Methods for expression of protein from synthetic mRNA delivered by injection

Abstract

The present disclosure relates to use of an oligosaccharide or a glucosidase to enhance protein expression from synthetic RNA delivered by injection. In particular, the present disclosure relates to use of chitosan or hyaluronidase to transfect skeletal muscle or skin cells. Other cells that can be effectively transfected with synthetic RNA / chitosan mixtures include bone marrow cells, peritoneal cavity cells, and retro-orbital sinus cells.

Description

METHODS FOR EXPRESSION OF PROTEIN FROM SYNTHETIC MRNA DELIVERED BY INJECTIONCROSS-REFERENCE TO RELATED APPLICATION^ )

[0001] This application claims priority benefit of U.S. Provisional Patent Application Nos. 63 / 575,510 filed on April 5, 2024, 63 / 681,682 filed on August 9, 2024, and 63 / 702,025 filed on October 1, 2024, each of which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to use of an oligosaccharide or a glucosidase to enhance protein expression from synthetic RNA delivered by injection. In particular, the present disclosure relates to use of chitosan or hyaluronidase to transfect skeletal muscle or skin cells. Other cells that can be effectively transfected with synthetic RNA / chitosan mixtures include bone marrow cells, peritoneal cavity cells, and retro-orbital sinus cells.BACKGROUND

[0003] Synthetic messenger RNA (synRNA) is now frequently used in therapeutic products, as well as in general tools for biomedical research. Typically, synRNAs are encapsulated in lipid nanoparticles (LNPs) before injection to protect the synRNA from degradation and to enhance the transfer of synRNA into the cytoplasm of target cells. However, LNPs are known to be toxic to mammalian cells and to induce an inflammatory response in vivo. This limits the dose of synRNA / LNP in formulations to be delivered locally (e.g., by injecting into tissues / organs) or systemically (e.g., by intravenous injection).

[0004] As such, there is a need in the art for tools for effectively expressing an exogenous protein of interest from a synRNA administered by injection. Methods for achieving sustained expression of an exogenous protein in vivo at the site of administration are especially desirable.BRIEF SUMMARY

[0005] The present disclosure relates to use of an oligosaccharide or a glucosidase to enhance protein expression from synthetic RNA delivered by injection. In particular, the present disclosure relates to use of chitosan or hyaluronidase to transfect skeletal muscle or skin cells.Other cells that can be effectively transfected with synthetic RNA / chitosan mixtures include bone marrow cells, peritoneal cavity cells, and retro-orbital sinus cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1A shows bioluminescent images of mice that received synthetic mRNA (synRNA) encoding luciferase by intramuscular injection in the presence or absence of hyaluronidase pretreatment. In brief, mice received pretreatment by intramuscular injection of hyaluronidase (+) or did not receive pretreatment [no hyaluronidase (-)], followed by the intramuscular injection of 20 pg of DENVm52A-LUC synRNA (Unmodified) mixed with Invivofectamine® 3.0 reagent (ThermoFisher Scientific), which forms a synRNA / li id complex. FIG. IB shows the change in luciferase activity of mice from Day 1 to Day 6 postinjection. For hyaluronidase (+) group, a single dose of 10 U (in 40 pL) of hyaluronidase was first injected into a single site of a mouse biceps femoris muscle. About 2 hours later, a single dose of 20 pg (in 60 pL) of DENVm52A-LUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent was injected into approximately the same site of the biceps femoris muscle. For hyaluronidase (-) group, a single dose of 20 pg (in 60 pF) of DENVm52A-EUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent was injected into a single site of a mouse biceps femoris muscle. Euciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).

[0007] FIG. 2 shows the change in luciferase activity of mice from Day 1 to Day 8 postinjection. For hyaluronidase (+) group, 10 U of hyaluronidase was mixed with 20 pg of DENVm52A-EUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent. Then, a single dose of a solution comprising hyaluronidase and DENVm52A-EUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent (20 pg synRNA and 10 U hyaluronidase in 100 pF solution) was injected into a single site of a mouse biceps femoris muscle. For hyaluronidase (-)a group, a single dose of 20 pg of DENVm52A-EUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent (in 100 pF solution) was injected into a single site of a mouse biceps femoris muscle. For hyaluronidase (-)b group, a single dose of 20 pg of DENVm52A-EUC synRNA (Unmodified) complexed with Invivofectamine® 3.0 reagent (in 60 pF solution) was injected into a single site of a mouse biceps femoris muscle.

[0008] FIG. 3 shows the change in luciferase activity in mice from Day 1 to Day 25 postinjection. A dose of 20 pg of TriLink Flue synRNA (Modified with 5moU) was either complexed with Invivofectamine® 3.0 reagent or mixed with chitosan oligosaccharides (0.09 pg for a final concentration of 1.5 pg / ml) and delivered by intramuscular injection. Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).

[0009] FIG. 4 shows the effect of synRNA / chitosan incubation conditions on the luciferase activity in recipient mice from Day 1 to Day 25 after intramuscular injection. For the chitosan RT group (5 min, room temperature), 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with chitosan (0.15 pg), kept at room temperature for 5 minutes, and injected intramuscularly into mice (assessed only from Day 1 to Day 7). For the chitosan ice group (18 min, on ice), 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with chitosan (0.09 pg), kept on ice for 18 minutes, and injected intramuscularly into mice. For the chitosan fridge group (overnight, 4°C), 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with chitosan (0.09 pg), kept at 4°C overnight, and injected intramuscularly into mice (assessed only from Day 1 to Day 7). Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ). Mean + SEM (n=3) are shown. A line at 106in Y axis indicates the detection limit of luciferase activity.

[0010] FIG. 5 shows the effect of poly-L-lysine on the luciferase activity of recipient mice from Day 1 to Day 24 after intramuscular injection of synRNA. 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with poly-L-lysine (7.98 pg) and injected intramuscularly into mice (Poly-L-lysine). As a control, 20 pg of TriLink Flue synRNA (modified with 5moU) alone was injected intramuscularly into mice (Control (-)). Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ). Mean + SEM (n=3) are shown. A line at 106in Y axis indicates the detection limit of luciferase activity.

[0011] FIG. 6A shows the effect of chitosan amount on the luciferase activity of recipient mice from Day 1 to Day 24 after intramuscular injection of synRNA. 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with various amount of chitosan (0.03 pg, 0.3 pg, 3 pg, 30 pg, 300 pg), injected intramuscularly into mice. FIG. 6B shows the effect ofchitosan amount on the luciferase activity of recipient mice from Day 1 to Day 24 after intradermal injection of synRNA. 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with various amount of chitosan (0.03 pg, 0.3 pg, 3 pg, 30 pg, 300 pg), injected intradermally into mice. Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ). Mean + SEM (n=3) are shown. A line at 106in Y axis indicates the detection limit of luciferase activity.

[0012] FIG. 7 A shows representative bioluminescent images of BALB / c mice 1 day after receiving an intramuscular injection of synRNA. Briefly, 20 pg of GLOB120-LUC synRNA (modified with m I ) was mixed with Invivofectamine® 3.0 (lipid) reagent (left) or chitosan (right). FIG. 7B shows a plot of the level of luciferase activity in mice receiving synRNA mixed with a lipid reagent or with chitosan (N = 3). When synRNA was administered with the lipid reagent, luciferase expression was detected not only at the injected site (muscle), but also in liver. By contrast, when synRNA was administered with chitosan, luciferase expression was detected primarily at the injected site (muscle).

[0013] FIG. 8A shows a summary of luciferase activity of five BALB / c mice 1 day after receiving an intramuscular injection of synRNA. Briefly, 20 pg of TriLink Flue synRNA (modified with 5moU) was mixed with Invivofectamine® 3.0 (lipid) reagent, chitosan, or vehicle alone (naked synRNA). Luciferase activity was measured separately at the injected site (muscle) and in the liver. FIG. 8B shows the fraction (%) of averaged luciferase activity in muscle (injected site) and liver of five mice / group. When lipid reagent was used to deliver synRNA- LUC, at Day 1 post-injection, 26% of luciferase activity was detected in liver (off-target). In sharp contrast, when chitosan was used to deliver synRNA-LUC, only a small fraction (0.6%) of luciferase activity was detected in liver (off-target), with the majority (99%) of activity detected at the injected site (muscle). Naked synRNA-LUC showed a low level of luciferase activity at the injected site and, thus, a relatively high level (13.6%) was observed in the liver.

[0014] FIG. 9 shows luciferase activity of three BALB / c mice after intramuscular injection of 20 pg, 100 pg, or 300 pg of synRNA-LUC (TriLink Flue synRNA, modified with 5mou) in 60 pL. synRNA mixed with chitosan was first lyophilized, and then reconstituted with water before the injection. Mean +SEM is shown.

[0015] FIG. 10 shows luciferase activity of three BALB / c mice after intramuscular injection of 300 |ig synRNA-LUC (TriLink Flue synRNA, modified with 5mou) in 60 |iL. synRNA mixed with chitosan in either Lactated Ringer’s Solution (LR, pH 6.1) or Lactated Ringer’s Solution (LR, pH 5.0, 5% dextrose) was first lyophilized, and then reconstituted with water before the injection. Mean +SEM is shown.

[0016] FIG. 11A shows representative biolumine scent images of BALB / c mice 1 day after receiving synRNA-LUC by retro-orbital (RO), intraperitoneal (IP), or intraosseous (IO) injection. Briefly, 20 pg of synRNA-LUC (Trilink Flue synRNA, modified with 5mou) was mixed with 0.3 pg of chitosan. Black arrows (for RO, IP, IO) indicate the location of luciferase activity. FIG. 11B shows a plot of the level of luciferase activity of recipients of RO, intranasal (IN), IP, and IO injections (N = 3).DETAILED DESCRIPTION

[0017] The recent dramatic success of synthetic mRNAs (synRNAs) for COVID-19 vaccines indicates that synRNA may also find use in a broad variety of therapeutic applications, including protein replacement / supplement therapy (Kowalski, Rudra et al. 2019, Jackson, Kester et al. 2020, Wadhwa, Aljabbari et al. 2020, Hou, Zaks et al. 2021, Fang, Liu et al. 2022). The current standard procedure is to encapsulate synRNA within lipid nanoparticle (LNPs) to protect synRNA from degradation and to enhance transfection into target cells. For vaccine applications, billions of doses of synRNA / LNP have been safely administered to skeletal muscles and multiple doses have already been administered to millions of human subjects without major health issues. However, compared to the vaccine applications, therapeutic applications require the delivery of synRNA / LNP to much broader areas of tissue and at much higher doses. For example, for the vaccine applications, synRNA / LNP needs to be delivered to muscle cells at the injection site, whereas for the therapeutic applications, synRNA / LNP needs to be delivered to as many muscle cells as possible in each target muscle. Also, for vaccine applications, relatively small doses of synRNA / LNP are sufficient to induce an immune reaction to the protein encoded by the synRNA, whereas for therapeutic applications, much higher doses are required as the protein encoded by the synRNA is the therapeutic agent. This is problematic because LNPs are known to have toxicity to cells and to induce inflammatory responses (Moghimi and Simberg 2022, Lee, Jeong et al. 2023) and therefore LNP content must be limited.

