Particles containing circular RNA

Abstract

The present invention refers to Particles comprising circular RNA and compositions thereof as well as methods of their use.

Description

GRAF VON STOSCHUnser Zeichen / Our Ref. Datum / DateAI02P055WO December 11, 2025Applicant:Altamira Therapeutics AGParticles containing Circular RNA

[0001] General Description of Invention

[0002] The invention is directed to a nucleic acid delivery technology designed to deliver therapeutic circular RNAs ("circRNA") into cells.

[0003] More specifically, the invention is directed to particles or nanoparticles comprising a circular RNA ("circRNA"), a peptide and, optionally, a macromolecule. Compositions comprising such particles nanoparticles are described herein as well as well as methods of manufacturing such nanoparticles or particles. The circular RNA represents the therapeutic component of the inventive particles.

[0004] In one embodiment of the particles or nanoparticles of the invention, the peptide as one of its components is a positively charged peptide. In another embodiment, the peptide contains at least one histidine residue. In one embodiment, the positively charged peptide contains at least 1 or at least 2 histidine residues. In one embodiment, the peptide has a length of at least 10 amino acids or at least 12 amino acids. In one embodiment, the peptide contains at least 15 amino acids. In another embodiment, the peptide contains at least 20 amino acids. In still another embodiment, the peptides contains 21 or more, typically 21 amino acids. The peptide may comprise at least 3 or at least 4 positively charged amino acid residues, e.g. Arginine. In one embodiment, the peptide contains 5 residues with positively charged side chains. In one embodiment, the positively charged amino acid residues are arginine residues. In one embodiment, the peptide contains at least one histidine residue. In another embodiment, the peptide contains two histidine residues. In one embodiment, the peptide contains 5 Arginine residues and two histidine residues and, typically, has a length of 21 amino acidresidues. More specifically, the peptide may contain or may consist of the peptide H-VLTTGLPALISWIRRRHRRHC-OH (designated herein as "p5RHH").

[0005] Circular RNA ("circRNA") does not have a 5' and a 3' end. They may be resistant towards exonucleolytic degradation by their circular structure, thereby implying enhanced durability of circRNAs arising from the absence of free 5' or 3' ends, present in linear forms. Circular RNA thus forms a covalently closed continuous loop. In circular RNA, the 3' and 5' ends normally present in a liner RNA molecule have been joined together.

[0006] Circular RNA can be prepared both in vitro and in vivo through a splicing reaction of rearranged group I introns in a sequence-dependent manner. Currently established methods of RNA circularization are based on chemical reactions, enzymatic reactions, or the activity of autocatalytic RNA sequences. The chemical circularization methods rely on short precursor RNA sequences, synthesized via phosphoramidite chemistry. Alternatively, RNA circularization can be carried out with ligases, such as T4 RNA ligase I and T4 RNA ligase II. Both enzymes require 5' monophosphorylated precursors that are first adenylated and subsequently ligated with the 3' OH of the ribose at the 3' end of the RNA. The ligases are strongly dependent on the target RNA length and sequence. Other methods of RNA circularization involve catalytic nucleic acids. Still, the most common option to obtain circular RNA in vitro is to prepare a 5'-phosphorylated linear RNA and to circularize it using ligase. The linear e.g. ssRNA is annealed by a guide nucleic acid, such as DNA, e.g. by a 10 to 30 nt DNA molecule being complementary to the 5' and 3' end portions of the linear ssRNA. By addition of a ligase catalyzing a linkage of the 3' and 5' ends of the linear ssRNA, thereby circularizing the linear ssRNA.

[0007] In one embodiment circular RNA is single-stranded. In one embodiment, it is synthetic; in another embodiment, it is a natural circular RNA. Circular RNA may be synthesized via the synthesis of one or multiple precursor linear RNA, followed by RNA circularization mediated by chemical or enzymatical ligation. The precursor linear RNAs used for circular RNA synthesis can be synthesized by chemical and enzymatic approaches. Chemical synthesis of linear RNAs are based on phosphoramidite chemistry and solid-based synthesis. Such synthesis methods may be applied when synthesizing circular RNA of less than 100 nucleotides. When synthesizing larger circular RNA, in vitro transcription (IVT) is initially advantageously employed for the synthesis, e.g. of long linear RNA of 1.000 to 10.000 nucleotides using DNA templates and RNA polymerases (e.g., T7 RNA polymerases). Thereafter, the linear RNA is circularized. Circularization may be carried out chemically or enzymatically, the latter using protein enzymes or ribozymes. For chemical RNA circularization, condensing agents such ascyanogen bromide (BrCN) or l-Ethyl-3-(3-dime-thylaminopropyl)carbodiimide (EDC) are often used to induce the condensation of S'-end phosphate with 3'-end hydroxyl on linear RNA. Alternatively, protein enzymatic RNA circularization may be utilized due to its generally high ligation efficiency. For example, DNA and RNA ligases originating from T4 bacteriophage are employed to ligate linear RNAs with terminal monophosphate. Due to the high selectivity of T4 DNA ligase, only RNAs that are entirely complementary to the corresponding DNA splints will be ligated. T4 DNA ligase-based RNA circularization may advantageously be employed to precisely synthesize circular RNA using precursor linear RNAs synthesized by IVT, in which the run-off properties of RNA polymerases often cause heterogeneity of RNA termini.

[0008] In one embodiment, the circular RNA is coding. A circular coding RNA (e.g. having the function of an mRNA) thereby contains at least one open reading frame. In one embodiment, the circular RNA comprises at least one internal ribosomal entry site (IRES). In another embodiment, the circular RNA contains an N6-methyladenosine (m6A) RNA modification in the 5' untranslated region (UTR) allowing protein translation. The open reading frame encodes a therapeutically active protein or peptide. The protein or peptide may e.g. be used for vaccination purposes, e.g. as an antigen, e.g. a pathogenic, e.g. a viral, bacterial or cancer antigen. In another embodiment, the circular RNA is a non-coding RNA, such as gRNA, siRNA or miRNA.

[0009] The macromolecule as an optional component of the inventive particle may serve for coating purposes. The particles are thereby advantageously covered by an outer shell composed of the macromolecule. Various natural or non-natural macromolecules may be employed. Examples of such macromolecules are albumin, e.g. Human Serum Albumin (HSA) or Hyaluronic acid (HAC).

[0010] The size of the particles typically ranges from 30 nm to 900 nm in diameter or 50 nm to 500 nm in diameter. They may be referred to as "nanoparticles".

[0011] In another aspect of the present invention, a composition is disclosed containing the particles of the invention. The composition may be a pharmaceutical composition. It may comprise pharmaceutically acceptable excipients. The composition may be solid of liquid. If liquid, the composition may be an aqueous solution. The aqueous solution may contain other ingredients, such as a buffer, salts etc. The aqueous solution may be saline.

[0012] In another aspect of the present invention, a method for preparing particles or nanoparticles according to the invention is disclosed. The particles may be prepared by mixing circular RNA and peptide at a desired ratio, e.g. w / w or molar ratio. The molar ratio (peptide: RNA) may be in the range of 2500 to 10.000 or 4000 to 9000 or 2500 to 7500, specifically 5.000 to 7.500 or as defined elsewhere herein. The resulting N / P ratio may typically be in the range of 5 to 15 or 5 to 20, such as 8 to 20, 6 to 12, 7 to 14, or 7 to 12 or 8 to 12 or 8 to 16 or 10 to 16, specifically 10 or 12, more specifically 12. Thereafter, the mixture (advantageously in an aqueous solution or a buffered aqueous solution, such as PBS) may be incubated in e.g. for approximately 60 minutes at approximately 20°C. The incubation period may range from 5 min to lOh. The incubation temperature may be selected from 5°C to 40°C. After the incubation, the formulation can be immediately used. Alternatively, it may be coated with the desired macromolecule, e.g. albumin or hyaluronic acid. Coating the formulation may entail adding the coating agent to the particles or nanoparticles as previously prepared and incubating the mixture for an additional period of e.g. at least 3 min, such as at least 5 min or at least 10 minutes.

[0013] The inventive particles or nanoparticles or compositions may be used for therapy, e.g. for vaccination or as therapeutics by administering them to a subject in need thereof. Particles or nanoparticles containing non-coding circular RNA may be used for silencing overexpressed genes, e.g. for the treatment of cancer or infectious disease as disclosed herein.

[0014] The present invention is based on the finding that circular mRNA packaged by the particles or nanoparticles according to the invention show significantly higher intracellular expression than linear mRNA analogues thereof packaged analogously.

[0015] Description of the Figures

[0016] Figure 1: Expression levels of firefly luciferase after transfection with uncoated peptide / circular Flue mRNA nanoparticles were measured. A peak level of expression (measured as luminescence of the encoded luciferase) is observed 15 min after the onset of incubation at 37°C (Figure 1A). Upon studying luciferase expression at shorter incubation periods (5 to 15 min) by an analogous experimental set-up, peak expression was observed 10 min after the onset of incubation at 37°C (Figure IB).

[0017] Figure 2: Expression levels of firefly luciferase after transfection (incubation of 24h in HEK293T cells) with peptide / circular Flue mRNA in uncoated nanoparticles were measured at differentN / P ratios. Increasing the N / P ratio from 8 to 10 resulted in increased transfection, as indicated by higher luciferase expression (luminescence; " R. L. U." as relative light unit). Lipofectamine-based transfection of the very same circular mRNA (about 50% dose, 100 ng (ri. bar)) vs. 227 ng of peptide / circRNA nanoparticles) used for the experiment (left bar and the bar in the middle) were compared with each other. Lipofectamine as an established transfection agent exhibited only 5% of the activity (at about 50% dose) which was measured for the peptide / circRNA complexes according to the invention.

[0018] Figure 3: Expression levels of firefly luciferase after transfection with peptide / circular Flue mRNA in uncoated nanoparticles were measured at different N / P ratios as compared to a standard linear mRNA formulation ("mRNA") upon 24h incubation (in HEK293T cells). Increasing the N / P ratio from 8 to 10 and further from 10 to 12 resulted in increasingly improved transfection efficiency, as indicated by higher luciferase expression (luminescence). Moreover, transfection efficiency was shown to be improved for circular mRNA (as compared to linear mRNA) when applying comparable N / P ratios (8.3 and 8) (Figure 3A). In Figure 3B, experimental results of an analogous experimental set-up are shown. Peptide / circRNA complexes at higher N / P ratios are compared with each other (N / P= 12, N / P=14, N / P=16 and N / P=18). Further increasing the N / P ratio beyond N / P=12 does not further enhance the transfection efficiency (plateu effect). No cellular toxicity was observed by increasing the peptide concentration.

[0019] Figure 4: Expression levels of firefly luciferase after transfection with peptide / circular Flue mRNA in HSA (human serum albumin) coated (in all instances, peptide to HSA ratio: 5:1) or uncoated nanoparticles were measured upon 24h incubation in HEK293T cells (as compared to linear "mRNA") (Figure 4A). Increasing the N / P ratio from 8 to 10 and further from 10 to 12 resulted in increasingly improved transfection efficiency, irrespective of whether the nanoparticles were HSA coated or not, as indicated by approximately identical luciferase expression levels (luminescence) for all studied samples of the same N / P ratio (Figure 4A). In Figure 4B, an analogous experimental set-up was chosen for studying the expression levels of firefly luciferase after transfection with peptide / circular Flue mRNA in HA (hyaluronic acid) coated (in all instances, peptide to HA ratio of 50:1) or uncoated nanoparticles upon 24h incubation in HEK293T cells as compared to linear "mRNA". Again, identical luciferase expression levels (luminescence) for all studied samples exhibiting the same N / P ratio show comparable transfection efficiency (Fig. 4B).

[0020] Figure 5: Expression levels of firefly luciferase after transfection with equimolar quantities of peptide / linear (mRNA; N / P=8.3) or peptide / circular Flue mRNA ("circRNA"), N / P=10) in uncoated nanoparticles ( NPs) were measured upon 24h, 48h and 72h incubation in HEK293T cells. The observed luminescence of linear mRNA / peptide complexes is significantly lower than for circular RNA / peptide complexes according to the invention (at all time points). The expression levels of the transfected peptide / circular RNA complexes remains essentially constant over the time course for an incubation period from 24h to 72h.

[0021] Figure 6: Expression levels of firefly luciferase after transfection with peptide / circular Flue mRNA in HA (hyaluronic acid) coated nanoparticles (N / P=12) were measured for a variety of distinct formulations characterized by distinct peptide to HA (peptide / HA) ratios (1.5, 1:20, 1:50 and 1:200 for low MW hyaluronic acid; 1:50 and 1:200 for medium MW hyaluronic acid) upon 24h incubation in HEK293T cells in comparison to uncoated nanoparticles. The peptide / HA ratio and the HA molecular weight (MW) have, if at all, only a marginal impact on the expression levels (transfection efficiency)), as depicted by Figures 6A and 6B. In addition, Figures 6A and 6B allow to compare the transfection efficiency of peptide / circRNA complexes according to the invention upon measuring the expression levels directly after lh incubation ("fresh samples") or upon measuring the expression levels of the samples incubated for 1 h only one week later (storage for one week at 4°C. After one week storage, the expression levels remained essentially stable for all stored samples as compared to the measured expression levels of the corresponding "fresh samples".

[0022] Figure 7: The luciferase expression levels were measured upon 24h incubation with Panc-1 cells by peptide / circRNA complexes (N / P ratio=12) according to the invention either coated with HSA at a peptide / HSA ratio of 1:20 or coated with HA at a peptide / HA ratio of 1:50. The observed expression levels are essentially unaltered. The expression levels correspond to the expression levels observed in HEK293T cells.

[0023] Figure 8: A non-linear dose-response was observed when doubling the dose by increasing the dose from 100 ng to 200 ng peptide / circRNA complexes (N / P ratio=8) according to the invention. Either uncoated or coated at a peptide / HSA nanoparticles of a ratio of 1:20 were studied in HEK293T cells after 24h incubation. Coated and uncoated samples do not show significantly different expression levels for 100 ng and 200 ng doses. The expression levels increased in a non-linear manner by using a dose of 200 ng instead of 100 ng for both coated and uncoated nanoparticles

[0024] Figure 9: Expression levels of peptide / circRNA complexes (N / P=8) of the invention are measured in HEK293T cells after 24h incubation (Figure 9A) and HepG2 cells (Figure 9B), respectively. The expression levels of the same nanoparticles (uncoated; green), coated with HSA (red) at a peptide / HSA ratio of 1:20 (middle bar) and coated with HSA (peptide / HSA ratio 1:20) in combination with polysorbate 20 (PS20) (right bar) were compared. The addition of PS20 increases luciferase expression as compared to nanoparticles coated with HSA (without the addition of PS20) slightly. The addition of PS20 to the incubation solution may thus increase transfection efficiency

[0025] Figure 10: Physico-chemical properties were measured by a DLS (dynamic light scattering) analysis (measurement of the nanoparticle size upon 2 min centrifugation at 3000 rpm, i.e. by determination of their hydrodynamic radius (nm)) for various samples of peptide / circRNA complexes according to the invention. They are characterized by the same N / P ratio (N / P ratio=12) and by distinct peptide / HSA ratios of 1:1, 1:5, 1:20, 1:100 and uncoated (" HSA 0"). The hydrodynamic size of HSA coated formulations does not show a significant change compared to uncoated formulations

[0026] Figure 11: Nucleotide sequence of circular RNA used for the experiments (SEQ ID NO: 29).

