CN116444685A - Efficient controllable RNA delivery system - Google Patents
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- CN116444685A CN116444685A CN202310396772.0A CN202310396772A CN116444685A CN 116444685 A CN116444685 A CN 116444685A CN 202310396772 A CN202310396772 A CN 202310396772A CN 116444685 A CN116444685 A CN 116444685A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
The invention relates to a high-efficiency controllable RNA delivery system. The highly efficient controllable RNA delivery system involves a transfer protein, a delivery enhancing factor, and a repressor protein. The present invention increases the efficiency of delivery of a specific cargo RNA by anchoring a transfer protein containing a delivery enhancing factor to the cell membrane; further by adding an extracellular repressor protein, by controlling the separation of the transfer protein from the repressor protein, for regulating the delivery of the nucleic acid. The RNA delivery system of the invention realizes high-efficiency delivery and controllable release of RNA, reduces toxic and side effects caused by continuous stimulation of drugs, and enhances the treatment effects of tumors, immunotherapy, single-gene mutation genetic diseases and polygenic mutation genetic diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a high-efficiency controllable RNA delivery system, and more particularly relates to a fusion protein, an isolated nucleic acid, an expression vector, a recombinant cell, a method for packaging viruses, virus particles, a nucleic acid delivery system, a method for delivering nucleic acid, a pharmaceutical composition and application thereof.
Background
Extracellular vesicles (Extracellular Vesicle, EV) refer to microvesicles released by living cells with a monolayer structure, including microvesicles (microvesicles) and virus-like particles (exosomes). EV expressing viral proteins (such as HIV viral Gag proteins) are also known as Virus-Like particles (VLP). EV-based drug delivery tools have evolved rapidly in recent years.
Endogenous viruses are a class of sequences found in the genomes of humans and other animals that generally have extremely high similarity to retroviruses, and are generally considered retroviruses that are domesticated after infection during evolution and integration into the host genome. Studies have reported that the Arc protein of mice and the dArc1 protein of drosophila exhibit very similar behavior to viruses, they are able to form a structure resembling the capsid of a virus, recognize and encapsulate self RNAs, release in neurons in the form of EVs, are received by downstream cells and translate the proteins. This transcellular transfer behavior of Arc and dArc1 was demonstrated to play a role in nervous system development.
In 2021, team Zhang Feng screened a large number of endogenous viruses in the mouse and human genomes based on similar ideas, and designed an RNA delivery system SEND (master SEND system) using the PEG10 protein with the strongest secretion capacity. PEG10 can specifically bind RNA with 3 'and 5' untranslated regions (3 'utr,5' utr) on its own RNA and encapsulate it into a capsid formed by the self-multimerization of PEG10 protein, released as VLP. The released VLPs can be collected by ultracentrifugation, and VLPs modified by a fusogenic protein (VSV-G, syncytin, etc.) can be taken up by recipient cells, so that RNA encapsulated by VLPs can be expressed in the cells. The SEND system has been demonstrated to deliver Cas9 and gRNA, with potential application prospects in the field of gene therapy, likely to be a competitor to the currently commercialized liposome/lentiviral vectors. The main use of the Zhang Feng team to examine SEND delivery efficiency is the Cre-DIO reporting system, where the recipient cells contain DIO-GFP elements that are recombined by Cre when the SEND system delivers Cre (cyclase) mRNA to the recipient cells and expresses Cre recombinase, and the recipient cells produce green fluorescence with a GFP positive cell ratio characterizing SEND delivery efficiency.
But current SEND systems deliver less efficient, for example: under the Cre-DIO detection system, the highest SEND efficiency is about 60%; except the Cre-DIO system, the Zhang Feng team adopts a second generation sequencing method to count the successful editing rate (index%) of the specific site of the receptor cell, verifies the delivery efficiency of SEND to Cas9 and gRNA, has the murine SEND efficiency of about 30 percent and the humanized PEG of about 40 percent; the SEND system is only one quarter to one fifth as efficient as the mature lentiviral vector.
Furthermore, SEND systems continue to express upon transfer into donor cells, and VLP production and release is continuous, but in practical clinical applications, it is desirable in many cases to be able to achieve targeted or timed administration. The VLP release step of the SEND system faces an uncontrollable problem.
Thus, there is a need in the art to develop an efficient and controllable RNA delivery system.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
studies have shown that SEND systems can deliver RNA into target cells and treat related diseases by targeting specific sites. But the problem of uncontrollable release of VLPs due to low RNA delivery efficiency presents a significant challenge for accurate treatment. Therefore, the invention solves the problems of difficult efficient and targeted drug delivery by constructing an efficient and accurate nucleic acid delivery system.
The present invention aims to solve at least one of the technical problems in the related art to some extent.
In a first aspect of the invention, the invention provides a fusion protein. According to an embodiment of the invention, the fusion protein transfer protein and delivery enhancing factor; the transfer protein is linked to a delivery enhancing factor. The inventors have found that the fusion proteins can deliver RNA across cells, can be used in the field of gene therapy, and by delivering RNA into specific cells or tissues, thereby improving therapeutic efficacy and reducing negative effects on healthy cells.
According to an embodiment of the present invention, the above fusion protein may further include at least one of the following technical features:
according to an embodiment of the invention, the transfer protein comprises at least one of PEG10 protein, arc protein, dArc1 protein.
According to an embodiment of the invention, the transfer protein comprises a PEG10 protein; PEG10 proteins that include only the first reading frame of the PEG10 protein are preferred. The inventors have experimentally verified that the RNA delivery efficiency of PEG10 proteins comprising only the first reading frame is significantly advantageous compared to PEG10 proteins comprising the first reading frame and the second reading frame.
According to an embodiment of the invention, the C-terminal of the transfer protein may be linked to a cargo protein comprising MS2.
According to an embodiment of the invention, the C-terminus of the delivery enhancing factor is linked to the N-terminus of the transfer protein.
According to an embodiment of the invention, a modification site is included between the delivery enhancing factor and the transfer protein.
According to an embodiment of the invention, the modification site comprises at least one of glycine-tryptophan connecting peptide, nuclear export signal.
According to an embodiment of the invention, the delivery enhancing factor comprises at least one of an acylated tag, a small molecule receptor, a classical membrane protein signal peptide, and a transmembrane region. The inventors found that the PEG10 protein can be anchored on the cell membrane by adding an acylation tag, a small molecule receptor, a classical membrane protein synthesis pathway and the like. By anchoring the multimerizing PEG10 RF1 to the membrane, the PEG10 protein can be efficiently loaded into EVs, with the best effect of the acylated tag.
According to an embodiment of the invention, the delivery enhancing factor is an acylated tag.
According to an embodiment of the invention, the acylated tag comprises at least one of Myr-tag, YES1-tag, lyn-tag, LM-tag, HIV Gag-tag, nef-tag, gap43-tag, src-tag, fen-tag.
According to an embodiment of the invention, the acylated tag is Myr-tag. The inventor finally obtains that the RNA delivery efficiency is highest by using Myr-tag as the acylation tag through screening 9 acylation tags.
According to an embodiment of the invention, the fusion protein is further linked at the N-terminus to the C-terminus of the transmembrane domain.
According to an embodiment of the invention, the transmembrane domain is further linked to an extracellular repressor protein. The inventor finds that the effect of controlling the timing of the drug to play a role can be achieved by expressing the fusion protein containing the repressor protein in recombinant cells and controlling the separation of the repressor protein and the transfer protein, and provides a new solution for avoiding the toxic and side effects caused by continuous stimulation of RNA drugs.
According to an embodiment of the invention, the repressor protein comprises at least one of EGFP protein, an axon protein 1b extracellular domain, a CD4 extracellular domain.
According to an embodiment of the invention, the repressor protein is no less than 140 amino acids. The inventors found through experiments that the extracellular vesicle loading-inducing inhibitory effect is independent of domain type, and is only size dependent. It was further verified that PEG10 function was inhibited when the repressor protein contained a larger extracellular region (not less than 140 amino acids).
According to an embodiment of the invention, a modification site is included between the delivery enhancing factor and the transmembrane domain.
According to an embodiment of the invention, the modification site comprises at least one of CIBN, hVOL 1.
According to an embodiment of the invention, the N-terminus of the repressor protein is further linked to the C-terminus of a membrane-localized signal peptide.
According to an embodiment of the invention, the membrane localization signal peptide is selected from at least one of an igκ signal peptide, a VSVg signal peptide, an axon protein 3b signal peptide.
According to an embodiment of the invention, the membrane localization signal peptide is selected from at least one of an igκ signal peptide, a VSVg signal peptide, an axon protein 3b signal peptide. According to the embodiment of the invention, the inventor selects the signal peptide (axon protein 3b signal peptide) at the N end of the classical membrane protein, and regulates the PEG10 protein to anchor the upper membrane through signal transduction.
According to an embodiment of the invention, the fusion protein further comprises a cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein. The inventors modified the original edition SEND system by adding repressor and protease cleavage site outside the cell, by controlling the excision of the extracellular repressor protein, or by cleaving the PEG10 protein with the acylation tag from the membrane protein with the repressor, and re-filming in a lipid anchored manner, and finally realizing the controlled release of the extracellular vesicles containing PEG10 protein and its cargo RNA.
According to an embodiment of the invention, the cleavage site is controlled by a light-sensitive protein, and can be cleaved by a corresponding protease only under illumination of a specific wavelength. The inventor has found through experimental verification that the RNA delivery function of the fusion protein can be recovered through protease cleavage after 470nm blue light irradiation.
According to an embodiment of the invention, the cleavage site comprises at least one of a TEV protease cleavage site and an MMP protease cleavage site.
