TW202200607A - Ribonucleoproteins for rna therapeutics delivery - Google Patents

Abstract

The present invention serves as a platform technology to deliver RNA therapeutics into cells. It provides a system for delivery of RNA molecules for biomedical purposes. The modular protein-based system described in this invention allows for customization of protein modules to achieve specificity in cell-targeting, thus having the ability to be optimized for treating different diseases. Examples of types of diseases that could adopt this technology for treatment include cancer, neurodegenerative diseases and viral infection.

Description

Ribonucleoproteins for ribonucleic acid drug delivery

The present invention relates to ribonucleoproteins for RNA drug therapy delivery. At the same time, the present invention provides the use of modified ribonucleoprotein complexes as RNA drug therapy delivery agents for gene therapy to treat various diseases.

RNA interference (RNAi) is a gene silencing process initiated by double-stranded RNA (dsRNA) in cells. RNAi plays an important role in cellular defense against viruses, which typically produce dsRNA during replication. The discovery of RNAi earned Craig Mello and Andrew Fire the 2006 Nobel Prize in Biomedicine. One of the main pathways of RNAi involves small interfering RNAs (siRNAs), short dsRNAs of about 21 nucleotides, which are generated by cleavage of longer dsRNAs (eg, shRNAs) via Dicer. The siRNA is unstripped into single-stranded RNA, one of which is loaded onto the RNA-induced silencing complex (RISC), which directs the degradation of the target RNA via sequence complementarity.

RNAi's ability to silence specific genes has prompted researchers to investigate its therapeutic potential in the treatment of cancer, viral infections and neurodegenerative diseases. However, siRNA delivery remains challenging. In order to effectively deliver siRNA drug therapeutics, several strategies have been devised to protect and stabilize siRNA and carry it into cells. By far, the most efficient approach is to use lipid-based carriers to encapsulate or bind siRNA and to pass it across cell membranes. This strategy has shown promise in clinical trials and is expected to be used in the treatment of hepatitis B, pancreatic cancer, hypercholesterolemia, and more. However, there are still some disadvantages including toxicity and eliciting a potential immune response. To protect siRNA from nuclease degradation, siRNA can be chemically modified, such as 2'O-methylation and 2'O-methyl phosphorodithioate. However, it has been reported that chemical modifications may reduce the effectiveness of siRNA. Since viruses are efficient gene delivery vehicles, viral vectors have also been designed as siRNA delivery agents. Despite their potential, viral vectors carry a high risk of eliciting an immune response.

Other proposed methods include siRNA coupling (e.g. polyethylene glycol (PEG), aptamer, cholesterol, N-acetylgalactosamine (Gal-NAc)) and the use of exosomes, inorganic materials and proteins as vector. In addition, N-acetylgalactosamine (GalNAc), which binds to hepatocyte receptors, has been successfully targeted for ligand-conjugates of the liver. Alnylam Pharmaceuticals, which developed the drug Patisiran, pioneered the use of GalNAc conjugates for siRNA delivery. Thus, siRNA delivery technology for the liver has made significant progress relative to delivery to other organs. Effective delivery of RNA therapeutics to organs other than the liver appears to be the current bottleneck for RNA therapy.

Therefore, an object of the present invention is to provide an RNA therapy delivery agent using an RNA-binding protein as a delivery system for gene therapy of various diseases.

In a first aspect, there is provided a method of delivering RNA into a cell, the method comprising: providing a modified nucleoside ribonucleoprotein (snRNP) complex into the cell, the modified nucleoribonucleoprotein (snRNP) complex The thing includes a core, wherein the core includes: one or more RNA molecules; one or more Sm proteins, or one or more LSm proteins, or any combination of Sm and LSm proteins, or any variant; and an Sm binding sequence, wherein the Sm binding sequence is linked to RNA, wherein the RNA binds to at least one Sm protein or at least one LSm protein, or any combination of modified snRNPs or variants thereof, and Wherein, at least one cellular receptor ligand is linked to at least one Sm protein or at least one LSm protein or any combination or variant thereof in the modified snRNP.

In a first embodiment of the first aspect, the one or more Sm proteins comprise SmD3, SmF, SmB, SmG, SmE, SmD1, and SmD2 in SEQ ID NOs: 1-7, respectively, or variants thereof, comprising SEQ ID NOs: 1-7 SmB' in ID NO: 9 and SmD1' in SEQ ID NO: 10.

In a second embodiment of the first aspect, the LSm proteins comprise LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10 and LSm11 in SEQ ID NOs: 11-20, respectively.

In a third embodiment of the first aspect, the cellular receptor ligand for receptor-mediated endocytosis includes epidermal growth factor (EGF) and any family member thereof, wherein EGF or any family member thereof is linked to SmD2 .

In a fourth embodiment of the first aspect, the RNA is part of a small hairpin ribonucleoprotein complex (shRNP) comprising 6-FAM attached to its 5' end Fluorescently labeled shRNA of KRA S of the target SEQ ID NO: 27, wherein, the shRNA of the egfp of the target SEQ ID NO: 28 is connected with DY547 dye at its 5' end.

In a fifth embodiment of the first aspect, the Sm binding sequence is attached to the RNA at its 3' or 5' end.

In a sixth embodiment of the first aspect, a cell receptor ligand is attached to the N-terminus, C-terminus or at the 3-strand 3 of said Sm protein or said LSm protein or any variant thereof and 4 in the ring.

