Nucleic acid ligand conjugates and their use for delivery to cells

Abstract

The invention relates to nucleic acid ligand conjugates and their use for delivery to cells. In particular, the present invention relates to a coupling product comprising a ligand linked to a nucleic acid. The invention also relates to methods of delivering nucleic acids to cells and treating diseases using the conjugate products. The invention also relates to a method of increasing uptake of a nucleic acid by a cell comprising coupling a nucleic acid with a ligand to form a coupling product of the invention.

Description

[0001] This application is a divisional application of the application filed on May 12, 2021, with application number 202180037230.3 and invention title "Nucleic Acid Ligand Conjugates and Their Use in Cell Delivery". Priority Statement

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 024,142, filed May 13, 2020, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to conjugate products comprising ligands linked to nucleic acids. The invention also relates to methods for delivering nucleic acids to cells and treating diseases using the conjugate products. Furthermore, the invention relates to methods for increasing cellular uptake of nucleic acids, comprising conjugating nucleic acids to ligands to form the conjugate products of this invention. Background of the Invention

[0004] The difficulty of blocking many important cancer targets using traditional methods prompted inventors and others to consider using RNA interference (RNAi) as a treatment (Pecot et al., Nat. Rev. Cancer 11:59 (2011)). The discovery of RNAi in 1998 (Fire et al., Nature Pecot et al. (391:806 (1998)) won the 2006 Nobel Prize for revealing that double-stranded RNA (dsRNA) can bind to mRNA sequences in the cytoplasm, subsequently leading to the degradation of mRNA by the RNA-induced silencing complex (RISC) or the inhibition of translation (Pecot et al.). Nat. Rev. Cancer 11:59 (2011)). Given that RNAi can effectively silence any RNA sequence, using "therapeutic RNAi" to target "undruggable" targets is highly desirable. The inventors have previously demonstrated proof-of-concept that RNAi can be used to target mutant KRAS (Pecot et al., Mol. Cancer Ther. 13:2876 (2014)).

[0005] Despite the enormous potential of RNAi, numerous practical challenges hinder the successful and efficient delivery of oligonucleotides for cancer therapy (Pecot et al., Nat. Rev. Cancer11:59 (2011)). Barriers include intravascular degradation caused by serum exonucleases and endonucleases, rapid oligonucleotide clearance, the need for endosome escape, and avoidance of immune stimulation. In recent years, the use of targetable, biocompatible ligands (e.g., GalNAC targeting the desialyl glycoprotein receptor (ASGPR)) and state-of-the-art chemically modified siRNAs (siRs) has achieved some success in circumventing many of these barriers (Foster et al., 11:59 (2011)). Mol. Ther. 26:708 (2018); Nair et al., J. Am. Chem. Soc. 136:16958 (2014)). Given the aforementioned challenges, many have turned to the use of nanoparticle carriers to deliver oligonucleotides to tumors; however, this has not yet led to clinical success, and there are no FDA-approved therapies for delivering nanoparticle-mediated oligonucleotides in cancer.

[0006] Despite advances in the field, there remains a need for improved systems capable of delivering nucleic acids into cells, such as cancer cells. Therefore, this invention overcomes these technical deficiencies by providing compositions and methods for delivering nucleic acids into cells using targeted ligands. Summary of the Invention

[0007] Due to the remarkable success of GalNAC-conjugated chemically optimized siRs, such as the FDA-approved givosiran targeting ASGPR in the liver (Sardh et al., N. Engl. J. Med. In 380:549 (2019), the inventors chose to evaluate whether a ligand-coupled approach could target cancer and eliminate the need for nanocarriers. Using the Encyclopedia of Cancer Cell Lines (CCLE) dataset, they found that epidermal growth factor receptor (EGFR) is highly expressed in most epithelial cancers. The GE11 ligand was chosen; it is a 12-amino acid peptide (Li) that is found to bind to EGFR but does not induce mitotic signaling. et al., FASEB J.19:1978 (2005)). A highly scalable and simple synthetic and click chemistry method for GE11 was developed, using a biocompatible polyethylene glycol (PEG) linker to conjugate GE11 to siRNA. In two cell lines from different cancer origins (LU65-lung cancer and HCT 116-colon cancer), both expressing EGFR, GE11-Cy5-siRNA was rapidly and extensively internalized into cells compared to unconjugated siRNA. Significant RNAi activity was also observed when siRNA was targeted using GE11-KRAS without the use of transfection reagents. The extent of siRNA uptake in both cell lines (reaching an increase of approximately >150-250-fold in just 24 hours) was remarkable and comparable to the levels observed when GalNAC-siRNA, designed to target hepatocytes (an increase of approximately 8-fold). (Nair) et al., J. Am. Chem. Soc. 136:16958 (2014)), which far exceeded expectations. Given the extent to which oligonucleotides are taken up into cancer cells, ligand-coupled nucleic acid delivery has real potential in the use of nucleic acids to treat cancer.

[0008] Therefore, one aspect of the present invention relates to a coupling product comprising: A polypeptide containing the epidermal growth factor receptor (EGFR) targeting portion; Connector; and Nucleic acid.

[0009] Another aspect of the invention relates to compositions, such as pharmaceutical compositions comprising the conjugate of the invention.

[0010] Another aspect of the invention relates to a method for delivering nucleic acids into cells, the method comprising contacting the cells with an effective amount of the conjugate or composition of the invention.

[0011] Another aspect of the invention relates to a method for treating a disease in a subject who requires it, the method comprising administering to the subject a therapeutically effective amount of the conjugate product or pharmaceutical composition of the invention, thereby treating the disease.

[0012] Another aspect of the invention relates to a method for increasing cellular uptake of nucleic acids, the method comprising conjugating the nucleic acid via a linker to a polypeptide containing an EGFR-targeting moiety to form a conjugated product, wherein the cells express EGFR, and wherein the uptake of the nucleic acid by the cells is increased relative to nucleic acids not conjugated to a polypeptide containing an EGFR-targeting moiety.

[0013] This application provides the following solutions: 1. A coupling product comprising: a) A polypeptide containing the epidermal growth factor receptor (EGFR) targeting portion; b) Connector; and c) Nucleic acid.

[0014] 2. The coupling product according to Scheme 1, wherein the polypeptide comprises GE11.

[0015] 3. The coupling product according to any one of Scheme 1 or 2, wherein the polypeptide is modified to include a cysteine ​​residue at its C-terminus.

[0016] 4. The coupling product according to any one of Schemes 1-3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0017] 5. The coupling product according to any one of claims 1-4, wherein the linker comprises polyethylene glycol (PEG).

[0018] 6. The coupling product according to any one of Schemes 1-5, wherein the linker comprises dibenzocyclooctylene-PEG4-maleimide.

[0019] 7. The coupling product according to any one of Schemes 1-4, wherein the joint is a hexylamino joint coupled with a cleavable disulfide bond or a non-cleavable stalk.

[0020] 8. The coupling product according to any one of Schemes 1-4, wherein the linker is succinimide 3-(2-pyridyldithio)-propionate, succinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid, or triethylene glycol.

[0021] 9. The coupling product according to any one of claims 1-8, wherein the polypeptide is covalently bound to the linker.

[0022] 10. The coupling product according to Scheme 9, wherein the polypeptide is covalently bound to a thiol group on a cysteine ​​residue.

[0023] 11. The coupling product according to any one of Schemes 1-10, wherein the nucleic acid is DNA.

[0024] 12. The coupling product according to any one of schemes 1-10, wherein the nucleic acid is RNA.

[0025] 13. The conjugation product according to any one of Schemes 1-12, wherein the nucleic acid is single-stranded.

[0026] 14. The coupling product according to any one of Schemes 1-12, wherein the nucleic acid is double-stranded.

[0027] 15. The conjugation product according to any one of schemes 1-14, wherein the nucleic acid is selected from the group consisting of: siRNA, microRNA, shRNA, antisense nucleic acid, ribonuclease, cytotoxic tRNA, guide RNA, long noncoding RNA, antisense miRNA oligonucleotide, and plasmid DNA.