According to an embodiment of the invention, the extracellular repressor selects EGFP protein having an amino acid sequence shown as SEQ ID NO. 1,
YPYDVPDYANPGTMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHHLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGGS(SEQ ID NO:1)。
according to an embodiment of the invention, the transmembrane domain has the amino acid sequence shown in SEQ ID NO. 2,
TEPGIRRVPGASEVIRESSSTTGMVVGIVAAAALCILILLYAMYKYRNRDGGGSGGGS(SEQ ID NO:2)。
according to an embodiment of the invention, the membrane-localized signal peptide has the amino acid sequence shown as SEQ ID NO. 3,
MHLRIHARRSPPRRPAWTLGIWFLFWGCIVSSVWSQLSSNVASSSSTSSSPGSH(SEQ ID NO:3)。
according to an embodiment of the invention, the fusion protein has the amino acid sequence shown in SEQ ID NO. 4,
MGCINSKRKDMAAAGGSSNCPPPPPPPPPNNNNNNNTPKSPGVPDAEDDDERRHDELPEDINNFDEDMNRQFENMNLLDQVELLAQSYSLLDHLDDFDDDDEDDDFDPEPDQDELPEYSDDDDLELQGAAAAPIPNFFSDDDCLEDLPEKFDGNPDMLGPFMYQCQLFMEKSTRDFSVDRIRVCFVTSMLIGRAARWATAKLQRCTYLMHNYTAFMMELKHVFEDPQRREAAKRKIRRLRQGPGPVVDYSNAFQMIAQDLDWTEPALMDQFQEGLNPDIRAELSRQEAPKTLAALITACIHIERRLARDAAAKPDPSPRALVMPPNSQTDPTEPVGGARMRLSKEEKERRRKMNLCLYCGNGGHFADTCPAKASKNSPPGNSPAPLDYKDDDDK(SEQ ID NO:4)。
in a second aspect of the invention, the invention provides an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid encodes the fusion protein according to the first aspect of the invention. The fusion protein coded by the isolated nucleic acid has stronger specificity, longer half-life and higher efficacy, can effectively treat or prevent cancers under the condition of less dosage, has low toxic and side effects and higher safety.
According to an embodiment of the present invention, the above isolated nucleic acid may further include at least one of the following technical features:
according to an embodiment of the invention, the isolated nucleic acid is DNA or RNA.
In a third aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries an isolated nucleic acid according to the second aspect of the invention. According to the embodiment of the invention, the expression vector can efficiently express the fusion protein in a proper receptor cell, and the fusion protein has stronger specificity, longer half-life period, higher efficacy, low toxic and side effects and higher safety.
According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:
according to an embodiment of the invention, the expression vector further comprises: a promoter operably linked to the isolated nucleic acid of the second aspect of the invention.
According to an embodiment of the invention, the promoter is selected from at least one of CMV, EF-1. Alpha., RSV.
According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
According to an embodiment of the invention, the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus and an adenovirus-associated virus.
In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell expresses the fusion protein according to the first aspect of the invention or carries the isolated nucleic acid according to the second aspect of the invention, the expression vector according to the third aspect of the invention. According to some embodiments of the invention, the recombinant cells can efficiently and abundantly express fusion proteins under appropriate conditions, the fusion proteins have stronger specificity, longer half-life and higher efficacy, and nucleic acid drugs can be delivered to target cells with smaller dosage, so that effective treatment or prevention of diseases is realized, the toxic and side effects are low, and the safety is higher.
According to an embodiment of the present invention, the recombinant cell may further include at least one of the following technical features:
according to an embodiment of the invention, the recombinant cell is a HEK293FT, HEK293T or BHK cell.
According to an embodiment of the invention, the recombinant cell is a HEK293FT cell.
In a fifth aspect of the invention, the invention provides a method of packaging a virus. According to an embodiment of the invention, the method comprises subjecting the recombinant cell according to the fourth aspect of the invention to a culture treatment under conditions suitable for protein expression, and subjecting the culture treatment product to the repressor removal treatment in order to obtain the virus. The inventor finds that the method for packaging viruses according to the embodiment of the invention can culture and obtain a large amount of high-purity viruses in a short time, and can realize controllable packaging and release of the viruses.
In a sixth aspect of the invention, the invention provides a method of packaging a virus. According to an embodiment of the present invention, the method comprises subjecting the recombinant cells of the fourth aspect of the present invention to a culture treatment under conditions suitable for protein expression, and subjecting the culture treatment product to a fusion protein excision treatment to obtain the virus. The inventor finds that the method for packaging viruses according to the embodiment of the invention can culture and obtain a large amount of high-purity viruses in a short time, and can realize controllable packaging and release of the viruses.
According to an embodiment of the present invention, the two methods for packaging viruses may further include at least one of the following technical features:
according to an embodiment of the invention, the fusion protein further comprises a cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein.
According to an embodiment of the invention, the repressor removal treatment comprises administering the culture treatment product during the culture to a protease treatment. After protease treatment, the fusion protein breaks, and the broken transfer protein breaks away from the control of the repressor protein and becomes a form capable of being effectively loaded into extracellular vesicles.
According to an embodiment of the invention, the intracellular region excision treatment comprises contacting the culture treatment product with a protease suitable for cleaving the cleavage site. After protease treatment, the transfer protein with the acylation tag is excised, becomes free, is modified by intracellular acyltransferase, and is coated again in a lipid-anchored manner to become a form capable of effectively entering extracellular vesicles.
In a seventh aspect of the invention, the invention provides a viral particle. According to an embodiment of the invention, the viral particles are obtained by packaging according to the method of the fifth or sixth aspect of the invention. According to the embodiment of the invention, the virus particles obtained through virus packaging are simple in acquisition method, easy to operate, short in time consumption and low in cost.
In an eighth aspect of the invention, the invention provides a nucleic acid delivery system. According to an embodiment of the invention, the system comprises a fusion protein according to the first aspect of the invention, an isolated nucleic acid according to the second aspect of the invention, an expression vector according to the third aspect of the invention, a recombinant cell according to the fourth aspect of the invention and a viral particle according to the seventh aspect of the invention. According to an embodiment of the invention, the nucleic acid delivery system has the following advantages:
1) High-efficiency conveying: the nucleic acid delivery system can accurately deliver the genetic drug into the target cell. It can have higher accuracy and efficiency than traditional chemical drugs;
2) Therapeutic is stronger: treatment of genetic diseases typically requires specific intervention against the patient's genes, while the nucleic acid delivery system is more specific therapeutic. It can treat genetic diseases by targeting specific genes, regulating gene expression, and the like;
3) The safety is high: compared with the traditional medicine, the gene medicine has higher safety and relatively fewer toxic and side effects. In addition, the nucleic acid delivery system can reduce adverse reactions of gene drugs, so that the therapeutic effect of the drugs is more remarkable;
4) The sustainability is strong: treatment of genetic disorders typically requires long-term intervention and treatment, and the nucleic acid delivery system may achieve sustainability of the treatment by multiple administrations or the like.
In a ninth aspect of the invention, the invention provides a method of delivering a nucleic acid. According to an embodiment of the invention, the method comprises contacting the nucleic acid delivery system according to the eighth aspect of the invention with the nucleic acid to be delivered. According to the embodiment of the invention, the nucleic acid delivery efficiency can be remarkably improved by adopting the method to deliver the nucleic acid, the acting speed of the medicine in cells is improved, and the expected curative effect can be achieved in the aspect of treating or preventing cancers.
According to an embodiment of the present invention, the above method of delivering a nucleic acid may further include at least one of the following additional technical features:
according to an embodiment of the invention, the nucleic acid to be delivered comprises at least one of a nucleic acid drug, mRNA and gRNA.
It should be noted that the nucleic acids to be delivered described in the present application include, but are not limited to, mRNA and gRNA, and since different nucleic acids have different advantages depending on the gene disease to be treated, the tumor type, the treatment regimen, and the like, it is necessary to selectively use nucleic acid drugs, mRNA, gRNA, and the like in clinical treatment to achieve optimal therapeutic effects.
In a tenth aspect of the invention, the invention provides a method of delivering a nucleic acid. According to an embodiment of the invention, the method comprises simultaneously expressing a receptor binding-deficient fusogenic protein and a polypeptide targeting a membrane epitope antigen in the nucleic acid delivery system of the eighth aspect of the invention. To achieve nucleic acid-specific delivery.
According to an embodiment of the invention, the membrane epitope antigen is CD3. According to embodiments of the present invention, target specific cargo delivery may be achieved using receptor binding defective fusogenic proteins (VSVg mutants) to co-express specific antibodies (CD 3 monoclonal antibodies).
In an eleventh aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the nucleic acid delivery system according to the eighth aspect of the invention and the nucleic acid drug to be delivered. As described above, the fusion protein according to the embodiment of the present invention can function in cells with high efficiency and control without causing toxic and side effects. Therefore, the pharmaceutical composition containing the above substances can also be effective in producing therapeutic effects in cells without causing toxic or side effects.
According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable carriers or excipients.
In a twelfth aspect of the invention, the invention provides the use of a pharmaceutical composition for the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a disease. According to the embodiment of the invention, the fusion protein and the nucleic acid drug to be delivered can be used for preparing the drug for treating or preventing diseases such as tumors and the like.
According to an embodiment of the present invention, the use of the above-mentioned pharmaceutical composition for the preparation of a medicament may further comprise at least one of the following additional technical features:
According to an embodiment of the invention, the disease comprises: tumor, immunotherapy, monogenic mutation, and polygenic mutation.
In a thirteenth aspect of the present invention, the present invention provides a gene editing system. According to an embodiment of the invention, the system comprises: the fusion protein according to the first aspect of the invention or the isolated nucleic acid according to the second aspect of the invention or the expression vector according to the third aspect of the invention or the recombinant cell according to the fourth aspect of the invention or the viral particle according to the seventh aspect of the invention or the nucleic acid delivery system according to the eighth aspect of the invention or the pharmaceutical composition according to the eleventh aspect of the invention. According to the embodiment of the invention, the gene editing system can be used as a means for gene editing and has the advantages of high efficiency, accuracy, programmability and the like.
