WO2018184267A1 - mRNA编码的纳米抗体及其应用 - Google Patents

mRNA编码的纳米抗体及其应用 Download PDF

Info

Publication number
WO2018184267A1
WO2018184267A1 PCT/CN2017/082096 CN2017082096W WO2018184267A1 WO 2018184267 A1 WO2018184267 A1 WO 2018184267A1 CN 2017082096 W CN2017082096 W CN 2017082096W WO 2018184267 A1 WO2018184267 A1 WO 2018184267A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nanobody
mrna
rna
cells
Prior art date
Application number
PCT/CN2017/082096
Other languages
English (en)
French (fr)
Inventor
谢维
郝睿
周雪晨
周云燕
苏志鹏
Original Assignee
东南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东南大学 filed Critical 东南大学
Priority to US16/484,977 priority Critical patent/US20200024365A1/en
Publication of WO2018184267A1 publication Critical patent/WO2018184267A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to mRNA-encoded Nanobodies and applications thereof, and belongs to the field of biomedicine, in particular to the field of nano-antibody drugs.
  • plasmid-represented DNA or virus as a medium, the expression of functional proteins in cells is a common means of gene therapy. Although this method has high transfection and expression efficiency, it cannot avoid the recombination of DNA and target cell genome. And the risk of other diseases. For example, exogenous DNA may be inserted inside a normal gene, causing mutation or even complete destruction of the expression of the gene. If the inserted gene is a more important functional gene, this change will have a significant impact on cell function. Destruction, and even lead to cell transformation to produce cancer. Using DNA-based plasmids or viruses as a tool, it is theoretically impossible to avoid the associated cancer risk, which is also an important reason why gene therapy methods have not been fully promoted.
  • RNA can't be reverse transcribed into DNA after it enters the cell, so it does not theoretically destroy the stability of the cell genome. It can be said that RNA therapy fundamentally avoids the cancer risk of gene therapy, and is more suitable. The safety needs of clinical medications.
  • DNA needs to enter the nucleus to be transcribed and expressed. It is difficult to transfect, and to some extent, the cells need to be in an active division cycle to enter the nucleus; and RNA can be expressed as long as it enters the cytoplasm, without nucleus transcription. It is also not dependent on the cell cycle. Under the same transfection conditions, RNA is more efficient than DNA in intracellular expression, and it is less difficult to apply in clinical practice.
  • the present invention proposes a novel idea and method system for regulating intracellular proteins by RNA-encoding nano-antibodies.
  • VHH variable domain of Heavy The chain antibody
  • sdAb single-domain antibody
  • Nanobodies are the smallest known antigen-binding proteins that can be stably expressed. The molecular size is only one-tenth the size of conventional IgG antibodies, and has the advantages of stable structure and strong specificity.
  • a mRNA-encoded Nanobody that encodes, encodes, and expresses a single-chain Nanobody that binds to a target protein. That is, a method and a tool system for expressing a specific Nanobody mRNA in a cell of interest are described in the present invention.
  • the tool system comprises: at least one in vitro transcribed RNA molecule encoding a Nanobody, a transfection tool that facilitates entry of the RNA molecule into the cell, and an auxiliary agent that enhances the transfection complex to recognize a particular cell and increase its transfection efficiency.
  • the RNA molecule After being transported inside the target cell, the RNA molecule can be recognized by a translational device within the cell to express a single-chain Nanobody that binds to a specific target protein. Depending on the way in which the Nanobody binds to the protein of interest, the conformation of the protein of interest undergoes different forms of modification, thereby effecting the inhibition or activation of the biological function of the protein of interest.
  • the present invention discloses the construction of an RNA molecule to encode a Nanobody that recognizes a specific target protein, by introducing certain chemical modifications and nucleotide sequences, increasing the stability and translation efficiency of the RNA in vivo, and transferring the RNA into a specific cell and Methods and tool systems for achieving intracellular expression of corresponding Nanobodies.
  • the method and tool system are particularly useful for medically treating diseases caused by dysfunction of specific proteins in cells.
  • the treatment of a cell with a specific protein function defect by the tool system described in the present invention differs according to the result of the binding of the produced Nanobody to the target protein, and the therapeutic applicability includes two aspects: one is that the RNA-encoded Nanobody is combined.
  • RNA-encoded Nanobodies stabilize the conformation of the target protein in a highly active state, thereby enhancing the protein.
  • the Nanobody referred to in the present invention refers to a variable region domain of a camel-derived heavy chain antibody which is capable of specifically binding to an antigenic epitope on a target protein. Since it is a single-chain protein and its structure is relatively stable, the usual Nanobodies are obtained by high expression of microorganisms such as Escherichia coli or yeast. In order to obtain a Nanobody that regulates the function of the target protein, the antigenic epitope of the purified antigen protein or protein is usually mixed with a suitable immunological adjuvant, and then the camel animal such as a Bactrian camel or an alpaca is injected multiple times.
  • the stimulated immune system produces B cells capable of secreting antigen-protein-specific heavy chain antibodies through cell activation, differentiation and maturation; collects total B cells from peripheral blood of immunized animals, extracts total RNA and reverses Record the cDNA library, or screen the B cells that can bind to the target antigen by flow cytometry, extract the total RNA and reverse-transcribe the cDNA library; use the cDNA library as a template and pass the nested PCR with specific primers (sequence 1).
  • the gene encoding the variable region of single-domain heavy chain antibody (VHH) was amplified, and the nano-antibody library capable of recognizing the target antigen protein was screened by phage display technology; these genes were transferred into E.
  • the nanobody antibody strain is determined by ELISA to determine the antibody strain having higher affinity with the antigen protein, and the coding genes of these nano antibodies are sequenced; the gene sequence of each clone is analyzed according to the sequence alignment software, and FR1, FR2 are removed. Redundant clones with identical FR3, FR4, CDR1, CDR2, and CDR3 sequences, and sequences with similar CDR3 regions (sequence similarity > 80%) were grouped together, and selected nanonucleotide clones with higher affinity in each group were in the large intestine. Highly expressed and purified in the bacillus to obtain the corresponding Nanobody; biochemical means to test the effect of different Nano-antibodies on the function of the antigenic protein, thereby obtaining activation Inhibiting the function of the protein Nanobodies.
  • a gene sequence encoding a functional Nanobody is obtained by sequencing, and the data can be used as a basic sequence information for synthesizing a nano-antibody RNA by biological means in the present invention.
  • Biological means generally refers to a method for obtaining RNA by in vitro transcription, which refers to a process in which an RNA is used as a template, NTP is used as a raw material, and RNA polymerase is used to mimic the in vivo transcription process to generate RNA in an in vitro cell-free system.
  • Nanobody gene sequences and corresponding RNAs are described in the method, either by flow cytometry and phage display technology, or by high-throughput sequencing technology (or Next Generation Sequencing, NGS).
  • the bioinformatics analysis method selects a DNA clone of a single domain heavy chain antibody variable region which can specifically bind to a target protein from a single-domain heavy chain antibody library of camel-derived immunity, and performs sequencing, expression, and viability identification of the obtained clone.
  • a functional Nanobody capable of specifically binding to a target protein and a gene sequence thereof are obtained, and RNA encoding the Nanobody is obtained by chemical synthesis or in vitro transcription according to the obtained gene sequence.
  • the nanobody gene sequences obtained by phage display technology are often located on phage display plasmids, including pHEN1, pHEN4, pMES4 or pMESy4, etc. Since the copy number of these plasmids is not high, the optimized method is to adopt the method of subcloning.
  • the gene encoding the Nanobody is transferred to other plasmids suitable for in vitro transcription, including pUC18, pUC19 and other high copy plasmids, pT7Ts, pGEM-1 series plasmid or pSP64 plasmid, etc.; also can be obtained by PCR for a large number of in vitro Transcribed DNA template.
  • the mRNA molecule carries at least one modification that enhances its stability.
  • a preferred embodiment is that the RNA molecule carries a variety of modifications to enhance its stability.
  • modifications that enhance stability include: replacing a naturally unmodified nucleotide with a chemically modified nucleotide (nucleotide), and increasing the GC in the RNA without affecting the amino acid composition of the encoded Nanobody.
  • the content of the base, and the introduction of a non-coding sequence (UTR) or the like which increases the stability at both ends of the RNA.
  • the increase in the content of GC bases in RNA mentioned here refers to comparison For AU base pairs, the proportion of GC bases should be increased as much as possible.
  • the present invention recommends the addition of specific non-coding sequences, i.e., 5'-UTR and 3'-UTR, upstream and downstream of the coding sequence, without affecting the amino acid sequence of the encoded protein.
  • specific non-coding sequences i.e., 5'-UTR and 3'-UTR
  • the length of the poly A tail is not less than 100, 200, 300 or 400 nucleotides.
  • the method of adding long pollin A tail includes the direct addition of polyadenylation at the end of the RNA transcript by poly A polymerase, or the direct introduction of poly A at the 3' end of the DNA template transcribed in vitro, or by RNA ligation.
  • the enzyme directly links poly A to the 3' end of the transcribed RNA product.
  • the RNA can be controlled to express the Nanobody in the cell by adjusting the length of the poly A tail carried by the RNA transcript. time.
  • polyA tail In addition to polyA tail, another common scenario is to introduce specific 5'-UTR and 3'-UTR regions downstream of the gene encoding the Nanobody, particularly C base repeats downstream of the mRNA stop codon. It has been shown to increase the stability of mRNA.
  • the present invention recommends non-coding regions of mRNA derived from other stable natural proteins, such as the 5'-UTR and 3'-UTR regions of protein mRNAs such as collagen, globin, actin, tubulin, GAPDH, and hison. The introduction of these non-coding regions upstream and downstream of the coding sequence also increases the stability of the corresponding RAN transcript.
  • homologous sequences were extracted from UTR regions upstream and downstream of various high-stable protein mRNA coding regions, and the following two new sequences (sequences) were obtained by screening to significantly improve mRNA stability under the same conditions.
  • sequences Two new sequences (sequences) were obtained by screening to significantly improve mRNA stability under the same conditions.
  • the addition of this non-coding sequence can more than double the intracellular half-life of mRNA.
  • the RNA in the tool system of the invention carries one or more modifications that enhance its stability, for extending the half-life of the RNA molecule, thereby increasing the translation and expression of the RNA molecule in the target cell to produce a Nanobody. s efficiency.
  • chemical modification of an RNA molecule refers to an element that constitutes RNA---nucleotides.
  • the modification is to replace the nucleotide without a modifying group in nature with a nucleotide having a modifying group.
  • the nucleotide consists of a nitrogen-containing base, a ribose, and a phosphoric acid.
  • modified bases include, but are not limited to, purines (adenine A, guanine G) and pyrimidines (cytosine C, uracil) with modified bases such as methylation, acetylation, hydrogenation, fluorination, and sulfuration.
  • purines adenine A, guanine G
  • pyrimidines cytosine C, uracil
  • modified bases such as methylation, acetylation, hydrogenation, fluorination, and sulfuration.
  • U an analog or derivative of thymine T).
  • modified bases include, but are not limited to, for example: pseudouridine ( ⁇ ), N6-methyladenosine (N6mA), inosine (I), methyluridine (mU) ), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (om5C), dihydrouridine (DHU, D), ribothymidine (rT), and 7 -methylguanosine (m7G) and the like.
  • one of the recommended scenarios of the present invention is to replace uracil with as much pseudo-purine as possible, and use as much methylcytosine as possible.
  • Substituted cytosine the ratio of U and C replaced by pseudo-uracil and methylcytosine in RNA transcripts is more than 20%, 30%, 40%, or even 100% ideally.
  • the present invention recommends base substitution for enhancing RNA stability in a coding sequence by molecular biological means without affecting the amino acid sequence of the encoded protein.
  • the stability of RNA molecules decreases with the increase of the number of cytosine cytidines (C) and uracil uridines (U), and the stability of RNA without C and U bases is relatively high.
  • C cytosine cytidines
  • U uracil uridines
  • a specific situation is that by using the degeneracy characteristics of multiple codons of the same amino acid, it is possible to reduce the number of codons with a large number of C and U by using codons encoding the same amino acid but having a small number of C and U.
  • the content of C and U in the entire RNA sequence makes the RNA molecule more stable.
  • the codon GGU or GGC of glycine (Gly) can be adjusted to GGA or GGG
  • the codon GCU or GCA of alanine (Ala) can be adjusted to GCA or GCG
  • the codon CUU or CUC of leucine can be adjusted to UUG or CUG
  • the codon AUU or AUC of iso-light can be adjusted to AUA
  • the codon UCU of serine or UCC can be adjusted to UCA or UCG
  • the codon CCU or CCC of proline can be adjusted to CCA or CCG
  • the codon ACU or ACC of threonine can be adjusted to ACA or ACG
  • arginine The codon CGU or CGC of Arg is adjusted to CGA or CGG, and so on, and these substitutions can reduce the C and U content of the coding region to increase the stability of the RNA.
  • the present invention still further preferably discloses that the mRNA molecule carries at least one modification which enhances its translation efficiency.
  • a preferred embodiment is that the RNA molecule is provided with a variety of modifications to enhance its translation efficiency.
  • modifications that increase translation efficiency include introducing a ribosome-capable hat (5'Cap) structure at the 5' end of the RNA, introducing a Kozak sequence and other sequences that enhance translation efficiency upstream of the Nanobody coding region, and Under the premise of not affecting the amino acid composition of the encoded Nanobody, it is preferred to use amino acid codons with higher translation efficiency.
