Compositions and methods for KRAS inhibition for the treatment of disease

Abstract

The present disclosure provides a pharmaceutical composition comprising a peptide-polynucleotide conjugate, wherein the peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and the polynucleotide is a small interfering RNA (siRNA) targeting human KRAS mRNA, wherein the target sequence of human KRAS mRNA does not encode G12, G13, or Q61 with reference to SEQ ID NO: 4, or does not encode a mutant amino acid at position 12, 13, or 61 with reference to SEQ ID NO: 4.

Description

Detailed Description of the Invention

[0001] 1. REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 486,339, filed February 22, 2023, and U.S. Provisional Application No. 63 / 624,088, filed January 23, 2024, which applications are incorporated herein by reference in their entireties.

[0002] [2. Explanation of electronically submitted text files] The contents of the electronic sequence listing (AURS_010_02WO_SeqList_ST26.xml; size 2,214,874 bytes; and created on February 10, 2024) are incorporated herein by reference in their entirety.

[0003] [3. Technical Field] The present disclosure generally relates to pharmaceutical compositions for knocking down KRAS. The present disclosure also relates to treating a disease or disorder in a subject using the pharmaceutical compositions disclosed herein.

[0004] [4. Background technology] The KRAS gene provides instructions for making a protein called KRAS, which plays a key role in cell division, cell differentiation, and cell self-destruction (apoptosis). Mutational activation of KRAS is a common cause of tumorigenesis.

[0005] [5. Overview] In one aspect, the present disclosure provides a pharmaceutical composition comprising a peptide-polynucleotide complex, wherein the peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and the polynucleotide is a small interfering RNA (siRNA) targeting human KRAS mRNA, wherein the target sequence of human KRAS mRNA does not encode G12, G13, or Q61 with reference to SEQ ID NO:4, or a mutant amino acid at position 12, 13, or 61 with reference to SEQ ID NO:4. In some embodiments, the peptide is non-lytic, non-cytotoxic, and can affect the release of the polynucleotide from a cellular endosome. In some embodiments, the peptide comprises two or more consecutive basic amino acids (a cationic region) and one or more histidine residues adjacent to the cationic region. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the siRNA comprises a sense strand and an antisense strand. In some embodiments, the sense strand and the antisense strand are each 16 to 24 bases in length. In some embodiments, the sense strand is 19 bases in length. In some embodiments, the antisense strand is 21 bases in length. In some embodiments, the sense strand and the antisense strand are modified. In some embodiments, the modification is selected from the group consisting of 2'-methoxy (2'-OMe), 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-O-MOE), 5'-vinylphosphonate, phosphorothioate (PTO), locked nucleic acid (LNA), locked nucleic acid (UNA), glycol nucleic acid (GNA), and DNA.In some embodiments, modifications of the sense strand include PTO at positions 1 and 2; 2'-F at positions 3, 7-9, 12, and 17; and 2'-OMe at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19. In some embodiments, modifications of the antisense strand include PTO at positions 1, 2, 19, and 20; 2'-F at positions 2 and 14; and 2'-OMe at positions 1, 3-13, and 15-21. In some embodiments, the last nucleotide of the sense strand is adenine (A). In some embodiments, the first nucleotide of the antisense strand is uracil (U). In some embodiments, the sense strand comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of any one of the antisense strands shown in Tables 1 and 2. In some embodiments, the ratio of peptide to polynucleotide is about 6:1 to about 18:1, where the ratio is the ratio of amine groups of the polymer, which may be positively charged, to phosphate groups of the nucleic acid, which are negatively charged. In some embodiments, the charge ratio of peptide to polynucleotide is about 12:1. In some embodiments, the ratio of peptide to polynucleotide is about 2:1 to about 3500:1, where the ratio is a molar ratio. In some embodiments, the ratio of peptide to polynucleotide is about 4:1 to about 1000:1. In some embodiments, the ratio of peptide to polynucleotide is about 5:1 to about 200:1. In some embodiments, the ratio of peptide to polynucleotide is about 50:1 to about 200:1. In some embodiments, the ratio of peptide to polynucleotide is about 5:1. In some embodiments, the ratio of peptide to polynucleotide is about 100:1.In some embodiments, the peptide-polynucleotide complex is a nanoparticle having a diameter of about 10 nm to about 300 nm. In some embodiments, the peptide-polynucleotide complex is coated with albumin and / or hyaluronic acid. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0006] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a blood cancer or a solid tumor cancer.

[0007] [6. Brief Description of the Drawings] Figure 1 shows exemplary modification patterns of the siRNAs disclosed herein. 2'-Methoxy refers to 2'-OMe, 2'-fluoro refers to 2'-F, and phosphorothioate refers to PTO.

[0008] Figures 2A-2F show the knockdown of KRAS by two representative siRNAs (XD-39946 and XD-39966) in NCI-H23 cells harboring the KRAS G12C mutation. Figure 2A shows the dose-response curve for XD-39946, and Figure 2B shows the dose-response curve for XD-39966. Figure 2C shows the relative mRNA expression levels of KRAS at different concentrations of XD-39946 and XD-39966. Figure 2D shows the relative mRNA expression levels of GAPDH at different concentrations of XD-39946 and XD-39966. Figure 2E shows the original data for the mRNA expression levels of KRAS and GAPDH at different concentrations of XD-39946 and XD-39966. Figure 2F shows a bar graph of the original data in Figure 2E.

[0009] Figures 3A-3C show knockdown of KRAS by two representative siRNAs (XD-39951 and XD-39947) in cell lines with wild-type or mutant KRAS. Figure 3A shows knockdown of KRAS in SW480 cells (G12V mutation). Figure 3B shows knockdown of KRAS in HT-29 cells (wild-type). Figure 3C shows knockdown of KRAS in LS174T cells (G12D mutation).

[0010] Figures 4A-4B show the knockdown of KRAS by representative siRNA XD-39951 and its effect on cell viability in other cell lines with additional KRAS mutations. Figure 4A shows KRAS knockdown in PDAC, NSCLC, and CRC cells with different KRAS mutations. Figure 4B shows cell viability after KRAS knockdown in PDAC, NSCLC, and CRC cells.

[0011] 7. Detailed Description of the Invention Disclosed herein are pharmaceutical compositions comprising peptide-polynucleotide complexes for the inhibition of KRAS for the treatment of diseases.

[0012] [7.1. Definition] 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 residues are artificial chemical mimetics of naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0013] The terms "homologous," "identical," or percent "identity," in the context of two or more peptides, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are identical (i.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 more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website www.ncbi.nlm.nih.gov / BLAST / , etc.). This definition also includes sequences that have deletions and / or additions, as well as substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and artificial variants. As described below, the algorithm can take gaps, etc. into account.

[0014] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it when found in its natural 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. In some embodiments, the term "purified" means that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel, meaning that the nucleic acid or protein is at least 85% pure, at least 95% pure, and at least 99% pure. In other embodiments, "purify" or "purification" refers to the removal of at least one contaminant from the composition being purified. Purification in this sense does not require that the purified compound be homogeneous, e.g., 100% pure.

[0015] The term "target sequence" refers to a sequence of nucleotides found in the mRNA of a target gene (e.g., the KRAS gene), which sequence of nucleotides is complementary to the antisense strand of the siRNA disclosed herein.

[0016] 7.2. Peptide-Polynucleotide Complexes One aspect of the present invention involves a peptide-polynucleotide complex. The peptide-polynucleotide complex of the present invention can efficiently transfect a polynucleotide associated with the peptide into the cytoplasm of a cell. The peptide, the polynucleotide, the peptide-polynucleotide complex, and the cell are described below.

[0017] 7.2.1. Peptides In one embodiment, the peptide-polynucleotide complexes of the present invention comprise a peptide. Generally, and as described in the Examples, the peptides of the present invention are derived from melittin and modified to reduce their cytotoxicity while maintaining their propensity to interact with membrane bilayers. Furthermore, the peptides are substantially non-lytic and non-cytotoxic to cells. The peptide-polynucleotide complexes of the present invention (1) have a function substantially similar to a peptide having the amino acid sequence of SEQ ID NO: 1 (VLTTGLPALISWIRRRHRRHC), SEQ ID NO: 2 (VLTTGLPALISWIRRRHRRHG), or SEQ ID NO: 3 (VLTTGLPALISWIKRKRQHRWRRRR), and (2) have an amino acid sequence similar or identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0018] As used herein, the phrase "functionally 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 can affect the release of a polynucleotide from an endosome. In some embodiments, the peptides of the present invention are non-lytic. The term "non-lytic" typically means that the lipid bilayer membrane of a cell is not compromised upon contact with the peptide. The integrity of the lipid bilayer membrane may be assessed by the inappropriate entry or exit of cells or extracellular components entering the cell. For example, cellular proteins and / or organelles may leak from cells with compromised lipid bilayer membranes. Alternatively, extracellular components (i.e., those that do not normally enter via gap junctions) may enter cells with compromised lipid bilayer membranes. However, it should be noted that even if the peptide is able to penetrate the lipid bilayer membrane of a cell and enter the interior of the cell, the integrity of the lipid bilayer membrane is not affected. In other embodiments, the peptides of the present invention are substantially non-cytotoxic. The term "non-cytotoxic" indicates that cells are typically not killed upon contact with the peptide. Typically, the peptides of the present invention reduce 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 certain embodiments, the peptides of the present invention are non-lytic and non-cytotoxic.

[0019] The peptides of the present invention are capable of associating with polynucleotides. Thus, in one embodiment, the peptides of the present invention comprise at least one cationic region that interacts with polynucleotides. Typically, the cationic region comprises two or more consecutive basic amino acids. Importantly, the peptides of the present invention also possess endosomolytic ability, which affects the release of polynucleotides from endosomes and allows them to enter the cytoplasm of cells. The term "endosomolytic" can be used to describe a substance that initiates or promotes endosomal lysis. As illustrated in the Examples, protonation of histidine residues in the peptides of the present invention promotes the degradation of the peptide-polynucleotide complex, thereby releasing the peptide and permeating the endosomal membrane to release the polynucleotide. Thus, in another embodiment, the peptides of the present invention comprise one or more histidine residues located adjacent to or within at least one cationic region of the peptide. By way of non-limiting example, if a peptide of the invention 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 regions of the peptide, at least one histidine adjacent to or within each of the first and third cationic regions of the peptide, at least one histidine adjacent to or within each of the second and third cationic regions of the peptide, or at least one histidine adjacent to or within each of the first, second, and third cationic regions of the peptide. The histidine residues adjacent to a cationic region may be located before or after the cationic region. In some embodiments, the histidine residues adjacent to a cationic region are immediately adjacent to the region.In other embodiments, the histidine residue adjacent to the 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 other embodiments, the histidine residue is within the cationic region. The endosomolytic ability of the peptides of the present invention obviates the need for additional endosomolytic substances, such as chloroquine, membrane-fusogenic peptides, inactivated adenovirus, and polyethyleneimine, to release transfected polynucleotides from endosomes and deliver them to the cytoplasm of cells. Such known endosomolytic substances can have adverse effects on cells and increase toxicity during transfection.