In a seventh embodiment of the first aspect, the one or more RNA molecules comprise small nuclear RNA.

In an eighth embodiment of the first aspect, the Sm binding sequence is one of SED ID Nos: 21-26.

In a second aspect of the present invention, there is provided a method of silencing a gene in a cell, comprising using a modified small nuclear ribonucleoprotein (snRNP) complex to deliver RNA containing shRNA and siRNA into the cell, the snRNP The complex includes a core that includes: one or more RNA molecules; one or more Sm proteins, or one or more LSm proteins, or any combination of Sm and LSm proteins, or any variant thereof; and an Sm binding sequence, wherein the Sm binding sequence is linked to the shRNA or siRNA, and wherein the shRNA or siRNA binds to at least one Sm protein or at least one LSm protein or any combination or variant thereof of the modified snRNP, and Wherein, at least one cellular receptor ligand is linked to at least one Sm protein or at least one LSm protein or any combination or variant thereof in the modified snRNP.

In a first embodiment of the second aspect, the one or more Sm proteins comprise SmD3, SmF, SmB, SmG, SmE, SmD1 and SmD2 of SEQ ID NOs: 1-7, respectively, or a variant thereof, the variant comprising SmB' in SEQ ID NO:9 and SmD1' in SEQ ID NO:10.

In a second embodiment of the second aspect, the LSm protein comprises LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10 and LSm11 of SEQ ID NOs: 11-20.

In a third embodiment of the second aspect, the at least one cell receptor ligand comprises epidermal growth factor (EGF) and any family member thereof, and wherein the EGF or any family member thereof is attached to SmD2.

In a fourth embodiment of the second aspect, the shRNP comprises a shRNA targeting KRAS of SEQ ID NO: 27 with a 6-FAM fluorescent tag attached to its 5'-end, and a targeting SEQ ID NO:28 eGFP shRNA with DY547 dye attached to its 5' end.

In a fifth embodiment of the second aspect, the Sm binding sequence is linked to the 3' or 5' end of the shRNA or siRNA.

In a sixth embodiment of the second aspect, the cell receptor ligand is attached to the N-terminus, C-terminus or the loop between said strands 3 and 4 of any one Sm protein or any one Sm protein or any variant Inside.

In a seventh embodiment of the second aspect, the one or more RNA molecules comprise small nuclear RNA.

In an eighth embodiment of the second aspect, the Sm binding sequence is one of SEQ ID NOs: 21-26.

Other aspects of the invention include providing gene therapy to treat a disease in a subject, comprising using a method according to the first or second aspect of the invention.

In any aspect of the invention, subjects receiving gene therapy according to the methods described herein include humans or other animals.

Those skilled in the art will appreciate that the invention described herein may be subject to changes and modifications in addition to those specifically described.

The present invention includes all such changes and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of these steps or features.

Throughout the specification, unless the context requires other explanations, the word "comprise" or variations, such as "comprises" or "comprising", can be understood as including the A reference to a whole or group of wholes does not exclude any other whole or group of wholes. It should also be noted that in this disclosure, and particularly in the scope and/or paragraphs of the claims, certain terms such as "comprises", "comprised", "comprising" and similar terms , may have meanings ascribed to U.S. patent law. For example, these terms may mean "includes", "included", "including" and similar terms; and such as "consisting essentially of" and "substantially consisting of" Terms such as "consists essentially of" have the meanings described in US patent law. For example, they allow elements not expressly recited, but exclude elements that are available in the prior art or that would affect the essential or novel characteristics of the invention.

In addition, in the entire specification and the scope of the patent application, unless the context requires other interpretations, the word "include" or its variations, such as "includes" or "including", may It is understood to include a whole or group of wholes mentioned, but not to exclude any other whole or group of wholes.

Other definitions of alternative terms used herein can be found in the Detailed Description of the Invention and translated throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as the subject matter commonly understood by one of ordinary skill in the art to which this invention belongs.

Other aspects and advantages of the present invention will be apparent to those of ordinary skill in the art upon review of the ensuing description.

The present invention serves as a platform technology for delivering RNA therapy delivery agents into cells (Figure 1). RNA therapy has huge application potential in the field of biotechnology. For example: gene silencing to treat chronic diseases and cancer, RNA-based inhibitors, and RNA-based vaccines. This invention provides an RNA molecule delivery system for the purpose of biomedicine. The modular protein-based system of the present invention allows for the customization of protein modules to facilitate specificity for cellular targets. Therefore, it can be used to modulate and treat different diseases. The types of diseases that can be treated with this technology include: cancer, neurodegenerative diseases and viral infections.

The present invention can also be used for RNA delivery for research purposes, for cell line and in vivo studies.