[0027] Figure 12: Nucleotide Sequence of linear mRNA used for the comparative experiments (SEQ ID NO: 30).

[0028] Introduction of the Invention

[0029] The present application refers to peptide-circRNA molecule complexes and compositions containing the same. It further refers to methods of using them same, such as methods of treatment or the use of the same in methods of treatment. The peptide-circRNA molecule complexes may be coated. In embodiments, the peptide-circRNA molecule complex is coated with albumin and / or hyaluronic acid. They may be characterized by a specific molar ratio of the two components of the complex. The peptide is a typically cationic or polycationic peptide, i.e. a peptide of net positive charge. In embodiments, the peptide-circRNA molecule complex is a nanoparticle with a diameter of about 10 nm to about 500 nm. In embodiments, the composition containing the same comprises a pharmaceutically acceptable carrier.

[0030] The disclosure provides methods of delivering a circRNA molecule into a cell, comprising contacting the cell with any one of the compositions disclosed herein. In embodiments, the RNA molecule is delivered into the cytoplasm of the cell. In embodiments, the RNA molecule is delivered into the nucleus of the cell. In embodiments, the contacting of the cell is performed in vitro, ex vivo or in vivo. In embodiments, the cell is a cancer cell. In embodiments, the cell is a cell affected by inflammation. In embodiments, the cell is an inflammatory cell, such as, for example, a mast cell, an eosinophil, a neutrophil, a basophil, a macrophage, a dendritic cell, a monocyte or a lymphocyte. In embodiments, the cell is a chondrocyte. In embodiments, the cell is a muscle cell.

[0031] The disclosure provides methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the compositions disclosed herein or the particles or nanoparticles or compositions disclosed herein for use for the treatment of a subject. In embodiments, the subject has a cancer, an autoimmune disease, an inflammation-related disease, inflammatory airway disease, rheumatoid arthritis, osteoarthritis, atherosclerosis, inflammatory bowel disease, systemic lupus erythematosus, type I diabetes, chronic obstructive pulmonary disease, asthma, or multiple sclerosis. In embodiments, the subject has a cancer. In embodiments, the cancer is a blood cancer or a solid tumor cancer. In embodiments, the subject is a human subject.

[0032] The disclosure provides methods of preventing a disease, comprising administering to the subject an effective amount of the composition disclosed herein or the peptide-circRNA complexes or nanoparticles disclosed herein. Prevention of a disease may refer to the prevention of a cancer, an autoimmune disease, an infectious disease, a metabolic disease, a muscular disease. In an embodiment, prevention refers to the prevention of an infectious disease.

[0033] DETAILED DESCRIPTION

[0034] Following long-standing patent law convention, the terms "a", “an", and "the" refer to "one or more" when used in this application, including the claims.

[0035] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0036] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term "about", when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0037] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, such as a mammal. The mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human. A subject's tissues, cells, or derivatives thereof, obtained in vivo or cultured in vitro are also encompassed. A human subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (1 month to 24 months), or a neonate (up to 1 month). In embodiments, the adults are seniors about 65 years or older, or about 60 years or older. In some embodiments, the subject is a pregnant woman or a woman intending to become pregnant.

[0038] As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. The term "treating" in one embodiment, includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in the patient that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); (3) relieving the condition (for example, by causing regression, or reducing the severity of the state, disorder or condition or at least one of its clinical or subclinical symptoms).

[0039] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to achieve an outcome, for example, to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like.

[0040] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

[0041] The terms "homologous," "identical," or percent "identity" in relation to two or more peptides, refers to two or more sequences or subsequences that have a specified percentage of amino acid residues that are the same ( / .e., about 60% identity, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection (see, e.g., NCBI website www.ncbi.nlm.nih.gov / BLAST / orthe like). The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants. The algorithms can account for gaps and the like.

[0042] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. The term "purified" in embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For instance, it means that the nucleic acid or protein is at least 85% pure, at least 95% pure, and most at least 99% pure. " Purify" or "purification" in embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.

[0043] The term "target sequence" refers to a sequence of nucleotides found within ta target gene (e.g., the p65 gene) of a target cell. Typically, it is an endogenous gene of the target cell. Such a sequence of nucleotides may be edited as disclosed herein.Peptide-circRNA molecule Complex

[0044] The disclosure provides particles and compositions containing such particles or nanoparticles, wherein the particles or nanoparticles represent a peptide-circRNA (molecule) complex ("peptide / circRNA complex"). The RNA is a circular single or double-stranded RNA, preferably coding a protein or RNA of interest. In embodiments, the peptide-circRNA complexes disclosed herein are capable of efficient transfection of the RNA molecule associated with the peptide into the cytoplasm of a cell or the nucleus of the cell. In an embodiment, the RNA molecule has an origin of replication. In another embodiment, the RNA molecule does not have an origin of replication. In embodiments, the RNA molecule of the peptide-circRNA complexes disclosed herein contains at least ORF (open reading frame) encoding the protein or RNA of interest. The expression of the encoded protein or RNA is under the control of a promotor. A circular plasmid containing an origin of replication may additionally contain a resistance gene, such as an ampicillin resistance gene.Peptide

[0045] In embodiments, the peptide-circRNA molecule complexes disclosed herein comprise a peptide. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is derived from melittin and modified to attenuate its cytotoxicity while maintaining its propensity for interacting with membrane bilayers. Furthermore, the peptide is substantially non-lytic and non- cytotoxic to cells.

[0046] In one embodiment of the complexes of the invention, the peptide as one of its components is a positively charged peptide. In another embodiment, the peptide contains at least one histidine residue. In one embodiment, the positively charged peptide contains at least 1 or at least 2 histidine residues. In one embodiment, the peptide has a length of at least 10 amino acids or at least 12 amino acids. In one embodiment, the peptide contains at least 15 amino acids.. In another embodiment, the peptide contains 18 to 24 amino acids. In still another embodiment, the peptide contains 18 to 22 amino acids. In another embodiment, the peptide contains 20 to 22 amino acids. In another embodiment, the peptide contains at least 20 amino acids. In still another embodiment, the peptides contains 21 or more, typically 21 amino acids. The peptide may comprise at least 3 or at least 4 positively charged amino acid residues, e.g. Arginine. In one embodiment, the peptide contains 5 residues with positively charged side chains. In one embodiment, the positively charged amino acid residues are arginine residues. In one embodiment, the peptide contains at least one histidine residue. In another embodiment, the peptide contains two histidine residues. In one embodiment, the peptide contains 5 Arginine residues and two histidine residues and, typically, has a length of 21 amino acidresidues. More specifically, the peptide may contain the sequence RRRHRR. In another embodiment, the peptide may be H-VLTTGLPALISWIRRRHRRHC-OH (designated herein as "p5RHH").

[0047] In embodiments, a peptide-circRNA molecule complex disclosed herein comprises a peptide that (1) has a function substantially similar to a peptide with an amino acid sequence of SEQ ID NO: 1 (VLTTGLPALISWIRRRHRRHC; pSRHH), SEQ. ID NO: 2 (VLTTGLPALISWIRRRHRRHG), or SEQ ID NO: 3 (VLTTGLPALISWIKRKRQHRWRRRR), and (2) has an amino acid sequence with similarity or identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. As used herein, the phrase "functions substantially similar to a peptide comprising SEQ ID NO: 1, 2, or 3" refers to a substantially non-lytic and / or non-cytotoxic peptide that is capable of affecting the release of the RNA molecule from an endosome. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is non-lytic. The term "non-lytic" means that the lipid bilayer of a cell typically is not compromised upon contact with the peptide. The integrity of the lipid bilayer may be assessed by the improper entry or exit of cellular or extracellular components into a cell. For example, cellular proteins and / or organelles may leak out of a cell with a compromised lipid bilayer. Alternatively, extracellular components ( / .e., those that normally do not enter via gap junctions, for example) may enter a cell with a compromised lipid bilayer. It should be noted, however, that the peptide may penetrate the lipid bilayer of a cell and enter the interior of the cell, but in doing so the integrity of the lipid bilayer is not affected. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is substantially non-cytotoxic. The term "non-cytotoxic" indicates that the cell typically is not killed upon contact with the peptide. Typically, a peptide of the peptide-circRNA molecule complexes disclosed herein decreases cell viability by no more than about 10%, no more than about 7%, no more than about 5%, or no more than about 3%. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is non-lytic and non-cytotoxic.

[0048] A peptide of the peptide-circRNA molecule complexes disclosed herein is capable of associating with a RNA molecule. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises at least one cationic region that interacts with a RNA molecule. As used herein, a "cationic region" has 2 or more contiguous, basic amino acids.

[0049] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein possesses an endosomolytic capacity, which allows it to affect the release of a RNA molecule from an endosome and into the cytoplasm of a cell. As used herein, the term "endosomolytic" describes substances that initiate or facilitate the lysis of endosomes. The endosomolytic capacity of a peptideof the peptide-circRNA molecule complexes disclosed herein obviates the need for additional endosomolytic agents, such as chloroquine, fusogenic peptides, inactivated adenoviruses and polyethyleneimine, for releasing transfected RNA molecules from endosomes for delivery into the cytoplasm of a cell. Such known endosomolytic agents have negative effects on cells and may increase cytotoxicity during transfection.

[0050] Without being bound by a theory, it is thought that the protonation of histidine residues of a peptide of the peptide-circRNA molecule complexes disclosed herein promotes disassembly of the peptide-circRNA molecule complex, which releases the peptide to permeabilize the endosomal membrane for RNA molecule release. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises one or more histid ine residues located adjacent to or within at least one cationic region of the peptide. By way of a non-limiting example, if a peptide of the peptide-circRNA molecule complexes disclosed herein comprises three cationic regions, the peptide may have at least one histidine adjacent to or within the first cationic region of the peptide, at least one histidine adjacent to or within the second cationic region of the peptide, at least one histidine adjacent to or within the third cationic region of the peptide, at least one histidine adjacent to or within each of the first and second cationic region of the peptide, at least one histidine adjacent to or within each of the first and third cationic region of the peptide, at least one histidine adjacent to or within each of the second and third cationic region of the peptide, or at least one histidine adjacent to or within each of the first, second and third cationic region of the peptide. A histidine residue adjacent to a cationic region may be positioned before or after the cationic region. In embodiments, a histidine residue adjacent to a cationic region is immediately adjacent to the region. In embodiments, a histidine residue adjacent to a cationic region is not immediately adjacent to the region. For example, the histidine residue may be within about 2, 3, 4 or 5 positions from the cationic region. In embodiments, a histidine residue is within a cationic region.

[0051] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises SEQ ID NO: 1 (19992). In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein consists of SEQ ID NO: 1. In embodiments, a peptide of the peptide- circRNA molecule complexes disclosed herein is a variant of SEQ ID NO: 1, wherein the variant comprises at least 10 contiguous amino acids of SEQ ID NO: 1 and functions substantially similar to a peptide comprising SEQ ID NO: 1. For instance, a peptide of the peptide-circRNA molecule complexes disclosed herein may encompass at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 1.

[0052] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least 80% identity to SEQ ID NO: 1, wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least about 80% identity to SEQ ID NO: 1 (for instance, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100%, including all values and subranges that lie therebetween), wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. The peptide comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 1, can have about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100% identity to the amino acid sequence of SEQ ID NO: 1.

[0053] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 1 may comprise one or more amino acids that have been conservatively substituted. For instance, one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids may be conservatively substituted.

[0054] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises SEQ ID NO: 2. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein consists of SEQ ID NO: 2. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is a variant of SEQ ID NO: 2, wherein the variant comprises at least 10 contiguous amino acids of SEQ ID NO: 2 and functions substantially similar to a peptide comprising SEQ ID NO: 2. For instance, a peptide of the peptide-circRNA molecule complexes disclosed herein may encompass at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 2.

[0055] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least 80% identity to SEQ ID NO: 2, wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least about 80% identity to SEQ ID NO: 2 (for instance, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100%, including all values and subranges that lie therebetween), wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. The peptidecomprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 2, can have about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100% identity to the amino acid sequence of SEQ ID NO: 2.

[0056] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 2 may comprise one or more amino acids that have been conservatively substituted. For instance, one, two, three, four, five, six, seven, eight, nine, or more than nine amino acids may be conservatively substituted.

[0057] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises SEQ ID NO: 3. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein consists of SEQ ID NO: 3. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein is a variant of SEQ ID NO: 3, wherein the variant comprises at least 10 contiguous amino acids of SEQ ID NO: 3 and functions substantially similar to a peptide comprising SEQ ID NO: 3. For instance, a peptide of the peptide-circRNA molecule complexes disclosed herein may encompass at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous amino acids of SEQ ID NO: 3.

[0058] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least 80% identity to SEQ ID NO: 3, wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least about 80% identity to SEQ ID NO: 3 (for instance, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100%, including all values and subranges that lie therebetween), wherein the peptide is non-lytic and is capable of affecting the release of a RNA molecule from an endosome of a cell. The peptide comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 3, can have about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0059] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 3 may comprise one or more amino acids that have been conservatively substituted. For instance, one, two, three, four, five, six, seven, eight, nine, or more than nine amino acids may be conservatively substituted.

[0060] In embodiments, a peptide of the peptide RNA molecule complexes disclosed herein may be modified at the N-terminus and / or C-terminus. The modification may be a faty acid modification, an acetylation and / or an amidation. In particular, the peptide may be modified at the C- terminus by an amidation ("amide").

[0061] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein comprises an amino acid sequence that has at least about 80% identity to any one of SEQ ID NOs: 4 to 219, including peptides Rn with 8<n<16 (for instance, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100% with any one of these sequences. The peptide-circRNA molecule complex may contain a peptide selected from any one of SEQ ID NOs. 1 to 28.. The sequences according to SEQ ID NOs: 4 to 28 are shown in the below table A:Peptide name Amino acid sequence SEQ ID NO: Mellitin GIGAVLKVLTTGLPALISWIKRKRQQ 4Peptide 5C VLTTGLPALISWIKRKRQQC 5Peptide 5RWR VLTTGLPALISWIKRKRQQRWRRRR 6Peptide 5RH VLTTGLPALISWIRRRHRRC 7Peptide 5RH-AA VAKVLTTGAPALISWIRRRHRRC 8Peptide 5RH-LL VLKVLTTLAPALISWIRRRHRRC 9TAT47-57 YGRKKRRQRRR 10 Penetratin RQIKIWFQNRRMKWKK 11 Transportan GWTLNSAGYLLGKINLKALAALAKKIL 12TP10 AGYLLGKINLKALAALAKKIL 13Rn9-RVG RRRRRRRRRRVG 14R9 RRRRRRRRR 15R15RRRRRRRRRRRRRRR 16 Dermaseptin S4 ALWMTLLKKVLKAA / l^AALNAVLVGANA 17MPG GALFLGFLGAAGSTMGAWSQPKKKRKV 18 MPGANLS GALFLGFLGAAGSTMGAWSQPKSKRKV 19MPG8 βAFLGWLGAWGTMGWSPKKKRK 20MPGa GALFLAFLAAALSLMGLWSQPKKKRKV 21CADY GLWRALWRLLRSLWRLLWRA 22Arg8 RRRRRRRR 23TAT GRKKRRQRRRPQ 24LMWP VS RRRRRRGGRRRR 25STR-Arg8 STRRRRRRRRR 26RALA WEARLARALARALARHLARALARALRACEA 27Pep-1 KETWWETWWTEWSQPKKKRKV 28

[0062] A peptide of the peptide-circRNA molecule complexes disclosed herein may be produced using a variety of techniques known in the art. The peptides may be isolated using standard techniques, may be synthesized using standard techniques, or may be purchased or obtained from a depository.

