Patent Application
20230063922
The present invention relates to ribonucleoproteins for RNA therapeutic delivery and gene silencing. In particular, the present invention provides RNA therapeutic delivery agents using modified ribonucleoprotein complexes as the delivery system for gene therapy to treat a variety of diseases and incorporating endosomal escape peptides (EEP) to facilitate the delivered payload to silence desired genes.
RNA interference (RNAi) is a gene silencing process initiated by double-stranded RNA (dsRNA) in the cell. RNAi plays an important role in cellular defense against viruses, which often generate dsRNAs during replication. The discovery of RNAi led to the 2006 Nobel Prize in Physiology and Medicine shared by Craig Mello and Andrew Fire. One of the main pathways of RNAi involves small interfering RNA (siRNA), a short -21 nucleotide dsRNA. siRNA is generated by Dicer from longer dsRNAs (e.g. shRNA). siRNA is then unwound into single-stranded RNAs, with one of the strands being loaded onto the RNA-induced silencing complex (RISC) to guide degradation of target RNAs through sequence complementarity.
The ability of RNAi to silence specific gene has prompted researchers to explore its therapeutic potential in treating cancer, viral infection, and neurodegenerative diseases. However, siRNA delivery remains challenging. To effectively deliver siRNA therapeutics, several strategies have been devised to protect and stabilize siRNA and carry it into cells. The most effective method to date uses lipid-based carriers to encapsulate or bind siRNA, and carry it across the plasma membrane. This strategy has shown promise resulting in clinical trials to treat hepatitis B, pancreatic cancer, hypercholesterolemia etc. However, there are some drawbacks, including toxicity and potential to elicit an immune response. To protect siRNA from nuclease degradation, chemical modifications have been added to siRNAs, e.g. 2′O-methylation and 2′O-methyl phosphorodithioate. Yet, there have been reports that chemical modifications could reduce siRNA effectiveness. Viral vectors have also been engineered as siRNA delivery agents because viruses are effective gene-delivery vehicles. Despite their potential, viral vectors carry a high risk in triggering an immune response.
Other proposed methods include siRNA conjugation (e.g. PEG, aptamer, cholesterol, Gal-NAc), and using exosomes, inorganic materials, and proteins as carriers. In particular, N-acetogalactosamine (GalNAc), which binds to liver cell receptors, has been successfully used as liver-targeting ligand conjugate. Alnylam Pharmaceuticals, which developed Patisiran, pioneered the use of GalNAc conjugate for siRNA delivery. Owing to this, liver siRNA delivery technology has made significant advances relative to delivery to other organs. Effective delivery of RNA therapeutics to organs other than liver appears to be a bottleneck for RNA therapy.
Accordingly, it is an objective of the present invention to provide an RNA therapeutic delivery agent using RNA binding proteins as the delivery system for gene therapy to treat a variety of diseases.
In a first aspect, there is provided an RNA therapeutic delivery agent including:
In a first embodiment of the first aspect, the one or more Sm proteins comprise SmD3, SmF, SmB, SmG, SmE, SmD1 and SmD2 of SEQ ID NOs: 1-7, respectively, or the variants thereof comprising SmB′ of SEQ ID NO: 9, SmD1′ of SEQ ID NO: 10, IGF-SmG of SEQ ID NO: 39, SmB1-95-GALAS of SEQ ID NO: 41, and H5E-SmF of SEQ ID NO: 42.
In a second embodiment of the first aspect, the LSm proteins comprise LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10, and LSm11 of SEQ ID NOs: 11-20, respectively.
In a third embodiment of the first aspect, the cell receptor ligand is for receptor-mediated endocytosis comprising epidermal growth factor (EGF) and any family members thereof, and wherein the EGF or any family members thereof is/are attached to any of the Sm proteins, or their variants, for example, SmD2.
In a further embodiment of the first aspect, the cell receptor ligand further comprises insulin-like growth factor (IGF), wherein the IGF or any family members thereof is/are attached to any of the Sm proteins, or their variants, for example, SmG.
In certain embodiments, the endosomal escape peptides include, but not limited to, H5E and GALAS.
In a fourth embodiment of the first aspect, the therapeutic RNA is incorporated into the short-hairpin ribonucleoprotein complex (shRNP) comprising an shRNA of SEQ ID NO: 27 or 36 attached with a 6-FAM fluorescent label at 5′-end thereof for targeting KRAS, an shRNA of SEQ ID NO: 28 attached with a DY547 dye at 5′-end thereof for targeting egfp, or a small-interfering RNA (siRNA) of SEQ ID NO: 37 and 38 for targeting KRAS, a pair of siRNAs of SEQ ID Nos: 45 and 46 for targeting a spike protein of a coronavirus, or a pair of siRNAs of SEQ ID Nos: 47 and 48 for targeting an envelope protein of the coronavirus.
In certain embodiments, other siRNAs targeting various viruses and/or cancers can also be incorporated into said shRNP for gene silencing.
In a fifth embodiment of the first aspect, the Sm binding sequence is attached to the RNA at either 3′-end or 5′-end thereof.
In a sixth embodiment of the first aspect, the cell receptor ligand is attached to N-terminus, C-terminus, or within a loop between strands 3 and 4 of any one of the Sm proteins or any one of the LSm proteins, or any variant thereof.
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 SEQ ID Nos: 21-26, where SEQ ID NO: 21 is 5′-AAUUUGUGG-3′; SEQ ID NO: 22 is 5′-GAUUUUUGG-3′; SEQ ID NO: 23 is 5′-AAUUUUUGA-3′; SEQ ID NO: 24 is 5′-AAUUUUUUG-3′; SEQ ID NO: 25 is 5′-UUUU-3′; SEQ ID NO: 26 is 5′-AAUUUGUCUAG-3′.