Modular RNA delivery agents are constructed using modified components of the small nuclear ribonucleoprotein (snRNP) complex. In one embodiment, the U4 snRNP is modified to form an RNA delivery agent. U4 snRNP is one of these snRNPs and can be obtained from eukaryotes; examples of other snRNPs can be used as other embodiments of the present invention. Including but not limited to: U1 snRNP, U2 snRNP, U5 snRNP, U6 snRNP, U7 snRNP, and Lsm1-7 loop, etc. U4 snRNP is a spliceosome that can tailor pre-messenger RNAs. Current snRNP complexes contain Sm proteins or LSm proteins, a core consisting of one or more RNA molecules, one of which contains an Sm-binding sequence, while the remaining RNAs can be fully or partially double-stranded or wild-type type or modified RNA. For example, the U4 snRNP core may consist of one 144 nucleotide long U4 small nuclear RNA (snRNA) (SEQ ID NO: 35) and seven Sm proteins. Seven of the Sm proteins are named SmD1 (SEQ ID NO: 6), SmD2 (SEQ ID NO: 7), SmG (SEQ ID NO: 4), SmE (SEQ ID NO: 5), SmF (SEQ ID NO: 5) 2), SmD3 (SEQ ID NO: 1) and SmB (SEQ ID NO: 3), or a variant thereof comprising SmB' (SEQ ID NO: 9) and SmD1' (SEQ ID NO: 10). Other snRNPs consist of various combinations of Sm proteins and Sm-like (LSm) proteins. All Sm and LSm proteins are from the same Sm fold family, have similar structures and form oligomeric ring structures. In other embodiments, U7 snRNP consists of LSm11 (SEQ ID NO: 20), LSm10 (SEQ ID NO: 19), SmG, SmE, SmF, SmD3 and SmB or SmB'; U6 snRNP consists of LSm2 (SEQ ID NO: 19) 12), LSm3 (SEQ ID NO: 13), LSm4 (SEQ ID NO: 14), LSm5 (SEQ ID NO: 15), LSm6 (SEQ ID NO: 16), LSm7 (SEQ ID NO: 17), and LSm8 (SEQ ID NO: 18); the LSm1-7 loop consists of LSm1 (SEQ ID NO: 11), LSm2, LSm3, LSm4, LSm5, LSm6 and LSm7. The Sm protein of U4 snRNP forms a doughnut-shaped ring around the Sm-binding site of U4 snRNA, as shown in the crystal structure (Fig. 2A). In order for one or more RNA molecules to form a complex with the Sm protein of the snRNP, the corresponding Sm binding sequence is linked to the RNA. Such possible junctions are exemplified below and are not limited to the 3' end or the 5' end of the RNA. In one embodiment, the Sm binding sequence of U4 snRNP (5'-AAUUUUUGA-3') (SEQ ID NO: 23) is linked to RNA such that the RNA binds to the modified U4 snRNP protein. Other snRNPs have variants in the Sm-binding sequence, as well as variant Sm proteins. Example: U1 snRNP (SEQ ID NO: 21), U2 snRNP (SEQ ID NO: 22), U5 snRNP (SEQ ID NO: 24), U6 snRNP (SEQ ID NO: 25), U7 snRNP (SEQ ID NO: 26 ) of the Sm-binding sequence. Attachment of cellular receptor ligands to a Sm protein makes uptake by cells via endocytosis. For example, epidermal growth factor (EGF) is attached to SmD2. EGF binds to the EGF receptor (EGFR) on the cell surface to facilitate the endocytic uptake of RNA delivery agents. Attachment of other ligands for receptor-directed endocytosis are other embodiments of the invention, including, but not limited to, attachment to other members of the epidermal growth factor family. For example: neuregulin-1, neuregulin-2, neuregulin-3, neuregulin-4, amphiregulin, epiregulin, epigen ), betacellulin, transforming growth factor-𝛼 (transforming growth factor-𝛼), etc. Members of the EGF family have similar structures and can bind to EGFR. Examples of possible points for ligand attachment are, without limitation, the N-terminus of any Sm/LSm protein, the C-terminus of any Sm/LSm protein, and within the loop between strands 3 and 4 of any Sm/LSm protein. The loop between 𝛽 strands 3 and 4 can be modified to optimize RNA protection against RNA degradation by nucleases. In nature, insertion of domains or long loops occurs at these three positions and does not affect their ability to form loops with other Sm/LSm proteins. (Example: LSm11 of U7 snRNP has a domain at the N-terminus of the Sm core and a long loop insertion between 𝛽 strands 3 and 4). In one embodiment of the present invention, EGF is attached to the C-terminus of SmD2 protein to form SmD2-EGF (SEQ ID NO: 8), and the Sm binding sequence is attached to the 5' end of small hairpin RNA (shRNA). The shRNA delivery agent is called the "small hairpin ribonucleoprotein complex" (shRNP) (Fig. 2B). Since RNAs containing Sm-binding sequences bind to Sm proteins, linking this short RNA sequence to various forms of RNA molecules (e.g. siRNA, saRNA, mRNA) should form ribonucleoprotein complexes with Sm proteins for delivery . Accordingly, various forms of RNA molecules are further embodiments of the present invention. In one embodiment of the present invention, shRNP is successfully recombined and shRNA is delivered into cells for gene silencing. Example