In a fourteenth aspect of the present invention, the present invention provides a gene editing method. According to an embodiment of the invention, the method comprises contacting the gene to be edited with a fusion protein according to the first aspect of the invention or an isolated nucleic acid according to the second aspect of the invention or an expression vector according to the third aspect of the invention or a recombinant cell according to the fourth aspect of the invention or a viral particle according to the seventh aspect of the invention or a nucleic acid delivery system according to the eighth aspect of the invention or a pharmaceutical composition according to the eleventh aspect of the invention or a gene editing system according to the thirteenth aspect of the invention. Editing the gene by the contacting treatment.
According to an embodiment of the invention, the gene to be edited is present in a cell.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic flow chart of a streaming data processing according to embodiment 1 of the present invention; wherein, transposon receptor cell line data processing procedure (above); lentiviral receptor cell line data processing scheme (below);
FIG. 2 is a graph of the detection of the delivery efficiency of acylated PEG10 RF1 RNA according to example 1 of the present invention; wherein, PEG10 various modification variants are structurally schematic (left); the activated mCherry positive cell duty cycle in the recipient cells (right);
FIG. 3 is a graph showing the fluorescence expression induced in lentiviral receptor cell lines in comparison of the master SEND and ultra-SEND systems according to example 1 of the present invention;
FIG. 4 is a graph showing the expression of fluorescence induced in transposon receptor cell lines, in comparison with the ultra-SEND system of example 1 according to the present invention;
FIG. 5 is an acylated tag screen result according to example 2 of this invention; wherein the name of the acylated tag and the amino acid sequence (left); lentiviral receptor cell line positive cell ratio (medium); transposon receptor cell line fluorescence mean increase (right);
FIG. 6 is a comparison of the SEND system and the ultra-SEND system in different donor cells according to example 3 of the invention;
FIG. 7 is a direct delivery fluorescent protein RNA effect according to example 4 of the present invention;
fig. 8 is a delivery efficiency test result for Cas9 and gRNA after PEG10 of example 5 connects MS2 at the C-terminus according to the invention; wherein, PEG10 various modification variants are structurally schematic (left); receptor cell BFP fluorescence intensity histogram (middle); the receptor cell BFP knockout rate (right);
FIG. 9 is a graph showing the change in BFP knockout rate of a recipient cell versus the amount of VLP administered according to example 5 of the present invention;
FIG. 10 is the inhibition of the RNA delivery function of PEG10 by the extracellular domain of the membrane-anchored protein according to example 6 of the present invention; wherein the PEG10 effect is anchored using CD4 with or without an extracellular domain (left); anchoring PEG10 effects with an extracellular domain-containing or extracellular domain-free axon protein 3b (right);
FIG. 11 is a series of extracellular domain sizes required to shorten the extracellular EGFP identification inhibitory effect according to example 7 of the present invention; wherein, extracellular carries the serial shortened EGFP membrane anchoring PEG10 structure schematic diagram (left); activated mCherry positive cell duty cycle in receptor cells (right)
FIG. 12 is a small molecule drug-induced membrane on PEG10 to enhance its RNA delivery efficiency according to example 8 of the present invention; wherein, the membrane anchors FRB and FKBP-PEG10 structure schematic diagram and the treatment condition (left); the activated mCherry positive cell duty cycle in the recipient cells (right);
FIG. 13 is a schematic illustration of the function of the resected extracellular domain-recoverable membrane-anchored PEG10 portion according to example 9 of the present invention; wherein, membrane anchoring PEG10 protein structure schematic diagram and treatment condition (left); receptor cell fluorescence mean (median); membrane proteins with TEV cleavage sites versus membrane proteins without cleavage sites, TEV enzyme treated (red) versus untreated (blue) PEG10 delivered RNA activated receptor cell fluorescence distribution histogram contrast (right);
FIG. 14 is a schematic diagram of the structure and control process of a fusion protein for controlling the function of PEG10 according to example 10 of the present invention by removing the acylated tag of PEG10 from the extracellular inhibitor-bearing membrane protein and then re-coating the membrane with a lipid anchor;
FIG. 15 is a comparison of the activation rates of lentiviral receptor cells for control of PEG10 function achieved by re-filming lipid anchored after excision of the acylated tag of PEG10 from the extracellular inhibitor bearing membrane protein in accordance with example 10 of the present invention;
FIG. 16 is a diagram showing the fluorescent expression of lentiviral receptor cells with control of PEG10 function by re-coating the membrane with lipid anchor after excision of the acylated tag of PEG10 from the membrane protein with extracellular inhibitor according to example 10 of the present invention;
FIG. 17 is a schematic diagram showing the structure and control process of a fusion protein for controlling the function of PEG10 by light according to example 11 of the present invention;
FIG. 18 is a comparison of lentiviral receptor cell activation rates for controlling PEG10 function by light illumination according to example 11 of the present invention;
FIG. 19 is a histogram of fluorescent expression levels of Jurkat receptor cells for achieving target specific delivery by using mutant fusogenic proteins and coexpression antibodies according to example 12 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In describing the present invention, the terms related thereto are explained and illustrated only for convenience of understanding the scheme and are not to be construed as limiting the protection scheme of the present invention.
Definition and description
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the isolated nucleic acid described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the isolated nucleic acid.
In this context, the ultra-SEND system is referred to as an optimized SEND system, and the non-optimized SEND system is referred to as a master SEND system (see background).
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other 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 belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Fusion proteins
In one aspect of the invention, the invention provides a fusion protein. The fusion protein comprises: transfer proteins and delivery enhancing factors; the transfer protein is linked to a delivery enhancing factor. According to some embodiments of the invention, the fusion protein has stronger specificity, longer half-life and higher efficacy, can effectively treat or prevent cancer with lower dosage, has low toxic and side effects and higher safety.
The transfer protein is a protein capable of forming a viral capsid structure or a similar viral capsid structure, recognizing and encapsulating self RNA, being released from cells in the form of Extracellular Vesicles (EVs), being received and translated by downstream cells.
It should be noted that the repressor protein refers to an extracellular domain of any protein (e.g., extracellular EGFP, etc.), and is independent of the type and is only related to the size of the extracellular domain of any protein. As used herein, a "repressor protein" refers to a protein that can limit the function of a transfer protein. In this application, the inventors have experimentally verified that the extracellular domain-induced extracellular vesicle loading-inhibiting effect is independent of domain type, and is only size dependent. The desired extracellular domain sequence contains at least 140 amino acids to produce the inhibitory effect.
In the examples of the present application, the membrane-localized signal peptide is an axon protein 3b signal peptide. The inventor selects the signal peptide (axon protein 3b signal peptide) of the N-terminal of classical membrane protein axon protein 3b, and regulates and controls the PEG10 protein to anchor the upper membrane.
According to an embodiment of the invention, the transfer protein comprises at least one of PEG10 protein, arc protein, dArc1 protein. The PEG10 protein, arc protein, dARc1 protein described herein are all capable of forming a virus-like capsid structure, recognizing and encapsulating RNA. All three proteins have been shown to have transcellular transfer behavior (Elissa D.Pastuzyn et al., "The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein That Mediates Intercellular RNA Transfer," Cell 172, no.1-2 (January 2018): 275-288.e18, https:// doi.org/10.1016/j.cell.2017.12.024; james Ashley et al., "retroviruses-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic Boutons," Cell 172, no.1-2 (January 2018): 262-274.e11, https:// doi.org/10.1016/j.cell.2017.12.022.). Preferably, the transfer protein in this application is a PEG10 protein, more preferably a PEG10 protein that expresses only its first reading frame (RF 1).
The PEG10 protein is a protein existing in a natural protein library in human body, and is derived from a Long Terminal Repeat (LTR) retrotransposon. The PEG10 protein contains two Reading Frames (RF), two lengths of proteins can be expressed by one ribosome-1 frameshift site, and RF1 contains a Capsid domain (Capsd, CA) and a Nucleocapsid domain (NC) which are respectively responsible for virus Capsid formation and RNA binding; RF2 contains, in addition to CA and NC, a Protease domain (PR) which cleaves PEG10 itself, dividing the CA, NC, PR, RT four domains into four segments, and a reverse transcriptase domain (Reverse Transcriptase, RT) which has been inactivated during evolution. In the master SEND system, the expressed protein comprises RF1 and RF2 (denoted RF1+ 2); in embodiments of the invention, the fusion protein does not comprise the RF2 reading frame, only the reading frame RF1 is expressed. The PEG10 is murine PEG10 (MmPEG 10), and the humanized PEG10 can achieve the effect similar to that achieved by the murine PEG 10.
According to an embodiment of the invention, the transfer protein comprises PEG10 RF1. According to embodiments of the present invention, the RNA delivery efficiency of PEG10 RF1 was significantly better than PEG10 RF1+2 after the addition of the acylated tag.
According to an embodiment of the invention, the delivery enhancing factor comprises at least one of an acylated tag, a small molecule receptor, a classical membrane protein signal peptide, and a transmembrane region. The inventors found that the PEG10 protein can be anchored on the cell membrane by adding an acylation tag, a small molecule receptor, a classical membrane protein synthesis pathway and the like. By anchoring the multimerizing PEG10 RF1 to the membrane, the PEG10 protein can be efficiently loaded into EVs, with the best effect of the acylated tag. Based on the optimal effect of the acylation tag, the inventor further screens Myr-tag, YES1-tag, lyn-tag, LM-tag, HIV Gag-tag, nef-tag, gap43-tag, src-tag and Fen-tag on the basis of the optimal effect of the acylation tag, and finally obtains the highest RNA delivery efficiency when the acylation tag is Myr-tag.