  • the method of the present invention recommends the use of a higher frequency tRNA codon in human cells instead of those having a lower frequency of use. a. For example, among the six codons encoding leucine (Leu), the most frequently used CUG is selected, and among the four codons encoding valine (Val), the most frequently used GUG is selected; in glutamine-encoded Among the two codons, choose the most frequently used CAG and so on.
  • the 5-terminal end of the RNA molecule needs to have a specific cap structure (5'-cap structure).
  • the capping at the 5' end not only increases the stability of the transcription product, but also increases the efficiency of recognition by the translation machinery, thereby increasing the expression level of the mRNA-encoding Nanobody in the cell.
  • Commonly used hat structure modification bases include, but are not limited to, 7-methylguanine (m7G(5')ppp(5')G, 7-methylguanylate) and Thermo Fisher's ARCA-cap (Anti-Reverse Cap Analog) ), the latter can increase the probability of RNA being synthesized in the right direction.
  • Adding a 5' end cap to RNA can be achieved by replacing some of the normal GTP with such modified guanine in an in vitro transcriptional reaction system. It is recommended to modify the guanine to a working concentration of 1 mM to 4 mM, which is the normal GTP concentration. 2-8 times.
  • the method for introducing a base modification in the present invention is to additionally add a nucleotide having a modifying group to the transcription system, or completely replace the natural unmodified nucleotide with a nucleotide having a modifying group, so that these The modified nucleotide is involved in the synthesis of RNA transcripts under the action of RNA polymerase.
  • the above base modifications may be used singly or in combination of various methods, and the purpose is to make the generated RNA have higher stability and translation efficiency.
  • RNA transcript encoding the Nanobody of the present invention a special substance may be added to the tool system to prolong the half-life of the RNA in the cell.
  • reagents may be specific proteins or nucleic acid fragments, such as polyA binding proteins for protecting the 3' end sequence of an RNA molecule, or DNA or RNA fragments complementary to a certain sequence of an RNA transcription molecule, and the like.
  • these substances are mixed with RNA, which can directly or indirectly interact with the transcribed RNA. After the mixture is transferred into the cells, the RNA that binds the protective molecule is reduced by the nuclease, so that Leave it in the cell for a longer period of time.
  • different protective molecules can be used alone or in combination, and the purpose is to make the generated RNA have higher stability and longer intracellular half-life.
  • the present invention also preferably discloses that the mRNA molecule carries at least one modification that reduces its immunogenicity.
  • a preferred embodiment is through a variety of modifications to reduce the likelihood that it will cause an immune response in the body.
  • modifications that reduce RNA immunogenicity include: treatment with a phosphatase to remove an immunogenic phosphorylation group, particularly a triphosphate group, at the 5' end of an RNA molecule that is transcribed or chemically synthesized in vitro; The introduction of N6-methyladenosine, 5-methylcytidine, and other 2-O-methylated nucleotides into the RNA molecule prevents the RNA molecule from forming a secondary structure of the immunogenic double-stranded RNA type.
  • RNA in the inventive tool system carries one or more modifications that reduce its immunogenicity, thereby increasing the efficiency with which the RNA molecule is translated and expressed in the target cell to produce the Nanobody.
  • the present invention recommends the use of phosphatase treatment to remove the 5'-end of the in vitro transcribed or chemically synthesized RNA molecule to cause an immunogenic phosphorylation group, particularly a triphosphate group, to reduce its immunogenicity.
  • RNA molecules in eukaryotic cells often carry some modified nucleotides, such as N6-methyladenosine, 5-methylcytidine, and other 2-O-methylated nucleotides, etc., although the number is small.
  • this RNA molecule can be prevented from activating the innate immune response.
  • the present invention recommends the introduction of modified nucleotides including N6-methyladenosine, 5-methylcytidine, and 2'-O-methylated nucleotides in an RNA molecule transcribed in vitro, which can prevent the RNA molecule from forming immunity.
  • the secondary structure of the predominantly double-stranded RNA type thereby reducing the degradation of the RNA molecule by the innate immune response and prolonging its half-life in the cell. It should be noted that different modification methods can be used either alone or in combination, and the purpose is to make the generated RNA have lower immunogenicity and longer intracellular half-life.
  • the present invention still further preferably discloses that the upstream and downstream modifications of the mRNA-encoded protein have an intracellular localization signal. That is to say, the nanobodies encoded by the RNA molecules can be localized to specific cell regions or organelles depending on the cell distribution of the target protein. For example, in some cases, the produced Nanobodies can be localized to the cytoplasm, nucleus, and mitochondria as needed. In a specific subcellular structure such as the endoplasmic reticulum or Golgi.
  • the coding region of the in vitro transcribed RNA molecule used in the present invention may comprise a specific cell localization signal, and the Nanobody produced by translation of the RNA molecule may be designated as a corresponding specific cell region depending on the region of the cell to which the target protein is bound. Increase the recognition effect of Nanobodies on their target proteins.
  • the localization signal here refers to a specific amino acid sequence obtained by RNA coding, such as a signal peptide, a leader peptide, a classification signal, a localization signal, etc., according to requirements.
  • a nuclear localization signal such as PKKKRKV, PQKKIKS, QPKKP, and RKKR
  • NES nuclear export signal
  • ER-retention signals such as KDEL, DDEL, DEEL, QEDL, RDEL, etc.
  • localization of the nanobody to the endocytic localization signal of the endocytosis eg, sequence MDDQRDLISNNEQLP
  • PTS peroxisome Peroxisomal targeting signals
  • the mRNA encoding the Nanobody enters the cell via a transport vector.
  • a transport vector that carries an RNA transcript into a target cell.
  • the target cell refers to a cell with a defective function of the target protein, and expression of the RNA-encoding Nanobody in these cells can regulate the activity of the target protein therein and restore the normal function of the cell.
  • the transport carrier includes various forms of a pharmaceutical carrier that aids in the uptake of the nucleic acid material, the composition of which can deliver RNA molecules of different sizes into the target cell.
  • an RNA molecule can be combined with one or more chemical agents to be packaged into a form that is capable of entering a target cell.
  • the selection of suitable chemical reagents should consider not only the biological and chemical properties of the transport RNA, but also the use of the therapeutic regimen, as well as the biological environment to which the RNA molecule is exposed.
  • the transport vector does not destroy the biological activity of the RNA while encapsulating the RNA.
  • the transport vector has a certain propensity for the target cell, and its ability to bind to the target cell is stronger than other non-target cells.
  • the transfection vector used in the invention can be specific for RNA. Transfer to target cells without affecting other normal cells that do not require regulation.
  • the transport vector can be a liposomal vector, or other agent that facilitates entry of the nucleic acid into the target cell, as desired.
  • Suitable transport vehicles include, but are not limited to, liposomes; nanoliposomes with ceramide; liposomes with specific lipoproteins, such as lipids with ligand proteins such as apolipoprotein-B or apolipoprotein-E
  • the ligand protein binds to a target cell expressing a low-density lipoprotein receptor, thereby increasing the RNA-carrying transporter into the corresponding target cell; nanoliposomes; nanoparticulates, including phosphoric acid Calcium nanoparticles, silica nanoparticles, nanocrystalline particulates, bio-nanoparticles, semiconductor nanoparticles, etc.; polyarginine; starch transport system; micelles; emulsion As well as capsid proteins of certain viruses, etc., certain polymeric compounds can also be used as transport carriers for RNA.
  • a transport vector system can be used to preferentially target RNA encoding a Nanobody to a variety of target cells, suitable for the purpose
  • Target cells include, but are not limited to, hepatocytes, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, nerve cells, cardiomyocytes, fat cells, immune cells, ovarian cells, testicular cells, tumor cells, etc. .
  • the transport vector of the present invention also includes an easily detectable biological indicator to show that the RNA encoding the Nanobody successfully enters the target cell.
  • detection indicators include isotope labeling, fluorescent labeling or other materials commonly used in in vitro and in vivo experiments.
  • the detection substance can be covalently linked to the RNA molecule for tracing of the RNA molecule in the cell or tissue; or can be encoded by an RNA sequence (such as a fluorescent protein, luciferase, etc.) for displaying the RNA molecule in the cell. Translation situation.
  • the transport vector is added with a ligand that binds to the cell surface marker to be regulated. That is to say, by adding a ligand that binds to a target cell surface marker protein to a transport vector to increase the targeting of the transport complex to the target cell, the RNA can be transferred to a specific cell of a specific tissue, and Nanobodies that express specific functional proteins are translated intracellularly.
  • vectors carrying RNA encoding RNA can enter tissues and cells in a passive manner through natural diffusion.
  • RNA-carrying vectors carry additional components that can be recognized by specific target cells. It is capable of selectively entering specific cells.
  • the RNA-carrying vector of the invention may be provided with a ligand capable of increasing the binding of the complex to one or more target cells.
  • the localization ligand is apolipoprotein-B or apolipoprotein-E. These ligands are capable of binding to a target cell expressing a low density lipoprotein receptor; in other cases, the localization ligand is an antibody capable of binding to a target cell surface marker protein. These ligands are capable of binding to a marker protein on the surface of a target cell, thereby promoting binding of the transport vector to the target cell, and increasing the efficiency of targeting the Nanobody RNA carried by the transport vector to the target cell.
  • the composition system can be used to preferentially target nano-antibody RNA to a plurality of target cells
  • suitable target cells include, but are not limited to, hepatocytes, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, and nerve cells. , cardiomyocytes, fat cells, immune cells, ovarian cells, testicular cells, tumor cells, and the like.
  • the methods and tooling systems described herein can be used to modulate the function of a variety of key functional proteins within a cell, including a variety of functional proteins associated with disease development.
  • the RNA-encoded Nanobody is expressed intracellularly and bound to the target protein.
  • the target protein is stabilized in the inactive conformation by the Nanobody, and its function is inhibited, which is used to treat diseases caused by excessive activation of the target protein.
  • the target protein is stabilized in the activation conformation by the Nanobody, and its function is activated. It is used to treat diseases caused by insufficient activity of target proteins.
  • Optional target proteins include, but are not limited to, cellular signaling pathway molecules, transcription factors, protein modifying enzymes (methylation, alkylation, acetylation, phosphorylation, ubiquitination, etc.), protein de-modification enzymes (demethylation) , dealkylation, deacetylation, dephosphorylation, deubiquitination, etc.), nucleic acid modification enzymes (methylation, phosphorylation), nucleic acid de-modification enzymes (demethylation, dephosphorization) Acylation), structural proteins, important functional complexes, and other important disease marker proteins.
  • Diseases that can be controlled or treated by the method include, but are not limited to, dysplasia, Metabolic related diseases, tumors and cancers, autoimmune diseases, infectious diseases, autoimmune diseases, muscle and motor dysfunction, and the like.
  • Nanobodies Use mRNA-encoded Nanobodies to target a list of potential intracellular target proteins (antibodies of target mutant proteins; try to pick up harmful proteins, regulate the relationship between function and disease, and position the development direction):
  • E3ligases include:
  • AMFR APC/Cdc20, Apc/Cdh1, C6orf157, Cbl, CBLL1, CHFR, CHIP, DTL (Cdt2), E6-AP, HACE1, HECTD1, HECTD2, HECDT3, HECW1, HECW2, HERC2, HECR3, HECR4, HECR5, HUWE1 ,HYD,ITCH,LNX1,mathogunin,MARCH-I,MARCH-II,MARCH-III,MARCH-IV,MARCH-V,MARCH-VI,MARCH-VII,MARCH-VIII,MARCH-X,MDM2,MEKK1,MIB1 , MIB2, MycBP2, NEDD4, NEDD4L, Parkin, PELI1, Pirh2, PJA1, PJA2, RFFL, RFWD2, Rictor, RNF5, RNF8, RNF19, RNF190, RNF20, RNF34, RNF40, RNF125, RNF128, RNF38, RNF68,
  • HDACs include:
  • HATs include:
  • methyltransferases including:
  • Non-SAM Dependent Methyltransferases A variety of demethyltransferase include:
  • a variety of disease-related signaling pathway proteins include:
  • infectious viral marker proteins or pathogen proteins include:
  • the regulation of the HDAC6-WT by the mRNA-encoded Nanobody is specifically disclosed.
  • the nucleotide sequence of the mRNA corresponds to the nucleotide sequence of HDAC6-CAT1, and the expressed nano The antibody binds to HDAC6-WT.
  • the Nanobody has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:
  • the VHH chain of the amino acid sequence shown in Figure 16 which encodes the nucleotide sequence as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: 16.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
  • SEQ ID NO: 15 SEQ ID NO: 16 which can be used to encode a Nanobody against HDAC6-CAT1.
  • a host cell which is capable of expressing a Nanobody against HDAC6-CAT1 is also included.
  • the mRNA-encoded Nanobody disclosed by the present invention is capable of efficiently and safely regulating a specific protein function inside a cell.
  • the method encodes a nano-antibody by an in vitro transcribed RNA with a modified base and a translated original, and physically introduces the RNA transcript into a specific cell, so that it can be translated and expressed in the cytoplasm with a specific target. Protein-bound Nanobody.
  • Nanobody is a single-chain polypeptide structure having a small molecular weight (only 15 kd), it is very suitable for in vitro transcription or chemistry.
  • One RNA strand synthesized is encoded and expressed, and it is much less difficult to express a Nanobody with RNA than to express a conventional multi-subunit IgG antibody with RNA.
  • the specificity and stability of Nanobody recognition target proteins are significantly increased compared to conventional IgG antibodies or IgG antibody fragments, and their reliability in scientific research and clinical use is very good.