Expression and Purification SmD1/SmD2-EGF

The structural code for SmD1 (synthesized by Genscript) was cloned into the pET28a-sumo vector, which encodes an N-terminal six histidine tag followed by a sumo tag. A construct encoding a SmD2-epidermal growth factor (SmD2-EGF) fusion protein was synthesized (Genscript) and cloned into MCS2 of pCDFDuet-1. The two plastids were co-transformed in E. coli Rosetta (DE3) and the cells were grown at 37°C in 2xYT medium containing 50 μg/ml kanamycin, 100 μg/ml streptavidin Streptomycin and 34 μg/ml chloramphenicol until OD ₆₀₀ reached 0.6-0.8. Cells were then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM and incubated at 20°C for 18 hours. Next, cells were collected by centrifugation. Resuspend the cell pellet in lysis buffer (20 mM Tris, pH 7.5, 1 M NaCl (sodium chloride), 10 mM imidazole, 5% (v/v) glycerol, 10 mM β- mercaptoethanol (BME) and 17.8 μg/mL phenylmethylsulfonyl fluoride (PMSF), and lysed cells by sonication. Cell lysates were cleared by centrifugation and loaded onto HisTrap HP columns (GE Healthcare). The column was washed with 20 column volumes (CV) of wash buffer (20 mM Tris, pH 7.5, 1 M NaCl, 40 mM imidazole) followed by elution buffer (20 mM Tris, pH 7.5, 1 M NaCl) , 500 mM imidazole) eluted. The eluted protein was diluted 3-fold with buffer solution A (20 mM HEPES, pH 7.5, 5 mM DTT). Afterwards, further purification was performed using a 5 ml HiTrapHeparin column (GE Healthcare) that had been equilibrated with 85% buffer solution A and 15% buffer solution B (20 mM HEPES, pH 7.5, 2 M NaCl, 5 mM DTT). Proteins were eluted with buffer B using a gradient from 15% to 100%. The relevant fractions were concentrated and stored at -80°C.

Expression and Purification SmD3/SmB

The construct for N-terminal six histidine-tagged SmD3 was cloned into pET28a, and the construct for SmB with only residues 1-95 (SmB _1-95 ) was cloned into MCS2 of the pCDFDuet-1 vector . The two plastids were then cotransformed into E. coli BL21(DE3) cells and grown at 37°C in 2xYT medium supplemented with 50 μg/ml kanamycin and 100 μg/ml streptomycin. Expression was induced with a final concentration of 0.5 mM IPTG when the _OD600 reached 0.6-0.8. Cells were grown at 20°C for 18 hours. Cells were harvested by centrifugation and cell pellets were resuspended in lysis buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 10 mM BME and 17.8 μg/mL PMSF) and Cells were lysed by sonication. Cell lysates were cleared by centrifugation and loaded onto 5 ml HisTrap HP columns (GE Healthcare). The column was then washed with 20x CV of wash buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole) followed by elution buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 500 mM) imidazole) eluted. Eluted proteins were further purified using 5 ml HiTrap Heparin tubes (GE Healthcare) that had been pretreated with 60% buffer solution A (20 mM HEPES, pH 7.5 and 5 mM DTT) and 40% buffer solution B (20 mM HEPES, pH 7.5, 1 M NaCl, 5 mM DTT) for equilibration. Proteins were eluted using buffer A using a gradient of 40% to 100% buffer B. The relevant fractions were concentrated and stored at -80°C.

Expression and purification SmG/SmE

The SmG construct with six histidine tags at the C-terminus was cloned into pET26b, and SmE was cloned into MCS1 of the pCDFDuet-1 vector. The two plastids were then co-transformed into E. coli BL21(DE3) cells at 37°C in 2xYT supplemented with 50 μg/ml kanamycin, 100 μg/ml streptomycin, and 34 μg/ml chloramphenicol grown in medium. When the _OD600 reached 0.6-0.8, cells were induced by adding a final concentration of 0.5 mM IPTG and grown at 20 °C for 18 h. Cells were harvested and complexes were purified as described above for the His-SmD3/SmB 1-95 complex.

In vivo recombination

Combine 2 molar equivalents of SmD3/SmB, SmG/SmE, and SmD1/SmD2-EGF complexes in 500 μL of reconstitution buffer (20 mM HEPES, pH 7.5, 750 mM NaCl, 5 mM ethylenediaminetetraaceticacid (EDTA), and 5 mM DTT) Mixed with 1 molar equivalent of shRNA (synthesized by Dharmacon, Horizon Discovery). Before mixing, the shRNA was re-adhered by heating to 90°C for 5 minutes and then rapidly cooling on ice for 10 minutes. The RNA/protein mixture was incubated at 30°C for 30 minutes, followed by 15 minutes at 37°C, and then cooled on ice for 10 minutes. The SUMO tag on SmD1 was removed by overnight incubation with ULP1 protease at 4°C. Recombinant complexes were purified by particle size chromatography (Superdex® 200 increase 10/300 GL column, GE Healthcare) in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM EDTA and 5 mM DTT. Relevant fractions were pooled, concentrated and stored at -80°C.

The shRNA sequences are as follows: 1) shRNA targeting KRAS (SEQ ID NO: 27) 5'-6-FAM- CAA UUU UUG ACC UUG ACG AUA CAG CUA AUU CCU CGA GGA AUU AGC UGU AUC GUC AAG G -3' 2) shRNA targeting egfp (SEQ ID NO: 28) 5'-DY547- CAA UUU UUG AGC AAG CUG ACC CUG AAG UUC ACU CGA GUG AAC UUC AGG GUC AGC UUG C -3'

Cell Lines and Media

Culture human lung adenocarcinoma cell line A549 at 37 °C, 5% _CO . and in RPMI-1640 medium (Gibco, Life Technologies) containing 2 g/L sodium bicarbonate (Sigma-Aldrich), supplemented with penicillin and streptomycin (Gibco, Life Technologies) and 10% fetal bovine Serum (Gibco, Life Technologies).