[0018] Chitosan is a polysaccharide obtained by acid-hydrolyzing chitin. The inventor has previously shown that chitosan oligosaccharide enhances protein expression from synRNA delivered intradermally in the absence of LNPs or other transfection agents (see, International Application Nos. PCT / US2022 / 034104, PCT / US2022 / 075789, PCT / US2023 / 080800, and PCT / US2024 / 015869 of Elixirgen Therapeutics, Inc.). However, prior to development of the present disclosure, it was not known whether chitosan oligosaccharide can facilitate the delivery of naked synRNA by other routes of administration. As used herein, the terms oligosaccharide and polysaccharide are used interchangeably to refer to a saccharide polymer comprising at least three monosaccharides.

[0019] Hyaluronidase is an enzyme that depolymerizes hyaluronic acid. Injection of hyaluronidase enzyme (protein) into tissues creates microchannels in the interstitial matrix that allows fluids to flow through (Murray and Zafar Gondal 2024). This process is transient and reversible. Thus, hyaluronidase has been used to facilitate the distribution of drugs to tissues (Buhren, Schrumpf et al. 2016, Jung 2020). Hyaluronidase has also been safely used to reduce muscle stiffness in myotonic dystrophy (Raghavan, Lu et al. 2016, Amir, Kim et al. 2022). Additionally, hyaluronidase has been used to enhance the transfer of adeno-associated virus (AAV) to rat skeletal muscle (Favre, Cherel et al. 2000), as well as to enhance electroporation of DNA into rat skeletal muscle (Akerstrom, Vedel et al. 2015). However, prior to development of the present disclosure, hyaluronidase has not been known to facilitate the distribution of synRNA / lipid complexes in tissues, including skeletal muscle.

[0020] The present disclosure relates to use of an oligosaccharide or a glucosidase to enhance protein expression from synthetic RNA delivered by injection, such as by intramuscular or intradermal injection. In particular, the present disclosure relates to use of chitosan or hyaluronidase to transfect skeletal muscle or skin cells.General Techniques and Definitions

[0021] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0022] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

[0023] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0024] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., about 500 nucleotides in length when used in reference to an RNA molecule refers to an RNA molecule that is from 450 to 550 nucleotides in length).

[0025] As used herein, the term “synthetic mRNA”, abbreviated as “synRNA” refers to a mRNA molecule comprising at least a 5’-UTR (5 ’-untranslated region), a CDS (coding sequence), and a 3’-UTR (3 ’-untranslated region), in which the CDS is heterologous to at least the 3’-UTR. As such, synRNAs are not naturally-occurring molecules.

[0026] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a certain length unless otherwise specified. Polypeptides may include natural amino acid residues or a combination of natural and nonnatural amino acid residues. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity (e.g., antigenicity).

[0027] As used herein in reference to a protein of interest, the terms “coding sequence,” “CDS”, “open reading frame” and “ORF” refer to the nucleotide sequences that encode the protein of interest. Due to the degeneracy of the genetic code, multiple distinct nucleotide sequences can encode the same amino acid sequence.

[0028] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed fromits original environment). The term “isolated,” when used in reference to a recombinant protein, refers to a protein that has been removed from the culture medium of the host cell that produced the protein. In some embodiments, an isolated protein is at least 75%, 90%, 95%, 96%, 97%, 98% or 99% pure as determined by HPLC.

[0029] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to affect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.

[0030] In the present disclosure, the terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats) and pets (e.g., dogs and cats). In some preferred embodiments, the subject is a human subject.

[0031] The term “dose” as used herein in reference to a composition comprising a mRNA encoding a protein of interest refers to a measured portion of the mRNA taken by (administered to or received by) a subject at any one time.

[0032] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, the phrases “higher level of protein expression” and “stronger protein expression” refer to a level of protein expression as a consequence of contacting a cell with a composition of the present disclosure comprising a mRNA encoding the protein that is greater than 1, preferably greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above a level of protein expression as a consequence of a control condition (e.g., administration of a comparator composition that either does not comprise the mRNA or comprises a control mRNA that does not encode the protein). The phrases “lower level of protein expression” and “weaker protein expression” refer to a level of protein expression as a consequence of a control condition (e.g., administration of a comparator composition that either does not comprise the mRNA or comprises a control mRNA that does not encode the protein) that is less than 1, preferably less than 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold below a level of protein expression as a consequence of administration of a composition of the present disclosure comprising an mRNA encoding the protein.

[0033] As used herein, “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antigen) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0034] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into an antigen of interest and the products screened for a desired activity, e.g., increased stability and / or immunogenicity .

[0035] Amino acids generally can be grouped according to the following common sidechain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro; and(6) aromatic: Trp, Tyr, Phe.

[0036] Conservative amino acid substitutions will involve exchanging a member of one of these classes with another member of the same class. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes with a member of another class.

[0037] As used herein, the term “excipient” refers to a compound present in a composition comprising an active ingredient (e.g., mRNA encoding a protein of interest).Pharmaceutically acceptable excipients are inert pharmaceutical compounds, and may include for instance, solvents, bulking agents, buffering agents, tonicity adjusting agents, and preservatives (Pramanick 2013). In some embodiments the compositions of the present disclosure comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).

[0038] The term “injection” as used herein refers to the administration of a composition to a mammalian subject via force. In some embodiments, “injection” involves administration of a composition using a needle, such as a hypodermic needle or a microneedle. In other embodiments, “injection” involves administration of a composition using a needle-free injection device, such as a PharmaJet® spring-powered injector. The composition may be administered locally via a route selected from but not limited to intramuscular injection, intradermal injection, transdermal injection, intravenous, intranasal, intravitreal, subretinal, suprachoroidal, intrathecal, intra-articular, intrauterine, and intratumoral injection.I. Hyaluronidase

[0039] As described in Example 1, hyaluronidase has been identified as a reagent that can enhance protein expression from synRNA / lipid complexes. As such hyaluronidase formulations are thought to be suitable for reducing the dose of a synRNA / lipid complex necessary to achieve a desired level of expression of an exogenous protein encoded by the synRNA.

[0040] Hyaluronidase is an enzyme that depolymerizes hyaluronic acid. Injection of hyaluronidase enzyme (protein) into tissues creates microchannels in the interstitial matrix that allows fluids to flow through (Murray and Zafar Gondal 2024). This process is transient, and the tissues return to prior state within a day. Thus, hyaluronidase has been used to facilitate the distribution of drugs to tissues (Buhren, Schrumpf et al. 2016, Jung 2020). Hyaluronidase has also been safely used to reduce the muscle rigidity in myotonic dystrophy (Raghavan, Lu et al. 2016, Amir, Kim et al. 2022). However, hyaluronidase has not been used to facilitate the distribution of synRNA / lipid complexes in tissues, including muscles. During development of the present disclosure, the use of hyaluronidase to enhance delivery of synRNA / lipid complexes to tissues has been tested.

[0041] First, hyaluronidase was delivered intramuscularly. Two hours later, synRNA / lipid complex was delivered intramuscularly (Example 1). The synRNA encodes luciferase, and thus, the protein production from this synRNA / lipid complex can be monitored by the in vivo luciferase assay. Although it was impossible to do the second injection exactly at the same location, an attempt was made to do so by marking the first injection site and then the second injection was done on the marked site. As shown in FIG. IB, mice received hyaluronidase injection showed the higher luciferase expression than the mice received no hyaluronidase injection. This indicates that hyaluronidase is a reagent that is not a component of LNPs but can enhance the delivery of synRNA / lipid complex into muscles.

[0042] For the convenience of delivery procedure, it would be ideal, if hyaluronidase can be mixed with synRNA / lipid complex for simultaneous delivery to skeletal muscle. To address this notion, hyaluronidase was added to synRNA / lipid complex before intramuscular injection (Example 1). As shown in FIG. 2, mice that received hyaluronidase in addition to synRNA / lipid complex showed higher luciferase expression than mice that received only synRNA / lipid complex. This indicates that hyaluronidase, pre-mixed with synRNA / lipid complex, can enhance the delivery of synRNA / lipid complex into muscles.II. Chitosan Oligosaccharides

[0043] As described in Example 2, chitosan oligosaccharides have been identified as a reagent that can enhance protein expression from synRNA. Importantly, chitosan oligosaccharides have been found to be a substitute for lipid-based transfection reagents, such as LNPs. That is, formulations comprising chitosan oligosaccharides are suitable for transfection of naked synRNA into skeletal muscle.

[0044] Chitosan is an oligosaccharide obtained by acid-hydrolyzing chitin. In previous patent applications, chitosan oligosaccharide has been shown to enhance protein expression from synRNA delivered intradermally without LNP or other transfection agents (i.e., naked synRNA) (see, International Application Nos. PCT / US2022 / 034104, PCT / US2022 / 075789, PCT / US2023 / 080800, and PCT / US2024 / 015869 of Elixirgen Therapeutics, Inc.). However, it is not known whether chitosan oligosaccharide can facilitate the delivery of naked synRNA to other tissues, such as muscles.