[0028] 16. The conjugation product according to any one of schemes 1-15, wherein the nucleic acid is a siRNA that silences KRAS.

[0029] 17. The coupling product according to any one of Schemes 1-16, wherein the adapter is covalently bound to the nucleic acid.

[0030] 18. The coupling product according to Scheme 17, wherein the nucleic acid is modified to provide a binding site with the adapter.

[0031] 19. The coupling product according to Scheme 18, wherein the nucleic acid is modified to include an azide group as the binding site.

[0032] 20. A composition comprising the coupling product and the carrier as described in any one of schemes 1-19.

[0033] 21. A pharmaceutical composition comprising the conjugate product of any one of schemes 1-19 and a pharmaceutically acceptable carrier.

[0034] 22. A method for delivering nucleic acids into cells, the method comprising contacting the cells with an effective amount of the composition described in scheme 20 or 21.

[0035] 23. The method according to Scheme 22, wherein the cell is a cancer cell.

[0036] 24. The method according to Scheme 23, wherein the cancer cells are selected from the group consisting of: non-small cell lung cancer cells, lung cancer cells, colon cancer cells, pancreatic cancer cells, and leukemia cells.

[0037] 25. The method according to any one of schemes 22-24, wherein the cells express high levels of EGFR.

[0038] 26. The method according to any one of claims 22-25, wherein the method does not include the use of a transfection reagent.

[0039] 27. A method for treating a disease in a subject who requires it, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of scheme 21, thereby treating the disease.

[0040] 28. The method according to scheme 27, wherein the disease is cancer.

[0041] 29. The method according to Scheme 28, wherein the cancer is selected from the group consisting of: non-small cell lung cancer, lung cancer, colon cancer, pancreatic cancer, and blood cancer.

[0042] 30. The method according to any one of claims 27-29, wherein the method does not include the use of a transfection reagent.

[0043] 31. A method for increasing cellular uptake of nucleic acids, the method comprising conjugating the nucleic acid via a linker to a polypeptide containing an EGFR-targeting moiety to form a conjugated product, wherein the cells express EGFR, and wherein the uptake of the nucleic acid by the cells is increased relative to nucleic acids not conjugated to a polypeptide containing an EGFR-targeting moiety.

[0044] 32. The method according to Scheme 31, wherein the polypeptide comprises GE11.

[0045] 33. The method according to any one of Scheme 31 or 32, wherein the polypeptide is modified to include a cysteine ​​residue at its C-terminus.

[0046] 34. The method according to any one of claims 31-33, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1.

[0047] 35. The method according to any one of claims 31-34, wherein the connector comprises polyethylene glycol (PEG).

[0048] 36. The method according to any one of claims 31-35, wherein the connector comprises dibenzocyclooctylene-PEG4-maleimide.

[0049] 37. The method according to any one of claims 31-36, wherein the polypeptide is covalently bound to the linker.

[0050] 38. The method according to Scheme 37, wherein the polypeptide is covalently bound to a thiol group on a cysteine ​​residue.

[0051] 39. The method according to any one of schemes 31-38, wherein the nucleic acid is DNA.

[0052] 40. The method according to any one of schemes 31-38, wherein the nucleic acid is RNA.

[0053] 41. The method according to any one of schemes 31-40, wherein the nucleic acid is single-stranded.

[0054] 42. The method according to any one of schemes 31-40, wherein the nucleic acid is double-stranded.

[0055] 43. The method according to any one of schemes 31-42, wherein the nucleic acid is selected from the group consisting of: siRNA, microRNA, shRNA, antisense nucleic acid, ribonuclease, cytotoxic tRNA, guide RNA, long noncoding RNA, antisense miRNA oligonucleotide, and plasmid DNA.

[0056] 44. The method according to any one of schemes 31-43, wherein the nucleic acid is a siRNA that silences KRAS.

[0057] 45. The method according to any one of schemes 31-44, wherein the adapter is covalently bound to the nucleic acid.

[0058] 46. ​​The method of claim 45, wherein the nucleic acid is modified to provide a binding site with the adapter.

[0059] 47. The method according to Scheme 46, wherein the nucleic acid is modified to include an azide group as the binding site.

[0060] These and other aspects of the invention will be set forth in more detail in the following description of the invention. Attached Figure Description

[0061] Figure 1 Many cancers show high levels of EGFR.

[0062] Figure 2 The results, confirmed by FACS, show that the LU65 lung cancer cell line has high EGFR expression.

[0063] Figure 3 The results, confirmed by FACS, showed that the HCT116 colon cancer cell line has high EGFR expression.

[0064] Figure 4 Show the synthetic scheme of the coupling product.

[0065] Figure 5 LC / MS confirmation of the GE11-PEG-KRAS Seq3 siRNA conjugate product.

[0066] Figure 6 LC / MS confirmation of the GE11-PEG-KRAS Seq3 siRNA conjugate product.

[0067] Figure 7 The GE11-conjugated Cy5-tagged siRNA was shown to be significantly time-dependently and freely taken up (without transfection reagent) into two different EGFR-expressing cancer cells (colon and lung cancer).

[0068] Figure 8The results showed that, for both siRNAs, GE11-conjugated KRAS-silencing siRNAs (Seq2 and Seq3) provided significant KRAS silencing against HCT116 (KRAS G13D mutant) colon cancer cells at 48 hours. No transfection reagent was used. This indicates that GE11-conjugated siRNAs can be taken up into cells and effectively silence mRNA.

[0069] Figure 9 This study demonstrates that GE11-conjugated Cy5-labeled siRNA enters cells via receptor-mediated endocytosis in EGFR-expressing cancer cells (HCT116 colon cancer). Cancer cells were first transfected with a GFP-labeled reporter plasmid, which localized to early (green, Rab5a) and late (green, Rab7a) endosomes or lysosomes (green, Lamp1) subcellular structures. Cells were then treated with GE11-siRNA in culture medium for 4 hours without transfection reagents. Cells were then washed three times with PBS and then imaged. The colocalization of Cy5 signaling (blue-green) with early and late endosomes and lysosomes indicates their entry into cells via receptor-mediated endocytosis.

[0070] Figure 10 This study demonstrates that GE11-conjugated Cy5-labeled siRNA enters cells via receptor-mediated endocytosis in EGFR-expressing cancer cells (HCT116 colon cancer). Cancer cells were first transfected with a GFP-labeled reporter plasmid, which localized to early (green, Rab5a) and late (green, Rab7a) endosomes or lysosomes (green, Lamp1) subcellular structures. Cells were then treated with GE11-siRNA in culture medium for 24 hours without transfection reagents. Cells were then washed three times with PBS and then imaged. The colocalization of Cy5 signaling (blue-green) with early and late endosomes and lysosomes indicates their entry into cells via receptor-mediated endocytosis.

[0071] Figure 11A-11B This demonstrates in vivo evidence of KRAS silencing in an HCT116 tumor (KRAS G13D). The tumor is approximately 125 mm in size. 3HCT116 (KRAS G13D) tumors were treated with PBS or with an EGFR-targeting ligand (GE11) conjugated to the KRAS siRNA sequence via the indicated linker. The linkers evaluated used hexylamino linkers conjugated to either a cleavable disulfide bond (SPDP) or a non-cleavable bond (SMCC or TEG) stalk. (SPDP: succinimide 3-(2-pyridinedithio)propionate; SMCC: trans-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide; TEG: triethylene glycol). Mice were treated subcutaneously with GE11-siRNA suspended in sterile PBS at 5 mg / kg (mpk). Tumors were harvested on days 3 (D3) and 7 (D7) following a single subcutaneous injection of GE11-siRNA dissolved in PBS (200 μL / mouse). Tumor RNA was isolated, and real-time qPCR was performed on the KRAS and 18S housekeeping genes. At the indicated time points, KRAS was found to be silenced in up to 50-70% of the tumors. Each group and time point shown represents 5 individual tumors. * P < 0.05 *** P < 0.01, **** P < 0.001. Mice were also given a one-time dose of 5 mpk or 10 mpk or a 5-day dose of 10 mpk (cumulative 50 mpk over 5 days), and no observable toxicity or weight loss was observed. Detailed Implementation

[0072] The invention will now be described with reference to the accompanying drawings, which illustrate preferred embodiments of the invention. However, the invention may be embodied in various forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided to make this disclosure comprehensive and complete, and to fully convey the scope of the invention to those skilled in the art.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used to describe this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0074] Nucleotide sequences are presented in single strand only in this document, 5' to 3' orientation, from left to right, unless otherwise specified. In accordance with 37 CFR §1.822 and established usage, nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature, or (for amino acids) with single-letter or three-letter codes. See, for example, the PatentIn User Manual, 99-102 (November 1990) (US Patent and Trademark Office).