[0020] In some embodiments, a peptide of the invention comprises SEQ ID NO: 1. In other embodiments, a peptide of the invention consists of SEQ ID NO: 1. In certain embodiments, a peptide of the invention is a variant of SEQ ID NO: 1, wherein the variant comprises at least 10 contiguous amino acids of SEQ ID NO: 1 and has substantially similar function to a peptide comprising SEQ ID NO: 1. For example, a peptide of the invention may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 1.

[0021] In some embodiments, peptides of the invention comprise SEQ ID NO: 2. In other embodiments, peptides of the invention consist of SEQ ID NO: 2. In certain embodiments, peptides of the invention are variants of SEQ ID NO: 2, where the variants comprise at least 10 contiguous amino acids of SEQ ID NO: 2 and have substantially similar function to peptides comprising SEQ ID NO: 2. For example, peptides of the invention may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 2.

[0022] In some embodiments, peptides of the invention comprise SEQ ID NO: 3. In other embodiments, peptides of the invention consist of SEQ ID NO: 3. In certain embodiments, peptides of the invention are variants of SEQ ID NO: 3, where the variants comprise at least 10 contiguous amino acids of SEQ ID NO: 3 and have substantially similar function to peptides comprising SEQ ID NO: 3. For example, peptides of the invention may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 3.

[0023] In some embodiments, a peptide of the present invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, wherein the peptide is non-lytic and can affect the release of a polynucleotide from a cellular endosome. The peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and may have about 80%, about 85%, about 90%, or about 95% identity to the amino acid sequence of SEQ ID NO: 1. A peptide of the present invention comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 may contain one or more conservatively substituted amino acids. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids may be conservatively substituted, so long as the resulting peptide has substantially similar function to a peptide comprising SEQ ID NO: 1.

[0024] In some embodiments, a peptide of the present invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2, wherein the peptide is non-lytic and can affect the release of a polynucleotide from a cellular endosome. The peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2, and may have about 80%, about 85%, about 90%, or about 95% identity to the amino acid sequence of SEQ ID NO:2. A peptide of the present invention comprising an amino acid sequence having at least 80% identity to SEQ ID NO:2 may contain one or more conservatively substituted amino acids. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids may be conservatively substituted, so long as the resulting peptide has substantially similar function to a peptide comprising SEQ ID NO:2.

[0025] In some embodiments, a peptide of the present invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, wherein the peptide is non-lytic and can affect the release of a polynucleotide from a cellular endosome. The peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, and may have about 80%, about 85%, about 90%, or about 95% identity to the amino acid sequence of SEQ ID NO:3. A peptide of the present invention comprising an amino acid sequence having at least 80% identity to SEQ ID NO:3 may contain one or more conservatively substituted amino acids. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids may be conservatively substituted, as long as the resulting peptide has substantially similar function to a peptide comprising SEQ ID NO:3.

[0026] The peptides of the present invention can be made using a variety of techniques known in the art: they can be isolated using standard techniques, synthesized using standard techniques, or purchased or obtained from a depository.

[0027] When the peptides of the present invention contain a C-terminal thiol in the form of a cysteine ​​residue, they can form disulfide bonds with other free thiol groups, such as free thiol groups from the same or different peptides. Those skilled in the art can readily determine whether dimerization improves plasmid DNA delivery. Without wishing to be bound by theory, dimerization may improve plasmid DNA delivery for certain peptides of the present invention by improving DNA condensation. Dimerization may be induced by incubation of free peptide in 20% DMSO for 24-72 hours or by other methods known in the art. As a non-limiting example, free thiols may be quantified by a colorimetric assay using Ellman's reagent.

[0028] The peptides of the present invention may be labeled. Non-limiting examples of suitable labels include fluorescent labels, chemiluminescent labels, radioactive labels, colorimetric labels, and resonance labels. Methods for labeling peptides are well known in the art.

[0029] The peptide may be bound to a cargo complex. As used herein, the term "cargo complex" refers to a molecule or agent that is carried by or bound to a peptide, other than the polynucleotide of the present invention. In other words, the peptide of the present invention may be bound to a cargo complex in addition to the polynucleotide of the present invention. For example, the cargo complex may be an imaging cargo, a therapeutic cargo, a cytotoxic cargo, or a targeting cargo.

[0030] Non-limiting examples of imaging cargo molecules and agents include any molecule, agent, or material that has a detectable physical or chemical property. Such imaging cargoes have been well developed in fields such as fluorescence imaging, magnetic resonance imaging, positron emission tomography, Raman imaging, optical coherence tomography, photoacoustic imaging, Fourier transform infrared imaging, or immunoassays, and generally, any label useful in these 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. Patent No. 4,391,904, which is incorporated herein by reference in its entirety.

[0031] Non-limiting examples of therapeutic cargoes may include any substance with biological activity, such as pharmacological agents, including analgesics, antipyretics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungals, antihypertensives, anti-inflammatory drugs, including nonsteroidal and steroidal drugs, antineoplastics, anti-anxiety drugs, immunosuppressants, antimigraine drugs, sedatives, hypnotics, antianginal drugs, antipsychotics, antimanic drugs, antiarrhythmic drugs, arthritis drugs, antigout drugs, anticoagulants, thrombolytic drugs, antifibrinolytic drugs, hemodynamic drugs, antiplatelet drugs, anticonvulsants, antiparkinsonian drugs, antihistamines, antirestenotic drugs, antipruritics, drugs useful for calcium regulation, antibacterial drugs, antiviral drugs, antifungals, anti-infective drugs, bronchodilators, steroid compounds, and hormones, and combinations thereof. Alternatively, the cargo complex may be in the form of a molecular complex or a pharmacologically acceptable salt component.

[0032] Cytotoxic cargo refers to a molecule or substance that is harmful to cells (e.g., by killing or damaging them). Examples include anti-microtubule agents such as taxol (paclitaxel, docetaxel) and vinca alkaloids (vincristine, vinblastine). For example, examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0033] The targeting cargo may be any molecule or substance that directs the peptide-polynucleotide complex of the present invention to a cell. The targeting cargo may be directed to a eukaryotic target cell or a prokaryotic target cell. Non-limiting examples of targeting substances may include antibodies or antibody fragments, receptor ligands, small molecules, peptides, polypeptides, lipids, carbohydrates, nucleic acids, siRNAs, shRNAs, antisense RNAs, dendrimers, microbubbles, or aptamers.

[0034] The means by which the cargo complex is attached to the peptide of the invention can vary depending on the embodiment. The cargo complex may be attached to the peptide of the invention by any means known in the art, including covalent or non-covalent attachment.

[0035] In another aspect, the peptide-polynucleotide complex of the present invention comprises a polynucleotide. The polynucleotide may be single-stranded, double-stranded, or a combination thereof. In some embodiments, the polynucleotide is double-stranded. In other embodiments, the polynucleotide is single-stranded. In still other embodiments, the polynucleotide is a combination of single-stranded and double-stranded.

[0036] Polynucleotides of the present invention may contain ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or a combination of RNA and DNA. Furthermore, polynucleotides may contain modified nucleobases, such as modified DNA or RNA bases. Modifications may occur at, but are not limited to, the 2'-position of the sugar, the C-5 position of pyrimidines, and the 8-position of purines. Examples of suitable modified DNA or RNA bases include 2'-fluoronucleotides, 2'-aminonucleotides, 5'-aminoallyl-2'-fluoronucleotides, and phosphorothioate nucleotides (monothiophosphates and dithiophosphates). Alternatively, polynucleotides may be nucleotide mimics. Examples of nucleotide mimics include locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and phosphorodiamidate morpholino oligomers (PMOs).

[0037] In some embodiments, the polynucleotide of the present invention is a combination of RNA and DNA. In other embodiments, the polynucleotide comprises DNA. When the polynucleotide is DNA, the polynucleotide may comprise an expression cassette. As used herein, an "expression cassette" refers to a nucleic acid construct comprising a nucleic acid sequence encoding a protein or peptide operably linked to a promoter. In certain embodiments, the nucleic acid construct further comprises additional regulatory sequences. Non-limiting examples of additional regulatory sequences include transcription termination sequences. Other additional regulatory sequences are known in the art. As used herein, the term "promoter" may refer to a synthetic or naturally occurring molecule capable of conferring or activating expression of a target nucleic acid sequence in a cell. The promoter may be the promoter normally associated with the DNA polynucleotide of the present invention or may be a heterologous promoter. Heterologous promoters may be derived from sources such as viruses, bacteria, fungi, plants, insects, and animals. The promoter may regulate the expression of the DNA sequence constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs. The promoter may regulate expression in response to external stimuli, such as developmental stages, physiological stress, pathogens, metal ions, or inducers or activators (i.e., inducible promoters). Non-limiting representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, HSP70 basal promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, promoters containing the nucleic acid sequence of a tetracycline response element (TRE), and the CMV IE promoter. In some alternatives of these embodiments, the DNA polynucleotides of the present invention are incorporated into vectors. Those skilled in the art will be able to construct vectors using standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996).(Both of which are incorporated herein by reference.) Vectors include, but are not limited to, plasmids, cosmids, transposable elements, viruses (bacteriophage, animal viruses, plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., those derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors, including replication-competent, replication-deficient, and gutless forms thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, and Rous sarcoma virus vectors.

[0038] In still other embodiments, the polynucleotide comprises RNA. Non-limiting examples of RNA sequences may include mRNA, which can encode proteins, as well as non-coding RNA, such as tRNA, rRNA, snoRNA, microRNA, siRNA, saRNA, piRNA, and long non-coding RNA (lncRNA). For example, the nucleic acid may comprise mRNA. In a preferred embodiment, when the nucleic acid comprises mRNA, the mRNA molecule may be 5'-capped, polyadenylated, or capped and polyadenylated. Alternatively, the mRNA molecule may comprise an internal ribosome entry site (IRES) for translating an internal open reading frame of the mRNA.

[0039] In certain embodiments, the polynucleotide comprises a non-coding RNA that can regulate or inhibit the expression of a nucleic acid sequence expressed in a cell. Non-limiting examples of non-coding RNA that can regulate or inhibit the expression of a nucleic acid sequence expressed in a cell include microRNA (also known as miRNA), siRNA, piRNA, and lncRNA. Generally, transfection of a cell with a non-coding RNA that can regulate or inhibit the expression of a nucleic acid sequence can lead to the cleavage of the nucleic acid sequence, can enhance, prevent, or disrupt the translation of the nucleic acid sequence into a protein, or can regulate the transcription of the nucleic acid sequence.