The human embryonic kidney cell line 293T with endogenous green fluorescent protein expression (293T _- eGFP) was maintained in DMEM, high glucose (Gibco, Life Technologies) with 3.7 g/ L sodium bicarbonate, supplemented with penicillin and streptomycin, 5% fetal bovine and 5% neonatal bovine serum (Gibco, Life Technologies).

gene expression analysis

A549 cells were seeded in 12-well flat-bottom tissue culture plates (Falcon BD) and cultured overnight. Supplement the medium with a final concentration of 123 nM (each) of protein subcomplexes (His-SUMO-SmD1/SmD2-EGF, SmD3/SmB1-95, SmG-His/SmE), 123 nM shRNP, and buffer solution is negative control. and incubated at 37°C, 5% _CO . After 4 days, total RNA was isolated using RNAzol RT (Molecular Research Center) and reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega) according to the experimental protocol provided by the manufacturer. Real-time polymerase chain reaction was performed on a StepOnePlus Real-Time PCR instrument (Applied Biosystems) using the synthesized cDNA and TB Green Premix Ex Taq II (Takara). The sequences of primers used in RT-PCR are as follows: KRAS 1 pre-primer (SEQ ID NO: 29) and inverse primer (SEQ ID NO: 30), KRAS 2 pre-primer (SEQ ID NO: 31) and inverse primer primer (SEQ ID NO: 32), GAPDH pre-primer (SEQ ID NO: 33) and reverse primer (SEQ ID NO: 34). Relative levels of KRAS expression were calculated using the ∆∆CT method with GAPDH as a normalized control.

cell viability assay

A549 cells were seeded in 96-well flat-bottom tissue culture plates (Nunc) and cultured overnight. Supplement the medium with final concentrations of 123 nM and 246 nM (each) of protein subcomplexes (His-SUMO-SmD1/SmD2-EGF, SmD3/SmB1-95, SmG-His/SmE), 123 nM and 246 nM shRNP, and buffer solution were negative controls and were incubated at 37°C, 5% _CO . After 3 days, with the addition of MTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide, 0.5 mg/ml, Sigma-Aldrich) Medium was replaced with medium and incubated for 2 hours. The ligand was then removed, and the formed formazan crystalline salt was dissolved with 100 μL of dimethyl sulfoxide (RCI-Labscan) while shaking for 30 minutes. Absorbance at 540 nm and reference absorbance at 690 nm were measured on a Biotek ELx800 Microplate Detector (Biotek). Calculate cell viability (%) according to the following formula: Cell viability (%) = (OD _{protein or shRNP complex} /OD _{buffer solution} ) × 100%

Conjugate Focus Microscopy

A549 cells were seeded in glass bottom tissue culture dishes and cultured overnight. 123 nM protein subcomplex, 123 nM shRNA, 123 nM shRNP and buffer solution were respectively added to the medium and incubated for 2 days, while the other was buffered only as a negative control. Before imaging, cells were co-stained with Hoechst 33342 nucleic acid stain (0.3ug/ml, Invitrogen). Images of fluorescently labeled cells were captured with a Nikon Eclipse Ti2 conjugated laser scanning microscope. The Hoechst 33342 signal is collected in the blue (λ _ex = 400 nm, λ _em = 410-480 nm) channel, while the 6-FAM signal is collected in the green (λ _ex = 491 nm, λ _em = 500-550 nm) channel .

293T-eGFP cells were seeded in glass bottom dishes and cultured overnight. 100 nM protein subcomplex, 100 nM shRNA, 100 nM shRNP and buffer solution were added to the medium and incubated for 2 days with buffer solution as negative control. Cells were co-stained with Hoechst 33342 nucleic acid stain prior to imaging. Images of fluorescently labeled cells were captured with a Nikon Eclipse Ti2 conjugated laser scanning microscope. The Hoechst 33342 signal was collected in the blue (λ _ex = 400 nm, λ _em = 410-480 nm) channel. The eGFP signal was collected in the green (λ _ex = 491 nm, λ _em = 510-540 nm) channel. The DY547 signal was collected in the green (λ _ex = 561 nm, λ _em = 570-600 nm) channel.

shRNP in vitro assembly of

The Sm protein can be engineered as an RNA delivery agent. The Sm protein is engineered from U4 snRNP, which can provide shRNA that targets KRAS gene knockout and is carried out by RNA interference. Recombinant proteins were co-expressed and purified for the subcomplex SmD1/SmD2-EGF complex, SmD3/SmB1-95 complex and SmG/SmE complex, respectively. Recombinant proteins were co-expressed and purified for the subcomplex SmD1/SmD2 EGF complex, SmD3/SmB _1-95 complex and SmG/SmE complex. The C-terminus of SmD2 is fused to epidermal growth factor (EGF) and acts as a ligand for the EGF receptor (EGFR), which can be taken up by cells through endocytosis. To improve the solubility of the SmD1/SmD2-EGF complex, SUMO was also used as a solubility tag and fused to the N-terminus of SmD1 for expression, which was subsequently removed by adding ULP1 protease during assembly. In order to make the shRNA targeting KRAS bind to the Sm protein, in the present invention, a Sm binding sequence is added to the 5' end of the shRNA. In addition, a 6-FAM fluorescent label was added to the 5' end of the shRNA so that it could be used to track the shRNA in cells.