[0063] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein contains a C-terminal thiol in the form of a cysteine residue. In embodiment, when a peptide of the peptide-circRNA molecule complexes disclosed herein contains a C-terminal thiol in the form of a cysteine residue, a peptide of the peptide-circRNA molecule complexes disclosed herein may be able to form a disulfide bond with another free thiol group, for example, with a free thiol group from the same or different peptide. A skilled artisan can readily determine whether dimer formation does or does not improve the delivery of plasmid RNA. Without wishing to be bound by theory, dimer formation may improve the delivery of plasmid RNA for certain peptides disclosed herein due to improved RNA condensation. Dimerization may be induced by incubation of free peptide in 20% DMSO for 24-72 hours, or by other methods known in other art. As a non-limiting example, free thiols may be quantified by colorimetric assays using Ellman's Reagent.

[0064] In embodiments, a peptide of the peptide-circRNA molecule complexes disclosed herein may be labeled. Non-limiting examples of suitable labels include fluorescent labels, chemiluminescent labels, radioactive labels, colorimetric labels, and resonance labels. Methods of labeling peptides are well known in the art.

[0065] In embodiments, a peptide may be bound to a cargo complex. As used herein, the term "cargo complex" may refer to any molecule or agent that may be carried by or bound to the peptide other than a RNA molecule disclosed herein. Stated another way, a peptide of the peptide-circRNA molecule complexes disclosed herein may be bound to a cargo complex in addition to a RNA molecule disclosed herein. For instance, a cargo complex may be an imaging cargo, a therapeutic cargo, a cytotoxic cargo, or a targeting cargo.

[0066] Non-limiting examples of imaging cargo molecules and agents may include any molecule, agent, or material having a detectable physical or chemical property. Such imaging cargos have been well-developed in the field of fluorescent imaging, magnetic resonance imaging, positron emission tomography, Raman imaging, optical coherence tomography, photoacoustic imaging, Fourier transform infrared imaging, or immunoassays and, in general, most any label useful in such methods may be applied to the present invention. For a review of various labeling or signal producing systems that may be used, see U. S. Pat. No. 4,391,904, incorporated herein by reference in its entirety.

[0067] Non-limiting examples of therapeutic cargo may include any substance that has a biological activity, such as pharmacological agents. Such therapeutic cargo may include analgesics, antipyretics, anti-asthmatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatories including non-steroidal and steroidal, antineoplastics, antianxiety agents, immunosuppressive agents, anti-migraine agents, sedatives, hypnotics, antianginal agents, antipsychotic agents, antimanic agents, antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants, thrombolytic agents, anti-fibrinolytic agents, hemorheologic agents, antiplatelet agents, anticonvulsants, anti-parkinson agents, antihistamines, anti-restenosis agents, anti-pruritics, agents useful for calcium regulation, antibacterial agents, antiviral agents, antimicrobials, anti-infectives, bronchodilators, steroidal compounds and hormones, and combinations thereof. Alternatively, a cargo complex may be in the form of components of molecular complexes or pharmacologically acceptable salts.

[0068] Cytotoxic cargo refers to a molecule or agent that is detrimental to (e.g., kills or damages) a cell. Examples may include anti-microtubule drugs such as the taxols (paclitaxel, docetaxel) and vinca alkaloids (vincristine, vinblastine). For instance, examples may include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin didne, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0069] A targeting cargo may be any molecule or agent that directs a peptide-circRNA molecule complex disclosed herein to a cell. A targeting cargo may be directed to a eukaryotic target cell or a prokaryotic target cell. Non-limiting examples of targeting agents may include an antibody or an antibody fragment, a receptor ligand, a small molecule, a peptide, a polypeptide, a lipid, a carbohydrate, a nucleic acid, a siRNA, a shRNA, an antisense RNA, a dendrimer, a microbubble, or an aptamer.

[0070] The means by which a cargo complex is bound to a peptide of the peptide-circRNA molecule complexes disclosed herein can and will vary depending on the embodiment. A cargo complex may be bound to a peptide of the peptide-circRNA molecule complexes disclosed herein by any means known in the art, including covalently or non-covalently.

[0071] Further details on the peptides of the peptide-nucleic acid molecule complexes disclosed herein are provided in U. S. Patent No. 9987371, U. S. Patent No. 10758627, and U. S. Patent No.11529388, the contents of each of which is incorporated herein by reference in their entireties for all purposes.RNA molecule

[0072] The peptide-circRNA complexes disclosed herein comprise a circular RNA molecule. The circular RNA molecule may be single stranded, double stranded, or a combination thereof. In embodiments, the RNA molecule is double stranded, in embodiments, the RNA molecule is single stranded. In embodiments, the RNA molecule is a combination of single stranded and double stranded. In an embodiment, the circular RNA molecule comprises an origin of replication. The RNA molecule may encode a protein or an RNA.

[0073] Additionally, the RNA molecule may comprise modified nucleic acid bases, such as modified RNA bases. Non-naturally or naturally occurring modifications may occur to e.g. dampen the immune response triggered by the peptide-circRNA complex and / or its encoded protein. Such modifications may prevent an overshooting immune response, such as for the treatment of autoimmune diseases. They may be employed to dampen CpG islands and CpG methylation. Modifications may include the base analogs 5-methyl dCTP or 2-F-sCTP., Nl-methylpseudoridine, pseudouridine, 5'-methoxyuridine, 4-Thio-dT, 4-Thio-Uridin, 5-Br-dC, 5-Br-dU, 5-lod-C, 5-Ethynyl-dU (5-EdU), 5-F-dU, 5-Formyl-dC, 5-l-dC, 5-l-dU, Inosin, and N6-Me-A (m6A), in particular Nl-methylpseudoridine.

[0074] In embodiments, the RNA molecule may encode a protein or it may encode a transfer RNA, a ribosomal RNA, small nuclear RNAs (snRNAs), small nucleolar RNAs (sno RNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs), PlWI-interacting RNAs (piRNAs), and long noncoding RNAs (IncRNAs), guide RNAor catalytic RNA. In general, transfection of a cell with a circular RNA molecule coding for a non-coding RNA may be capable of regulating or inhibiting the expression of a nucleic acid sequence or may lead to cleavage of the nucleic acid sequence, or may enhance, prevent, or disrupt translation of the nucleic acid sequence into a protein, or regulate the transcription of a nucleic acid sequence. Transfection of a cell with a RNA molecule encoding a guide RNA may be used for gene editing, thereby e.g. allowing a defective gene to be repaired, such as in gene therapy. Transfection of a cell with a RNA molecule encoding a protein may allow to provide a protein concentration which is not reached by the body cells due to a defective cellular gene or insufficient gene expression by the cell. The disclosure thus also provides a method of treating of a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of the peptide-circRNA molecule complex as disclosed herein or the composition as disclosed herein.

[0075] In embodiments, a RNA molecule disclosed herein encodes a non-coding RNA capable of disrupting expression of a nucleic acid sequence expressed in a ceil. As used herein, "disrupting expression of a nucleic acid sequence" may be used to describe any decrease in the expression level of a nucleic acid sequence (such as, an mRNA), or a protein translated from the nucleic acid sequence, when compared to a level of expression of the nucleic acid sequence in a cell that was not treated with a peptide-circRNA molecule complex disclosed herein. In embodiments, the RNA molecule may encode a short interfering RNA (siRNA).

[0076] In embodiments, an encoded siRNA may comprise a double-stranded RNA molecule that ranges from about 15 to about 29 nucleotides in length. In embodiments, the encoded siRNA is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length. In embodiments, the encoded siRNA is about 16 to about 18, about 17 to about 19, about 21 to about 23, about 24 to about 27, or about 27 to about 29 nucleotides in length. In embodiments, the encoded siRNA is about 21 nucleotides in length. An encoded siRNA may optionally further comprise one or two single-stranded overhangs, e.g., a 5' overhang on one or both ends, a 3' overhang on one or both ends, or a combination thereof. The encoded siRNA may be formed from two RNA molecules that hybridize together or, alternatively, may be generated from a short hairpin RNA (shRNA). In embodiments, the two strands of the encoded siRNA may be completely complementary, such that no mismatches or bulges exist in the duplex formed between the two sequences. In embodiments, the two strands of the encoded siRNA may be substantially complementary, such that one or more mismatches and / or bulges may exist in the duplex formed between the two sequences. In embodiments, one or both of the 5' ends of the encoded siRNA may have a phosphate group, while in embodiments one or both of the 5' ends lack a phosphate group. In embodiments, one or both of the 3' ends of the encoded siRNA may have a hydroxyl group, while in embodiments one or both of the 5' ends lack a hydroxyl group.

[0077] One strand of the encoded siRNA, which is referred to as the "antisense strand" or "guide strand," includes a portion that hybridizes with a target transcript. A target transcript refers to a nucleic acid sequence (such as, an mRNA), the expression of which is targeted for disruption by the siRNA. In embodiments, the antisense strand of the encoded siRNA is completely complementary with a region of the target transcript, i.e., it hybridizes to the target transcript without a single mismatch or bulge over a target region. In embodiments, the target region is between about 15 and about 29 nucleotidesin length. In embodiments, the target region is at least 16 nucleotides in length. In embodiments, the target region is about 18-20 nucleotides in length. In embodiments, the antisense strand is substantially complementary to the target region, i.e., one or more mismatches and / or bulges may exist in the duplex formed by the antisense strand and the target transcript. In embodiments, encoded siRNAs are targeted to exonic sequences of the target transcript. Those of skill in the art are familiar with programs, algorithms, and / or commercial services that design siRNAs for target transcripts. Nonlimiting examples are the Rosetta siRNA Design Algorithm (Rosetta Inpharmatics, North Seattle, Wash.), MISSION® siRNA (Sigma-Aldrich, St. Louis, Mo.) and siGENOME siRNA (Thermo Scientific). The encoded siRNA may be enzymatically synthesized in vitro using methods well known to those of skill in the art. Alternatively, the encoded siRNA may be chemically synthesized using oligonucleotide synthesis techniques that are well known in the art.

[0078] Nucleic acid sequences disclosed herein may be obtained using a variety of different techniques known in the art. The nucleic acid sequences, as well as homologous sequences, may be isolated using standard techniques, may be synthesized using standard techniques, or may be purchased or obtained from a depository. Once the nucleic acid sequence is obtained, it may be amplified for use in a variety of applications, using methods known in the art.Polypeptide-circRNA molecule Complex

[0079] In embodiments, the polypeptide disclosed herein and the RNA molecule disclosed herein associate to form a complex. As used herein, the term "associate" may refer to the interaction of a peptide and a RNA molecule through non-covalent bonds, or to the covalent bonding of a peptide and a RNA molecule. In embodiments, a polypeptide and a RNA molecule disclosed herein associate through non-covalent bonds such as a hydrogen bond, an ionic bond, a bond based on Van der Waals, a hydrophobic bond, or electrostatic interactions. For instance, a peptide of the peptide-circRNA molecule complexes disclosed herein may have an overall net positive charge, which may allow the peptide to associate with a RNA molecule disclosed herein through electrostatic interactions to form a complex disclosed herein.

[0080] The ratio of peptide to RNA molecule at which a peptide of the peptide-circRNA molecule complexes disclosed herein associates with a RNA molecule disclosed herein can and will vary depending on the peptide, the RNA molecule composition, or the size of the RNA molecule, and may be determined experimentally. In essence, a suitable molar ratio of a peptide of the peptide-circRNA molecule complexes disclosed herein to a RNA molecule of the peptide-circRNA molecule complexes disclosed herein may be a molar ratio wherein the peptide completely complexes the RNA molecule,while minimizing exposure of a subject (who is administered the peptide-circRNA molecule complexes disclosed herein) to the peptide.

[0081] In embodiments, the ratio is the molar ratio. In embodiments, the molar ratio of peptide to RNA molecule is about 2.500:1 to about 30.000:1. Depending on the size of the RNA-molecule, the molar ratio may vary. The molar ratio may be in the range of about 2.500:1 to about 10.000:1, more specifically in the range of 2.500:1 to 8.000:1, such as 4.000 to 10.00 or 6.000 to 10.000. In another embodiment, molar ratio is about 2.500:1 to about 30.000:1, about 5.000:1 to about 20.000:1, about 10.000:1 to about 25.000:1, about 10.000:1 to about 20.000:1, or about 15.000:1 to about 20.000:1, in particular about 5.000:1 to 10.000:1. In an embodiment, the molar ratio of peptide to circRNA molecule is about 2.500:1 to about 15.000:1. In another embodiment, the molar ratio of peptide to circRNA molecule is about 5.000:1 to about 10.000:1. In still another embodiment, the molar ratio of peptide to circRNA molecule is about 15.000:1 to about 20.000:1.

[0082] In embodiments, the ratio is the charge ratio. As used herein, the "charge ratio" is the ratio of positively-chargeable polymer amine groups to negatively-charged nucleic acid phosphate groups. In embodiments, the charge ratio of peptide to RNA molecule is about 6:1 to about 18:1 or 6:1 to 12:1 or 8:1 to 12:1. In embodiments, the charge ratio of peptide to RNA molecule is about 6:1. In embodiments, the charge ratio of peptide to RNA molecule is about 7:1. In embodiments, the charge ratio of peptide to RNA molecule is about 8:1. In embodiments, the charge ratio of peptide to RNA molecule is about 9:1. In embodiments, the charge ratio of peptide to RNA molecule is about 10:1. In embodiments, the charge ratio of peptide to RNA molecule is about 11:1. In embodiments, the charge ratio of peptide to RNA molecule is about 12:1. In embodiments, the charge ratio of peptide to RNA molecule is about 13:1. In embodiments, the charge ratio of peptide to RNA molecule is about 14:1. In embodiments, the charge ratio of peptide to RNA molecule is about 15:1. In embodiments, the charge ratio of peptide to RNA molecule is about 16:1. In embodiments, the charge ratio of peptide to RNA molecule is about 17:1. In embodiments, the charge ratio of peptide to RNA molecule is about 18:1.

[0083] In an embodiment, the N / P ratio of the peptide-pRNA complex may typically be in the range of 5 to 20, such as 6 to 18, 6 to 114, 8 to 14 or 8 to 12. The N to P ratio (N / P ratio) describes the stoichiometry between protonatable nitrogen ( / V) in the peptide component peptide-circRNAmolecule complexes and anionic phosphate groups (P) in the RNA molecule peptide-circRNA molecule complexes.