[0045] Chitosan has also been used to deliver nucleic acids such as DNA and RNA, but it requires the elaborate procedures to form chitosan nanoparticles. By contract, during development of the present disclosure, chitosan oligosaccharide was simply mixed with synRNA before administration by intramuscular injection (Example 2). As shown in FIG. 3, chitosan oligosaccharide, not only enhanced the initial (Day 1) luciferase expression from naked synRNA to the level comparable to that of synRNA / lipid complex, but also sustained the luciferase expression for much longer than synRNA / lipid complex. The comparator LNP used here was Invivofectamine® 3.0 reagent (ThermoFisher Scientific), also referred to herein as “lipid reagent”, but the rapid reduction of protein expression has also been reported for other LNPs. For example, synRNA / Invivofectamine® 3.0 reagent complex took 4 days (from Day 1 to Day 5) for 10-fold reduction of luciferase activity (FIG. 3), whereas a previously reported synRNA / lipid complex took only 2 days (from Day 1 to Day 3) for 10-fold reduction of luciferase activity (Pardi, Tuyishime et al. 2015). On the other hand, synRNA / chitosan mixture took 16 days (from Day 1 to Day 17) for 10-fold reduction of luciferase activity (FIG. 3). Therefore, the duration of luciferase protein production (i.e., the indication of synRNA stability) by chitosan is 4- to 8-fold longer than LNPs. Notably, the luciferase activity increased from Day 1 to Day 3 for synRNA / chitosan mixture, whereas the luciferase activity dramatically decreased during the same period (FIG. 3).

[0046] This was a surprising finding, as such a sustained protein expression from RNA has not been achieved by synRNA. Sustained expression has been achieved only by selfreplicating RNA (srRNA) or self-amplifying RNA (saRNA), which can replicate and increase / maintain RNAs. In sum, this indicates that chitosan oligosaccharide can enhance the delivery of synRNA without LNPs nor transfection reagents (i.e., naked mRNA) into muscles, and sustain the protein production for long time. Considering the fact that chitosan oligosaccharide was simply mixed with synRNA before injection into muscles, whereas LNP has to be complexed with synRNA by an elaborate method, the effect of chitosan oligosaccharides was even more surprising.

[0047] As described above, one unique feature of the current disclosure is that this procedure does not rely on the elaborate procedure to form nanoparticles between chitosan and synRNA, which often includes hyaluronic acid and other components (Chuan, Jin et al. 2019, Soliman, Alameh et al. 2020). For example, one report used the mixing of chitosan and nucleicacids (DNA) by a stirrer for about 2 hours at room temperature (Sato, Ishii et al. 2001). Another report used vigorous mixing of chitosan with a homogenizer at 7000 rpm for 2 min (Van Bavel, Issler et al. 2023). By sharp contrast, the methods of the current disclosure simply involve mixing chitosan and synRNA. To test the duration of the chitosan effects after mixing, chitosan and synRNAs were mixed and then incubated for 5 min at room temperature, for 18 minutes on ice, or for overnight at 4°C (Example 3; FIG. 4). The results of incubation for 5 minutes at room temperature (around 25 °C) or 18 minutes on ice (around 0°C) were similar to the results obtained after injection immediately after mixing (Example 3). On the other hand, the results of overnight incubation in a refrigerator (around 4°C) reduced the effect of chitosan dramatically (Example 3; FIG. 4). These results indicate that chitosan / synRNA mixture was stable on ice or at room temperature for a short time, but not stable after prolonged storage at 4°C.

[0048] It is well known that chitosan, which is positively charged due to its nitrogen, binds electrostatically to nucleic acids, which are negatively charged due to its phosphate. To test whether other positively charged polymers have the same effect as chitosan, another well-known positively charged polymer, poly-L-lysine, was tested (Example 4). The result showed that poly- L-lysine (7.98 pg for 20 pg of synRNA) also enhanced the expression of synRNAs delivered by injection (FIG. 5). Without being bound by theory, this observation supports the notion that positively charged molecules can enhance the expression of negatively charged synRNAs.

[0049] It is unlikely that chitosan simply works as an RNase-inhibitor, as had been shown previously using in vitro enzyme assays (Yakovlev, Mitkevich et al. 2007). This is because poly-L-lysine, which is not an RNase inhibitor, also enhanced expression of synRNA delivered by injection (Example 4).

[0050] One of the critical parameters of nanoparticles that deliver nucleic acids to cells, tissues, and organs, is the nitrogen / phosphate (N / P) ratio. The N / P ratio reported to be optimal for transfection / gene delivery is around 5 (Gary, Min et al. 2013). In the context of chitosan / DNA nanoparticles, one group found that the best N / P ratio is 5, followed by 10, whereas a N / P ratio of 20 is much less effective (Ishii, Okahata et al. 2001, Sato, Ishii et al. 2001). N / P ratios of 2 or below do not work (Sato, Ishii et al. 2001). Thus, the working N / P ratio range is rather narrow. To test whether this optimal N / P ratio rule also applies to the chitosan / synRNA mixtures of the present disclosure, a range of chitosan amounts (0.03 pg, 0.3pg, 3 pg, 30 pg, 300 pg) were mixed with 20 pg of synRNA, and injected into muscle and skin cells (Example 5). The N / P ratio is calculated to be 0.003 for 0.03 pg, 0.03 for 0.3 pg, 0.3 for 3 pg, 3 for 30 pg, and 30 for 300 pg. Surprisingly, a broad N / P ratio range was found to be effective (N / P ratio of 0.003 to 3), except for 300 pg group (N / P ratio of 30), for both intramuscular (FIG. 6A) and intradermal injection (FIG. 6B). This is a sharp contrast to chitosan / nucleic acid nanoparticles, whose optimum N / P ratio is around 5. This further supports the notion that the chitosan / synRNA mixtures of the present disclosure are different from the chitosan / nucleic acid particles of previous reports.

[0051] One reason for a N / P ratio of chitosan / DNA to be around 5 (Ishii, Okahata et al. 2001, Sato, Ishii et al. 2001) or even 60 (Koping-Hoggard, Varum et al. 2004) is to make chitosan / nucleic acid nanoparticle be positively-charged rather than charge-neutral. With this background, it is surprising that the very low N / P ratios described in Example 5 are effective for expression of synRNA from highly negatively-charged chitosan / synRNA mixtures delivered by injection. A low N / P ratio (below 1) may provide advantages to chitosan / synRNA mixture for the delivery and expression of synRNA in vivo. Although chitosan is known to be biodegradable, safe, and non-allergic, it is still desirable to limit the dose of chitosan for medical use, and thus, a low dose of chitosan required for a low N / P ratio is advantageous for therapeutic uses.

[0052] As noted above, the present disclosure defined a broad range of N / P ratios that are effective for expression of synRNA from chitosan / synRNA mixtures delivered by injection. However, there is contemplated to be an optimum N / P ratio for each tissue type. For example, for intramuscular injection, a N / P ratio of 0.03 (0.3 pg chitosan for 20 pg of synRNA) was the best condition among those tested (FIG. 6A). On the other hand, for intradermal injection, a N / P ratio of 3 (30 pg chitosan for 20 pg of synRNA) was one of the best conditions, although other conditions were also suitable for synRNA expression (FIG. 6B).

[0053] In some embodiments the N / P ratio of the synRNA / positively-charged polymer mixture of the compositions of the present disclosure is from about 0.03 to about 100. In some embodiments, the N / P ratio is equal to or greater than (lower limit) about 0.03, 0.1, 0.3, 1.0, 3.0 or 10, and less than or equal to (upper limit) about 100, 30, 10, 3.0, 1.0, 0.3 or 0.1, provided that the lower limit is less than the upper limit. In some embodiments, the N / P ration is from about 0.03 to about 10, or from about 0.03 to about 3.0, or from about 0.03 to about 1.0. In someembodiments, the N / P ratio is from about 0.1 to about 10, from about 0.1 to about 3.0, or from about 0.1 to about 1.0. In some preferred embodiments, the positively-charged polymer is chitosan. In other embodiments, the positively-charged polymer comprises poly-L-lysine.A. Localized Delivery of synRNA by Chitosan

[0054] A well-known problem with synRNA administered by intramuscular injection in a lipid-based delivery system is that it enters the circulation, and becomes trapped in the liver where it is expressed (Pardi, Tuyishime et al. 2015, Carrasco, Alishetty et al. 2021). This phenomenon was reproduced herein (Example 6, FIG. 7A, left and FIG. 7B, left). In this example, synRNA delivered as a mixture with a lipid reagent did not remain at the injection site (i.e., muscle), but accumulated in a remote location (i.e., liver). In fact, nearly half (46.4%) of luciferase activity due to expression of the synRNA was detected in liver (off-target site).

[0055] Surprisingly, synRNA delivered as a mixture with chitosan remained at the injection site (FIG. 7A, right). Luciferase expression levels from synRNA / chitosan were comparable to those from synRNA / lipid reagent at the injection site (muscles) (FIG. 7B). In contrast, luciferase expression levels from synRNA / chitosan were very low or undetectable (background level) in liver (off-target site) (FIG. 7B), while luciferase expression levels from synRNA / lipid reagent were very high in liver. Only a small fraction (1.9%) of luciferase activity was detected in liver (off-target site) (FIG. 7C) after intramuscular injection of synRNA / chitosan.

[0056] Chitosan’s ability to localize synRNA and expression of protein(s) it encodes at the intended site (muscle in this case) was further demonstrated in Example 7. Both naked synRNA and synRNA / chitosan, when injected intramuscularly, remained at the injected site as determined by measuring luciferase activity, with an enhancement of expression observed in the synRNA / chitosan group as compared to the naked synRNA group (FIG. 8A). For naked synRNA, 13.6% of luciferase activity was located in liver (off-target site), whereas for synRNA / chitosan, only 0.6% of luciferase activity were located in liver (FIG. 8B). The lipid reagent failed to localize synRNA at the injection site (FIG. 8A), resulting in 25.7% of luciferase activity located in liver (off-target site) (FIG. 8B).B. Lyophilization of synRNA / Chitosan Mixtures

[0057] Lyophilization of chitosan / synRNA mixtures was also accomplished. After reconstitution with Lactated Ringer’s solution, the lyophilized chitosan / synRNA mixture showed similar efficiency in terms of expression of synRNA delivered by injection.