The Sm protein subcomplex was mixed with shRNA, and the shRNP complex was purified by particle size chromatography (Figure 3). The 260/280 ratio of the purified complex was 1.5 (>1), indicating the presence of RNA. In this version of the shRNA complex, a 6-Sm protein-shRNA complex was obtained. And usually U4 snRNP contains 7 Sm proteins (excluding SmF). Next, the presence of these six Sm proteins was confirmed by SDS-PAGE analysis (Fig. 2).

cellular uptake

To investigate whether shRNP can be taken up by cells, it was tested on the A549 human lung adenocarcinoma cell line. A549 cells were K-Ras positive. Cells were grown in medium containing shRNP at a final concentration of 123 nM. As a control, cells were cultured in medium with corresponding amounts of Sm protein used to assemble shRNP complexes. Two days later, the medium was replaced with fresh medium and examined by conjugate focus fluorescence microscopy. Under a conjugate focus microscope, cells cultured with shRNP were observed to contain diffused FAM fluorescent signals in the cytoplasm, indicating that FAM-labeled shRNA had entered the cytoplasm (Figure 4). In the control with only Sm protein, only a small amount of background fluorescent signal was observed.

To further confirm that the observation of the protein-only control was only background noise, repeated tests were performed using shRNA (not complexed with Sm protein), and the protein-only and buffer solution controls. All of these control cells also emitted similar background speckle fluorescence when using FAM channels that did not spread on the cytoplasm (Figure 5), suggesting that the cytoplasmic fluorescent signal diffused in cells incubated with shRNP came from after entering the cells FAM-tagged shRNA (Figure 4). From these experiments it can be concluded that the shRNP complex is capable of delivering shRNA into cells.

KRAS gene expression analysis

To investigate whether shRNA targeting KRAS entering A549 cells in the form of shRNP could silence KRAS by RNA interference, and quantitative polymerase chain reaction (qPCR) was used to examine KRAS gene expression. A549 cells were first cultured with 123 nM shRNP, the corresponding amount of Sm protein and buffer control for 4 days, and then qPCR was performed on KRAS RNA using two sets of different primer pairs. The results of the qPCR study showed that both cells incubated with shRNP and Sm proteins had decreased KRAS RNA levels relative to the buffer control group (Figures 6A and 6B). The percentage change in KRAS expression of cells cultured with shRNP was 84% (primer pair 1) (Fig. 6A) and 74% (primer pair 2) (Fig. 6B) of the buffer control group. However, the results showed that the Sm protein-only samples showed a greater reduction in KRAS gene expression (55% for primer pair 1 and 50% for primer pair 2) compared to the buffer solution control group (Figures 6A and 6B). . Sm protein without shRNA was observed to decrease KRAS expression, but no additive effect when shRNA was present. It can only be speculated that there is an interference between the expression of the ligand EGF and KRAS , since K-Ras (the product of the KRAS gene) and EGFR are in the same cellular signaling pathway. To study the RNAi effects of shRNPs unperturbed by this, another target that is not directly affected by ligand and cellular receptor binding and activation is required. These results also provide new ideas for the optimal design of shRNPs to avoid the use of ligands that might interfere with the silenced target.

cell activity ( Cell Viability )

To investigate the effect of shRNP on the viability of A549 cells, A549 cells cultured with 123nM and 246nM shRNP, the corresponding Sm protein (without shRNA) and buffer solution control group were detected by MTT assay. After four days of culture, the MTT assay was performed again. Compared with the buffer solution control group (100%), the cell viability of cells incubated with 123 nM shRNP was 93.33%, while that of the protein-only control group was 108.55% (Fig. 7). For cells incubated with 246 nM shRNP, the activity was only 23.53% compared to the buffer-only control. However, the cell viability of the corresponding cells cultured with the protein control group was also significantly reduced, at 34.56%, indicating that the protein carrier may be toxic to A549 cells at higher concentrations. These results suggest that at lower concentrations of shRNP that are nontoxic to the protein carrier, there is only a slight negative effect on the survival of A549 lung cancer cells. It has been previously reported that only KRAS shRNA knockdown in A549 cells showed no significant reduction in cell viability (Singh et al., 2009, A Gene Expression Signature Associated with "K-Ras Addiction" Reveals Regulators of EMT and Tumor Cell Survival. Cancer Cell), supporting the observations described herein.

egfp as silent target

Due to the interference between EGFR activation by EGF ligand and KRAS gene expression, shRNP cannot be conclusively demonstrated to have an RNAi effect. To show that shRNAs delivering shRNPs into cells can lead to gene silencing, shRNPs carrying shRNAs targeting eGFP were assembled. Since the product of the eGFP gene is enhanced green fluorescent protein (eGFP), which produces green fluorescence, eGFP can act as a reporter gene; if eGFP gene silencing occurs, a reduction in green fluorescence is expected. In this assembly, the same Sm protein and modifications were used, including fusion with an EGF ligand at the C-terminus of SmD2. The designed shRNA contains the eGFP mRNA sequence fragment, and a Sm binding sequence is attached to its 5' end. The fluorescent marker has been changed to DY547 (red) so it does not interfere with the fluorescent signal from eGFP (green). Using a similar approach as described above, shRNP complexes containing the target eGFP were assembled and validated by particle size sieve chromatography, 260/280 ratio (1.5) and SDS-PAGE analysis (Figure 8).