  • the Nanobody can activate or inhibit the function of the target protein, thereby achieving bidirectional regulation of protein function.
  • the Nanobody can be inserted into the recessed space of the surface of the target protein, and if the binding-induced conformational change results in the more active center of the target protein, the function of the target protein is more active; This binding causes the active center of the target protein to be blocked or distorted, thereby inhibiting the function of the protein.
  • the RNA-encoded Nanobody is used as a tool to regulate the function of intracellular proteins, and has the characteristics of easy operation, stable performance, and controllable effect, and has good clinical application prospects.
  • the “Nanobody-encoded Nanobody regulates intracellular target protein function” of the present invention discloses a method capable of efficiently and safely regulating the function of a specific protein inside a cell.
  • the method encodes a nano-antibody by an in vitro transcribed RNA with a modified base and a translated original, and physically introduces the RNA transcript into a specific cell, so that it can be translated and expressed in the cytoplasm to be associated with a specific target protein.
  • the bound Nanobody depending on the way in which the Nanobody binds to the target protein, the method can be used to inhibit the activity of the target protein as well as to promote the function of the target protein.
  • the method can effectively and specifically intervene in the function of the disease-related protein inside the cell, and thereby achieve the purpose of treating the disease.
  • Most of the disease-related proteins studied by the predecessors are located inside the cells.
  • the invention solves the problem that the nano-antibodies targeting intracellular proteins cannot be used for the treatment of clinical diseases, and fundamentally broadens the applicable range of the nano-antibody drugs. .
  • this method eliminates the risk of alteration of the host cell genome sequence, is safe and easy to remove, and is very suitable for the design and production of clinical drugs. .
  • Figure 1 is a restriction diagram of recombinant plasmid digestion, wherein: 1: DL2000 DNA standard molecular weight; 2: pET32a-CAT1-JD recombinant plasmid double digestion product, indicating that the HDAC6-CAT1-JD target fragment was successfully cloned into the pET32a (+) vector.
  • Figure 2 shows the SDS-PAGE analysis of the recombinant protein-inducing product, wherein: M: protein standard; 1: BL21/CAT1-JD cell-induced product inclusion body purification product, size 48 ku, indicating successful expression of HDAC6-CAT1-JD recombinant protein And the purification effect is better.
  • Figure 3 is a test diagram of ELISA antibody levels of immune camels, wherein the serum antibody titer after immunization reached 104, indicating that the HDAC6-CAT1-JD recombinant protein has a better immune effect.
  • Figure 4 is a diagram showing the expression and purification of CAT1-JD recombinant protein-specific Nanobodies in the host strain Escherichia coli, wherein: 2 strains of Nanobody SEQ ID NO: 4 and SEQ ID NO: 6 were not successfully purified, and the remaining 14 Nanobodies Both were well purified and the size was 15 kDa.
  • Figure 5 is a ELISA diagram of the affinity of each clone selected for HDAC6-WT, wherein: 9 Nanobodies responded well to HDAC6-WT, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID, respectively. NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16.
  • Figure 6 is a representation of Nanobodies against CAT1-JD recombinant protein in eukaryotic hosts, wherein: 16 Nanobodies are expressed in eukaryotic hosts.
  • Figure 7 is a Western-blot diagram showing the effect of Hela cells on the level of substrate acetylation, wherein: 1 strain of Nanobody SEQ ID NO: 5 is expressed in eukaryotic cells, the substrate acetylation level is increased, and HDAC6 is removed. The role of acetylase inhibitors.
  • Figure 8 is an IF diagram showing the effect of CHO-K1 cell expression on the level of substrate acetylation, wherein the cells transfected with the eukaryotic recombinant plasmid of SEQ ID NO: 5 are more red-light than those not transfected with the recombinant plasmid.
  • the expression level of acetylation of the substrate was increased after the expression of a Nanobody SEQ ID NO: 5 in eukaryotic expression, and the effect of HDAC6 deacetylase inhibitor was again demonstrated.
  • Figure 9 is an in vitro transcription method and each step effect diagram, wherein: 1 is a PCR product; 2 is mRNAs capping for 0.5 h; 3 is mRNAs capping for 1.5 h; 4 is Dnase 1 treated mRNA; 5 is mRNA plus The tail poly(A) indicates that the in vitro mRNA transcription is better.
  • Figure 10 is a diagram showing the in vivo binding of an in vitro transcript anti-EGFP antibody to EGFP, wherein the anti-actin antibody containing the GFP tag emits green fluorescence, and the His-tagged anti-GFP Nanobody emits red fluorescence, and the two fluorescences coincide. It indicated that the mRNA of anti-GFP Nanobody transcribed in vitro was successfully transfected into cells, which replaced the effect of plasmid transfected cells and could correctly recognize GFP protein and play a certain biological function.
  • Figure 11-12 shows the in vivo expression of in vitro transcripts compared to DNA, in which the mRNA results are shown in Figure 11, and the DNA results are shown in Figure 12, indicating that mRNA is expressed 3 h after transcription, while DNA is post-transcriptionally. Expression began only after 12 h, and mRNA expression time was earlier than DNA.
  • Figure 13 is a graph showing changes in acetylation after RNA is transfected into cells, wherein: mRNA of SEQ ID NO: 5 is successfully expressed in HeLa cells, and the level of Tubulin acetylation of cells transfected with the mRNA of SEQ ID NO: 5 is increased, indicating mRNA It can replace DNA and act as a HDAC6 deacetylase inhibitor.
  • the reagents and starting materials used in the present invention are either commercially available or can be prepared according to literature methods.
  • the test methods for the specific conditions not specified in the examples of the present invention are in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer.
  • the application method of the present invention is described by taking a Nanobody against the cytoplasmic deacetylase HDAC6 as an example.
  • Example 1 Expression and purification of HDAC6-CAT1 truncated protein:
  • PCR primers were designed by using Primer Primer5.0 software: CAT1-JD-5-sal1 (cgaGTCGACgagcagttaaatgaattccattg) and CAT1-JD-3-not1 (gcgGCGGCCGCggcggccatctcacccttggggtcc), and the length of the amplified gene fragment was 801 bp; 2) Using CAT1-JD-5-sal1 and CAT1-JD-3-not1 as primers, the HDAC6-WT recombinant plasmid was used as a template to amplify 6-272 amino acids of HDAC6-Cat1.
  • the PCR reaction system was 25 ⁇ L, and contained 22.5 ⁇ L of PCR Mix (containing enzyme), 1 ⁇ L of upstream primer, 1 ⁇ L of downstream primer, 1 ⁇ L of DNA template, and 9.5 ⁇ L of double distilled water.
  • the cycling parameters of PCR were: pre-denaturation at 95 ° C for 8 min; denaturation at 95 ° C for 40 s, annealing at 57 ° C for 40 s, extension at 72 ° C for 50 s, 35 cycles, and finally at 72 ° C for 10 min. Observed by 1% agarose gel electrophoresis.
  • each PCR amplified gene fragment was recovered by using a commercially available DNA gel recovery kit, and the purified 10 ⁇ L gene fragment and 10 ⁇ L pET-32a (+) vector were respectively Double digestion with SalI and XhoI restriction enzymes, the digestion reaction can be carried out in a 0.5 mL EP tube, and a 37 ° C water bath for 4-5 h. After gene digestion, each gene fragment and vector are recovered and purified.
  • PCR amplification was carried out by aspirating 1 ⁇ L of the corresponding bacterial solution as a template under sterile conditions, and after PCR, the PCR product was identified by 1.0% agarose gel electrophoresis.
  • the bacterial solution corresponding to the sample with the correct and specific PCR result is selected, and the recombinant plasmid is extracted by the plasmid extraction kit.
  • 1 ⁇ g of each recombinant plasmid was double-digested with 0.5 ⁇ L of Sal I and 0.5 ⁇ L of Not I restriction endonuclease, and the correct recombinant plasmid was identified by double enzyme digestion and sent to Shanghai Invitrogen for sequencing.
  • the digestion reaction can be carried out in a 0.5 mL EP tube, and the digestion process is a 37 ° C water bath for 4 hours.
  • 10 ⁇ L of the digested product was subjected to 1% agarose gel electrophoresis, and the results are shown in Fig. 1.
  • the CAT1-JD fragment obtained by digestion showed the expected size, which was 801 bp, and Sequencing is completely correct, indicating the HDAC6-CAT1-JD target fragment
  • the clone was successfully cloned into the pET32a(+) vector.
  • the correct recombinant plasmid pET32a-CAT1-JD was transformed into BL21 competent bacteria, and the obtained bacteria were named BL/CAT1-JD.
  • Single colonies of different recombinant bacteria were picked into 3 mL LB liquid medium containing Kana antibiotic (content 50 ⁇ g/mL), shaken at 37 ° C, shaken for 2-3 hours, until the OD600 of the bacterial solution reached 0.8-1.0
  • IPTG isopropylthiogalactoside
  • the cells were centrifuged at 8000 rpm for 3 min, and the bacteria were collected and washed twice with PBS solution. After the cells were resuspended in 500 ⁇ L of PBS, the cells were ultrasonically disrupted, and the parameters were 200 W, time 10 min (working 3 s, interval 7 s), then 8000 rpm. After centrifugation for 5 min, the supernatant was collected while resuspending the pellet with 500 ⁇ L of PBS.
  • lane 2 is the purified product of BL21/CAT1-JD cell-inducing product inclusion body with a size of 48 ku, indicating that HDAC6-CAT1-JD recombinant protein is successfully expressed and purified. better.
  • Protein immunization effect is better; and total RNA is extracted; (3) cDNA is synthesized and VHH is amplified by nested PCR; (4) 20 ug pMECS phage display vector and 10 ug VHH are digested with restriction endonucleases PstI and NotI and two (5) The ligation product was transformed into electroporation competent cell TG1, and the human antibody Fc fragment Nanobody phage display library was constructed and the storage capacity was determined. The size of the storage capacity was about 1.2 ⁇ 10 8 .
  • Example 3 Nanobody screening process for CAT1-JD recombinant protein:
  • TG1 cells were cultured in 2 ⁇ TY medium, 40uL helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage. The next day, phage was precipitated by PEG/NaCl, and the amplified phage was collected by centrifugation.
  • Example 4 Screening for specific positive clones by ELISA using phage enzyme-linked immunosorbent assay (ELISA):
  • IPTG was added to a final concentration of 1 mM, and cultured at 28 ° C overnight;
  • crude antibody was obtained by osmotic expansion method, and the antibody was transferred to an antigen-coated ELISA plate and allowed to stand at room temperature for 1 h;
  • Unbound antibody was washed away with PBST, and 100 ul of Mouse anti-HA tag antibody (murine anti-HA antibody, purchased from Covens) diluted 1:2000 was added and allowed to stand at room temperature for 1 h;
  • the bound antibody was added with 100 ul of Anti-mouse alkaline phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, purchased from Sigma) diluted at 1:2000, and allowed to stand at room temperature for 1 h;
  • washed away with PBST The bound antibody is added with alkaline phosphatase coloring solution, and after 5-10 minutes, the absorption value is read at 405 wavelength on the microplate
  • the amino acid sequence of the antibody is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
  • the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 region shown in Figure 16 constitutes the entire VHH.
  • Example 5 Expression of CAT1-JD recombinant protein-specific Nanobodies in host strain Escherichia coli, Purification and identification
  • coli is collected by centrifugation, and the crude extract of the antibody is obtained by the osmotic expansion method; (5) by nickel column affinity chromatography The antibody was purified to obtain a high-purity Nanobody, as shown in Figure 4, in which two Nanobodies SEQ ID NO: 4 and SEQ ID NO: 6 were not successfully purified, and the remaining 14 Nanobodies were well purified and sized. It is 15kDa.
  • the antibody was transferred to an HDAC6-WT coated ELISA plate and allowed to stand at room temperature for 1 h; (7) Unbound antibody was washed away with PBST, and 100 ul of the Mouse anti-HA tag antibody diluted 1:2000 was added.
  • Mouse anti-HA antibody purchased from Covens
  • Unbound antibody was washed away with PBST, and 100 ul of Anti-mouse alkaline phosphatase conjugate diluted 1:2000 was added (goat anti-mouse base)
  • the phosphatase-labeled antibody was purchased from Sigma and allowed to stand at room temperature for 1 h.
  • Nanobodies reacted well with HDAC6-WT which are SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ, respectively.
  • ID NO: 8 SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16.
  • Example 6 Expression of Nanobody against CAT1-JD Recombinant Protein in Eukaryotic Host and Effect of In Vivo Expression on Substrate Acetylation Level
  • the plasmids of the different clones obtained by the above sequencing analysis were ligated in tandem with EGFP to construct the eukaryotic recombinant plasmid pcDNA.1, and the eukaryotic recombinant plasmid and the pcDNA3.1 empty plasmid were transfected into Hela cells using liposome 2000. After 36 h, the cells were lysed and identified by Western-blot. The protein was transferred to the NC membrane, and 10 mL of the mouse anti-His Tag antibody diluted 1:2000 (purchased from Sigma) was added at room temperature.
  • Nanobodies were expressed in eukaryotic hosts; (2) in (1) The cell lysate was identified by Western-blot, the protein was transferred to the NC membrane, and 10 mL of 1:2000 diluted Rabbit anti-Tubulin K40 antibody (rabbit anti-Tubulin K40 antibody, purchased from Abcam) was added and allowed to stand at room temperature for 1 h; Unbound antibody was washed away with PBST, and 10 mL of 1:2000 diluted Goat Anti-rabbit HRP conjugate (goat anti-mouse horseradish peroxidase-labeled antibody, purchased from Sigma) was added and allowed to stand at room temperature for 1 h; The unbound antibody was washed away and exposed. As shown in Fig.
  • Nanobody SEQ ID NO: 5 was expressed in eukaryotic cells, and the level of acetylation of the substrate was elevated, and there was an HDAC6 deacetylase inhibitor.
  • Rabbit anti-Tubulin K40 antibody (rabbit anti-Tubulin K40 antibody, purchased from Abcam), was allowed to stand at room temperature for 1 h; unbound antibody was washed away with PBST, and 100 ⁇ L of Goat Anti-rabbit A568conjugate diluted 1:2000 was added (goat anti-small) Rat red light labeled antibody, From Sigma, left at room temperature for 1 h; unbound antibody was washed away with PBST and observed with a fluorescence microscope. As shown in Figure 8, transfected with SEQ ID NO: 5 compared to cells not transfected with the recombinant plasmid.
  • Example 6 the mRNA of SEQ ID NO: 5 was transcribed in vitro using the recombinant pcDNA3.1 plasmid of SEQ ID NO: 5 as a template. The method was the same as in Example 5, and the Western-blot was identified. As a result, as shown in Fig. 12, the mRNA of SEQ ID NO: 5 was successfully expressed in HeLa cells, and the level of Tubulin acetylation of the cells transfected with the mRNA of SEQ ID NO: 5 increased, indicating that mRNA can replace DNA and function as HDAC6. The role of acetylase inhibitors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