[0084] Methods of determining the ratio wherein the peptide is capable of completely complexing the RNA molecule are known in the art and may include gel retardation assays. Methods of determining a molar ratio wherein exposure of a subject to the peptide is minimized are known in the art and may include cytotoxicity measurements using increasing doses of the polypeptide.

[0085] In embodiments, the peptide-circRNA molecule complexes disclosed herein may be about 10 nm to about 500 nm in diameter. In embodiments, the diameter of the peptide-circRNA molecule complex is about 10 nm to about 800 nm or 50 nm to 800 nm or lOOnm to 800 nm or 200 nm to 800 nm or 300 nm to 500 nm.. In embodiments, the diameter of the peptide-circRNA molecule complex is at least about 10 nm. In embodiments, the diameter of the peptide-circRNA molecule complex is at most about 500 nm. In embodiments, the diameter of the peptide-circRNA molecule complex is about 10 nm to about 500 nm, about 10 nm to about 300 nm, about 10 nm to about 150 nm, about 10 nm to about 200 nm, about 10 nm to about 250 nm, about 10 nm to about 300 nm, about 50 nm to about 500 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, about 50 nm to about 250 nm, about 50 nm to about 300 nm, about 100 nm to about 500 nm, about 100 nm to about 300 nm, about 100 nm to about 250 nm, about 100 nm to about 300 nm, about 150 nm to about 200 nm, about 150 nm to about 250 nm, about 150 nm to about 300 nm, about 200 nm to about 250 nm, about 200 nm to about 300 nm, or about 250 nm to about 300 nm. In embodiments, the diameter of the peptide-circRNA molecule complex is about 10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, or about 300 nm or about 500 nm.

[0086] In embodiments, the peptide-circRNA molecule complex is a nanoparticle with a diameter of about 10 nm to about 800 nm. In embodiments, the nanoparticles disclosed herein may be further modified to enhance stability of the nanoparticle. For instance, a nanoparticle disclosed herein may be coated with albumin and / or hyaluronic acid to enhance stability. In embodiments, the nanoparticles disclosed herein coated with albumin may be about 5 to about 500 nm or more in diameter, such as 300 nm to 500 nm.

[0087] Particle size and / or charge may be assessed using methods known in the art. Non-limiting examples of methods of measuring the size of a particle may include dynamic light scattering, light scattering, multi-angle light scattering, field-flow fractionation systems, laser diffraction, electrozone(electric sensing zone), light obscuration— also referred to as photozone and single particle optical sensing (SPOS), sieve analysis, aerodynamic measurements, air permeability diameter, sedimentation, nanoparticle tracking analysis, electron microspcopy, atomic force microscopy, small-angle X ray scattering, flow cytometry, measuring the zeta potential of the particle, analytical ultracentrifugation or combinations thereof. In embodiments, particle size is assessed by dynamic light scattering. In embodiments, particle charge is assessed by measuring the zeta potential of the particle. In embodiments, particle size and / or charge is assessed by dynamic light scattering or by measuring the zeta potential of the particle.

[0088] In embodiments, the nanoparticles disclosed herein may have a zeta potential of about –15 to about 20 mV, about 0 mV or more. For instance, the nanoparticle may have a zeta potential of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 mV or more. In embodiments, the nanoparticle has a zeta potential of about 1, about 2, about 3, about 4, or about 5 mV. In embodiments, the nanoparticle has a zeta potential of about 10, 11, 12, 13, or about 14 mV. In embodiments, the nanoparticle has a zeta potential of about 11, about 12, about 13, about 14, or about 15 mV. In embodiments, the nanoparticle has a zeta potential of about 1, about 2, about 3, about 4, or about 5 mV. In embodiments, the nanoparticle has a zeta potential of about 10, about 11, 12, about 13, or about 14 mV. In embodiments, the nanoparticle has a zeta potential of about 3.72 mV. In embodiments, the nanoparticle has a zeta potential of about 12 mV. In embodiments, the nanoparticle has a zeta potential of about 13.1 mV.

[0089] In embodiments, the peptide-circRNA molecule complexes disclosed herein are capable of efficient release of the RNA molecule into the cytoplasm of a cell. In other embodiments, the peptide-circRNA molecule complexes disclosed herein are capable of efficient release of the RNA molecule into the nucleus of a cell. The peptide RNA molecule complexes may pass the cytoplasm and enter into the nucleus. In embodiments, the peptide-circRNA molecule complex may also be capable of protecting the RNA molecule from degradation upon administration in a subject. In embodiments, the peptide-circRNA molecule nanoparticle disclosed herein may remain stable in the presence of serum. In embodiments, the nanoparticle may remain stable in the presence of serum for about 10, 20, 30, 40, 50, 60 minutes, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 hours, about 1, 2, 3, 4, 5, 6, 7 days or longer. In embodiments, the nanoparticle may remain stable in the presence of about 5, 10, 15, 25, 50, 100, 150, 200, or about 300 pg / ml or more human serum albumin. Stability of the nanoparticle may be determined by measuring the ability of a nanoparticle to maintain the activity ofa RNA molecule of the peptide-circRNA molecule complex of the nanoparticle, or by measuring changes in the size of a nanoparticle over time.

[0090] Methods of preparing the peptide-circRNA molecule complexes disclosed herein generally comprise contacting a peptide of the peptide-circRNA molecule complexes disclosed herein with a RNA molecule disclosed herein to form the peptide-circRNA molecule complex. Typically, a peptide and a RNA molecule are contacted by incubating under conditions suitable for a peptide-circRNA molecule complex disclosed herein to form. Exemplary conditions suitable for a peptide-circRNA molecule complex disclosed herein to form are described in the examples. Typically, such conditions may comprise a temperature of about 30°C to about 40°C, and incubation times of between about 20 sec to about 60 min or more. Suitable temperatures may also be lower than about 30°C. For example, incubation may occur on ice. One skilled in the art will appreciate that the length and temperature of incubation can and will vary depending on the peptide and the RNA molecule and may be determined experimentally.

[0091] In embodiments, the peptide-circRNA molecule nanoparticle disclosed herein may be further modified to enhance stability of the nanoparticle. For instance, a peptide-circRNA molecule complex disclosed herein may be crosslinked to enhance the stability of nanoparticles. One of ordinary skill in the art would recognize that a suitable cross-linker can and will vary depending on the composition of the nanoparticle and the antibody or antibody fragment. In embodiments, the peptide-circRNA molecule nanoparticle disclosed herein may be chemically crosslinked using chemical cross-linkers such as glutaraldehyde, bis-carboxylic acid spacers, bis-carboxylic acid-active esters, using a bis-linker amine / acid by carbodiimide coupling protocol, or using a click chemistry protocol, carbodiimide-coupling chemistry, acylation, active ester coupling, or alkylation.

[0092] In embodiments, the peptide-circRNA molecule nanoparticle disclosed herein may be coated with a compound capable of enhancing the stability of nanoparticles. Methods of modifying a nanoparticle to enhance stability are known in the art. Further details are provided in Nicolas et al., 2013 Acta Biomater. 9:4754-4762, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0093] As used herein, the term "coating" may refer to the interaction of a peptide-circRNA molecule complex disclosed herein with a compound through non-covalent bonds, or to the covalent bonding of a peptide-circRNA molecule complex disclosed herein with a compound. In embodiments,a peptide-circRNA molecule complex disclosed herein and a coating compound associate through non- covalent bonds such as a hydrogen bond, an ionic bond, a bond based on Van der Waals, a hydrophobic bond, or electrostatic interactions. For instance, a peptide-circRNA molecule complex disclosed herein may have an overall net positive charge, and a coating compound may have an overall negative charge which may allow the peptide-circRNA molecule complex and compound to associate through electrostatic interactions to form a complex disclosed herein,

[0094] Non-limiting examples of compounds that may be used to coat a nanoparticle to enhance stability of the nanoparticle include polymeric compounds, such as proteins, peptides, or polysaccharides. Further Examples of such compounds are albumin, in particular human serum albumin, bovine serum albumin, hyaluronic acid, fatty acids such as oleic acid, polyethylene glycol, polysaccharides such as chitosan, heparin or heparans and other glycosaminoglycans, or other published coating materials known to those skilled in the art. In embodiments, stability of a peptide-circRNA molecule complex disclosed herein may be enhanced by coating nanoparticles with a fatty acid. In embodiments, the stability of a peptide-circRNA molecule complex disclosed herein may be enhanced by coating nanoparticles with a polysaccharide, such as hyaluronic acid.

[0095] In embodiments, stability of the peptide-circRNA molecule nanoparticle disclosed herein may be enhanced by coating nanoparticles with albumin, in particular human serum albumin. Albumins are negatively charged globular proteins commonly found in blood serum. Without being bound by a theory, it is thought that coating nanoparticles disclosed herein with albumin may enhance stability of nanoparticles by preventing flocculation. In embodiments, the albumins that may be used to coat a nanoparticle comprising a peptide-circRNA molecule complex disclosed herein are serum albumins, and may include bovine serum albumin and human serum albumin. In exemplary embodiments, stability of a nanoparticle comprising a peptide-circRNA molecule complex disclosed herein may be enhanced by coating nanoparticles with human serum albumin and / or hyaluronic acid.

[0096] In essence, a nanoparticle is coated with albumin by incubating the nanoparticle with a solution comprising albumin. Nanoparticles may be incubated in a solution comprising about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 mg / ml or more albumin. In embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 0.1, 0.3, 0.5, 0.7, or 0.9 mg / ml albumin. In embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 1.0, 1.2, 1.4, 1.6,or 1.8 mg / ml albumin. In embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 2.0, 2.2, 2.4, 2.6, or 2.8 mg / ml albumin. In embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 3.0, 3.2, 3.4, 3.6, or 3.8 mg / ml albumin. In additional embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 4.0, 4.2, 4.4, 4.6, 4.8, or 5.0 mg / ml albumin. In embodiments, nanoparticles comprising a peptide-circRNA molecule complex disclosed herein may be incubated in a solution comprising about 4.0 mg / ml albumin.

[0097] In embodiments, the peptide-circRNA molecule complexes disclosed herein may be incubated with albumin for about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 minutes or more to coat the peptide-circRNA molecule complex. In embodiments, a particle comprising a peptide-circRNA molecule complex disclosed herein is incubated with albumin for about 5, 10, 15, or about 20 minutes. In embodiments, a particle comprising a peptide-circRNA molecule complex disclosed herein is incubated with albumin for about 20, 25, 30, or about 35 minutes. In embodiments, a particle comprising a peptide-circRNA molecule complex disclosed herein is incubated with albumin for about 35, 40, 45, or about 50 minutes. In embodiments, a particle comprising a peptide-circRNA molecule complex disclosed herein is incubated with albumin for about 50, 55, or about 60 minutes or more. In embodiments, a particle comprising a peptide-circRNA molecule complex disclosed herein is incubated with albumin for about 25, 30, or about 35 minutes.

[0098] In embodiments, the peptide-circRNA molecule complex disclosed herein may be incubated with hyaluronic acid for about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 minutes or more to allow the hyaluronic acid coat the peptide-circRNA molecule complex or integrate into the peptide-circRNA molecule complex. In embodiments, a peptide-circRNA molecule complex disclosed herein may be incubated with hyaluronic acid for about 1, 2, 3, 4, 5, 10, 12, 18, or 24 hours or more, to allow the hyaluronic acid coat the peptide-circRNA molecule complex or integrate into the peptide- circRNA molecule complex. In embodiments, a peptide-circRNA molecule complex disclosed herein may be incubated with hyaluronic acid for about 45 minutes. Shorter times could be used in embodiments, for example, when using flow processes or microfluidic devices.Cells

[0099] In embodiments, a peptide-circRNA molecule complex disclosed herein is capable transfecting the RNA molecule into the cytoplasm of a cell. In embodiments, a cell is a prokaryotic cell.In embodiments, a cell is a eukaryotic cell. The cell may be in vitro, in vivo (such as, in a subject), in situ, or ex vivo. The cell may be a single cell or may be part of a tissue or an organ.

[0100] In embodiments, a peptide-circRNA molecule complex disclosed herein may be administered to a ceil in vitro by incubating a cell in the presence of a peptide-circRNA molecule complex disclosed herein under conditions suitable for transfection of a RNA molecule of a peptide- circRNA molecule complex. Conditions suitable for transfection of a RNA molecule in a peptide- circRNA molecule complex disclosed herein may be as described in the examples. One skilled in the art will appreciate that the length of incubation can and will vary depending on the peptide-circRNA molecule complex, and the cells. Typically, such conditions may comprise incubation times of between about ten minutes and 24 hours, transfection conditions may comprise incubation times of between about 15 minutes and 3 hours.

[0101] A peptide-circRNA molecule complex disclosed herein may be administered to a cell in vivo (such as, in a subject) by administering to a subject a composition (such as, a pharmaceutical composition) comprising a peptide-circRNA molecule complex disclosed herein.Compositions

[0102] In embodiments, a peptide-circRNA molecule complex disclosed herein may be incorporated into a composition. In embodiments, the composition is a pharmaceutical composition suitable for administration to a subject. A pharmaceutical composition disclosed herein may be used to disrupt, induce or modify the expression of one or more nucleic acid sequences either normally expressed in a cell or “de novo". A pharmaceutical composition as disclosed herein may also serve for vaccination purposes. The pharmaceutical composition may thus also be a vaccine. For instance, a pharmaceutical composition disclosed herein may be used to disrupt the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences normally expressed in a cell. In embodiments, the encoded protein(s) allow(s) to replace or to supplement a cell function or may serve as an antigen for eliciting an immune response. In embodiments, expression of an encoded protein which is either not expressed by the subject or e.g. expressed at lower than required concentrations leads to treatment of a disease or a disorder.

[0103] When a pharmaceutical composition disclosed herein is used to induce the expression of more than one nucleic acid sequence, a pharmaceutical composition may be formulated using a mixture of more than one peptide-circRNA molecule complex, wherein each complex comprises a RNA molecule capable of inducing the expression of a different nucleic acid sequence or additionalexpression is by the encoded protein is achieved. In embodiments, more than one RNA molecule may be used for generating a mixture of peptide-circRNA molecule complexes, wherein each RNA molecule is capable of inducing the expression of a different nucleic acid sequence.

[0104] A pharmaceutical composition disclosed herein may also comprise one or more non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles as desired. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with nanoparticles disclosed herein, use thereof in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0105] A pharmaceutical composition disclosed herein may be formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, polysorbates, poloxamers or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride, glucose or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. A pharmaceutical composition disclosed herein may particularly comprise a non-ionic tensid, more particularly a polysorbate or a mixture of polysorbates, such as polysorbate 20 (PS2O: polyoxyethylen(20)-sorbitan-monolaurat); PS40 (polyoxyethylene (20) sorbitan monopalmitate), PS60 (polyoxyethylene (20) sorbitan monostearate) or PS80 (polyoxyethylene (20) sorbitan monooleate), in particular PS20.