[0058] Importantly, lyophilization and reconstitution in a smaller volume is effective for concentration of synRNA / chitosan, which makes it possible to inject high amounts of synRNA in a limited volume. Indeed, this has been an issue for the therapeutic application of synRNAs. There are obvious limitations of injection volume for each tissue / organ. For example, the standard formulation of synRNA is 1 mg / mL or lower due to the standard procedure of in vitro transcription, purification, and manufacturing process of synRNAs. For testing in mice, the injection volume in muscles is typically limited to 60 pL, in which case only up to 60 pg of synRNA can be injected. Methods for lyophilization and reconstitution of synRNA were tested (Example 8). A simplified reconstitution procedure was adopted involving the inclusion of Lactated Ringer’s solution before lyophilization, and subsequent reconstitution of the lyophilized synRNA / chitosan with sterile water. The volume of Lactated Ringer’s solution was adjusted to the reconstituted volume, so that the final concentration of Lactated Ringer’s solution was lx of the original solution. For 60 pL injection volume, 20 pg (final concentration 0.33 pg / pL), 100 pg (final concentration 1.67pg / pl), 300 pg (final concentration 5.00 pg / pl) synRNA were tested. As shown in FIG. 9, luciferase activity increased from 20 pg to 300 pg. This demonstrates that chitosan can be used in lyophilized synRNA formulations. This permits the concentration of synRNA / chitosan to be increased in a limited injection volume.

[0059] Two types of Lactated Ringer’s solution (USP) are commonly available. One is the standard Lactated Ringer’s Solution (pH 6.5, [6.0 to 7.5]), and the other is Lactated Ringer’s Solution, 5% dextrose (pH 5.0 [4.0 to 6.5]). Both Lactated Ringer’s solutions were tested (Example 9). As shown in FIG. 10, luciferase expression from synRNA formulated in the Lactated Ringer’s Solution, 5% dextrose (pH 5.0 [4.0 to 6.5]) was higher than that formulated in the standard Lactated Ringer’s Solution (pH 6.5, [6.0 to 7.5]).III. Therapeutic Uses of synRNA-Delivered by Intramuscular Injection

[0060] There are many diseases caused by a shortage or loss-of-function of proteins, which are potential targets for protein replacement therapies employing synRNA. On the otherhand, overproduction or aberrant production of proteins causes other types of diseases. synRNAs encoding the dominant-negative form of these proteins are potential therapeutics for these diseases. The technologies presented in this patent application will allow the delivery of synRNAs encoding muscle proteins to muscles in patients with muscle diseases such as myopathies.

[0061] For example, according to the UniProt database, many human proteins are involved in myopathies. These genes include, but not limited to, AAC1, AARF, ABCC9, ABHD5, ABPL, ACAC, ACACA, ACADS, ACC1, ACCA, ACTA, ACTA1, ACTA2, ACTA3, ACTG2, ACTL3, ACTN2, ACTSA, ACTSG, ACTVS, ADCK2, ADL, ADSS1, ADSSL1, AGAT, AGK, AGL, ALDA, ALDOA, ALG14, ALR, AMPD1, AMPHL, ANO5, ANTI, ANX11, ANXA11, APEG1, ATGL, ATP2A1, ATP6, ATPASE6, BAG3, BHLHC1, BHLHC4, BINI, BIS, BM-043, C10orf2, C14orfl0, C14orfl27, C16orf25, C17orf89, C19orf3, Clorf69, C1QBP, C20orfl8, C20orf34, C20orf7, C20orf72, C22orfl6, C3orfl, C3orf29, C3orf31, C3orf60, C3orf9, C4orf41, C6orfl42, C6orf66, C6orf98, C8orf38, CACH1, CACN1, CACNA1S, CACNL1A3, CALC, CANP, CANP3, CANPL3, CAPN3, CASQ, CASQ1, CAV3, CAVIN1, CBARA1, CCDC78, CDA016, CDK5RAP1, CFL2, CGI, CG2, CGI-04, CGI-05, CGI-39, CGI-58, CGL65, CHAC, CHCHD10, CHETK, CHIP, CHKB, CHKL, CIA30, Cip, CLP46, CMYA4, CNBP, CNTN1, COB, COI, COII, COIII, COL12A1, COL12A1L, COL6A1, COL6A2, COL6A3, COX2, COXI, COXII, COXIII, CRACM1, CRYA2, CRYAB, CRYAC, CXorf6, CYTB, D9S57E, DAG2, DAK, DAP13, DBP2, DDK1, DDX16, DES, DGK, DGUOK, DHX16, DNA2, DNA2L, DNAJB4, DNAJB6, DNAJW, DNM2, DPZF, DYN2, DYSF, E2IG1, ECGF1, EDMD, EFE2, EMD, ENO3, EPG5, ETFDH, FAM111B, FCMD, FDX1L, FDX2, FER1L1, FHL1, FKBP14, FKBP22, FKRP, FKSG13, FKTN, FLAD1, FLC3A, FLC3B, FLN2, FLNC, FMT, FMT1, FOXRED1, FP17425, FP17548, FP634, FRIGG, FXR1, G4.5, G5PR, GAA, GABARAP, GABARAPL1, GABARAPL2, GATM, GBE1, GC1QBP, GCLC, GDD1, GDE, GEC1, GEF2, GFER, GIG46, GIPC, GIPC1, GLCL, GLCLC, GLCNE, GNE, GOK, GOLGA2, GOSR2, GRIM19, GS27, GYG, GYG1, HABP1, HACD1, HAD, HADI, HADH, HADHA, HADHB, HADHSC, HBRR, HCCS4, HDNB1, HEL-220, HEL-S-70, HERV1, HLC1, HLJ1, HMGCR, HNRNPA1, HNRNPA2B1, HNRNPDL, HNRPA1, HNRPA2B1, HNRPDL, HPO, HRAS, HRAS1, HRPAP20, HSJ2, HSP22, HSPB5, HSPB8, HSPC125, HSPC167, HT004, HT007, HT2A, HUP1, HXBL, IBA57, IBM2, IGSF9B, IPLA22, IPLA2G, IPO 12,ISCU, ITGA7, ITSN, ITSN1, JFP10, JKTBP, KBTBD10, KBTBD13, KBTBD5, KIAA0083, KIAA0371, KIAA0577, KIAA0613, KIAA0668, KIAA0684, KIAA0723, KIAA0796, KIAA0866, KIAA0986, KIAA1011, KIAA1030, KIAA1073, KIAA1210, KIAA1262, KIAA1297, KIAA1354, KIAA1632, KIAA1642, KIAA1682, KIAA1756, KIAA1780, KIAA2034, KLHL40, KLHL41, KLHL9, KRP1, KTELC1, KY, LAMA2, LAMM, LAMP2, LAP1, LDB3, LMN1, LMNA, LMOD1, LMOD2, LMOD3, LRP12, LSTOO5, LYRM3, LYRM6, MAMLD1, MAP1ALC3, MAP1LC3B, MAP3K20, MAP7D3, MATR3, MCT1, MDP1, MDP3, MDS010, MDSRP, MEAX, MECP2, MEGF10, MGME1, MICU1, MID49, MIEF2, MIG10, MLC2, MLCK, MLCK1, MLIP, MLK7, MLTK, MRF4, MRJ, MRLC1, MSAP, MS JI, MSTO1, MSTP029, MTAP, MTATP6, MT-ATP6, MTCO1, MT-CO1, MTCO2, MT-CO2, MTCO3, MT-CO3, MTCYB, MT-CYB, MTFMT, MTM1, MTMR1, MTMR12, MTMR14, MTMR2, MTMR3, MTND3, MT-ND3, MTPOLB, MTU1, MULK, MVK, My013, MYBPC1, MYBPCS, MYF3, MYF6, MYH11, MYH14, MYH2, MYH3, MYH7, MYHCB, MYHSA2, MYL1, MYL2, MYL3, MYL9, MYLK, MYLK1, MYMX, MYNE1, MYO18B, MYOD, MYODI, MYOP, MYOT, MYPN, MYRL2, NADH3, NADHB14, NCIE2, NCL1, ND3, NDUFA1, NDUFA10, NDUFA11, NDUFA12, NDUFA12L, NDUFA13, NDUFA2, NDUFA6, NDUFA8, NDUFA9, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFAF8, NDUFB10, NDUFB11, NDUFB3, NDUFB7, NDUFB8, NDUFB9, NDUFC2, NDUFS1, NDUFS2, NDUFS2L, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEB, NIFUN, NOTCH2NLC, NUA, NUBPL, OCTN2, OK / SW-cl.110, ORAI1, ORCA, OSIL, OTEX, P53R2, PAB2, PABP2, PABPN1, PAX7, PDZD8, PDZK8, PE01, PEPP1, PFKM, PFKX, PGK1, PGKA, PIP3AP, PNPLA2, PNPLA8, POGLUT1, POLG, POLG1, POLG2, POLGA, POMT1, PPI 131, PP1629, PP591, PP8985, PPP2R3C, PRP2, PTPLA, PTRF, PUS1, PYGM, PYROXD1, RAB5IP, RBCK1, RGS19IP1, RHOXF1, RILPL1, RLP1, RNASEH1, RNF163, RNF54, RNH1, RRM2B, RYDR, RYR1, RYR3, SCHAD, SCN4A, SDH2, SDHA, SDHF, SELENON, SELN, SEPN1, SF2P32, SGCA, SGCB, SGCD, SGCG, SH3D1A, SIL1, SLC16A1, SLC22A5, SLC25A4, SLC25A42, SLIM1, SLTP005, SMCR7, SMPX, SPEG, SPTBN3, SPTBN4, SQSTM1, SRMX, SRYP, ST7, STA, STAC3, STIM1, STUB1, SUN2, SUR2, SVIL, SYNE1, SYNE2, TAFAZZIN, TAMM41, TAZ, TCAP, TEX4, TIA1, TIM22, TIMM22, TIMMDC1, TK2, TKFC, TMEM126B, TMEM142A, TMEM16E, TMEM43, TMEM65, TMEM70, TMOD, TMOD1, TMOD4, TMSB, TNNC2, TNNT1, TNPO3,TNT, TNX, TNXB, TNXB1, TNXB2, T0P3, T0P3A, T0R1AIP1, TPM2, TPM3, TRAPPCI 1, TRIM32, TRMT1, TRMU, TTID, TTN, TWNK, TYMP, UBCE7IP3, UBE4B, UFD2, UNC45, UNC45B, UNC84B, UNQ111 / PRO1064, UNQ247 / PRO284, UNQ2564 / PRO6244, UNQ322 / PRO381, UNQ406 / PRO768, UNQ490 / PR01006, UNQ545 / PRO836, UQOR1, UQOR22, VCP, VMA21, VPS13A, XAP3, XAP4, XB, XMEA, YARS2, ZAK, ZASP, ZBTB20, ZFYVE10, ZNF288, ZNF9.IV. Therapeutic Uses of synRNA Delivered to Any Tissue or Organ

[0062] As described above, the chitosan / synRNA mixtures of the present disclosure (also referred to herein as chitosan / synRNA compositions and chitosan / synRNA formulations) are suitable for administration not only to muscle and skin cells, but to other cell types, tissues, and organs. Cell and tissue types in which synRNA can be expressed from chitosan / synRNA delivered by injection include, but are not limited to, muscle, skin, bone marrow, central nervous system, lung, heart, eye, ear, liver, pancreas, and joints. Chitosan / synRNA mixtures can be delivered by a variety of injection methods, including, but not limited to, intramuscular, intradermal, intravenous, intranasal, intravitreal, subretinal, suprachoroidal, intrathecal, intraarticular injections, and intrauterine injections.