100 nM shRNPs containing the target eGFP were incubated with 293T-eGFP cells expressing endogenous eGFP. Cells were set up to be incubated with each 100 nM shRNA-free Sm protein subcomplex (protein only), 100 nM shRNA only, and a buffer control group. After two days in culture, the medium was replaced with fresh medium and studied using a conjugate focus microscope (Figure 9). In the shRNP complex samples, a DY547 channel fluorescent signal was observed in the cytoplasm of most cells, but no DY547 signal was found in the protein-only and buffer solution controls. In the control group, most cells had an eGFP fluorescent signal. Notably, the number of cells with eGFP signal was significantly reduced in samples incubated with shRNP complexes compared to buffer control and protein-only controls. It was also observed in the shRNP complex samples that cells displaying DY547 fluorescent signal had no observable or reduced eGFP fluorescent signal, whereas cells without DY547 signal showed high intensity eGFP signal. These observations show that delivery of shRNP complexes containing the target eGFP into cells can result in a reduction in eGFP levels in cells. However, in the shRNA-only samples, DY547 signal was also present in some cells, but eGFP signal in these cells was not significantly reduced. This observation revealed that the expression of egfp was not silenced only in the shRNA samples. The DY547 signal observed in the shRNA-only samples can be attributed to the presence of DY547 fluorescent dye molecules isolated from degraded RNA. Cy3 dyes containing Cy3-tagged RNA may enter cells when RNA is degraded by nucleases. DY547 is an alternative to Cy3 and is structurally similar to Cy3. DY547 and Cy3 have a positive charge at neutral pH, which facilitates their entry into cells. Based on the lack of reduction in eGFP signal in cells stained with DY547 only in shRNA samples, it is inferred that the DY547 signal is from the DY547 dye and is the result of RNA degradation; it is likely that RNA did not enter the cells and therefore eGFP fluorescence was not reduced. It was concluded that shRNAs have gene silencing effects only when delivered via shRNPs. Industrial applicability

The present invention serves as a platform technology for delivering RNA therapy delivery agents into cells. Since RNA therapy delivery agents have enormous potential applications in biotechnology, such as gene silencing for chronic disease and cancer treatment, RNA-based inhibitors, and RNA-based vaccines, the present invention provides delivery for biomedical purposes A system of RNA molecules. The modular protein-based system described in the present invention allows for the customization of protein modules to achieve specificity for target cells, and thus has the ability to be optimized for the treatment of different diseases. The types of diseases that can be treated with this technology include cancer, neurodegenerative diseases and viral infections. The present invention can also be used for RNA delivery for research purposes, for cell line and in vivo studies.

none

The invention will become apparent from the ensuing description of the invention, taken in conjunction with the accompanying drawings, in which:

[Figure 1] Shows the barriers encountered in RNAi therapy and provides an overview of shRNA delivery and RNA interference. [Figure 2A] A partial model of the crystal structure of the U4 snRNP core is shown. [Fig. 2B] shows the design of the small hairpin ribonucleoprotein complex (shRNP). The N-terminus, C-terminus, and inserts between strands 3 and 4 of each Sm protein can be used for customized ligand fusions and protein modifications. [Figure 3] shows the SDS-PAGE analysis chart of particle size sieve chromatography (Superdex® 200 Increase) and shRNP-labeled KRAS . The UV absorption light at 280 nm (smooth line) and the UV absorption light at 254 nm (dashed line) were analyzed by SDS-PAGE and the corresponding wave fronts were fractionated and collected. [ Fig. 4 ] Shown is a conjugate focus microscope image of shRNP extracted in A549 cells. A549 cells were co-cultured with shRNP complexes (bottom panel) or with only the protein component (top panel). Nuclei stained with Hoerscht 33342 show signal (left); signal shown by FAM-labeled shRNA (middle); nuclei stained with FAM-labeled shRNA image overlay (right); ruler 100 μm. [Fig. 5] shows the background fluorescence value of A549 cells. A549 cells were co-cultured with buffer control only (top row), with only protein components (middle row), or only shRNA (bottom row). Nuclei after staining with Hoerscht 33342 showing signal (left column); signal after background fluorescence using FAM fluorescence channel (middle column); nuclear staining and FAM channel images superimposed (right column). Fluorescent background speckle signals were present in the three control groups, but did not appear to disperse into the cytoplasm. Ruler gauge 100 μm. [ FIG. 6A ] The PCR quantification of KRAS expressed by primer pair 1 is shown. [Fig. 6B] shows the PCR quantification of KRAS expressed by primer pair 2. [Fig. [ Fig. 7 ] Shows the results of detecting the viability of A549 cells under different treatments by MTT (cell viability assay). [Figure 8] shows the analysis chart of particle size sieve chromatography (Superdex® 200 Increase) and SDS-PAGE of shRNP -targeted eGFP. The UV absorption light at 280 nm (smooth line) and the UV absorption light at 254 nm (dashed line) were analyzed by SDS-PAGE and the corresponding wave fronts were fractionated and collected. [Fig. 9] shows the decrease of eGFP signal in eGFP-expressing HEK293T cells after shRNP extraction. HEK293T cells expressing eGFP were co-cultured with buffer control (top row), used to assemble the protein component of shRNP (second row), shRNA alone (third row), or shRNP complexes with target GFP (bottom row). . The shRNA is labeled with DY547 dye at the 5'-end. Nuclei stained with Hoerscht 33342 show signal (first column); eGFP signal (second column); DT547 or shRNA signal (third column); overlapping nuclear staining, signals for eGFP and DY547 (fourth column). In representative cells, both the DY547-shRNA signal and the reduced eGFP signal of the shRNP entry RNAi effect are indicated by arrows (fourth row). Ruler gauge 100 μm.