[0106] Oral compositions generally may include an inert diluent or an edible carrier. Oral compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier foruse as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, and the like, may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0107] In embodiments, a pharmaceutical composition disclosed herein is formulated to be compatible with parenteral administration. For instance, pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, balanced salt solution, bacteriostatic water, Cremophor EL (BASF; Parsippany, N. J.), or phosphate buffered saline (PBS). In exemplary embodiments, a pharmaceutical composition disclosed herein is formulated with phosphate buffered saline (PBS).

[0108] In embodiments, the composition is sterile and fluid to the extent that allows easy syringeability. In embodiments, the composition is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0109] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0110] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and may include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0111] In embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, chitosans, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These may be prepared according to methods known to those skilled in the art, for example, as described in U. S. Pat. No. 4,522,811.

[0112] Additional formulations of pharmaceutical compositions may be in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y. (1980). Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners.

[0113] One of skill in the art will recognize that the concentration of a peptide-circRNA molecule complex disclosed herein in a pharmaceutical composition can and will vary depending in part on the route of administration, the subject, and the reason for the administration, and may be determined experimentally. Methods of experimentally determining the concentration of an active agent such as nanoparticles disclosed herein in a pharmaceutical composition are known in the art. In general, a pharmaceutical composition may be formulated to comprise about 0.1 nM to about 50 μM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. For example, a pharmaceutical composition may be formulated to comprise about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 86 nM, 87 nM, 88 nM, 89 nM, 90 nM, 91 nM, 92 nM, 93 nM, 94 nM, 95 nM, 96 nM, 97 nM, 98 nM, 99 nM, 100 nM, 101 nM, 102 nM, 103 nM, 104 nM, 105 nM, 106 nM, 107 nM, 108 nM, 109 nM, 110 nM, 111 nM, 112 nM, 113 nM, 114 nM, 115 nM, 116 nM, 117 nM, 118 nM, 119 nM, 120 nM, 121 nM, 122 nM, 123 nM, 124 nM, 125 nM, 126 nM, 127 nM, 128 nM, 129 nM, 130 nM, 131 nM, 132 nM, 133 nM, 134 nM, 135 nM, 136 nM, 137 nM, 138 nM, 139 nM, 140 nM, 141 nM, 142 nM, 143 nM, 144 nM, 145 nM, 146 nM, 147 nM, 148 nM, 149 nM, 150 nM, 151 nM, 152 nM, 153 nM, 154 nM, 155 nM, 156 nM, 157 nM, 158 nM, 159 nM, 160 nM, 161 nM, 162 nM, 163 nM, 164 nM, 165 nM, 166 nM, 167 nM, 168 nM, 169 nM, 170 nM, 171 nM, 172 nM, 173 nM, 174 nM, 175 nM, 176 nM, 177 nM, 178 nM, 179 nM, 180 nM, 181 nM, 182 nM, 183 nM, 184 nM, 185 nM, 186 nM, 187 nM, 188 nM, 189 nM, 190 nM, 191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197 nM, 198 nM, 199 nM, 200 nM, 201 nM, 202 nM, 203 nM, 204 nM, 205 nM, 206 nM, 207 nM, 208 nM, 209 nM, 210 nM, 211 nM, 212 nM, 213 nM, 214 nM, 215 nM, 216 nM, 217 nM, 218 nM, 219 nM, 220 nM, 221 nM, 222 nM, 223 nM, 224 nM, 225 nM, 226 nM, 227 nM, 228 nM, 229 nM, 230 nM, 231 nM, 232 nM, 233 nM, 234 nM, 235 nM, 236 nM, 237 nM, 238 nM, 239 nM, 241 nM, 242 nM, 243 nM, 244 nM, 245 nM, 246 nM, 247 nM, 248 nM, 249 nM, 251 nM, 252 nM, 253 nM, 254 nM, 255 nM, 256 nM, 257 nM, 258 nM, 259 nM, 261 nM, 262 nM, 263 nM, 264 nM, 265 nM, 266 nM, 267 nM, 268 nM, 269 nM, 271 nM, 272 nM, 273 nM, 274 nM, 275 nM, 276 nM, 277 nM, 278 nM, 279 nM, 281 nM, 282 nM, 283 nM, 284 nM, 285 nM, 286 nM, 287 nM, 288 nM, 289 nM, 291 nM, 292 nM, 293 nM, 294 nM, 295 nM, 296 nM, 297 nM, 298 nM, 299 nM, 300 nM, 301 nM, 302 nM, 303 nM, 304nM, 305 nM, 306 nM, 307 nM, 308 nM, 309 nM, 310 nM, 311 nM, 312 nM, 313 nM, 314 nM, 315 nM, 316 nM, 317 nM, 318 nM, 319 nM, 320 nM, 321 nM, 322 nM, 323 nM, 324 nM, 325 nM, 326 nM, 327 nM, 328 nM, 329 nM, 330 nM, 331 nM, 332 nM, 333 nM, 334 nM, 335 nM, 336 nM, 337 nM, 338 nM, 339 nM, 340 nM, 341 nM, 342 nM, 343 nM, 344 nM, 345 nM, 346 nM, 347 nM, 348 nM, 349 nM, 350 nM, 351 nM, 352 nM, 353 nM, 354 nM, 355 nM, 356 nM, 357 nM, 358 nM, 359 nM, 360 nM, 361 nM, 362 nM, 363 nM, 364 nM, 365 nM, 366 nM, 367 nM, 368 nM, 369 nM, 370 nM, 371 nM, 372 nM, 373 nM, 374 nM, 375 nM, 376 nM, 377 nM, 378 nM, 379 nM, 380 nM, 381 nM, 382 nM, 383 nM, 384 nM, 385 nM, 386 nM, 387 nM, 388 nM, 389 nM, 390 nM, 391 nM, 392 nM, 393 nM, 394 nM, 395 nM, 396 nM, 397 nM, 398 nM, 399 nM, 400 nM, 401 nM, 402 nM, 403 nM, 404 nM, 405 nM, 406 nM, 407 nM, 408 nM, 409 nM, 410 nM, 411 nM, 412 nM, 413 nM, 414 nM, 415 nM, 416 nM, 417 nM, 418 nM, 419 nM, 420 nM, 421 nM, 422 nM, 423 nM, 424 nM, 425 nM, 426 nM, 427 nM, 428 nM, 429 nM, 430 nM, 431 nM, 432 nM, 433 nM, 434 nM, 435 nM, 436 nM, 437 nM, 438 nM, 439 nM, 440 nM, 441 nM, 442 nM, 443 nM, 444 nM, 445 nM, 446 nM, 447 nM, 448 nM, 449 nM, 450 nM, 451 nM, 452 nM, 453 nM, 454 nM, 455 nM, 456 nM, 457 nM, 458 nM, 459 nM, 460 nM, 461 nM, 462 nM, 463 nM, 464 nM, 465 nM, 466 nM, 467 nM, 468 nM, 469 nM, 470 nM, 471 nM, 472 nM, 473 nM, 474 nM, 475 nM, 476 nM, 477 nM, 478 nM, 479 nM, 480 nM, 481 nM, 482 nM, 483 nM, 484 nM, 485 nM, 486 nM, 487 nM, 488 nM, 489 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, 509 nM, 510 nM, 511 nM, 512 nM, 513 nM, 514 nM, 515 nM, 516 nM, 517 nM, 518 nM, 519 nM, 520 nM, 521 nM, 522 nM, 523 nM, 524 nM, 525 nM, 526 nM, 527 nM, 528 nM, 529 nM, 530 nM, 531 nM, 532 nM, 533 nM, 534 nM, 535 nM, 536 nM, 537 nM, 538 nM, 539 nM, 540 nM, 541 nM, 542 nM, 543 nM, 544 nM, 545 nM, 546 nM, 547 nM, 548 nM, 549 nM, 550 nM, 551 nM, 552 nM, 553 nM, 554 nM, 555 nM, 556 nM, 557 nM, 558 nM, 559 nM, 560 nM, 561 nM, 562 nM, 563 nM, 564 nM, 565 nM, 566 nM, 567 nM, 568 nM, 569 nM, 570 nM, 571 nM, 572 nM, 573 nM, 574 nM, 575 nM, 576 nM, 577 nM, 578 nM, 579 nM, 580 nM, 581 nM, 582 nM, 583 nM, 584 nM, 585 nM, 586 nM, 587 nM, 588 nM, 589 nM, 590 nM, 591 nM, 592 nM, 593 nM, 594 nM, 595 nM, 596 nM, 597 nM, 598 nM, 599 nM, 600 nM, 601 nM, 602 nM, 603 nM, 604 nM, 605 nM, 606 nM, 607 nM, 608 nM, 609 nM, 610 nM, 611 nM, 612 nM, 613 nM, 614 nM, 615 nM, 616 nM, 617 nM, 618 nM, 619 nM, 620 nM, 621 nM, 622 nM, 623 nM, 624 nM, 625 nM, 626 nM, 627 nM, 628 nM, 629 nM, 630 nM, 631 nM, 632 nM, 633 nM, 634 nM, 635 nM, 636 nM, 637 nM, 638 nM, 639 nM, 640 nM, 641 nM, 642 nM, 643 nM, 644 nM, 645 nM, 646 nM, 647 nM, 648 nM, 649 nM, 650 nM, 651 nM, 652 nM, 653 nM, 654 nM, 655 nM, 656 nM, 657 nM, 658 nM, 659 nM, 660 nM, 661 nM, 662 nM, 663 nM, 664 nM, 665 nM, 666 nM, 667 nM, 668 nM, 669 nM, 670 nM, 671 nM, 672 nM, 673 nM, 674 nM, 675 nM, 676 nM, 677 nM, 678 nM, 679 nM, 680 nM, 681 nM, 682 nM, 683 nM, 684 nM, 685 nM, 686 nM, 687 nM, 688 nM, 689 nM, 690 nM, 691 nM, 692 nM, 693 nM, 694 nM, 695nM, 696 nM, 697 nM, 698 nM, 699 nM, 700 nM, 701 nM, 702 nM, 703 nM, 704 nM, 705 nM, 706 nM, 707 nM, 708 nM, 709 nM, 710 nM, 711 nM, 712 nM, 713 nM, 714 nM, 715 nM, 716 nM, 717 nM, 718 nM, 719 nM, 720 nM, 721 nM, 722 nM, 723 nM, 724 nM, 725 nM, 726 nM, 727 nM, 728 nM, 729 nM, 730 nM, 731 nM, 732 nM, 733 nM, 734 nM, 735 nM, 736 nM, 737 nM, 738 nM, 739 nM, 740 nM, 741 nM, 742 nM, 743 nM, 744 nM, 745 nM, 746 nM, 747 nM, 748 nM, 749 nM, 750 nM, 751 nM, 752 nM, 753 nM, 754 nM, 755 nM, 756 nM, 757 nM, 758 nM, 759 nM, 760 nM, 761 nM, 762 nM, 763 nM, 764 nM, 765 nM, 766 nM, 767 nM, 768 nM, 769 nM, 770 nM, 771 nM, 772 nM, 773 nM, 774 nM, 775 nM, 776 nM, 777 nM, 778 nM, 779 nM, 780 nM, 781 nM, 782 nM, 783 nM, 784 nM, 785 nM, 786 nM, 787 nM, 788 nM, 789 nM, 790 nM, 791 nM, 792 nM, 793 nM, 794 nM, 795 nM, 796 nM, 797 nM, 798 nM, 799 nM, 800 nM, 801 nM, 802 nM, 803 nM, 804 nM, 805 nM, 806 nM, 807 nM, 808 nM, 809 nM, 810 nM, 811 nM, 812 nM, 813 nM, 814 nM, 815 nM, 816 nM, 817 nM, 818 nM, 819 nM, 820 nM, 821 nM, 822 nM, 823 nM, 824 nM, 825 nM, 826 nM, 827 nM, 828 nM, 829 nM, 830 nM, 831 nM, 832 nM, 833 nM, 834 nM, 835 nM, 836 nM, 837 nM, 838 nM, 839 nM, 840 nM, 841 nM, 842 nM, 843 nM, 844 nM, 845 nM, 846 nM, 847 nM, 848 nM, 849 nM, 850 nM, 851 nM, 852 nM, 853 nM, 854 nM, 855 nM, 856 nM, 857 nM, 858 nM, 859 nM, 860 nM, 861 nM, 862 nM, 863 nM, 864 nM, 865 nM, 866 nM, 867 nM, 868 nM, 869 nM, 870 nM, 871 nM, 872 nM, 873 nM, 874 nM, 875 nM, 876 nM, 877 nM, 878 nM, 879 nM, 880 nM, 881 nM, 882 nM, 883 nM, 884 nM, 885 nM, 886 nM, 887 nM, 888 nM, 889 nM, 890 nM, 891 nM, 892 nM, 893 nM, 894 nM, 895 nM, 896 nM, 897 nM, 898 nM, 899 nM, 900 nM, 901 nM, 902 nM, 903 nM, 904 nM, 905 nM, 906 nM, 907 nM, 908 nM, 909 nM, 910 nM, 911 nM, 912 nM, 913 nM, 914 nM, 915 nM, 916 nM, 917 nM, 918 nM, 919 nM, 920 nM, 921 nM, 922 nM, 923 nM, 924 nM, 925 nM, 926 nM, 927 nM, 928 nM, 929 nM, 930 nM, 931 nM, 932 nM, 933 nM, 934 nM, 935 nM, 936 nM, 937 nM, 938 nM, 939 nM, 940 nM, 941 nM, 942 nM, 943 nM, 944 nM, 945 nM, 946 nM, 947 nM, 948 nM, 949 nM, 950 nM, 951 nM, 952 nM, 953 nM, 954 nM, 955 nM, 956 nM, 957 nM, 958 nM, 959 nM, 960 nM, 961 nM, 962 nM, 963 nM, 964 nM, 965 nM, 966 nM, 967 nM, 968 nM, 969 nM, 970 nM, 971 nM, 972 nM, 973 nM, 974 nM, 975 nM, 976 nM, 977 nM, 978 nM, 979 nM, 980 nM, 981 nM, 982 nM, 983 nM, 984 nM, 985 nM, 986 nM, 987 nM, 988 nM, 989 nM, 990 nM, 991 nM, 992 nM, 993 nM, 994 nM, 995 nM, 996 nM, 997 nM, 998 nM, 999 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or about 50 μM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 0.1 nM to about 1.0 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 nM to about 10nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 nM to about 100 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 nM to about 200 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 nM to about 50 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 10 nM to about 100 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 10 nM to about 200 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 50 nM to about 100 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 50 nM to about 200 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 100 nM to about 200 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 150 nM to about 200 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 200 nM to about 100 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 500 nM to about 1000 nM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 μM to about 50 μM of a RNA molecule in a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition may be formulated to comprise about 1 to about 100 nM, in particular about 1 to about 50 nM or about 10 to about 20 nM. A concentration of peptide in a peptide-circRNA molecule complex disclosed herein may be calculated based on the desired concentration of RNA molecule and the ratio of peptide to RNA molecule in the peptide-circRNA molecule complex disclosed herein.

