the ability of the oligonucleotide to enter cells and perform functions such as inhibiting gene expression as discussed above.

[0233] Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the backbone of the analog presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those that have a substantially uncharged, phosphorus-containing backbone.

[0234] As discussed in more detail below, in a preferred embodiment, the oligonucleotide is a morpholino oligonucleotide.

[0235] 1. Heterocyclic bases The majority of naturally occurring nucleotides contain uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases. Oligonucleotides can contain chemical modifications to their nucleobase components. Chemical modifications of heterocyclic bases or heterocyclic base analogs can be effective in increasing binding affinity or stability when binding to target sequences. Chemically modified heterocyclic bases include, but are not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5- and 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.

[0236] 2. Glycomodification Polynucleotides may also contain nucleotides with modified sugar moieties or sugar moiety analogs, including, but not limited to, 2'-O-aminoethoxy, 2'-O-aminoamonioethyl (2'-OAE), 2'-O-aminoethoxy, 2'-O-amino ... These include 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME), and 2'-O-(N-(methyl)acetamido) (2'-OMA). The 2'-O-aminoethyl sugar moiety substitution is particularly preferred because it is protonated at neutral pH, thus suppressing charge repulsion between the TFO and the target duplex. This modification stabilizes the C3'-endo conformation of the ribose or dexyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.

[0237] The polynucleotide may be a morpholino oligonucleotide. Morpholino oligonucleotides are typically composed of two or more morpholino monomers containing purine or pyrimidine base-pairing moieties that are effective in binding to bases in a polynucleotide through base-specific hydrogen bonds, linked together by a 1-3 atom long phosphorus-containing linkage that connects the morpholino nitrogen of one monomer to the 5' exocyclic carbon of the adjacent monomer. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil, or thymine. The synthesis, structure, and binding characteristics of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0238] Important properties of morpholino-based subunits typically include: the ability to link in oligomeric form via stable, uncharged backbone linkages; the ability of the polymers formed to have high T even for oligomers as short as 10-14 bases; mThe ability to bear nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, or inosine) such that it can hybridize with a complementary base target nucleic acid, including a target RNA having a base; the ability of the oligomer to be actively transported into mammalian cells; and the ability of the oligomer:RNA heteroduplex to resist RNAse degradation. In some embodiments, the oligonucleotide employs morpholino-based subunits bearing base-pairing moieties connected by uncharged linkages.

[0239] 3. Internucleotide linkages An internucleotide bond refers to the chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides can increase the binding affinity or stability of the polynucleotide or reduce its susceptibility to nuclease digestion. Cationic modifications, including but not limited to, diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP), can be particularly useful due to their reduced electrostatic repulsion between the oligonucleotide and the target. Modifications to the phosphate backbone can also include substituting a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate internucleoside linkage instead of a phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.

[0240] Examples of modified nucleotides with reduced charge include modified internucleotide linkages such as phosphate analogs with achiral and uncharged intersubunit linkages (e.g., Sterchak, et al., Organic Chem., 52:4202, (1987)), and uncharged morpholino-based polymers with achiral intersubunit linkages as discussed above (see, e.g., U.S. Patent No. 5,034,506). Some internucleotide linkage analogs include morpholidates, acetals, and polyamide-linked heterocycles.

[0241] In another embodiment, the oligonucleotide is comprised of a locked nucleic acid. Locked nucleic acids (LNAs) are modified RNA nucleotides (see, e.g., Braasch, et al., Chem. Biol., 8(1):1-7 (2001). LNA forms a hybrid with DNA that is more stable than a DNA / DNA hybrid, similar in nature to a peptide nucleic acid (PNA) / DNA hybrid. Therefore, LNA can be used as a PNA molecule. In some embodiments, LNA binding efficiency can be increased by adding a positive charge to it. LNA is produced using a commercially available nucleic acid synthesizer and standard phosphoramidite chemistry.

[0242] In some embodiments, the oligonucleotide is composed of peptide nucleic acid. Peptide nucleic acid (PNA) is a synthetic DNA mimic in which the phosphate backbone of an oligonucleotide is entirely replaced by repeating N-(2-aminoethyl)-glycine units, and the phosphodiester bond is typically replaced by a peptide bond. Various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNA maintains the spacing of heterocyclic bases similar to that of conventional DNA oligonucleotides, but is an achiral, neutrally charged molecule. Peptide nucleic acid is composed of peptide nucleic acid monomers.

[0243] Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone components of oligonucleotides such as PNAs can be peptide linkages, or they can be non-peptide peptide linkages. Examples include acetyl caps, amino spacers (referred to herein as O-linkers) such as 8-amino-3,6-dioxaoctanoic acid, and amino acids such as lysine are particularly useful when a positive charge is desired in PNAs. Methods for chemical assembly of PNAs are well known. For example, see U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571.

[0244] Polynucleotides may optionally contain one or more terminal residues or modifications at either or both ends to increase the stability and / or affinity of the oligonucleotide to its target. Commonly used positively charged moieties include the amino acids lysine and arginine, but other positively charged moieties may also be useful. For example, lysine and arginine residues can be added to bis-PNA linkers or to the carboxy or N-terminus of PNA chains. Polynucleotides may also be modified to be end-capped using 3'-propylamine groups to prevent degradation. Procedures for 3'- or 5'-capping oligonucleotides are well known in the art.

[0245] VI. Coating Agents for Polyplexes The efficiency of polynucleotide delivery using polymers can be affected by the positive charge on the polyplex surface. For example, the zeta potential of polyplexes, +8.9 mV, can attract and bind to negatively charged plasma proteins in the blood during circulation, leading to rapid clearance by the reticuloendothelial system (RES). Efficiency can also be affected by the instability of polyplex nanoparticles. For example, as discussed in the Examples below, polyplex particles incubated in NaAc buffer containing 10% serum nearly doubled in size within 15 minutes and increased more than 10-fold after 75 minutes. As a result of this size increase, the expanded polyplexes may be removed from the circulation by uptake in the liver. Therefore, in some embodiments, polyplexes are treated or coated to improve polynucleotide delivery efficiency. In some embodiments, the coating improves cell-specific targeting of polyplexes, improves stability (i.e., stabilizes the size of polyplexes in vivo), increases the half-life of polyplexes in vivo (i.e., in the systemic circulation), or a combination thereof, compared to controls. In some embodiments, the control is uncoated polyplexes.

[0246] An exemplary polyplex coating for targeting tumor cells is polyE-mRGD. As used herein, polyE-mRGD refers to a synthetic peptide containing three segments: a first segment containing a polyglutamic acid (polyE) stretch that is negatively charged at physiological pH and can therefore electrostatically bind to the positively charged surface of polyplexes; a second segment containing a neutral polyglycine stretch that serves as a neutral linker; and a polyplex containing RGD and α v β3 and α v The third segment contains an RGD sequence that binds to tumor endothelium through interaction with β5.

[0247] As discussed in more detail below, the polyE-mRGD used in the Examples reversed the surface charge of III-20% PDL / pLucDNA polyplexes. When polyE-mRGD was added at a peptide / DNA weight ratio of 5:1, the zeta potential of the polyplexes changed from +8.9 mV to -5.8 mV. The peptide-coated polyplexes were stable and resistant to aggregation upon incubation in NaAc buffer containing 10% serum, demonstrating that the modified polyplexes can avoid clearance by the RES during in vivo circulation.

[0248] In one embodiment, polyE-mRGD comprises the sequence EEEEEEEEEEEEEEEEGGGGGGRGDK (SEQ ID NO: 1), or RGDKGGGGGGEEEEEEEEEEEEEEEE (SEQ ID NO: 2), or a variant thereof having 85%, 90%, 95%, or greater than 95% sequence identity to SEQ ID NO: 1 or 2.

[0249] Another exemplary coating that can be used to prepare charge-neutral or negatively charged particles that maintain their size in vivo is described in Harris, et al., Biomaterials, 31:998-1006 (2010), and is an amino acid for non-specific systemic administration. The polypeptides used in the coating may include the acid sequence GGGGGGEEEEEEEEEEEEEEEE (SEQ ID NO: 3, poly-E), or the amino acid sequence GdPdLGdVdRG-GGGGGG-EEEEEEEEEEEEEEEE-CONH2 (SEQ ID NO: 4, poly-E-Cat), which contain polycationic sequences that increase targeting to the spleen, spine, sternum, and femur. In some embodiments, the polypeptide used in the coating is a variant of SEQ ID NO: 3 or 4 that has 85%, 90%, 95%, or greater than 95% sequence identity to SEQ ID NO: 3 or 4.

[0250] In vitro studies have shown that adsorption of immunoglobulin G (IgG) and complement protein C3 to nanoparticles increases their uptake by Kupffer cells, and incubation in serum increases hepatic uptake in vivo after hepatic perfusion (Nagayama, et al., Int. J. Pharm., 342:215-21 (2007)). Reports also indicate that galactose can be used to guide polymeric gene delivery particles to hepatocytes via the asialoglycoprotein receptor (Zhang, et al., J. Controlled Release, 102:749-63 (2005)).

[0251] A. Compositions for Modifying Surface Charge The polynucleotide delivery efficiency of polyplexes can be improved by coating the particles with a negatively charged agent at physiological pH. Preferably, the negatively charged agent can electrostatically bind to the positively charged surface of the polyplex. The negatively charged agent can neutralize or reverse the charge of the polyplex. Thus, in some embodiments, the negatively charged agent confers a net negative charge on the polyplex.

[0252] In some embodiments, the negatively charged agent is a negatively charged polypeptide. For example, the polypeptide can contain aspartic acid, glutamic acid, or a combination thereof, such that the overall charge of the polypeptide is negative at neutral pH. In some embodiments, the polypeptide is a polyaspartic acid polypeptide consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 aspartic acid residues. In some embodiments, the polypeptide is a polyglutamic acid polypeptide consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 glutamic acid residues. Other negatively charged molecules include small molecules (i.e., MW less than 1500, 100, 750, or 500 Daltons) such as hyaluronic acid.

[0253] Increasing the negative charge on the particle surface can reduce or prevent the negative interactions described above, where more positively charged particles attract and bind to negatively charged plasma proteins in the blood during circulation, resulting in rapid clearance by the reticuloendothelial system (RES). In some embodiments, the particle's zeta potential is about -15 mV to about 10 mV, preferably about -15 mV to about 8 mV, more preferably about -10 mV to about 8 mV, more preferably about -8 mV to about 8 mV. The zeta potential can be more negative or more positive than the above range, provided the particle is stable (i.e., does not aggregate) and is not easily removed from the bloodstream. The zeta potential can be manipulated by coating or functionalizing the particle surface with one or more moieties that alter the surface charge. Alternatively, the monomer itself can be functionalized and / or additional monomers can be introduced into the polymer, thereby altering the surface charge.

[0254] B. Targeting moiety In some embodiments, the polyplexes contain targeting domains or targeting signals specific to a cell type or cell state. Examples of moieties that may or may not be linked to polyplexes include targeting moieties that provide delivery of molecules to specific cells. The targeting signals or sequences may be specific to a host, tissue, organ, cell, organelle, non-nuclear organelle, or cellular compartment. For example, the compositions herein can be modified with galactosyl-terminated polymers to target the compositions to the liver or liver cells. The modified compositions selectively enter hepatocytes after interaction of the galactose residues of the carrier with the asialoglycoprotein receptor, which is abundantly present with high affinity only on these cells. The compositions herein can also target other specific intercellular regions, compartments, or cell types.

[0255] In one embodiment, the targeting signal binds to its ligand or receptor located on the surface of the target cell, bringing the vector and the cell membrane close enough to each other to allow the vector to penetrate the cell. Additional embodiments of the present disclosure are directed to specifically delivering polynucleotides to specific tissues or cell types, where the polynucleotide can encode a polypeptide or can prevent the expression of a different polynucleotide. The polynucleotide delivered to the cell can encode a polypeptide that can enhance or contribute to the function of the cell.

[0256] The targeting moiety can be an antibody or antigen-binding fragment thereof, an antibody domain, an antigen, a T-cell receptor, a cell surface receptor, a cell surface adhesion molecule, a major histocompatibility locus protein, a viral envelope protein, and a peptide selected by phage display that specifically binds to a defined cell.

[0257] Those skilled in the art will recognize that the targeting properties of the described polyplexes can be altered simply by changing the targeting signal. It is known in the art that nearly every cell type in a mammalian tissue has a unique cell surface receptor or antigen. Therefore, nearly any ligand for a cell surface receptor or antigen can be incorporated as a targeting signal. For example, peptidyl hormones can be used as targeting moieties to target delivery to those cells that have receptors for such hormones. Chemokines and cytokines can similarly be used as targeting signals to target delivery of complexes to their target cells. Various techniques have been developed to identify genes preferentially expressed in certain cells or cell states, and those skilled in the art can use such techniques to identify targeting signals that are preferentially or uniquely expressed in a target tissue of interest.

[0258] Tumor targeting In one embodiment, the targeting signal is used to selectively target tumor cells. Tumor cells express cell surface markers that may be expressed only in tumors or that are present in non-tumor cells but are preferentially displayed on tumor cells. Such markers can be targeted to increase delivery of polyplexes to cancer cells.

[0259] For example, in some embodiments, the targeting moiety is a polypeptide comprising an arginine-glycine-aspartic acid sequence. For example, the targeting moiety comprises an RGD sequence, and v β3 and α vThe targeting moiety may be arginine-glycine-aspartic acid-lysine (RGDK, mRGD) other than the polypeptide that can bind to tumor endothelium through interaction with β5. In some embodiments, the targeting moiety comprises the polypeptide sequence R / KxxR / K (where "x" is any amino acid), which allows binding to neuropilin-1. Binding to integrins or neuropilin-1 are two approaches to improve tumor-targeted delivery and tissue-penetrating delivery to tumors in vivo. Similar approaches have been reported to promote ligand-specific gene delivery in vitro and targeted gene delivery to the liver, spleen, and bone marrow in vivo.