According to an embodiment of the invention, the cleavage site is controlled by a light-sensitive protein, and can be cleaved by a corresponding protease only under illumination of a specific wavelength. In one embodiment of the present application, the RNA delivery function of the fusion protein can be restored by cleavage with a protease after irradiation with 470nm blue light.
According to an embodiment of the invention, the fusion protein further comprises a protease cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein. The inventors modified the original edition SEND system by adding repressor and protease cleavage site outside the cell, by controlling the excision of the extracellular repressor protein, or by cleaving the PEG10 protein with the acylation tag from the membrane protein with the repressor, and re-filming in a lipid anchored manner, and finally realizing the controlled release of the extracellular vesicles containing PEG10 protein and its cargo RNA.
According to an embodiment of the invention, the cleavage site comprises at least one of a TEV protease cleavage site and an MMP protease cleavage site. Herein, the cleavage site is satisfied that it is used under conditions of sequence specificity (i.e., does not recognize the rest of the fusion protein) and cleavage of the remaining first amino acid to glycine, not limited to TEV protease cleavage site and MMP protease cleavage site.
The inventors found that the most efficient Myr tag carries a single myristoylation modification and a single palmitoylation modification by screening the acylated tag, and that the myristoylation modification is located on the amino group of the glycine at the first position, so that it is necessary that the glycine at the first position after cleavage. However, at the same time, the efficiency of PEG10 is also improved by the dipalmitoyl modification (Gap 43 tag), which occurs on the cysteine side chain, and therefore in this case the cleavage site need not be limited to glycine at the first position. The adoption of the TEV enzyme cutting site can ensure that the first amino acid after cutting is glycine, while the adoption of the MMP enzyme cutting site does not necessarily ensure that the first amino acid is glycine.
Isolated nucleic acids
In another aspect of the invention, the invention provides an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid encodes the fusion protein described above.
According to an embodiment of the invention, the isolated nucleic acid is DNA or RNA.
Those skilled in the art will appreciate that the features and advantages described above for fusion proteins are equally applicable to isolated nucleic acids and will not be described in detail herein.
The isolated nucleic acid means a substance which is obtained by separating DNA or RNA in a cell or a tissue from other biomolecules under a certain condition. Such materials can be used in a variety of laboratory procedures, such as PCR, gene cloning, and sequencing.
Expression vector
In yet another aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned isolated nucleic acid.
It should be noted that the expression vector may refer to a cloning vector or a recombinant vector, and may be obtained by operably linking the nucleic acid to a commercially available vector (e.g., a plasmid or a viral vector), and commonly used plasmids include pcdna3.1. The plasmid used in the examples of the present invention was a plasmid obtained by optimizing pcDNA3.1 by the inventors.
According to an embodiment of the invention, the expression vector further comprises: a promoter operably linked to the isolated nucleic acid of the second aspect of the invention.
According to an embodiment of the invention, the promoter is selected from at least one of CMV, EF-1. Alpha., RSV.
According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
It should be noted that, the non-pathogenic viral vector refers to a viral vector used in the process, and the modified virus has lost pathogenicity and does not cause diseases to cells or human bodies.
According to an embodiment of the invention, the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus and an adenovirus-associated virus.
Recombinant cells
In yet another aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell expresses the fusion protein of the invention as described above or carries the isolated nucleic acid as described above or the expression vector as described above.
According to an embodiment of the invention, the recombinant cell is a HEK293FT, HEK293T or BHK cell.
The recombinant cells of the invention can be mammalian cells such as BHK cells, CHO cells, COS cells, HEK293FT, HEK293T, myeloma cells and the like. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, more preferably BHK, HEK293FT or HEK293T cells, most preferably HEK293FT cells.
Method for packaging viruses
In yet another aspect of the invention, the invention provides a method of packaging a virus. According to an embodiment of the invention, the method comprises subjecting the recombinant cells of the invention described previously to a culture treatment under conditions suitable for the expression of the protein, and subjecting the culture treatment product to said repressor removal treatment, so as to obtain said virus.
The method for packaging the virus has the following advantages:
1) High purity: the amount and time of expression of the target protein can be controlled when the recombinant cells are used for culturing, thereby obtaining the target protein with high purity.
2) Safety: the possible residual repressor in the recombinant cells can be removed by the repressor removal treatment, so that the potential risk of virus infection is reduced, and the safety of virus preparation is improved.
3) High yield: the target protein expressed by the recombinant cells can be produced in large quantities, so that more viruses can be obtained.
Method for packaging viruses
In yet another aspect of the invention, the invention provides another method of packaging a virus. According to an embodiment of the present invention, the method comprises subjecting the aforementioned recombinant cells of the present invention to a culture treatment under conditions suitable for protein expression, and subjecting the culture treatment product to an intracellular domain excision treatment, so as to obtain the virus. The inventor finds that the method can be used for efficiently obtaining the virus product with high purity, and has simple operation and low cost. In addition, the method can realize accurate regulation and control of the genome of the virus by a directional gene editing technology, thereby obtaining the virus with specific biological functions.
According to an embodiment of the invention, the fusion protein further comprises a cleavage site located between the extracellular domain and the transmembrane region or between the transmembrane region and the intracellular region.
According to an embodiment of the invention, the fusion protein further comprises a cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein.
According to an embodiment of the invention, the repressor removal treatment comprises administering the culture treatment product during the culture to a protease treatment. After protease treatment, the fusion protein breaks, and the broken transfer protein breaks away from the control of the repressor protein and becomes a form capable of being effectively loaded into extracellular vesicles.
According to an embodiment of the invention, the intracellular region excision treatment comprises contacting the culture treatment product with a protease suitable for cleaving the cleavage site. After protease treatment, the transfer protein is excised and becomes free, modified with intracellular acyltransferase, and then coated again in a lipid-anchored manner to become a form capable of efficiently entering extracellular vesicles.
The cleavage site as used herein refers to a specific sequence of an amino acid in a protein, and a protease recognizes the sequence and cleaves the protein into two fragments.
Virus particles
In yet another aspect of the invention, the invention provides a viral particle. According to an embodiment of the invention, the viral particles are obtained by packaging according to the method of the invention as described previously. According to the embodiment of the invention, the virus particles obtained through virus packaging are simple in acquisition method, easy to operate, short in time consumption and low in cost.
Herein, the cells of the invention or cells capable of secreting virus-like particles (for delivery of nucleic acids) include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or essentially any other type of cell source. Cells of the invention include cells that produce virus-like particles in vivo. Cells according to the invention may be selected from a wide range of cells and cell lines, such as mesenchymal stem cells or stromal cells (obtainable from, for example, bone marrow, adipose tissue, wharton's jelly, perinatal tissue, placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells and more specifically amniotic epithelial cells optionally expressing various early markers, bone marrow-suppressive cells, M2-polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human Embryonic Kidney (HEK) cells, endothelial cell lines such as microvascular endothelial cells or lymphoid endothelial cells, erythrocytes, erythroid progenitor cells, chondrocytes, MSCs of different origin, amniotic cells, amniotic Epithelial (AE) cells, any cells obtained by amniocentesis or from placenta, airway epithelial cells or alveolar epithelial cells, fibroblasts, endothelial cells, and the like. In addition, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic Cells (DCs) are also within the scope of the invention, and essentially any type of cell capable of producing extracellular vesicles is also included herein. In general, virus-like particles can be derived from essentially any cell source, whether primary or immortalized cell lines. The virus-like particle cells may be any embryonic, fetal, and adult somatic stem cell type, including induced pluripotent stem cells (ipscs) and other stem cells derived by any method. When treating neurological disorders, it is possible to consider the use of, for example: primary neural cells, astrocytes, oligodendrocytes, microglia and neuro progenitor cells are used as source cells. For the patient to be treated, the cells may be allogeneic, autologous, or even xenogeneic in nature, i.e., the cells may be from the patient himself or from unrelated, matched or unmatched donors.
Nucleic acid delivery system
In yet another aspect of the invention, the invention provides a nucleic acid delivery system. According to an embodiment of the invention, the system comprises the fusion proteins, isolated nucleic acids, expression vectors, recombinant cells and viral particles of the invention as described previously. According to the embodiment of the invention, the nucleic acid delivery system greatly improves the nucleic acid delivery efficiency, and the controllable release and the safety are higher.
Method of delivering nucleic acids
In yet another aspect of the invention, the invention provides a method of delivering a nucleic acid. According to an embodiment of the invention, the method comprises contacting the nucleic acid delivery system of the invention as described previously with the nucleic acid to be delivered. According to the embodiment of the invention, the nucleic acid delivery efficiency can be remarkably improved by adopting the method to deliver the nucleic acid, the acting speed of the medicine in cells is improved, and the expected curative effect can be achieved in the aspect of treating or preventing cancers.
Herein, the nucleic acid to be delivered includes at least one of a nucleic acid drug, mRNA, and gRNA.
Methods of introducing isolated nucleic acids into viral particles or cells are known to those of skill in the art herein, including but not limited to lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, biolistics, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. Ultrasound procedures using, for example, the Sonitron 2000 system (Rich-Mar) can also be used for the delivery of exogenous nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by amaxa biosystems (Cologne, germany), maxcyte corporation (Rockville, maryland), BTX molecule delivery systems (holiston, MA) and Copernicus Therapeutics corporation (see, e.g., US 6008336).
Method of delivering nucleic acids
In yet another aspect of the invention, the invention provides a method of delivering a nucleic acid. According to an embodiment of the invention, the method comprises simultaneously expressing a receptor binding defective fusogenic protein and a polypeptide targeting a membrane epitope antigen in the aforementioned nucleic acid delivery system of the invention.