[0075] Unless otherwise stated, standard methods known to those skilled in the art can be used to construct recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK. et al. , MOLECULARCLONING: A LABORATORY MANUAL 4th Edition (Cold Spring Harbor, NY, 2012); AUSUBEL et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0076] Furthermore, the present invention also considers that, in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted.

[0077] To further illustrate, for example, if the specification indicates that a particular amino acid can be selected from A, G, I, L, and / or V, the language also indicates that the amino acid can be selected from any subset of these amino acids (e.g., A, G, or I or L; A, G, I, or V; A or G; L only; etc.), as each such sub-combination is explicitly described herein. Furthermore, this language also indicates that one or more particular amino acids can be omitted. For example, in a particular embodiment, the amino acid is not A, G, or I; not A; not G or V; etc., as each such possible omission is explicitly described herein.

[0078] definition The following terms are used in the description herein and in the appended claims.

[0079] The singular form “a / kind (a, an)” is also intended to include the plural form unless the context clearly indicates otherwise.

[0080] Furthermore, when referring to measurable values ​​such as the length of a polynucleotide or polypeptide sequence, dosage, time, temperature, etc., the term “about” as used herein means a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0081] As used in this article, “and / or” means and includes any and all possible combinations of one or more of the related listed items, but not combinations when interpreted as alternatives (“or”).

[0082] As used herein, the transitional phrase “consistent with…” should be interpreted as including “the materials or steps mentioned herein, as well as materials or steps that do not substantially affect the essential and novel features of the claimed invention (e.g., nucleic acid delivery)”. Therefore, the term “consistent with…” as used herein should not be interpreted as equivalent to “comprising / including”.

[0083] When applied to the polynucleotide or polypeptide sequences of the present invention, the term "consistently composed of" (and grammatical variations) means a polynucleotide or polypeptide consisting of the sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids at the 5' and / or 3' or N-terminus and / or C-terminus of the sequence (such that the function of the polynucleotide or polypeptide is not substantially altered). The sum of the ten or fewer additional nucleotides or amino acids includes the total number of the two additional nucleotides or amino acids added together. When applied to the polynucleotides of the present invention, the term "substantially altered" means that the ability to modify the expression of the target nucleic acid is increased or decreased by at least about 50% or more compared to the expression level of the polynucleotide consisting of the sequence. When applied to the polypeptides of the present invention, the term "substantially altered" means that the enzyme activity is increased or decreased by at least about 50% or more compared to the activity of the polypeptide consisting of the sequence.

[0084] The term “enhancement” or “increase” refers to an increase of at least approximately 1.25 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, or even 15 times in a specific parameter.

[0085] As used herein, the terms “inhibition” or “reduction” or their grammatical variations refer to a reduction or attenuation of a particular level or activity by at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In certain embodiments, inhibition or reduction results in little or virtually no detectable activity (at most, insignificantly, for example, less than about 10% or even 5%).

[0086] As used herein, the term “polypeptide” includes both peptides and proteins, unless otherwise stated.

[0087] As used herein, the terms “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and include RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a nucleotide chain, regardless of chain length. Nucleic acids can be double-stranded or single-stranded. In the case of a single-stranded nucleic acid, it can be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (such as inosine or phosphate-thioester nucleotides). For example, such oligonucleotides can be used to prepare nucleic acids with altered base-pairing capabilities or increased resistance to nucleases. The present invention also provides nucleic acids that are complementary sequences (which can be fully or partially complementary) to the nucleic acids, nucleotide sequences, or polynucleotides of the present invention. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and other bases, can also be used for antisense, dsRNA, and ribonuclease pairing when synthesizing dsRNA. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are effective antisense inhibitors of gene expression. Other modifications can also be made, such as modifying the phosphodiester backbone or the 2'-hydroxyl group in the RNA riboglycosylation.

[0088] As used herein, the term "sequence identity" has its standard meaning in the art. As is known in the art, many different procedures can be used to determine whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity can be determined using standard techniques known in the art (including, but not limited to, Smith & Waterman, ...). Adv. Appl. Math. 2 The local sequence identity algorithm of :482 (1981), by Needleman & Wunsch, J. Mol. Biol. 48 The sequence identity comparison algorithm of :443 (1970), through Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85 The similarity search method of :2444 (1988), and the implementation of these algorithms by computer (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI), Devereux et al. , Nucl. Acid Res. 12 The best-fit sequence procedure described in :387 (1984) is preferably used with the default settings or determined by inspection.

[0089] A useful example of an algorithm is PILEUP. PILEUP creates multiple sequence alignments from a set of related sequences using progressive pairwise alignments. It can also draw a tree to show the clustering relationships used to create the alignments. PILEUP employs Feng & Doolittle... J. Mol. Evol. 35 A simplified version of the progressive alignment method of :351 (1987); this method is similar to that of Higgins & Sharp, CABIOS 5 The method described in :151 (1989).

[0090] Another example of a useful algorithm is the BLAST algorithm, which is used in Altschul et al. , J. Mol. Biol . 215 :403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90 As described in :5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program, which is derived from Altschul et al. , Meth. Enzymol. , 266 :460 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are preferably set to their default values. These parameters are dynamic values, established by the program itself based on the composition of a specific sequence and the composition of a specific database of the target sequences being searched; however, these values ​​can be adjusted to increase sensitivity.

[0091] Another useful algorithm is Altschul et al., Nucleic Acids Res. 25 :3389 (1997) reported BLAST with vacancies.

[0092] The percentage amino acid sequence identity value is determined by dividing the number of identical matched residues by the total number of residues in the "longer" sequence within the alignment region. The "longer" sequence is the sequence with the most actual residues in the alignment region (gap introduced by WU-Blast-2 to maximize the alignment score is ignored).

[0093] In a similar manner, the percentage of nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotides in a polynucleotide specifically disclosed herein.

[0094] Alignment may include introducing gaps in the sequence to be aligned. Furthermore, for sequences containing more or fewer nucleotides than those specifically disclosed herein, it should be understood that, in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides relative to the total number of nucleotides. Thus, for example, in one embodiment, the number of nucleotides in the shorter sequence will be used to determine the sequence identity of sequences shorter than those specifically disclosed herein. In the percentage of identity calculation, relative weights are not assigned to various manifestations of sequence variation (e.g., insertions, deletions, substitutions, etc.).

[0095] In one implementation, only identical scores are positive (+1), and all forms of sequence variation, including gaps, are assigned a value of "0," eliminating the need for the weighted scale or parameters used for sequence similarity calculations as described below. For example, the percentage of sequence identity can be calculated by dividing the number of matching identical residues by the total number of residues in the "shorter" sequence in the alignment region and multiplying by 100. The "longer" sequence is the sequence with the most actual residues in the alignment region.

[0096] As used herein, the terms “substantially identical” or “corresponding to” mean that two nucleic acid sequences have at least 60%, 70%, 80%, or 90% sequence identity. In some embodiments, two nucleic acid sequences may have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0097] As used herein, a “separate” polynucleotide (e.g., “separate DNA” or “separate RNA”) means a polynucleotide that is separate from or substantially free of at least some other components of a naturally occurring organism or virus, such as cellular or viral structural components, or other polypeptides or nucleic acids typically found linked to that polynucleotide.

[0098] Similarly, an "isolated" polypeptide refers to a polypeptide that is isolated from or substantially free of at least some other components of a naturally occurring organism or virus, such as cellular or viral structural components or other polypeptides or nucleic acids typically found linked to the polypeptide.