[0260] Other exemplary tumor-specific cell surface markers include, but are not limited to, alpha-fetoprotein (AFP), C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer antigen-125 associated with ovarian cancer (CA-125), cancer antigen 15-3 associated with breast cancer (CA15-3), cancer antigen-19 associated with gastrointestinal cancer (CA-19) and cancer antigen-242, carcinoembryonic antigen (CEA), cancer-associated antigen (CAA), chromogranin A, epithelial mucin antigen (MC5), human epithelial specific antigen (HSA), and IgG4-associated antigen (IGMA). (HEA), Lewis(a) antigen, melanoma antigen, melanoma-associated antigen 100, 25, and 150, mucin-like cancer-associated antigen, multidrug resistance-associated protein (MRPm6), multidrug resistance-associated protein (MRP41), Neu oncogene protein (C-erbB-2), neuron-specific enolase (NSE), P-glycoprotein (mdr1 gene product), multidrug resistance-associated antigen, p170, multidrug resistance-associated antigen, prostate-specific antigen (PSA), CD56, NCAM, EGFR, CD44, and folate receptor. In one embodiment, the targeting signal consists of an antibody specific for a tumor cell surface marker.

[0261] antibody Another embodiment provides an antibody or antigen-binding fragment thereof bound to a polyplex that acts as a targeting signal. The antibody or antigen-binding fragment thereof is useful for directing the polyplex to a cell type or cell state. In one embodiment, the polyplex is coated with a polypeptide that is an antibody-binding domain, for example, a polypeptide derived from a protein known to bind to antibodies, such as protein A and protein G from Staphylococcus aureus. Other domains known to bind to antibodies are known in the art and can be substituted. The antibody-binding domain links the antibody or antigen-binding fragment thereof to the polyplex.

[0262] In certain embodiments, the antibody that functions as the targeting signal is a polyclonal antibody, a monoclonal antibody, a linear antibody, a humanized antibody, a chimeric antibody, or a fragment thereof. Exemplary antibody fragments are those fragments that bind to the antibody-binding portion of the non-viral vector, and include Fab, Fab', F(ab'), Fv diabodies, linear antibodies, single-chain antibodies, and bispecific antibodies, which are known in the art.

[0263] In some embodiments, the targeting signal comprises all or part of an antibody that directs the polyplex to a desired target cell type or cell state. The antibody can be monoclonal or polyclonal, but is preferably monoclonal. For human gene therapy purposes, the antibody can be derived from a human gene, specific for a cell surface marker, and produced to reduce potential immunogenicity to a human host, as is known in the art. For example, transgenic mice containing the entire human immunoglobulin gene cluster can produce "human" antibodies that can be utilized. In one embodiment, a fragment of such a human antibody is used as the targeting signal. In a preferred embodiment, a single-chain antibody modeled on a human antibody is prepared in prokaryotic culture.

[0264] In one embodiment, the targeting signal is directed to cells of the nervous system, including the brain and peripheral nervous system. Cells in the brain comprise several types and states, each with its own cell surface molecule specific to that type. Furthermore, cell types and states can be further characterized and grouped by the display of common cell surface molecules.

[0265] In one embodiment, targeting signal is directed to specific neurotransmitter receptors that are expressed on the surface of cells in the nervous system.The distribution of neurotransmitter receptors is well known in the art, and those skilled in the art can use neurotransmitter receptor specific antibodies as targeting signal to target the described compositions.Furthermore, considering the specificity of neurotransmitters to their receptors, in one embodiment, targeting signal is composed of neurotransmitters or ligands that can specifically bind to neurotransmitter receptors.

[0266] In one embodiment, the targeting signal is specific to cells of the nervous system, which may include astrocytes, microglia, neurons, oligodendrites, and Schwann cells. These cells can be further divided by their function, location, shape, neurotransmitter class and pathological state. Cells of the nervous system can also be identified by their differentiation state, for example, stem cell. Exemplary markers specific to these cell types and states are well known in the art and include, but are not limited to, CD133 and neurospheres.

[0267] In one embodiment, the targeting signal is directed to cells of the musculoskeletal system. Muscle cells include several types and have unique cell surface molecules specific to their type and state. Furthermore, cell types and states can be further characterized and grouped by the display of common cell surface molecules.

[0268] In one embodiment, the targeting signal is directed to a specific neurotransmitter receptor expressed on the surface of muscle cells. The distribution of neurotransmitter receptors is well known in the art, and those skilled in the art can target the described compositions by using neurotransmitter receptor-specific antibodies as targeting signals. Furthermore, given the specificity of neurotransmitters to their receptors, in one embodiment, the targeting signal is composed of a neurotransmitter. Exemplary neurotransmitters expressed in muscle cells that can be targeted include, but are not limited to, acetylcholine and norepinephrine.

[0269] In one embodiment, the targeting signal is specific to muscle cells, which are divided into two major groups: type I and type II. These cells can be further divided according to their function, location, shape, myoglobin content and pathological state. Muscle cells can also be identified according to their differentiation state, such as muscle stem cells. Exemplary markers specific to these cell types and states are well known in the art and include, but are not limited to, MyoD, Pax7, and MR4.

[0270] C. Linker In some embodiments, the polyplexes can be coated with both a negatively charged drug and a targeting moiety. In some embodiments, the negatively charged drug and the targeting moiety are linked together by a linker. The linker can be a polypeptide or any other suitable linker known in the art, such as polyethylene glycol (PEG).

[0271] In some embodiments, the linker is a polypeptide that has approximately neutral charge at physiological pH. In some embodiments, the linker polypeptide is polyglycine. For example, in some embodiments, the linker is composed of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more glycine residues. In a preferred embodiment, the linker is a 6-residue polyglycine.

[0272] In some embodiments, the negatively charged agent, alone or in combination with a targeting moiety, is linked to the polyplex by electrostatic interactions. In some embodiments, the negatively charged agent, targeting moiety, or combination thereof is linked to the polyplex by covalent conjugation to the polymer backbone or to side chains attached to the polymer backbone.

[0273] D. Methods for Reducing Polyplex Size and Aggregation Resistance to aggregation can be important because maintaining a small particle size limits hepatic clearance and preserves the ability of polyplex particles to transfect target cells. Thus, in preferred embodiments, polyplexes are resistant to aggregation. Preferably, polyplexes, whether coated or uncoated, have a radius of about 1 nm to about 1000 nm, more preferably about 1 nm to about 500 nm, and most preferably about 15 nm to about 250 nm. For example, in some embodiments, coated polyplexes loaded with polynucleotides have a radius of about 150 nm to 275 nm.

[0274] The ratio of polynucleotide weight to polymer weight (polynucleotide:polymer), the content and amount of polyplex coating, or a combination thereof, can be used to adjust the size of the polyplex.

[0275] For example, the examples below show that, in some embodiments, the transfection efficiency of particles with a polymer to DNA ratio of 25:1 is lower than the transfection efficiency of particles with polymer to DNA ratios of 50:1, 100:1, 150:1, and 200:1. The most preferred polymer:polynucleotide ratio for a particular formulation can be determined empirically using methods known in the art, such as those described in the examples below. In general, the weight:weight ratio of polymer:polynucleotide is preferably greater than about 10:1, more preferably greater than about 50:1, and most preferably greater than about 100:1. The weight:weight ratio of polymer:polynucleotide is preferably between about 10:1 and 500:1, more preferably between about 25:1 and 250:1, and most preferably between about 50:1 and 150:1. In some embodiments, the weight ratio of polymer:polynucleotide is about 100:1. Preferably, the polyplexes have a spherical shape.

[0276] The Examples also show, in some embodiments, the transfection efficiency of particles as a function of the ratio of coating agent molecules to polynucleotide molecules (coating agent:polynucleotide). Ratios are expressed by weight. The most preferred coating agent:polynucleotide ratio for a particular formulation can be empirically determined using methods known in the art, such as those described in the Examples below. Generally, the coating agent:polynucleotide ratio is greater than 0, preferably less than about 50:1, more preferably less than about 25:1, and most preferably less than about 10:1. The coating agent:polynucleotide ratio is preferably between about 1:1 and 10:1, more preferably between about 2.5:1 and 7.5:1. In some embodiments, the coating agent:polynucleotide ratio is about 5:1. Coating agent:polynucleotide ratios of 10:1, 5:1, and 2.5:1 are also referred to herein as 10x, 5x, and 2.5x, respectively. Preferably, the polyplexes are spherical in shape.

[0277] E. PEG-blocking polymers The polymers can be used in drug delivery, e.g., forming particles, such as microparticles or nanoparticles, or micelles, which can release one or more therapeutic, prophylactic, and / or diagnostic agents in a controlled release manner over a desired period of time.

[0278] pH-responsive micellar nanocarriers are often formed through the self-assembly of amphiphilic block copolymers, consisting of a hydrophilic outer shell (e.g., PEG) and a hydrophobic inner core that can respond to the medium pH. Typically, upon changing the medium pH from neutral or slightly basic to slightly acidic, the micellar core undergoes accelerated disassembly, becoming completely soluble in water or substantially swollen in aqueous media. As a result, drug-encapsulated micelles with slow drug release rates at physiological pH can be triggered by acidic pH to rapidly unload drug molecules. Polymer segments constituting the micellar core in previous reports include poly(orthoester), poly(β-aminoester), poly(L-histidine), etc. A major drawback of most previous micellar systems is the multiple steps required to prepare the copolymers, as well as the difficulty of controlling the polymer molecular weight and adjusting the polymer composition during copolymer synthesis.

[0279] The copolymers exhibited varying release rates as a function of pH. The in vitro drug release behavior of DTX-encapsulated micelles of PEG2K-PPMS copolymer samples (PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL) was studied in PBS solution at both physiological pH 7.4 and acidic pH 5.0. In general, DTX release from all micellar samples followed biphasic release kinetics and exhibited significant pH dependence. DTX-loaded PEG2K-PPMS copolymer micelles rapidly released 25–45% of the drug during the first 12 hours, followed by a slower release of an additional 25–40% over the next 132 hours. The effect of medium pH on the drug release rate is significant. For example, at the end of the incubation period (144 h), the cumulative DTX released from micelles of PEG2K-PPMS-11% PDL, PEG2K-PPMS-30% PDL, and PEG2K-PPMS-51% PDL copolymers was 66%, 60%, and 55%, respectively, at a physiological pH of 7.4, which correspondingly increased to 85%, 81%, and 75% at an acidic pH of 5.0. The observed pH-triggered acceleration of DTX release from PEG2K-PPMS copolymer micelles is consistent with previous observations that changing the medium pH from 7.4 to 5.0 causes significant swelling of the micelles due to protonation and size increase of the micellar PPMS core. This pH-triggered micelle size expansion would certainly facilitate the diffusion and release of entrapped DTX from the micelle core into the aqueous medium. At a given pH, the DTX release rate is likely controlled by interactions between the drug in the micelle core and the PPMS matrix. Because PDL-rich PEG2K-PPMS copolymers are expected to form strong hydrophobic domains in their micelle inner cores to better capture and retain hydrophobic DTX molecules, drug release from such copolymer micelles should be slower and more sustained. This hypothesis is supported by experimental results showing that the DTX release rate from PEG2K-PPMS copolymer micelles decreases with increasing PDL content in the PPMS chain segments of the copolymer at both pH 7.4 and 5.0.

[0280] Upon uptake of micelles by tumor cells, the micellar particles are known to undergo entrapment in endosomes at pHs ranging from 5.5 to 6.0 and in lysosomes at pHs ranging from 4.5 to 5.0. As the above results clearly demonstrate, these acidic environments inevitably trigger rapid DTX release from PEG2K-PPMS copolymer micelles, thus enhancing the cytotoxicity of the drug-loaded micelles. The amino groups in the copolymer may act as proton sponges to facilitate endosomal escape. Therefore, the pH-responsive properties exhibited by PEG2K-PPMS copolymer micelles are highly desirable, making them excellent carriers for the delivery of anticancer drugs.

[0281] VII. Formulations The formulation is prepared using a pharmaceutically acceptable "carrier" composed of materials considered to be safe and effective, and can be administered to an individual without causing undesired biological side effects or unwanted interactions. A "carrier" is any component present in a pharmaceutical formulation other than the active ingredient or ingredients. The term "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, and coating compositions.

[0282] For detailed information on materials, equipment, and processes for preparing tablets and delayed-release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989) and Ansel et al., Pharmaceutical See Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, PA: Williams & Wilkins, 1995).

[0283] VIII. Methods for Preparing Polyplexes A. Methods for Making Particles The particles can be prepared using a variety of techniques known in the art, which may depend on a variety of factors, including the polymer used to form the nanoparticles, the desired size range of the resulting particles, and compatibility with the material to be encapsulated.

[0284] Methods known in the art that can be used to prepare nanoparticles include, but are not limited to, polyelectrolyte condensation (see Suk et al., Biomaterials, 27, 5143-5150 (2006)); single and double emulsions; nanoparticle molding, and electrostatic self-assembly (e.g., polyethyleneimine-DNA or liposomes).

[0285] In one embodiment, the loaded particles are typically prepared by mixing a solution of the polymer in an organic solvent with the polynucleotide of interest. The polymer solution is prepared by dissolving or suspending the polymer in the solvent. The solvent must be selected so as not to adversely affect (e.g., destabilize or decompose) the encapsulated nucleic acid. Suitable solvents include, but are not limited to, DMSO and methylene chloride. The concentration of the polymer in the solvent can be varied as needed. In some embodiments, the concentration is, for example, 25 mg / ml. The polymer solution can also be diluted with a buffer solution, such as a sodium acetate buffer.

[0286] The polymer solution is then mixed with the drug to be encapsulated, such as a polynucleotide. The drug can be dissolved in a solvent to form a solution before mixing it with the polymer solution. In some embodiments, the drug is dissolved in a physiological buffer before mixing it with the polymer solution. The ratio of polymer solution volume to drug solution volume can be 1:1. The combination of polymer and drug is typically incubated for several minutes to form particles before using the solution for its desired purpose, such as transfection. For example, the polymer / polynucleotide solution can be incubated for 2, 5, 10, or longer than 10 minutes before using the solution for transfection. Incubation can be at room temperature.

[0287] In some embodiments, the particles are also incubated with a solution containing a coating agent before use. The particle solution can be incubated with the coating agent for 2, 5, 10, or longer than 10 minutes before using the polyplexes for transfection. Incubation can be at room temperature.