mRNA编码的纳米抗体及其应用，特别是纳米抗体药物领域。为了解决以上问题，用RNA编码纳米抗体调控胞内蛋白的新思路和方法系统。mRNA编码的纳米抗体，该mRNA携带的编码信息在细胞内被识别、翻译、表达出可以与靶蛋白结合的单链纳米抗体。采用该方法可以高效、特异性地干预细胞内部疾病相关蛋白的功能，并以此实现治疗疾病的目的。同时该方法和以DNA或病毒为载体在细胞内表达纳米抗体的常用策略不同，该方法摒除了对宿主细胞基因组序列造成改变的风险，具有安全和容易清除的特点，非常适合临床药物设计生产的需要。

Description

mRNA编码的纳米抗体及其应用 技术领域
本发明涉及mRNA编码的纳米抗体及其应用，属于生物医学领域，特别是纳米抗体药物领域。
背景技术
随着生物医学研究的进步，大量与疾病发生相关的蛋白功能失调被发现，其中绝大多数(90％以上)致病性蛋白均位于细胞内部。然而，由于普通的单克隆抗体难以有效进入细胞内部，目前已有的抗体药物均通过结合位于细胞表面或细胞外的靶蛋白来实现其治疗作用。细胞外靶蛋白的数量非常有限，受此限制，市场已有抗体药物识别的目标蛋白高度集中，这一方面大大限制了抗体药物的适应症范围，很多疾病缺乏合适的抗体类药物；另一方面，有限的标靶也导致制药公司的研发工作高度同质化，造成行业内的过度竞争和社会资源的浪费。一种能够方便、高效、安全、特异性、并双方向(包括活化和抑制)地调控细胞内功能蛋白活性的方法，将开启生物制药产业发展的新阶段。
用质粒为代表的DNA或者病毒为媒介，在细胞内表达功能蛋白是基因治疗的常用手段，该方法虽然具有较高的转染和表达效率，但却无法避免DNA与靶细胞的基因组发生重组，而导致其他疾病的风险。举例而言，外源DNA可能被插入某个正常的基因内部，从而导致突变甚至完全破坏该基因的表达，如果这个被插入的基因是比较重要的功能基因，这种改变将对细胞功能造成重大破坏，甚至导致细胞转化产生癌症。以基于DNA的质粒或病毒为工具，在理论上无法避免相关的致癌风险，这也是基因治疗方法未能充分推广的重要原因。相比而言，RNA进入细胞后，无法反转录成DNA,因此在理论上不会对细胞基因组的稳定性造成破坏，可以说，RNA疗法从根本上规避了基因疗法的致癌风险，更适合临床用药的安全需求。此外，DNA需要进入细胞核才能被转录和表达，转染难度较大，且在一定程度上需要细胞处于活跃的分裂周期才能进入细胞核；而RNA只要进入细胞质内就可以表达，不需进细胞核转录，也不依赖细胞周期，同样的转染条件下RNA比DNA在胞内表达的效率更高，在临床应用的难度更小。
发明内容
为了解决以上问题，本发明提出用RNA编码纳米抗体调控胞内蛋白的新思路和方法系统。
二十世纪九十年代，人们在从软骨鱼和骆驼科动物的体内发现天然缺失轻链的抗体，并发现此类抗体往往以重链二聚体的形式存在，此类抗体被称为重链抗体。进一步的研究发现，驼源重链体抗体中，与抗原结合的重链可变区可以单独稳定表达，并具有良好的抗原结合的特异性和亲和力，该重链可变区(variable domain of Heavy chain antibody,VHH)被称为单域抗体(single-domain antibody,sdAb)或纳米抗体(nanobody)。纳米抗体是已知最小的能够稳定表达的抗原结合蛋白，分子大小仅为常规IgG抗体的十分之一，具有结构稳定、特异性强的优点。
基于此，我们首先在本发明中公开了mRNA编码的纳米抗体，该mRNA携带的编码信息在细胞内被识别、翻译、表达出可以与靶蛋白结合的单链纳米抗体。也就是说，在本发明中描述了一种在目的细胞内表达编码特定纳米抗体mRNA的方法和工具系统。该工具系统包括：至少一种体外转录的编码纳米抗体的RNA分子，协助该RNA分子进入细胞内部的转染工具，以及增强该转染复合物识别特定细胞并提高其转染效率的辅助试剂。RNA分子被运送到目的细胞内部后，可以被细胞内的翻译机器识别，表达出与特定靶蛋白结合的单链纳米抗体。根据该纳米抗体与目的蛋白结合方式的不同，目的蛋白的构象发生不同形式的改变，从而实现抑制或者活化目的蛋白生物学功能的作用。
本发明公开的构建RNA分子以编码识别特定目标蛋白的纳米抗体，通过引入一定的化学修饰和核苷酸序列，增加该RNA在体内的稳定性和翻译效率，以及将该RNA转入特定细胞并实现相应纳米抗体在胞内表达的方法和工具系统。该方法和工具系统特别适用于医学临床上治疗因细胞内特定蛋白功能失调造成的疾病。用本发明描述的工具系统处理带有特定蛋白功能缺陷的细胞，根据产生的纳米抗体与目标蛋白结合造成结果的不同，其治疗的适用症包括两个方面：一是RNA编码的纳米抗体通过结合来抑制目的蛋白的功能，用于治疗细胞内致病基因高表达或过度活化而导致的疾病，；二是RNA编码的纳米抗体将目标蛋白的构象稳定于高活性的状态，从而增强该蛋白的功能，用于治疗细胞内目标蛋白表达不足或活力下降而造成的功能缺陷。
本发明提到的纳米抗体是指驼源重链抗体的可变区结构域，其能够特异性地与目标蛋白上的抗原表位发生紧密结合。由于是单链蛋白,且结构比较稳定，通常的纳米抗体均采用大肠杆菌或酵母等微生物高表达获得。为了得到调控目标蛋白功能的纳米抗体，通常将纯化得到的抗原蛋白或者蛋白的抗原表位结构域与合适的免疫佐剂混合后，多次注射双峰驼、羊驼等驼类动物，该动物被刺激的免疫系统经过细胞活化、分化和成熟的过程，产生能够分泌抗原蛋白特异性重链抗体的B细胞；收集免疫动物外周血中的总B细胞，抽提总RNA并反转 录cDNA文库，或者通过流式细胞仪筛选能够与靶抗原结合的B细胞，抽提其总RNA并反转录cDNA文库；以cDNA文库为模板，通过特定的引物(序列1)经套式PCR扩增单域重链抗体可变区(VHH)的编码基因，通过噬菌体展示技术筛选获得能够识别目的抗原蛋白的纳米抗体库；将这些基因转入大肠杆菌中，建立能在大肠感觉中高效表达的纳米抗体株，通过ELISA的方式确定与抗原蛋白有较高亲和力的抗体株，并对这些纳米抗体的编码基因进行测序；根据序列比对软件分析各个克隆株的基因序列，去除FR1、FR2、FR3、FR4、CDR1、CDR2、CDR3序列相同的冗余克隆，并将CDR3区域相似(序列相似性>80％)的序列归为一组，选择各组中亲和力较高的纳米抗体克隆，在大肠杆菌中高表达并纯化得到相应的纳米抗体；用生化手段检验不同纳米抗体对抗原蛋白功能产生的影响，从而获得能够活化或抑制目的蛋白功能的纳米抗体。
通过测序得到编码功能性纳米抗体的基因序列，该数据可以作为本发明中通过生物手段合成编码纳米抗体RNA的基本序列信息。生物手段通常指通过体外转录获得RNA的方法，该方法指在体外无细胞系统中，以DNA作为模板，以NTP为原料，依赖RNA聚合酶，模仿体内转录过程生成RNA的过程。
方法中描述了获取特异性纳米抗体基因序列以及相应RNA的方法，优选方案是利用流式细胞术和噬菌体展示技术，或者利用高通量测序技术(或下一代测序技术Next Generation Sequencing,NGS)结合生物信息学分析方法，从驼源免疫的单域重链抗体库中筛选能与靶蛋白特异结合的单域重链抗体可变区的DNA克隆，对获得的克隆进行测序、表达、和活力鉴定，获得能与目标蛋白特异性结合的功能性纳米抗体及其基因序列，根据获得的基因序列，通过化学合成或体外转录的方法得到编码该纳米抗体的RNA。
一般来讲，通过噬菌体展示技术获得的纳米抗体基因序列往往位于噬菌体展示质粒上，包括pHEN1，pHEN4，pMES4或pMESy4等，由于这些质粒的拷贝数不高，优化的方法是通过亚克隆的方法将编码纳米抗体的基因转移到更适合体外转录的其他质粒上，包括pUC18，pUC19等高拷贝质粒，pT7Ts,pGEM-1系列质粒或pSP64质粒等等；也可以通过PCR的方法得到大量的用于体外转录的DNA模板。
进一步地，在本发明中还公开了mRNA分子带有至少一种增强其稳定性的修饰。优选方案是该RNA分子上带有多种修饰用以增强其稳定性。在某些情境下，增强稳定性的修饰包括：用带有化学修饰的核苷酸(nucleotide)代替天然的没有修饰的核苷酸，在不影响编码纳米抗体氨基酸组成的情况下增加RNA中GC碱基的含量，以及在RNA的两端引入增加其稳定性的非编码序列(UTR)等。这里提到的增加RNA中GC碱基的含量，是指相较 于AU碱基对，应当尽量增加GC碱基的比例。
譬如，为了增加RNA转录产物的稳定性，本发明推荐在不影响编码蛋白氨基酸序列的前提下，在编码序列的上游和下游添加特定的非编码序列，即5’-UTR和3’-UTR。相关的情境之一是在RNA的3-末端增加较长的多聚腺嘌呤尾巴(poly A tail)，从而增加RNA的稳定性。在一定的情境下，poly A tail的长度不少于100，200，300或者400个核苷酸。添加长ploy A tail的方法即包括通过poly A polymerase在RNA转录产物末端直接添加多聚腺嘌呤，也可以通过在体外转录的DNA模板的3’端直接引入poly A来实现，还可以通过RNA连接酶将poly A与转录RNA产物的3’端直接相连得到。特别地，由于poly A tail的长度可以调节RNA转录产物在体内的半衰期，在某些情境下，可以通过调整RNA转录产物带有的poly A tail的长度，控制该RNA在细胞内表达纳米抗体的时间。除了polyA tail，另一种比较常用的情境是在编码纳米抗体的基因上下游引入特定的5’-UTR和3’-UTR区，特别是在mRNA终止密码子下游较多的C碱基重复，被证明可以提高mRNA的稳定性。本发明推荐来源于其他稳定性较高的天然蛋白的mRNA的非编码区，例如collagen、globin、actin、tubulin、GAPDH、histone等蛋白mRNA的5’-UTR和3’-UTR区，在纳米抗体编码序列的上下游引入这些非编码区，也可以提高相应RAN转录产物的稳定性。根据我们的研究，从多种高稳定性蛋白mRNA编码区上下游的UTR区提取同源性序列，通过筛选获得以下两条新序列(序列如下)可以显著提高mRNA的稳定性,在同等条件下，该非编码序列的加入可将mRNA在胞内的半衰期提高一倍以上。
5’UTR添加序列：
Figure PCTCN2017082096-appb-000001
3’UTR添加序列：
Figure PCTCN2017082096-appb-000002
需要说明的是，以上四种优化RNA分子编码序列的方法既可以单独使用，也可以多种方法合并使用，其目的都是使产生的RNA具有更高的稳定性和翻译效率。
在多数情境下，该发明的工具系统中的RNA带有一种或多种增强其稳定性的修饰，用于延长RNA分子的半衰期，从而增加该RNA分子在靶细胞内被翻译并表达产生纳米抗体的效率。在这里，对RNA分子的化学修饰是指对构成RNA的元件---核苷酸(nucleotides) 的修饰，即用带有修饰基团的核苷酸代替天然情况下没有修饰基团的核苷酸。具体而言，核苷酸由含氮碱基、核糖与磷酸三个部分组成，为了不影响体外转录时RNA的生成，此处的化学修饰主要位于其碱基部分。常用的修饰碱基包括，但不限于，带有甲基化、乙酰化、氢化、氟化以及硫化等修饰碱基的嘌呤(腺嘌呤A，鸟嘌呤G)和嘧啶(胞嘧啶C，尿嘧啶U，胸腺嘧啶T)的类似物或衍生物。最常用的修饰碱基包括但不限于，例如：假尿嘧啶(pseudouridine，ψ)，N6-甲基腺腺嘌呤(N6mA)，次黄嘌呤(inosine，I)，甲基尿嘧啶(methyluridine,mU)，5-甲基胞嘧啶(5-methylcytosine,5mC)，5-羟甲基胞嘧啶(5-hydroxymethylcytosine,om5C)，二氢尿嘧啶(dihydrouridine，DHU，D)，ribothymidine(rT)，以及7-甲基鸟嘌呤(7-methylguanosine，m7G)等等。
特别地，由于U、C碱基数量与RNA稳定性成反比，本发明推荐的情境之一是用尽量多的假尿嘧啶(pseudouridine，ψ)取代尿嘧啶，而用尽量多的甲基胞嘧啶取代胞嘧啶，是RNA转录产物中U、C被假尿嘧啶和甲基胞嘧啶替代的比率超过20％，30％，40％，甚至理想情况下100％。
本发明推荐在不影响编码蛋白氨基酸序列的前提下，通过分子生物学的手段在编码序列中进行增强RNA稳定性的碱基替换。RNA分子的稳定性随着其中含有胞嘧啶cytidines(C)和尿嘧啶uridines(U)的数量升高而下降，不含有C、U碱基的RNA稳定性相对较高。这里所说的意思是，在AU碱基对中应尽量增加A的含量，而在CG碱基对中，则应尽量增加G的含量。也即是说，在本专利推荐的情境下，可以在不影响蛋白氨基酸序列的前提下，降低RNA序列中C、U碱基的含量。一个特定的情境是，利用同一个氨基酸具有多个密码子的简并性特点，通过用编码相同氨基酸但C、U数量较少的密码子替代原来C、U数量较多的密码子，可以降低整条RNA序列中的C、U含量，从而使RNA分子更为稳定。例如，甘氨酸(Gly)的密码子GGU或GGC可以调整为GGA或GGG，丙氨酸(Ala)的密码子GCU或GCA可以调整为GCA或GCG，缬氨酸(Val)的密码子GUU或GUC可以调整为GUA或GUG，亮氨酸(Leu)的密码子CUU或CUC可以调整为UUG或CUG，异亮(Iso)氨酸的密码子AUU或AUC可以调整为AUA,丝氨酸的密码子UCU或UCC可以调整为UCA或UCG，脯氨酸(Pro)的密码子CCU或CCC可以调整为CCA或CCG，苏氨酸(Thr)的密码子ACU或ACC可以调整为ACA或ACG，精氨酸(Arg)的密码子CGU或CGC调整为CGA或CGG，等等，这些替换均可降低编码区的C、U含量以增加RNA的稳定性。