Pharmaceutical composition
In yet another aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the aforementioned nucleic acid delivery system and the nucleic acid drug to be delivered. As described above, the fusion protein according to the embodiment of the present invention can function in cells with high efficiency and control without causing toxic and side effects. Therefore, the pharmaceutical composition containing the above substances can also be effective in producing therapeutic effects in cells without causing toxic or side effects.
According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable carriers or excipients.
Herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the extracellular vesicles administered in the composition. The pharmaceutically acceptable carrier may enhance or stabilize the composition or may be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers can include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier may be selected to minimize adverse side effects and/or minimize degradation of one or more active ingredients in the subject. Adjuvants may also be included in any of these formulations.
Herein, the term "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Formulations for parenteral administration may, for example, contain excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, ethylene vinyl acetate copolymer particles, and surfactants (including, for example, polysorbate 20).
In this context, the invention provides pharmaceutical compositions comprising a therapeutic agent and a fusion protein as described previously coupled to the therapeutic agent. The manner in which the fusion protein is coupled to the therapeutic agent may be in a conventional manner.
In this context, the pharmaceutical compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, e.g. with a chemotherapeutic agent. Thus, the pharmaceutical composition may also contain a chemotherapeutic agent.
Use of pharmaceutical compositions for the preparation of a medicament
In a further aspect of the invention, the invention proposes the use of a pharmaceutical composition for the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a disease. According to the embodiment of the invention, the fusion protein can be used for preparing medicines which have the same efficacy as the fusion protein and can be used for treating or preventing diseases such as tumors and the like. According to an embodiment of the invention, the disease comprises: genetic diseases of tumors and monogenic mutations.
In this context, the pharmaceutical compositions according to the invention may be administered by a variety of methods known in the art. The route and/or manner of administration may vary depending on the desired result. In some embodiments, the administration is intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous. The pharmaceutically acceptable carrier should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, the pharmaceutical composition comprising at least one virus-like particle and a pharmaceutically acceptable carrier or excipient may be in a form suitable for parenteral administration. In some embodiments, the pharmaceutical composition may be in the form of a sterile injectable aqueous or suspension, which may be formulated according to known procedures. The sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally-acceptable buffer.
As used herein, the term "treatment" refers to alleviating the severity and/or frequency of symptoms, eliminating symptoms and/or root causes, preventing the occurrence of symptoms and/or root causes thereof, and ameliorating or remedying the damage. Tumors are non-limiting examples of conditions that may be treated using the compositions and methods described herein. Thus, "treatment" includes: preventing a disease or condition from occurring in a mammal, particularly when such a mammal is susceptible to the condition but has not yet been diagnosed as having the disease or condition; inhibiting a disease or condition, i.e., arresting its development; alleviating a disease or condition, i.e., causing regression of the disease or condition; and alleviating or eliminating symptoms caused by a disease or condition, i.e., alleviating pain and treating or not treating the underlying disease or condition.
Gene editing system
In yet another aspect of the present invention, the present invention provides a gene editing system. According to an embodiment of the invention, the system comprises: the fusion protein or the isolated nucleic acid or the expression vector or the recombinant cell or the viral particle or the nucleic acid delivery system or the pharmaceutical composition. According to the embodiment of the invention, the gene editing system can be used as a means for gene editing, and has important significance in treating diseases caused by gene mutation and chromosome aberration.
Gene editing method
In yet another aspect of the present invention, the present invention provides a method of gene editing. According to an embodiment of the invention, the method comprises contacting the gene to be edited with the fusion protein or the isolated nucleic acid or the expression vector or the recombinant cell or the viral particle or the nucleic acid delivery system or the pharmaceutical composition or the gene editing system. Editing the gene by the contacting treatment.
According to an embodiment of the invention, the gene to be edited is present in a cell.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.
It should be noted that FKBP (FK 506-binding protein) and FRB (FKBP 12-rapamycin binding domain) are two commonly used "small molecule response binding proteins", and FKBP can bind to FRB after rapamycin (rapamycin) is added. In the following examples, FKBP-PEG10 RF1 is: PEG10 RF1 was attached to the C-terminus of FKBP (right half of the left protein structure example of FIG. 12).
Example 1: the addition of an acylation tag anchors PEG10 to the cell membrane, improving RNA delivery efficiency
1. Cell culture, transfection and virus-like particle (VLP) extraction
4X 10 cells per dish in 10cm cell culture dishes 6 Cell density HEK293FT cells were inoculated, medium was supplemented with 10% (total volume of medium) fetal bovine serum using DMEM, 37 ℃ +5% CO 2 The incubator cultures the cells. After 24h of inoculation, when the cells have grown to 70-90% confluence, the fresh medium is replaced and Neofect transfection reagent (Cat.: TF 20121201) is used to transfect the following plasmids, 3.33ug each:
1) MmPEG10rc4 (Addgene #174858, plasmid for original SEND), or a plasmid expressing the following modified optimized PEG 10: PEG10 RF1, LM-PEG10 RF1+2, LM-PEG10 RF1, myr-PEG10 RF1+2, myr-PEG10 RF1. Wherein LM and Myr are two different acylated tags, RF1 means to express only the first frame of PEG10, and rf1+2 means to express the full length of PEG10 and produce two different lengths of PEG10 protein (RF 1 and RF 2) from its intrinsic ribosomal frameshift mechanism;
2) The fusogenic protein VSVg (adedge # 12259);
3) The RNA cargo MmCargo (Cre) (Addgene # 174862).
The examples of the present invention used two control groups, the background control group and the empty vesicle control group. Wherein, the background control group only expresses the fusion promoting protein VSVg and RNA cargo, and no PEG10 related plasmid is used for representing the nonspecific random loading of the VLP to the cytoplasmic Cre RNA; the empty vesicle control group expressed only the fusogenic protein VSVg, without PEG10 or Cre-related plasmid, used as a fluorescence intensity reference value.
After 16h after transfection, the transfection medium was removed, cells were rinsed with 2mL of PBS buffer, cultured continuously with 25mL of fresh medium to collect VLPs, the supernatant was collected after 48h, centrifuged at 2000 Xg for 10min to remove cells and cell debris, filtered using a 0.45 μm filter, ultracentrifuged at 4℃for 120,000Xg for 2h (Beckman Coulter, XPN-100), the supernatant was discarded, and the pellet was resuspended in 100. Mu.L of PBS buffer to complete the VLP extraction.
2. Recipient cell construction and treatment
The examples of the present invention used two recipient cells, each based on HEK293T cells to construct stable transgenic cell lines. One constructed using lentiviral transfection and the other constructed using the PiggyBac transposon stable rotation method, both with the insertion of CMV:: DIO-mCherry reporter element for response to Cre and with EF1 alpha::: BFP-T2A-Puror for labelling and screening. The piggyBac transposon constructed recipient cell line has more inserted CMV than the lentivirus constructed recipient cell line, i.e., the DIO-mCherry elements are more numerous, and can characterize the Cre RNA delivery efficiency difference in a wider range, while the lentivirus constructed recipient cell line has higher sensitivity for detecting small amounts of Cre RNA difference. RNA delivery efficiency was evaluated in the examples of the present invention using mainly lentiviral receptor cell lines, which were used mainly to distinguish the difference in high-level RNA delivery in the case of lentiviral receptor cell lines approaching saturation (> 90% activation rate).
24 hours prior to the experiment, 200uL of medium per well, 3X 10, was used in 48 well cell culture plates 4 Cell density inoculation of two receptor cells, after VLP extraction was completed, 10uL of VLP extract was added per well for transposon receptor cell line; for lentiviral receptor cell lines, 20 μl of VLP extract was added per well. Each experimental group was replicated 3 times. After the transposon receptor cell line is continuously cultured for 24 hours and the lentiviral receptor cell line is continuously cultured for 72 hours, the collected cells are subjected to flow cytometry to detect the mCherry fluorescent protein expression level, and flow data are obtained.
3. Streaming data analysis
The obtained flow datse:Sup>A were analyzed using FlowJo 10.0 software, datse:Sup>A processing flow is shown in fig. 1, the example selects SSC-FSC primary colonies to remove cell debris, selects FSC-H-FCS-se:Sup>A linear portions to select single cells, selects BP450 channel BFP labeled recipient cells, and counts the ECD channel mCherry fluorescence mean or positive cell rate:
for transposon receptor cell lines, the RNA delivery efficiency is characterized in the following manner: counting the mCherry fluorescence intensity mean value of the receptor cells, calculating the fluorescence intensity ratio of each experimental group to the empty vesicle control group, and marking the fluorescence intensity ratio as the fluorescence mean value increment;
For lentiviral receptor cell lines, the RNA delivery efficiency was characterized in the following manner: defining the threshold value of mCherry positive cells, counting the positive cell proportion higher than the threshold value in each experimental group based on the positive cell proportion of the empty vesicle control group being less than 0.1%, and marking the positive cell proportion as the percentage of mCherry positive cells.
4. Analysis of results
The results show that Myr-tag performance is better than LM-tag in the two groups of different acylated tags tested; for each modified tag, the RNA delivery efficiency of PEG10RF1 is also generally higher than PEG10 RF1+2. Among them, the most efficient of adding the acylation tag Myr-tag and expressing PEG10RF1 was named ultra-SEND system based on this optimized RNA delivery system of PEG10, which was able to successfully activate more than 95% of the receptor cells in the lentiviral receptor cell line (FIG. 2).
Photographing two kinds of receptor cells under a confocal microscope (Zeiss LSM980, the laser intensity is identical with the fluorescence gain value), and finding that the number of receptor cells which can be activated by an ultra-SEND system is significantly higher than that of the non-optimized SEND system in lentiviral receptor cells (figure 3); in transposon receptor cell lines, the relative intensity of the fluorescent signal activated by the ultra-SEND system is significantly higher than that of the unoptimized SEND system (fig. 4). The above results indicate that the RNA delivery efficiency of the ultra-SEND system is much higher than that of the unoptimized SEND system.