[0063] As described herein, chitosan can localize synRNA and expression of protein(s) it encodes at the injection site. This is a sharp contrast to lipid delivery vehicles, which apparently facilitates systemic distribution of synRNA via the recipient’s circulatory system, and subsequent retention in the liver (Everton, Rizvi et al. 2021). Localized delivery of synRNA by chitosan was tested in additional modes of administration, namely retro-orbital (RO) injection, intraperitoneal (IP) injection, intraosseous (IO) injection, and intranasal (IN) instillation. IP, and IO are routes for deliver of pharmaceutical compositions to systemic circulation and may be employed as an alternative to intravenous (IV) injection (Example 10). RO injection delivers a pharmaceutical composition to the retro-orbital sinus, and subsequently to systemic circulation (Yardeni, Eckhaus et al. 2011). Indeed, it has been shown that synRNA / LNPs accumulate in the liver after RO injection (Rizvi, Everton et al. 2021). IP injection delivers a pharmaceutical composition to microcapillaries and subsequently to systemic circulation (Al Shoyaib, Archie et al. 2019). synRNA / LNPs have also been shown to accumulated in the liver after IP injection (Pardi, Tuyishime et al. 2015). IO injection efficiently delivers a pharmaceutical composition tosystemic circulation and is used as an alternative vascular access (Zhang, Liu et al. 2016, Dornhofer and Kellar 2024). IN instillation is used to deliver pharmaceutical compositions to the upper and lower respiratory tract in mice (Southam, Dolovich et al. 2002).

[0064] As shown in FIG. 11A and FIG. 11B, synRNA / chitosan did not enter systemic circulation, but remained at the injection site as determined by measuring expression of luciferase encoded by the synRNA. The expression by IN instillation was low or undetectable. These data suggest that chitosan can be used to localize expression of synRNA in cells of the retro-orbital sinus by RO injection, cells of the peritoneal cavity (including immune cells such as macrophages, B cells, and T cells, as well as mesothelial cells) by IP injection (Ray and Dittel 2010), and cells of the bone marrow cavity (including hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial stem cells, osteoblast cells, and osteoclast cells) by IO injection (Nombela-Arrieta and Manz 2017).V. Therapeutic Uses of Chitosan / synRNA Mixture for Any Gene

[0065] As described above, there are many diseases caused by a shortage or loss-of- function of proteins, which are potential targets for protein replacement therapies employing synRNA. On the other hand, overproduction or aberrant production of proteins causes other types of diseases. synRNAs encoding the dominant-negative form of these proteins are potential therapeutics for these diseases. The delivery method disclosed herein may be applicable to any artificial genetic constructs, such as genetic constructs for genome editing / engineering such as CRISPR / CAS9 and ribonucleoproteins such as TERT / TERC for telomerase.VI. Therapeutic Uses of Any Chitosan / RNA Mixture

[0066] Chitosan is contemplated to enhance expression of other types of RNA molecules. Such RNA molecules include, but are not limited to, siRNA, tRNA with or without modifications, circular RNA, microRNA, self-replicating RNA (also called self-amplifying RNA) and their variants, such as c-srRNA (controllable self-replicating RNA), any viral RNA, any cellular mRNA made naturally or in vitro, long-noncoding RNA (IncRNA), synthetic mRNA (synRNA), or combinations of these RNA molecules.VII. Enumerated Embodiments1. A method of expressing a protein in a muscle of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with chitosan in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by intramuscular injection under conditions effective for expression of the protein in the muscle, wherein the pharmaceutical composition is devoid of a lipid transfection reagent.2. The method of embodiment 1, wherein expression of the protein in the muscle is detectable for at least fourteen days post-injection, optionally for three weeks or longer postinjection.3. The method of embodiment 1 or embodiment 2, wherein level of expression of the protein in the muscle is higher than from a comparator composition comprising an identical amount of the synRNA mixed with the transfection reagent in the physiologically acceptable solution administered by intramuscular injection, wherein the comparator composition is devoid of chitosan.4. The method of any one of embodiments 1-3, wherein duration of expression of the protein in the muscle is longer than from a comparator composition comprising an identical amount of the synRNA mixed with the transfection reagent in the physiologically acceptable solution administered by intramuscular injection, wherein the comparator composition is devoid of chitosan5. A method of expressing a protein in a muscle of a mammalian subject, comprising:(i) administering hyaluronidase into the muscle of the subject by injection; and(ii) administering a synthetic RNA (synRNA) encoding the protein to the muscle of subject by injection under conditions effective for expression of the protein in the muscle.6. The method of embodiment 5, wherein the hyaluronidase injection is performed for a period of time before the synRNA injection.7. The method of embodiment 5, wherein the hyaluronidase injection and the synRNA injection are performed concurrently.8. The method of embodiment 5, wherein the hyaluronidase and the synRNA are both present in a pharmaceutical composition comprising a physiologically acceptable solution delivered to the muscle by a single injection.9. The method of any one of embodiments 5-8, wherein the synRNA is complexed with a lipid transfection reagent.10. The method of any one of embodiments 1-9, wherein the muscle is skeletal muscle.11. The method of embodiment 10, wherein the physiologically acceptable solution is a saline solution.12. The method of embodiment 11, wherein the saline solution is selected from the group consisting of Ringer’s lactate solution, phosphate-buffered saline, TRIS-buffered saline, and a combination thereof.13. The method of any one of embodiments 1-12, wherein the synRNA comprises at least one modified nucleoside.14. The method of embodiment 13, wherein the at least one modified nucleoside comprises “5mC+'P”, “ml'P”, “5moU” or “ ”, optionally wherein the at least one modified nucleoside comprises “ml'P”, optionally wherein the at least one modified nucleoside comprises “5mC+T”.15. A composition comprising a mixture of a synthetic RNA (synRNA) and a positively- charged polymer, wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), and wherein N / P ratio of the composition is from about 0.03 to about 100.16. The composition of embodiment 15, wherein the N / P ratio of the composition is from about 0.03 to about 1.17. The composition of embodiment 15 or embodiment 16, wherein the positively charged polymer comprises chitosan.18. The composition of embodiment 15 or embodiment 16, wherein the positively charged polymer comprises poly-L-lysine.19. The composition of any one of embodiments 15-18, wherein the composition is devoid of a lipid transfection reagent.20. The composition of any one of embodiments 15-19, wherein the mixture is present in a physiologically acceptable solution.21. A method of expressing a protein in tissue of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with a positively-charged polymer in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by injection into the tissue under conditions effective for expression of the protein in the tissue, wherein the synRNA is not self-replicating, and wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), optionally wherein the expression level of the protein on day one post-injection is at least 10-fold higher than the expression level of the protein on day one post-injection of a comparator composition devoid of the positively-charged polymer but otherwise identical to the pharmaceutical composition, or optionally wherein the expression level of the protein on day one post-injection is from about 10-fold higher to about 10,000-fold higher, from about 10-fold higher to about 1,000-fold higher, or from about 10-fold higher to about 100-fold higher, than the expression level of the protein on day one post-injection of a comparator composition devoid of the positively-charged polymer but otherwise identical to the pharmaceutical composition.22. The method of embodiment 21, wherein the expression level of the protein is higher on day two and / or day three post-injection than on day one post-injection.23. The method of embodiment 21, further comprising incubating the pharmaceutical composition at -4°C to 25°C for up to 20 minutes before administration.24. A method of expressing a protein in tissue of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with a positively-charged polymer in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by injection into the tissue under conditions effective for expression of the protein in the tissue, optionally wherein the synRNA is not self-replicating, wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), wherein the tissue is not the subject’s liver and the expression level of the protein in the liver on day one post-injection is at least 10-fold lower than the expression level of the protein on day one post-injection of a comparator composition comprising an identical amount of the synRNA as the pharmaceutical composition mixed with a lipid transfection reagent but devoid of the positively-charged polymer; and / or wherein the tissue is not the subject’s liver and the expression level of the protein in the liver on day one post-injection is from 50 to 500-fold lower (or at least 100-fold lower) than the expression level of the protein in the tissue.25. The method of embodiment 21 or embodiment 24, further comprising after step (i): lyophilizing the pharmaceutical composition to form a lyophilized composition; and reconstituting the lyophilized composition with a sterile aqueous solution to form a reconstituted pharmaceutical composition before administration, optionally wherein the reconstituted pharmaceutical composition is administered within 30 minutes of the reconstitution.26. The method of any one of embodiments 21-25, wherein the positively-charged polymer comprises chitosan.27. The method of any one of embodiments 21-25, wherein the positively-charged polymer comprises poly-L-lysine.28. The method of any one of embodiments 21-27, wherein N / P ratio of the pharmaceutical composition is from about 0.03 to about 100.29. The method of embodiment 28, wherein the N / P ratio of the pharmaceutical composition is from about 0.03 to about 1.30. The method of any one of embodiments 21-29, wherein the pharmaceutical composition is devoid of a lipid transfection reagent.31. The method of any one of embodiments 21-30, wherein the injection is intramuscular injection.32. The method of any one of embodiments 21-30, wherein the injection is intradermal injection.33. The method of any one of embodiments 21-30, wherein the injection is intraosseous injection.34. The method of any one of embodiments 21-30, wherein the injection is intraperitoneal injection.35. The method of any one of embodiments 21-30, wherein the injection is retro-orbital injection.36. The method of any one of embodiments 21-35, wherein expression of the protein in the tissue is detectable for at least 10 days post-injection, optionally for about two to three weeks post-injection.37. The method of any one of embodiments 21-36, wherein the expression level of the protein on day three post-injection is from about 30% to about 300% of the expression level of the protein on day one post-injection.EXAMPLES