[0288] In some embodiments, when the agent is a polynucleotide, the polynucleotide is first complexed with a polycation before mixing with the polymer. Complexation can be achieved by mixing the polynucleotide and polycation in an appropriate molar ratio. When using a polyamine as the polycation species, it is useful to determine the molar ratio of polyamine nitrogen to polynucleotide phosphate (N / P ratio). In a preferred embodiment, the inhibitory RNA and polyamine are mixed together at an N / P ratio of approximately 1:1 to 1:25, preferably about 8:1 to 15:1, to form the complex. The volume of polyamine solution required to achieve a particular molar ratio is calculated using the following formula:

number

[0289] The term "polycation" refers to a compound that has a positive charge, preferably at least two positive charges, at a selected pH, preferably physiological pH. The polycationic moiety has about 2 to about 15 positive charges, preferably about 2 to about 12 positive charges, and more preferably about 2 to about 8 positive charges, at a selected pH value. Many polycations are known in the art. Suitable components of polycations include basic amino acids and their derivatives, such as arginine, asparagine, glutamine, lysine, and histidine; cationic dendrimers; and aminopolysaccharides. Suitable polycations can be linear, e.g., linear tetralysine, branched, or dendrimeric in structure.

[0290] Exemplary polycations include, but are not limited to, synthetic polycations based on acrylamide and 2-acrylamido-2-methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similar quaternized polypyridines, diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate, lipopolyamines, poly(allylamine), such as the strong polycation poly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene, and polypeptides such as protamine, histone polypeptides, polylysine, polyarginine, and polyornithine.

[0291] In some embodiments, the polycation is a polyamine. A polyamine is a compound having two or more primary amine groups. Suitable naturally occurring polyamines include, but are not limited to, spermine, spermidine, cadaverine and putrescine. In a preferred embodiment, the polyamine is spermidine.

[0292] In another embodiment, the polycation is a cyclic polyamine.Cyclic polyamines are known in the art and are described in, for example, U.S. Patent No. 5,698,546, WO1993 / 012096 and WO2002 / 010142.Exemplary cyclic polyamines include, but are not limited to, cyclenes.

[0293] Spermine and spermidine are derivatives of putrescine (1,4-diaminobutane), which is produced from L-ornithine by the action of ornithine decarboxylase (ODC). L-ornithine is the product of L-arginine degradation by arginase. Spermidine is a triamine structure produced by spermidine synthase (SpdS), which catalyzes the monoalkylation of putrescine (1,4-diaminobutane) with the decarboxylated S-adenosylmethionine (dcAdoMet) 3-aminopropyl donor. Formal alkylation of both amino groups of putrescine with the 3-aminopropyl donor yields the symmetrical tetraamine spermine. Spermine biosynthesis proceeds to spermidine through the action of spermine synthase (SpmS) in the presence of dcAdoMet. The 3-aminopropyl donor (dcAdoMet) is derived from S-adenosylmethionine by the sequential conversion of L-methionine by methionine adenosyltransferase, followed by decarboxylation by AdoMetDC (S-adenosylmethionine decarboxylase). Thus, putrescine, spermidine, and spermine are metabolites derived from the amino acids L-arginine (L-ornithine, putrescine) and L-methionine (dcAdoMet, aminopropyl donor).

[0294] IX. Methods of Using Particles / Micelles A. Drug Delivery The particles can be used to deliver an effective amount of one or more therapeutic, diagnostic, and / or prophylactic agents to a patient in need of such treatment. The amount of agent to be administered can be readily determined by the prescribing physician and will depend on the age and weight of the patient and the disease or disorder being treated.

[0295] The particles are useful in drug delivery (as used herein, "drug" includes therapeutic, nutritional, diagnostic, and prophylactic agents), whether injected intravenously, subcutaneously, or intramuscularly, administered to the nasal or pulmonary system, injected into a tumor environment, administered to a mucosal surface (vaginally, rectally, buccal, sublingual), or encapsulated for oral delivery. The particles can be administered as a dry powder, in an aqueous suspension (in water, saline, buffered saline, etc.), in a hydrogel, organogel, or liposome, capsule, tablet, lozenge, or other standard pharmaceutical excipient.

[0296] B. Transfection The composition may be for cell transfection of polynucleotides. As discussed in more detail below, transfection can occur in vitro or in vivo and can be applied in applications including gene therapy and disease treatment. The composition may be more efficient, less toxic, or a combination thereof, when compared to a control. In some embodiments, the control is cells treated with an alternative transfection reagent, such as LIPOFECTAMINE 2000 or polyethyleneimine (PEI).

[0297] Transfection is performed by contacting cells with a solution containing polyplexes. For in vivo methods, the contact typically occurs in vivo after the solution is administered to a subject. For in vitro methods, the solution is typically added to a cell culture and allowed to contact the cells for a few minutes, hours, or days. The cells can then be washed to remove excess polyplexes.

[0298] The specific polynucleotide delivered by polyplex can be selected by those skilled in the art depending on the condition or disease to be treated. The polynucleotide can be, for example, a gene of interest or cDNA, mRNA, functional nucleic acid such as inhibitory RNA, tRNA, rRNA, or an expression vector encoding the gene of interest or cDNA, functional nucleic acid, tRNA, or rRNA. In some embodiments, two or more polynucleotides are administered in combination.

[0299] The compositions can be used in methods for delivering polynucleotides to cells in vitro. For example, polyplexes can be used for in vitro transfection of cells. The methods typically involve contacting cells with a polyplex containing a polynucleotide in an effective amount to introduce the polynucleotide into the cytoplasm of the cells. In some embodiments, the polynucleotide is delivered to the cells in an effective amount to change the genotype or phenotype of the cells. The cells can be primary cells isolated from a subject or cells of an established cell line. The cells can be of a homogenous cell type or a heterogeneous mixture of different cell types. For example, polyplexes can be introduced into the cytoplasm of cells from a heterogeneous cell line with different cell types, e.g., in feeder cell cultures or mixed cultures of various states of differentiation. The cells can be transformed cell lines that can be maintained indefinitely in cell culture. Exemplary cell lines include those available from the American Type Culture Collection, including tumor cell lines.

[0300] Any eukaryotic cell can be transfected to generate cells, e.g., primary cells and established cell lines, that express a particular nucleic acid, e.g., a metabolic gene. Suitable types of cells include, but are not limited to, stem cells, totipotent cells, pluripotent cells, embryonic stem cells, inner mass cells, adult stem cells, bone marrow cells, cells derived from umbilical cord blood, The present invention also includes undifferentiated or partially differentiated cells, including cells derived from ectoderm, mesoderm, or endoderm.Suitable differentiated cells include somatic cells, nerve cells, skeletal muscle, smooth muscle, pancreatic cells, hepatic cells, and cardiac cells.In another embodiment, siRNA, antisense polynucleotide (including siRNA or antisense polynucleotide) or inhibitory RNA can be transfected into cells using the composition.

[0301] The method is particularly useful in the field of personalized therapy, for example, to repair defective genes, dedifferentiate cells, or reprogram cells. For example, target cells are first isolated from a donor using methods known in the art, contacted with a polyplex containing a polynucleotide that induces in vitro (ex vivo) changes, and then administered to a patient in need thereof. The source or cell includes cells directly collected from a patient or an allogeneic donor. In a preferred embodiment, The target cells administered to the subject are autologous, e.g., derived from the subject, or syngeneic. Allogeneic cells can also be isolated by using target cells obtained or derived from an antigenically matched, genetically unrelated donor (identified through national registration), or from a genetically related sibling or parent.

[0302] Cells can be selected by positive and / or negative selection techniques. For example, antibodies that bind to specific cell surface proteins can be conjugated to magnetic beads and immunogenic procedures utilized to recover the desired cell type. It may be desirable to enrich target cells before transient transfection. As used herein in the context of a composition enriched for a specific target cell, "enriched" refers to a higher proportion of the desired element (e.g., target cell) than that found in the natural source of the cells. The cell composition may be enriched by at least one order of magnitude, preferably two or three orders of magnitude, more preferably 10, 100, 200, or 1000 orders of magnitude, than the natural source of the cells. Once the target cells are isolated, they may be propagated by growing them in a suitable medium according to established methods known in the art. Established cell lines may also be useful for the method. Cells can be cryopreserved, if necessary, before transfection.

[0303] The cells are then contacted in vitro with the composition to repair, dedifferentiate, redifferentiate, and / or reprogram the cells. The cells can be monitored and the desired cell type can be selected for therapeutic administration.

[0304] After repair, dedifferentiation, and / or redifferentiation and / or reprogramming, the cells are administered to a patient in need thereof. In a most preferred embodiment, the cells are isolated from the same patient and administered back to the same patient. In an alternative embodiment, the cells are isolated from one patient and administered to a second patient. The method can also be used to generate frozen stocks of modified cells that can be stored long-term for later use. In one embodiment, fibroblasts, keratinocytes, or hematopoietic stem cells are isolated from a patient and repaired, dedifferentiated, or reprogrammed in vitro to provide therapeutic cells for the patient.

[0305] C. In vivo methods The composition can be used in methods for delivering polynucleotides to cells in vivo. The polymer has been found to be more efficient and / or less toxic for systemic in vivo transfection of polynucleotides than alternative transfection reagents, including LIPOFECTAMINE 2000, PEI, and even other PMSCs. Thus, in some embodiments, cell-specific polyplexes containing therapeutic polynucleotides are administered systemically in vivo to treat diseases, such as cancer.

[0306] In some in vivo approaches, the composition is administered to the subject in a therapeutically effective amount.As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or otherwise provide desired pharmacological and / or physiological effects.The exact dosage varies according to various factors, such as subject-dependent variables (for example, age, immune system health, etc.), disease, and treatment to be carried out.

[0307] Pharmaceutical compositions may be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or using bioerodible inserts, and can be formulated into dosage forms suitable for each route of administration.

[0308] In some embodiments, the compositions are administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective to deliver the composition to targeted cells. Other possible routes include transdermal or oral.

[0309] In certain embodiments, the composition is administered locally, for example, by direct injection into the site to be treated. In some embodiments, the composition is injected or otherwise administered directly into one or more tumors. Typically, local injection results in an increased local concentration of the composition that is higher than can be achieved by systemic administration. In some embodiments, the composition is delivered locally to appropriate cells by using a catheter or syringe. Other means of locally delivering such compositions to cells include using an infusion pump (e.g., manufactured by Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (see, e.g., P. Johnson and J.G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987) which , which may result in a sustained release of polyplexes to the area immediately surrounding the implant.

[0310] Polyplexes can be provided to cells directly, such as by contacting them with cells, or indirectly, such as through the action of any biological process. For example, polyplexes can be formulated in a physiologically acceptable carrier or vehicle and injected into tissues or fluids surrounding the cells. Polyplexes can cross cell membranes by simple diffusion, endocytosis, or any active or passive transport mechanism.

[0311] The compositions can be used in gene therapy protocols for the treatment of gene-related diseases or disorders. Cellular dysfunction can also be treated or reduced using the compositions and methods. In some embodiments, diseases suitable for gene therapy are specifically targeted. The disease can be in children, e.g., individuals under the age of 18, typically under the age of 12, or in adults, e.g., individuals aged 18 or older. Thus, embodiments of the present disclosure are directed to treating a host diagnosed with a disease by transfecting a polyplex containing a polynucleotide into affected cells, where the polynucleotide encodes a therapeutic protein. In another embodiment, inhibitory RNA is directed to a specific cell type or condition to reduce or eliminate protein expression, thereby achieving a therapeutic effect. The present disclosure encompasses manipulating, augmenting, or replacing genes to treat diseases caused by genetic defects or abnormalities.

[0312] Suitable genetically-based diseases that can be treated with the compositions herein include, but are not limited to:

[0313] Mitochondrial Disorders: Alpers disease; Barth syndrome; beta-oxidation deficiency; carnitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; cytochrome c oxidase (COX) deficiency; LHON-Leber hereditary optic neuropathy; MM-mitochondrial myopathy; LIMM-fatal infantile mitochondrial myopathy; MMC-maternal myopathy and cardiomyopathy; NARP-neurogenic muscle weakness, ataxia, and retinitis pigmentosa; Leigh disease; FICP-fatal infantile cardiomyopathy plus Plus), MELAS-associated cardiomyopathy; MELAS - mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; LDYT - Leber's hereditary optic neuropathy and dystonia; MERRF - myoclonic epilepsy and ragged-red muscle fibers; MHCM - maternally inherited hypertrophic cardiomyopathy; CPEO - chronic progressive external ophthalmoplegia; KSS - Kearns-Sayre syndrome; DM - diabetes mellitus; DMDF - diabetes mellitus plus hearing loss; CIPO - chronic intestinal pseudo-obstruction with myopathy and ophthalmoplegia; DEAF - maternally inherited hearing loss or aminoglycoside-induced hearing loss; PEM - progressive encephalopathy; SNHL - sensorineural hearing loss; encephalomyopathy; mitochondrial cytopathy; dilated cardiomyopathy; GER - gastrointestinal reflux; DEMCHO - dementia and chorea; AMDF - ataxia, myoclonus, and exercise intolerance; ESOC - epilepsy, stroke, optic atrophy, and cognitive decline; FBSN Familial bilateral striatal necrosis; FSGS focal segmental glomerulosclerosis; LIMM fatal infantile mitochondrial myopathy; MDM myopathy and diabetes mellitus; MEPR myoclonic epilepsy and psychomotor regression; MERME MERRF / MELAS overlap; MHCM maternally inherited hypertrophic cardiomyopathy; MICM maternally inherited cardiomyopathy; MILS maternally inherited Leigh syndrome; mitochondrial encephalocardiopathy; multisystem mitochondrial disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION non-arteritic anterior ischemic optic neuropathy; NIDDM non-insulin-dependent diabetes mellitus; PEM progressive encephalopathy; PME progressive myoclonic epilepsy; RTT Rett syndrome; SIDS sudden infant death syndrome; MIDD maternally inherited diabetes and hearing loss.and MODY (Maturity-Onset Diabetes of the Young).