In a second aspect of the present invention, there is provided a modified small nuclear ribonucleoprotein (snRNP) complex for gene silencing in a cell, where the snRNP complex comprises a core, and where the core comprises:
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 the variant thereof comprising SmB′ of SEQ ID NO: 9, SmD1′ of SEQ ID NO: 10, IGF-SmG of SEQ ID NO: 39, SmB1-95-GALA3 of SEQ ID NO: 41, and H5E-SmF of SEQ ID NO: 42.
In a second embodiment of the second aspect, the LSm proteins comprise LSm1, LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, LSm8, LSm10, and LSm11 of SEQ ID Nos: 11-20, respectively.
In a third embodiment of the second aspect, the at least one cell receptor ligand comprises an epidermal growth factor (EGF) and any family members thereof, and wherein the EGF or any family members thereof is/are attached to any of the Sm proteins, or their variants, for example, SmD2.
In a further embodiment of the second aspect, the cell receptor ligand further comprises IGF, wherein the IGF or any family members thereof is/are attached to any of the Sm proteins, or their variants, for example, SmG.
In certain embodiments, the endosomal escape peptides include, but not limited to, H5E and GALAS.
In a fourth embodiment of the second aspect, the shRNP comprises an shRNA of SEQ ID NO: 27 or 36 attached with a 6-FAM fluorescent label at 5′-end thereof for targeting KRAS, an shRNA of SEQ ID NO: 28 attached with a DY547 dye at 5′-end thereof for targeting egfp, an siRNA of SEQ ID NOs: 37 and 38 for targeting KRAS, a pair of siRNAs of SEQ ID NOs: 45 and 46 for targeting a spike protein of a coronavirus, or a pair of siRNAs of SEQ ID NOs: 47 and 48 for targeting an envelope protein of the coronavirus.
In certain embodiments, other siRNAs targeting various viruses and/or cancers can also be incorporated into said shRNP for gene silencing.
In a fifth embodiment of the second aspect, the Sm binding sequence is attached to the shRNA or siRNA at either 3′-end or 5′-end thereof.
In a sixth embodiment of the second aspect, the cell receptor ligand is attached to N-terminus, C-terminus, or within a loop between strands β and 4 of any one of the Sm proteins or any one of the Sm proteins, or any variant thereof.
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, where SEQ ID NO: 21 is 5′-AAUUUGUGG-3′; SEQ ID NO: 22 is 5′-GAUUUUUGG-3′; SEQ ID NO: 23 is 5′-AAUUUUUGA-3′; SEQ ID NO: 24 is 5′-AAUUUUUUG-3′; SEQ ID NO: 25 is 5′-UUUU-3′; SEQ ID NO: 26 is 5′-AAUUUGUCUAG-3′.
Other aspects of the present invention include providing a gene therapy to treat diseases in a subject comprising using the agent or complex or alike according to the first or second aspect of the present invention or described herein.
In certain embodiments, the diseases include, but not limited to, cancers, neurodegenerative disease, and viral infections.
In any aspects of the present invention, the subject receiving the gene therapy according to the method described herein includes human or other animals.
In an exemplary embodiment, the agent or complex described herein is configured to deliver one or more therapeutic RNAs via a non-viral/non-lipid-based transfection method.
Preferably, the one or more therapeutic RNAs is/are delivered to a target cell or tissue of the subject via a protein-based delivery system including the agent or complex described herein, wherein the modified snRNP complex is formed ex vivo with the one or more RNA molecules, the one or more therapeutic RNAs in the presence of the corresponding Sm binding sequences, and one or more cell binding ligands before being delivered to the subject.
In certain embodiments, one or more endosomal escape peptides (EEPs) is further attached to one or more Sm/LSm proteins, or any combination or variants thereof, of the modified snRNP complex before being delivered to the subject.
In certain embodiments, the agent or complex is administered to the subject via one or more of intratumoral, intramuscular, intraperitoneal, and intravenous injections in the absence of any viral/lipid-based vector/carrier.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.
The present invention includes all such variation and modifications. The invention also includes all the steps and features referred to or indicated in the specification, individually or collectively, and any and all combination or any two or more of the steps or features.
Throughout the present specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the present invention.
Furthermore, throughout the present specification and claims, unless the context requires otherwise, the word “include” or variations such as “includes” or “including”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the present invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
Other aspects and advantages of the present invention will be apparent to those skilled in the art from a review of the ensuing description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:
The present invention serves as a platform technology to deliver RNA therapeutics into cells (
This invention could also be applied to RNA delivery for research purposes, be used in cell lines and in vivo studies.