[0067] Abbreviations: 3’-UTR (3 ’-untranslated region); 5’-UTR (5 ’-untranslated region); 5mC+T (5-methylcytosine and pseudouridine); 5moU (5-methoxyuridine); CDS (coding sequence); DENV (dengue virus); Flue (firefly luciferase); GOI (gene of interest); GS-linker (Glycine-Serine Linker); IM (intramuscular); IN (intranasal); IO (intraosseous); IP (intraperitoneal); IVT (in vitro transcription); kb (kilobase); lactated Ringer’s solution (LR); lipid reagent (Invivofectamine® 3.0 reagent); LUC (luciferase); m I T (N1 -methylpseudouridine);MCS (multiple cloning site); N / P (nitrogen / phosphate); nt (nucleotide); O / N (overnight); ORF (open reading frame); PCR (polymerase chain reaction); T (pseudouridine); RO (retro-orbital); RT (room temperature); synRNA (synthetic mRNA); and Unm (unmodified).Example 1. Hyaluronidase Enhances Delivery of synRNA into Muscle

[0068] This example describes the use of hyaluronidase to facilitate transfection of muscle with synthetic mRNA (synRNA) encoding a protein of interest.Materials and Methods

[0069] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0070] DENVm52A-LUC synthetic mRNA: The backbone of this synthetic mRNA (DENVm) is described in PCT Application No. PCT / US2024 / 015869 of Elixirgen Therapeutics, Inc. DENVm is based on the 5’ and 3’ untranslated regions (UTRs) of Dengue virus 2 (DENV) (NC_001474.2 and (Kinney, Butrapet et al. 1997). The CDS (coding sequence) of DENV RNA is replaced with a multiple cloning site (MCS). Four mutations were introduced to remove two ATG start codons of DENV, upstream of the MCS, and to preserve the secondary RNA structure. The synRNA used here encodes a stretch of 52 adenines at its 3’ -end, followed by Ndel restriction enzyme site. The CDS of luciferase (exemplary protein of interest) was inserted in the MCS to form pDENVm52A-LUC.

[0071] Production of synthetic RNA by in vitro transcription: pDENVm52A-LUC plasmid DNA was linearized with Ndel restriction enzyme and used as a template DNA for IVTusing the MEGAscript T7 Kit (ThermoFisher Scientific), according to the manufacturer’s instructions. A 5’-Cap was incorporated using CleanCap® AG analog (TriLink). DENVm52A- LUC synRNA was made without any nucleoside modifications (Unmodified) or with nucleoside modification(s), such as mlT*.

[0072] Preparation of synRNA / lipid complex'. Invivofectamine® 3.0 reagent (ThermoFisher Scientific) is an animal-origin-free lipid-based transfection reagent. The synRNA / lipid complex was prepared according to the manufacturer’s instructions. In brief, synRNA was mixed with Invivofectamine® 3.0 reagent using a vortex mixer and incubated for 30 minutes at 50°C. The synRNA / lipid complex was diluted with PBS prior to use.

[0073] Hyaluronidase: Hyaluronidase protein was purchased from Stemcell Technologies (Catalog No. 07461). Hyaluronidase (lyophilized powder) was reconstituted with Lactated Ringer’s solution (LR).

[0074] Injection of hyaluronidase before injection of synRNA / lipid complex into muscles: For the hyaluronidase (+) group, a single dose of 10 U (in 40 pL of LR) of hyaluronidase was first injected into a single site of the biceps femoris muscle of BALB / c mice. About two hours later, a single dose of 20 pg (in 60 pL) of DENVm52A-LUC synRNA / lipid complex (Unmodified) was injected into approximately the same site of the biceps femoris muscle. For the hyaluronidase (-) group, a single dose of 20 pg (in 60 pL of LR) of DENVm52A-LUC synRNA / lipid complex (Unmodified) was injected into a single site of the biceps femoris muscles of BALB / c mice.

[0075] Simultaneous injection of hyaluronidase and synRNA / lipid complex into muscles: For the hyaluronidase (+) group, 10 U (in 40 pL of LR) of hyaluronidase was mixed with 20 pg (in 60 pL) of DENVm52A-LUC synRNA / lipid complex (Unmodified). Then, a single dose of hyaluronidase / DENVm52A-LUC synRNA / lipid complex (Unmodified) containing 20 pg synRNA and 10 U hyaluronidase in 100 pL solution was injected into a single site of the biceps femoris muscle of BALB / c mice. For the hyaluronidase (-) group a, a single dose of 20 pg (in 100 pL) of DENVm52A-LUC synRNA / lipid complex (Unmodified) was injected into a single site of the biceps femoris muscle of BALB / c mice. For the hyaluronidase (-) group b, a single dose of 20 pg (in 60 pL) of DENVm52A-LUC synRNA / lipid complex (Unmodified) was injected into a single site of the biceps femoris muscle of BALB / c mice. 1

[0076] Measurement of Luciferase activity: Luciferase activity was assessed by the AMI HTX Biolumine scent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0077] The effect of hyaluronidase pre-treatment was first assessed. As shown in FIG. 1A and FIG. IB, intramuscular injection of hyaluronidase two hours before intramuscular injection of a solution comprising a synRNA / lipid complex enhanced delivery of the synRNA / lipid complex into muscle, resulting in higher levels of luciferase activity.

[0078] Next, the effect of simultaneous administration of hyaluronidase and a synRNA / lipid complex was assessed. As shown in FIG. 2, intramuscular injection of hyaluronidase in a solution comprising a synRNA / lipid complex enhanced delivery of the synRNA / lipid complex into muscle, resulting in higher levels of luciferase activity.

[0079] In summary, the use of hyaluronidase in methods of transfecting skeletal muscle in vivo is advantageous for promoting expression of an exogenous protein of interest from a synRNA / lipid complex administered intramuscularly.Example 2. Chitosan Enhances Delivery of synRNAs into Muscle

[0080] This example describes the use of chitosan to facilitate transfection of muscle with synRNA encoding a protein of interest.Materials and Methods

[0081] Mice: C57BL / 6 mice and BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0082] TriLink Flue synRNA (5moU): This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), modified with 5 -methoxy uridine (5moU), and a poly(A) tail. Although the length of the poly(A) tail of this synRNA is not known, it is likely to be about the same length as TriLink’ s EPO synRNA product, which is reported to be 124 ± 15 nt (Gilar, Doneanu et al. 2023).

[0083] Preparation and intramuscular injection of synRNA / lipid complex'. Invivofectamine® 3.0 reagent (ThermoFisher Scientific) is an animal-origin-free lipid-based transfection reagent. The synRNA / lipid complex was prepared according to the manufacturer’s instructions. In brief, synRNA was mixed with Invivofectamine® 3.0 reagent using a vortex mixer and incubated for 30 minutes at 50°C. The synRNA / lipid complex was diluted with PBS prior to use. The final synRNA / lipid complex containing 20 pg synRNA complexed with Invivofectamine® 3.0 reagent in 200 pL solution was injected into three sites (about 66 pL each) of biceps femoris muscles of C57BL / 6 mice.

[0084] Preparation and intramuscular injection of synRNA / chitosaw. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting and kept on ice until use. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide (1.5 pg / ml final concentration) in 60 pL Lactated Ringer’s solution was injected into a single site of biceps femoris muscles of BALB / c mice.

[0085] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0086] As shown in FIG. 3, a formulation comprising synRNA and chitosan oligosaccharide, in the absence of a lipid transfection reagent, was effective in delivering synRNA into skeletal muscle. Surprisingly, the inclusion of chitosan oligosaccharide in the formulation resulted in sustained luciferase expression. Strikingly in the first week postinjection, there was very little reduction in luciferase expression from formulations comprising chitosan oligosaccharide. This is thought to be the first instance in which a protein encoded by a non-self- replicating synRNA, including a circular RNA, has been expressed for an extended period of time.

[0087] In summary, the use of chitosan oligosaccharide in methods of transfecting skeletal muscle in vivo is advantageous for promoting expression of an exogenous protein of interest from a synRNA administered intramuscularly. Remarkably, the inclusion of chitosanoligosaccharide in the transfection formulation resulted in prolonged protein expression (over 2 weeks), which was far superior to the lipid-containing transfection formulation.Example 3. Preparation of Chitosan / synRNA Mixtures

[0088] This example indicates that the effect of chitosan on synRNA expression does not require the formation of chitosan / synRNA nanoparticles.Materials and Methods

[0089] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0090] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), modified with 5 -methoxy uridine (5moU), and a 120 poly(A) tail. However, the length of the poly(A) tail of this synRNA is likely to be 124 ± 15 nt (Gilar, Doneanu et al. 2023).

[0091] Preparation and intramuscular injection of synRNA / chitosan'. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide (0.15 pg or 0.09 pg) in 60 pL Lactated Ringer’s solution was kept at room temperature for 5 minutes, on ice for 18 minutes, or at 4°C overnight, and injected into a single site of biceps femoris muscle of BALB / c mice.

[0092] Measurement of Luciferase activities: Luciferase activity was assessed by theAMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0093] As shown in FIG. 4, chitosan enhances the delivery and expression of synRNA, when administered by intramuscular injection, this is consistent with the results presented in Example 2, in which a simple mixing of synRNA and chitosan was sufficient. However,incubation of the synRNA / chitosan mixture overnight at 4°C reduced the effect of chitosan on syn RNA expression.

[0094] In summary, the enhancement of delivery and expression of synRNAs to skeletal muscle by chitosan can be achieved by simply mixing chitosan and synRNAs.Example 4. Poly-L-Lysine Enhances Delivery and Expression of synRNAs

[0095] This example demonstrates that mixing poly-L-lysine with synRNA also enhances the delivery and expression of synRNAs in vivo.Materials and Methods

[0096] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0097] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), modified with 5 -methoxy uridine (5moU), and a 120 poly(A) tail. However, the length of the poly(A) tail of this synRNA is likely to be 124 ± 15 nt (Gilar, Doneanu et al. 2023).

[0098] Preparation and intramuscular injection of synRNA / poly-L-lysine'. Poly-L-lysine hydrobromide (molecular weight 1 kDa - 5 kDa) was purchased from Sigma- Aldrich (Product No. P0879). The synRNA / poly-L-lysine mixture was prepared by mixing synRNA with poly-L- lysine by gentle pipetting. As a control, 20 pg of TriLink Flue synRNA (modified with 5moU) alone was injected intramuscularly into mice (Control (-)). The final synRNA / poly-L-lysine mixture containing 20 pg synRNA and poly-L-lysine (7.98 pg) in 60 pL Lactated Ringer’s solution or a control synRNA was kept on ice until use and injected into a single site of the biceps femoris muscle of BALB / c mice.