[0114] A pharmaceutical composition may also be formulated to comprise about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or about 700 μg / ml or more of a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition is formulated to comprise 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about100 pg / ml of a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition is formulated to comprise 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 pg / ml of a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition is formulated to comprise 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 pg / ml of a peptide-circRNA molecule complex disclosed herein. In embodiments, a pharmaceutical composition is formulated to comprise 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or about 700 pg / ml or more of a peptide-circRNA molecule complex disclosed herein.

[0115] In case of the pharmaceutical composition being a vaccine, it may be advantageously be employed for prevention of infectious dieses or cancer diseases or for treating a cancer disease by therapeutic vaccination. Thereby, the "pharmaceutical composition" may advantageously be administered intramuscularly, in particular when being used for prevention of an infectious disease. The infectious diseases may be selected from an infectious disease as disclosed herein.Methods of Use

[0116] The disclosure provides methods of using the peptide-circRNA molecule complexes (or compositions containing the same) disclosed herein or peptide-circRNA molecule complexes (or compositions containing the same), e.g. for in vitro use or for use in a method of preventing and treating a subject. In embodiments, the disclosure provides methods for using a peptide-circRNA molecule complex disclosed herein or its use to transfect the RNA molecule into the cytoplasm of a cell. In embodiments, the cell is in vitro. In embodiments, the cell is in vivo (such as, in a subject). The disclosure also provides methods for using a peptide-circRNA molecule complex disclosed herein or a peptide-circRNA molecule complex disclosed herein for use in a method to transfect the RNA molecule into the cytoplasm of a cell in a subject in need thereof. In embodiments, the methods disclosed herein comprises contacting a cell with a peptide-circRNA molecule complex disclosed herein under conditions suitable for transfection of a RNA molecule. In embodiments, the methods disclosed herein comprise administering a pharmaceutical composition comprising a peptide-circRNA molecule complex disclosed herein to a subject in need thereof.

[0117] In embodiments, the disclosure provides methods for treating a condition in a subject or the use in methods for treating a condition in a subject. In embodiments, the methods for treating a condition in a subject disclosed herein comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a peptide-circRNAmolecule complex disclosed herein. A peptide-circRNA molecule complex disclosed herein is capable of efficiently transfecting, or delivering, the RNA molecule of the peptide-circRNA molecule complex into a cell of the subject.

[0118] In embodiments, a RNA molecule disclosed herein comprises a coding double-stranded RNA or a RNA encoding a non-coding RNA capable of inducing, modifying, regulating or inhibiting expression of a nucleic acid sequence. By efficiently transfecting a RNA molecule capable of inducing, modifying, regulating or inhibiting expression of a nucleic acid sequence, a method disclosed herein may be used to treat any condition that can be treated by regulating or inhibiting the expression of a nucleic acid sequence normally expressed in a cell.Administration to a Subject in Need Thereof

[0119] In embodiments, the disclosure provides methods or the use in methods of administering a therapeutically effective amount of a peptide-circRNA molecule complex or a pharmaceutical composition disclosed herein to a subject in need thereof. As used herein, the phrase "a subject in need thereof" refers to a subject in need of preventative or therapeutic treatment. A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, a subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another embodiment, a subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In still another embodiment, a subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, a subject may be a zoological animal. As used herein, a "zoological animal" refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In embodiments, a subject is a mouse. In embodiments, a subject is a human.

[0120] As described herein, a peptide-circRNA molecule complexes or a pharmaceutical composition disclosed herein is formulated to be compatible with its intended route of administration. Suitable routes of administration include parenteral, oral, pulmonary, transdermal, transmucosal, and rectal administration. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intrathecal, or intrasternal injection, or infusion techniques. Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation {e.g., via an aerosol), buccal e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo), parenteral {e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal,diaphragm and / or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain). In some embodiments, the administration is by injection into the central nervous system. The most suitable route in any given case will depend on the nature and severity of the condition being treated and / or prevented and on the nature of the composition that is being used. In embodiments, a pharmaceutical composition or a peptide-circRNA molecule complexes disclosed herein is administered by injection. The pharmaceutical composition may be injected intramuscularly, in particular in case of treating or preventing an infectious disease ora cancer disease by vaccination.

[0121] One of skill in the art will recognize that the amount and concentration of the composition or the peptide-circRNA molecule complex administered to a subject will depend in part on the subject and the reason for the administration. Methods for determining optimal amounts are known in the art. In general, the concentration of a peptide-circRNA molecule complex disclosed herein in a pharmaceutical composition may be as described herein.

[0122] Compositions disclosed herein are typically administered to a subject in need thereof in an amount sufficient to provide a benefit to the subject. This amount is defined as a "therapeutically effective amount." A therapeutically effective amount may be determined by the efficacy or potency of the particular composition, the disorder being treated, the duration or frequency of administration, the method of administration, and the size and condition of the subject, including that subject's particular treatment response. A therapeutically effective amount may be determined using methods known in the art, and may be determined experimentally, derived from therapeutically effective amounts determined in model animals such as the mouse, or a combination thereof. Additionally, the route of administration may be considered when determining the therapeutically effective amount. In determining therapeutically effective amounts, one skilled in the art may also consider the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject.

[0123] When a pharmaceutical composition disclosed herein is administered to a subject by injection, a composition may be administered to the subject in a bolus in an amount of about 0.1 mg / kg to about 100 mg / kg or more. In embodiments, a pharmaceutical composition disclosed herein is administered to a subject in an amount of about 0.1 mg / kg to about 5 mg / kg. In embodiments, apharmaceutical composition disclosed herein is administered to a subject in an amount of about 5 mg / kg to about 15 mg / kg. In embodiments, a pharmaceutical composition disclosed herein is administered to a subject in an amount of about 15 mg / kg to about 30 mg / kg. In embodiments, a pharmaceutical composition disclosed herein is administered to a subject in an amount of about 30 mg / kg to about 45 mg / kg. In additional embodiments, a pharmaceutical composition disclosed herein is administered to a subject in an amount of about 45 mg / kg to about 100 mg / kg or more. In embodiments, a composition is administered to the subject in a bolus in an amount of about 0.5 to about 1.5 mg / kg.

[0124] A composition may also be administered by injecting more than one bolus into the subject over a period of time. For instance, a composition may be administered by injecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more boluses into the subject. In embodiments, a composition is administered by injecting 1, 2, 3, 4, or 5 boluses into the subject. In embodiments, a composition is administered by injecting 5, 6, 7, 8, 9, 10 or more boluses into the subject. In embodiments, a composition is administered by injecting 2, 3, or 4 boluses into the subject. The boluses may be injected about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about every 12 hours, or they may be injected about every 1, 2, 3, 4, 5, 6, or about every 7 days. In embodiments, boluses may be injected about every day.Treatment Methods

[0125] The disclosure provides methods of treating a subject in need thereof or the use of a pharmaceutical composition disclosed herein or a peptide-circRNA molecule complexes in methods, comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein or a peptide-circRNA molecule complexes as disclosed herein to a subject in need thereof. The disclosure also provides methods of preventing a disease in a subject or the use of a pharmaceutical composition disclosed herein or a peptide-circRNA molecule complexes in methods of preventing a disease, comprising administering an effective amount of a pharmaceutical composition disclosed herein or a peptide-circRNA molecule complexes as disclosed herein to a subject in need thereof.

[0126] In embodiments, the subject has a disease or a disorder, such as, for example, a cancer or is at risk of a disease, such as an infectious disease, in embodiments, the disclosure provides methods or the use in methods of treating a neoplasm or cancer. The neoplasm may be malignant or benign, the cancer may be primary or metastatic; the neoplasm or cancer may be early stage or late stage. The cancer may be a blood cancer or a solid tumor cancer. A cancer or a neoplasm may be treated by delivering a nucleic acid sequence to a cancer tumor in a subject. The cancer or neoplasm may betreated by slowing cancer cell growth, killing cancer cells or reducing the spreading of cancer cells to generate metastases.

[0127] In embodiments, the methods or the use in methods comprise delivering a composition or a RNA molecule of the nanoparticles disclosed herein to a cancer cell or a healthy body cell, such as a muscle cell, an immune cell, or a liver cell in a subject in vivo. A healthy cell should allow to express the protein encoded by the RNA molecule of the complex (protein replacement therapy), in particular in case of genetic diseases. By protein replacement therapy, a protein is replaced or supplemented in patients in whom that particular protein is deficient or absent. By circRNA, the capacity of coding circRNA allows to encode peptides or proteins and spark their transient cellular expression. It allows to restore protein deficiencies through circRNA protein replacement therapy or to achieve antigen presentation through circRNA vaccination. It has long been established that mRNA is generated through the transcription of the genomic DNA to mediate the delivery of the genetic information to the cellular translational machinery. It may be applied for rare monogenic diseases or endocrinological diseases. These conditions are owed to single-gene defects present in the human coding genome that cause the encoded proteins to be defective or even missing. Despite their rarity, these so-called "orphan" diseases are numerous.

[0128] In embodiments, the methods or the use in methods comprise treating a cancer or a neoplasm by delivering a RNA molecule of the nanoparticles disclosed herein to cells of the tumor microenvironment or to other cells in the surroundings of a tumor. In embodiments, the methods or the use in methods comprising prophylactic or therapeutic cancer vaccination of a subject having a cancer or a neoplasm being at risk of having or being at risk of a cancer disease by delivering a RNA molecule of the nanoparticles disclosed herein to cells of the tumor microenvironment or to other cells in the surroundings of a tumor. Non-limiting examples of neoplasms or cancers that may be treated with a method disclosed herein may include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenomas / carcinoids, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumors (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancers (non-small cell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesotheliomas (adult malignant, childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia (chronic), myeloid leukemias (adult acute, childhood acute), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary (metastatic), stomach cancer, supratentorial primitive neuroectodermal tumor (childhood), T-celllymphoma (cutaneous), T-cell leukemia and lymphoma, testicular cancer, throat cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site (adult, childhood), ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor (childhood). In embodiments, a method disclosed herein is used to treat T-cell leukemia and lymphoma. In an exemplary embodiment, a method disclosed herein is used to treat Human T-Lymphotropic Virus-1 (HTLV-1) induced adult T- cell leukemia / lymphoma (ATLL).

[0129] In embodiments, the subject has a disease or a disorder, such as, for example, an inflammatory disease. In embodiments, the disclosure provides methods or the use in methods of treating an inflammatory disease. Thus, the disclosure provides use in methods or methods of treating and / or delaying the onset of an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of any one or more of the compositions disclosed herein.

[0130] In embodiments, the methods the use in methods comprise delivering a RNA molecule of the nanoparticles disclosed herein to an inflammatory cell in a subject in vivo. In embodiments, the cell is an inflammatory cell, such as, for example, a mast cell, an eosinophil, a neutrophil, a basophil, a macrophage, a monocyte or a lymphocyte. In embodiments, the methods or the use in methods comprise delivering a RNA molecule of the nanoparticles disclosed herein to a cell affected by inflammation in a subject in vivo. In embodiments, the methods or the use in methods comprise treating an inflammatory disease in a subject by administering a RNA molecule of the nanoparticles disclosed herein to the subject.

[0131] Non-limiting examples of inflammatory diseases include autoimmune diseases (e.g. rheumatoid arthritis, psoriatic arthritis, gouty arthritis, lupus, psoriasis, ankylosing spondylitis), cardiovascular diseases (e.g. high blood pressure, heart disease), gastrointestinal diseases (e.g., inflammatory bowel disease, Crohn's disease, ulcerative colitis), lung disease (e.g., chronic obstructive pulmonary disease (COPD), asthma), mental illness (e.g., depression, anxiety), metabolic illness (e.g., Type 2 diabetes mellitus, Type 1 diabetes mellitus), neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease), fatty liver disease, endometriosis, allergy, and cancer.

[0132] Other examples of inflammatory diseases include Encephalitis, Myelitis, Meningitis, Arachnoiditis, Neuritis, Dacryoadenitis, Scleritis, Episcleritis, Keratitis, Retinitis, Chorioretinitis, Blepharitis, Conjunctivitis, Uveitis, Otitis externa, Otitis media, Labyrinthitis, Mastoiditis, Carditis, Endocarditis, Myocarditis, Pericarditis, Vasculitis, Arteritis, Phlebitis, Capillaritis, Sinusitis, Rhinitis, Pharyngitis, Laryngitis, Tracheitis, Bronchitis, Bronchiolitis, Pneumonitis, Pleuritis, Mediastinitis, Stomatitis, Gingivitis, Gingivostomatitis, Glossitis, Tonsillitis, Sialadenitis / Parotitis, Cheilitis, Pulpitis, Gnathitis, Esophagitis, Gastritis, Gastroenteritis, Enteritis, Colitis, Enterocolitis, Duodenitis, Ileitis, Caecitis, Appendicitis, Proctitis, Hepatitis, Ascending cholangitis, Cholecystitis, Pancreatitis, Peritonitis, Dermatitis, Folliculitis, Cellulitis, Hidradenitis. Arthritis, Dermatomyositis, Myositis, Synovitis / Tenosynovitis, Bursitis, Enthesitis, Fasciitis, Capsulitis, Epicondylitis, Tendinitis, Panniculitis, Osteochondritis: Osteitis / Osteomyelitis, Spondylitis, Periostitis, Chondritis, Urinary system, Nephritis, Glomerulonephritis, Pyelonephritis, Ureteritis, Cystitis, Urethritis, Oophoritis, Salpingitis, Endometritis, Parametritis, Cervicitis, Vaginitis, Vulvitis, Mastitis, Orchitis, Epididymitis, Prostatitis, Seminal vesiculitis, Balanitis, Posthitis, Balanoposthitis, Chorioamnionitis, Funisitis, Omphalitis, Insulitis, Hypophysitis, Thyroiditis, Parathyroiditis, Adrenalitis, Lymphangitis, and Lymphadenitis.

[0133] In embodiments, the methods or the use comprise delivering a RNA molecule of a peptide-circRNA molecule complex disclosed herein for preventing an infectious disease orfor use as a vaccine. In such embodiments, the RNA molecule of the peptide-circRNA complex encodes at least one antigen, e.g. one, two or three antigen(s). An encoded antigen may be derived from a pathogen associated with infectious disease and / or which may be considered as being associated with the induction of an infectious disease. Said antigen may typically be a peptide or protein antigen, or a fragment, variant and / or derivative of said peptide or protein antigen. The antigen may be viral, bacterial or protozoological origin, in particular of viral origin. Exemplary viral antigens may be derived from influenza virus, RSV, West Nile virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Lassa virus, Herpes simplex, AIDS virus, Rhinovirus, Ebola virus, and Yellow fever virus. Bacterial antigens may be derived from Enterococcus, Chlamydia, Borrelia and Salmonella. Protozoological infections may be derived from Trypanosoma, Leishmania and Plasmodium. The antigen is typically a surface protein of an infectious agent, such as a viral or bacterial surface protein. The encoded protein may comprise the full-length surface protein or a fragment thereof. The fragment is specifically suitable, if it comprises at least one epitope.