[0314] Nuclear Disease: Muscular dystrophy, Ellis-van Creveld syndrome, Marfan syndrome, myotonic dystrophy, spinal muscular atrophy, achondroplasia, amyotrophic lateral sclerosis, Charcot-Marie-Tooth syndrome, Cockayne syndrome, flexor dysplasia, Duchenne muscular dystrophy, Ellis-van Creveld syndrome, fibrodysplasia ossificans progressiva, Alzheimer's disease, Angelman syndrome, epilepsy, essential tremor, Fragile X syndrome, Friedreich's ataxia, Huntington's disease, Niemann-Pick disease, Parkinson's disease, Prader-Willi syndrome, Rett syndrome, spinal cord injury Cerebral atrophy, Williams syndrome, ataxia-telangiectasia, anemia, sickle cell anemia, Burkitt's lymphoma, Gaucher disease, hemophilia, leukemia, paroxysmal nocturnal hemoglobinuria, porphyria, thalassemia, Crohn's disease, alpha-1-antitrypsin deficiency, cystic fibrosis, hearing loss, Pendred syndrome, glaucoma, gyrate retinochoroidal atrophy, adrenal hyperplasia, adrenoleukodystrophy, Cockayne syndrome, long QT syndrome, immunodeficiency with hyper-IgM, Alport syndrome, Ellis-van Creveld syndrome, fibrodysplasia ossificans progressiva, Waardenburg syndrome, Werner syndrome.

[0315] Infectious diseases: Viruses - AIDS, AIDS-related syndrome, chickenpox, common cold, cytomegalovirus infection, Colorado tick fever, dengue fever, Ebola hemorrhagic fever, mumps, influenza, hand, foot and mouth disease, hepatitis - herpes simplex, shingles, HPV, influenza, Lassa fever, measles, Marburg hemorrhagic fever, infectious mononucleosis, mumps, poliomyelitis, progressive multifocal leukencephalopathy, rabies, rubella , SARS, smallpox, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease - yellow fever; Bacterial - anthrax, bacterial meningitis, brucellosis, bubonic plague, campylobacteriosis, cat scratch disease, cholera, diphtheria, typhus, gonorrhea, leprosy, legionellosis, leprosy, leptospirosis, listeriosis, Lyme disease, melioidosis, MRSA infection, nocardiosis, whooping cough, pneumococcal pneumonia, psittacosis, Q fever, Rocky Mountain spotted fever or RMSF, salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhoid fever, whooping cough; Parasitic - Af Trypanosomiasis, amebiasis, ascariasis, babesiosis, Chagas disease, clonorchiasis, cryptosporidiosis, cysticercosis, diphyllobothriasis, dracunculiasis, echinococcosis, intestinal enterobiasis, fascioliasis, filariasis, free-living amebiasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, kala-azar, leishmaniasis, malaria, trichosis, myiasis, onchocerciasis, pediculosis, pinworm infection, scabies, schistosomiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis.

[0316] Cancer: Breast and ovarian cancer, Burkitt's lymphoma, chronic myeloid leukemia, colon cancer, lung cancer, malignant melanoma, multiple endocrine neoplasia, neurofibromatosis, p53 LieFrauMeni, pancreatic cancer, prostate cancer, retinoblastoma, von Hippel-Lindau syndrome, polycystic kidney disease, tuberous sclerosis.

[0317] Metabolic Disorders: Adrenoleukodystrophy, Atherosclerosis, Best Disease, Gaucher Disease, Glucose-Galactose Malabsorption, Gyrate Atrophy, Early-Onset Diabetes Mellitus, Obesity, Paroxysmal Nocturnal Hemoglobinuria, Phenylketonuria, Refsum Disease, Tangier Disease, Tay-Sachs Disease, Adrenoleukodystrophy, Type 2 Diabetes Mellitus, Gaucher Disease, Hereditary Hemochromatosis, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, Menkes Syndrome, Niemann-Pick Disease, Pancreatic Cancer, Prader-Willi Syndrome, Porphyria, Refsum Disease, Tangier Disease, Wilson Disease, Zellweger Syndrome, Progeria, SCID.

[0318] Autoimmune disorders: such as autoimmune polyglandular syndrome, lupus, type 1 diabetes, scleroderma, multiple sclerosis, Crohn's disease, chronic active hepatitis, rheumatoid arthritis, Graves' disease, myasthenia gravis, myositis, antiphospholipid syndrome (APS), uveitis, polymyositis, Raynaud's phenomenon, and demyelinating neuropathies, as well as polymyalgia rheumatica, temporal arteritis, Sjogren's syndrome, Bechet's disease, Churg-Strauss syndrome, and Takayasu's arteritis A rare disorder.

[0319] Inflammatory disorders: alopecia, varicose vein dysplasia, Ellis-van Creveld syndrome, asthma, arthritis, e.g., osteoarthritis, rheumatoid arthritis, and spondyloarthropathy.

[0320] Age-related disorders: Alzheimer's disease, Parkinson's disease, atherosclerosis, age-related macular degeneration, and age-related osteoporosis.

[0321] The methods and compositions can also be used to treat, manage, or reduce symptoms associated with aging, in tissue regeneration / regenerative medicine, stem cell transplantation, induction of reversible genetic modifications, expression of inhibitory RNA, cognitive enhancement, performance enhancement, and cosmetic modifications to humans or non-human animals.

[0322] D. Transgenic Non-Human Animals The compositions and methods can also be used to generate transgenic non-human animals. In particular, zygote microinjection, nuclear transfer, blastomere electrofusion, and blastocyst injection of embryonic stem (ES) cell cybrids each provide a viable strategy for generating transgenic animals. In one embodiment, embryonic stem (ES) cells are transfected and injected into blastocysts of mammalian embryos as a means of generating chimeric mice. In another embodiment, embryonic stem (ES) cells are first prepared, followed by blastocyst injection into embryos. The use of cells carrying specific genes and modifications of interest allows for the creation of transfected DNA and the study of its results. Theoretically, this technique offers the possibility of transferring any polynucleotide into a whole organism. For example, the methods and compositions can be used to generate mice carrying polynucleotides delivered into specific cell types or cell states.

[0323] A unicellular or multicellular non-human organism, preferably a non-human mammal, more preferably a mouse, can be transfected with the composition of the present disclosure by administering the composition to the non-human organism.In one embodiment, the polynucleotide remains episomal and is not stably integrated into the genome of the host organism.In another embodiment, the polynucleotide prevents the expression of the target gene.Therefore, the expression of the polynucleotide in a specific cell of the host can be controlled by the amount of polynucleotide administered to the host.

[0324] Transfected non-human organisms have several advantages over conventional transgenic organisms. For example, the transfected organisms of the present invention can be generated without sexual reproduction and in a shorter time than conventional transgenic organisms. Furthermore, the expression of a polynucleotide of interest in a host can be directly regulated by the amount of the polynucleotide of interest administered to the host. The dosage-controlled expression of a polynucleotide of interest can be correlated with the phenotype and changes observed in transfected animals. In addition, an inducible expression and / or replication control element can be included in the polynucleotide of interest to provide inducible and dosage-dependent expression and / or replication. Suitable inducible expression and / or replication control elements are known in the art. Furthermore, the effects of genes and gene modifications on specific cell types and conditions can be studied without affecting the entire cell population of an animal.

[0325] X.Kit Also disclosed are kits or packs that provide the elements necessary to perform eukaryotic or prokaryotic transfection, particularly transfection of specific cell types or cell states. According to one embodiment, a kit is provided that includes a polymer and, optionally, a polyplex coating, e.g., a target-specific coating. The polymer can be combined with a user-selected polynucleotide to form a complex that can be used to transfect a host or host cell. The polyplex can be further mixed with a coating to provide cell-type or cell-state-specific targeting.

[0326] The individual components of the kit can be packaged in various containers, e.g., vials, tubes, microtiter well plates, bottles, etc. Other reagents, e.g., positive control samples, negative control samples, buffers, cell culture media, etc., can be included in separate containers and provided with the kit. Preferably, the kit will also include instructions for use. [Example]

[0327] The present invention will be further understood by reference to the following non-limiting examples.

[0328] Problem Solved: Protein therapeutics can be used as highly effective medical treatments for a wide range of diseases (Harris et al., Pharmacol. Biochem. Behav. 150e151 48e56 (2016); Jiang et al., J. Control Release 213 36e44 (2015); Natarajan et al., Transl. Res. 188 10e26 (2017); Efremenko et al., J. Control Release 247 175e181 (2017); Jiang et al., Funct. Mater. 28 (6) 1703982 (2018); Jiang et al., J. Control Release 231 38e49 (2016)). However, the clinical use of this therapeutic class has been limited by their cost and instability after systemic administration, highlighting the need for new approaches to ensure sustained, efficient, and safe delivery (Carter, Exp. Cell Res. 317 (9) 1261e1269 (2011)). Gene therapy is attractive due to the promise of sustained protein secretion with low administration frequency (Weissman, Expert Rev. Vaccines 14 (2) 265e281 (2015)).

[0329] Although virus-based DNA therapy offers the possibility of long-term protein expression, it also raises many safety concerns, particularly regarding the risk of insertional mutagenesis, the induction of severe immune responses, and the difficulty of controlling protein expression levels.In contrast to DNA, mRNA induces the expression of encoded proteins without entering the nucleus, thus demonstrating higher efficiency in transfecting non-dividing cells while reducing the risk of insertional mutagenesis.An additional advantage of mRNA therapy is that its dosage is scalable and treatment is transiently related to mRNA stability in the cytosol.

[0330] A solution to the problem: A "top-down" approach to synthesizing a library of polymeric materials is provided herein. Poly(amine-co-ester) (PACE) was used for gene delivery. PACE:pDNA polyplexes are one of the most efficient and least toxic non-viral vectors (Zhou et al., Nat. Mater. 11 (1) 82e90 (2012)). PACE polymers were designed with several key features: (1) biodegradability provided by ester linkages in the polymer backbone; (2) low cationic charge density that allows electrostatic complexation with nucleic acids while avoiding the toxicity associated with highly cationic polymers; and (3) hydrophobicity provided by lactone groups that stabilize the polyplexes. The resistance of lipase chemistry offers high chemical versatility, allowing modulation of polymer architecture through monomer selection to easily generate a family of distinct materials. Such versatility holds promise for the translation of PACE technology for mRNA delivery.

[0331] Further modifications have been made to chemistries and delivery systems that have been shown to be effective in delivering drugs to the pulmonary system.

[0332] Example 1 PACE polymers can deliver mRNA material and method material u-Pentadecalactone (PDL), diethyl sebacate (DES), sebacic acid, N-methyldiethanolamine (MDEA), chloroform, dichloromethane, hexane, chloroform-d, chromium(III) acetylacetonate, ethylenediamine, ethanolamine, glycine, and 1,10-carbonyldiimidazole (CDI) were purchased from Sigma-Aldrich (Saint Louis, MO) and used as received. 2-[(2-aminoethyl)(methyl)amino]ethanol (AEMAE) was purchased from ChemBridge Corporation (San Diego, CA). Immobilized Candida antarctica lipase B (CALB) (Novozym 435) supported on acrylic resin was also obtained from Sigma-Aldrich and dried at 2.0 mmHg and 50°C for 20 hours before use. The TransIT-mRNA transfection kit was purchased from Mirus Bio LLC (Madison, WI). Modified Fluc mRNA encoding luciferase and mouse erythropoietin (EPO) mRNA encoding EPO were purchased from TriLink Biotechnologies (San Diego, CA). HEK293, Daoy, and SH-SY5Y cells were purchased from ATCC (Manassas, VA).

[0333] Polymer Synthesis PACE synthesis was performed as described by Zhou et al., Nat. Mater. 11 (1) 82–90 (2012), with some modifications to generate polymers with different structures. Briefly, (1) diethyl sebacate or sebacic acid was used for polymerization to obtain either classical or acidic PACE. (2) Different percentages of PDL (10% or 20%) were added to the reaction mixture to vary the hydrophobicity of classical PACE. (3) To vary the molecular weight (MW) of classical PACE containing 10% PDL, the reaction time of the second step was varied from 8 to 72 hours to obtain polymers with MWs ranging from 2 kDa to 20 kDa.

[0334] result PACE is a family of terpolymers formed by enzymatic copolymerization of diesters / diacids with amino-substituted diols and lactones. PACE can efficiently deliver pDNA (Zhou et al., Nat. Mater. 11 (1) 82e90 (2012)), microRNA (Adams et al., Cancer Res. 76 (4) 927e939 (2016)), and siRNA (Cui et al., Nat. Commun. 8 (1) 191 (2017)). However, given the structural differences between mRNA and these other nucleic acids (Kauffman et al., Nano Lett. 15 (11) 7300e7306 (2015)), modification of existing PACE structures and / or development of new PACE variants have been considered as a means to improve delivery. The high tolerance of lipase catalysts offers structural versatility, particularly in terms of hydrophobicity and molecular weight. Starting from one of the most efficient PACE compositions for DNA delivery and transfection (classical PACE), different polymers with varying MW and lactone content were synthesized to specifically improve mRNA delivery and transfection. [ka]

[0335] The PACE synthesis was modified to vary different parameters in the final polymer. Diethyl sebacate (R = CH2CH3) or sebacic acid (R = H) were used to generate classical (ester / OH)-terminated or acidic (COOH / OH)-terminated PACE, respectively. The duration of the second step of the synthesis was varied from 8 to 72 hours to alter the MW of the classical PACE. The PDL content (10% or 20%) was varied to modify the hydrophobicity of the polymer.

[0336] Gel permeation chromatography (GPC) was used to determine the MW of the polymers. All PACE polymers were able to complex mRNA and form nanosized polyplexes with neutral or negative surface charges.

[0337] When evaluated using the RIBOGREEN® assay, all polymers were capable of encapsulating mRNA with efficiencies ranging from 55 to 76%. The ability of different PACE polymers to transfect mRNA expressing luciferase (LUC) in HEK-293 cells was used to screen for in vitro transfection performance. None of these PACE polymers, representing different structural modifications, provided transfection levels comparable to those of TRANSIT®, a commercially available drug used as a positive control (Figures 1A-1C). However, a clear trend in transfection levels with polymer MW was observed. As the polymer MW decreased from 20 kDa to 5 kDa, transfection efficiencies increased by two orders of magnitude (1.1 x 10, respectively). 5 RLU / mg and 4.4 × 10 7 An increase in RLU / mg was observed. However, this trend appears to plateau at 5 kDa, as the level of transfection was dramatically reduced using PACE with a MW of 2 kDa, comparable to that obtained with free mRNA. Previous studies have reported that the strength and stability of electrostatic complexation between polycations and polyanions increases exponentially with the length of the polycation (Akinc et al., Bioconj. Chem. 14 (5) 979e988 (2003); Tsuchida & Osada, Makromol. Chem. 175 (2) 593e601 (1974); Choosakoonkriang et al. al., J. Pharmaceut. Sci. 92 (8) 1710e1722 (2003), Schaffer et al., Biotechnol. Bioeng. 67 (5) 598e606 (2000)).