[0040] In some embodiments, the polynucleotide of the present invention comprises a non-coding RNA that can disrupt the expression of a nucleic acid sequence expressed in a cell.As used herein, "disrupting the expression of a nucleic acid sequence" can be used to describe any reduction in the expression level of a nucleic acid sequence or the protein translated from the nucleic acid sequence, when compared with the expression level of the nucleic acid sequence in cells that are not treated with the peptide-polynucleotide complex of the present invention.In some alternatives of this embodiment, the polynucleotide comprises a short interfering RNA (siRNA).

[0041] Generally, siRNAs comprise double-stranded RNA molecules ranging from about 15 to about 29 nucleotides in length. In some embodiments, siRNAs may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides in length. In other embodiments, siRNAs may be 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 some embodiments, siRNAs may be about 21 nucleotides in length. siRNAs may optionally further comprise one or two single-stranded overhangs, e.g., one single-stranded or two single-stranded 5'-end overhangs, one single-stranded or two single-stranded 3'-end overhangs, or a combination thereof. siRNAs may be formed from two RNA molecules that hybridize together, or may be generated from short hairpin RNAs (shRNAs) (see below). In some embodiments, the two strands of the siRNA may be completely complementary, such that there are no mismatches or bulges in the duplex formed between the two sequences. In other embodiments, the two strands of the siRNA may be substantially complementary, such that there may be one or more mismatches and / or bulges in the duplex formed between the two sequences. In certain embodiments, one or both 5' ends of the siRNA may have a phosphate group, while in other embodiments, one or both 5' ends lack a phosphate group. In other embodiments, one or both 3' ends of the siRNA may have a hydroxyl group, while in other embodiments, one or both 3' ends lack a hydroxyl group.

[0042] One strand of an siRNA, called the "antisense strand" or "guide strand," contains a portion that hybridizes with a target transcript. A target transcript refers to a nucleic acid sequence expressed in a cell that corresponds to the desired expression to be disrupted. In the context of the therapeutic compositions of the present invention, disrupting the expression of a target transcript may have a beneficial effect. In some embodiments, the antisense strand of an siRNA may be perfectly complementary to a region of the target transcript, i.e., it hybridizes to a target transcript that is between about 15 and about 29 nucleotides in length, at least 16 nucleotides in length, and does not contain a single mismatch or bulge in a target region of about 18 to 20 nucleotides in length. In other embodiments, the antisense strand may be substantially complementary to the target region, i.e., one or more mismatches and / or bulges may be present in the duplex formed by the antisense strand and the target transcript. Typically, an siRNA targets an exon sequence of the target transcript. Those skilled in the art are familiar with programs, algorithms, and / or commercial services that design siRNA corresponding to target transcripts.Representative examples are Rosetta siRNA design algorithm (Rosetta Inpharmatics, North Seattle, Wash.), MISSION® siRNA (Sigma-Aldrich, St. Louis, Mo.), and siGENOME siRNA (Thermo Scientific).siRNA can be enzymatically synthesized in vitro using methods known to those skilled in the art.Alternatively, siRNA can be chemically synthesized using oligonucleotide synthesis techniques known in the art.

[0043] In some embodiments, the polynucleotide of the present invention comprises a non-coding RNA capable of inhibiting the expression of a nucleic acid sequence encoding KRAS. In some embodiments, the non-coding RNA is an siRNA. In some embodiments, the target sequence of human KRAS mRNA does not encode G12, G13, or Q61 relative to the wild-type human KRAS protein, or does not encode a mutant amino acid present at position 12, 13, or 61 relative to the wild-type human KRAS protein. In some embodiments, the amino acid sequence of wild-type human KRAS protein is as follows: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 4).

[0044] Exemplary siRNAs compatible with the polypeptide-polynucleotide complexes disclosed herein are shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3]

[0045] In some embodiments, the siRNA disclosed herein is modified, in some embodiments, the modification is selected from the group consisting of 2'-methoxy (2'-OMe), 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-O-MOE), 5'-vinylphosphonate, phosphorothioate (PTO), locked nucleic acid (LNA), locked nucleic acid (UNA), glycol nucleic acid (GNA), and DNA.

[0046] In some embodiments, the modification of the sense strand includes PTO at position 1 and / or 2. In some embodiments, the modification of the sense strand includes 2'-F at one or more of positions 3, 7-9, 12, and 17. In some embodiments, the modification of the sense strand includes 2'-OMe at one or more of positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19. In some embodiments, the modification of the antisense strand includes PTO at one or more of positions 1, 2, 19, and 20. In some embodiments, the modification of the antisense strand includes 2'-F at position 2 and / or 14. In some embodiments, the modification of the antisense strand includes 2'-OMe at any of positions 1, 3-13, and 15-21.

[0047] In some embodiments, the last nucleotide of the sense strand is adenine (A). In some embodiments, the first nucleotide of the antisense strand is uracil (U). In some embodiments, the last nucleotide of the sense strand is uracil (U). In some embodiments, the first nucleotide of the antisense strand is adenine (A). In some embodiments, the last nucleotide of the sense strand is cytosine (C). In some embodiments, the first nucleotide of the antisense strand is guanine (G). In some embodiments, the last nucleotide of the sense strand is guanine (G). In some embodiments, the first nucleotide of the antisense strand is cytosine (C).

[0048] Exemplary modified siRNAs compatible with the polypeptide-polynucleotide complexes disclosed herein are shown in Table 2 below, where n=2'O-methyl RNA, Nf=2'-fluoro RNA, and s=phosphorothioate. [Table 2-1] [Table 2-2] [Table 2-3]

[0049] In some embodiments, the sense strand comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having at least 98% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having at least 99% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2. In some embodiments, the sense strand comprises a nucleotide sequence having 100% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2.

[0050] In some embodiments, the antisense strand comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 98% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 99% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises a nucleotide sequence having 100% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2.

[0051] Generally speaking, the promoter utilized to drive the in vivo expression of one or more siRNA or shRNA transcription units may be an RNA polymerase III (Pol III) promoter. Certain Pol III promoters, such as the U6 or H1 promoters, do not require cis-acting regulatory elements within the transcribed region and are therefore particular embodiments. In other embodiments, Pol II promoters may be used to drive the expression of one or more siRNA or shRNA transcription units. In some embodiments, tissue-specific, cell-specific, or inducible Pol II promoters may be used.

[0052] Constructs providing templates for siRNA or shRNA synthesis can be generated using standard recombinant DNA techniques and inserted into any of a wide variety of different vectors suitable for eukaryotic expression. Guidance can be found in Current Protocols in Molecular Biology (Ausubel et al., John Wiley & Sons, New York, 2003) or Molecular Cloning: A Laboratory Manual (Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001). Those skilled in the art will also recognize that vectors may contain additional regulatory sequences (e.g., termination sequences, translational regulatory sequences, etc.) as well as selectable marker sequences. DNA plasmids, including those based on pBR322, PUC, etc., are well known in the art. Many expression vectors already contain a suitable promoter or promoters; therefore, it is only necessary to insert a nucleic acid sequence encoding the desired RNAi agent at the appropriate location relative to the promoter. Viral vectors can also be used to provide intracellular expression of RNAi agents. Suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, etc. In some embodiments, the RNAi expression vector is an shRNA lentiviral vector or lentiviral particle, such as those provided in MISSION® TRC's shRNA products (Sigma-Aldrich).

[0053] The nucleic acid sequences of the present invention may be obtained using a variety of different techniques known in the art. Nucleotide sequences and homologous sequences may be isolated using standard techniques, synthesized using standard techniques, or purchased or obtained from a depository. Once the nucleotide sequence is obtained, it may be amplified for use in a variety of applications using methods known in the art.

[0054] 7.2.3. Polypeptide-Polynucleotide Complexes In another aspect of the present invention, the polypeptide and polynucleotide of the present invention associate to form a complex. As used herein, the term "associate" may refer to a non-covalent interaction between the peptide and polynucleotide, or a covalent bond between the peptide and polynucleotide. In some embodiments, the polypeptide and polynucleotide of the present invention associate through a non-covalent bond, such as a hydrogen bond, an ionic bond, a bond based on van der Waals forces, a hydrophobic bond, or an electrostatic interaction. For example, the peptide of the present invention may have an overall net positive charge, which allows the polynucleotide of the present invention to associate with the peptide through an electrostatic interaction to form a complex of the present invention. Methods for forming the polypeptide-polynucleotide complex of the present invention are known in the art and are described in further detail herein.

[0055] When the peptide of the present invention is associated with the polynucleotide of the present invention, the ratio of the peptide to the polynucleotide may vary depending on the peptide, the polynucleotide composition, or the size of the polynucleotide, and may be determined experimentally. Essentially, a suitable molar ratio of the peptide of the present invention to the polynucleotide of the present invention may be a molar ratio in which the peptide completely complexes with the polynucleotide while minimizing the subject's exposure to the peptide.

[0056] In some embodiments, the ratio is a molar ratio. In some embodiments, the molar ratio is about 2:1 to about 3500:1. In some embodiments, the molar ratio of peptide to polynucleotide (peptide:polynucleotide) is about 4:1 to about 1000:1. In some embodiments, the molar ratio of peptide to polynucleotide is about 10:1 to about 500:1. In some embodiments, the molar ratio of peptide to polynucleotide is about 5:1 to about 200:1. In some embodiments, the molar ratio of peptide to polynucleotide is about 50:1 to about 200:1. In some embodiments, the molar ratio of peptide to polynucleotide is about 100:1. In some embodiments, the molar ratio of peptide to polynucleotide is about 5:1.

[0057] In some embodiments, the ratio is the ratio of amine groups of the polymer, which may be positively charged, to phosphate groups of the nucleic acid, which are negatively charged. In some embodiments, the charge ratio between the peptide and the polynucleotide (peptide:polynucleotide) is about 6:1 to about 18:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 6:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 7:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 8:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 9:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 10:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 11:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 12:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 13:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 14:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 15:1. In some embodiments, the charge ratio between the peptide and the polynucleotide is about 16:1. In some embodiments, the charge ratio of the peptide to the polynucleotide is about 17: 1. In some embodiments, the charge ratio of the peptide to the polynucleotide is about 18:1.

[0058] Methods for determining the ratio at which the peptide can fully complex with the polynucleotide are known in the art and may include gel shift assays as described in the Examples. Methods for determining the molar ratio that minimizes exposure of a subject to the peptide are known in the art and may include cytotoxicity measurements using increasing doses of the polypeptide.

[0059] The peptide-polynucleotide complex of the present invention may have a diameter of about 10 nm to about 500 nm. In some embodiments, the diameter of the peptide-polynucleotide complex is about 10 nm to about 300 nm. In some embodiments, the diameter of the peptide-polynucleotide complex is at least about 10 nm. In some embodiments, the diameter of the peptide-polynucleotide complex is at most about 300 nm. In some embodiments, the diameter of the peptide-polynucleotide complex is about 10 nm to about 50 nm, about 10 nm to about 100 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 100 nm, about 50 nm to about 150 nm, about 50 nm to about 200 nm, or about 50 nm to about 250 nm. nm, about 50 nm to about 300 nm, about 100 nm to about 150 nm, about 100 nm to about 200 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 some embodiments, the diameter of the peptide-polynucleotide complex is about 10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, or about 300 nm.