[0134] The vaccine may be an adjuvanted vaccine. The vaccine may thus contain one or more adjuvant(s) for increasing the immune response. The adjuvant may support or trigger an innateimmune response. It may be a compound which activates a pattern recognition receptor, such as e.g. a receptor selected from the Toll-like receptor (TLR) family, including e.g. a Toll like receptor selected from human TLR1 to TLR10.

[0135] The peptide / circRNA complex may be administered such that it enters into a healthy body cell, such as a muscle cell, allowing to express the protein encoded by the RNA molecule. Expression of the protein, typically an antigen, allows the protein to be presented to the immune system eliciting an immune response against the encoded protein. Depending on the body cell intended envisaged to express the encoded antigen, the administration route may be chosen. For expression by a muscle cell, intramuscular administration, typically intramuscular injection, is the administration route of choice.

[0136] In embodiments, the methods or the use comprise delivering a RNA molecule of a peptide-circRNA molecule complex disclosed herein to a cancer cell in vitro. For instance, a RNA molecule of a peptide-circRNA molecule complex disclosed herein may be delivered to a cancer cell line in vitro. A cancer cell may be a cancer cell line cultured in vitro. In embodiments, a cancer cell line may be a primary cell line that is not yet described. Methods of preparing a primary cancer cell line utilize standard techniques known to individuals skilled in the art. In other alternatives, a cancer cell line may be an established cancer cell line. A cancer cell line may be adherent or non-adherent, or a cell line may be grown under conditions that encourage adherent, non-adherent or organotypic growth using standard techniques known to individuals skilled in the art. A cancer cell line may be contact inhibited or non-contact inhibited.

[0137] In embodiments, the cancer cell line may be an established human cell line derived from a tumor. Non-limiting examples of cancer cell lines derived from a tumor include the osteosarcoma cell lines 143B, CAL-72, G-292, HOS, KHOS, MG-63, Saos-2, and U-2 OS; the prostate cancer cell lines DU145, PC3 and Lncap; the breast cancer cell lines MCF-7, MDA-MB-438 and T47D; the myeloid leukemia cell line THP-1, the glioblastoma cell line U87; the neuroblastoma cell line SHSY5Y; the bone cancer cell line Saos-2; the colon cancer cell lines WiDr, COLO 320DM, HT29, DLD-1, COLO 205, COLO 201, HCT-15, SW620, LoVo, SW403, SW403, SW1116, SW1463, SW837, SW948, SW1417, GPC-16, HCT-8, HCT 116, NCI-H716, NCI-H747, NCI-HSO8, NCI-H498, COLO 320HSR, SNU-C2A, LS 180, LS 174T, MOLT-4, LS513, LS1034, LS411N, Hs 675. T, CO 88BV59-1, Co88BV59H21-2, Co88BV59H21-2V67-66, 1116-NS-19-9, TA 99, AS 33, TS 106, Caco-2, HT-29, SK-CO-1, SNU-C2B and SW480; the non-small cell lung cancer (NSCLC) cell lines H358, H2122, H441, H727, SK-Lu-1, H2009, the melanoma cell line B16-F10, the macrophage cell line RAW264.7, the F8 cell line, and the pancreatic carcinoma cell lines Panel, PANC 10.05, CAPAN-1, CAPAN-2, PSN1, MIA-PaCa2. In an exemplary embodiment, a peptide- circRNA molecule complex disclosed herein may be administered to a F8 cell line. In embodiments, a peptide-circRNA molecule complex disclosed herein may be administered to a B16-F10 cell line.

[0138] In embodiments, the nanoparticles or compositions disclosed herein are used for specific delivery to tumors. Without being bound by a theory, it is thought that the leaky vasculature of tumors allows for the extravasation of the nanoparticles disclosed herein due to its physicochemical characteristics. The coating of the nanoparticles disclosed herein with albumin enriches the local concentration of the nanoparticles through binding to the receptors pg60 and / or SPARC. These receptors are upregulated in certain tumors. Coating the nanoparticle with hyaluronic acid can have the same effect on other tumor types through the CD44 receptor. Thus, the nanoparticles may be coated by serum albumin, in particular human serum albumin. Alternatively, hyaluronic acid, preferably hyaluronic acid of a medium or low molecular weight may be used for coating such that the nanoparticles have a hyaluronic acid coating.

[0139] In embodiments, the methods comprise delivering a RNA molecule of a peptide-circRNA molecule complex disclosed herein to an inflammatory cell or a cell affected by inflammation in vitro. In embodiments, the nanoparticles or compositions disclosed herein are used for specific delivery to sites of inflammation. Without being bound by a theory, it is thought that the leaky vasculature induced by inflammation allows for the extravasation of the nanoparticles disclosed herein due to its physicochemical characteristics. The coating of the nanoparticles disclosed herein with albumin enriches the local concentration of the nanoparticles through binding to the receptors pg60 and / or SPARC. These receptors are upregulated in response to inflammation. Coating the nanoparticle with hyaluronic acid can have the same effect on other inflammation sites through the CD44 receptor.Kits

[0140] The disclosure provides kits comprising: a first composition comprising a peptide of the peptide-circRNA molecule complexes disclosed herein, and optionally a second composition comprising a RNA molecule. Alternatively, a RNA molecule of interest may be provided by a user of the kit. By following directions provided by the kit, a user of the kit may mix the composition comprising a peptide of the peptide-circRNA molecule complexes disclosed herein and a composition comprising a RNA molecule to form a peptide-circRNA molecule complex. The directions of the kit may include instructions to mix the peptide and RNA molecule at a suitable ratio. The kit may alsoinclude suitable buffers, water, cross-linking reagents or albumin. The kit may be used for the methods described above.EXAMPLES

[0141] It is to be understood that the description above as well as the examples that follow are intended to illustrate, and not limit, the scope of the invention.* * * * *

[0142] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

[0143] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. The incorporated patents include, but are not limited to, US Patent No. 9,987,371, US Patent No. 10,758,627, and US Patent No. 11,529,388. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0144] The following materials were used for the experiments according to the Examples:

[0145] Peptide used in all experiments unless otherwise specified: p5RHH (also denominated as „PEP-C" herein): H-VLTTGLPALISWIRRRHRRHC-OH (SEQ ID NO:1):PeptideName Sequence MWp5RHH (" PEP-C") H-VLTTGLPALISWIRRRHRRHC-OH Free base 2542.0Acetate salt: 3022.43 g / mol

[0146] Circular mRNA used in all experiments according to SEQ ID NO: 29 (Figure 11).Circular mRNANameSequence (ORF) Modification and CAP Length MWGenescript Figure 11 No modification and 2570 nt 828321 g / mol Firefly luciferase no CAPcircular RNACircular mRNA according to Figure 11 is available by GenScript (https: / / www.genscript.com / gsfiles / techfiles / Catalog_F-Luc_Circular_RNA.pdf?=20240612?44615805)

[0147] Linear mRNA used in all experiments is according to SEQ. ID NO: 30 (Figure 12). The " U" nucleotides of the linear mRNA are represented as " T" nucleotides by the sequences protocol.Linear mRNAName Sequence (ORF) Modification and CAP Length (nt) MW > firefly igure 12 Fully substituted with N1-Methyl- = Full-length 637566 g / mol luciferase Pseudo-U, Cleancap' sequence:1929 ntThe ORF sequence according to Figure 12 of the full-length mRNA is available as L-8102 https: / / www.trilinkbiotech.com / cleancap-m6-fluc-mrna-nlmepsu.html)Coating MaterialsName Production Manufacturer Grade MW Human Serum Albumin Recombinant Albumedix GMP like, 67500 Da> 99.0% purityFurther Materials• PBS buffer (Gibco, Cat. No. 10010-023)• p5RHH (4.3 mM, dissolved in water)• HEK293T cells (ECACC, Cat. No. 12022001)• DMEM medium (high glucose, Gibco, Cat. No. 11965092)• Bright-Glo Luciferase Assay System (Promega, Cat. No. E2620)

[0148] The following protocol was applied for all Examples studying uncoated peptide - circular RNA nanoparticles (uncoated "peptide / circRNA complexes"), unless indicated herein otherwise: For formulating uncoated peptide - circular RNA ("circRNA") nanoparticles, particles were prepared by diluting circRNA (1 mg / ml in citric acid) to the desired concentration in PBS. The cationic peptide used for complexation (" PEP-C" synonymous to "p5RHH") was diluted in nuclease free water to the desired concentration in PBS and kept frozen. Equal volumes of diluted circRNA and " PEP-C" were mixed together in a 1500 uL Eppendorf tube and contents were mixed by pipetting up and down three times; thereafter the tubes were incubated for 60 minutes at approximately 20°C or 37°C.

[0149] The following protocol was applied for all Examples studying coated peptide - circular RNA nanoparticles (coated "peptide / circRNA complexes"), unless indicated herein otherwise: For coating, 70 uL of uncoated nanoparticles were coated for each experiment. The procedure was carried out by adding to the uncoated formulation the desired amount of coating agent (HA (hyaluronic acid) or HSA (human serum albumin)). The coating agent was added in a volume of less than 10% of the uncoated formulation volume. If needed, the coating agents were diluted in nuclease free water to reach the required concentration. Once the coating agent had been added, the samples were mixed by pipetting up and down three times and thereafter incubated at approximately 20°C for 10 minutes.

[0150] The following protocol was applied for all Examples measuring the transfection efficiency of nanoparticles containing the circular RNA encoding luciferase (expressed in HepG2, Panc-1 cells and HEK293T cells) by luminescence. For cell transfection: 20.000 cells for HEK-293T or PANC-1 cells or 15.000 cells in the case of HepG2 cells were seeded in complete medium (DMEM supplemented with 10% FBS) per well and allowed to attach to the well plate for at least 24h in an incubator set at 37°C and 5% CO2. For time course experiments the number of cells seeded was reduced to 50%, if incubations were carried out for 48h or 25% if incubations were carried out for 72h. On the day of transfection, the cell culture media was exchanged for fresh complete medium; thereafter the formulation (approximately 5 uL) was added to 100 uL of culture media and the cells were returned to the incubator to allow the transfection to take place. As indicated, expression levels were measured 24, 48 or 72 h after transfection. For doing so, the plates were taken out from the incubator and processed to quantify the expression of luciferase following the instructions of the Promega's " Bright- Glo Luciferase Assay System" kit; finally, samples were read using a GloMax plate reader.

[0151] Further experimental details are described for the individual experiments as described by Examples 1 and 2.

[0152] Example 1: Measurement of Transfection Efficiency

[0153] Example 1.1Expression levels of firefly luciferase after transfection with circular Flue mRNA uncoated nanoparticles were measured by luminescence upon incubation at 5 min, 10 min and 15 min at 37°C.The results are shown in Figures 1A and IB and described by the legend of Figure 1.

[0154] Example 1.2Peptide / circRNA complexes of N / P ratios 8 (100 ng) and 10 (227 ng) were studied in comparison to a circRNA complexed with lipofectamine by measuring the expression level (by luminescence measurement) of the transfected peptide / circRNA nanoparticles after transfection of HEK293T cells.More detailed experimental conditions are represented by below Table 1:Table 1Working solution Final concentration Nanoparticle{PM) (pM) preparationMolar ng mRNAConditions Ratio added to ceils Vol added to Vol medium in Exposure ceils well toNPs mRNA PEP-C mRNA PEP-C Time Temp.(min) (°C)circRNAN / P 8 450 0.055 225 4100 2270.11 60 RT 5 100 24h circRNAN / P 10 576 0.055 288 5150 227The results are shown in Figure 2 and described by the legend of Figure 2.

[0155] Example 1.3Nanoparticles of peptide / circular RNA (prepared with different N / P ratios) were analysed in terms of their expression levels as compared to linear mRNA (N / P=8.3) after transfection of HEK293T cells by the luminescence of the expressed luciferase.More detailed experimental conditions are represented by below Tables 2 and 3:Table 2 _Working solution Final concentration Nanoparticle(PM) (PM) preparation mRNAConditions Molar added to Vol added Vol medium Exposure to Ratio cells to cells in well NPs mRNA PEP-C mRNA PEP-C Time Temp, (ng)(min) comRNAN / P 8.3 350 0.055 175 3188 175circRNAN / P 8 450 0.055 225 4100 227circRNAN / P 10 0.11 566 0.055 283 5150 60 RT 227 5 100 24hcircRNAN / P 12 676 0.055 338 6150 227Table 3Working solution Nanoparticle(MM) Final cone (pM) preparation mRNAConditions Molar added to Vol added to Vol medium Exposure Ratio ceils cells in well toNPs (ngmRNA PEP- Time Temp. )C mRNA PEP-C (min) (°C)circRNAN / P 12 676 338 6150 227circRNAN / P 14 792 395 7200 2270.11 0.055 60 RT 5 100 24h circRNAN / P 16 902 450 8200 227circRNAN / P 18 1018 508 9250 227The results are shown in Figures 3A and 3B and described by the legend of Figure 3.

[0156] Example 1.4The expression levels of human serum albumin (HSA) or hyaluronic acid (HA) coated peptide - circularRNA nanoparticles (compared to peptide / linear mRNA nanoparticles) were measured after transfection of HEK293T cells by the luminescence of the expressed luciferase.More detailed experimental conditions of the experiments at distinct N / P ratios are represented by below Tables 4 and 5.Table 4 _Working Final Nanoparticlsolution concentration e CoatingmRN (PM) (pM) preparationA Vol Vol Conditions Molar added added medium Exposure to ceils in well Ratio to to NPs Final Final Ratio Coating Coating cells (pL) (pL) mRNA Time Temp Coater mRNA PEP-C PEP-C cone, con. Temp Peptide / Time (ng) (min) (°C) (mg / ml) HSA (min) (pM) (°C)mRNA 350 175 3188 175 N / P 8.3circRNA 350 175 227 3188 N / P 6.2circRNA N / A 24h 0.11 450 0.055 225 60 RT 227 5 100 4100 N / P 8circRNA 566 283 227 5150 N / P 10circRNA 676 338 227 6150 N / P 12mRNAN / P 8.3 350 175 3188 HSA 2.3 35 5 10 RT 175HSAcircRNAN / P 6.2 350 175 3188 HSA 2.3 35 5 10 RT 227HSAcircRNAN / P 8 450 225 4100 HSA 2.9 45 5 10 RT 227HSAcircRNAN / P 10 566 283 5150 HSA 3.7 57 5 10 RT 227HSAcircRNAN / P 12 676 338 6150 HSA 4.4 68 5 10 RT 227HSATable 5Working solution Final(UM) concentration Nanoparticle(pM) preparation CoatingmRNA vol Vol Conditions Molar added to added medium Exposure Ratio the cells to cells in well toNPs Final Final Coating CoatingmRNA (pL)PEP-C mRNA PEP-C Time Temp Ratio (i>9) (PM (min) CC) Coater cone, cone Peptide / HA Time Temp.(mg / ml) (PM) (min) CC)mRNAN / P 8.3 350 175 3188 175circRNAN / P 6.2 350 175 3188 227circRNAN / P 8 450 225 4100 N / A227circRNAN / P 10 566 283 5150 227circRNAN / P 12 676 338 6150 227mRNAN / P 8.3 HAHA 0.11 350 0.055 175 3188 60 RT mid 0.5 4 50 10 RT 175 5 100 24h circRNAN / P 6.2 HAHA 350 175 3188 mid 0.5 4 50 10 RT 227circRNAN / P 8 HAHA 450 225 4100 mid 0.6 5 50 10 RT 227circRNAN / P 10 HAHA 566 283 5150 mid 0.8 6 50 10 RT 227circRNAN / P 12 HAHA 676 338 6150 mid 1.0 7 50 10 RT 227The results are shown in Figures 4A and 4B and described by the legend of Figure 4.