[0338] These results indicate that very short polymers (2 kDa) are inefficient for transfection, likely due to insufficient mRNA complexation. Above the 2 kDa threshold, shorter polymers (5 kDa) are more efficient at delivering mRNA than higher MW polymers (10 kDa or 20 kDa), likely due to inefficient release of mRNA from high-MW PACE polyplexes inside cells. High-MW PACE chains contain numerous positively charged and hydrophobic domains, which result in thermodynamically stable binding with mRNA and prevent its release from the polyplex. The results indicate that efficient mRNA delivery derives from a delicate balance of polymer MW; the length of the polymer chain must be long enough to ensure mRNA complexation and polyplex stabilization, yet short enough for mRNA release inside the cell. On the other hand, increasing the PDL content of the 10 kDa PACE polymer from 10% to 20% did not significantly affect the transfection efficiency ( Figure 1 B), which is the opposite of what was observed with pDNA, confirming that the polymer structure benefits from modulation of the respective genetic material.

[0339] This indicates that polymers having a molecular weight of more than 2 kDa to less than 10 kDa, more preferably in the range of 3 kDa to 8 kDa, and most preferably 5 kDa, will be most effective.

[0340] Example 2 PACE transfection efficiency is highly dependent on its end-group composition material and method Polymer end group modification To prepare PACE with different end groups, the parent polymer was synthesized using sebacic acid instead of diethyl sebacate, which gave PACE with a mixture of hydroxyl and carboxyl end groups. Both end groups were then activated with CDI in a 1:40 molar ratio by stirring overnight at room temperature in dry dichloromethane. The mixture was washed three times with deionized water, followed by evaporation of DCM under vacuum to give the reaction product PACE-CDI (see scheme below).

[0341] PACE-CDI was reacted with amine-containing molecules to yield PACE with new terminal groups. Specifically, glycine and AEMAE were used to generate PACE-COOH and PACE-MAE, respectively. For conjugation, 5 mM glycine or AEMAE was reacted with 0.5 mM PACE-CDI in DMSO for 40 hours at room temperature under constant stirring. After the reaction, the mixture was washed with 10 volumes of deionized water and extracted with DCM, followed by evaporation of the DCM under vacuum to yield PACE-COOH and PACE-MAE. Using this protocol, 5 kDa PACE-CDI and ethylenediamine were added in a precise 2:1 molar ratio to synthesize 10 kDa acidic PACE. [ka]

[0342] As shown above, acidic PACE was subjected to CDI activation (1, 2) followed by nucleophilic substitution with amine-containing molecules (3, 4; R = glycine, or AEMAE) to obtain PACE with -COOH or -MAE terminal groups.

[0343] Polymer characterization 1H and 13C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. For inverse-gated 13C NMR, samples were prepared at 50 mg / mL of polymer in chloroform-d, and chromium(III) acetylacetonate (Cr(acac)3) was added as a relaxation agent at a concentration of 5 mg / mL. Signals were recorded using a T1 relaxation time of 10 seconds. The molecular weight (MW) of the polymer was determined using a microSTYRAGEL column (mixed bed; pore size 100 Å to 10 6 The MW was measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a 5000 Å (1000 Å) column. Chloroform containing 0.2 wt% triethylamine was used as the eluent at a flow rate of 1 mL / min. A sample concentration of 2 mg / mL and an injection volume of 100 μL were used. The MW of the polymers was determined based on a conventional calibration curve generated with narrow polydispersity polystyrene standards from Sigma Aldrich (Saint Louis, MO). Empower II GPC software was used to run the GPC instrument and perform MW calculations.

[0344] Polyplex preparation and characterization Unless otherwise specified, polymer:mRNA polyplexes were prepared at a polymer:mRNA weight ratio of 100:1 in 25 mM sodium acetate buffer (pH 5.8). For in vitro experiments, a 10 μg mRNA / mL solution was prepared: 1 μL of polymer solution (100 mg / mL in DMSO) was first diluted with 50 μL of sodium acetate buffer. After brief vortexing, the polymer solution was mixed with 1 μg of mRNA diluted in 50 mL of sodium acetate buffer and vortexed again. The polymer:mRNA mixture was incubated at room temperature for 10 min before use. For in vivo experiments, a 100 μg mRNA / mL solution in sodium acetate buffer was prepared by the same method.

[0345] The hydrodynamic diameter of the polyplexes was measured by dynamic light scattering (DLS) using a Malvern Nano-ZS (Malvern Instruments, UK) after dilution of the polyplexes in DI water at a concentration of 2 μg / mL of mRNA. To measure the zeta potential, the same solution was loaded into a disposable capillary cell and analyzed in the Malvern Nano-ZS.

[0346] The encapsulation efficiency (EE) of mRNA in polyplexes was measured using the Quant-IT RiboGreen RNA kit (Invitrogen, no. R11491) according to the manufacturer's instructions. Because the RiboGreen assay measures the amount of free mRNA in solution, this amount was subtracted from the initial amount added to form the polyplexes to obtain the amount of mRNA complexed within the polyplexes.

[0347] cell culture HEK293 and SH-SY5Y cells were cultured in 4.5 g / L glucose DMEM medium (Gibco No. 11965) supplemented with 10% FBS and 1% pen / strep at 37°C and 5% CO. Daoy cells were cultured in 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg / L sodium bicarbonate EMEM medium (ATCC No. 30-2003) supplemented with 10% FBS and 1% pen / strep at 37°C and 5% CO.

[0348] In vitro transfection For in vitro transfection of Fluc mRNA, cells were seeded into 24-well plates at a density of 75,000 cells / well in 500 μL of medium and incubated overnight to ensure adhesion. The medium was replaced with 400 μL of transfection medium (culture medium containing 10% FBS but without pen / strep), and 100 μL of polyplex (1 μg total mRNA) was added to each well. For the free mRNA control, 1 μg of mRNA was diluted in 100 μL of acetate buffer and added to the well. A commercially available mRNA transfection kit, TransIT, was used as a control. Briefly, 1 μg of mRNA was mixed with 0.7 μL of Boost Reagent and 1.1 μL of TransIT Reagent in 100 μL of OPTIMEM medium (Gibco #11058021). 24 hours after transfection, luciferase expression was measured. Cells were washed and lysed using 200 μL of 1× lysis buffer (Promega, No. E397A) and subjected to one freeze-thaw cycle at −80° C. 20 μL of the lysate was then mixed with 100 μL of luciferase reporter reagent (Promega, No. E1483), and luminescence was read in a Glomax luminometer (Promega). The protein content of the lysate was measured using the Pierce BCA Protein Assay Kit (ThermoFisher, No. 23225). All experiments were performed in duplicate, with three independent experiments.

[0349] result 2 is a bar graph showing the transfection efficiency of PACE-COOH and PACE-MAE with MW of either 5 kDa or 10 kDa, synthesized by the method described in Example 2. Statistical significance was determined by Student's t-test (denoted as follows: *, P<0.05; ***, P<0.001).

[0350] PACE, synthesized by terpolymerization of diethyl sebacate, PDL, and MDEA, contains a mixture of methyl (from diethyl sebacate) and hydroxyl (from MDEA) end groups. When the monomer diethyl sebacate was replaced by sebacic acid for PACE synthesis to form acidic PACE, its mRNA transfection efficiency doubled (Figure 2). Different end groups on the same polymer can dramatically affect pDNA transfection efficiency (Sunshine et al., Biomacromolecules 12 (10) 3592e3600 (2011); Sunshine et al., Mol. Pharm. 9 (11) 3375e3383). (2012)). Experiments were designed to determine whether end group composition could also affect mRNA transfection, and more specifically, whether modified end groups on PACE could improve mRNA delivery.

[0351] Acidic PACE, containing a mixture of carboxyl and hydroxyl end groups with a molecular weight of around 5 kDa, was used as the starting material. Both of these end groups could be activated by 1,10-carbonyldiimidazole (CDI), which was further substituted with amine-containing molecules, including glycine or AEMAE, to form carboxyl (eCOOH) end groups (PACE-COOH) or (methylamino)ethanol (-MAE) end groups (PACE-MAE), respectively.

[0352] Acidic PACEs, PACEs bearing -COOH or -MAE terminal groups, were obtained by CDI activation followed by nucleophilic substitution with amine-containing molecules (3, 4; R = glycine, or AEMAE).

[0353] These two monomers were chosen to mimic the naturally occurring mix of end groups found in acidic PACE in order to identify end groups with higher transfection efficiency. The reaction mechanism ensured >90% conversion for both end groups, as confirmed by NMR spectroscopy.

[0354] We next evaluated the ability of these polymers to transfect mRNA in vitro. Figure 3A is a bar graph showing the effect of MW (average LUC per mg of protein) using classical PACE with 10% PDL content and molecular weights of 2 kDa, 5 kDa, 10 kDa, and 20 kDa. Figure 3B is a bar graph showing the effect of PDL content (average LUC per mg of protein) using 10 or 20% PDL for a 10 kDa classical PACE polymer. Figure 3C is a bar graph showing the transfection efficiency (average LUC per mg of protein) of acidic PACE and classical PACE with 5 kDa MW and 10% PDL content. Results are presented as the mean ± SD of three independent experiments performed in duplicate.

[0355] Significant differences in transfection efficiency were observed between PACEs with different end groups, as the 5 kDa PACE-MAE polymer demonstrated a transfection efficiency two orders of magnitude higher than that of PACE-COOH of the same MW (Figure 3C). This effect can be explained by the difference in EE of these two polymers, as PACE-MAE encapsulated 98% of the total mRNA, while PACE-COOH encapsulated only 18%. Acidic PACEs with molecular weights higher than 5 kDa were not directly synthesized due to technical challenges (difficulty in removing the water byproduct as the polymer chains grow longer). However, 10 kDa acidic PACEs can be obtained by CDI activation of the 5 kDa polymer followed by crosslinking of the two polymer chains with ethylenediamine molecules. When 10 kDa acidic PACE was modified with either carboxyl or hydroxyl end groups, its transfection efficiency was significantly improved compared with its 5 kDa counterpart (Figure 3A), implying that for PACE-COOH and PACE-MAE polymers, increasing MW leads to increased transfection efficiency.

[0356] This behavior differs dramatically from classical PACE, where transfection efficiency decreases as MW increases, further demonstrating the importance of the terminal group in transfection efficiency. While the EE of PACE-COOH increased from 18% to 45% when MW was increased from 5 kDa to 10 kDa, the EE of PACE-MAE did not change significantly (98% at 5 kDa vs. 95% at 10 kDa), indicating that increased EE is not the only explanation for the improved transfection efficiency.

[0357] These data indicate that the contributions of PACE end groups and MW are more than additive. The properties of the PACE end groups likely contribute to its ability to complex with mRNA, and MW may also play a role in complexation and may be involved in downstream biological steps such as endosomal escape and / or mRNA release in the cytosol.

[0358] Example 3 "Top-down" activation of PACE for mRNA delivery activated PACE polymer material and method Polymer activation Polymers (20-30 mg) with different starting MW were spread evenly on the inner surface of glass vials to form a thin film that ensured efficient air permeation. The vials were then incubated at a controlled temperature (typically 37 °C) with exposure to humid airflow for different lengths of time.

[0359] in vitro toxicity To evaluate the cytotoxicity of PACE polymer and TransIT, HEK293 cells were seeded in 96-well plates at a density of 10,000 cells / well in 100 μL of medium and incubated overnight to ensure adhesion. Polymer:mRNA polyplexes or TransIT / mRNA complexes were formed using the same w:w ratio as for the transfection experiments and diluted in transfection medium at different concentrations. 100 μL of polyplex-containing medium was added to the wells to achieve final mRNA concentrations ranging from 0.01 to 20 μg / mL. After 24 hours of incubation, cell viability was measured using the MTT assay. All experiments were performed in duplicate in three independent experiments.

[0360] Lyophilization of polyplexes PACE:mRNA polyplexes were prepared using sodium acetate buffer, and different concentrations of trehalose solution (30 mg / mL or 60 mg / mL in 25 mM sodium acetate buffer, pH = 5.8) were added to the polyplex suspension at a 1:1 volume ratio to obtain final trehalose concentrations of 0, 15, or 30 mg / mL. The mixture was then flash-frozen in liquid nitrogen and lyophilized for 2 days. At the end of lyophilization, the polyplexes were resuspended in sodium acetate buffer, and transfection efficiency was evaluated in HEK293 cells. Characterization of transfection and gene expression was performed using the methods described above.

[0361] result After initial screening by "bottom-up" synthesis of a library of PACEs, the polymer's MW and end-group composition were identified as two key parameters determining the transfection efficiency of this material for mRNA. The effect of MW appeared to significantly affect transfection efficiency within a narrow range of 5 kDa to 10 kDa. A "top-down" approach was adapted to simultaneously vary the MW and end-group of PACE by controlled hydrolysis of high-MW polymers. By doing this, PACE end-groups and MWs with higher transfection efficiency were modulated and identified.

[0362] This method generated a new family of materials called actuated PACE (aPACE). These biodegradable aPACE polymers conferred high transfection levels both in vitro and in vivo with negligible toxicity.

[0363] aPACE was produced by controlled hydrolysis of the ester backbone. Exposure to air at moderate temperatures provides mild conditions for the hydrolysis of PACE, thus reducing its MW and exposing hydroxyl and carboxyl end groups.

[0364] To confirm this, aPACE was characterized by NMR spectroscopy and GPC. NMR analysis of aPACE demonstrated that the initiation process resulted in the exposure of hydroxyl and carboxyl end groups, as observed by an increase in the area under the hydroxyl peak (58.2 and 58.9 for aPACE and classical PACE, respectively) and the appearance of a distinct carboxyl peak at 178 ppm in aPACE compared to classical, uninitiated PACE. GPC showed that the initiation process reduced the MW of all PACE polymers (Figure 4).

[0365] The hydrolysis of polyesters is well known. These reactions typically proceed at elevated temperatures, pressures, and / or in the presence of a catalyst. It has been observed that initiation can be accelerated when carried out at 100°C compared to 37°C. However, the desired molecular weight of aPACE was obtained using milder temperatures, which provided for a more reproducible process.