[0099] The term "fragment" applied to polynucleotides will be understood to mean a nucleotide sequence that is shortened relative to a reference nucleic acid or nucleotide sequence and comprises, is substantially identical to (e.g., 90%, 92%, 95%, 98%, 99% identity) a consecutive nucleotide sequence of the reference nucleic acid or nucleotide sequence, and is composed primarily of and / or consists of that nucleotide sequence. Where appropriate, such nucleic acid fragments according to the invention may be included in larger polynucleotides in which they are components. In some embodiments, such fragments may comprise, are substantially composed of and / or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive nucleotides of the nucleic acid or nucleotide sequence according to the invention.

[0100] The term "fragment" applied to polypeptides will be understood to mean an amino acid sequence that is shortened relative to the length of a reference polypeptide or amino acid sequence, and that comprises, is predominantly composed of, and / or consists of an amino acid sequence that is identical or nearly identical (e.g., 90%, 92%, 95%, 98%, 99% identity) to the reference polypeptide or amino acid sequence. Where appropriate, such polypeptide fragments according to the invention may be included in larger polypeptides in which they are components. In some embodiments, such fragments may comprise, predominantly composed of, and / or consist of, a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive amino acids of the length of the polypeptide or amino acid sequence according to the invention.

[0101] A "fusion protein" is a polypeptide generated when two heterologous nucleotide sequences or fragments thereof encoding two (or more) different polypeptides not found to fuse together in nature are fused together in the correct translation reading frame. Exemplary fusion polypeptides include the polypeptide (or fragment thereof) of the present invention fused with all or part of glutathione S-transferase, maltose-binding protein or reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitopes, etc.

[0102] The term "expression" for a polynucleotide coding sequence means that the sequence is transcribed and optionally translated. Typically, according to the invention, expression of the coding sequence of the invention will result in the production of the polypeptide of the invention. Fully expressed polypeptides or fragments can also function in intact cells without purification.

[0103] As used herein, the term "gene" refers to a nucleic acid molecule capable of producing mRNA, antisense RNA, miRNA, etc. Genes may or may not be used to produce functional proteins. Genes may include coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). Genes can be "isolated," meaning that the nucleic acid is substantially or essentially free of the components that are normally linked to nucleic acids in their natural state. These components include other cellular material, culture media from recombinant production systems, and / or the various chemicals used in the chemical synthesis of nucleic acids.

[0104] As used herein, “complementary” polynucleotides are those polynucleotides that can pair bases according to the standard Watson-Crick complementarity rules. Specifically, purines will pair bases with pyrimidines to form guanine and cytosine (G:C) combinations, and in the case of DNA, adenine pairs with thymine (A:T), or in the case of RNA, adenine pairs with uracil (A:U). For example, the sequence “AGT” binds to the complementary sequence “TCA”. It should be understood that two polynucleotides can hybridize even if they are not perfectly complementary, as long as each polynucleotide has at least one region that is substantially complementary to the other.

[0105] As used herein, the term "complementarity" or "complementarity" refers to the natural binding of polynucleotides through base pairing under permissible salt and temperature conditions. Complementarity between two single-stranded molecules can be "partial," where only some nucleotides bind, or it can be complete when there is perfect complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0106] As used herein, the terms "substantially complementary" or "partially complementary" mean that at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in two nucleic acid sequences are complementary. In some embodiments, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the nucleotides in the two nucleic acid sequences may be complementary. The terms "substantially complementary" or "partially complementary" can also mean that two nucleic acid sequences are capable of hybridizing under highly stringent conditions, and such conditions are well known in the art.

[0107] As used herein, "heterologous" refers to a nucleic acid sequence derived from another species or from the same species or organism but modified from its original form or the form primarily expressed in cells. Therefore, a nucleotide sequence derived from an organism or species different from the cell in which the nucleotide sequence was introduced is heterologous to that cell and its progeny. Furthermore, heterologous nucleotide sequences include nucleotide sequences of the same origin and insertion type as the native original cell type, but which exist in a non-natural state, such as having a different copy number, and / or being controlled by regulatory sequences different from those found in nature.

[0108] As used herein, the terms “contact,” “introduction,” and “application” are used interchangeably and refer to the process of delivering the conjugate product of the present invention or the polynucleotide of the present invention into cells to inhibit or alter or modify the expression of a target gene or cellular process. The conjugate product can be applied in a variety of ways, including but not limited to extracellular introduction into cavities, intercellular spaces, or introduction into the circulation of the organism.

[0109] In the context of cells or organisms, "introduction" refers to the presentation of a nucleic acid molecule to an organism and / or cell in a manner that allows the nucleic acid molecule to enter the cell. When more than one nucleic acid molecule is to be introduced, these nucleic acid molecules may be assembled as part of a polynucleotide or nucleic acid construct, or as different polynucleotides or nucleic acid constructs, and may be located on the same or different nucleic acid constructs. Thus, these polynucleotides may be introduced into the cell in a single transformation event or in different transformation events. Therefore, as used herein, the term "transformation" refers to the introduction of a heterologous nucleic acid into a cell. Cellular transformation can be stable or transient.

[0110] In the case of polynucleotides, "transient conversion" refers to the introduction of polynucleotides into cells without their integration into the cell's genome.

[0111] In the case of introducing a polynucleotide into a cell, "stable introduction" or "being stably introduced" means that the introduced polynucleotide is stably incorporated into the cell's genome, and thus the cell is stably transfected to have that polynucleotide.

[0112] As used in this article, "stable transformation" or "stable transformation" refers to the introduction of nucleic acid molecules into cells and their integration into the cellular genome. Therefore, the integrated nucleic acid molecules can be inherited by their offspring, and more specifically, by offspring across multiple generations. The term "genome" as used in this article includes both the nuclear genome and the mitochondrial genome, and therefore includes the integration of nucleic acids into, for example, the mitochondrial genome. Stable transformation as used in this article can also refer to transgenes that remain outside of chromosomes, for example, as miniature chromosomes.

[0113] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or protein blotting, which detects the presence of peptides or polypeptides encoded by one or more transgenes introduced into the organism. Stable transformation of cells can be detected, for example, by DNA blot hybridization of cellular genomic DNA having nucleotide sequences that specifically hybridize with the nucleotide sequences of the transgene introduced into the organism. Stable transformation of cells can be detected, for example, by RNA blot hybridization of cellular RNA having nucleic acid sequences that specifically hybridize with the nucleotide sequences of the transgene introduced into the organism. Stable transformation of cells can also be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions known in the art, using specific primer sequences that hybridize with the target sequence of the transgene, resulting in the amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols known in the art.

[0114] As used herein, a “transfection reagent” is any compound or molecule that enhances the delivery of nucleic acids into cells by contacting cells before and / or simultaneously with contacting nucleic acids, or by contacting nucleic acids before contacting cells. Transfection reagents do not covalently bind to nucleic acids.

[0115] A "therapeutic peptide" or "therapeutic nucleic acid" is a peptide or nucleic acid that can alleviate or reduce symptoms caused by a deficiency, insufficiency, excess, or loss of protein or nucleic acid in cells or a subject. Additionally, a "therapeutic peptide" may be a peptide that originally provides benefits to a subject, such as anti-cancer effects or improved graft survival.

[0116] As used herein, “RNAi” or “RNA interference” refers to a sequence-specific post-transcriptional gene silencing process mediated by double-stranded RNA (dsRNA). As used herein, “dsRNA” refers to partially or fully double-stranded RNA. Double-stranded RNA is also known as small interfering RNA (siRNA), small interfering nucleic acid (siNA), microRNA (miRNA), etc. In RNAi, dsRNA containing a first (antisense) strand complementary to a portion of the target gene and a second (sense) strand fully or partially complementary to the first antisense strand is introduced into the organism. After introduction, the target gene-specific dsRNA is processed into relatively small fragments (siRNA) and subsequently distributed throughout the organism, resulting in loss-of-function mutations in one generation that may have phenotypes very similar to those caused by complete or partial deletion of the target gene.