[0060] The nanoparticles of the present invention may be further modified to improve the stability of the nanoparticles. For example, the nanoparticles of the present invention may be coated with albumin and / or hyaluronic acid to improve the stability. The nanoparticles of the present invention coated with albumin may have a diameter of about 5 nm to about 90 nm or more.

[0061] Particle size and / or charge may be assessed using methods known in the art. Non-limiting examples of methods for measuring particle size include dynamic light scattering, light scattering, multi-angle light scattering, field flow fractionation systems, laser diffraction, electrozone (electrical sensing zone), light blocking—also known as photozone—and single particle optical sensing (SPOS), sieve analysis, aerodynamic force measurement, air permeability diameter, sedimentation, nanoparticle tracking analysis, electron microscopy, atomic force microscopy, small-angle X-ray scattering, flow cytometry, measuring the zeta potential of particles, analytical ultracentrifugation, or a combination thereof. In some embodiments, particle size is assessed by dynamic light scattering. In some embodiments, particle charge is assessed by measuring the zeta potential of the particles. In further embodiments, particle size and / or charge is assessed by dynamic light scattering or by measuring the zeta potential of the particles.

[0062] The nanoparticles of the present invention may have a zeta potential of about -15 to about 20 mV, or about 0 mV or greater. For example, the nanoparticles 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 greater. In some embodiments, the nanoparticles have a zeta potential of about 1, about 2, about 3, about 4, or about 5 mV. In other embodiments, the nanoparticles have a zeta potential of about 10, 11, 12, 13, or about 14 mV. In still other embodiments, the nanoparticles have a zeta potential of about 11, about 12, about 13, about 14, or about 15 mV. In exemplary embodiments, the nanoparticles have a zeta potential of about 1, about 2, about 3, about 4, or about 5 mV. In other embodiments, the nanoparticles have a zeta potential of about 10, about 11, 12, about 13, or about 14 mV. In exemplary embodiments, the nanoparticles have a zeta potential of about 3.72 mV. In other embodiments, the nanoparticles have a zeta potential of about 12 mV. In yet other embodiments, the nanoparticles have a zeta potential of about 13.1 mV.

[0063] The peptide-polynucleotide complex can efficiently release the polynucleotide into the cytoplasm of cells. The peptide-polynucleotide complex can also protect the polynucleotide from degradation upon administration to a subject. Therefore, the peptide-polynucleotide nanoparticles of the present invention can be stably maintained in the presence of serum. The nanoparticles can be stably maintained in the presence of serum for about 10, 20, 30, 40, 50, or 60 minutes, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 hours, about 1, 2, 3, 4, 5, 6, or 7 days or more. The nanoparticles can be stably maintained in the presence of about 5, 10, 15, 25, 50, 100, 150, 200, or about 300 μg / ml or more of human serum albumin. The stability of a nanoparticle may be determined by measuring the ability of the nanoparticle to maintain the activity of the polynucleotide of the peptide-polynucleotide complex, or by measuring the change in size of the nanoparticle over time. Methods for measuring nanoparticle size may be as described in this section.

[0064] Methods for preparing peptide-polynucleotide complexes of the present invention generally involve contacting a peptide of the present invention with a polynucleotide of the present invention to form a peptide-polynucleotide complex. Typically, the peptide and polynucleotide are contacted by incubation under conditions suitable for the formation of a peptide-polynucleotide complex. Suitable conditions for the formation of a peptide-polynucleotide complex are as described in the Examples. Typically, such conditions may include a temperature of about 30°C to about 40°C and an incubation time of about 20 seconds to about 60 minutes, or longer. Suitable temperatures may also be lower than about 30°C. For example, incubation may be performed on ice. Those skilled in the art will recognize that the length and temperature of incubation may vary depending on the peptide and polynucleotide and may be determined empirically.

[0065] Nanoparticles containing the peptide-polynucleotide complexes of the present invention may be further modified to enhance the stability of the nanoparticles. For example, the peptide-polynucleotide complexes of the present invention may be crosslinked to enhance the stability of the nanoparticles. Those skilled in the art will recognize that suitable crosslinkers will vary depending on the composition of the nanoparticles and their antibodies or antibody fragments. In some embodiments, the peptide-polynucleotide complexes of the present invention may be chemically crosslinked using chemical crosslinkers such as glutaraldehyde, biscarboxylic acid spacers, biscarboxylic acid-active esters, bislinker amines / acids via carbodiimide coupling procedures, or using click chemistry procedures, carbodiimide coupling chemistry, acylation, active ester coupling, or alkylation.

[0066] Alternatively, the peptide-polynucleotide complex of the present invention may be coated with a compound that can increase the stability of the nanoparticle. Methods for modifying nanoparticles to increase stability are known in the art and may be as described in Nicolas et al., 2013 Acta Biomater. 9:4754-4762, the disclosure of which is incorporated herein by reference in its entirety.

[0067] As used herein, the term "coating" may refer to the interaction of a compound with a peptide-polynucleotide complex via a non-covalent bond, or the covalent bond between the peptide-polynucleotide complex and a compound. In some embodiments, the peptide-polynucleotide complex of the present invention and the coating compound are associated via a non-covalent bond, such as a hydrogen bond, an ionic bond, a bond based on van der Waals forces, a hydrophobic bond, or an electrostatic interaction. For example, the peptide-polynucleotide complex of the present invention may have an overall net positive charge, and the coating compound may have an overall negative charge, which may allow the peptide-polynucleotide complex and the compound to associate via electrostatic interaction to form the complex of the present invention.

[0068] Non-limiting examples of compounds that may be used to coat nanoparticles to enhance their stability include albumin, fatty acids such as oleic acid, polyethylene glycol, polysaccharides such as chitosan, heparin or heparan, and other glycosaminoglycans, or other published coating materials known to those of skill in the art. In some embodiments, the stability of the peptide-polynucleotide complexes of the invention may be enhanced by coating the nanoparticles with fatty acids. In other embodiments, the stability of the peptide-polynucleotide complexes of the invention may be enhanced by coating the nanoparticles with polysaccharides.

[0069] In some embodiments, the stability of nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be enhanced by coating the nanoparticles with albumin. Albumin is a negatively charged globular protein commonly found in serum. Without wishing to be bound by theory, it is believed that coating the nanoparticles of the present invention with albumin may enhance the stability of the nanoparticles by preventing aggregation. Preferably, the albumin that can be used to coat nanoparticles comprising the peptide-polynucleotide complexes of the present invention is serum albumin, and may include bovine serum albumin and human serum albumin. In an exemplary embodiment, the stability of nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be enhanced by coating the nanoparticles with human serum albumin.

[0070] Essentially, nanoparticles are coated with albumin by incubation in a solution containing albumin. Nanoparticles may be incubated in a solution containing 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 of albumin. In some embodiments, nanoparticles containing the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 0.1, 0.3, 0.5, 0.7, or 0.9 mg / ml of albumin. In other embodiments, nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 1.0, 1.2, 1.4, 1.6, or 1.8 mg / ml of albumin. In still other embodiments, nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 2.0, 2.2, 2.4, 2.6, or 2.8 mg / ml of albumin. In other embodiments, nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 3.0, 3.2, 3.4, 3.6, or 3.8 mg / ml of albumin. In further embodiments, nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 4.0, 4.2, 4.4, 4.6, 4.8, or 5.0 mg / ml of albumin. In some embodiments, nanoparticles comprising the peptide-polynucleotide complexes of the present invention may be incubated in a solution containing about 4.0 mg / ml of albumin.

[0071] The peptide-polynucleotide complexes 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-polynucleotide complexes. In some embodiments, particles containing peptide-polynucleotide complexes of the invention are incubated with albumin for about 5, 10, 15, or about 20 minutes. In other embodiments, particles containing peptide-polynucleotide complexes of the invention are incubated with albumin for about 20, 25, 30, or about 35 minutes. In still other embodiments, particles containing peptide-polynucleotide complexes of the invention are incubated with albumin for about 35, 40, 45, or about 50 minutes. In other embodiments, particles containing peptide-polynucleotide complexes of the invention are incubated with albumin for about 50, 55, or about 60 minutes or more. In some embodiments, particles containing peptide-polynucleotide complexes of the invention are incubated with albumin for about 25, 30, or about 35 minutes.

[0072] The peptide-polynucleotide complexes can 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 coat the peptide-polynucleotide complexes with hyaluronic acid or incorporate hyaluronic acid into the peptide-polynucleotide complexes. The peptide-polynucleotide complexes can be incubated with hyaluronic acid for about 1, 2, 3, 4, 5, 10, 12, 18, or 24 hours or more to coat the peptide-polynucleotide complexes with hyaluronic acid or incorporate hyaluronic acid into the peptide-polynucleotide complexes. In some embodiments, the peptide-polynucleotide complexes can be incubated with hyaluronic acid for about 45 minutes. In some embodiments, shorter incubation times can be used, for example, when using a flow process or microfluidic device.

[0073] [7.2.4. Cells] In another aspect of the present invention, the peptide-polynucleotide complex of the present invention is capable of transfecting the polynucleotide into the cytoplasm of a cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. The cell may be in vitro, in vivo, in situ, or ex vivo. The cell may be a single cell or may comprise a tissue or organ. The term "cell" also refers to a cell in a subject.

[0074] The peptide-polynucleotide complexes of the present invention may be administered to cells in vitro by incubating the cells in the presence of the peptide-polynucleotide complexes of the present invention under conditions suitable for transfection of the polynucleotide in the peptide-polynucleotide complex. Conditions suitable for transfection of the polynucleotide in the peptide-polynucleotide complex may be as described in the Examples. Those skilled in the art will appreciate that the length of incubation may vary depending on the peptide-polynucleotide complex and the cells. Typically, such conditions may include an incubation time of about 10 minutes to 24 hours, and transfection conditions may include an incubation time of about 15 minutes to 3 hours.

[0075] The peptide-polynucleotide complexes of the invention may be administered to cells in vivo (ie, within a subject) by administering to the subject a composition comprising the peptide-polynucleotide complexes of the invention.

[0076] 7.3. Pharmaceutical Compositions In another embodiment of the present invention, the peptide-polynucleotide complexes of the present invention may be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical compositions of the present invention may be used to disrupt the expression of two or more nucleic acid sequences normally expressed in a cell. For example, the pharmaceutical compositions of the present invention may be used to disrupt the expression of one, two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid sequences normally expressed in a cell. Those skilled in the art will understand that pharmaceutical compositions are administered to treat disease, prevent disease, or promote health. Thus, the pharmaceutical compositions of the present invention may be used to disrupt the expression of any nucleic acid sequence normally expressed in a cell, such that disruption of expression has a measurable and beneficial effect (i.e., a noticeable effect) on the subject to whom the composition is administered.