Example 2: acylated tag screening
In this example, 7 additional typical acylation tags (YES 1-tag, lyn-tag, HIV Gag-tag, nef-tag, gap43-tag, src-tag, fen-tag) were selected to modify PEG10 RF1 (Myr-tag in example 1 was replaced), and the amino acid sequence of the selected acylation tag is shown in FIG. 5. The modified plasmid was introduced into cells for culturing, transfection, VLP collection, and treatment of recipient cells (HEK 293T lentiviral and transposon recipient cell lines) (the same amount of the plasmid was used as in example 1 instead of the PEG 10-related plasmid in example 1, and the RNA cargo plasmid was unchanged from the fusogenic plasmid).
Flow cytometry assays were performed on treated receptor cells (HEK 293T lentiviral receptor cell line and transposon receptor cell line) to obtain flow cytometry test off-machine data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
Analysis of the PEG10 protein containing Myr-tag and LM-tag of example 1 showed that the PEG10 protein added with Myr-tag (MGCINSKRKD) tag had the best RNA delivery efficiency, followed by YES1-tag (MGCIKSKEN) and Lyn-tag (MGCIKSKGK) tags (FIG. 5). In the tested acylation tags, the LM-tag, myr-tag, gap43-tag, lyn-tag, YES1-tag and Src-tag can reach more than 90% of activation efficiency in a lentiviral receptor cell line.
Example 3: comparison of VLP production efficiency from different donor cells
According to the examples of the present invention, HEK293T, HEK293FT and BHK cells were used as donor cells for VLP production, and the steps of cell culture, transfection, VLP collection and recipient cell treatment were the same as in example 1, and all three donor cells were seeded 24h before transfection at a seeding density of 4X 10 per 10cm dish 6 Each cell, three donor cells were subjected to the following 4 experimental groups:
(1) Non-optimized SEND: transfection of VSVg, mmCargo (Cre) and MmPEG10rc4
(2) ultra-SEND: transfection of VSVg, mmCargo (Cre) and Myr-PEG10 RF1
(3) Background control group: transfection of VSVg and MmCargo (Cre), use of empty plasmid to make up total transfected plasmid quantity
(4) Empty vesicle control group: VSVg was transfected and the total transfected plasmid amount was made up using empty plasmid
Flow cytometry detection was performed on the treated receptor cells (HEK 293T lentiviral receptor cells) to obtain flow cytometry test off-machine data.
By comparing the activation efficiency of VLPs produced by three different donor cells to the recipient cells, the results indicate that the ultra-SEND system is superior to the non-optimized SEND system in all three donor cells, the increase in RNA delivery efficiency by the ultra-SEND system is common among the different donor cells, and HEK293FT is the optimal donor cell (fig. 6).
Example 4: directly delivering fluorescent protein RNA, and proving that the ultra-SEND efficiency is higher under the condition of no amplification
According to the examples of the present invention, an RNA cargo plasmid, designated MmCargo (mNeonGreen), was constructed before and after PEG10 '/3' -UTR was linked to mNanGreen (the highest brightness monomeric green fluorescent protein that has been reported). Using HEK293T cells as donor cells, the procedure of cell transfection, VLP collection was the same as in example 1, transfected with 3.33ug each of the following plasmids:
(1) MmPEG10rc4 (Addgene # 174858) (non-optimized SEND system), or Myr-PEG10 RF1 (ultra-SEND system);
(2) The fusogenic protein VSVg (adedge # 12259);
(3) And RNA cargo MmCargo (mNeonGreen).
The background control group only expresses the fusion promoting protein VSVg and RNA cargo, and no PEG10 related plasmid is used for representing the nonspecific random loading of VLP to cytoplasmic RNA; the empty vesicle control group expressed only the fusogenic protein VSVg, without PEG10 and cargo RNA, used as a fluorescence intensity reference value.
The recipient cells were incubated with HEK293T cells without stable transformation, and the collected VLPs were added to the recipient cells in proportion (as in example 1) for two additional days, followed by flow cytometry detection of the collected cells to obtain flow cytometry test off-machine data. Similarly, the flow cytometry test run-down data obtained were analyzed using FlowJo 10.0 software, the first two steps of the data processing procedure (selection of primary colonies and single cells) were identical to fig. 1, and FITC channel green fluorescence intensity of selected cells was counted in the third step.
The plasmid constructed according to this example enables RNA of mNanGreen to be recognized by PEG10 and packaged into VLPs, delivering mNanGreen directly using ultra-SEND and original edition SEND, without amplification by Cre-DIO system, and observing the expression of green fluorescence directly. The results indicate that the ultra-SEND system can directly detect its increased green fluorescence expression by flow cytometry, while the RNA delivery capacity of master SEND is not sufficient to distinguish from the control group (fig. 7), indicating that the RNA delivery efficiency of ultra-SEND is much higher than that of master SEND.
Example 5: connecting MS2 protein at C end of PEG10 RF1, efficiently delivering gRNA and Cas9 for gene editing
This example uses the BFP knockout system to characterize gene editing efficiency: HEK293T receptor cells constructed using lentiviral transfection as described in example 1, in which the inserted EF 1. Alpha. BFP-T2A-Puror cell can express BFP (blue fluorescent protein), a gRNA (guide RNA) designed to target the BFP can be guided to knock out BFP in the receptor cells by ultra-SEND if it is able to deliver the gRNA together with Cas9 into the receptor cells, and the ratio of the reduced fraction of BFP fluorescence intensity in the receptor cells (BFP knock-out rate) can be detected using flow cytometry, reflecting the efficiency of the ultra-SEND delivery CRISPR system for gene editing.
The following plasmids were constructed in this example:
PEG10 related plasmids: MS2 protein (Myr-PEG 10RF 1-MS 2) was attached to the C-terminus of Myr-PEG10 RF1, or MS2 protein (PEG 10RF 1+2-MS 2) was attached to the C-terminus of PEG10RF1 +2. The MS2 protein is capable of binding to RNA with MS2 stem loop structure.
gRNA plasmid: two MS2 stem loop structures (2×MS 2-stemloop) were added to the gRNA backbone sequence (gRNA scaffold), designated BFP-gRNA-2×MS2-stemloop, whose nucleotide sequence is shown in SEQ ID NO:5, and the gRNA was expressed using the U6 promoter.
GGGGTGAACTTCACATCCAAGTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTGCAGG GCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCAACATGAGGATCACCCATGTCTGC AGGGCCAAGTGGCACCGAGTCGGTGC(SEQ ID NO:5)。
Using HEK293FT cells as donor cells, the cell culture, transfection, VLP collection procedure was the same as in example 1, transfected with 2.5ug each of the following plasmids:
(1) Cas9 RNA capable of being recognized and loaded by PEG 10: mmCargo (Cas 9) (Addgene# 174865);
(2)BFP-gRNA-2×MS2-stemloop；
(3) The fusogenic protein VSVg (adedge # 12259);
(4) PEG 10-related plasmids, each experimental group was: PEG10RF1+2, PEG10 RF1+2-MS2, myr-PEG10 RF1,
Myr-PEG10 RF1-MS2。
wherein, the background control group only expresses the fusion proteins VSVg, gRNA and MmCargo (Cas 9), without PEG 10-related plasmid, for characterizing the non-specific random loading of VLPs to cytoplasmic RNAs; the empty vesicle control group expressed only the fusogenic protein VSVg, used as a fluorescence intensity reference value.
The recipient cells (HEK 293T lentiviral recipient cells) were seeded in the same manner and seeding density as in example 1 and the incubation time was extended to 5 days to allow adequate degradation of BFP accumulated prior to VLP addition to the cells. And performing flow cytometry detection on the treated receptor cells to obtain flow cytometry test machine-setting data. Similarly, the flow cytometry test run-down data obtained were analyzed using FlowJo 10.0 software, the first two steps of the data processing procedure (selecting the main community and single cells) were identical to those of fig. 1, and the proportion of BFP fluorescence reduction part of BP450 channel in the experimental group was counted based on the empty vesicle control group in the third step.
The results indicate that PEG10 RF1 with an acylation modification and attached MS2 is able to efficiently deliver Cas9 and gRNA into the recipient cell, knockout target gene (BFP), knockout efficiency about 75%, far higher than editing efficiency previously reported by master SEND. Whereas none of PEG10 RF1+2, myr-PEG10 RF1 lacking MS2, or PEG10 RF1+2-MS2 that was not membrane anchored, achieved efficient delivery (FIG. 8).
Further, the above experimental procedure was repeated using the most efficient Myr-PEG10 RF1-MS2, and the amount of VLPs added to the recipient cells was changed in a gradient, and 2.5uL, 5uL, 10uL, 15uL, 20uL, 25uL, 30uL or 50uL of the extracted VLPs were added to the recipient cells, respectively, and BFP knockout rate-VLP dose curves were made, and it was found that a small amount of VLPs (2.5 uL) could achieve about 50% gene editing efficiency, and the editing efficiency was nearly saturated at about 15uL (FIG. 9).
Example 6: inhibition of PEG10 by Membrane Anchor protein extracellular Domain
This example constructs the following plasmids according to conventional methods:
(1) The signal peptide expressing the classical type I membrane protein neuroxin 3b (axon protein 3 b) was attached to the transmembrane domain followed by PEG10 RF1.
(2) EGFP (green fluorescent protein) was added between the Neurexin 3b signal peptide and the transmembrane domain, as in the plasmid of (1).
(3) The membrane localization signal peptide Igkappa is expressed with the extracellular domain, transmembrane region and intracellular region of the classical type I membrane protein CD4 (CD 4 amino acid sequences 26-458) followed by the attachment of PEG10 RF1.