[0157] Example 1.5The expression levels of uncoated peptide - circular RNA nanoparticles were measured by the luminescence of the expressed luciferase as a function of time at 24h, 48h and 72h after onset of incubation with HEK293T cells as compared to peptide / linear mRNA nanoparticles.More detailed experimental conditions are represented by below Table 6:Table 6Working solution Final concentration Nanoparticle Vol (pM) (pM) preparation ngMol added Vol medium Condition ar mRNA Exposure to Ratio to in well cells NPs added Time Temp, mRNA PEP-C PEP-C mRNA (PL) to cells (pL) (min) comRNAN / P 8.3 350 175 3188 175 24h, 48h, 0.11 0.055 RT 5 60 100 72h circRNAN / P 10 566 283 5150 227The results are shown in Figure 5 and described by the legend of Figure 5.

[0158] Example 1.6Coated peptide / circular RNA nanoparticles with different HA coatings (based on medium molecular weight (mid MW) hyaluronic acid or low molecular weight (low MW) hyaluronic acid) and with distinct peptide to HA ratios ) were analysed in terms of their expression levels as compared to uncoated nanoparticles upon transfection of HEK293T cells by the luminescence of the expressed luciferase. The measurement results of freshly prepared samples (Fig. 6A) are compared to samples which were stored for one week after their preparation (Figure 6B).More detailed experimental conditions are represented by below Table 7:Final NanopartiWorkingsolution (pM) concentration cleprepar Coating(pM) ationMolar mRNARa added Vol Vol Condition tioto the added medium Exposure cells to cells in well toNPs Time Temp Final Final Ratio Coating Coating (ng) (Pt) (PL) mRNA PEP-C mRNA PEP-C (mi ■ CC) Coater cone, cone. Peptide Time Temp.n) (mg / ml) (pM) / HA (min) (»C)circRNAN / P 12 N / AcircRNAN / P 12 HAHA 1:50 mid 0.95 7 50 10 RTcircRNAN / P 12 HAHA 1:200 mid 0.24 2 197 10 RTcircRNAN / P 12 HaHA 1:5 0.11 676 0.055 338 6150 60 RT low 1.01 67 5 10 RT 227 5 100 24h circRNAN / P 12 HaHA 1:20 low 0.25 17 20 10 RTcircRNAN / P 12 HaHA 1:50 low 0.1 7 51 10 RTcircRNAN / P 12 HaHA 1:200 low 0.03 2 169 10 RTThe results are shown in Figures 6A and 6B and described by the legend of Figure 6.

[0159] Example 1.7Coated peptide / circular RNA nanoparticles (coated with HSA (peptide / HSA ratio: 1:20)) or HA (peptide / HA ratio: 1:50)) were analysed in terms of their expression levels as compared to uncoated peptide / circular RNA complexes upon incubation in and transfection of Panc-1 cells by the luminescence of the expressed luciferase.More detailed experimental conditions are represented by below Table 8:Table 8Working Finalconcentrat Nanoparticlesolution (pM) ion preparation Coating_ (BMI _mRNAMolar added Vol Vol Condition Ratio to the added medium Exposure Time Temp, Final Final Coating Coating ce to cells in well toNPs mRNA PEP-C mRNA PEP-C Coater Ratio lls(min) rq cone, cone (uL) (uL(m Peptide / HA Time Temp ) g / ml) (PM) (min) ro (ng)circRNAN / P 12 N / AcircRNAN / P 12 615 20HSA 0.11 676 0.055 338 0 60 RT HSA 1.11 17 10 RT 227 5 100 24h circRNAN / P 12 50HA HAmid 7 0.95 RT 10The results are shown in Figure 7 and described by the legend of Figure 7.

[0160] Example 1.8HSA coated peptide / circular RNA nanoparticles (peptide / HSA ratio: 1:20) were analysed in terms of their expression levels as compared to uncoated peptide / circular RNA complexes upon incubation in and transfection of HEK293T cells (at distinct doses: 100 ng or 200 ng) by the luminescence of the expressed luciferase.More detailed experimental conditions are represented by below Table 9:Table 9Final Nanoparticl Working concentration e Coating solution (pM) preparation _ » _Vol mRNA Vol medi Molar added to added Exposure Condition urn in Ratio Final the cells to cells toNPs Final Coating Rato Coating PEP- Time Tern Coat well mRNA PEP-C mRNA cone, cone. Peptide / HS Time Temp. (ng) (pL) C (min) (°C) er (pL) (mg / ml) (pM) A (min) (°C)circRNAN / A N / P 8circRNA 0.11 444 0.055 222 4050 60 RT 100 / 200 4.4 100 24h N / P 8 HS 0.7 11 20 10 RT HSA A1:20The results are shown in Figure 8 and described by the legend of Figure 8.

[0161] Example 1.9HSA coated peptide / circular RNA nanoparticles (peptide / HSA ratio: 1:20) were analysed in terms of their expression levels as compared to uncoated peptide / circular RNA complexes upon incubation in and transfection of HEK293T cells (Figure 9A) or HepG2 cells (Figure 9B) either with or without addition of PS20 (polysorbate 20) by the luminescence of the expressed luciferase.More detailed experimental conditions are represented by below Table 10:Table 10Nanoparticl Working Finalsolution (pM) concentration e Coating(pM) preparationmRNA VolMolar added added Vol me Condition t dium Exposure Ratio o the toTim in well toNPs Final PS20 Final Coating Coating cells cells(«L) Temp. Ratio e (uL) (ng) mRNA PEP-C mRNA PEP-C Coater cone, (mg / mL) cone. Time Temp. Peptide / HA (mi (°C) (mg / mL) in form. (min) (pM) (°C) n)circRNA N / A N / P 8circRNAN / P 8 N / A24h HSA 444 222 RT 3.3 100 0.11 0.055 4050 60 150HSA 1.11 17 10 RT 20 circRNAN / P 8 0.0075 HSAPS20The results are shown in Figures 9A and 9B and described by the legend of Figure 9.

[0162] Example 2: Physicochemical analysis of circRNA nanoparticles

[0163] The size (hydrodynamic radius) of the prepared nanoparticles peptide / circRNA particles (N / P=12) was measured for samples of different peptide: HSA ratios; hereby, the plate was initially centrifuged for 2 min at 3000 rpm and thereafter the samples were analyzed by dynamic light scattering.

[0164] Starting Materials1. 1 mg / mL Flue circRNA solution, kept on ice2. 5 mM Bachem peptide solution in water, kept on ice3. PBS (Gibco #10010-023), room temperature4. 100 mg / mL HSA solution, room temperature5. 10 mg / ml HSA solution in PBS, room temperature6. 5 mg / ml HSA solution in PBS, room temperatureTable 11HSA:peptideSample name circRNA (nM) peptide (uM) HSA (uM) ratiocFLUC-01-001 (55-6150-HSA-18.1) 1:1 45 275 273 cFLUC-01-002 (55-6150-HSA-4.2) 1:5 52 321 64 cFLUC-01-003 (55-6150-HSA-1.0) 1:20 49 302 15 cFLUC-01-004 (55-6150-HSA-0.2) 1:100 52 322 3cFLUC-01-005 (55-6150-HSA-0.0) — 55 336 0

[0165] The samples were prepared in 200 ul Eppendorf tubes by the follwing protocol.1. In Tube 1 3.18 µL of 1 mg / mL circRNA were added to 31.8 pL of PBS, mix by pipetting (3x, 35 ul pipetting volume). 10 tubes were prepared.2. In Tube 2 4.7 µL of PEP-C were added to 30.3 pL PBS, mix by pipetting (3x, 35 ul pipetting volume). 10 tubes were prepared.3. 35 pL of Tube 2 were added to 35 pL with circRNA in Tube 1, mix by pipetting (3x, 70 ul pipetting volume).4. Samples were incubated for Ih at RT5. The uncoated formulations are finished. The remaining eight formulations were coated with HSA:1. For 1:1 ratio add 15.5 pL of 100 mg / mL HSA x22. For 1:5 ratio add 3.1 pL of 100 mg / mL HSA x23. For 1:20 ratio add 7.7 pL of 10 mg / mL HSA x24. For 1:100 ratio add 3.0 pL of 5 mg / mL HSA x25. Mix by pipetting (3x, pipetting volume=tot sample volume)6. The tubes of each formulation were pooled

[0166] The results are shown in Figure 10 and described by the legend of Figure 10.

Claims

Claims1. A particle or nanoparticle comprising a peptide, a circular RNA (circRNA) and, optionally, a macromolecule.

2. The particle or nanoparticle according to claim 1, wherein the circular RNA is coding or non-coding RNA, optionally modified by non-naturally occurring nucleotides.

3. The particle or nanoparticle according to claim 1 or 2, wherein the circular RNA is coding and contains an IRES element.

4. The particle or nanoparticle according to claim 1, 2 or 3, wherein the peptide has a length of at least 15 amino acids5. The particle or nanoparticle according to any one of the preceding claims, wherein the peptide has a length of from 18 to 24, preferably 18 to 22 amino acids, more preferably 20 to 22, most preferably 21 amino acids.

6. The particle or nanoparticle according to any one of the preceding claims, wherein the peptide is positively charged.

7. The particle or nanoparticle according to any one of the preceding claims, wherein the peptide comprises at least 3 positive charges by their amino acid side chains.

8. The particle or nanoparticle according to any one of the preceding claims, wherein the peptide has a length of 21 amino acids and comprises 5 positive side chain amino acid charges.

9. The particle or nanoparticle according to any one of the preceding claims, wherein the peptide contains or consists of the amino acid sequence according to any one of SEQ ID NOs: 1 to 28, in particular containing or consisting of the amino acid sequence H-VLTTGLPALISWIRRRHRRHC-OH.

10. The particle or nanoparticle according to any one of the preceding claims, wherein the N / P ratio is in the range of 5 to 20, 5 to 15, 8 to 20, 8 to 16, 7 to 14, 7 to 12 or 8 to 12, preferably 10 or 12.

11. The particle or nanoparticle according to any one of claims 1 to 10, wherein the molar ratio of peptide to circRNA molecule is about 2.500:1 to about 30.000:1, about 5.000:1 to about 20.000:1, about 10.000:1 to about 25.000:1, about 10.000:1 to about 20.000:1, or about 15.000:1 to about 20.000:1, in particular about 5.000:1 to 10.000:1.

12. The particle or nanoparticle according to any one of claims 1 to 11, wherein the molar ratio of peptide to circRNA molecule is about 2.500:1 to about 15.000:1.

13. The particle or nanoparticle according to any one of claims 1 to 12, wherein the molar ratio of peptide to circRNA molecule is about 5.000:1 to about 10.000:1.

14. The particle or nanoparticle according to any one of claims 1 to 11, wherein the molar ratio of peptide to circRNA molecule is about 15.000:1 to about 20.000:1.

15. The particle or nanoparticle according to any one of the preceding claims, wherein the macromolecule is a coating macromolecule which covers the surface of the particle.

16. The particle or nanoparticle according to claim 15, wherein the macromolecule is albumin and / or hyaluronic acid.

17. The particle or nanoparticle according to any one of the preceding claims, wherein the particles has a diameter in the range of 50 to 500 nm, or 200 to 500 nm or 300 to 500 nm.

18. A composition comprising a particle or nanoparticle according to any one of claims 1 to 17.

19. The composition according to claim 18, wherein the composition comprises a pharmaceutically acceptable excipient.

20. A method of preparing a particle or nanoparticle according to any one of claims 1 to 17 comprising:- providing a circular RNA and a peptide;- mixing the circular RNA and the peptide;- incubating the solution; and, optionally,- adding the coating macromolecule and incubating the mixture.

21. A method of delivering a circRNA molecule into a cell, comprising contacting the cell with the particle or nanoparticle according to any one of claims 1 to 17 or a composition according to claim 18 or 19.

22. The method according to claim 21, wherein the circRNA molecule is delivered into the cytoplasm or the nucleus of the cell.

23. The method according to claim 21 or claim 22, wherein the contacting of the cell is performed in vitro, ex vivo or in vivo.

24. The method according to any one of claims 21 to 23, wherein the cell is a cancer cell.

25. The method according to any one of claims 21 to 24, wherein the cell is a cell affected by inflammation.

26. A method of treatment by administering a particle or nanoparticle according to any one of claims 1 to 17 or a composition according to claim 18 or 19 to a subject in need thereof.

27. The method according to claim 26, wherein the subject has a cancer, an autoimmune disease, an inflammation-related disease, inflammatory airway disease, rheumatoid arthritis, osteoarthritis, atherosclerosis, inflammatory bowel disease, systemic lupus erythematosus, type I diabetes, chronic obstructive pulmonary disease, asthma, or multiple sclerosis.

28. The method according to claim 26, wherein the subject has a cancer or an infectious disease.

29. The method according to claim 28, wherein the cancer is a blood cancer or a solid tumor cancer.

30. The method according to claim 28, wherein the subject has a viral, bacterial or protozoological infectious disease.

31. The method according to claim 30, wherein the subject has a viral infectious disease.

32. The method according to claim 26 or claim 27, wherein the subject has an inflammation-related disease.

33. The method according to claim 32, wherein the inflammation-related disease is rheumatoid arthritis, psoriatic arthris, gouty arthritis, lupus, psoriasis, ankylosing spondylitis, high blood pressure, heart disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), asthma, depression, anxiety, Type 2 diabetes meilitus, Type 1 diabetes meilitus, fatty liver disease, endometriosis, or allergy.

34. The method according to claim 26, wherein the subject has a genetic disease requiring protein replacement therapy or an endocrinological disease requiring protein replacement therapy.

35. The method according to any one of claims 26 to 34, wherein the subject is a human subject.

36. A particle or nanoparticle according to any one of claims 1 to 17 or a composition according to claim 18 or 19 for use in a method in a method of any one of claims 26 to 35, in particular for use in a method of treatment a disease or disorder.

37. The particle or nanoparticle or the composition according to claim 36 for use in a method of treating a cancer, an autoimmune disease, an inflammation-related disease, inflammatory airway disease, rheumatoid arthritis, osteoarthritis, atherosclerosis, inflammatory bowel disease, systemic lupus erythematosus, type I diabetes, chronic obstructive pulmonary disease, asthma, or multiple sclerosis.

38. A particle or nanoparticle according to any one of claims 1 to 17 or a composition according to claim 18 or 19 for use in a method of vaccination.

39. The particle or nanoparticle or the composition according to claim 38 for use in a method of vaccination against a cancer or an infectious disease.

40. An in vitro method of modifying a cell, comprising administering to the cell an effective amount of the particle or nanoparticle according to any one of claims 1 to 17 or the composition of claim 18 or 19.

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