[0077] In some embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence normally expressed in a cell. In some embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence encoding KRAS. In some embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence encoding STAT3. In some embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence encoding JNK2. In further embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence encoding the p65 subunit of the canonical NFκB signaling pathway. In some embodiments, the pharmaceutical composition of the present invention is used to disrupt the expression of a nucleic acid sequence encoding the p100 / p52 subunit of the canonical NFκB signaling pathway.

[0078] In other embodiments, the pharmaceutical compositions of the invention are used to disrupt the expression of two nucleic acid sequences normally expressed in cells: a nucleic acid sequence encoding the p65 subunit of the canonical NFκB signaling pathway, and a nucleic acid sequence encoding the p100 / p52 subunit of the canonical NFκB signaling pathway.

[0079] When the pharmaceutical compositions of the present invention are used to disrupt the expression of two or more nucleic acid sequences normally expressed in a cell, the pharmaceutical compositions may be formulated using a mixture of two or more peptide-polynucleotide complexes, each complex containing a polynucleotide capable of disrupting the expression of a different nucleic acid sequence normally expressed in the cell. Alternatively, two or more polynucleotides may be used to generate a mixture of peptide-polynucleotide complexes, each polynucleotide capable of disrupting the expression of a different nucleic acid sequence normally expressed in the cell.

[0080] The pharmaceutical compositions of the present invention may also contain one or more non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles, as needed. As used herein, the term "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 the nanoparticles of the present invention, its use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0081] The pharmaceutical composition of the present invention can be formulated to suit its intended route of administration.Suitable routes of administration include parenteral administration, oral administration, intrapulmonary administration, transdermal administration, transmucosal administration and rectal administration.The term parenteral used herein includes subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection or intrasternal injection or infusion technique.

[0082] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain ingredients such as a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate; and agents for adjusting tonicity such as sodium chloride, glucose, or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0083] Oral compositions generally may contain 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 an excipient and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier may be applied orally, gargled, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain optional ingredients such as binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterol; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavoring; or compounds of a similar nature. For administration by inhalation, the compound is 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.

[0084] In some embodiments, pharmaceutical compositions of the present invention are formulated for parenteral administration. For example, pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, balanced salt solutions, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ), or phosphate buffered saline (PBS). In an exemplary embodiment, pharmaceutical compositions of the present invention are formulated in phosphate buffered saline (PBS).

[0085] In all cases, the composition may be sterile and fluid to the extent that easy syringability exists. The composition may be stable under the conditions of manufacture and storage and may be 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 polyethylene glycol, and the like), and suitable mixtures thereof. 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, the composition may also contain isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by including in the injectable compositions substances that delay absorption, for example, aluminum monostearate and gelatin.

[0086] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent, optionally with one or a combination of the above-listed ingredients, and then filter sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile injectable powder, the preparation method is vacuum drying and freeze-drying, which produces a powder of active compound plus any additional desired ingredients from the above-mentioned previously sterile-filtered solution.

[0087] Systemic administration may also be achieved 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, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration may also be achieved by the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art. The compound 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.

[0088] In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body, such as a sustained-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, chitosan, and polylactic acid. Methods for preparing such formulations are readily apparent to those skilled in the art. These may be prepared according to methods known to those skilled in the art, for example, the methods described in U.S. Patent No. 4,522,811.

[0089] Further formulations of pharmaceutical compositions are found, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY (1980). Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16th Ed ISBN: 0-912734-04-3, latest edition, the entire contents of which are incorporated herein by reference. These provide a summary of formulation techniques commonly known to practitioners.

[0090] Those skilled in the art will recognize that the concentration of the peptide-polynucleotide complexes of the present invention in pharmaceutical compositions may vary depending, in part, on the route of administration, the subject, and the reason for administration, and may be determined empirically. Methods for empirically determining the concentration of active agents, such as the nanoparticles of the present invention, in pharmaceutical compositions are known in the art. Generally, pharmaceutical compositions may be formulated to contain about 0.1 nM to about 50 μM of polynucleotide in the peptide-polynucleotide complexes of the present invention. For example, pharmaceutical compositions may contain 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, or 24 nm of polynucleotide in the peptide-polynucleotide complexes of the present invention. m, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41n m, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm , 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm, 75nm, 76nm, 77nm, 78nm, 79nm , 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm, 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm, 111nm, 112nm, 113nm, 114nm, 115nm, 116nm, 117nm, 118nm, 119nm, 120nm, 121nm, 122nm, 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130nm,131nm、132nm、133nm、134nm、135nm、136nm、137nm、138nm、139nm、140nm、14 1nm、142nm、143nm、144nm、145nm、146nm、147nm、148nm、149nm、150nm、151nm m、152nm、153nm、154nm、155nm、156nm、157nm、158nm、159nm、160nm、161nm、 162nm、163nm、164nm、165nm、166nm、167nm、168nm、169nm、170nm、171nm、172 nm、173nm、174nm、175nm、176nm、177nm、178nm、179nm、180nm、181nm、182nm 、183nm、184nm、185nm、186nm、187nm、188nm、189nm、190nm、191nm、192nm、1 93nm, 194nm, 195nm, 196nm, 197nm, 198nm, 199nm, 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229nm, 230nm, 231nm, 232nm, 233nm, 234nm m, 235nm, 236nm, 237nm, 238nm, 239nm, 241nm, 242nm, 243nm, 244nm, 245nm, 246nm, 247nm, 248nm, 249nm, 251nm, 252nm, 253nm, 254nm, 255nm, 256nm, 257 nm, 258nm, 259nm, 261nm, 262nm, 263nm, 264nm, 265nm, 266nm, 267nm, 268nm, 269nm, 271nm, 272nm, 273nm, 274nm, 275nm, 276nm, 277nm, 278nm, 279nm, 2 81nm, 282nm, 283nm, 284nm, 285nm, 286nm, 287nm, 288nm, 289nm, 291nm, 292nm, 293nm, 294nm, 295nm, 296nm, 297nm, 298nm, 299nm, 300nm, 301nm, 302nm303nm, 304nm, 305nm, 306nm, 307nm, 308nm, 309nm, 310nm, 311nm, 312nm, 313nm, 314nm, 315nm, 316nm, 317nm, 318nm, 319nm, 320nm, 321nm, 322nm, 323nm m, 324nm, 325nm, 326nm, 327nm, 328nm, 329nm, 330nm, 331nm, 332nm, 333nm, 334nm, 335nm, 336nm, 337nm, 338nm, 339nm, 340nm, 341nm, 342nm, 343nm, 344 nm, 345nm, 346nm, 347nm, 348nm, 349nm, 350nm, 351nm, 352nm, 353nm, 354nm, 355nm, 356nm, 357nm, 358nm, 359nm, 360nm, 361nm, 362nm, 363nm, 364nm, 3 65nm, 366nm, 367nm, 368nm, 369nm, 370nm, 371nm, 372nm, 373nm, 374nm, 375nm, 376nm, 377nm, 378nm, 379nm, 380nm, 381nm, 382nm, 383nm, 384nm, 385nm 386nm, 387nm, 388nm, 389nm, 390nm, 391nm, 392nm, 393nm, 394nm, 395nm, 396nm, 397nm, 398nm, 399nm, 400nm, 401nm, 402nm, 403nm, 404nm, 405nm, 406nm m、407nm、408nm、409nm、410nm、411nm、412nm、413nm、414nm、415nm、416nm、 417nm、418nm、419nm、420nm、421nm、422nm、423nm、424nm、425nm、426nm、427 nm、428nm、429nm、430nm、431nm、432nm、433nm、434nm、435nm、436nm、437nm 、438nm、439nm、440nm、441nm、442nm、443nm、444nm、445nm、446nm、447nm、4 48nm、449nm、450nm、451nm、452nm、453nm、454nm、455nm、456nm、457nm、458 nm、459nm、460nm、461nm、462nm、463nm、464nm、465nm、466nm、467nm、468nm、469nm、470nm、471nm、472nm、473nm、474nm、475nm、476nm、477nm、478nm、47 9nm、480nm、481nm、482nm、483nm、484nm、485nm、486nm、487nm、488nm、489nm m、490nm、491nm、492nm、493nm、494nm、495nm、496nm、497nm、498nm、499nm、 500nm、501nm、502nm、503nm、504nm、505nm、506nm、507nm、508nm、509nm、510 nm、511nm、512nm、513nm、514nm、515nm、516nm、517nm、518nm、519nm、520nm 、521nm、522nm、523nm、524nm、525nm、526nm、527nm、528nm、529nm、530nm、5 31nm, 532nm, 533nm, 534nm, 535nm, 536nm, 537nm, 538nm, 539nm, 540nm, 541nm, 542nm, 543nm, 544nm, 545nm, 546nm, 547nm, 548nm, 549nm, 550nm, 551nm 552nm, 553nm, 554nm, 555nm, 556nm, 557nm, 558nm, 559nm, 560nm, 561nm, 562nm, 563nm, 564nm, 565nm, 566nm, 567nm, 568nm, 569nm, 570nm, 571nm, 572nm m、573nm、574nm、575nm、576nm、577nm、578nm、579nm、580nm、581nm、582nm、 583nm、584nm、585nm、586nm、587nm、588nm、589nm、590nm、591nm、592nm、593 nm、594nm、595nm、596nm、597nm、598nm、599nm、600nm、601nm、602nm、603nm 、604nm、605nm、606nm、607nm、608nm、609nm、610nm、611nm、612nm、613nm、6 14nm、615nm、616nm、617nm、618nm、619nm、620nm、621nm、622nm、623nm、624 nm、625nm、626nm、627nm、628nm、629nm、630nm、631nm、632nm、633nm、634nm、635nm、636nm、637nm、638nm、639nm、640nm、641nm、642nm、643nm、644nm、64 5nm、646nm、647nm、648nm、649nm、650nm、651nm、652nm、653nm、654nm、655nm m、656nm、657nm、658nm、659nm、660nm、661nm、662nm、663nm、664nm、665nm、 666nm、667nm、668nm、669nm、670nm、671nm、672nm、673nm、674nm、675nm、676 nm、677nm、678nm、679nm、680nm、681nm、682nm、683nm、684nm、685nm、686nm 、687nm、688nm、689nm、690nm、691nm、692nm、693nm、694nm、695nm、696nm、6 97nm, 698nm, 699nm, 700nm, 701nm, 702nm, 703nm, 704nm, 705nm, 706nm, 707nm, 708nm, 709nm, 710nm, 711nm, 712nm, 713nm, 714nm, 715nm, 716nm, 717nm 718nm, 719nm, 720nm, 721nm, 722nm, 723nm, 724nm, 725nm, 726nm, 727nm, 728nm, 729nm, 730nm, 731nm, 732nm, 733nm, 734nm, 735nm, 736nm, 737nm, 738nm m, 739nm, 740nm, 741nm, 742nm, 743nm, 744nm, 745nm, 746nm, 747nm, 748nm, 749nm, 750nm, 751nm, 752nm, 753nm, 754nm, 755nm, 756nm, 757nm, 758nm, 759 nm, 760nm, 761nm, 762nm, 763nm, 764nm, 765nm, 766nm, 767nm, 768nm, 769nm, 770nm, 771nm, 772nm, 773nm, 774nm, 775nm, 776nm, 777nm, 778nm, 779nm, 7 80nm, 781nm, 782nm, 783nm, 784nm, 785nm, 786nm, 787nm, 788nm, 789nm, 790nm, 791nm, 792nm, 793nm, 794nm, 795nm, 796nm, 797nm, 798nm, 799nm, 800nm801nm、802nm、803nm、804nm、805nm、806nm、807nm、808nm、809nm、810nm、811nm、812nm、813nm、814nm、815nm、816nm、817nm、818nm、819nm、820nm、821nm、822nm、823nm、82、 4nm, 825nm, 826nm, 827nm, 828nm, 829nm, 830nm, 831nm, 832nm, 833nm, 834nm, 835nm, 836nm, 837nm, 838nm, 839nm, 840nm, 841nm, 842nm, 843nm, 844nm, 8 45nm、846nm、847nm、848nm、849nm、850nm、851nm、852nm、853nm、854nm、855 nm、856nm、857nm、858nm、859nm、860nm、861nm、862nm、863nm、864nm、865nm、 866nm, 867nm, 868nm, 869nm, 870nm, 871nm, 872nm, 873nm, 874nm, 875nm, 876nm, 877nm, 878nm, 879nm, 880nm, 881nm, 882nm, 883nm, 884nm, 885nm, 886nm 887nm, 888nm, 889nm, 890nm, 891nm, 892nm, 893nm, 894nm, 895nm, 896nm, 897nm, 898nm, 899nm, 900nm, 901nm, 902nm, 903nm, 904nm, 905nm, 906nm, 907nm m, 908nm, 909nm, 910nm, 911nm, 912nm, 913nm, 914nm, 915nm, 916nm, 917nm, 918nm, 919nm, 920nm, 921nm, 922nm, 923nm, 924nm, 925nm, 926nm, 927nm, 928 nm, 929nm, 930nm, 931nm, 932nm, 933nm, 934nm, 935nm, 936nm, 937nm, 938nm, 939nm, 940nm, 941nm, 942nm, 943nm, 944nm, 945nm, 946nm, 947nm, 948nm, 94 9nm, 950nm, 951nm, 952nm, 953nm, 954nm, 955nm, 956nm, 957nm, 958nm, 959nm, 960nm, 961nm, 962nm, 963nm, 964nm, 965nm, 966nm, 967nm, 968nm, 969nm, 9 70nm, 971nm, 972nm, 973nm, 974nm, 975nm, 976nm, 977nm, 978nm, 979nm, 980nm, 981nm, 982nm, 983nm, 984nm, 985nm, 986nm, 987nm, 988nm, 989nm, 990nm991nm, 992nm, 993nm, 994nm, 995nm, 996nm, 997nm, 998nm, 999nm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μ m, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 21μm, 22μm, 23μm, 2 The pharmaceutical composition may be formulated to contain 4 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 polynucleotide. In some embodiments, the pharmaceutical composition may be formulated to contain about 0.1 nM to about 1.0 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 1 nM to about 10 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 1 nM to about 100 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 1 nM to about 200 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 1 nM to about 50 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 10 nM to about 100 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 10 nM to about 200 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 50 nM to about 100 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 50 nM to about 100 nM of polynucleotide in the peptide-polynucleotide complex of the present invention.The peptide-polynucleotide complex of the present invention may be formulated to contain about 50 nM to about 200 nM of polynucleotide. In other embodiments, the pharmaceutical composition may be formulated to contain about 100 nM to about 200 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 150 nM to about 200 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 200 nM to about 100 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 500 nM to about 1000 nM of polynucleotide in the peptide-polynucleotide complex of the present invention. In other embodiments, the pharmaceutical composition may be formulated to contain about 1 μM to about 50 μM of polynucleotide in the peptide-polynucleotide complex of the present invention. The peptide concentration in the peptide-polynucleotide complex of the present invention may be calculated based on the desired polynucleotide concentration and the ratio of peptide to polynucleotide in the peptide-polynucleotide complex of the present invention.