同时，本发明还进一步优选公开了mRNA分子带有至少一种增强其翻译效率的修饰。 优选方案是该RNA分子上带有多种修饰用以增强其翻译效率。在某些情境下，增加翻译效率的修饰包括：在RNA的5’端引入招募核糖体的帽子(5’Cap)结构，在纳米抗体编码区上游引入Kozak序列和其他增强翻译效率的序列，以及在不影响编码纳米抗体氨基酸组成的前提下，选择使用翻译效率更高的氨基酸密码子等。
考虑到纳米抗体编码区所用密码子对应tRNA的使用频率，为了增加RNA转录产物的翻译效率，本发明的方法推荐用在人类细胞中使用频率较高的tRNA密码子，替代那些使用频率较低的密码子。例如，在编码亮氨酸(Leu)的六个密码子中选择使用频率最高的CUG，在编码缬氨酸(Val)的四个密码子中选择使用频率最高的GUG；在编码谷氨酰胺的两个密码子中选择使用频率最高的CAG等等。
此外，为了促进核糖体对RNA转录产物的识别，增加翻译效率，RNA分子的5-末端需要带有特定的帽子结构(5’-cap structure)。5’端加帽不但可以增加转录产物的稳定性，还可以提高翻译机器对其识别的效率，从而增加mRNA编码纳米抗体在细胞内的表达水平。常用的帽子结构修饰碱基包括，但不限于，7-甲基鸟嘌呤(m7G(5')ppp(5')G，7-methylguanylate)和Thermo Fisher公司的ARCA-cap(Anti-Reverse Cap Analog)等，后者可以增加RNA向正确方向合成的几率。为RNA添加5’端帽子，可以通过在体外转录的反应体系中用此类修饰的鸟嘌呤代替部分正常GTP来实现，推荐修饰鸟嘌呤的工作浓度为1mM-4mM，该用量为正常GTP浓度的2-8倍。
本发明中引入碱基修饰的方法，是在转录体系中额外添加带有修饰基团的核苷酸，或者用带有修饰基团的核苷酸完全替代天然无修饰的核苷酸，使这些修饰的核苷酸在RNA聚合酶的作用下，参与RNA转录产物的合成。需要说明的是，以上碱基修饰既可以单独使用，也可以多种方法合并使用，其目的都是使产生的RNA具有更高的稳定性和翻译效率。
在某些情形下，为了进一步提高本发明中编码纳米抗体的RNA转录产物的稳定性，可以向工具系统中加入特殊物质，以延长RNA在细胞内的半衰期。此类试剂可以是特殊的蛋白或者核酸片段,例如用于保护RNA分子3’端序列的polyA结合蛋白，或者是与RNA转录分子的一定序列互补的DNA或RNA片段等等。在RNA转染细胞之前，将此类物质与RNA混合，它们可以直接或间接与转录RNA发生作用，将此混合物转入细胞后，结合了保护分子的RNA被核酸酶降解的机会下降，从而能够在细胞内存留更长的时间。需要说明的是，不同的保护分子既可以单独使用，也可以多种方法合并使用，其目的都是使产生的RNA具有更高的稳定性和更长的胞内半衰期。
更进一步地，本发明还优选公开了mRNA分子带有至少一种降低其免疫原性的修饰。 优选方案是通过多种修饰以降低其造成机体免疫反应的可能性。在某些情境下，降低RNA免疫原性的修饰包括：用磷酸酶处理去除体外转录或化学合成的RNA分子5’端造成免疫原性的磷酸化基团，特别是三磷酸基团；通过向RNA分子中引入N6-methyladenosine,5-methylcytidine,以及其他2-O-位甲基化的核苷酸，阻止该RNA分子形成免疫原性较强的双链RNA型的二级结构等。
细胞存在一套天然免疫系统(innate immune system)，在受到病原分子侵染的时候，能够通过激活一系列复杂的炎症反应将入侵物降解掉。很多哺乳动物细胞表达了多种模式识别受体(pattern recognition receptor,PRRs)，可以识别、结合并降解进入细胞的外源性RNA，这是体外转录RNA在细胞内翻译效率低下的重要原因。在多数情境下，该发明的工具系统中的RNA带有一种或多种降低其免疫原性的修饰，从而增加该RNA分子在靶细胞内被翻译并表达产生纳米抗体的效率。
有报道指出，外源RNA分子5’端的磷酸化修饰可以影响RNA对天然免疫系统的免疫原性，带有5’-三磷酸化修饰的RNA比没有磷酸化修饰的RNA更容易被降解。据此特性，本发明推荐用磷酸酶处理去除体外转录或化学合成的RNA分子5’端造成免疫原性的磷酸化基团，特别是三磷酸基团，以降低其免疫原性。此外，真核细胞中的天然mRNA往往带有一些修饰的核苷酸，例如N6-methyladenosine,5-methylcytidine,以及其他2-O-位甲基化的核苷酸等等，尽管数量不多，但可以阻止该RNA分子激活天然免疫反应。据此，本发明推荐在体外转录的RNA分子中引入包括N6-methyladenosine,5-methylcytidine,以及2’-O-methylated nucleotides在内的修饰核苷酸，这些修饰碱基可以阻止该RNA分子形成免疫原性较强的双链RNA型的二级结构,从而降低天然免疫反应对RNA分子的降解，延长其在细胞内的半衰期。需要说明的是，不同的修饰方法既可以单独使用，也可以多种方法合并使用，其目的都是使产生的RNA具有更低的免疫原性和更长的胞内半衰期。
同时，本发明还进一步优选公开mRNA编码蛋白的上下游修饰有细胞内定位信号。这也就是说，RNA分子编码的纳米抗体可以根据靶蛋白的细胞分布情况，定位于特殊的细胞区域或者细胞器，譬如在某些情境下，产生的纳米抗体可以根据需要定位于细胞质、细胞核、线粒体、内质网、或高尔基体等特定的亚细胞结构中。
本发明中所用体外转录RNA分子的编码区可以包含特定的细胞定位信号，根据结合目标蛋白所处细胞区域的不同，该RNA分子翻译产生的纳米抗体可以被定为于相应特定的细胞区域，以增加纳米抗体对于其目标蛋白的识别效果。这里的定位信号是指有RNA编码翻译得到的特殊的氨基酸序列，例如信号肽、前导肽、分类信号、定位信号等，根据需求可以 位于纳米抗体的N-端或C-端，包括但不限于：将纳米抗体定位于细胞核内的核定位信号(nuclear localization signal,NLS，如PKKKRKV,PQKKIKS,QPKKP及RKKR等序列)；将纳米抗体定位于细胞质中的核输出信号(nuclear export signal,NES，如LxxxLxxLxL)；将纳米抗体定位于内质网上的内质网定位(ER-retention)信号(如KDEL,DDEL,DEEL,QEDL及RDEL等序列)；将纳米抗体定位于内吞体的内吞体定位信号(如序列MDDQRDLISNNEQLP)；将纳米抗体定位于线粒体的线粒体定位信号(mitochondrial targeting signal)；将纳米抗体定位于过氧化物酶体的过氧化物酶体靶向信号(peroxisomal targeting signals,PTS，如SKL)等等。
作为另一公开，我们在本发明中进一步公开了前述的mRNA编码的纳米抗体在制备用于调节细胞内靶蛋白功能制剂中的应用。
并且进一步地，我们公开了所述编码纳米抗体的mRNA通过转运载体进入细胞内。
本发明的工具系统中还包括携带RNA转录物进入目标细胞的转运载体。目标细胞是指带有靶蛋白功能缺陷的细胞，RNA编码纳米抗体在这些细胞内的表达能够调控其中靶蛋白的活性，并恢复细胞的正常功能。在这里，转运载体包括多种形式的帮助核酸类物质摄入的药物载体，该载体的组成可以将大小不同的RNA分子递送进入目标细胞。在转运载体中，RNA分子可以与一种或多种化学试剂结合，从而被包装成为能够进入目标细胞的形式。合适的化学试剂的选择，不但要考虑其转运RNA的生物和化学性质，也要结合治疗方案实施时的使用情况，以及RNA分子施用后所暴露的生物环境等因素。在施用当中，转运载体在包裹RNA的同时并不破坏RNA的生物活性。在某些情境下，转运载体对目标细胞要具有一定的倾向性，及其结合目标细胞的能力要强于其他非目标细胞，在理想的情况下，发明中使用的转染载体可以将RNA特异性的转入目标细胞，而不影响其他无需调控的正常细胞。
根据需要，转运载体可以是脂质体(liposomal)载体，或者其他有助于核酸进入目标细胞的试剂。合适的转运载体包括但不限于：脂质体；带有神经酰胺的纳米脂质体；带有特殊脂蛋白的脂质体，例如带有apolipoprotein-B或者apolipoprotein-E等配体蛋白的脂质体,这些配体蛋白能够与表达低密度脂蛋白受体的目标细胞结合，从而提高载有RNA的转运载体进入相应的目标细胞；纳米脂质体(nanoliposomes)；纳米颗粒(nanoparticulates)，包括磷酸钙纳米颗粒，二氧化硅纳米颗粒,纳米晶体颗粒(nanocrystalline particulates),生物纳米颗粒，半导体纳米颗粒等；多聚精氨酸；淀粉类的转运系统；微胶束(micelles)；乳状液(emulsion)；以及某些病毒的衣壳蛋白等等，某些多聚化合物也可以用作RNA的转运载体。转运载体系统可以用于将编码纳米抗体的RNA优先靶向多种目标细胞，合适的目 标细胞包括但不限于：肝细胞、上皮细胞、内皮细胞、肺细胞、骨细胞、干细胞、间叶细胞、神经细胞、心肌细胞、脂肪细胞、免疫细胞、卵巢细胞、睾丸细胞、肿瘤细胞等等。
在某些情境中，为了便于观察，本发明的转运载体中还要包括一个易于检测的生物指标，以显示编码纳米抗体的RNA成功进入目标细胞。常用的检测指标包括同位素标记、荧光标记或者其他体内体外实验常用的材料。该检测物质即可以与RNA分子共价连接，用于RNA分子在细胞或组织中的示踪；也可以由RNA序列编码(如荧光蛋白、荧光素酶等)，用于显示RNA分子在细胞内的翻译情况。
并且更进一步地，我们优选公开了转运载体添加有与目标待调节细胞表面标志相结合的配体。这也即是说，可以通过向转运载体中添加与目标细胞表面标志蛋白相结合的配体，以增加转运复合物对目的细胞的靶向性，可以将RNA转入特定组织的特定细胞，并在细胞内翻译表达出特定功能蛋白的纳米抗体。某些情境下，携带编码纳米抗体RNA的载体可以通过自然扩散的被动方式进入组织和细胞，其他的情境下，携带RNA的载体带有附加的、可以被特定的靶细胞识别的组成成分，使其能够选择性地进入特定细胞。优选方案是，发明中携带RNA的载体可以带有能够增加该复合物与一种或多种目标细胞结合的配体，某些情境下，该定位用配体是apolipoprotein-B或者apolipoprotein-E,这些配体能够与表达低密度脂蛋白受体的目标细胞结合；在其他的情况下，该定位配体是能够与目标细胞表面标志蛋白结合的抗体。这些配体能够与目标细胞表面的标志蛋白结合，从而促进转运载体与目标细胞的结合，增加将该转运载体携带的纳米抗体RNA靶向目标细胞的效率。该组成系统可以用于将纳米抗体RNA优先靶向多种目标细胞，合适的目标细胞包括但不限于：肝细胞、上皮细胞、内皮细胞、肺细胞、骨细胞、干细胞、间叶细胞、神经细胞、心肌细胞、脂肪细胞、免疫细胞、卵巢细胞、睾丸细胞、肿瘤细胞等等。
本发明描述的方法和工具系统可以用于调控细胞内部多种关键功能蛋白的功能，包括多种与疾病发生相关的功能蛋白，优选方案是RNA编码的纳米抗体在细胞内表达并与目标蛋白结合。某些情境下，目标蛋白被纳米抗体稳定于失活构象，其功能被抑制，用于治疗目标蛋白过度活化导致的疾病；其他情境下，目标蛋白被纳米抗体稳定于活化构象，其功能被激活，用于治疗目标蛋白活力不足造成的疾病。可选目标蛋白包括但不限于：细胞信号通路分子、转录因子、蛋白修饰酶(甲基化、烷基化、乙酰化、磷酸化、泛素化等等)、蛋白去修饰酶(去甲基化、去烷基化、去乙酰化、去磷酸化、去泛素化等等)、核酸修饰酶(甲基化、磷硫酰化)、核酸去修饰酶(去甲基化、去磷硫酰化)、结构蛋白、重要的功能性复合物、以及其他重要的疾病标志蛋白等。该方法可以控制或治疗的疾病包括但不限于：发育异常、 代谢相关疾病、肿瘤和癌症、自身免疫性疾病、传染性疾病、自身免疫性疾病、肌肉及运动功能失调等等。
用mRNA编码纳米抗体靶向潜在的胞内目标蛋白的列表(target突变蛋白的抗体；尽量挑有害蛋白，功能调控与疾病的关系，定位发展方向)：
多种E3ligases包括：