[0099] Measurement of Luciferase activities: Luciferase activity was assessed by theAMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0100] As shown in FIG. 5, poly-L-lysine (7.98 pg for 20 pg synRNA) enhanced the delivery and expression of synRNA, when administered by intramuscular injection.

[0101] In summary, delivery and expression of synRNAs in skeletal muscle can be enhanced by poly-L-lysine.Example 5. Chitosan Enhances Expression of synRNA at a Broad Dose Range

[0102] This example describes that chitosan enhances the delivery and expression of synRNA at broad dose ranges when administered by intramuscular or intradermal injection.Materials and Methods

[0103] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0104] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), modified with 5 -methoxy uridine (5moU), and a 120 poly(A) tail. However, the length of the poly(A) tail of this synRNA is likely to be 124 ± 15 nt (Gilar, Doneanu et al. 2023).

[0105] Preparation and intramuscular injection of synRNA / chitosan'. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide in 60 pL Lactated Ringer’s solution was kept on ice until use and injected into a single site of the biceps femoris muscle or skin of BALB / c mice.

[0106] Measurement of Luciferase activities: Luciferase activity was assessed by theAMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0107] As shown in FIG. 6A, chitosan enhances the delivery and expression of synRNA, when administered by intramuscular injection. Interestingly, the effective range of chitosan was very broad (e.g., 0.03 pg to 30 pg chitosan per 20 pg of synRNA).

[0108] Similarly, as shown in FIG. 6B, chitosan enhances the delivery and expression of synRNA, when administered by intradermal injection. Interestingly, the effective range of chitosan was very broad (e.g., 0.03 pg to 30 pg chitosan per 20 pg of synRNA).

[0109] In summary, chitosan enhances the delivery and expression of synRNAs in both skeletal muscle and skin at a broad dose range.Example 6. Chitosan Localizes Expression of synRNA in Muscle

[0110] This example describes the effect of chitosan on synRNA delivered intramuscularly.Materials and Methods

[0111] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0112] GLOB120-LUC synRNA (modified with ml )’. This construct was used previously described (PCT / US2024 / 015869), and synRNA was made according to published protocols (Warren, Manos et al. 2010, Mandal and Rossi 2013). The 3’-UTR sequence of GLOB120-LUC synRNA is identical to the Mus musculus hemoglobin alpha, adult chain 1 (Hba-al), mRNA (NM_008218.2). The RNA was made with CleanCapOAG analog (Capl: TriLink) and included nucleoside modification with ml . A poly(A) tail of 120 consecutive adenine nucleotides was added to the 3’ end by tail-PCR.

[0113] Preparation and intramuscular injection of synRNA / lipid complex or synRNA / chilosaiv. Invivofectamine® 3.0 reagent (ThermoFisher Scientific) is an animal-origin- free, lipid-based transfection reagent (lipid reagent). The synRNA / lipid complex was prepared according to the manufacturer’s instructions. In brief, synRNA was mixed withInvivofectamine® 3.0 reagent using a vortex mixer and incubated for 30 minutes at 50°C. The synRNA / lipid complex was diluted with PBS prior to use. The final synRNA / lipid complex containing 20 pg synRNA complexed with Invivofectamine® 3.0 reagent in 200 pL solution was injected into three sites (about 66 pL each) of mouse forelimb muscles. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide in 60 pL Lactated Ringer’s solution was kept on ice until use and injected into four sites of mouse forelimb muscles.

[0114] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0115] synRNAs encoding luciferase were mixed with either lipid reagent or chitosan, and then injected into forelimb muscles of BALB / c mice. One day after the intramuscular injection, bioluminescent imaging of luciferase activity was performed. As shown in FIG. 7A, synRNA delivered with the lipid reagent did not remain at the injection site (i.e., muscle), but moved to a remote location (i.e., liver). In contrast, synRNA delivered with chitosan largely remained at the injection site (muscle). FIG. 7B shows levels of luciferase activity at the injected site and the liver of mice receiving synRNA with lipid reagent or with chitosan by intramuscular injection (3 mice / group).

[0116] In summary, synRNA delivered with chitosan remains in the injected muscles, whereas appreciable amounts of synRNA delivered with lipid reagent is carried to the liver (off- target site).Example 7. Chitosan Enhances Expression of synRNA In Situ

[0117] This example describes the effect of chitosan synRNA administered by intramuscular injection.Materials and Methods

[0118] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0119] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), is modified with 5-methoxyuridine (5moU), and includes a 120 poly(A) tail.

[0120] Preparation and intramuscular injection of synRNA / lipid complex, synRNA / chitosan, or naked synRNA: Invivofectamine® 3.0 reagent (ThermoFisher Scientific) is an animal-origin-free, lipid-based transfection reagent (lipid reagent). The synRNA / lipid complex was prepared according to the manufacturer’s instructions. In brief, synRNA was mixed with Invivofectamine® 3.0 reagent using a vortex mixer and incubated for 30 minutes at 50°C. The synRNA / lipid complex was diluted with PBS prior to use. The final synRNA / lipid complex containing 20 pg synRNA complexed with Invivofectamine® 3.0 reagent in 200 pL solution was injected into three sites (about 66 pL each) of mouse forelimb muscles. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide in 60 pL Lactated Ringer’s solution was kept on ice until use and injected into four sites of mouse forelimb muscles. Naked synRNA in Lactated Ringer’s solution was prepared without lipid reagent or chitosan.

[0121] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0122] synRNAs encoding luciferase gene were mixed with lipid reagent or chitosan, or vehicle alone (naked synRNA), and then injected into forelimb muscles of BALB / c mice (5 mice / group). One day after the intramuscular injection, bioluminescent imaging of luciferase activity was performed at the injection site (muscle) and the liver. As shown in FIG. 8A and FIG. 8B, synRNA delivered with lipid reagent resulted in luciferase expression both at theinjection site (74.3%) and in the liver (25.7%). By contrast, synRNA delivered with chitosan resulted in luciferase expression mainly at the injection site (99.4%). In the case of naked synRNA, luciferase expression at the injection site was much lower than from synRNA delivered with the lipid reagent or with chitosan. As such, despite the low level of off-target luciferase expression in the liver, this level amounted to an appreciable percentage (13.6%) of total luciferase activity.

[0123] In summary, chitosan enhances the expression of synRNAs at the injection site compared to naked synRNA. Moreover, unlike delivery with a lipid reagent, chitosan results in retention of synRNA at the site of injection with very low off-target expression target site (liver).Example 8. synRNA / Chitosan Mixtures Can Be Lyophilized

[0124] This example describes the lyophilization and reconstitution of synRNA / chitosan mixtures, which allows for injection of high doses of synRNA.Materials and Methods

[0125] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0126] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), is modified with 5-methoxyuridine (5moU), and includes a 120 poly(A) tail.

[0127] Lyophilization of synRNA / chitosa . Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). For lyophilization, FreeZone Plus 2.5 Liter Cascade Benchtop Freeze Dry System (Labconco) was used. The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting, followed by addition of trehalose (Sigma- Aldrich) and Lactated Ringer’s solution. For 20 pg synRNA, the mixture of 0.3pg chitosan, 20 pg synRNA, 72 pg trehalose, and 60 pL Lactated Ringer’s solution was freeze-dried. For 100 pg synRNA, the mixture of 1.5 pg chitosan, 100 pg synRNA, 360 pg trehalose, and 60 pL Lactated Ringer’ssolution was freeze-dried. For 300 pg synRNA, the mixture of 4.5 pg chitosan, 300 pg synRNA, 1080 pg trehalose, and 60 pL Lactated Ringer’s solution was freeze-dried.

[0128] Reconstitution and intramuscular injection of synRNA / chitosan: Freeze-dried synRNA / chitosan mixture was reconstituted with 60 pL water, and intramuscularly injected into four sites of the forelimbs of BALB / c mice. The final concentration of synRNA was 0.33 pg / pL for 20 pg synRNA, 1.67pg / pl for 100 pg synRNA, and 5.00 pg / pl for 300 pg synRNA.

[0129] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0130] FIG. 9 shows levels of luciferase activity for the 20 pg, 100 pg, or 300 pg synRNA-injected groups at Day 2 of post-intramuscular injection (3 mice / group). Average luciferase activities were proportional to the amount of synRNA injected.

[0131] In summary, synRNA / chitosan can be lyophilized and reconstituted to high concentrations, which makes it possible to inject high doses of synRNA, resulting in high levels of expression of the protein encoded by the synRNA.Example 9. Comparison of Lactated Ringer’s Solutions

[0132] This example describes the testing of two Lactated Ringer’ s Solution, one with 5% dextrose (pH 5.0 [4.0 to 6.5]), and the other without dextrose (pH 6.5, [6.0 to 7.5]).Materials and Methods

[0133] Mice'. BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0134] TriLink Flue synRNA (5moU): This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), is modified with 5-methoxyuridine (5moU), and includes a 120 poly(A) tail.

[0135] Lyophilization of synRNA / chitosan: Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No.44009). For lyophilization, FreeZone Plus 2.5 Liter Cascade Benchtop Freeze Dry System (Labconco) was used. The synRNA / chitosan mixture was prepared by mixing 300 pg synRNA with 4.5 pg chitosan by gentle pipetting, followed by addition of 1080 pg trehalose (Sigma- Aldrich) and 60 pL Lactated Ringer’s solution, followed by freeze-drying. pH was measured for Lactated Ringer’s solution: Lactated Ringer’s and 5% Dextrose Injection, USP (pH 5.0) (NDC 0990-7929-09, ICU Medical); Lactated Ringer’s Solution (pH 6.1) (NDC 13985-803-60, Nova- Tech, Inc.).

[0136] Reconstitution and intramuscular injection of synRNA / chitosan'. Freeze-dried synRNA / chitosan mixture was reconstituted with 60 pL water, and intramuscularly injected into four sites of the forelimbs of BALB / c mice. The final concentration of synRNA was 5.00 pg / pl synRNA.

[0137] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0138] FIG. 10 shows levels of luciferase activity of recipients of synRNA in Lactated Ringer’s Solution from Day 1 to Day 7 post-intramuscular injection (3 mice / group). synRNA formulated in the Lactated Ringer’s Solution, 5% dextrose (pH 5.0) showed slightly better luciferase expression than synRNA formulated in the Lactated Ringer’s Solution without dextrose (pH 6.1).