[0091] Pharmaceutical compositions may also be formulated to contain 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 the peptide-polynucleotide complexes of the invention. In some embodiments, pharmaceutical compositions may be formulated to contain 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 μg / ml of the peptide-polynucleotide complexes of the invention. In other embodiments, pharmaceutical compositions may be formulated to contain 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 μg / ml of the peptide-polynucleotide complexes of the invention. In yet other embodiments, pharmaceutical compositions may be formulated to contain 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 μg / ml of the peptide-polynucleotide complexes of the invention. In still other embodiments, pharmaceutical compositions may be formulated to contain 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or about 700 μg / ml or more of the peptide-polynucleotide complexes of the invention.

[0092] [7.4. How to use] In another aspect, the present invention encompasses methods of using the peptide-polynucleotide complexes of the present invention to transfect a polynucleotide into the cytoplasm of a cell. In some embodiments, the cell is in vitro. In other embodiments, the cell is in vivo. Accordingly, the present invention also provides methods of using the peptide-polynucleotide complexes of the present invention to transfect a polynucleotide into the cytoplasm of a cell in a subject in need of treatment. Generally speaking, the methods of the present invention involve contacting a cell with a peptide-polynucleotide complex of the present invention under conditions suitable for transfection of the polynucleotide. Suitable cells and conditions are described above. In embodiments in which the cell is in vivo, the methods of the present invention typically involve administering to the subject in need of treatment a pharmaceutical composition comprising the peptide-polynucleotide complex of the present invention. Suitable pharmaceutical compositions are described herein.

[0093] In another aspect, the present invention encompasses a method of treating a condition in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a peptide-polynucleotide complex. The peptide-polynucleotide complex of the present invention can efficiently transfect or deliver the polynucleotide of the peptide-polynucleotide complex to cells of the subject.

[0094] In some embodiments, the polynucleotides of the present invention comprise non-coding RNAs capable of regulating or inhibiting the expression of a nucleic acid sequence expressed in a cell. By efficiently transfecting a polynucleotide capable of regulating or inhibiting the expression of a nucleic acid sequence expressed in a cell, the methods of the present invention 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. In some embodiments, the present invention encompasses methods of administering a peptide-polynucleotide complex of the present invention to a subject to treat an NFκB-mediated condition. In some embodiments, the present invention encompasses methods of administering a peptide-polynucleotide complex of the present invention to a subject to treat a condition associated with overexpression or aberrant expression of KRAS in a subject. In some embodiments, the present invention encompasses methods of administering a peptide-polynucleotide complex of the present invention to a subject to treat a condition associated with STAT3 dysregulation in a subject. In some embodiments, the present invention encompasses methods of administering a peptide-polynucleotide complex of the present invention to a subject to treat a condition associated with JNK2 dysregulation in a subject.

[0095] The peptides, polynucleotides, and peptide-polynucleotide complexes are as described herein. Pharmaceutical compositions containing the peptide-polynucleotide complexes of the present invention are as described herein. Methods for administering the peptide-polynucleotide complexes of the present invention and methods for treating diseases are described below.

[0096] 7.4.1. Administration to Subjects in Need In one aspect, the present invention encompasses administering a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. As used herein, the term "subject in need of treatment" refers to a subject in need of preventive or therapeutic treatment. The subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, such as a mouse, rat, guinea pig, etc. In other embodiments, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals include pigs, cows, horses, goats, sheep, llamas, and alpacas. In yet other embodiments, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet other embodiments, the subject may be a zoological animal. As used herein, "zoological animal" refers to animals that may be found in a zoo. Such animals may include non-human primates, big cats, wolves, and bears. In some embodiments, the subject is a mouse. In some preferred embodiments, the subject is a human.

[0097] As described herein, pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Suitable routes of administration include parenteral, oral, pulmonary, transdermal, transmucosal, and rectal administration. In some embodiments, pharmaceutical compositions of the present invention are administered by injection.

[0098] Those skilled in the art will recognize that the amount and concentration of the composition administered to a subject will depend, in part, on the subject and the reason for administration. Methods for determining optimal amounts are known in the art. Generally, the concentration of the peptide-polynucleotide complex of the present invention in a pharmaceutical composition will be as described herein.

[0099] The compositions of the present invention are typically administered to a subject in need of treatment in an amount sufficient to provide benefit to the subject. Such an amount is defined as a "therapeutically effective amount." A therapeutically effective amount may be determined by the potency or efficacy of the particular composition, the disease being treated, the duration or frequency of administration, the method of administration, and the subject's physical condition and condition, including the subject's particular response to treatment. A therapeutically effective amount may be determined using methods known in the art, or may be determined experimentally by deriving therapeutically effective amounts determined in animal models such as mice, or by a combination of these. Furthermore, the route of administration may be taken into consideration when determining the therapeutically effective amount. In determining a therapeutically effective amount, one skilled in the art may also consider the presence, nature, and extent of side effects associated with the administration of a particular compound in a particular subject.

[0100] When the pharmaceutical composition of the present invention is administered to a subject by injection, the composition may be administered as a bolus injection to a subject in an amount of about 0.1 mg / kg to about 100 mg / kg or more. In some embodiments, the pharmaceutical composition of the present invention is administered to a subject in an amount of about 0.1 mg / kg to about 5 mg / kg. In other embodiments, the pharmaceutical composition of the present invention is administered to a subject in an amount of about 5 mg / kg to about 15 mg / kg. In still other embodiments, the pharmaceutical composition of the present invention is administered to a subject in an amount of about 15 mg / kg to about 30 mg / kg. In other embodiments, the pharmaceutical composition of the present invention is administered to a subject in an amount of about 30 mg / kg to about 45 mg / kg. In further embodiments, the pharmaceutical composition of the present invention is administered to a subject in an amount of about 45 mg / kg to about 100 mg / kg or more. In some embodiments, the composition is administered as a bolus injection to a subject in an amount of about 0.5 to about 1.5 mg / kg.

[0101] The composition may also be administered to the subject in two or more bolus infusions over a period of time. For example, the composition may be administered to the subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bolus infusions. In some embodiments, the composition is administered to the subject in 1, 2, 3, 4, or 5 bolus infusions. In other embodiments, the composition is administered to the subject in 5, 6, 7, 8, 9, 10, or more bolus infusions. In some embodiments, the composition is administered to the subject in 2, 3, or 4 bolus infusions. The boluses may be administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about every 12 hours, or they may be administered about every 1, 2, 3, 4, 5, 6, or about every 7 days. In some embodiments, the boluses may be administered approximately daily.