In this invention, a modular RNA delivery agent is constructed using modified components of small nuclear ribonucleoprotein (snRNP) complex. In one embodiment, U4 snRNP is modified to become an RNA delivery agent. U4 snRNP is one of the many snRNPs which can be found in eukaryotic organisms; examples of other snRNPs, which can be other embodiments of the present invention, include but not limited to, U1 snRNP, U2 snRNP, U5 snRNP, U6 snRNP, U7 snRNP, Lsm1-7 ring etc. U4 snRNP is part of spliceosome that splices pre-messenger RNAs. The present snRNP complex includes a core which is comprised of Sm or LSm proteins, of one or more RNA molecules where the RNA molecules contain a Sm binding sequence and the rest of the RNA can be fully or partially double-stranded and either wild-type or modified RNA. For example, in the U4 snRNP core, it can be composed of a 144-nucleotide long U4 small nuclear RNA (snRNA) (SEQ ID NO: 35) and 7 Sm proteins, namely SmD1 (SEQ ID NO: 6), SmD2 (SEQ ID NO: 7), SmG (SEQ ID NO: 4), SmE (SEQ ID NO: 5), SmF (SEQ ID NO: 2), SmD3 (SEQ ID NO: 1) and SmB (SEQ ID NO: 3) or their variant including SmB′ (SEQ ID NO: 9) and SmD1′ (SEQ ID NO: 10). Other snRNPs comprise of different combinations of Sm and like-Sm (LSm) proteins. All Sm and LSm proteins are from the same Sm-fold family, having similar structures and form oligomeric ring structures. In another embodiment, U7 snRNP is composed of LSm11 (SEQ ID NO: 20), LSm10 (SEQ ID NO: 19), SmG, SmE, SmF, SmD3 and SmB or SmB′; U6 snRNP is composed of LSm2 (SEQ ID NO: 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); LSm1-7 ring is composed of LSm1 (SEQ ID NO: 11), LSm2, LSm3, LSm4, LSm5, LSm6, and LSm7. The Sm proteins for U4 snRNP form a doughnut-shaped ring around the Sm binding site on the U4 snRNA, as revealed by crystal structure (
The following examples are intended to aid the understanding and enablement of certain embodiments of the present invention, which should not be intended to limit the scope of the present invention.
In Vitro Assembly of shRNP
As proof-of-principle that the Sm proteins can be engineered as RNA delivery agent, the Sm proteins were engineered from the U4 snRNP to deliver an shRNA that targets KRAS gene for knockdown via RNA interference. Separately, recombinant proteins were co-expressed and purified for the subcomplexes SmD1/SmD2-EGF complex, SmD3/SmB1-95 complex, and SmG/SmE complex. The C-terminus of SmD2 was fused with an epidermal growth factor (EGF) as ligand for EGF receptor (EGFR) for cellular uptake via endocytosis. To improve solubility of the SmD1/SmD2-EGF complex, a solubility tag SUMO was also fused onto the N-terminus of SmD1 for expression and the tag was later removed by addition of ULP1 protease during assembly. In order for the shRNA targeting KRAS to bind to the Sm proteins, a Sm binding sequence was added at the 5′-end of the shRNA in the present design. Additionally, a 6-FAM fluorescent label was added at the 5′-end of the shRNA for tracking of the shRNA in cells.
The Sm protein subcomplexes were mixed with the shRNA and the shRNP complex purified by size exclusion chromatography (
To investigate if shRNP can be uptaken into cells, it was tested on A549 lung adenocarcinoma cells. A549 cells are K-Ras positive. The cells were incubated in media containing shRNP at 123 nM final concentration. As control, the cells were incubated in media with the corresponding amounts of the Sm proteins used to assemble the shRNP complex. After two days, the media were replaced with fresh media and confocal fluorescence microscopy was performed. Under confocal microscopy, it was observed that cells incubated with shRNP contained diffused FAM fluorescence signals in the cytoplasm, suggesting that the FAM-tagged shRNA had entered the cell cytoplasm (
To further confirm that the observation for the protein only control was just background noise, the tests were repeated with shRNA only (without complexation with Sm protein), protein only, and buffer controls. Cells from all these controls also gave similar background speckled fluorescence when using FAM channel that did not diffuse over the cell cytoplasm (
To investigate if the shRNA targeting KRAS that entered A549 cells in the form of shRNP can silence KRAS via RNA interference, quantitative polymerase chain reaction (qPCR) was performed to check for KRAS gene expression. A549 cells were first incubated for 4 days with 123 nM shRNP, corresponding amounts of Sm proteins, and buffer control, then qPCR was performed on KRAS RNA using two different sets of primer pairs. Results from our qPCR studies showed that KRAS RNA levels, relative to buffer control, decreased for both cells incubated with shRNP and Sm proteins (
To investigate the effects of shRNP on A549 cell viability, MTT assays were performed on A549 cells incubated with 123 nM and 246 nM shRNP, corresponding Sm proteins only (without shRNA), and buffer control. After 4 days of incubation, MTT assays were performed. Compared to buffer control (100%), cell viability for cells incubated with 123 nM shRNP was 93.33% while protein only control was 108.55% (
egfp as Target for Silencing
Due to interference between EGFR activation by EGF ligand and KRAS gene expression, it could not be conclusively shown that shRNP has RNAi effect. In order to show that the shRNA delivered by shRNP into cells can lead to gene silencing, shRNP carrying a shRNA targeting egfp was assembled. As the product of egfp gene is enhanced green fluorescence protein (eGFP) that produces green fluorescence, eGFP can serve as a reporter; green fluorescence reduction is expected if egfp gene silencing occurs. In this assembly, the same set of Sm proteins and modifications, including the EGF ligand fusion at the C-terminus of SmD2, were used. The shRNA containing a fragment of egfp mRNA sequence was designed and attached with an Sm binding sequence on its 5′-end. The fluorescence tag was changed to DY547 (red) so that it will not interfere with the fluorescence signal from eGFP (green). Using similar methods as described above, the shRNP complex targeting egfp was assembled and verified by size exclusion chromatography, 260/280 ratio (1.5) and SDS-PAGE analysis (
100 nM of shRNP targeting egfp was incubated with 293T-eGFP cells that express endogenous eGFP. Cells were set up for incubation with 100 nM each of Sm protein subcomplexes without shRNA (protein only), 100 nM shRNA only and Buffer Control. After two days of incubation, the media were replaced with fresh media, and confocal microscopy studies were performed (
In the previous embodiments of the present invention, the inventors showed that shRNP containing 6 Sm proteins (SmD1, SmD2-EGF, SmD3, SmB1-95, SmG and SmE) and an shRNA either targeting KRAS or egfp can be uptaken by cells.