[0139] In summary, synRNA / chitosan can be formulated in either of two commercially available Lactated Ringer’s Solutions.Example 10. Chitosan Localizes Expression of synRNA In Situ

[0140] This example describes the effect of chitosan on protein expression from synRNA administered by different routes.Materials and Methods

[0141] Mice: BALB / c mice were purchased from the Jackson Laboratory. Mice were housed and handled according to a protocol approved by the institutional animal care and use committee (IACUC).

[0142] TriLink Flue synRNA (5moU) This synRNA, which was purchased from TriLink (Catalog No. L-7202), contains a CleanCap® 5’ cap (Capl), is modified with 5-methoxyuridine (5moU), and includes a 120 poly(A) tail.

[0143] Preparation and intramuscular injection of synRNA / chitosan'. Chitosan oligosaccharides (molecular weight < 5 kDa, >75% deacetylated) were purchased from Heppe Medical Chitosan GmbH (Product No. 44009). The synRNA / chitosan mixture was prepared by mixing synRNA with chitosan by gentle pipetting. The final synRNA / chitosan mixture containing 20 pg synRNA and chitosan oligosaccharide in 60 pL Lactated Ringer’s solution was kept on ice until use and then injected into mice via retro-orbital (RO) injection, intranasal (IN) instillation, intraperitoneal (IP) injection, or intraosseous (IO) injection (3 mice / group). RO injection was performed according to a published procedure (Yardeni, Eckhaus et al. 2011). IN instillation was performed according to a published procedure (Southam, Dolovich et al. 2002). IP injection was performed according to a standard procedure (Al Shoyaib, Archie et al. 2019). IO injection (to tibia) was performed according to a published procedure (Zhang, Liu et al. 2016).

[0144] Measurement of Luciferase activities: Luciferase activity was assessed by the AMI HTX Bioluminescent Imaging system (Spectral Instruments Imaging, Tucson, AZ).Results and Conclusions

[0145] RO injection, IP injection, and IO injection are well-established routes for systemic delivery of pharmaceutical compositions to mammalian subjects. IN instillation is a well-established route for localized delivery of pharmaceutical compositions to the upper and lower respiratory tract of mammalian subjects. To test these delivery routes, synRNA-LUC was mixed with chitosan and then delivered to mice via RO, IP or IO injection, or IN instillation. Luciferase activity was monitored at Day 1 post-administration. As shown in FIG. 11A, for RO, IP, and IO injections, chitosan localized luciferase expression from synRNA at the injectionsites. FIG. 11B shows levels of luciferase activity from synRNA delivered by RO, IP, and IO injections. Luciferase activity from synRNA delivered by IN instillation was not above background.

[0146] In summary, chitosan can localize synRNA and its expression at the injection site with little to no off-target expression.ReferencesAkerstrom, T., K. Vedel, J. Needham, P. Hojman, E. Kontou, Y. Hellsten and J. F. P.Wojtaszewski (2015). "Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle." Biochem Biophys Rep 4: 342- 350.Al Shoyaib, A., S. R. Archie and V. T. Karamyan (2019). "Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies?" Pharm Res 37(1): 12.Amir, A., S. Kim, A. Stecco, M. P. Jankowski and P. Raghavan (2022). "Hyaluronan homeostasis and its role in pain and muscle stiffness." PM R 14(12): 1490-1496.Buhren, B. A., H. Schrumpf, N. P. Hoff, E. Bolke, S. Hilton and P. A. Gerber (2016). "Hyaluronidase: from clinical applications to molecular and cellular mechanisms." Eur J Med Res 21: 5.Carrasco, M. J., S. Alishetty, M. G. Alameh, H. Said, L. Wright, M. 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Claims

CLAIMSWe claim:

1. A method of expressing a protein in tissue of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with a positively-charged polymer in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by injection into the tissue under conditions effective for expression of the protein in the tissue, wherein the synRNA is not self-replicating, and wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), optionally wherein the expression level of the protein on day one post-injection is at least 10-fold higher than the expression level of the protein on day one post-injection of a comparator composition devoid of the positively-charged polymer but otherwise identical to the pharmaceutical composition, or optionally wherein the expression level of the protein on day one post-injection is from about 10-fold higher to about 10,000-fold higher, from about 10-fold higher to about 1,000-fold higher, or from about 10-fold higher to about 100-fold higher, than the expression level of the protein on day one post-injection of a comparator composition devoid of the positively-charged polymer but otherwise identical to the pharmaceutical composition.

2. The method of claim 1, wherein the expression level of the protein is higher on day two and / or day three post-injection than on day one post-injection.

3. The method of claim 1 or claim 2, further comprising incubating the pharmaceutical composition at -4°C to 25°C for up to 20 minutes before administration.

4. A method of expressing a protein in tissue of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with a positively-charged polymer in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by injection into the tissue under conditions effective for expression of the protein in the tissue,optionally wherein the synRNA is not self-replicating, wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), wherein the tissue is not the subject’s liver and the expression level of the protein in the liver on day one post-injection is at least 10-fold lower than the expression level of the protein on day one post-injection of a comparator composition comprising an identical amount of the synRNA as the pharmaceutical composition mixed with a lipid transfection reagent but devoid of the positively-charged polymer; and / or wherein the tissue is not the subject’s liver and the expression level of the protein in the liver on day one post-injection is from 50 to 500-fold lower (or at least 100-fold lower) than the expression level of the protein in the tissue.

5. The method of claim 1 or claim 4, further comprising after step (i): lyophilizing the pharmaceutical composition to form a lyophilized composition; and reconstituting the lyophilized composition with a sterile aqueous solution to form a reconstituted pharmaceutical composition before administration, optionally wherein the reconstituted pharmaceutical composition is administered within 30 minutes of the reconstitution.

6. The method of any one of claims 1-5, wherein the positively-charged polymer comprises chitosan.

7. The method of any one of claims 1-5, wherein the positively-charged polymer comprises poly-L-lysine.

8. The method of any one of claims 1-7, wherein N / P ratio of the pharmaceutical composition is from about 0.03 to about 100.

9. The method of claim 8, wherein the N / P ratio of the pharmaceutical composition is from about 0.03 to about 1.

10. The method of any one of claims 1-9, wherein the pharmaceutical composition is devoid of a lipid transfection reagent.

11. The method of any one of claims 1-10, wherein the injection is intramuscular injection.

12. The method of any one of claims 1-10, wherein the injection is intradermal injection.

13. The method of any one of claims 1-10, wherein the injection is intraosseous injection.

14. The method of any one of claims 1-10, wherein the injection is intraperitoneal injection.

15. The method of any one of claims 1-10, wherein the injection is retro-orbital injection.

16. The method of any one of claims 1-15, wherein expression of the protein in the tissue is detectable for at least 10 days post-injection, optionally for about two to three weeks postinjection.

17. The method of any one of claims 1-16, wherein the expression level of the protein on day three post-injection is from about 30% to about 300% of the expression level of the protein on day one post-injection.

18. A method of expressing a protein in a muscle of a mammalian subject, comprising:(i) mixing a synthetic RNA (synRNA) encoding the protein with chitosan in a physiologically acceptable solution to prepare a pharmaceutical composition; and(ii) administering the pharmaceutical composition to the mammalian subject by intramuscular injection under conditions effective for expression of the protein in the muscle, wherein the pharmaceutical composition is devoid of a lipid transfection reagent.

19. The method of claim 18, wherein expression of the protein in the muscle is detectable for at least fourteen days post-injection, optionally for three weeks or longer post-injection.

20. The method of claim 18 or claim 19, wherein level of expression of the protein in the muscle is higher than from a comparator composition comprising an identical amount of the synRNA mixed with the transfection reagent in the physiologically acceptable solution administered by intramuscular injection, wherein the comparator composition is devoid of chitosan.

21. The method of any one of claims 18-20, wherein duration of expression of the protein in the muscle is longer than from a comparator composition comprising an identical amount of the synRNA mixed with the transfection reagent in the physiologically acceptable solution administered by intramuscular injection, wherein the comparator composition is devoid of chitosan22. A method of expressing a protein in a muscle of a mammalian subject, comprising:(i) administering hyaluronidase into the muscle of the subject by injection; and(ii) administering a synthetic RNA (synRNA) encoding the protein to the muscle of subject by injection under conditions effective for expression of the protein in the muscle.

23. The method of claim 22, wherein the hyaluronidase injection is performed for a period of time before the synRNA injection.

24. The method of claim 22, wherein the hyaluronidase injection and the synRNA injection are performed concurrently.

25. The method of claim 22, wherein the hyaluronidase and the synRNA are both present in a pharmaceutical composition comprising a physiologically acceptable solution delivered to the muscle by a single injection.

26. The method of any one of claims 22-25, wherein the synRNA is complexed with a lipid transfection reagent.

27. The method of any one of claims 18-26, wherein the muscle is skeletal muscle.

28. The method of claim 27, wherein the physiologically acceptable solution is a saline solution.

29. The method of claim 28, wherein the saline solution is selected from the group consisting of Ringer’s lactate solution, phosphate-buffered saline, TRIS-buffered saline, and a combination thereof.

30. The method of any one of claims 18-29, wherein the synRNA comprises at least one modified nucleoside.

31. The method of claim 30, wherein the at least one modified nucleoside comprises “5mC+'P”, “ml'P”, “5moU” or “ ”, optionally wherein the at least one modified nucleoside comprises “ml'P”, optionally wherein the at least one modified nucleoside comprises “5mC+'P”.

32. A composition comprising a mixture of a synthetic RNA (synRNA) and a positively- charged polymer, wherein the synRNA comprises phosphate (P) and the polymer comprises nitrogen (N), and wherein N / P ratio of the composition is from about 0.03 to about 100.

33. The composition of claim 32, wherein the N / P ratio of the composition is from about 0.03 to about 1.

34. The composition of claim 32 or claim 33, wherein the positively charged polymer comprises chitosan.

35. The composition of claim 32 or claim 33, wherein the positively charged polymer comprises poly-L-lysine.

36. The composition of any one of claims 32-35, wherein the composition is devoid of a lipid transfection reagent.

37. The composition of any one of claims 32-36, wherein the mixture is present in a physiologically acceptable solution.

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