[0102] 7.4.2. Cancer Treatment In some embodiments, the methods of the present invention are used to treat tumors or cancer. Tumors can be malignant or benign, cancers can be primary or metastatic, and tumors or cancers can be early-stage or late-stage. Cancers can be blood cancers or solid tumor cancers. Cancers or tumors can be treated by delivering nucleic acid sequences to a subject's cancer tumor. Cancers or tumors can be treated by slowing cancer cell growth, killing cancer cells, or reducing the spread of cancer cells that cause metastasis. In some embodiments, the cancer cells express KRAS or a mutant form of KRAS. The present invention is particularly suitable for treating patients with one or more diverse KRAS mutations because the nucleic acid sequences of the present invention were carefully selected to target KRAS positions outside known KRAS mutation hotspots, such as the region of amino acid G12, G13, and Q61 mutations. The conjugates of the present invention are designed to selectively target cancer cells by their ability to penetrate cancer tissue, thereby specifically targeting cancer cells regardless of the nature of the KRAS mutation.

[0103] In some embodiments, the polynucleotide of the peptide-polynucleotide complex of the invention may treat cancer or tumors by delivering the polynucleotide on nanoparticles to cancer cells in a subject in vivo. In some embodiments, the polynucleotide of the peptide-polynucleotide complex of the invention may treat cancer or tumors by delivering the polynucleotide on nanoparticles to cells in the tumor microenvironment or other cells surrounding the tumor. Non-limiting examples of tumors or cancers that may be treated with the methods of the invention include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (pediatric cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway glioma and thalamic glioma). Lower glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor (pediatric type, gastrointestinal type), cancer of unknown primary site, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's family of tumors Ewing's sarcoma, extracranial germ cell tumors (pediatric type), extragonadal germ cell tumors, extrahepatic bile duct cancer, eye tumors (intraocular melanoma, retinoblastoma), gallbladder cancer, stomach (abdominal) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, germ cell tumors (pediatric extracranial type, extragonadal type, ovarian type), gestational trophoblastic tumors, gliomas (adult type, pediatric brainstem type, pediatric cerebral astrocytic type, pediatric visual pathway type and hypothalamic type), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer , Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (pediatric type), intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lip and oral cancer, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system),Macroglobulinemia (Waldenstrom's), malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma (pediatric type), melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma (adult malignant type, pediatric type), metastatic squamous cell carcinoma of the neck with unknown primary origin, oral cancer, multiple endocrine neoplasia syndrome (pediatric type), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, myeloid leukemia (chronic), myeloid leukemia (acute adult, acute pediatric), multiple myeloma, myeloproliferative disease (chronic), nasal cavity and sinonasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germinoma, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), sinonasal and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumor (pediatric type), pituitary adenoma, plasma cell tumor, pleuropulmonary blastoma tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, rhabdomyosarcoma (pediatric type), salivary gland cancer, sarcoma (Ewing family tumor type, Kaposi's type, soft tissue type, uterine type), Sézary syndrome, skin cancer (non-melanoma, melanoma), skin cancer (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of occult primary (metastatic), gastric cancer, supratentorial primitive neuroectodermal tumor (pediatric type), T-cell lymphoma (skin) , T-cell leukemia and lymphoma, testicular cancer, throat cancer, thymoma (pediatric type), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (pediatric type), transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor (gestational), site of unknown primary site (adult type, pediatric type), transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (pediatric type), vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor (pediatric type). In some embodiments, the methods of the present invention are used to treat T-cell leukemia and lymphoma. In an exemplary embodiment, the methods of the present invention are used to treat human T-lymphotropic virus-1 (HTLV-1)-induced adult T-cell leukemia / lymphoma (ATLL).

[0104] In other embodiments, the polynucleotide of the peptide-polynucleotide complex of the present invention may be delivered to cancer cells in vitro. For example, the polynucleotide of the peptide-polynucleotide complex of the present invention may be delivered to a cancer cell line in vitro. The cancer cells may be a cancer cell line cultured in vitro. In some alternative embodiments, the cancer cell line may be a primary cell line that has not yet been described. Methods for generating primary cancer cell lines utilize standard techniques known to those of skill in the art. In other alternatives, the cancer cell line may be an established cancer cell line. The cancer cell line may be adherent or non-adherent, or the cell line may be grown under conditions that promote adherent, non-adherent, or organotypic growth using standard techniques known to those of skill in the art. The cancer cell line may be contact-inhibited or non-contact-inhibited.

[0105] In some embodiments, the cancer cell line may be an established human cell line derived from a tumor. Non-limiting examples of tumor-derived cancer cell lines include osteosarcoma cell lines 143B, CAL-72, G-292, HOS, KHOS, MG-63, Saos-2, and U-2 OS; prostate cancer cell lines DU145, PC3, and Lncap; breast cancer cell lines MCF-7, MDA-MB-438, and T47D; myeloid leukemia cell line THP-1; glioblastoma cell line U87; neuroblastoma cell line SHSY5Y; bone cancer cell line Saos-2; and 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; non-small cell lung cancer (NSCLC) cell lines H358, H2122, H441, H727, SK-Lu-1, and H2009; melanoma cell line B16-F10; macrophage cell line RAW264.7; F8 cell line; and pancreatic cancer cell lines Panc1, PANC 10.05, CAPAN-1, CAPAN-2, PSN1, and MIA-PaCa2. In an exemplary embodiment, the peptide-polynucleotide complexes of the present invention may be administered to the F8 cell line. In another exemplary embodiment, the peptide-polynucleotide complexes of the present invention may be administered to the B16-F10 cell line.

[0106] [7.5. Kit] Another aspect of the present invention includes a kit. The kit includes a first composition comprising a peptide of the present invention and, optionally, a second composition comprising a polynucleotide. Alternatively, the polynucleotide of interest may be provided by the user of the kit. By following the instructions provided with the kit, the user of the kit may mix a composition comprising a peptide of the present invention with a composition comprising a polynucleotide to form a peptide-polynucleotide complex. The instructions for the kit may include instructions for mixing the peptide and polynucleotide in an appropriate ratio. The kit may also include an appropriate buffer, water, a cross-linking reagent, or albumin.

[0107] 8. Examples

[0108] The following are descriptions of various methods and materials used in this study. These are intended to provide a complete disclosure and description of how one of ordinary skill in the art can make and use the invention, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent all experiments that have been performed or could be performed. The exemplary descriptions written in the present tense are not necessarily performed, but are descriptions that could be performed to generate data, etc. relevant to the teaching of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0109] Example 1: siRNA targeting KRAS siRNAs were developed to avoid the mutation sites, aiming to use the same compound to knockdown KRAS both in the mutated and unmutated forms. The avoided mutation sites are G12, G13, and Q61.

[0110] We started with in silico evaluation. In the bioinformatics approach, a standard siRNA structure was assumed. Positions 2 to 18 (5'-3') of the sense and antisense strands were used for specificity calculations. Positions 1 to 19 (5'-3') of the antisense strand were used for cross-reactivity and human SNP analysis. The following parameters were evaluated: Species cross-reactivity in humans, cynomolgus monkeys, rhesus monkeys, and mice Analyses based on standard siRNA designs using 19 and 17 bases (without considering positions 1 and 19) for cross-reactivity assessment. Perfect match and single mismatch analyses were included. Predicted specificity in humans, rhesus monkeys, cynomolgus monkeys, and mice The sense and antisense strands were analyzed separately. Identity of siRNA seed regions and seed regions of known miRNAs Identification of siRNAs targeting regions with known SNPs through analysis of the human SNP database (NCBI-DB-SNP). Information included the location of the SNP within the target sequence and the minor allele frequency (MAF) when data were available. · siRNA activity prediction based on standard siRNA design

[0111] Initially, 96 sequences were selected for in vitro analysis, which are shown in Table 1 above.

[0112] The 96 selected sequences were further modified, and the modification patterns are shown in Figure 1. The modified sequences are shown in Table 2 above.

[0113] These 96 sequences were synthesized and tested at two different doses (0.1 and 10 nM) in NCI-H23 cells harboring the KRAS G12C mutation, and the results of this analysis are shown in Table 3 below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0114] The 24 best performing siRNAs were selected and subjected to dose-response studies to evaluate Kras silencing. The results of the dose-response studies (IC50 values ​​and percent inhibition) are shown in Table 4 below. In screening these siRNAs, the dose-response curves showed highly variable shapes: some reached a plateau of 100% target expression, while some surprisingly appeared to reach saturation / maximal knockdown at all doses tested. [Table 4]

[0115] Two siRNAs (shown in Table 5) had unexpectedly high activity and were tested at lower doses, achieving a concentration of 0.00002 nM (20 fM). In this dose-response curve, maximal knockdown was observed at all doses. The results of this analysis are shown in Figures 2A-2F. [Table 5]

[0116] Finally, we evaluated two candidates (XD-39951 and XD-39947) in cell lines with different KRAS mutations. These evaluations were performed by transfecting siRNA into cells with either wild-type KRAS or KRAS mutations (HT-29 cells: wild-type; SW480: G12V mutation; LS174T: G12D mutation). As shown in Figure 3A-3C, both sequences were able to knockdown KRAS regardless of whether it was mutated or not.

[0117] The XD-39951 candidate was further tested in cell lines harboring additional KRAS mutations. These evaluations were performed by transfecting siRNA into cells harboring additional KRAS mutations. As shown in Figure 4A, in addition to G12V and G12D, XD-39951 was able to knock down KRAS mutations G12C, G12R, G12A, and A146T. As shown in Figure 4B, knockdown of KRAS led to decreased cell viability in some cases.

[0118] This formulation allows for specific delivery to tumors, since tumors typically have leaky blood vessels and the physicochemical properties of the nanoparticles disclosed herein allow for their extravasation.

[0119] Coating nanoparticles with albumin enriches their local concentration by binding to the receptors pg60 and / or SPARC, which are upregulated in certain tumors.

[0120] Coating nanoparticles with hyaluronic acid may exert a similar effect on other tumor types via the CD44 receptor.

[0121] Example 2: Materials and Methods 1.1 Knockdown analysis of Kras in NCI-H23 cells after transfection with different siRNAs [Table 6]

[0122] <Method> NCI-H23 cells (ATCC) at a density of 20,000 cells per well were transfected with increasing concentrations of Kras siRNA (0.00002 nM to 50 nM) using RNAiMax transfection agent (Invitrogene) according to the manufacturer's instructions. Kras knockdown was analyzed 24 hours posttransfection using the Quantigene® branched DNA assay.

[0123] 1.2 Knockdown analysis of Kras in HT-29, SW480, and LS174T cells after transfection with different siRNAs

[0124] [Table 7]

[0125] [Table 8]

[0126] [Table 9]

[0127] [Table 10]

[0128] <Device> 1) Applied Biosystems Inc. (ABI), fast PCR system 7900H, 384-well format Device ID:BEPCR0030 2) Data analysis software: ABI SDS2.4 3) Nanodrop(TM) 2000 Spectrophotometer Device ID:BENOP0020 4) Qiagen Tissue Lyser II Device ID: BETIS0010

[0129] <Method>

[0130] siRNA resuspension (Aims 1-3) a) Centrifuge screw-capped vials briefly at low speed (up to 4000 x g) to ensure all material in the vial or well has settled to the bottom before opening. b) Carefully remove the screw cap. c) Add nuclease-free water to adjust the stock solution concentration to 100 μM. d) Allow the vial or plate to stand at ambient temperature for a few minutes. e) Resuspend by gently pipetting up and down 5 times. f) Repeat steps d and e. g) Aliquot the resuspended siRNA into multiple tubes or plates to minimize the number of freeze-thaw cycles. Store at -80°C. h) NOTE: The siRNA solution was kept on ice when preparing the transfection reaction.