[0366] To investigate the effect of the initiation protocol on PACE transfection efficiency for mRNA, we tested aPACE polymers generated from different starting MWs at different initiation times. Self-assembled polyplexes generated from aPACE and mRNA were highly effective in transfecting HEK293 cells, resulting in luciferase expression levels comparable to those of the positive control, TransIT (Figures 5A-5B). Transfection efficiency depended on the initiation time and the initial MW of the initiation polymer, with an optimal initiation time for each initial MW (Figure 5A: 5 days for 5 kDa polymers, 10 days for 10 kDa polymers, and 30 days for 20 kDa polymers). With these improved initiation times, all aPACE polymers provided levels of transfection comparable to TransIT and significantly higher transfection levels compared to their non-initiation counterparts (Figure 2, p<0.005).

[0367] When tested for cytotoxicity at different concentrations, TransIT induced significant cell death, whereas all aPACE formulations were not cytotoxic for similar amounts of delivered mRNA (Figure 5C).

[0368] An accelerated activation process in which the 20 kDa polymer was activated at 100°C for 6 hours resulted in similar transfection levels as the 20 kDa polymer activated at 37°C for 30 days (Figure 5D). Despite the accelerated activation process, a similar kinetic profile was observed when activation was performed at 100°C compared to 37°C. Both processes exhibited an optimal increase in transfection efficiency followed by a decrease in activity. This observation indicates that a similar time-dependent process is occurring at both temperatures.

[0369] The MWs of these aPACEs were close to each other, ranging from 6 kDa to 8 kDa. Considering the differences in their transfection efficiencies, the data demonstrate a rapid change in transfection efficiency within a narrow range of aPACE MWs, highlighting the value of using a "top-down" priming approach for screening. Because delivery vehicles should be tailored for transport of different genetic materials, a controlled "top-down" priming process constitutes a simple and powerful method for optimizing the MW and end-group combinations for each genetic material. Advantages of this process include the precision of fine-tuning key factors such as MW and the simplicity of synthesizing separate libraries of polymers in the priming step from the same starting material. Overall, the results confirmed that the approach provided optimal combinations of MW and end-groups tailored for mRNA delivery and transfection.

[0370] To ensure the broad efficacy of aPACE, these polymers were also tested for transfection in Daoy cells, a human medulloblastoma cell line, and SH-SY5Y cells, a human neuroblastoma cell line. As observed in HEK293 cells, the activation process significantly increased PACE transfection efficiency in both cell lines.

[0371] The effect of lyophilization on aPACE:mRNA polyplexes was also examined. Trehalose was used as a cryoprotectant, and transfection efficiency was evaluated in HEK293 cells after polyplex reconstitution. Lyophilized polyplexes prepared with aPACE were as efficient as fresh polyplexes, even in the absence of a cryoprotectant, whereas the addition of a high concentration of trehalose (6%) slightly increased transfection efficiency.

[0372] Example 4 aPACE is effective for mRNA delivery in vivo material and method in vivo studies All animal work was completed at Yale University in accordance with the guidelines of the Yale Animal Resource Center (YARC) and the Institutional Animal Care and Use Committee (IACUC). Female BALB / c mice (20 g, Charles River, Willimantic, CT, USA) were used for the experiments.

[0373] PACE:mRNA polyplexes (0.1 mg / mL in mRNA, N=3), aPACE:mRNA polyplexes (0.1 mg / mL in mRNA, N=3), TransIT:mRNA complexes (0.1 mg / mL in mRNA, N=3), free mRNA diluted in sodium acetate buffer (0.1 mg / mL in mRNA, N=3), or sodium acetate buffer (25 mM, pH 5.8, N=3) were administered intravenously through the tail vein in a volume of 200 μL.

[0374] Retroorbital blood samples (50 μL) were collected before particle administration and 6, 24, 48, 72, and 7 days after injection. Immediately after collection, plasma was separated by centrifugation (3000 g, 10 min) and frozen at -80°C until further analysis. Plasma EPO concentrations were measured using an ELISA kit (R&D Systems). 24 hours and 7 days after injection, livers, kidneys, and spleens were collected, processed for H&E staining, and scored by an external pathologist for any abnormal cellular morphology. GraphPad Software (La Jolla, California, USA, www.graphpad.com / ) was used for graphing and statistical analysis. Statistical significance was tested using a two-tailed, unpaired Student's t-test at a 95% confidence level.

[0375] result To evaluate aPACE polymers for delivery of therapeutically relevant mRNA, their ability to deliver EPO-expressing mRNA was evaluated in mice. Two aPACE polymers (5 kDa PACE with 5-day onset and 10 kDa PACE with 10-day onset) were tested, along with a positive control, TransIT, which was chosen for its proven efficacy for in vivo mRNA delivery (Kariko et al., Mol. Ther. 2014). (5) 948e953 (2012)). The 20 kDa PACE, which was activated for 30 days, was not tested because the polyplexes formed were not sufficiently stable in terms of size at the high concentrations required for in vivo administration.

[0376] EPO mRNA:aPACE polyplexes were intravenously injected into wild-type mice to deliver a total dose of 20 mg of mRNA, and blood was collected at different time points after injection to measure EPO levels by ELISA. The mRNA polyplexes using the optimized aPACE polymer demonstrated high efficacy in delivering EPO mRNA, as reflected in the subsequent EPO production.

[0377] Six hours after injection, the best non-activating polymer, 5 kDa non-activating PACE, produced high levels of EPO (530 ng / mL), higher than the positive control, TRANSIT® (170 ng / mL). 10 kDa non-activating PACE produced lower EPO levels (14 ng / mL) at 6 hours, confirming the trend observed in vitro for non-activating polymers, where polyplexes formed from polymers with MW greater than 5 kDa provided lower transfection efficiencies.

[0378] Figure 5A is a line graph showing luciferase mRNA transfection efficiency (mean LUC per mg of protein) as a function of prime time (days), demonstrating the preferred prime time for each MW (lower data point at day 0, identified as 20 kDa; middle data point at day 0, identified as 10 kDa; upper data point at day 0, identified as 5 kDa). Primed 5 kDa PACE did not significantly increase EPO production compared to the non-primed form, whereas primed 10 kDa over 10 days at 37°C significantly increased EPO levels to 1100 ng / mL (Figure 5A, p<0.0001).

[0379] Figure 5B is a bar graph showing the transfection efficiency (average LUC per mg of protein) using non-primed PACE and primed PACE of different initial MW with their preferred primed times (5 days for 5 kDa polymers, 10 days for 10 kDa polymers, and 30 days for 20 kDa polymers; **p<0.005). Figure 5C is a line graph showing the cytotoxicity profile (% cell viability) of mRNA:aPACE polyplexes (5 kDa 5 days, 10 kDa 10 days, and 20 kDa 30 days (cluster of upper lines)) compared with mRNA:TransIT complexes (lower lines). Blood chemistry and histology analyses demonstrated that aPACE did not induce systemic toxicity 24 h or 7 days after administration. Figure 5D is a bar graph showing the transfection efficiency (average LUC per mg of protein) of aPACE using different primed temperatures (**p<0.005).

[0380] Figure 6A is a bar graph showing EPO blood concentrations 6 hours after IV administration of mRNA (20 mg total) using TRANSIT®, 5 kDa non-activated PACE, 5-day activated 5 kDa aPACE, 10 kDa non-activated PACE, or 10-day activated 10 kDa aPACE. Results are presented as mean ± SD for N=3 animals (****p<0.0001). Figure 6B is a line graph showing the time course of EPO production after IV administration of mRNA (20 mg total) using vehicle (lower line), TransIT (middle line), or 10-day activated 10 kDa aPACE (upper line). Results are presented as mean ± SD for N=3 animals (***p<0.001 and *p<0.05). Figures 6C-6F are bar graphs showing blood chemistry (AST (Figure 6C), ALT (Figure 6D), urea (Figure 6E), creatinine (Figure 6F)) analysis 24 h and 7 days after IV administration of acetate buffer, free mRNA, or mRNA:aPACE polyplexes. Results are presented as mean ± SEM for N = 3 animals.

[0381] For both TRANSIT® and aPACE polyplexes, EPO production peaked 6 hours after injection. Although repeated injections of acetate buffer (vehicle) followed by bleeding induced a slight increase in EPO blood levels over time, EPO levels after administration of the 10 kDa aPACE polyplex were significantly higher than those obtained after administration of TRANSIT® for up to 48 hours (p<0.001 at 6 hours and p<0.05 at 24 and 48 hours).

[0382] Using the best aPACE polymer (10 kDa activated for 10 days), significant EPO production was obtained for up to 48 hours, significantly longer than the blood half-life of free EPO (approximately 2 hours).

[0383] These results demonstrate that the end group and MW of PACE influence its mRNA transfection. The combination of MW and end group in the initiator polymer enabled efficient mRNA complexation and transfection in vitro and in vivo, while the low cation density of PACE ensured a low toxicity profile.

[0384] Example 5 PACE end groups affect messenger RNA loading onto polyplexes material and method Synthesis of end-group modified PACE polymers and polyplexes The PACE scaffold, made from a cationic diol, diethyl sebacate, and lactone, has a mixture of hydroxyl and methyl end groups (Zhou, et al., Nature Materials, 11:82-90 (2012). [PMCID: PMC4180913]).

[0385] To modify PACE with different end groups, the parent polymer was synthesized using sebacic acid instead of diethyl sebacate, which gave PACE with a mixture of hydroxyl and carboxyl end groups. Both end groups were activated with carbodiimidazole (CDI) in a 1:40 molar ratio by stirring overnight at room temperature in dry dichloromethane (DCM). The mixture was washed three times with deionized water, followed by evaporation of the DCM under vacuum to yield the reaction product PACE-CDI: CDI activation (1, 2) followed by nucleophilic substitution with amine-containing molecules (3, 4), as shown below and in Examples 1–4. [ka]

[0386] For conjugation, 5 mM of the amine-containing molecule was reacted with 0.5 mM of PACE-CDI in DMSO under constant stirring at room temperature for 40 hours. After the reaction, the mixture was washed with 10 volumes of deionized water and extracted with DCM, followed by evaporation of the DCM under vacuum to give PACE with a new terminal group.

[0387] The chemical structure of the end group is shown below. [ka] [ka]

[0388] Ribogreen mRNA loading assay Ribogreen is a small dye that fluoresces after binding to messenger RNA. After complexing with PACE, the messenger RNA becomes inaccessible to the dye, and a lower fluorescence signal is observed. The mRNA, polyplexes, and ribogreen were mixed, and fluorescence was monitored.

[0389] Cy5 mRNA / polyplex uptake assay The mRNA encoding the ddRLuc-Fc protein and FLuc was first coencapsulated by PACE polymer, and then co-transfected with a proteasome inhibitor, FLuc. Expi293F cells were fed for 7 hours in the presence of epoxomycin. Cells were then checked by dual luciferase assay to reveal RLuc activity (n=4). Data were normalized to TRANSIT® (set to 1).

[0390] Cy5-labeled mRNA formed polyplexes with PACE having different terminal groups and delivered them to HEK293 cells, which were monitored for fluorescence.

[0391] Endosomal escape assay Using PACE with different end groups, endosomal escape of polyplexes prepared with fLuc mRNA was quantified with the ddRLuc system.

[0392] result PACE end groups affect messenger RNA loading onto polyplexes. The results of the ribogreen assay are shown in Figure 7 and are in close agreement with the corresponding gel retardation assay.

[0393] The PACE end group affects the cellular uptake of polyplexes. The results of the cellular uptake assay are shown in Figures 8A-8D. Figure 8A shows a schematic of what occurred to generate the results in Figures 8B, 8C, and 8D, showing the efficiency of mRNA encapsulation, uptake, and endosomal escape, as well as transfection efficiency. The efficiency of mRNA encapsulation was quantified by Ribogreen assay (n = 4). Polymers encapsulating Cy5-labeled mRNA were delivered to Expi293F cells, followed by FACS to quantify the uptake level (n = 3) (Figure 8B). Statistical differences were analyzed by unpaired Student's t-test between each polymer with an mRNA group. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0394] PACE with different terminal groups demonstrated different abilities to be taken up by cells. The PACE terminal group affects the endosomal escape of mRNA. The results of the endosomal escape assay using fLuc mRNA and PACE with different terminal groups are shown in Figure 8C.

[0395] The PACE end group chemistry affects the transfection efficiency of the polymeric delivery system. The transfection efficiency of PACE was measured by the fluc activity produced by HEK293 cells. The results are shown in Figure 8D.

[0396] Figures 9A-9C show the linear correlation between transfection efficiency (expressed as flux activity) and different steps of transfection, including mRNA loading (Figure 9A), uptake (Figure 9B), and endosomal escape (Figure 9C). As the biological step approaches protein production, the R value of the linear correlation increases, indicating better predictive power.

[0397] Example 6 Quantifying Endosomal Escape of a Library of Polymers for mRNA Delivery material and method Polymer Synthesis PACE synthesis was performed as described above, with some modifications to generate PACE polymers with different end groups. The parent polymer was synthesized using sebacic acid, which gave PACE with a mixture of hydroxyl and carboxyl end groups. Both end groups were activated with CDI in a 1:40 molar ratio by stirring overnight at room temperature in dry dichloromethane. The mixture was washed three times with deionized water, followed by evaporation of the DCM under vacuum to yield the product PACE-CDI.

[0398] PACE-CDI was then reacted with amine-containing compounds (Table 1) to yield PACEs with new terminal groups. For conjugation, 5 mM of each compound was reacted with 0.5 mM of PACE-CDI in DMSO for 40 hours at room temperature under constant stirring. After the reaction, the mixture was washed with 10 volumes of deionized water and extracted with DCM, followed by evaporation of the DCM under vacuum to yield new polymers designated P1–P31 (Table 1).

[0399] Polymer characterization 1H NMR spectra were recorded on an Agilent DD2 400 MHz NMR spectrometer. Signals were recorded using a T1 relaxation time of 10 seconds. The molecular weights (MW) of the polymers were measured by gel permeation chromatography (GPC) using a Waters HPLC system equipped with a microSTYRAGEL column (mixed bed; pore size 100 Å–106 Å). Chloroform containing 0.2 wt% triethylamine was used as the eluent at a flow rate of 1 mL / min. A sample concentration of 2 mg / mL and an injection volume of 100 μL were used. The MW of the polymers was determined based on a conventional calibration curve generated with narrow polydispersity polystyrene standards from Sigma Aldrich (Saint Louis, MO, USA). The GPC instrument was run and MW calculations were performed using Empower II GPC software.