(4) The membrane localization signal peptide igκ was expressed with the transmembrane and intracellular domains of the classical type I membrane protein CD4 (CD 4 amino acid sequences 387-458) followed by the attachment of PEG10 RF1.
The constructed plasmid was introduced into cells for culturing, transfection, VLP collection, and treatment of recipient cells (HEK 293T lentiviral recipient cell line) (the same amount of the plasmid was used as in example 1 instead of the PEG 10-related plasmid in example 1, and the RNA cargo plasmid and the fusogenic plasmid were unchanged). And performing flow cytometry detection on the treated receptor cells to obtain flow cytometry test machine-setting data.
Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1. The results of comparing the effect of the classical membrane protein synthesis pathway with the effect of the addition of the acylation tag to anchor PEG10 show that the membrane-anchored PEG10 synthesized by the classical membrane protein pathway has a certain effect as well, but if it has a larger (more than 140 amino acids, see example 7) extracellular domain (in this case EGFP or CD4 extracellular domain), the function of PEG10 will be inhibited (fig. 10).
Example 7: determination of minimum amino acid number of extracellular repressor
According to the examples of the present invention, extracellular EGFP series truncated plasmids of EGFP-Neuraxin 3b TM-PEG10 RF1 fusion protein were constructed and introduced into cells for culturing, transfection, VLP collection, and receptor cell treatment (same as in example 1). Flow cytometry detection was performed on the treated receptor cells (HEK 293T lentiviral receptor cell line) to obtain flow cytometry off-machine data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
The results indicate that starting from GFP a 1-187, the progressively shorter extracellular domain has not been able to completely inhibit PEG10 function, and considering the length of the neuroxin 3b signal peptide and its accessory sequence used to express the fusion protein, the shortest extracellular domain capable of producing a complete inhibition effect (GFP a 1-135) requires about 140 amino acids (fig. 11), which is the shortest size required for the extracellular domain to induce PEG10 loading inhibition effect.
Example 8: the small molecule receptor anchors PEG10 to cell membrane, improving its RNA delivery efficiency
According to an embodiment of the invention, the cell culture, VLP collection and recipient cell (HEK 293T lentiviral recipient cell line) processing steps are the same as example 1. Wherein the cell transfection procedure was modified using plasmids: each 10cm dish was transfected with 2.5ug of the following plasmid:
(1) Membrane anchored FRB: neurexin3b TM-FRB, or EGFP-Neurexin3b TM-FRB, or membraneless anchored FRB and the total transfected plasmid amount was filled in using an equal amount of empty plasmid.
(2)FKBP-PEG10 RF1。
(3) The fusogenic protein VSVg (Addgene # 12259).
(4) The RNA cargo MmCargo (Cre) (Addgene # 174862).
Wherein, the background control group only expresses the fusion promoting protein VSVg and RNA cargoes, and no PEG10 related plasmid and FRB related plasmid are used for representing the nonspecific random loading of the VLP to the cytoplasmic Cre RNA; the empty vesicle control group expressed only the fusogenic protein VSVg, without PEG10 or Cre or FRB-related plasmid, used as a fluorescence intensity reference value.
The experimental group was charged with rapamycin (solarbio.r8140, dissolved in DMSO stock solution at a concentration of 2.5 mM) at a final concentration of 200nM at the time of VLP collection with 25mL of fresh medium, for recruitment of free FKBP-PEG10 RF1 onto cell membranes. An equal volume of DMSO (dimethyl sulfoxide) was added to the control group.
And performing flow cytometry detection on the treated receptor cells to obtain flow cytometry test machine-setting data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
The results indicate that the recruitment of PEG10 RF1 to the cell membrane using rapamycin enhances its RNA delivery capacity, whereas if the protein on the cell membrane used to recruit PEG10 RF1 had a larger extracellular domain (EGFP), the induction of PEG10 RF1 to the upper membrane did not increase its RNA delivery capacity, further verifying that there was an inhibitory effect of the extracellular domain on EV loading of PEG10 (fig. 12).
Example 9: membrane protein extracellular domain excision
This example uses the cleavage method (TEV protease) to cleave off the extracellular domain. The following plasmids were constructed: contains the full-length classical type I membrane protein Neuroxin 1b (axon 1b, nrxn1 b) followed by PEG10 RF1 and the addition of a TEV cleavage site (TEVcs), designated Nrxn1b-TEVcs-PEG10, between the extracellular domain of Neuroxin 1b and the transmembrane region.
The constructed plasmid was introduced into cells using HEK293T cells as donor cells for cell culture, VLP collection, and recipient cell treatment (same as in example 1), wherein TEV protease (Beyotime, P2308) was added to the experimental group at a final concentration of 10U/mL while 25mL fresh medium was changed to collect VLPs, and EGFP-Nrxn3bTM-PEG10 without TEV cleavage site was used for the control group.
Flow cytometry detection was performed on the treated receptor cells (HEK 293T transposon receptor cells) to obtain flow cytometry test off-machine data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
The results indicate that the removal of the extracellular domain by TEV can restore part of PEG10 function (fig. 13).
Example 10: PEG10 is excised from membrane proteins with extracellular inhibitors and then re-coated in a lipid anchored manner to effect control of PEG10 function
The present example constructs the following fusion proteins: the N-terminus is a membrane-localized signal peptide, followed by an extracellular repressor (EGFP in this case), followed by a transmembrane region (Neurexin 3b transmembrane region in this case), followed by a TEV cleavage site, followed by an acylation tag and PEG10RF1, designated EGFP-Nrxn3b TM-TEVcs-Myr-PEG10 RF1. An example of the structure of the fusion protein is shown in FIG. 14.
PEG10 function was inhibited by the presence of an extracellular repressor before TEV protease cleaved the fusion protein. When the TEV protease is expressed to cleave the fusion protein, the acylation tag and PEG10 are separated from the inhibition of the repressor protein, the acylation tag is in a free state in the cell, the acylation tag can be modified by the endogenous acylase in the cell and a fatty chain is added, so that the PEG10 is coated again in a lipid anchoring manner, and the lipid anchoring PEG10 can effectively play a role in RNA delivery (example 1), thereby realizing active control of the function of the PEG10 (FIG. 14).
The TEV protease used in this example was the high efficiency TEV protease ultra-TEV protease (uTEVp) reported previously, and the amino acid sequence was referenced to CMV-GFP-CaM-uTEVp (Addgene # 163028).
The constructed plasmid was introduced into cells for cell culture, VLP collection, and recipient cell treatment (as in example 1), and the transfection procedure was changed to: the following plasmids were transfected at 2.5ug each:
(1)EGFP-Nrxn3b TM-TEVcs-Myr-PEG10 RF1
(2) uTEVp, or equivalent empty plasmid
(3) Fusion promoting protein VSVg (Addgene # 12259)
(4) The RNA cargo MmCargo (Cre) (Addgene # 174862).
Flow cytometry detection was performed on the treated receptor cells (HEK 293T lentiviral receptor cells) to obtain flow cytometry test off-machine data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
The results indicate that the extracellular repressor (EGFP in this case) is capable of inhibiting the function of PEG10 in the uncleaved case; expression of uTEVp cleaves the acylated tag Myr and PEG10 RF1 from the membrane anchored protein with the extracellular repressor, PEG10 is able to resume RNA delivery function (fig. 15). The above results demonstrate that the controlled release of cargo RNA can be achieved by adding an extracellular repressor to inhibit PEG10 function, cleaving the acylated tagged PEG10 with a protease to release the repressor, and re-membrane to resume function in a lipid anchored manner.
Taking a picture of the receptor cells under a confocal microscope (Zeiss LSM980, the laser intensity is exactly the same as the fluorescence gain value), it can be seen that more receptor cells are activated in the experimental group expressing the uTEVp, while the activation level of the experimental group not expressing the uTEVp for cutting is similar to that of the background control group (fig. 16), indicating that the active control of the release of the cargo RNA can be achieved by the above method.
Example 11: control of PEG10 function by light-operated cutting
The present example constructs the following fusion proteins: the addition of the photoactive proteins CIBN and hLOV1 between the transmembrane region (Nrxn 3b TM) and the TEV cleavage site (TEVcs) of the EGFP-Nrxn3b TM-TEVcs-Myr-PEG10 RF1 fusion protein described in example 10, with TEVcs immediately adjacent to the C-terminus of hLOV1, denoted EGFP-Nrxn3b TM-CIBN-hLOV1-TEVcs-Myr-PEG10 RF1; the N-terminal of uTEVp is added with a photoprotein CRY2, which is denoted as CRY2-uTEVp. An example of the structure of two fusion proteins is shown in FIG. 17.
CIBN, CRY, hLVO1 the hLOV1 can embed the C-terminal TEVcs in the center of the hLOV1 under the condition of no blue light irradiation in response to 470nm blue light irradiation, so as to prevent uTEVp from cutting the TEVcs; under blue light irradiation, hLOV1 releases TEVcs, allowing it to be cleaved by uTEVp, and CIBN binds to CRY, recruiting uTEVp to the vicinity of the fusion protein, efficiently cleaving TEVcs, releasing the acylated-tag-bearing PEG10 RF1, restoring its RNA delivery function (fig. 17).
The constructed plasmid was introduced into cells for cell culture, VLP collection, and recipient cell treatment (as in example 1), and the transfection procedure was changed to: the following plasmids were transfected at 2.5ug each:
(1)EGFP-Nrxn3b TM-CIBN-hLOV1-TEVcs-Myr-PEG10 RF1
(2)CRY2-uTEVp
(3) Fusion promoting protein VSVg (Addgene # 12259)
(4) The RNA cargo MmCargo (Cre) (Addgene # 174862).