[0117] MicroRNAs (miRNAs) are non-protein-coding RNAs, typically ranging from about 18 to about 25 nucleotides in length. These miRNAs directly cleave trans-expressing target transcripts and negatively regulate gene expression involved in various regulatory and developmental pathways (Bartel, Cell 116:281-297 (2004); Zhang et al., Dev. Biol. 289:3-16 (2006)). Therefore, miRNAs have been shown to participate in various aspects of growth and development, as well as signal transduction and protein degradation. Since the first miRNAs were discovered in plants (Reinhart...), et al., Genes Dev. 16:1616-1626 (2002), Park et al.. Curr. Biol. 12:1484-1495 (2002)), hundreds of microRNA genes (MIR genes) have been identified. Many microRNA genes have been identified and are publicly available in databases (miRBase; microRNA.sanger.ac.uk / sequences). miRNAs are also described in U.S. Patent Publications 2005 / 0120415 and 2005 / 144669A1, the entire contents of which are incorporated herein by reference.

[0118] Genes encoding miRNAs produce primary miRNAs (called "pri-miRNAs") ranging in length from 70 bp to 300 bp, which can form imperfect stem-loop structures. A single pri-miRNA may contain one or more miRNA precursors. In animals, pri-miRNAs are processed in the nucleus into shorter hairpin RNAs (pre-miRNAs) of approximately 65 nt in length by the RNase III enzyme Drosha and its cofactors DGCR8 / Pasha. The pre-miRNAs are then exported to the cytoplasm, where they are further processed by another RNase III enzyme, Dicer, releasing miRNAs / miRNAs of approximately 22 nt in size. * Dimers. There are many reviews on microRNA biosynthesis and function; for example, see Bartel. Cell 116:281-297 (2004), Murchison et al., Curr. Opin. Cell Biol. 16:223-229 (2004), Dugas et al., Curr. Opin. Plant Biol. 7:512-520 (2004) and Kim, Nature Rev. Mol. Cell Biol. 6:376-385 (2005).

[0119] As used herein, when applied to polynucleotide or polypeptide sequences, the term "modified" refers to a sequence that differs from the wild-type sequence due to one or more deletions, additions, substitutions, chemical modifications, or any combination thereof.

[0120] As used in this article, “isolation” or “purification” (or grammatical equivalent) of a viral vector means that the viral vector is at least partially separated from at least some of the other components in the starting material.

[0121] The terms “treat,” “treating,” or “treatment of” (and their grammatical variations) refer to a reduction, at least partial improvement, or stabilization of the severity of a subject’s condition, and / or a reduction, relief, decrease, or stabilization of at least one clinical symptom, and / or a slowing of the progression of the disease or condition.

[0122] The terms “prevent,” “preventing,” and “prevention” (and their grammatical variations) refer to preventing and / or delaying the onset of a disease, symptom, and / or clinical symptom in a subject and / or reducing the severity of the onset of a disease, symptom, and / or clinical symptom relative to what would occur without the method of the present invention. Prevention can be complete, such as the complete absence of disease, symptom, and / or clinical symptom. Prevention can also be partial, such that the occurrence and / or severity of a disease, symptom, and / or clinical symptom in the subject is lower than what would occur without the present invention.

[0123] The term "therapeuticly effective" as used herein refers to a quantity sufficient to provide some improvement or benefit to the subject. In other words, a "therapeuticly effective" quantity is a quantity that provides some relief, reduction, decrease, or stabilization of at least one clinical symptom in the subject. Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as it provides some benefit to the subject.

[0124] As used herein, "preventive effectiveness" means an amount sufficient to prevent and / or delay the onset of a subject's disease, condition, and / or clinical symptoms and / or reduce and / or delay the severity of such onset relative to the severity of the onset of the subject's disease, condition, and / or clinical symptoms occurring without the method described in this invention. Those skilled in the art will understand that the level of prevention need not be complete, provided that the subject receives some benefit.

[0125] Coupled products One aspect of the present invention relates to a coupling product comprising: A polypeptide containing the epidermal growth factor receptor (EGFR) targeting portion; Connector; and Nucleic acid.

[0126] The polypeptide containing the EGFR targeting moiety can be any targeting moiety known in the art or subsequently identified. In some embodiments, the polypeptide comprises the amino acid sequence of dodecapeptide GE11 (YHWYGYTPQNVI (SEQ ID NO:1)) or a sequence that is at least 80% identical thereto (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), constitutes predominantly of said sequence, or is composed of said sequence. The GE11 sequence can be modified by any combination of addition, deletion, and / or substitution, and can include naturally occurring and non-naturally occurring amino acids.

[0127] The GE11 sequence or any other EGFR-targeting moiety can be modified to provide a reaction site for preparing the coupling product. In one embodiment, the peptide is modified to include a cysteine ​​residue at its C-terminus. For GE11, the additional cysteine ​​residue forms the sequence YHWYGYTPQNVIC (SEQ ID NO:2). In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:2 or a sequence that is at least 80% identical thereto (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), constitutes predominantly of said sequence, or is composed of said sequence.

[0128] In some implementations, the EGFR targeting moiety may be a carborane-containing macrocyclic peptide, such as CbaP5 and CbaP14 as described in Yin et al., J.Am. Chem. Soc. 141:19193 (2019), the entire contents of which are incorporated herein by reference.

[0129] The linker can be any linker suitable for covalently or nonvalently linking peptides and nucleic acids. In some embodiments, the linker is a pharmaceutically acceptable linker, such as, but not limited to, polyethylene glycol (PEG) linkers, reducible disulfide linkers, acid-labile oxime linkers, reactive oxygen species (ROS) sensitive borate ester linkers, peptide linkers, or hydrazone linkers. In some embodiments, the linker is a hexylamino linker coupled to a cleavable disulfide bond (e.g., succinimide 3-(2-pyridyldithio)propionate (SPDP)) or a non-cleavable linker (e.g., 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) or triethylene glycol (TEG)).

[0130] In some embodiments, the connector may comprise polyethylene glycol (PEG). In some embodiments, the connector may comprise dibenzocyclooctyn-PEG4-maleimide, consist primarily of dibenzocyclooctyn-PEG4-maleimide, or consist of dibenzocyclooctyn-PEG4-maleimide.

[0131] In some implementations, the peptide is covalently bound to the linker, for example, to a thiol group on a cysteine ​​residue.

[0132] Nucleic acids can be any nucleic acid that is intended to be introduced into cells, either in vitro or in vivo. Nucleic acids can be nucleic acids used for research or therapeutic purposes. Nucleic acids can be nucleic acids that can be used to modify (increase or decrease) the levels of nucleic acids or proteins in cells.

[0133] In some implementations, the nucleic acid is DNA, RNA, or a hybrid of DNA and RNA. In some implementations, the nucleic acid is double-stranded or single-stranded. In the case of single-stranded nucleic acid, it can be a sense strand or an antisense strand.

[0134] Nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions according to procedures known in the art. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or the physical stability of the double strand formed between the nucleic acid and the target nucleotide sequence (e.g., phosphate thioester derivatives and acridine-substituted nucleotides can be used). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl-Q nucleoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl Aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannose-Q nucleoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), weidingoside, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, nucleic acids can be generated using expression vectors containing nucleic acids that have been cloned and encode nucleic acids.

[0135] Nucleic acids may also comprise nucleotide sequences in which at least one or all of the internucleotide bridging phosphate residues are modified phosphate esters, such as methyl phosphonate, methyl thiophosphonate, morpholinate phosphate, piperazine phosphate, and aminophosphate. For example, each or every other internucleotide bridging phosphate residue may be modified as described. In another non-limiting example, the nucleic acid is a nucleotide sequence in which at least one or all nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). In another example, one or more nucleotides may be 2'-fluoronucleotides, 2'-O-methylnucleotides, or locked nucleic acid nucleotides. For example, each or every other nucleotide may be modified as described. See also Furdon. et al., Nucleic Acids Res. 17:9193 (1989); Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al., Nucleic Acids Res. 18:3537 (1990); Sprout et al., Nucleic Acids Res. 17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988); the entire contents of which are incorporated herein by reference for guidance on the preparation of polynucleotide molecules, including those containing modified nucleotide bases.