[0131] <Cell seeding and TO plate reading (Objectives 1-3)> a) 24 hours before transfection (day 0), cells are seeded at a predetermined density in 90 µL of culture medium in a 96-well plate. The next day, cells reach 30-50% confluence. b) Remove the plate from the TO group (day 1) and add 10 μL of culture medium to each well to take a TO reading. c) Add 100 μL of CellTiter-Glo reagent to each well. d) Agitate on an orbital shaker for 20 minutes to promote cell lysis. e) Incubate the plate at room temperature for 10 minutes to stabilize the luminescence signal. (Note: Variations in the luminescence signal within the standard plate may be caused by temperature gradients, uneven cell seeding, edge effects in multi-layer plates, etc.) f) Attach a black seal as a back seal to the bottom of each plate. g) Record the luminescence using an EnVision Multi Label reader.

[0132] <siRNA transfection (Objectives 1-3)> a) On the day of transfection (Day 1), replace the culture medium with 90 μL of fresh culture medium. b) Transfect the cells with siRNA at the final concentration ranges shown in the attached table, and perform it in triplicate. Prepare the siRNA-lipid complex as follows.

Table 11

[0133] <Recovery of qPCR samples (Objectives 1-3)> a) 24 hours before transfection (Day 0), seed the cells at a pre-determined density in 2.25 mL of culture medium in a 6-well plate. The cells will reach 30-50% confluent the next day. b) On the day of transfection (day 1), replace the medium with 2.25 mL of fresh growth medium. c) Transfect cells with siRNA at the range of final concentrations shown in the Appendix. Prepare siRNA-lipid complexes as follows. [Table 12] d) Add 125 μL of diluted siRNA to 125 μL of diluted Lipofectamine and incubate the mixture at RT for 5 minutes. Add the siRNA-lipid complexes dropwise to each well and mix gently by rocking the plate back and forth. Allow the cells to incubate for the specified time before harvesting. If necessary, replace the medium with fresh medium after 24 hours of incubation. e) Remove the medium and freeze the transfected cells in liquid nitrogen and store at -80°C.

[0134] <Sample information> 366 samples obtained from the in vitro efficacy study were used for gene expression detection. Detailed information of the samples is shown in Table 6 below. [Table 13]

[0135] <Total RNA extraction> 1) Place stainless steel beads (average diameter 5 mm) and 350 µL of Buffer RLT into the 2 mL microcentrifuge tube containing the cells. Place the tube into the TissueLyser adapter set and run the TissueLyser at 20 Hz for 5 minutes. Continue with RNA extraction. 2) Add 1 volume of 70% ethanol to the lysate and mix well by pipetting. Do not centrifuge. Proceed immediately to step 3. 3) Place a sample containing any precipitate, up to 700 μL, into the RNeasy Mini spin column placed in a 2 mL collection tube (included). Close the lid and centrifuge at 8000 × g or more for 15 seconds. Discard the flow-through. Transfer the remaining lysate to the same tube and repeat step 3. 4) Add 350 μL of Buffer RW1 to the RNeasy column. Close the lid and centrifuge at 8000 × g for 15 seconds. Discard the flow-through. 5) Add 10 μL of DNase I stock solution to 70 μL of Buffer RDD. Invert the tube gently to mix and centrifuge briefly. 6) Add the DNase I reaction mixture (80 μL) directly to the membrane of the RNeasy column and let the tube stand on the bench top (20 - 30 °C) for 15 minutes. 7) Add 350 μL of Buffer RW1 to the RNeasy column. Close the lid and centrifuge at 8000 × g for 15 seconds. Discard the flow-through. 8) Add 500 μL of Buffer RPE to the spin column. Close the lid gently and centrifuge at 8000 × g for 2 minutes to wash the spin column membrane. Discard the flow-through. 9) Add another 500 μL of Buffer RPE to the spin column. Close the lid gently and centrifuge at 8000 × g for 2 minutes to wash the spin column membrane. 10) Place the RNeasy spin column into a new 1.5 mL collection tube (included). Add 30 - 50 μL of RNase-free water directly to the center of the spin column membrane. Close the lid gently and centrifuge at maximum speed for 1 minute to elute the RNA.

[0136] <RNA Quantification> Total RNA quantification using a Nanodrop™ 2000 spectrophotometer.

[0137] <cDNA Synthesis (Reverse Transcription, RT)> 1) Set up the RT reaction as follows.

Table 14

Table 15

[0138] <Real-time PCR reaction (TaqMan method)> 1) Prepare the real-time PCR as follows: [Table 16] 2) Real-time PCR procedure [Table 17] 3) ddH2O was used as a no template control (NTC). RNA samples were used as no reverse transcription controls (No RT). Three technical replicates were performed for each sample.

[0139] Those skilled in the art will appreciate that modifications may be made to the present embodiments without departing from the broad inventive concept thereof, and therefore, it is understood that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

[0140] Throughout the Background and specification, various publications, articles, and patents are cited or discussed, and each of these references is incorporated herein by reference in its entirety. Incorporated patents include, but are not limited to, U.S. Patent No. 9,987,371, U.S. Patent No. 1,075,827, and U.S. Patent No. 1,152,9388. Any discussion of documents, statutes, materials, devices, articles, and the like included herein is for the purpose of explaining the context of the present invention. No admission is made that any or all of the matters in such discussion constitute part of the prior art with respect to any invention(s) disclosed or claimed. [Brief explanation of the drawings]

[0141] [Figure 1] 1 shows exemplary modification patterns of the siRNAs disclosed herein. [Figure 2A] 1 shows the dose-response curve for XD-39946. [Figure 2B] 1 shows the dose-response curve for XD-39966. [Figure 2C] The relative mRNA expression levels of KRAS at different concentrations of XD-39946 and XD-39966 are shown. [Figure 2D] The relative mRNA expression levels of GAPDH at different concentrations of XD-39946 and XD-39966 are shown. [Figure 2E] The original data on the mRNA expression levels of KRAS and GAPDH at different concentrations of XD-39946 and XD-39966 are shown. [Figure 2F] A bar graph of the original data is shown in Figure 2E. [Figure 3A] Knockdown of KRAS in SW480 cells (G12V mutation) is shown. [Figure 3B] Knockdown of KRAS in HT-29 cells (wild type) is shown. [Figure 3C] 1 shows knockdown of KRAS in LS174T cells (G12D mutation). [Figure 4A]1 shows knockdown of KRAS in PDAC, NSCLC, and CRC cells with different KRAS mutations. [Figure 4B] 1 shows cell viability following knockdown of KRAS in PDAC, NSCLC, and CRC cells.

Claims

1. 1. A pharmaceutical composition comprising a peptide-polynucleotide complex, The peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and A pharmaceutical composition, wherein the polynucleotide is a short interfering RNA (siRNA) targeting human KRAS mRNA, and the target sequence of human KRAS mRNA does not encode G12, G13, or Q61 with reference to SEQ ID NO: 4, or does not encode a mutant amino acid at position 12, 13, or 61 with reference to SEQ ID NO:

4.

2. 10. The pharmaceutical composition of claim 1, wherein the peptide is non-lytic, non-cytotoxic, and capable of affecting the release of a polynucleotide from the endosome of a cell.

3. 3. The pharmaceutical composition of claim 1, wherein the peptide comprises two or more consecutive basic amino acids (cationic region) and one or more histidine residues adjacent to the cationic region.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the siRNA comprises a sense strand and an antisense strand.

6. The pharmaceutical composition according to claim 5, wherein the sense strand and the antisense strand each have a length of 16 to 24 bases.

7. 7. The pharmaceutical composition of claim 5 or 6, wherein the sense strand is 19 bases in length.

8. 7. The pharmaceutical composition of claim 5 or 6, wherein the antisense strand is 21 bases in length.

9. The pharmaceutical composition according to any one of claims 5 to 8, wherein the sense strand and the antisense strand are modified.

10. 10. The pharmaceutical composition of claim 9, wherein the modification is selected from the group consisting of 2'-methoxy (2'-OMe), 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-O-MOE), 5'-vinylphosphonate, phosphorothioate (PTO), locked nucleic acid (LNA), locked nucleic acid (UNA), glycol nucleic acid (GNA), and DNA.

11. The modification of the sense strand is (1) PTO in the first and second positions; (2) 2'-F at positions 3, 7-9, 12, and 17; and (3) The pharmaceutical composition of claim 10, which contains 2'-OMe at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19.

12. The modification of the antisense strand is (1) PTO in positions 1, 2, 19, and 20; (2) 2'-F at positions 2 and 14; and (3) The pharmaceutical composition of claim 10, which contains 2'-OMe at positions 1, 3 to 13, and 15 to 21.

13. The pharmaceutical composition according to any one of claims 5 to 12, wherein the last nucleotide of the sense strand is adenine (A).

14. The pharmaceutical composition according to any one of claims 5 to 12, wherein the first nucleotide of the antisense strand is uracil (U).

15. 15. The pharmaceutical composition of any one of claims 5 to 14, wherein the sense strand comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of any one of the sense strands shown in Table 1 and Table 2.

16. The pharmaceutical composition of any one of claims 5 to 14, wherein the antisense strand comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of any one of the antisense strands shown in Table 1 and Table 2.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the ratio of peptide to polynucleotide is from about 2:1 to about 3500:1, said ratio being a molar ratio.

18. 18. The pharmaceutical composition of claim 17, wherein the ratio of peptide to polynucleotide is from about 5:1 to about 200:

1.

19. 18. The pharmaceutical composition of claim 17, wherein the ratio of peptide to polynucleotide is about 100:

1.

20. 18. The pharmaceutical composition of claim 17, wherein the ratio of peptide to polynucleotide is about 5:

1.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the ratio of peptide to polynucleotide is from about 6:1 to about 18:1, said ratio being the ratio of amine groups of the polymer, which may be positively charged, to phosphate groups of the nucleic acid, which are negatively charged.

22. 22. The pharmaceutical composition of claim 21, wherein the charge ratio of the peptide to the polynucleotide is about 12:

1.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the peptide-polynucleotide complex is a nanoparticle having a diameter of about 10 nm to about 300 nm.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the peptide-polynucleotide complex is coated with albumin and / or hyaluronic acid.

25. The pharmaceutical composition according to any one of claims 1 to 24, further comprising a pharmaceutically acceptable carrier.

26. A method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 25.

27. 27. The method of claim 26, wherein the disease or disorder is cancer.

28. 28. The method of claim 27, wherein the cancer is a blood cancer or a solid tumor cancer.

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