[0400] The PACE end-group modification reaction was monitored using NMR spectroscopy. The characteristic peaks of PACE were distributed in the 1-4.5 ppm region. When the MDEA monomer was the end group, its terminal hydrogens shifted from 4.2 (g) to 3.6 (g') ppm and 2.7 (e) to 2.6 (e') ppm, respectively. CDI activation resulted in the formation of split peaks in the 7-8.5 ppm region, due to the formation of two different PACE-CDI conjugations (Figure S2). The terminal hydrogens of the MDEA monomer shifted from 3.6 (g') to 4.4 (g'') ppm and 2.6 (e') to 2.8 (e'') ppm, respectively. The terminal hydrogens of the sebacic acid monomer shifted from 2.3 (c) to 2.4 (c'') ppm and 1.5 (b) to 1.7 (b'') ppm, respectively. The conjugation reactions of 31 small molecules were then confirmed by the disappearance of the split peak in the 7-8.5 ppm region and the shift of the g'', e'', c'', and b'' peaks. [Table 1-1] [Table 1-2]

[0401] Polyplex preparation and characterization Unless otherwise specified, polymer:mRNA polyplexes were prepared at a polymer:mRNA weight ratio of 100:1 in 25 mM sodium acetate buffer (pH 5.8). For in vitro experiments, a 10 μg mRNA / mL solution was prepared: 1 μL of polymer solution (100 mg / mL in DMSO) was first diluted in 50 μL of sodium acetate buffer. After brief vortexing, the polymer solution was mixed with 1 μg of mRNA diluted in 50 μL of sodium acetate buffer and vortexed again. The polymer:mRNA mixture was incubated at room temperature for 10 minutes before use. For in vivo experiments, a 100 μg mRNA / mL solution in sodium acetate buffer was prepared by the same method.

[0402] The hydrodynamic diameter of the polyplexes was measured by dynamic light scattering (DLS) using a Malvern Nano-ZS (Malvern Instruments, UK) after dilution of the polyplexes in deionized water at a concentration of 2 μg / mL of mRNA. To measure the zeta potential, the same solution was loaded into a disposable capillary cell and analyzed in the Malvern Nano-ZS.

[0403] Transmission electron microscope (TEM) 2 μL of nanoparticles were applied to a TEM grid (Electron microscopy sciences, catalog number CF400-Cu-TH). After 1 minute, the liquid was carefully removed. Then, one drop of staining solution (Nanoprobes, NANO-W) was added to the top of the grid. After 1 minute, the liquid was removed. The grid was allowed to air dry. Images were taken using a TEM (FEI Tecnai Osiris 200 kV TEM).

[0404] cell culture Expi293F™ cells (Thermo Fisher, Cat. No. A14527) were maintained at 37°C and 8% CO2 in Expi293™ Expression Medium (Thermo Fisher, Cat. No. A1435102) with constant shaking on a microplate shaker (VWR, Cat. No. 89032-092).

[0405] ddRLuc-Fc purification The plasmid encoding ddRLuc-Fc was transfected into Expi293F cells using linear polyethyleneimine (Polysciences Inc., catalog no. 23966). Sixty hours after transfection, cells were harvested, and ddRLuc-Fc was purified from the cell lysate. Cells were lysed in TBS (10 mM Tris, 150 mM NaCl, pH 7.5) supplemented with 0.5-1% Triton® X-100 (American Bio, catalog no. AB02025) and EDTA-free protease inhibitor cocktail (Roche, catalog no. 11873580001) for 1 hour at 4°C. The cell lysate was then clarified by centrifugation at 110,000 × g at 4 ° C for 1 hour in an Optima L-100K ultracentrifuge (Beckman Coulter) followed by filtration through a 0.22 μm filter (EMD Millipore, catalog number SCGPT01RE). Protein A-SEPHAROSE® beads (GE healthcare, catalog number 17-0780-01) were added to the filtered cell lysate and incubated for 3 hours at 4 ° C with rotation for protein binding. The beads were washed in TBS, and the protein was eluted with 100 mM glycine (pH 3.3). The eluate was then neutralized, concentrated, and buffer-exchanged into PBS.

[0406] Co-encapsulation of FLuc mRNA and ddRLuc-Fc by PACE polymer An agarose gel retardation assay was performed to check the co-encapsulation efficiency of FLuc-encoding mRNA and ddRLuc-Fc probe: free mRNA and protein were allowed to migrate in the gel while the encapsulated ones were retained in the loading well.

[0407] Horizontal agarose gel electrophoresis Five micrograms of ddRLuc-Fc and 1 microgram of mRNA encoding FLuc were mixed with 100 micrograms of polymer in a total volume of 18 microliters and incubated at room temperature for 10 minutes. Two microliters of 10x DNA loading buffer was added to the polyplexes, followed by loading onto a 1% agarose gel. mRNA migration was detected using UV light, and protein migration was detected by Coomassie blue staining (Bio-Rad, catalog number 161-0786).

[0408] Luciferase assay Expi293F cells were first treated with 200 nM epoxomicin (ApexBio Technology, Catalog No. A2606) for 30 minutes at 37°C and then added at 200,000 cells per well to a 96-V bottom plate containing free mRNA (negative control), TRANSIT® (Mirus, Catalog No. MIR2250, positive control), or polymer co-encapsulating ddRLuc-Fc and FLuc mRNA. Seven hours after transfection, cells were washed three times with PBS and lysed in 50 μL of passive lysis buffer (Promega, Catalog No. E1910) supplemented with 20 μM z-VAD(OMe)-FMK (Abcam, Catalog No. ab120487) for 20 minutes at room temperature. Lysates were then lysed using a Centro XS 3Assays were performed using an LB 960 high-sensitivity microplate luminometer (Berthold, Cat. No. 46970). RLuc luminescence was measured for 1 second using native-Coelenterainze (Nanolight Technology, Cat. No. 303), and FLuc luminescence was measured for 3 seconds using a luciferase assay system (Promega, Cat. No. E1501). All independent experiments were performed in triplicate.

[0409] In vitro translation (IVT) IVT of mRNA-loaded polyplexes was performed using a rabbit reticulocyte lysate system (Promega, catalog no. L4960) according to the manufacturer's instructions. Polyplexes loaded with 1 μg of mRNA encoding FLuc were incubated in 50 μL of lysate mixture at 37°C for 75 minutes. 2 μL samples were taken from the reaction mixture every 15 minutes and immediately frozen on dry ice. After sample collection, FLuc activity in all samples was measured using a luciferase assay system (Promega, catalog no. E1501). The rate of IVT for a particular polyplex was evaluated as the slope of the linear regression between FLuc activity and the time of sample collection.

[0410] Measurement of encapsulation efficiency The encapsulation efficiency of mRNA in polyplexes was measured using the Quant-IT RiboGreen RNA kit (Invitrogen, no. R11491) according to the manufacturer's instructions. Because the RIBOGREEN® assay measures the amount of free mRNA in solution, this amount was subtracted from the initial amount added to form the polyplexes to obtain the amount of mRNA complexed within the polyplexes.

[0411] Fluorescence-activated cell sorting (FACS) Expi293F cells were seeded into 24-well plates at a density of 5,000 cells / well. After 12 hours, nanoparticles encapsulating Cy5 mRNA were added to each well. After 1 hour, cells were collected for FACS analysis (Life Technologies, Attune NxT).

[0412] result The results are shown in Figures 10A-10B. The IVT rate of all polyplexes was measured by the production of FLuc activity per hour. Simple linear regressions between FLuc mRNA transfection results and IVT rate, as well as IVT rate and encapsulation efficiency, were performed using the quantified and normalized data. Each data point represents a PACE polymer with a unique end group. The straight line represents the best fit. R 2 Label the values.

[0413] Effect of polymer end group and route of administration The in vitro translation rate of mRNA polyplexes does not correlate with transfection efficiency. Figures 10A and 10B are graphs demonstrating that the in vitro translation rate of mRNA polyplexes does not correlate with transfection efficiency. The IVT rate of all polyplexes was measured by the production of FLuc activity per hour. Simple linear regressions between (Figure 10A) FLuc mRNA transfection results and IVT rate, and (Figure 10B) IVT rate and encapsulation efficiency were performed using quantified and normalized data. Each data point represents a PACE polymer with a unique end group. The straight line represents the best fit. R 2 Label the values.

[0414] Example 7 Preparation of PACE-PEG blend polyplexes and pulmonary delivery of PACS-polyplex formulations Although mRNA-based vaccines are a promising strategy that can be easily adapted to emerging viral strains such as SARS-CoV-2, more research is needed into delivery strategies to develop optimal mRNA vaccine formulations. A library of cationic poly(amine-co-ester)s, or PACEs, with different terminal groups has demonstrated that they are efficient and safe mRNA delivery vehicles.

[0415] Recent events due to diseases such as COVID-19 and resulting lung diseases have highlighted the importance of improved formulations for pulmonary delivery. The following study screened PACE polyplexes in vitro to identify promising formulations for inhaled delivery of mRNA to the lung. This study investigated the effect of polyethylene glycol (PEG) on mRNA transfection efficiency and its role in stabilizing the nanoparticles. Additional studies demonstrated high levels of transfection in the lungs after pulmonary delivery of the polyplexes.

[0416] material and method A library of PACE polymers with different end groups was screened for size, stability, and transfection efficiency of EGFP mRNA in A459 human lung cells, as described in the previous examples and in Table 1. To optimize the formulation, polyplexes were prepared in buffer solutions ranging in pH from 4.8 to 6, and a small volume of PEG-conjugated PACE (PACE-PEG) was incorporated to improve polyplex stability.

[0417] Polymer Synthesis The PACE polymer was synthesized as described above.

[0418] Polyplex formulation All PACE polyplexes were formed at a 1:100 PACE-to-mRNA weight ratio. The polymer was dissolved in DMSO (100 mg / mL) overnight at 37°C with shaking. To form polyplexes, mRNA was diluted to a concentration of 20–200 μg / mL in 25 mM sodium acetate buffer (pH 4.8–6.0). In a separate tube, dissolved PACE was diluted to a concentration of 2–20 mg / mL in 25 mM sodium acetate buffer (pH 4.8–6.0) and vortexed for 15 seconds. The diluted mRNA and PACE were then combined and vortexed for 25 seconds. The solution was left at room temperature for 10 minutes before use to allow polyplex formation. The size and zeta potential of the polyplexes were characterized by dynamic light scattering (DLS, Zetasizer Pro, Malvern Analytical).

[0419] To formulate PACE-PEG blend polyplexes, PACE-PEG and PACE were separately dissolved in DMSO at a concentration of 100 mg / mL. The dissolved PACE-PEG was diluted with the dissolved PACE (using intermediate dilutions, if necessary) to create a blend polymer solution with the desired PACE-PEG concentration. Polyplexes were then prepared as described above.

[0420] Polyplexes coated with DSPE-PEG (PEG2k, Nano OCS) were formed by dissolving DSPE-PEG in DMSO (10 mg / mL). The dissolved DSPE-PEG was then combined with PACE in DMSO (100 mg / mL) at a volume ratio of 1:10. Intermediate DSPE-PEG dilutions in DMSO were performed as necessary to achieve the desired final ratio of DSPE-PEG to PACE. Polyplexes were then formed as described above by substituting the DSPE-PEG / PACE blend in DMSO for PACE in DMSO.

[0421] Polyplex stability studies To assess polyplex stability, polyplexes were formulated in sodium acetate buffer to a final concentration of 1 mg / mL as described above. 20 μL of polyplex solution was added to a tube along with 1 mL of phosphate-buffered saline (PBS). Samples were then placed in a shaking incubator (300 rpm, 37°C) and removed at various time points for DLS sizing. Jiang Y, Lu Q, Wang Y, et al. Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery. Nano Lett. 2020;20(2):1117-1123. doi:10.1021 / acs.nanolett.9b04426

[0422] In vitro screening The A549 human lung cell line (ATCC) was used for all in vitro screening experiments. Cells were grown in a 1:1 solution of DMEM:F12 (GE Healthcare) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals) and 50 μg / mL gentamicin and stored in a 37°C incubator under 5% CO2. 24 hours before polyplex delivery, cells were seeded at 50,000 cells per well in 24-well plates. Immediately before polyplex delivery, the cell medium was refreshed and cells were treated with 0.5 μg of EGFP mRNA (Trilink Biotechnologies) using PACE polyplex. 24 hours after treatment, cells were rinsed with PBS, dissociated with TrypLE Express enzyme (Thermo Fisher Scientific) for 15 minutes, and then transferred to a 96-well round-bottom plate. Cells were washed once with 2% bovine serum albumin (BSA) in PBS and then stained with Zombie Live / Dead Fixable Near IR (Biolegend) according to the manufacturer's instructions. After staining, cells were washed once more with 2% BSA in PBS and then analyzed by flow cytometry (Attune). NxT) and the results were analyzed in FlowJo version 10.6.2.

[0423] In vivo assay All animal work was completed at Yale University in accordance with the guidelines of the Yale Animal Resource Center (YARC) and the Institutional Animal Care and Use Committee (IACUC). Male BALB / c mice (20 g, Charles River, Willimantic, CT, USA) were used for the experiments. PACE:mRNA polyplexes (0.1 mg / mL mRNA, N=3) diluted in sodium acetate buffer were administered intravenously or intraperitoneally in a volume of 200 μL. In vivo bioluminescence (photons / second / cm) was measured. 2 / sr) were detected and quantified by IVIS Spectrum (PerkinElmer) 6 hours after injection.

[0424] inhalation delivery In vivo experiments were performed in 10- to 30-week-old C57BL / 6 mice (EGFP mRNA delivery) and BALB / c mice (FLuc mRNA delivery). Polyplex solutions (10 mg / mL polymer) were prepared immediately before administration. For inhalation delivery, mice were anesthetized with isoflurane and suspended by their incisors. The tongue was retracted with forceps, and 5 μg of mRNA in PACE polyplex was administered to the back of the mouth. The tongue was held in the retracted position for 10 breaths while the polyplex was inhaled.