[0136] In some implementations, the nucleic acid may be selected from the group consisting of: siRNA, microRNA, shRNA, antisense nucleic acid, ribonuclease, cytotoxic tRNA, guide RNA, long noncoding RNA, antisense miRNA oligonucleotide, and plasmid DNA.

[0137] In one embodiment, the nucleic acid is a siRNA that silences KRAS or an antisense oligonucleotide, as described in U.S. Patent No. 10,619,159 or U.S. Publication No. 2020 / 0248185.

[0138] In some implementations, the adapter covalently binds to the nucleic acid. The nucleic acid can be modified to provide an adapter binding site, for example, by including an azide group as the binding site.

[0139] Another aspect of the invention relates to compositions comprising the conjugate product of the invention and a carrier. In some embodiments, the composition is a pharmaceutical composition comprising the conjugate product of the invention and a pharmaceutically acceptable carrier.

[0140] Another aspect of the invention relates to a method for increasing cellular uptake of nucleic acids, the method comprising conjugating the nucleic acid via a linker to a polypeptide containing an EGFR-targeting moiety to form a conjugated product, wherein the cells express EGFR, and wherein the uptake of the nucleic acid by the cells is increased relative to nucleic acids not conjugated to a polypeptide containing an EGFR-targeting moiety.

[0141] The nucleic acid, adapter, and EGFR targeting moiety can be any of the above. The conjugate can be prepared by any method known in the art and can be prepared as described above and in the examples.

[0142] How to use This document provides various methods of using the conjugates and / or compositions of the present invention. Therefore, one aspect of the invention relates to a method of delivering nucleic acids into cells, the method comprising contacting the cells with an effective amount of the conjugates or compositions of the present invention. The cells may be in vitro, ex vivo, or in vivo cells. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are selected from the group consisting of: non-small cell lung cancer cells, lung cancer cells, colon cancer cells, pancreatic cancer cells, and leukemia cells. In some embodiments, the cells express EGFR, for example, at a higher level of EGFR relative to other cells. In one embodiment, the cells are cancer cells that express EGFR at a higher level than non-cancer cells from the same subject or relative to the average level of EGFR found in the general population.

[0143] In some implementations, methods for increasing cellular uptake of nucleic acids do not include the use of transfection reagents separated from the conjugate.

[0144] Another aspect of the invention relates to a method of treating a disease in a subject who requires it, the method comprising administering to the subject a therapeutically effective amount of the conjugate or pharmaceutical composition of the invention, thereby treating the disease. The disease may be one in which diseased cells express EGFR, for example, at higher levels of EGFR relative to other cells. In some embodiments, the disease is cancer, for example, cancer selected from the group consisting of non-small cell lung cancer, lung cancer, colon cancer, pancreatic cancer, and blood cancer. In one embodiment, the cancer comprises a mutated human KRAS gene containing one or more missense mutations G12C, G12D, G12V, and G13D. Cancers containing a mutated human KRAS gene (containing one or more missense mutations G12C, G12D, G12V, and G13D) are cancers such as tumors in which one or more cells express the mutated KRAS gene.

[0145] In some implementations, the method of treating the disease does not include the use of a transfection reagent separated from the conjugate.

[0146] In one embodiment of each of these aspects, the subject may be a subject who has already been diagnosed with a disease (e.g., cancer). In another embodiment, the subject may be a person at risk of developing a disease (e.g., cancer) (e.g., susceptible due to genetic factors, smoking, viral infection, exposure to chemicals, etc.). In yet another embodiment, the subject may be a subject who has been identified as carrying a mutated KRAS gene and has been or has not yet been diagnosed with cancer.

[0147] The conjugates or compositions of the present invention can be delivered to cells by contact with cells using any method known in the art. In one embodiment, the conjugates or compositions of the present invention are administered directly to a subject. Typically, the conjugates of the present invention are resuspended in a pharmaceutically acceptable carrier (e.g., saline) and administered orally, topically, or via intravenous infusion, or by subcutaneous, intramuscular, intracranial, intrathecal, intraperitoneal, rectal, intravaginal, intranasal, intragastric, intratracheal, or intrapulmonary administration. Preferably, they are delivered directly to the site of disease or ailment, such as the lungs, intestines, or pancreas. The conjugates or implants can be delivered to the tumor by intratumoral injection or injection into the blood vessels supplying the tumor. The required dose depends on the chosen route of administration; the nature of the formulation; the nature of the patient's disease; the subject's body type, weight, surface area, age, and sex; other medications being administered; and the judgment of the attending physician. Suitable dose ranges from 0.01 μg / kg to 100.0 μg / kg. Given the varying efficiencies of different routes of administration, the required dose is expected to vary considerably. For example, oral administration is expected to require higher doses than intravenous administration (e.g., 2, 3, 4, 6, 8, 10 times; 20, 50, 100, 150, or more times). These dose levels can be optimized using standard empirical procedures, as is well known in the art. Administration can be a single or multiple administrations. Encapsulating the inhibitor in a suitable delivery carrier (e.g., polymeric microparticles or implantable devices) can improve delivery efficiency, particularly for oral delivery.

[0148] The conjugates or compositions of the present invention may optionally be delivered together with other therapeutic agents. Additional therapeutic agents may be delivered concurrently with the conjugates or compositions of the present invention. As used herein, the word “concurrently” means that they are close enough in time to produce a combined effect (i.e., concurrently can mean simultaneously, or it can mean two or more events occurring within a short time period before or after each other). In one embodiment, the conjugate or composition of the present invention is administered in combination with an agent for treating cancer, such as: 1) vinca alkaloids (e.g., vincristine, vinblastine); 2) epipodophyllotoxin (e.g., etoposide and teniposide); 3) antibiotics (e.g., daunorubicin (actinomycin D), doxorubicin (daunorubicin; erythromycin), doxorubicin, bleomycin, procainoxam (scintillans), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-α); 6) platinum coordination complexes (e.g., cisplatin and carboplatin); 7) anthraquinones (e.g., mitoxantrone); 8) substituted... 9) Ureas (e.g., hydroxyurea); 10) Methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH); 11) Adrenocortical inhibitors (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 12) Adrenocortical steroids (e.g., prednisone); 13) Progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 14) Estrogens (e.g., diethylstilbestrol and ethinylestradiol); 15) Anti-estrogens (e.g., tamoxifen); 16) Androgens (e.g., testosterone propionate and flumethasone); 17) Anti-androgens (e.g., flutamide); and 18) Gonadotropin-releasing hormone analogs (e.g., leuprorelin).In another embodiment, the compounds of the present invention are administered in combination with anti-angiogenic agents, such as anti-VEGF antibodies (e.g., bevacizumab (Avastin), ranibizumab (LUCENTIS) and other angiogenesis promoters (e.g., bFGF, angiopoietin-1), anti-α-v / β-3 angiotonic antibodies (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF-α conjugate, cyclophosphamide, compritillary A4 phosphate, dimethylflavone acetate, docetaxel, lenalidomide, enzatolin, Paclitaxel, albumin-bound nanoparticle paclitaxel formulation (Abraxane), soy isoflavones (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, valtarani base, everolimus, suramin, sunitinib malate, XL184, ZD6474, ATN-161, silengiptide, and celecoxib, or any combination thereof.

[0149] As used herein, the term "cancer" refers to any abnormal growth of cells, whether benign or malignant. Examples include, but are not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, kidney cancer, etc. Adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, piloblastic leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, idiopathic thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteosarcoma, primary macroglobulinemia, and retinoblastoma. In some implementation schemes, the cancer is selected from the group of tumor-forming carcinomas.

[0150] Pharmaceutical Composition Another aspect of the invention relates to pharmaceutical formulations and methods of administering them to achieve any of the therapeutic effects discussed above (e.g., treating cancer). Pharmaceutical formulations may comprise any of the agents discussed above in a pharmaceutically acceptable carrier.

[0151] "Pharmaceutical acceptable" means a material that is biologically or otherwise harmless, meaning that the material can be administered to a subject without causing any unwanted biological effects, such as toxicity.