Claims (20)

A method of delivering RNA into cells, comprising: A modified nucleoside ribonucleoprotein (snRNP) complex is provided into a cell, the modified nucleoside ribonucleoprotein (snRNP) complex comprising a core, wherein the core comprises: one or more RNA molecules; one or more Sm proteins, or one or more LSm proteins, or any combination of Sm and LSm proteins, or any variant; and an Sm binding sequence, wherein the Sm binding sequence is linked to RNA, wherein the RNA binds to at least one Sm protein or at least one LSm protein, or any combination of modified snRNPs or variants thereof, and Wherein, at least one cellular receptor ligand is linked to at least one Sm protein or at least one LSm protein or any combination or variant thereof in the modified snRNP. The method for delivering RNA into cells of claim 1, wherein the one or more Sm proteins comprise SmD3, SmF, SmB, SmG, SmE, SmD1, and SmD2 in SEQ ID NOs: 1-7, respectively, or variants thereof, including SmB' in SEQ ID NO: 9 and SmD1' in SEQ ID NO: 10. The method for delivering RNA into cells as claimed in claim 1, wherein the LSm proteins comprise LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10 and LSm11 in SEQ ID NOs: 11-20, respectively . The method of delivering RNA into a cell of claim 1, wherein the cellular receptor ligand for receptor-mediated endocytosis includes epidermal growth factor (EGF) and any family member thereof, wherein, EGF or any member of its family is linked to SmD2. The method of delivering RNA into a cell of claim 1, wherein the RNA is part of a small hairpin ribonucleoprotein complex (shRNP) comprising a marker The shRNA of KRAS of target SEQ ID NO:27 is attached with 6-FAM fluorescent label at its 5' end, wherein, the shRNA of target SEQ ID NO:28 of egfp is connected with DY547 dye at its 5' end . The method of delivering RNA into a cell as claimed in claim 1, wherein the Sm binding sequence is attached to the 3' end or the 5' end of the RNA. The method of delivering RNA into a cell of claim 1, wherein the cellular receptor ligand is attached to the N-terminal, C - the end or within the loop between said 𝛽 strands 3 and 4. The method of delivering RNA into a cell of claim 1, wherein the one or more RNA molecules comprise small nuclear RNA. The method for delivering RNA into cells of claim 1, wherein the Sm binding sequence is one of SED ID NOs: 21-26. A method of silencing a gene in a cell, comprising utilizing a modified small nuclear ribonucleoprotein (snRNP) complex containing shRNA and siRNA RNA for delivery into the cell, the snRNP complex comprising a core comprising a : one or more RNA molecules; one or more Sm proteins, or one or more LSm proteins, or any combination of Sm and LSm proteins, or any variant thereof; and an Sm binding sequence, wherein the Sm binding sequence is linked to the shRNA or siRNA, and wherein the shRNA or siRNA binds to at least one Sm protein or at least one LSm protein or any combination or variant thereof of the modified snRNP, and Wherein, at least one cellular receptor ligand is linked to at least one Sm protein or at least one LSm protein or any combination or variant thereof in the modified snRNP. The method of claim 10, wherein the one or more Sm proteins comprise SmD3, SmF, SmB, SmG, SmE, SmD1 and SmD2 of SEQ ID NOs: 1-7, respectively, or variants thereof, The body includes SmB' in SEQ ID NO:9 and SmD1' in SEQ ID NO:10. The method of claim 10, wherein the LSm protein comprises LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10, and LSm11 of SEQ ID NOs: 11-20. The method of claim 10, wherein the at least one cell receptor ligand comprises epidermal growth factor (EGF) and any family member thereof, and wherein the EGF or any family member thereof is attached to SmD2. The method of claim 10, wherein the shRNP comprises a shRNA targeting KRAS of SEQ ID NO: 27, and a 6-FAM fluorescent tag is attached to its 5'-end, and a target SEQ ID NO :28 eGFP shRNA with DY547 dye attached to its 5' end. The method of claim 10, wherein the Sm binding sequence is linked to the 3' end or the 5' end of the shRNA or siRNA. The method of claim 10, wherein the cell receptor ligand is attached to the N-terminus, C-terminus or between the strands 3 and 4 of any Sm protein or any Sm protein or any variant within the ring. The method of claim 10, wherein the one or more RNA molecules comprise small nuclear RNAs. The method of claim 10, wherein the Sm binding sequence is one of SEQ ID NOs: 21-26. A method of providing gene therapy for treating a disease in a subject comprising using the method of any one of claims 1 to 9. A method of providing gene therapy for treating a disease in a subject comprising using the method of any one of claims 10 to 18.

Images