AMFR,APC/Cdc20,Apc/Cdh1,C6orf157,Cbl,CBLL1,CHFR,CHIP,DTL(Cdt2),E6-AP,HACE1,HECTD1,HECTD2,HECDT3,HECW1,HECW2,HERC2,HECR3,HECR4,HECR5,HUWE1,HYD,ITCH,LNX1,mathogunin,MARCH-I,MARCH-II,MARCH-III,MARCH-IV,MARCH-V,MARCH-VI,MARCH-VII,MARCH-VIII,MARCH-X,MDM2,MEKK1,MIB1,MIB2,MycBP2,NEDD4,NEDD4L,Parkin,PELI1,Pirh2,PJA1,PJA2,RFFL,RFWD2,Rictor,RNF5,RNF8,RNF19,RNF190,RNF20,RNF34,RNF40,RNF125,RNF128,RNF38，RNF68，SCF/beta-Trcp,SCF/FBW7,SCF/Skp2,SHPRH,SIAH1,SIAH2,SMURF1,SMURF2,TOPORS,TRAF6,TRAF7,TRIM63,UBE3B,UBE3C,UBR1,UBR2,UHRF2,VHL,WWP1,WWP2,ZNRF1.Et al.
多种HDACs包括：
◆ HDAC1-HDAC11
◆ Sirtuin1–Sirtuin7
多种HATs包括：
◆ GCN5
◆ PCAF
◆ Tip60
◆ MORF
◆ MOZ
◆ HBO1
◆ P300
◆ CBP
◆ SRC-1
◆ ACTR(RAC3,AIB1,TRAM-1)
◆ TIF-2(GRIP1)
◆ SRC-3
◆ TFIIIC(p220,p110,p90)
◆ CLOCK
多种methyltransferase,包括：
◆ histone methyltransferase
◆ DNA/RNA methyltransferase
◆ N-terminal methyltransferase
◆ Non-SAM Dependent Methyltransferases多种demethyltransferase包括：
◆ KDM1family
◆ KDM2family
◆ KDM3family
◆ KDM4family
◆ KDM5family
◆ KDM6family
◆ protein-glutamate methylesterase
多种疾病相关信号通路蛋白包括：
◆ p53
◆ KRAS
◆ BRAF
◆ BCR/ABL
◆ BCL1
◆ BCL2
◆ PML-RARa
◆ c-KIT
◆ EGFR
◆ EGFR(FISH)
◆ HER2/neu(FISH)
◆ JAK2
◆ ALK(FISH)
◆ DPD/TYMS
◆ TPMT
多种感染性病毒标志蛋白或病原体蛋白包括：
◆ RSV的组成(呼吸道合胞体病毒)
在本发明中特别地公开了该mRNA编码的纳米抗体对于HDAC6-WT的调控作用，在这一应用中，mRNA的核苷酸序列对应于HDAC6-CAT1的核苷酸序列，其表达后的纳米抗体与HDAC6-WT结合。
具体地，纳米抗体具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列的VHH链，其编码核苷酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16所示。
还包括有生成这一mRNA的模板DNA分子，其核苷酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16所示，可以用以编码针对HDAC6-CAT1的纳米抗体。
进一步地，在这一应用中，我们还进一步公开了一种表达载体，这一表达载体包含有前述的DNA分子的核苷酸序列。
并且，进一步地，还包括有一个宿主细胞，该宿主细胞能够表达针对HDAC6-CAT1的纳米抗体。
本发明公开的mRNA编码的纳米抗体能够高效、安全地调控细胞内部特定蛋白功能的方法。该方法通过体外转录的、带有修饰碱基以及翻译原件的RNA编码纳米抗体，并通过物理方法将该RNA转录产物导入特定的细胞，使其能在细胞质内翻译表达出可以与特定靶 蛋白相结合的纳米抗体。
同时，相较于用RNA为载体在细胞内部表达常规IgG抗体全长或IgG抗体的片段的方法，由于纳米抗体为分子量较小(仅15kd)的单链多肽结构，非常适合用体外转录或化学合成的一条RNA链进行编码和表达，用RNA表达纳米抗体的难度远远小于用RNA表达常规的多亚基IgG抗体。此外，和常规IgG抗体或IgG抗体片段相比，纳米抗体识别靶蛋白的特异性和稳定性都显著升高，其在科学研究和临床使用中的可靠性很好。更重要的是，根据与目标蛋白的结合方式的不同，不同的纳米抗体可以活化或者抑制目标蛋白的功能，从而实现对蛋白功能的双向调节。具体而言，由于其体积小，纳米抗体可以插入靶蛋白表面凹陷的空隙中，如果这种结合诱发的构象改变导致目标蛋白活性中心更开放，则使目标蛋白的功能更为活跃；反之，如果这种结合导致目标蛋白的活性中心被屏蔽或扭曲，则使该蛋白的功能被抑制。综上所述，以RNA编码的纳米抗体为工具调节胞内蛋白的功能，具有易于操作、性能稳定、效果可控的特点，临床应用前景良好。
本发明“用mRNA编码的纳米抗体调节细胞内靶蛋白功能”公开了一种能够高效、安全地调控细胞内部特定蛋白功能的方法。该方法通过体外转录的、带有修饰碱基以及翻译原件的RNA编码纳米抗体，并通过物理方法将该RNA转录产物导入特定的细胞，使其能在细胞质内翻译表达出可以与特定靶蛋白相结合的纳米抗体；根据纳米抗体与目标蛋白结合方式的不同，该方法既能用于抑制目标蛋白的活力，也能用于促进目标蛋白的功能。采用该方法可以高效、特异性地干预细胞内部疾病相关蛋白的功能，并以此实现治疗疾病的目的。前人研究的绝大多数疾病相关的蛋白都位于细胞内部，该发明解决了靶向胞内蛋白的纳米抗体无法用于临床疾病治疗的难题，从根本上拓宽了纳米抗体类药物的可适用范围。此外，和以DNA或病毒为载体在细胞内表达纳米抗体的常用策略不同，该方法摒除了对宿主细胞基因组序列造成改变的风险，具有安全和容易清除的特点，非常适合临床药物设计生产的需要。
附图说明
图1为重组质粒双酶切鉴定图，其中：1:DL2000DNA标准分子量；2:pET32a-CAT1-JD重组质粒双酶切产物，说明HDAC6-CAT1-JD目的片段成功克隆进入pET32a(+)载体。图2为重组蛋白诱导产物SDS-PAGE分析，其中：M:蛋白标样；1:BL21/CAT1-JD菌体诱导产物包涵体纯化产物，大小为48ku，说明HDAC6-CAT1-JD重组蛋白成功表达且纯化效果较好。
图3为免疫骆驼的ELISA抗体水平检测图，其中：免疫后血清抗体效价达104，，说明HDAC6-CAT1-JD重组蛋白免疫效果较好。
图4为CAT1-JD重组蛋白特异性纳米抗体在宿主菌大肠杆菌中的表达和纯化图，其中：2株纳米抗体SEQ ID NO:4和SEQ ID NO:6未成功纯化，其余14株纳米抗体均得到较好的纯化，大小为15kDa。
图5为所选各克隆与HDAC6-WT的亲和效果ELISA图，其中：9株纳米抗体与HDAC6-WT有较好的反应，分别为SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
图6为针对CAT1-JD重组蛋白的纳米抗体在真核宿主中的表达，其中：16株纳米抗体均在真核宿主中表达。
图7为Hela细胞表达后对底物乙酰化水平的影响Western-blot图，其中：1株纳米抗体SEQ ID NO:5在真核体内表达后，对底物乙酰化水平升高，有HDAC6去乙酰化酶抑制剂的作用。
图8为CHO-K1细胞表达后对底物乙酰化水平的影响IF图，其中：与没有转染进重组质粒的细胞相比，转染SEQ ID NO:5真核重组质粒的细胞红光更明显，说明1株纳米抗体SEQ ID NO:5在真核体内表达后，对底物乙酰化水平升高，再一次证明HDAC6去乙酰化酶抑制剂的作用。
图9为体外转录方法及各步效果图，其中：1为PCR产物；2为mRNAs加帽孵育0.5h；3为mRNAs加帽孵育1.5h；4为Dnase 1处理后的mRNA；5为mRNA加尾poly(A)，说明体外mRNA转录效果较好。
图10为体外转录产物抗EGFP抗体体内结合EGFP图，其中：含有GFP标签的抗actin抗体发绿色荧光，含有His标签的抗GFP纳米抗体发红色荧光，且两种荧光重合。说明体外转录的抗GFP纳米抗体的mRNA成功转染进细胞，替代了质粒转染细胞的作用，且能正确识别GFP蛋白，起到一定的生物学功能。
图11-12为体外转录产物与DNA相比的体内表达图，其中：mRNA结果如图11所示，DNA结果如图12所示，说明mRNA在转录后3h便开始表达，而DNA在转录后12h才开始表达，mRNA表达时间早于DNA。
图13为RNA转入细胞后乙酰化改变图，其中：SEQ ID NO:5的mRNA在Hela细胞中成功表达，且转染有SEQ ID NO:5的mRNA的细胞Tubulin乙酰化水平上升，说明mRNA可以替代DNA，起到HDAC6去乙酰化酶抑制剂的作用。
具体实施方式
为了更好的理解本发明，下面我们结合具体的实施例和附图对本发明进行进一步的阐述，但值得注意的是本发明的实施不仅限于此。
本发明所用试剂和原料均市售可得或者可以按照文献方法制备。本发明实施例中未注明的具体条件的试验方法均按照常规条件，或者按照制造厂商所建议的条件。
以抗胞质内去乙酰化酶HDAC6的纳米抗体为例，介绍本发明的应用方法。
实施例1：HDAC6-CAT1截短蛋白的表达与纯化：
(1)根据HDAC6的基因序列，运用Premier Primer5.0软件设计PCR引物：CAT1-JD-5-sal1(cgaGTCGACgagcagttaaatgaattccattg)和CAT1-JD-3-not1(gcgGCGGCCGCggcggccatctcacccttggggtcc)，扩增基因片段长度为801bp；(2)以CAT1-JD-5-sal1与CAT1-JD-3-not1为引物，以HDAC6-WT重组质粒为模板，扩增出HDAC6-Cat1的6-272个氨基酸。PCR反应体系25μL，含2×PCR Mix(含酶)12.5μL、上游引物1μL、下游引物1μL、DNA模板1μL、双蒸水9.5μL。PCR的循环参数为：预变性95℃8min；变性95℃40s,退火57℃40s，延伸72℃50s，35个循环；最后72℃延伸10min。1％琼脂糖凝胶电泳观察。(3)构建pET32a-Cat1-JD重组质粒，方法如下：用市售DNA胶回收试剂盒回收各PCR扩增的基因片段，将纯化后得到的10μL基因片段及10μL pET-32a(+)载体分别用SalⅠ和XhoⅠ限制性内切酶进行双酶切处理，酶切反应可在0.5mL的EP管中进行，37℃水浴4-5h。酶切后回收、纯化各基因片段和载体。将酶切回收后的7μL各基因片段分别与1μL酶切后载体利用1μL T4连接酶进行连接，连接体系置16℃6-8h或过夜，得连接产物。DH5α感受态细胞制备与连接产物转化，根据上述平板上菌落的生长情况挑取分散的单菌落若干，接种于3mL含50μg/mL氨苄青霉素的LB液体培养基中，37℃200rpm振荡培养过夜。在无菌条件下吸取相应的菌液1μL作为模板进行PCR扩增，PCR结束后，用1.0％的琼脂糖凝胶电泳鉴定PCR产物。选取PCR结果正确且特异的样品所对应的菌液，利用质粒提取试剂盒提取重组质粒。(4)将1μg各重组质粒分别用0.5μL Sal I和0.5μL Not I限制性内切酶进行双酶切处理，将双酶切鉴定正确的重组质粒送上海Invitrogen公司测序。酶切反应可在0.5mL的EP管中进行，酶切过程为37℃水浴，4小时。酶切完成后，取10μL酶切后的产物进行1％琼脂糖凝胶电泳，结果见图1，从图中可以看出，酶切所得CAT1-JD片段与预期大小相符，为801bp,，且测序完全正确，说明HDAC6-CAT1-JD目的片段 成功克隆进入pET32a(+)载体。(5)测序正确的重组质粒pET32a-CAT1-JD转化至BL21感受态细菌中，获得的细菌分别命名为BL/CAT1-JD。分别挑取不同重组细菌的单菌落到3mL含有卡那抗生素(含量为50μg/mL)的LB液体培养基中，37℃摇床，振摇2-3小时，待菌液OD600达到0.8-1.0左右时，加入终浓度为1.0mM的IPTG(异丙基硫代半乳糖苷)，继续进行振摇培养5小时。培养结束后，8000rpm离心3min，收集细菌，并用PBS溶液洗涤2次，最终用500μL的PBS重悬菌体后，进行超声波破碎，参数为功率200W，时间10min(工作3s，间隔7s)，然后8000rpm离心5min，收集上清液，同时用500μL的PBS将沉淀重悬。(6)各取100μL 1.7中诱导表达所得的上清液和沉淀，加入25μL 5×Loading Buffer，100℃水浴煮样8min，然后加到已配好的聚丙烯酰胺凝胶上样孔中，每孔20μL。电泳时，以80V电压跑过浓缩胶，以120V电压跑完分离胶。然后将凝胶用考马斯亮蓝染色液染色3小时，换脱色液3-5次，每次1小时，至背景颜色洗脱干净，进行拍照记录。SDS-PAGE结果见图2，从图中可以看出，其中泳道2为BL21/CAT1-JD菌体诱导产物包涵体纯化产物，大小为48ku，说明HDAC6-CAT1-JD重组蛋白成功表达且纯化效果较好。