[0152] The formulations of the present invention may optionally include a medicinal agent, a pharmaceutical agent, a carrier, an adjuvant, a dispersant, a diluent, etc.

[0153] The conjugates or compositions of the present invention can be formulated according to known techniques for administration in a drug carrier. See, for example, Remington. The Science And Practice of Pharmacy (9 th Ed. 1995). In the manufacture of pharmaceutical formulations according to the invention, the conjugate (including its physiologically acceptable salts) is typically mixed with a particularly acceptable carrier. The carrier may be solid or liquid, or both, and is preferably formulated together with the conjugate or composition into a unit-dose formulation, such as a tablet, which may contain 0.01% or 0.5% to 95% or 99% (by weight) of the conjugate or composition. One or more conjugates or compositions may be incorporated into the formulations of the invention, which may be prepared by any known pharmaceutical technique.

[0154] Another aspect of the invention is a method of treating a subject in vivo, comprising administering a pharmaceutical composition of a conjugate or composition of the invention contained in a pharmaceutically acceptable carrier to the subject, wherein the pharmaceutical composition is administered in a therapeutically effective amount. The conjugate or composition of the invention can be administered to human subjects or animals in need of it by any of the methods of compound administration known in the art.

[0155] Non-limiting examples of the formulations of this invention include those suitable for oral, rectal, oral (e.g., sublingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm and smooth muscle, intradermal, intravenous, intraperitoneal), local (i.e., skin and mucous membrane surfaces, including airway surfaces), intranasal, percutaneous, intra-articular, intracranial, intrathecal, and inhalation administration, intravenous delivery to the liver via portal vein, and direct organ injection (e.g., injection into the liver, into a limb, into the brain or spinal cord for delivery to the central nervous system, injection into the pancreas, or injection into a tumor or tissue surrounding a tumor). In any given case, the most suitable route will depend on the nature and severity of the condition being treated, and the nature of the particular conjugate being used. In some embodiments, local delivery of the formulation may be necessary to avoid any side effects associated with systemic administration. For example, local administration can be accomplished by direct injection at the desired treatment site, or by intravenous injection at a site near the desired treatment site (e.g., into a blood vessel supplying the treatment site). In some embodiments, the formulation can be locally delivered to ischemic tissue. In some implementations, the formulation may be a sustained-release formulation, for example, in the form of a sustained-release library.

[0156] For injection, the carrier is typically a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate buffer, antimicrobial water, or Cremophor EL (BASF, Parsippany, NJ). For other administration methods, the carrier can be solid or liquid.

[0157] For oral administration, the conjugate can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The conjugate can be encapsulated in gelatin capsules along with inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Other inactive ingredients may be added to provide desired color, taste, stability, buffering capacity, dispersibility, or other known desired properties; examples of such inactive ingredients are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide continuous release of the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air, or compressed tablets can be enteric-coated for selective disintegration in the gastrointestinal tract. Oral liquid formulations may contain colorings and flavorings to increase patient acceptance.

[0158] Formulations suitable for oral (sublingual) administration include lozenges containing conjugates in a flavoring matrix (typically sucrose and gum arabic or tragacanth); and palstilles containing conjugates in an inert matrix such as gelatin and glycerin or sucrose and gum arabic.

[0159] The formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injectable solutions of a conjugate, preferably isotonic with the blood of the intended recipient. These formulations may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickeners. The formulations may be provided in single / dose or multi-dose containers, for example, in sealed ampoules and vials, and may be stored under lyophilized conditions requiring only immediate addition of a sterile liquid carrier, such as saline or water for injection, prior to use.

[0160] Temporary injectable solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. For example, in one aspect of the invention, an injectable, stable, sterile composition comprising the conjugate of the invention is provided in a sealed container in unit dose form. The conjugate or salt is presented in lyophilized form, which is reconstituteable with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. Unit dosage forms typically contain about 10 mg to about 10 g of the conjugate or salt. When the conjugate or salt is substantially insoluble in water, a sufficient amount of a pharmaceutically acceptable emulsifier can be used to emulsify the conjugate or salt in an aqueous carrier. Phosphatidylcholine is such a useful emulsifier.

[0161] Formulations suitable for rectal administration are preferably presented as unit-dose suppositories. These can be prepared by mixing the conjugate with one or more conventional solid carriers (e.g., cocoa butter) and then shaping the resulting mixture.

[0162] Formulations suitable for topical application to the skin are preferably in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Carriers that can be used include petrolatum, lanolin, polyethylene glycol, alcohols, transdermal penetration enhancers, and combinations of two or more of these.

[0163] Formulations suitable for transdermal administration can be presented as discrete patches designed to maintain close contact with the recipient's epidermis for extended periods. Formulations suitable for transdermal administration can also be delivered via iontophoresis (e.g., see Tyle). Pharm Res. 3:318 (1986)), and is usually in the form of an optional buffered aqueous solution of the conjugate. Suitable formulations contain citrate or bis / tris buffer (pH 6) or ethanol / water, and contain 0.1 M to 0.2 M of the conjugate.

[0164] Alternatively, the conjugate can be formulated for nasal or other administration to the lungs of a subject, for example, via an aerosol suspension containing inhalable particles inhaled by the subject. The inhalable particles can be liquid or solid. The term "aerosol" includes any airborne suspension capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosols include airborne droplet suspensions that can be generated in a metered inhaler, nebulizer, or fogging machine. Aerosols also include dry powder compositions suspended in air or other carrier gases, which can be delivered, for example, by blowing from an inhaler device. See Ganderton & Jones, Drug Delivery to the Respiratory Tract , Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). As those skilled in the art will know, liquid particulate aerosols containing couplings can be produced by any suitable means, such as using a pressure-driven aerosol sprayer or an ultrasonic sprayer. See, for example, U.S. Patent No. 4,501,729. Solid particulate aerosols containing couplings can also be produced using any solid particulate pharmaceutical aerosol generator with techniques known in the pharmaceutical field.

[0165] Alternatively, it can be administered locally rather than systemically, for example, in the form of a reservoir or sustained-release formulation.

[0166] Furthermore, this invention provides liposome formulations of the conjugates and their salts disclosed herein. Techniques for forming liposome suspensions are well known in the art. When the conjugate or its salt is a water-soluble salt, it can be incorporated into lipid vesicles using conventional liposome techniques. In this case, due to the water solubility of the conjugate or salt, the conjugate or salt will be substantially encased within the hydrophilic center or core of the liposome. The lipid layer used can be any conventional composition and may or may not contain cholesterol. When the conjugate or salt of interest is water-insoluble, conventional liposome formation techniques are again used to substantially encase the salt within the hydrophobic lipid bilayer forming the liposome structure. In either case, the resulting liposomes can be reduced in size using standard sonication and homogenization techniques.

[0167] Liposome formulations containing conjugates disclosed herein or their salts may be lyophilized to produce lyophilized products that can be reconstituted with a pharmaceutically acceptable carrier (such as water) to regenerate the liposome suspension.

[0168] In the case of water-insoluble conjugates, pharmaceutical compositions containing water-insoluble conjugates can be prepared, for example, in an aqueous emulsion. In this case, the composition will contain a sufficient amount of a pharmaceutically acceptable emulsifier to emulsify the desired amount of conjugate. Particularly useful emulsifiers include phosphatidylcholine and lecithin.

[0169] In a particular implementation, a therapeutically effective amount of the conjugate is administered to the subject, as defined above. The dosage of the pharmaceutically active compound can be determined by methods known in the art, see, for example... Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa). The therapeutically effective dose of any specific compound will vary from compound to patient and will depend on patient condition and route of delivery. As a general recommendation, doses of approximately 0.001 mg / kg to approximately 50 mg / kg will be therapeutically effective, all weights are based on the weight of the compound, including the use of saline. Higher levels of toxicity may limit intravenous doses to lower levels, such as up to approximately 10 mg / kg, all weights are based on the weight of the compound, including the use of saline. Oral administration may be administered at doses of approximately 10 mg / kg to 50 mg / kg. Typically, intramuscular injection may be administered at doses of approximately 0.5 mg / kg to 5 mg / kg. The specific doses for intravenous or oral administration of the compound are approximately 1... mol / kg to 50 mol / kg, and more specifically about 1 mol / kg. mol / kg to approximately 22 mol / kg and about 1 mol / kg to 33 mol / kg.