In this embodiment of the present invention, the inventors show that an shRNP consisting of (1) 7 Sm proteins, (2) a different ligand for receptor-mediated endocytosis, (3) and the receptor ligand being on a different Sm protein can also be reconstituted and elicit a gene knockdown effect. The inventors performed in vitro reconstitution of another shRNP complex consisting of SmD1, SmD2, SmD3, SmB1-95 (a variant of SmB (SEQ ID NO: 3), IGF-SmG (SEQ ID NO: 39) (SmG with its N-terminus fused with insulin-like growth factor 1, IGF1, residues 49-118 (SEQ ID NO: 40)), SmE, and SmF and an shRNA targeting KRAS labelled with FAM on the 5′-end (SEQ ID NO: 36) (This shRNA also contains a 2-nucleotide overhang on the 3′-end that does not form base-pairing) (
The inventors treated A549 cells that are K-Ras positive with 100 nM shRNP, siRNP, shRNA only, siRNA only, protein carrier only, or buffer control and incubated the cells for 48 hours before performing quantitative PCR to investigate KRAS gene expression (
Because the inventors previously were able to observe cellular uptake of shRNPs by confocal microscopy but their gene silencing effects were not definitively demonstrated by qPCR, the present disclosure proposes that the uptaken shRNPs can be trapped in endosomes. Endosomal escape is a known hurdle in drug delivery. Endosomal escape peptides (EEP) have been reported to be able to disrupt endosomes allowing drugs to be released into the cytoplasm. To investigate if fusing EEPs to shRNP proteins will allow RNA to break free from endosomes and elicit stronger RNAi effects, the inventors fused two different EEPs onto the two different Sm proteins: GALA3 (SEQ ID NO: 44) onto the C-terminus of SmB1-95 (SmB1-95-GALA3 (SEQ ID NO: 41)) and H5E (SEQ ID NO: 43) onto the N-terminus of SmF (H5E-SmF (SEQ ID NO: 42)). The inventors reconstituted two versions of shRNPs: (1) shRNP with SmB1-95-GALA3 as well as H5E-SmF (SmD1, SmD2, SmD3, SmB1-95-GALA3, IGF-SmG, SmE, and H5E-SmF, FAM-shRNAKRAS) (
The inventors then treated the A549 cells with 100 nM shRNPs from different peaks along with buffer, protein, and shRNA controls. The inventors performed qPCR on these samples to investigate their gene knockdown effects (
To confirm cellular uptake of shRNPs containing EEPs with IGF as receptor ligand, the inventors performed confocal microscopy studies on cells incubated with 100 nM shRNP containing both GALA3 and H5E (Peak 2) for 48 hours. The confocal microscopy images showed that cells incubated with shRNP containing GALA3, H5E and IGF had a stronger diffused fluorescence staining corresponding to FAM relative to buffer, protein and shRNA controls, which had weaker background fluorescence signals, suggesting that the shRNP complex aided cellular uptake of shRNA (
To show that the ribonucleoprotein core can also carry an siRNA for gene silencing, the inventor reconstituted a siRNP-IGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95, IGF-SmG, SmE, and H5E-SmF) and a small-interfering RNA (siRNA) targeting KRAS (SEQ ID NOS: 37 and 38). The inventor then treated A549 cells with 100 nM siRNP-IGF-H5E along with shRNP-IGF-H5E, buffer, protein, siRNA, and shRNA only controls for 48 hours. The inventors performed qPCR on these samples to investigate their gene knockdown effects (
To investigate whether using different ligands could result in different levels of internalization of the shRNPs into cells, the inventors used flow cytometry to quantify the levels of fluorescent-tagged shRNA in cells delivered by different shRNPs. The inventors assembled three versions of shRNPs: (1) shRNP-IGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95, IGF-SmG, SmE, and H5E-SmF and a DY547-tagged shRNA targeting KRAS (SEQ ID NO: 27); (2) shRNP-IGF/EGF-H5E consisting of IGF-SmD1, SmD2, SmD3, SmB1-95-H5E, IGF-SmG, SmE-EGF, and SmF and a DY547-tagged shRNA targeting KRAS (SEQ ID NO: 27); (3) shRNP-EGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95-H5E, SmG, SmE-EGF, and SmF and an DY547-tagged shRNA targeting KRAS (SEQ ID NO: 27). The inventors treated A549 cells with 100 nM of each shRNP and performed flow cytometry to quantify the fluorescence intensity of each sample at time points 1 h, 2 h, and 4 h post-treatment with shRNP (
To investigate whether silencing KRAS will have an effect on cancer cell viability, the inventors investigated KRAS silencing and cell viability effects of siRNPs on a human colorectal carcinoma cell line HCT116. HCT116 is K-Ras-dependent for progression. The inventors assembled three versions of siRNPs: (1) siRNP-IGF-H5E consisting of SmD1, SmD2-IGF, SmD3, SmB-H5E, SmG, SmE, and SmF, and a small-interfering RNA (siRNA) targeting KRAS (SEQ ID NOs: 37 and 38); (2) siRNP-EGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95-H5E, SmG, SmE-EGF, and SmF and a small-interfering RNA (siRNA) targeting KRAS (SEQ ID NOs: 37 and 38); and (3) siRN4P-IGF-H5E, a ribonucleoprotein complex consisting of 4 Sm proteins instead of 7 (SmD1, SmD2-IGF, SmD3, SmB-H5E and SmE). The inventors treated HCT116 cells with 100 nM of each of the siRNP, along with siRNA, corresponding proteins, and buffer only controls for 48 hours. After that, the inventors performed qPCR to investigate KRAS expression levels (
To investigate the dose-dependent effects of siRNP on KRAS gene expression and HCT116 cell viability, the inventors treated HCT116 with different concentrations of siRNP-EGF-H5E (100 nM, 200 nM, 500 nM and 1000 nM), along with siRNA and protein only controls with corresponding concentrations for 48 hours. After that, the inventors performed qPCR to quantify KRAS gene expression (