实施例2：针对HDAC6-CAT1纳米抗体文库的构建：
(1)将1mg CAT1-JD重组蛋白抗原与弗氏佐剂等体积混合，免疫一只新疆双峰驼，每周一次，共连续免疫7次，免疫过程中刺激B细胞表达特异性的纳米抗体；(2)7次免疫结束后，提取骆驼外周血淋巴细胞100ml，进行免疫骆驼的ELISA抗体水平检测，结果如图3所示，免疫后血清效价达104，说明HDAC6-CAT1-JD重组蛋白免疫效果较好；并提取总RNA；(3)合成cDNA并利用套式PCR扩增VHH；(4)利用限制性内切酶PstⅠ及NotⅠ酶切20ug pMECS噬菌体展示载体及10ug VHH并连接两种片段；(5)将连接产物转化至电转感受态细胞TG1中，构建人源抗体Fc片段纳米抗体噬菌体展示文库并测定库容，库容的大小约为1.2×108。
实施例3：针对CAT1-JD重组蛋白的纳米抗体筛选过程：
(1)取200uL重组TG1细胞至2×TY培养基中培养，期间加入40uL辅助噬菌体VCSM13侵染TG1细胞，并培养过夜以扩增噬菌体，次日利用PEG/NaCl沉淀噬菌体，离心收集扩增噬菌体；(2)将溶解在100mM pH 8.2NaHCO3中 的CAT1-JD重组蛋白200ug偶联在酶标板上，4℃放置过夜，同时设立负对照；(3)第二天加入100ul的3％BSA，室温封闭2h；(4)2h后，加入100ul扩增噬菌体(2×1011tfu免疫骆驼纳米抗体噬菌体展示基因库)，室温作用1h；(5)用PBS+0.05％Tween-20洗5遍，以洗掉结合的噬菌体；(6)用终浓度为25mg/ml的胰蛋白酶将于人源抗体Fc片段特异性结合的噬菌体解离下，并感染处于对数生长期的大肠杆菌TG1细胞，37℃培养1h，产生并收集噬菌体用于下一轮的筛选，相同筛选过程重复3轮，逐步的到富集。
实施例4：用噬菌体的酶联免疫方法(ELISA)筛选特异性阳性克隆：
(1)从上述3轮筛选后细胞培养板中，挑选175个单菌落分别接种于含100ug/mL氨苄青霉素的TB培养基的96深孔板中，并设置空白对照，37℃培养至对数期后，加入终浓度为1mM的IPTG，28℃培养过夜；(2)利用渗透胀破法获得粗提抗体，并将抗体转移至经抗原包被的ELISA板上，室温放置1h；(3)用PBST洗去未结合的抗体，加入100ul经1:2000稀释后的Mouse anti-HA tag antibody(鼠抗HA抗体，购自科文斯)，在室温放置1h；(4)用PBST洗去未结合的抗体，加入100ul经1:2000稀释后的Anti-mouse alkaline phosphatase conjugate(山羊抗小鼠碱性磷酸酶标记抗体，购自于西格玛)，在室温放置1h；(5)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，反应5-10min后于酶标仪上405波长处，读取吸收值；(6)当样品孔OD值大于对照孔5倍以上时，判定为阳性克隆孔；(7)将阳性克隆孔的菌转摇在含有100ug/ul氨苄青霉素的LB培养基中以便提取质粒并进行测序。
根据序列比对软件Vector NTI分析各个克隆株的基因序列，把FR1、FR2、FR3、FR4、CDR1、CDR2、CDR3序列相同的株视为同一克隆株，而序列不同的株视为不同克隆株，最终获得16株CAT1-JD重组蛋白特异性纳米抗体。其抗体的氨基酸序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16所示的FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4区，构成整个VHH。
实施例5：CAT1-JD重组蛋白特异性纳米抗体在宿主菌大肠杆菌中的表达、 纯化及鉴定
(1)将上述测序分析所获得不同克隆株的质粒点转化到大肠杆菌WK6中，并将其涂布在LB+amp+glucose即含有氨苄青霉素和葡萄糖的培养平板上，37℃培养过夜；(2)挑选单个菌落接种在5ml含有氨苄青霉素的LB培养液中，37℃摇床培养过夜；(3)接种1mL的过夜培养菌种至330mLTB培养液中，37℃摇床培养，培养到OD600nm值达到0.6-0.9时，加入1M IPTG，28℃摇床培养过夜；(4)离心，收集大肠杆菌，利用渗透胀破法，获得抗体粗提液；(5)通过镍柱亲和层析法纯化出抗体，获得高纯度的纳米抗体，如图4所示，其中2株纳米抗体SEQ ID NO:4和SEQ ID NO:6未成功纯化，其余14株纳米抗体均得到较好的纯化，大小为15kDa。(6)将抗体转移至经HDAC6-WT包被的ELISA板上，室温放置1h；(7)用PBST洗去未结合的抗体，加入100ul经1:2000稀释后的Mouse anti-HA tag antibody(鼠抗HA抗体，购自科文斯)，在室温放置1h；(8)用PBST洗去未结合的抗体，加入100ul经1:2000稀释后的Anti-mouse alkaline phosphatase conjugate(山羊抗小鼠碱性磷酸酶标记抗体，购自于西格玛)，在室温放置1h；(9)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，反应5-10min后于酶标仪上405波长处，读取吸收值，结果如图5所示，其中9株纳米抗体与HDAC6-WT有较好的反应，分别为SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
实施例6：针对CAT1-JD重组蛋白的纳米抗体在真核宿主中的表达及体内表达后对底物乙酰化水平的影响
(1)将上述测序分析所获得不同克隆株的质粒，与EGFP串联，构建真核重组质粒pcDNA.1，利用脂质体2000，将真核重组质粒与pcDNA3.1空质粒转染到Hela细胞中，36h后裂解细胞，进行Western-blot鉴定，将蛋白转印至NC膜上，加入10mL经1:2000稀释后的Mouse anti-His Tag antibody(鼠抗His抗体，购自西格玛)，在室温放置1h；用PBST洗去未结合的抗体，加入10mL经1:2000稀释后的Goat Anti-rabbit HRP conjugate(山羊抗小鼠辣根过氧化物酶标记抗体，购自于西格玛)，在室温放置1h；用PBST洗去未结合的抗体，曝光，结果如图6所示，16株纳米抗体均在真核宿主中表达；(2)将(1)中的 细胞裂解物进行Western-blot鉴定，将蛋白转印至NC膜上，加入10mL经1:2000稀释后的Rabbit anti-Tubulin K40antibody(兔抗Tubulin K40抗体，购自Abcam)，在室温放置1h；用PBST洗去未结合的抗体，加入10mL经1:2000稀释后的Goat Anti-rabbit HRP conjugate(山羊抗小鼠辣根过氧化物酶标记抗体，购自于西格玛)，在室温放置1h；用PBST洗去未结合的抗体，曝光，结果如图7所示，1株纳米抗体SEQ ID NO:5在真核体内表达后，对底物乙酰化水平升高，有HDAC6去乙酰化酶抑制剂的作用；(3)将SEQ ID NO:5真核重组质粒转染进入CHO-K1细胞，36h后进行间接免疫荧光，用PBST洗去细胞培养液，固定细胞后，加入100μL经1:2000稀释后的Rabbit anti-Tubulin K40antibody(兔抗Tubulin K40抗体，购自Abcam)，在室温放置1h；用PBST洗去未结合的抗体，加入100μL经1:2000稀释后的Goat Anti-rabbit A568conjugate(山羊抗小鼠红光标记抗体，购自于西格玛)，在室温放置1h；用PBST洗去未结合的抗体，用荧光显微镜观察，结果如图8所示，与没有转染进重组质粒的细胞相比，转染SEQ ID NO:5真核重组质粒的细胞红光更明显，说明1株纳米抗体SEQ ID NO:5在真核体内表达后，对底物乙酰化水平升高，再一次证明HDAC6去乙酰化酶抑制剂的作用。
实施例7：体外转录方法及各步效果图
设计体外转录PCR引物：T7-5UTR-GFP-VHH-pcDNA3.1-F(TAATACGACTCACTATAGGGGAGACCCAAGCTGGCTA)，3URT-GFP-VHH-pcDNA3.1-R(AGAATAGAATGACACCTACTC)，包含T7启动子，5’UTR和3’UTR区，以抗EGFP纳米抗体重组pcDNA3,1质粒为模板，PCR获得大量的DNA模板；NaAc回收50μL PCR反应体系，转移至新的PCR管；用HiScribeTM T7ARCA mRNA Kit(with Tailing)(mRNA体外转录试剂盒，购自NEB公司)，将DNA转录成mRNA，如图9所示，其中：1为PCR产物；2为mRNAs加帽孵育0.5h；3为mRNAs加帽孵育1.5h；4为Dnase 1处理后的mRNA；5为mRNA加尾poly(A)，说明体外mRNA转录效果较好。
实施例8：体外转录产物体内表达图
(1)将体外转录的mRNA 15ug加lipo2000 8uL，溶于250uL DMEM中，转染稳定表达含有GFP标签的抗actin抗体的CHO-K1细胞，36h后进行间接 免疫荧光，用PBST洗去细胞培养液，固定细胞后，加入100μL经1:2000稀释后的Mouse anti-His antibody(鼠抗His抗体，购自西格玛)，在室温放置1h；用PBST洗去未结合的抗体，加入100μL经1:2000稀释后的Goat Anti-mouse A568conjugate(山羊抗小鼠红光标记抗体，购自于西格玛)，在室温放置1h；用PBST洗去未结合的抗体，用荧光显微镜观察，结果如图10所示，含有GFP标签的抗actin抗体发绿色荧光，含有His标签的抗GFP纳米抗体发红色荧光，且两种荧光重合。说明体外转录的抗GFP纳米抗体的mRNA成功转染进细胞，替代了质粒转染细胞的作用，且能正确识别GFP蛋白，起到一定的生物学功能。
(2)同时，在转染后不同的时间点收集细胞，观察mRNA出现的时间，以DNA为对照。分别以0h、3h、6h、10h、12h、24h为时间点，收集细胞，进行间接免疫荧光，用PBST洗去细胞培养液，固定细胞后，加入100μL经1:2000稀释后的Mouse anti-His antibody(鼠抗His抗体，购自西格玛)，在室温放置1h；用PBST洗去未结合的抗体，加入100μL经1:2000稀释后的Goat Anti-mouse A568conjugate(山羊抗小鼠红光标记抗体，购自于西格玛)，在室温放置1h；用PBST洗去未结合的抗体，用荧光显微镜观察，mRNA结果如图11所示，DNA结果如图12所示，说明mRNA在转录后3h便开始表达，而DNA在转录后12h才开始表达，mRNA表达时间早于DNA。
实施例9：RNA转入细胞后乙酰化改变图
(1)方法同实施例6，以SEQ ID NO:5重组pcDNA3.1质粒为模板，体外转录SEQ ID NO:5的mRNA。方法同实施例5，进行Western-blot鉴定。结果如图12所示，SEQ ID NO:5的mRNA在Hela细胞中成功表达，且转染有SEQ ID NO:5的mRNA的细胞Tubulin乙酰化水平上升，说明mRNA可以替代DNA，起到HDAC6去乙酰化酶抑制剂的作用。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应当了解，在本发明不受上述实施例的限制，上述实施例和说明书中的描述只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。
Figure PCTCN2017082096-appb-000003
Figure PCTCN2017082096-appb-000004
Figure PCTCN2017082096-appb-000005
Figure PCTCN2017082096-appb-000006
Figure PCTCN2017082096-appb-000007
Figure PCTCN2017082096-appb-000008
Figure PCTCN2017082096-appb-000009
Figure PCTCN2017082096-appb-000010