[0425] For analysis of EGFP expression by flow cytometry, mice were euthanized 24 hours later. The lungs were perfused with 10 mL of PBS followed by 1 mL of dispase (50 U / mL, Corning Life Sciences). The trachea was then intubated with a blunt needle, and the lungs were inflated with 1 mL of dispase (50 U / mL). A suture was then tied along the trachea. The lungs were removed and placed in ice-cold PBS until further processing. To prepare single-cell suspensions for flow cytometry, lung tissue was minced into 2-3 mm pieces with scissors and transferred to a 15 mL tube with 5 mL of DNase 1 (1 mg / mL, Roche Diagnostics) and collagenase 1 (5 mg / mL, Worthington Biochemical) in PBS and incubated for 30 min at 37°C with shaking. The tissue pieces were then drawn into a 5 mL syringe with an 18-gauge needle 3-5 times. At the final aspiration, the fluid was drawn onto a pre-wetted 70 μm filter. Using a syringe plunger, any remaining tissue on the filter was gently disrupted. The filter was rinsed with 2 mL of 0.5% BSA in PBS, and the resulting cell suspension was centrifuged at 1,200 rpm for 5 minutes. To lyse any remaining red blood cells, the cells were resuspended in 2 mL of ammonium chloride-potassium (ACK) lysis buffer and incubated at room temperature for 4 minutes. The buffer was then neutralized with 4 mL of 10% FBS in PBS, and the cells were refiltered through a 70 μm filter and centrifuged at 1,200 rpm for 5 minutes. The cells were resuspended in 2% BSA in PBS, transferred to a 96-well round-bottom plate, and washed once more with 2% BSA in PBS. The cells were stained with Zombie Live / Dead Fixable Near IR (Biolegend) according to the manufacturer's instructions. The cells were washed once more with 2% BSA in PBS and then analyzed by flow cytometry. Results were analyzed in FlowJo version 10.6.2.

[0426] Luciferase expression in the lungs was analyzed using an In Vivo Imaging System (IVIS, PerkinElmer) and direct measurement of extracted lung protein. Six minutes after mRNA delivery, mice were intraperitoneally injected with 150 mg / kg D-luciferin and anesthetized with isoflurane. Luminescence signals were detected by IVIS 32 minutes later. Twenty-four hours later, mice were reinjected with D-luciferin (150 mg / kg) and imaged 15 minutes later. Lungs, spleens, livers, kidneys, and hearts were removed, and ex vivo organ luminescence was detected by IVIS 30 minutes after the initial injection. Lung and spleen tissues were then minced and placed in Precellys lysis tubes (Bertin Instruments) with 1 mL of Glo lysis buffer (Promega) and homogenized twice at 6500 rpm for 30 seconds. The lysates were transferred to Eppendorf tubes and centrifuged at 21,000 × g for 10 minutes to remove cellular debris. 20 μL of lysate was combined with 100 μL of D-luciferin (Promega), and luminescence was measured for 10 seconds in a luminometer. Luminescence values ​​were normalized to total protein concentration measured by Pierce BCA Protein Assay (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0427] result pH Figure 11 shows the percent transfection efficiency for polymers 2, 4, 14, 15, 16, 17, 20, 25, 27, and 31 in Table 1 at pH 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, and 6.0. The results demonstrate the optimal sodium acetate buffer pH for PACE polyplexes with different end groups. PACE transfection efficiency is strongly dependent on buffer pH, with the optimal pH varying depending on the polymer end group.

[0428] PEG concentration As shown by Figure 12A, the presence of PACE-PEG increased polyplex stability.

[0429] Figure 12B is a graph of transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 in Table 1 as a function of PACE-PEG content (%): 0, 0.05, 0.1.0, 0.25, 0.50, and 1. Figure 12C is a graph of EGFP expression for the same PACE-PEG mixtures, showing that PEG increased EGFP expression. PACE-PEG concentrations as low as 0.05 wt% reduced the mRNA transfection efficiency of most polymers in vitro, although one polymer (P14) maintained high transfection efficiency over a range of PEG concentrations.

[0430] Developing mRNA-based vaccine formulations for inhalation delivery poses inherent challenges, particularly in overcoming the mucus barrier while maintaining high transfection capacity. PEG coating is often used to increase penetration through mucus; however, PEG coverage must be optimized to ensure that transfection efficiency is not lost.

[0431] Polyplexes coated with DSPE-PEG (PEG2k, Nano OCS) were formed by dissolving DSPE-PEG in DMSO (10 mg / mL). The dissolved DSPE-PEG was then combined with PACE in DMSO (100 mg / mL) at a volume ratio of 1:10. Intermediate DSPE-PEG dilutions in DMSO were performed as necessary to achieve the desired final ratio of DSPE-PEG to PACE. Polyplexes were then formed as described above by substituting the DSPE-PEG / PACE blend in DMSO for PACE in DMSO.

[0432] The effect of PEG on transfection efficiency and gene expression is shown in Figures 13A-13C. Figure 13A is a graph of the effectiveness of 0%, 0.01%, 0.1%, 1%, or 10% DSPE-PEG on size (nm) and polydispersity. Figure 13B is a graph of the effectiveness of DSPE-PEG content on transfection efficiency (%) for polymers 14, 15, 16, 17, 25, 27, and 31 in Table 1. Figure 13C is a graph of the effect of DSPE-PEG concentration (0, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, and 5%) on EGFP expression.

[0433] Route of administration affects tissue distribution As shown by Figures 14A and 14B, the bioluminescence distribution before dissection 6 hours after IV and IP injections shows that the polyplexes, when injected, were primarily distributed in the spleen first, followed by the spleen, liver, and intestine.

[0434] In contrast, as shown by Figure 15, administration to the lung surprisingly resulted in high lung concentrations and little expression in other tissues.

[0435] This established that this formulation, when administered to the lung, is an effective and highly selective means for delivery of nucleic acids, such as mRNA, to the lung. In certain embodiments, for example, the following items are provided: (Item 1) Formula I: [ka] wherein n is an integer from 1 to 30; m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; R is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof; polymer. (Item 2) R1 and / or R2 are [ka] Item 1. The polymer according to item 1, wherein the polymer is not (Item 3) The polyplexes or particles formed from the polymers may be such that R1 and / or R2 are: [ka] 2. The polymer of claim 1, which exhibits improved loading, improved cellular transfection, improved intracellular endosomal release of mRNA, or a combination thereof, compared to a corresponding polyplex or particle consisting of (Item 4) The polymer has Formula II: [ka] wherein J1 and J2 are independently a linking moiety or absent, and R3 and R4 are substituted alkyls containing hydroxyl groups, primary amine groups, secondary amine groups, tertiary amine groups, or combinations thereof. Item 1. The polymer according to item 1. (Item 5) The polymer has Formula III: [ka] Item 1. The polymer according to item 1, having the structure: (Item 6) Item 1. The polymer according to item 1, wherein Z is the same as Z'. (Item 7) 2. The polymer of claim 1, wherein n is 4, 10, 13, or 14. (Item 8) Item 1, wherein m is 5, 6, or 7. (Item 9) R x is a substituted or unsubstituted alkyl. (Item 10) 2. The polymer according to item 1, wherein the weight average molecular weight, as measured by gel permeation chromatography using narrow polydispersity polystyrene standards, is about 2,000 daltons to 20,000 daltons, preferably about 2,000 daltons to about 10,000 daltons, and most preferably about 2,000 daltons to about 7,000 daltons. (Item 11) Item 5. The polymer according to item 4, wherein R3 is the same as R4. (Item 12) R3 and / or R4 are independently [ka] [ka] 5. The polymer according to item 4, selected from the group consisting of: (Item 13) 5. The polymer according to item 4, wherein J1 is —O— or —NH, and J2 is —C(O)NH— or —C(O)O—, or a combination thereof. (Item 14) 5. The polymer of claim 4, wherein R3, R4, or both contain a primary amine group and optionally one or more secondary or tertiary amine groups. (Item 15) 5. The polymer of claim 4, wherein R3, R4, or both, contain a hydroxyl group and optionally one or more amine groups. (Item 16) 5. The polymer of claim 4, wherein R3, R4, or both, contain hydroxyl groups and no amine groups. (Item 17) 5. The polymer according to item 4, wherein at least one of R3 and R4 does not contain a hydroxyl group. (Item 18) R3, R4, or both are unsubstituted C1-C 10 Alkylene-Aq-Unsubstituted C1-C 10 Alkylene-Bq, -unsubstituted C1-C 10 Alkylene-Aq-substituted C1-C 10 Alkylene-Bq, -substituted C1-C 10 Alkylene-Aq-Unsubstituted C1-C 10 Alkylene-Bq or -substituted C1-C 10 Alkylene-Aq-substituted C1-C 10 5. The polymer of claim 4, wherein Aq is absent or -NR5-, Bq is hydroxyl, primary amine, secondary amine, or tertiary amine, and R5 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. (Item 19) The poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] wherein n is an integer from 1 to 30; m, o, and p are independently integers from 1 to 20; x, y, and q are independently integers from 1 to 1000; Rx is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof; Item 1. The polymer according to item 1. (Item 20) 20. The polymer of item 19, wherein Z is the same as Z'. (Item 21) 21. The polymer of item 20, wherein Z is O and Z' is O, Z is NR' and Z' is NR'. (Item 22) 20. The polymer according to item 19, wherein Z is O and Z' is NR', or Z is NR' and Z' is O. (Item 23) 20. The polymer according to item 19, wherein Z′ is O and n is an integer from 1 to 24, for example, 4, 10, 13, or 14, and optionally Z is also O. (Item 24) 24. The polymer according to item 23, wherein m is an integer from 1 to 10, for example 4, 5, 6, 7, or 8, and optionally Z is also O. (Item 25) 20. The polymer according to item 19, wherein Z' is O, n is an integer from 1 to 24, for example, 4, 10, 13, or 14, m is an integer from 1 to 10, for example, 4, 5, 6, 7, or 8, o and p are the same integer from 1 to 6, for example, 2, 3, or 4, and optionally Z is also O. (Item 26) 20. The polymer according to item 19, wherein Z' is O, n is an integer from 1 to 24, for example, 4, 10, 13, or 14, m is an integer from 1 to 10, for example, 4, 5, 6, 7, or 8, R is alkyl, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, or homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl, or aryl, for example, phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl, or xylyl, and optionally Z is also O. (Item 27) 20. The polymer according to item 19, wherein n is 14, optionally pentadecalactone (PDL), m is 7, optionally sebacic acid, and o and p are 2, optionally N-methyldiethanolamine (MDEA). (Item 28) Formula II: [ka] Item 1. The polymer according to item 1, having the structure: J1 and J2 are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R3 and R4 in Formula II are independently a substituted alkyl containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. polymer. (Item 29) J1 is -O- or -NH-; J2 is —C(O)—, —C(O)NH—, or —C(O)O—; 29. The polymer according to item 28. (Item 30) 29. The polymer according to item 28, wherein R3 is the same as R4, and optionally R3 and / or R4 are linear. (Item 31) 31. The polymer of item 30, wherein R3, R4, or both, contain a hydroxyl group, and optionally R3, R4, or both, contain a hydroxyl group and one or more amine groups, optionally secondary amine groups or tertiary amine groups. (Item 32) 29. The polymer of item 28, wherein R3, R4, or both, contain hydroxyl groups and no amine groups. (Item 33) 29. The polymer according to item 28, wherein at least one of R3 and R4 does not contain a hydroxyl group. (Item 34) Formula III: [ka] Item 1. The polymer according to item 1, having the structure: J1 and J2 are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R3 and R4 in Formula II are independently a substituted alkyl containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. polymer. (Item 35) J1 is -O- or -NH-; J2 is —C(O)—, —C(O)NH—, or —C(O)O—; 35. The polymer according to item 34. (Item 36) 35. The polymer according to item 34, wherein R3 is the same as R4, and optionally R3 and / or R4 are linear. (Item 37) 35. The polymer of claim 34, wherein R3, R4, or both, contain a hydroxyl group, and optionally R3, R4, or both, contain a hydroxyl group and one or more amine groups, optionally secondary amine groups or tertiary amine groups. (Item 38) 35. The polymer of item 34, wherein R3, R4, or both, contain hydroxyl groups and no amine groups. (Item 39) 35. The polymer according to item 34, wherein at least one of R3 and R4 does not contain a hydroxyl group. (Item 40) 30. The polymer according to any one of items 19 to 39, wherein the weight average molecular weight of the polymer is greater than 20,000 daltons, greater than 15,000 daltons, greater than 10,000 daltons, greater than 5,000 daltons, or greater than 2,000 daltons. (Item 41) 41. The polymer according to item 40, wherein the weight average molecular weight of the polymer is from about 2,000 daltons to about 20,000 daltons, more preferably from about 5,000 daltons to about 10,000 daltons. (Item 42) 42. The polymer according to any of items 19 to 41, prepared from one or more lactones, one or more amine-diols (Z and Z′═O) or triamines (Z and Z′═NR′), and one or more diacids or diesters, wherein when two or more different lactones, diacids or diesters, and / or triamine or amine-diol monomers are used, the values ​​of n, o, p, and / or m can be the same or different. (Item 43) 43. The polymer according to any of items 19 to 42, formed into a polyplex or particle thereof. (Item 44) 44. The polymer according to item 43, formed into a polyplex or particle thereof having one or more encapsulated nucleic acids. (Item 45) The polyplex or particle may comprise R1 and / or R2, [ka] 45. The polymer of claim 44, formed from a polymer that does not consist of or contain: (Item 46) 45. The polymer according to claim 44 formed into a polyplex or particle thereof, wherein the poly(amine-co-ester) or poly(amine-co-amide) is in a mixture containing the poly(amine-co-ester) or poly(amine-co-amide) conjugated to polyethylene glycol (PEG), i.e., a PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide). (Item 47) The PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide) has the structure [ka] wherein m' and m'' are independently 0 or 1, with the proviso that m'+m'' is 1 or 2; J1 and J2 in formula XI are independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; In some forms of Formula XI, J1 is -O- or -NH-, and in some forms of Formula XI, J2 is -C(O)-, -C(O)NH-, or -C(O)O-. 47. The polymer according to item 46. (Item 48) 48. A method for administering one or more therapeutic, diagnostic, or prophylactic nucleic acid agents, or a combination thereof, in vivo, comprising administering to a cell or an individual in need thereof a polymer in the form of a polyplex or particle according to any of items 1 to 47. (Item 49) 49. The method of claim 48, comprising transfecting a cell by contacting the cell with the particle or polyplex. (Item 50) 49. The method of claim 48, wherein the polyplexes or particles are administered to the lung.

Claims

[Claim 1] The invention described in this specification.

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