To demonstrate that the invention can also be used as potential antiviral therapeutics, the inventors assembled siRNPs targeting Human Coronavirus 229E (HCoV-229E) Spike (5) and Envelope (E) genes. The S gene encodes for the coronavirus Spike protein while the E gene encodes for the coronavirus Envelope protein. To target the HCoV-229E S gene, the inventors assembled si(S)RNP-IGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95, IGF-SmG, SmE, and H5E-SmF, and a small-interfering RNA (siRNA) targeting the Spike (5) gene (SEQ ID Nos: 45 and 46 for si(S)-(+) and si(S)-(−)). To target the HCoV-229E E gene, the inventors assembled si(E)RNP-IGF-H5E consisting of SmD1, SmD2, SmD3, SmB1-95, IGF-SmG, SmE, and H5E-SmF, and a small-interfering RNA (siRNA) targeting E (SEQ ID Nos: 47 and 48 for siE-(+) and siE-(−)). To investigate whether the siRNPs can silence viral genes in cells subjected to viral infection, the inventors first treated VeroE6 Cells with HCoV-229E by incubating the cells with the virus for 2 hours, after which the inventors removed the virus-containing media and replenished fresh media containing 500 nM of each siRNP (si(S)RNP-IGF-H5E and si(E)RNP-IGF-H5E), along with buffer controls. The inventors also set up controls with VeroE6 cells without being exposed to HCoV-229E but treated with 500 nM of each siRNP (si(S)RNP-IGF-H5E and si(E)RNP-IGF-H5E) or buffer. After 48 hours of treatment with siRNPs or corresponding controls, the RNA from each sample was extracted for qPCR analysis (
Overall, the inventors demonstrated that their modular and customizable RNAi delivery agent can be modified to enhance its gene knockdown efficacies. With these results, this embodiment of the present invention demonstrated that 1) a 7-Sm or 4-Sm protein shRNP or siRNP complex can be reconstituted for RNA delivery, 2) the receptor ligand can be moved to another Sm protein and still allows for siRNP and shRNP to deliver RNA and knockdown genes, 3) cellular uptake can still occur using a different receptor ligand, 4) adding an EEP(s) to shRNP can enhance its gene silencing effects, 5) an oncogene silenced by the siRNP can reduce cancer cell viability, and 6) viral genes can be silenced in infected cells using the siRNPs.
Construct encoding for SmD1 (synthesized by Genscript) was cloned into pET28a-sumo vector encoding for an N-terminal hexahistidine-tag followed by a SUMO-tag. Construct encoding for SmD2 or SmD2-epidermal growth factor (SmD2-EGF) (synthesized by Genscript) was cloned into MCS2 of pCDFDuet-1. Both plasmids were then co-transformed in E. coli BL21(DE3) and the cells were grown in 2×YT media at 37° C. with 50 μg/ml kanamycin, 100 μg/ml streptomycin, and until OD600 reached 0.6-0.8. The cells were then induced using a final concentration of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and were grown at 20° C. for 18 h. The cells were harvested by centrifugation. The cell pellets were resuspended in lysis buffer (20 mM Tris, pH7.5, 1 M NaCl, 10 mM imidazole, 5% (v/v) glycerol, 10 mM 3-mercaptoethanol (BME) and 17.8 μg/mL phenylmethylsulfonyl fluoride (PMSF)) and lysed by sonication. Cell lysate was cleared by centrifugation and loaded onto a HisTrap HP column (Cytiva). The column was then washed with 20 column volumes (CV) of wash buffer (20 mM Tris, pH 7.5, 1 M NaCl, 40 mM imidazole) and eluted with elution buffer (20 mM Tris, pH 7.5, 1 M NaCl, 500 mM imidazole). The eluted protein was diluted 3 times with buffer A (20 mM HEPES, pH 7.5, 5 mM DTT) and further purified using a 5 ml HiTrap Heparin column (GE Healthcare) equilibrated with 85% buffer A and 15% buffer B (20 mM HEPES, pH 7.5, 2 M NaCl, 5 mM DTT). The protein was eluted using a gradient from 15% to 100% buffer B. The SUMO-tag on SmD1 was then removed by incubating with ULP1 protease overnight at 4° C. The untagged SmD1/SmD2 complex was subsequently purified by reverse His-tag purification using 1 ml HisTrap HP column (Cytiva). Fractions of interest were concentrated and store at −80° C.
Expression and Purification of His-SmD3/SmB1-95-GALA3, His-SmD3/SmB1-95-H5E and His-SmD3/SmB1-95
The construct for N-terminal hexahistidine tagged SmD3 was cloned into pET28a and the construct encoding for SmB residues 1-95 (SmB1-95), SmB1-95-GALA3 fusion or His-SmD3/SmB1-95-H5E protein was cloned into MCS2 of pCDFDuet-1. Both plasmids were then co-transformed into E. coli BL21 (DE3) cells and grown in 2×YT media supplemented by 50 μg/ml kanamycin and 100 μg/ml streptomycin at 37° C., expression was induced with a final concentration of 0.5 mM IPTG when its OD600 reached 0.6-0.8. The cells were grown at 20° C. for 18 hours. Cells were harvested by centrifugation and the 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 lysed by sonication. The cell lysate was cleared by centrifugation and loaded onto a 5 ml HisTrap HP column (GE Healthcare). The column was then washed with 20 CV of wash buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole) and eluted with elution buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 500 mM imidazole). The eluted protein was further purified by using a 5m1 HiTrap Heparin column (GE Healthcare) equilibrated with 60% buffer A (20 mM HEPES, pH 7.5 and 5 mM DTT) and 40% buffer B (20 mM HEPES, pH 7.5, 1 M NaCl, 5 mM DTT). The protein was eluted with a gradient from 40% to 100% buffer B. Fractions of interest were concentrated and stored at −80° C.