[0170] In a particular embodiment of the invention, a therapeutic effect can be achieved by applying the medication more than once (e.g., twice, three times, four times or more) at various time intervals (e.g., every hour, every day, every week, every month, etc.).

[0171] This invention can be used in veterinary and medical applications. Suitable subjects include birds and mammals, preferably mammals. The term "birds" as used herein includes, but is not limited to, chickens, ducks, geese, quails, turkeys, and pheasants. The term "mammals" as used herein includes, but is not limited to, humans, cattle, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects include newborns, infants, adolescents, and adults. In other embodiments, subjects are animal models of diseases such as cancer. In some embodiments, subjects have a disease or are at risk of developing a disease, such as cancer.

[0172] The following examples are not intended to limit the scope of the claims to the invention, but are intended as examples of certain embodiments. Any variations of the exemplary methods that would occur to those skilled in the art are within the scope of the invention. As will be understood by those skilled in the art, each aspect of the claimed invention has several embodiments and elements, and all combinations of different elements are contemplated herein; therefore, the specific combinations illustrated herein should not be construed as limiting the scope of the claimed invention. If a particular element is removed or added to a group of elements available in the combination, that group of elements will be interpreted as incorporating such a change.

[0173] Example 1 Development of nucleic acid conjugates method Flow cytometry: For EGFR expression analysis, detached LU65 or HCT116 cells were resuspended in PBS to form a single-cell solution. Fluorescently conjugated EGFR or control IgG antibody was added to an equal volume of the cell solution, and the cells were incubated on ice in the dark for 30 minutes. Excess antibody was washed away from the cells. The samples were then run on a flow cytometer to detect fluorescence signals and identify the proportion of antibody-labeled positive cells.

[0174] To assess free siRNA uptake, fluorescently labeled siRNA (uncoupled or coupled with GE11) was incubated with LU65 or HCT116 cells at specified time points. Cells were then washed with PBS to remove excess siRNA and fixed in 2% paraformaldehyde. Samples were then run on a flow cytometer to detect fluorescence signals and identify the proportion of cells that took up fluorescent siRNA.

[0175] GE11 Synthesis and siRNA Conjugation: GE11 was synthesized using a solid-phase peptide synthesis method. Figure 4 An additional cysteine ​​amino acid was added to the end of the peptide chain (GE11C). The free thiol group on the c-terminal cysteine ​​reacted with a dibenzocyclooctylene (DBCO)-PEG4-maleimide reagent to generate the GE11C-DBCO-PEG4-maleimide conjugate. A copper-free click reaction was performed to conjugate the DBCO-containing product with azide-coupled siRNA to generate the final GE11C-siRNA conjugate. The product was then run using an RNAClean & Concentrator kit to remove excess uncoupled GE11 peptide.

[0176] Liquid chromatography / mass spectrometry (LC / MS): GE11-conjugated siRNA was analyzed by LC / MS to confirm successful peptide-siRNA conjugation. LC / MS was used to display the mass fractions of all substances present in the submitted sample.

[0177] mRNA expression analysis: HCT116 or LU65 cells were incubated with control or GE11-conjugated antisense KRAS siRNA at the specified dose for 48 hours. At 48 hours, the culture medium containing excess siRNA was removed, and cells were lysed in RNA lysis buffer. RNA was isolated from the lysed samples using an RNA isolation kit. RNA was then quantified using a Nanodrop spectrophotometer, and an equal volume of RNA from each sample was loaded into a cDNA synthesis reaction. The resulting cDNA was analyzed by quantitative PCR using primers for KRAS and "housekeeper" gene primers to normalize the results.

[0178] result Figure 1 The study demonstrated positive EGFR expression in hundreds of cancer cell lines derived from solid tumors, highlighting the potential of EGFR-mediated cancer targeting.

[0179] Figure 2 and Figure 3 The results showed that staining the lung cancer LU65 cell line or the colon cancer HCT116 cell line with EGFR antibodies (respectively) resulted in a rightward shift (or an increase) of fluorescence signal relative to cells stained with control IgG antibodies. This indicates high EGFR-positive expression in both cancer cell lines.

[0180] exist Figure 5 The presence of a mass product (8878) equal to the sum of the mass of GE11 (1642), the mass of the DBCO-PEG4-maleimide linker (674), and the mass of the siRNA antisense strand (6562) indicates the presence of a successfully coupled product.

[0181] Figure 6 The results showed that, when conjugated with GE11, EGFR-expressing cancer cells exhibited a significant increase in siRNA uptake over time compared to unconjugated siRNA. The siRNA used was Seq2-DV22, which targets KRAS. These data indicate that the presence of the GE11 peptide significantly enhances siRNA delivery to cancer cells.

[0182] Seq2-DV22 Sensitive chain (SEQ ID NO:3)

[0183] Antisense chain (SEQ ID NO:4)

[0184] 2'-O-methyl group on m-glycosyl group 2fl-2'-fluorine on the sugar group * - Phosphothiophosphate between nucleotides Figure 7 and Figure 8 This demonstrates robust knockout of target gene expression when treated with GE11-conjugated siRNA. The siRNAs used were Seq2-DV22 and Seq3-DV22, which target KRAS. Since no transfection reagent was used, siRNA entry into cells was entirely receptor-mediated uptake. These data provide a proof-of-principle that GE11 conjugation enables efficient and effective siRNA uptake, which retains functional activity upon internalization into the cell.

[0185] Seq3-DV22 Sensitive chain (SEQ ID NO:5) Antisense chain (SEQ ID NO:6)

[0186] Figure 9 and Figure 10 This indicates that in EGFR-expressing cancer cells (HCT116 colon cancer), GE11-conjugated Cy5-tagged siRNAs enter cells via receptor-mediated endocytosis. The siRNA used was Seq2-DV22, which targets KRAS.

[0187] Figure 11A-11B In vivo evidence of gene silencing using siRNA conjugated with GE11 was presented. HCT116 (KRAS G13D) tumors were established in mice and then treated with PBS or with an EGFR-targeting ligand (GE11) conjugated to the KRAS siRNA sequence with the indicated linker (5 mg / kg). The siRNA used was D2-G13D-Hi2F, which targets KRAS. We found that up to 50% to 70% of KRAS in the tumors were silenced at the indicated time point. Mice were also given a single dose of 5 mg / kg or 10 mg / kg or 5 days of 10 mg / kg (cumulative 50 mg / kg over 5 days) without any observable toxicity or weight loss.

[0188] D2-G13D-Hi2F Sensitive chain (SEQ ID NO:7)

[0189] Antisense chain (SEQ ID NO:8)

[0190] The foregoing is illustrative of the invention and should not be construed as limiting it. The invention is defined by the appended claims, including equivalents of the claims.

Claims

1. A coupling product comprising: a) A polypeptide containing the epidermal growth factor receptor (EGFR) targeting portion; b) Connector; and c) Nucleic acid.

2. The coupling product according to claim 1, wherein the polypeptide comprises GE11.

3. The coupling product according to any one of claims 1 or 2, wherein the polypeptide is modified to include a cysteine ​​residue at its C-terminus.

4. The coupling product according to any one of claims 1-3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

5. The coupling product according to any one of claims 1-4, wherein the connector comprises polyethylene glycol (PEG).

6. The coupling product according to any one of claims 1-5, wherein the connector comprises dibenzocyclooctylene-PEG4-maleimide.

7. The coupling product according to any one of claims 1-4, wherein the joint is a hexylamino joint coupled with a cleavable disulfide bond or a non-cleavable stalk.

8. The coupling product according to any one of claims 1-4, wherein the linker is succinimide 3-(2-pyridyldithio)-propionate, succinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid, or triethylene glycol.

9. The coupling product according to any one of claims 1-8, wherein the polypeptide is covalently bound to the linker.

10. The coupling product of claim 9, wherein the polypeptide is covalently bound to a thiol group on a cysteine ​​residue.

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