Two groups of dishes were transfected in parallel under the same conditions, light and dark groups, respectively. Immediately after the addition of the transfection reagent, both groups of dishes were wrapped with aluminum foil for shading cultivation, operated strictly in the dark during subsequent treatment of the dark group, and illuminated with 660nm red light which did not activate the hLOV1 protein. After starting to collect VLPs by changing 25mL of fresh medium, the light group removed the aluminum foil and was given 470nm blue light at 60mW/cm 3 The 10 second light and the 5 second dark alternate, each light lasting for 10 minutes, once per hour.
Flow cytometry detection was performed on the treated receptor cells (HEK 293T lentiviral receptor cells) to obtain flow cytometry test off-machine data. Similarly, the flow cytometry test run-down data obtained was analyzed using FlowJo 10.0 software, and the data processing flow is shown in fig. 1.
The results showed that the light-treated experimental group had higher activation rate than the dark group of receptor cells (fig. 18), indicating that the above design can manipulate protease cleavage or no, and achieve controlled release of RNA by light.
Example 12: co-expression of specific antibodies using receptor binding defective fusogenic proteins for targeted specific cargo delivery
According to an embodiment of the present invention, a mutant of the fusogenic protein VSVg (K47Q, R354A) (Addgene # 182229) is used that fails to actively bind to the receptor of VSVg itself (low density lipoprotein receptor LDLRs) on the receptor cell membrane, but retains the ability to promote membrane fusion. If antibodies are expressed simultaneously on the surface of the VLP, the VLP may be directed into a receptor cell with the corresponding antigen, enabling targeted specific cargo delivery.
This example uses the RNA cargo plasmid MmCargo (mNeonGreen) of example 4. Using HEK293T cells as donor cells, the procedures of cell transfection, VLP collection, etc. were the same as in example 1, with 3.33ug of each of the following plasmids:
(1) Myr-PEG10RF1 (ultra-SEND system);
(2) The fusion-promoting protein VSVg mutant VSVg (K47Q, R354A) (Addgene# 182229);
(3) RNA cargo MmCargo (mNeonGreen);
(4) CD3 monoclonal antibodies (specifically recognizing CD3 antigen on Jurkat cells).
The control group expressed only Myr-PEG10RF1, VSVg (K47Q, R354A) and RNA cargo, without CD3 monoclonal antibody-related plasmid, to characterize background recognition of VLPs to specific cells without antibody guidance.
The recipient cells used were Jurkat cells whose surface expressed CD3 protein. The collected VLPs were added to the recipient cells in proportion (as in example 1) for two days following which the cells were collected for flow cytometry detection to obtain flow cytometry off-machine data. Similarly, the flow cytometry test run-down data obtained were analyzed using FlowJo 10.0 software, the first two steps of the data processing procedure (selection of primary colonies and single cells) were identical to fig. 1, and FITC channel green fluorescence intensity of selected cells was counted in the third step.
The results showed that the expression of green fluorescence of the recipient cells was higher than that of the control group in the presence of CD3 monoclonal antibodies (fig. 19), demonstrating that specific delivery of RNA cargo to Jurkat cells could be achieved using VSVg mutants and CD3 monoclonal antibodies.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (30)
1. A fusion protein comprising: transfer proteins and delivery enhancing factors; the transfer protein is linked to a delivery enhancing factor.
2. The fusion protein of claim 1, wherein the transfer protein comprises at least one of PEG10 protein, arc protein, dArc1 protein;
preferably, the transfer protein comprises a PEG10 protein; more preferably, the transfer protein comprises only the first reading frame of the PEG10 protein;
optionally, the C-terminus of the transfer protein may be linked to a cargo protein, including MS2.
3. The fusion protein of claim 1, wherein the C-terminus of the delivery-enhancing factor is linked to the N-terminus of the transfer protein;
optionally, a modification site is included between the delivery enhancing factor and the transfer protein;
optionally, the modification site comprises at least one of a glycine-tryptophan linker peptide, a nuclear export signal;
Optionally, the delivery enhancing factor comprises at least one of an acylated tag, a small molecule receptor, a classical membrane protein signal peptide, and a transmembrane region;
preferably, the delivery enhancing factor is an acylated tag;
optionally, the acylation tag comprises at least one of Myr-tag, YES1-tag, lyn-tag, LM-tag, HIV Gag-tag, nef-tag, gap43-tag, src-tag, fen-tag;
preferably, the acylated tag is Myr-tag.
4. The fusion protein of claim 1, wherein the fusion protein is further linked at the N-terminus to the C-terminus of the transmembrane domain;
optionally, the transmembrane domain is further linked to an extracellular repressor protein;
optionally, the repressor protein comprises at least one of an EGFP protein, an axon protein 1b extracellular domain, a CD4 extracellular domain;
optionally, the repressor protein is no less than 140 amino acids;
optionally, a modification protein is included between the delivery enhancing factor and the transmembrane domain;
optionally, the modified protein comprises at least one of CIBN, hVOL 1.
5. The fusion protein of claim 4, wherein the N-terminus of the repressor protein is further linked to the C-terminus of a membrane-localized signal peptide;
Optionally, the membrane-localized signal peptide is selected from at least one of an igκ signal peptide, a VSVg signal peptide, an axon protein 3b signal peptide.
6. The fusion protein of claim 5, further comprising a cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein;
optionally, the cleavage site is controlled by a light-sensitive protein, capable of being cleaved by the corresponding protease only under illumination of a specific wavelength;
optionally, the cleavage site comprises at least one of a TEV protease cleavage site and an MMP protease cleavage site.
7. The fusion protein of claim 4 or 5, wherein the repressor protein has an amino acid sequence as set forth in SEQ ID No. 1;
optionally, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO. 2;
optionally, the membrane-localized signal peptide has an amino acid sequence as shown in SEQ ID NO. 3.
8. The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence set forth in SEQ ID NO. 4.
9. An isolated nucleic acid encoding the fusion protein of any one of claims 1-8.
10. The isolated nucleic acid of claim 9, wherein the isolated nucleic acid is DNA or RNA.
11. An expression vector carrying the isolated nucleic acid of claim 9 or 10.
12. The expression vector of claim 11, further comprising: a promoter operably linked to the isolated nucleic acid of claim 9 or 10.
13. The expression vector of claim 12, wherein the promoter is selected from at least one of CMV, EF-1 a, RSV.
14. The expression vector of claim 12, wherein the expression vector is a non-pathogenic viral vector.
15. The expression vector of claim 14, wherein the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, and an adenovirus-associated virus.
16. A recombinant cell expressing the fusion protein of any one of claims 1 to 8 or carrying the isolated nucleic acid of claim 9 or 10 or the expression vector of any one of claims 11 to 15.
17. The recombinant cell of claim 16, wherein the recombinant cell is a HEK293FT, HEK293T or BHK cell;
Preferably, the recombinant cell is a HEK293FT cell.
18. A method of packaging a virus, characterized in that the recombinant cell of claim 16 or 17 is subjected to a culture treatment under conditions suitable for protein expression;
subjecting the culture treated product to said repressor removal treatment so as to obtain said virus.
19. A method of packaging a virus, characterized in that the recombinant cell of claim 16 or 17 is subjected to a culture treatment under conditions suitable for protein expression;
the culture treated product is subjected to fusion protein excision treatment to obtain the virus.
20. The method of claim 18 or 19, wherein the fusion protein further comprises a cleavage site located between the repressor protein and the transmembrane domain or between the transmembrane domain and the fusion protein;
optionally, the repressor removal treatment comprises contacting the culture treatment product with a protease suitable for cleaving the cleavage site;
optionally, the fusion protein excision treatment comprises contacting the culture treatment product with a protease suitable for cleaving the cleavage site.
21. A viral particle obtainable by packaging according to the method of any one of claims 18 to 20.
22. A nucleic acid delivery system comprising the fusion protein of any one of claims 1 to 8, the isolated nucleic acid of claim 9 or 10, the expression vector of any one of claims 11 to 15, the recombinant cell of claim 16 or 17, or the viral particle of claim 21.
23. A method of delivering a nucleic acid, wherein the nucleic acid delivery system of claim 22 is contacted with the nucleic acid to be delivered;
optionally, the nucleic acid to be delivered comprises at least one of a nucleic acid drug, mRNA, and gRNA.
24. A method of delivering a nucleic acid, wherein the polypeptide of the receptor binding-deficient fusogenic protein and the membrane-targeting epitope antigen are expressed simultaneously in the nucleic acid delivery system of claim 22;
optionally, the membrane epitope antigen is CD3.
25. A pharmaceutical composition comprising the nucleic acid delivery system of claim 22 and a nucleic acid drug to be delivered;
optionally, further comprising: pharmaceutically acceptable carriers or excipients.
26. Use of the pharmaceutical composition of claim 25 in the manufacture of a medicament for the treatment or prevention of a disease.
27. The use according to claim 26, wherein the disease comprises tumors, immunotherapy, monogenic mutations and polygenic mutated genetic diseases.
28. A gene editing system, the system comprising: the fusion protein of any one of claims 1 to 8 or the isolated nucleic acid of claim 9 or 10 or the expression vector of any one of claims 11 to 15 or the recombinant cell of claim 16 or 17 or the viral particle of claim 21 or the nucleic acid delivery system of claim 22 or the pharmaceutical composition of claim 25.
29. A method of gene editing comprising contacting a gene to be edited with the fusion protein of any one of claims 1 to 8 or the isolated nucleic acid of claim 9 or 10 or the expression vector of any one of claims 11 to 15 or the recombinant cell of claim 16 or 17 or the viral particle of claim 21 or the nucleic acid delivery system of claim 22 or the pharmaceutical composition of claim 25 or the gene editing system of claim 28.
30. The method of claim 29, wherein the gene to be edited is present in a cell.
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| CN202310396772.0A CN116444685A (en) | 2023-04-13 | 2023-04-13 | Efficient controllable RNA delivery system |
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