The construct for C-terminal hexahistidine-tagged SmG was cloned into pET26b and SmE was cloned into MCS1 of pCDFDuet-1 vector. Both plasmids were then co-transformed into E. coli Rosetta (DE3) pLysS cells and grown in 2×YT media supplemented by 50 μg/ml kanamycin, 100 μg/ml streptomycin and 34 μg/ml chloramphenicol at 37° C. When the OD600 the culture reached 0.6-0.8, the cells were induced by adding a final concentration of 0.5 mM IPTG and grown at 20° C. for 18 h. The cells were harvested, and the complex was purified as described for the His-SmD3/SmB 1-95 complex.
The construct for C-terminal hexahistidine-tagged SmG, or hexahistidine-tagged SmG with insulin-like growth factor 1 residues 49-118 (IGF) fused to its N-terminus, was cloned into pET26b, while SmE or SmE-EGF and SmF or H5E-SmF was cloned into MCS1 and MCS2 of pCDFDuet-1 respectively. Both plasmids were then co-transformed into E. coli BL21 cells and grown in 2×YT media supplemented by 50 μg/ml kanamycin and 100 μg/ml streptomycin at 37° C., expression was induced with a final concentration of 0.5 mM IPTG when its OD600 reached 0.6-0.8. The cells were grown at 20° C. for 18 hours. Cells were harvested by centrifugation and the 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 lysed by sonication. The cell lysate was cleared by centrifugation and loaded onto a 5 ml HisTrap HP column (Cytiva). The column was then washed with 20 CV of wash buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole) and eluted with elution buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 500 mM imidazole). The eluted protein was further purified by using a 5 ml HiTrap Q HP column (Cytiva) equilibrated with 85% buffer A (20 mM HEPES, pH 7.5 and 5 mM DTT) and 15% buffer B (20mM HEPES, pH 7.5, 1 M NaCl, 5 mM DTT). The protein was eluted with a gradient from 15% to 60% buffer B. Fractions of interest were concentrated and stored at −80° C.
In Vitro Reconstitution and SUMO-Tag Cleavage for shRNP
2 molar equivalents of SmD3/SmB, SmG/SmE, and SUMO-SmD1/SmD2-EGF complexes were mixed with 1 molar equivalent of shRNA (synthesized by Dharmacon, Horizon Discovery) in 500 μL reconstitution buffer (20 mM HEPES, pH 7.5, 750 mM NaCl, 5 mM ethylenediaminetetraaceticacid (EDTA), and 5 mM DTT). Prior to mixing, the shRNA was reannealed by heating to 90° C. for 5 min and snap-cooling on ice for 10 min. The RNA/Protein mixture was incubated at 30° C. for 30 min, followed by 37° C. for 15 min and then cooled on ice for 10 min. The SUMO-tag on SmD1 was then removed by incubating with ULP1 protease overnight at 4° C. The reconstituted complex was purified by size exclusion chromatography (Superdex® 200 increase 10/300 GL column, GE Healthcare) with 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 store at −80° C.
The shRNA sequences used for shRNP were as follows:
CCU CGA GGA AUU AGC UGU AUC GUC AAG G-3’
ACU CGA GUG AAC UUC AGG GUC AGC UUG C-3’
In Vitro Reconstitution for shRNP, siRNP, shRNP-IGF and siRNP-IGF
Equimolar amounts of SmD1/SmD2, His-SmD3/SmB1-95 or SmB1-95, IGF-SmG-His/SmE/SmF complexes and shRNA or siRNA were mixed in 750 μL reconstitution buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 5 mM DTT). Prior to mixing, the shRNA or siRNA was reannealed by heating to 90° C. for 5 min and snap-cooling on ice for 10 min. The RNA/Protein mixture was incubated at 30° C. for 30 min, followed by 37° C. for 15 min and then cooled on ice for 10 min. The reconstituted complex was purified by size exclusion chromatography (Superdex® 200 increase 10/300 GL column, Cytiva) with 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 store at −80° C.
In vitro reconstitution for shRNP containing EEPs (shRNP-IGF-GALA3-HSE, shRNP-IGF, shRNP-IGF-EGF, shRNP-EGF, siRNP-IGF-HSE, siRNP-EGF-H5E)
1.5 molar equivalents of SmD1/SmD2, His-SmD3/SmB1-95 or SmB1-95-GALA3 or SmB1-95-H5E, IGF-SmG-His/SmE/H5E-SmF or SmG-His/SmE-EGF/SmF complexes and 1 molar equivalent of shRNA or siRNA were mixed in 750 μL reconstitution buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM EDTA, and 5 mM DTT). Prior to mixing, the shRNA or siRNA was reannealed by heating to 90° C. for 5 min and snap-cooling on ice for 10 min. The RNA/Protein mixture was incubated at 30° C. for 30 min, followed by 37° C. for 15 min and then cooled on ice for 10 min. The reconstituted complex was purified by size exclusion chromatography (Superdex® 200 increase 10/300 GL column, Cytiva) with a buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 10 mM EDTA, and 5 mM DTT. Relevant Fractions were pooled, concentrated and store at −80° C.
Human lung adenocarcinoma cell line A549 was maintained in RPMI-1640 medium (Gibco, Life Technologies) with 2 g/L sodium bicarbonate(Sigma-Aldrich) and supplemented with penicillin and streptomycin(Gibco, Life Technologies), and 10% fetal bovine serum (Gibco, Life Technologies) at 37° C., 5% CO2.
Human embryonic kidney cell line 293T with endogenous enhanced green fluorescent protein expression (293T-eGFP) was maintained in DMEM, high glucose (Gibco, Life Technologies) with 3.7 g/L sodium bicarbonate and supplemented with penicillin and streptomycin, 5% fetal bovine and 5% Newborn calf serum(Gibco, Life Technologies) at 37° C., 5% CO2.
Gene Expression Analysis for shRNP
A549 cells were seeded in 12-well flat-bottomed tissue culture plate (Falcon BD) and incubated overnight. Culture medium was replenished with medium supplemented with a final concentration 123 nM (each) protein subcomplex (His-SUMO-SmD1/SmD2-EGF, SmD3/SmB1-95, SmG-His/SmE), 123 nM shRNP, and buffer negative control and incubated at 37° C., 5% CO2. After 4 days, total RNA was isolated using RNAzol RT (Molecular Research Center) and reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega) according to the manufacturer's protocol. Quantitative real-time PCR reactions were performed using synthesized cDNA with TB Green Premix Ex Taq II (Takara) on a StepOnePlus Real-Time PCR instrument (Applied Biosystems). The sequences of the primers used in the RT-PCR were as follows: KRAS 1 forward primer (SEQ ID NO: 29) and reverse primer (SEQ ID NO: 30), KRAS 2 forward primer (SEQ ID NO: 31) and reverse primer (SEQ ID NO: 32), GAPDH forward primer (SEQ ID NO: 33) and reverse primer (SEQ ID NO: 34). Relative levels of KRAS expression were calculated using the ΔΔCT method with GAPDH as the normalization control.
Gene Expression Analysis for Cell Treatment with shRNP-H5E and shRNP-GALA3-H5E
A549 cells were seeded in 12-well flat-bottomed tissue culture plate (Falcon BD) and incubated overnight. The culture media were replenished with medium supplemented with protein control (100 nM of each subcomplex: SmD1/SmD2, His-SmD3/SmB1-95 or His-SmD3/SmB1-95-GALA3, IGF-SmG-His/SmE/H5E-SmF), 100 nM shRNA (targeting KRAS), 100 nM shRNP, and buffer control. The cells were then incubated at 37° C., 5% CO2. After 2 days, total RNA was isolated using RNAzol RT (Molecular Research Center) and reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega) according to the manufacturer's protocol. Duplicate quantitative real-time PCR reactions were performed using synthesized cDNA with iTaq Universal SYBR Green Supermix (Bio-Rad) on a StepOnePlus Real-Time PCR instrument (Applied Biosystems). The sequences of the primers used in the RT-PCR were as follows: KRAS 2 forward (SEQ ID NO: 31) and reverse (SEQ ID NO: 32), GAPDH forward (SEQ ID NO: 33) and reverse (SEQ ID NO: 34). Relative levels of KRAS expression were calculated using the ΔΔCT method with GAPDH as the normalization control.
Gene Expression Assay for Cell Treatment with siRNPs
HCT116 cell was purchased from American Type Culture Collection (ATCC). HCT116 cells were seeded in 24-well flat-bottomed tissue culture plate (JET Biofil) and incubated overnight. The culture media were replenished with medium supplemented with buffer, RNA (100-1000 nM), protein (100-1000 nM) controls and siRNP (100-1000 nM) respectively. The cells were then incubated at 37° C., 5% CO2. The cells were lysed in TRIzol (Invitrogen) after 48 h and total RNA was extracted using the Direct-zol RNA kit (Zymo Research) following the manufacturer's protocol. The extracted RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara). Duplicate quantitative real-time PCR was then performed on the synthesized cDNA using the iTaq Universal SYBR Green Supermix (Bio-Rad) on a ViiA 7 Real-Time PCR system (Applied Biosystems). The sequences of the primers used in the RT-PCR for KRAS and GAPDH were the same as above (SEQ ID NOS: 31-34). Relative levels of KRAS expression were calculated using the ΔΔCT method with GAPDH as the normalization control.
Human coronavirus strain 229E (HCOV-229E) was purchased from ATCC, and propagated in MRCS cells. After 5 days of inoculation, the infected media were collected and centrifuged at 4000 rpm at 4° C. for 10 min. The supernatant was aliquoted and stored at −80° C. before use. For testing the effectiveness of the siRNP complexes, VeroE6 cells (JCRB Cell Bank) were first infected by HCOV-229E by replenishing the media with 250 μL of infected media from the MRCS cells, and incubated for 2 h. The infected media were then discarded, the cells washed with PBS, and 200 μL media supplemented with 500 nM of siRNP(S)-IGF-H5E, or siRNP(E)-IGF-H5E, or buffer were added to the cells. The uninfected (mock) cells were washed with PBS, and media supplemented with 500 nM of siRNP(S)-IGF-H5E, or siRNP(E)-IGF-H5E, or buffer were added to the cells. After 48 h post-infection, the cells were harvested for total RNA using RNAiso Plus (Takara), subjected to reverse transcription (Takara PrimeScript RT Reagent Kit), and quantitative (q)PCR to examine S and E gene expression. The sequences of the primers were:
Relative levels of S and E expression were calculated using the ΔΔCT method with ACTB gene encoding for beta-actin as the normalization control.
For the Investigations of shRNPKRAS on A549 Cells
A549 cells were seeded in glass-bottomed tissue culture dish and incubated overnight. Culture medium was replenished with a 123 nM each protein subcomplex-,123 nM shRNA-, 123 nM shRNP- and buffer-added medium and incubated for 2 days, while another with a buffer only as negative control. Cells were co-stained with Hoechst 33342 nucleic acid stain (0.3 ug/ml, Invitrogen) before imaging. Images of fluorescence-labelled cells were captured with a Nikon Eclipse Ti2 confocal laser-scanning microscope. Hoechst 33342 signal was captured in the blue (λex=400 nm, λem=410-480 nm) channel and 6-FAM signal was captured in the green (λex=491 nm, λem=500-550 nm) channel.
For the Investigations of shRNPKRAS Containing Endosomal Escape Peptide on A549 Cells
A549 cells were seeded in glass-bottomed tissue culture dish and incubated overnight. The culture media were replenished with media supplemented with protein control (100 nM of each subcomplex: SmD1/SmD2, His-SmD3/SmB1-95 or His-SmD3/SmB1-95-GALA3, IGF-SmG-His/SmE/H5E-SmF), 100 nM shRNA targeting KRAS, 100 nM shRNP and buffer control and incubated for 2 days. The cells were costained with Hoechst 33342 nucleic acid stain (0.3 ug/ml, Invitrogen) before imaging. Images of fluorescence-labelled cells were captured with a Nikon Eclipse Ti2 confocal laser-scanning microscope. Hoechst 33342 signal was captured in the blue (λex=400 nm) channel and 6-FAM signal was captured in the green (λex=491 nm) channel.
For the Investigations of shRNP, on 293T-eGFP Cells
293T-eGFP cells were seeded in glass-bottomed culture dish and incubated overnight. Culture medium was replenished with 100 nM each protein subcomplex-,100 nM shRNA-, 100 nM shRNP- and buffer-added medium and incubated for 2 days, with buffer as negative control. Cells were co-stained with Hoechst 33342 nucleic acid stain before imaging. Images of fluorescence-labelled cells were captured with a Nikon Eclipse Ti2 confocal laser-scanning microscope. Hoechst 33342 signal was captured in the blue (λex=400 nm, λem=410-480 nm) channel. eGFP signal was captured in the green (λex=491 nm, λem=510-540 nm) channel. DY547 signal was captured in the red (λex=561 nm, λem=570-600 nm) channel.
A549 cells were seeded in 96-well flat-bottomed tissue culture plate (Nunc) and incubated overnight. Culture medium was replenished with medium supplemented with a final concentration 123 nM and 246 nM (each) protein subcomplex (His-SUMO-SmD1/SmD2-EGF, SmD3/SmB1-95, SmG-His/SmE), 123 nM and 246 nM shRNP, and buffer negative control and incubated at 37° C., 5% CO2. After 3 days, culture medium was replaced by medium added with MTT reagent (3-(4, 5-dimethylthiazol-2, 5-diphenyltetrazolium bromide, 0.5 mg/ml, Sigma-Aldrich) and incubated for 2 h. The media was then removed and the formazan salt crystals formed were dissolved with 100 μL dimethyl sulfoxide (RCI Labscan) with 30 min shaking. Absorbance was measured at 540 nm wavelength with reference at 690 nm wavelength on a Biotek ELx800 microplate reader (Biotek). Cell viability (%) was calculated according to the equation:
Cell Viability(%)=(ODprotein or shRNP-complex/ODbuffer)×100%
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide) assay was performed to determine the cytotoxicity of siRNP on HCT116 cells. HCT116 cells (3×103/well in 100 μl DMEM) were seeded in 96-well tissue culture plate (SPL) and allowed to attach overnight at 37° C. The culture media were replenished with media supplemented with buffer, protein (100-1000 nM), RNA (100-1000 nM) control and siRNP (100-1000 nM) respectively. After 72 h of treatment, 10 μl of MTT solution (5 mg/ml) was added to each well and incubated at 37° C., 5% CO2 for 2 hours. The medium was then aspirated, and 100 pi of DMSO was added to dissolve the formed formazan crystals. The absorbance was measured at 570 nm and referenced at 630 nm by a microplate spectrophotometer (BD Biosciences, USA). The assays were carried out in duplicate.
A549 cells (3×104/well in 500 μl RPMI 1640) were seeded in 24-well tissue culture plate (SPL) and allowed to attach overnight at 37° C. The culture media were replenished with media supplemented with 100 nM Dy547-shRNA, Dy547-shRNP-EGF-HSE, Dy547-shRNP-IGF-HSE, Dy547-shRNP-IGF/EGF-H5E respectively, and buffer as negative control. After incubation of 1 h, 2 h, and 4 h, the cells were trypsinized, centrifuged and washed with PBS for 3 times. The cell pellets were resuspended with 500 μL, PBS and analyzed by BD ACCURI™ C6 Plus Flow Cytometer (BD Biosciences) utilizing 10,000 events. The mean fluorescence intensities (MFI) were calculated by FlowJo (Version 10).
The present invention serves as a platform technology to deliver RNA therapeutics into cells. As RNA therapeutics has immense potential applications in biotechnology, for example gene silencing for chronic disease and cancer treatment, RNA-based inhibitors as well as RNA-based vaccines, this invention 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. This invention could also be applied to RNA delivery for research purposes, be used in cell lines and in vivo studies.
This application claims priority from (1) U.S. Provisional Patent Application Ser. No. 63/242,013 filed on Sep. 8, 2021, and is a continuation-in-part under 35 U.S.C. 111(a) of (2) International Patent Application Number PCT/CN2021/080443 filed on Mar. 12, 2021 with a priority claim from U.S. Provisional Patent Application Ser. No. 62/988,929 filed on Mar. 13, 2020, which are hereby incorporated by reference in their entirety for all purposes. The sequence listing file under the file name “Sequence Listing 034590-000026.xml” submitted in ST.26 XML file format with a file size of 50 KB on Sep. 7, 2022 is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63242013 | Sep 2021 | US | |
| 62988929 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/080443 | Mar 2021 | US |
| Child | 17930088 | US |