Claims (10)

  1. mRNA编码的纳米抗体，其特征是，该mRNA携带的编码信息在细胞内被识别、翻译、表达出可以与靶蛋白结合的单链纳米抗体。
  2. 根据权利要求1所述的mRNA编码的纳米抗体，其特征是，mRNA分子带有至少一种增强其稳定性的修饰。
  3. 根据权利要求1所述的mRNA编码的纳米抗体，其特征是，mRNA分子带有至少一种增强其翻译效率的修饰。
  4. 根据权利要求1所述的mRNA编码的纳米抗体，其特征是，mRNA分子带有至少一种降低其免疫原性的修饰。
  5. 根据权利要求1所述的mRNA编码的纳米抗体，其特征是，mRNA编码蛋白的上下游修饰有细胞内定位信号。
  6. 权利要求1-5所述的mRNA编码的纳米抗体在制备用于调节细胞内靶蛋白功能制剂中的应用。
  7. 根据权利要求6所述的应用，其特征是，编码纳米抗体的mRNA通过转运载体进入细胞内。
  8. 根据权利要求7所述的应用，其特征是，转运载体添加有与目标待调节细胞表面标志相结合的配体。
  9. 根据权利要求6所述的应用，其特征是，mRNA的核苷酸序列对应于HDAC6-CAT1的核苷酸序列，其表达后的纳米抗体与HDAC6-WT结合。
  10. 根据权利要求9所述的应用，其特征是，纳米抗体具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列的VHH链，其编码核苷酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16所示。
PCT/CN2017/082096 2017-04-07 2017-04-27 mRNA编码的纳米抗体及其应用 WO2018184267A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/484,977 US20200024365A1 (en) 2017-04-07 2017-04-27 mRNA-ENCODED NANOBODY AND APPLICATION THEREOF

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710224513.4A CN106929513A (zh) 2017-04-07 2017-04-07 mRNA编码的纳米抗体及其应用
CN201710224513.4 2017-04-07

Publications (1)

Publication Number Publication Date
WO2018184267A1 true WO2018184267A1 (zh) 2018-10-11

Family

ID=59425936

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/082096 WO2018184267A1 (zh) 2017-04-07 2017-04-27 mRNA编码的纳米抗体及其应用

Country Status (3)

Country Link
US (1) US20200024365A1 (zh)
CN (1) CN106929513A (zh)
WO (1) WO2018184267A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109734808A (zh) * 2018-12-29 2019-05-10 南京融捷康生物科技有限公司 针对HDAC6-cat1区域的纳米抗体及其应用
EP4047084A4 (en) * 2019-10-17 2024-01-17 Univ Nagoya City Public Univ Corp ARTIFICIALLY SYNTHESIZED MRNA AND USE THEREOF
CN110981961B (zh) * 2019-11-28 2022-07-29 青岛大学 一种特异结合癌细胞蛋白b7-h4的纳米抗体h6的序列和应用
CN117794570A (zh) * 2021-06-02 2024-03-29 西韦克生物技术有限责任公司 抗体、抗体衍生物和多肽向真核细胞的细菌递送

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007147868A2 (en) * 2006-06-21 2007-12-27 Ens - Ecole Normale Superieure De Lyon Prevention of muscle atrophy
WO2009005673A1 (en) * 2007-06-28 2009-01-08 Schering Corporation Anti-igf1r
US20100136024A1 (en) * 2008-11-14 2010-06-03 Bartels Stephen P Selective Inhibition of Histone Deacetylase 6 for Ocular Neuroprotection or for Treatment or Control of Glaucoma
WO2010124009A2 (en) * 2009-04-21 2010-10-28 Schering Corporation Fully human anti-vegf antibodies and methods of using
CN104404630A (zh) * 2014-12-11 2015-03-11 东南大学 一种天然的双峰驼源噬菌体展示纳米抗体文库、构建方法及用途
WO2015189816A1 (en) * 2014-06-13 2015-12-17 Friedrich Miescher Institute For Biomedical Research New treatment against influenza virus
WO2016060520A1 (ko) * 2014-10-16 2016-04-21 차의과학대학교 산학협력단 줄기 세포의 신경 전구 세포로의 분화용 조성물 및 이를 이용한 방법

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0739981A1 (en) * 1995-04-25 1996-10-30 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
DE102004035227A1 (de) * 2004-07-21 2006-02-16 Curevac Gmbh mRNA-Gemisch zur Vakzinierung gegen Tumorerkrankungen
WO2009150539A2 (en) * 2008-06-10 2009-12-17 Institut Pasteur Variable domains of camelid heavy-chain antibodies directed against androctonus autralis hector toxins
EP2625189B1 (en) * 2010-10-01 2018-06-27 ModernaTX, Inc. Engineered nucleic acids and methods of use thereof
CN103396482B (zh) * 2013-05-17 2016-08-10 东南大学 一种前白蛋白纳米抗体、其编码序列及应用
CN103333248B (zh) * 2013-06-07 2014-07-09 东南大学 一种cd25纳米抗体、其编码序列及应用
EP2899208A1 (en) * 2014-01-28 2015-07-29 F.Hoffmann-La Roche Ag Camelid single-domain antibody directed against phosphorylated tau proteins and methods for producing conjugates thereof
CN106046164B (zh) * 2015-07-20 2019-10-25 广西医科大学 抗CTLA-4的纳米抗体Nb36及其制备方法与应用
CN106008709B (zh) * 2016-03-14 2019-08-06 西北农林科技大学 一种特异性结合PRRS病毒非结构蛋白Nsp4纳米抗体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007147868A2 (en) * 2006-06-21 2007-12-27 Ens - Ecole Normale Superieure De Lyon Prevention of muscle atrophy
WO2009005673A1 (en) * 2007-06-28 2009-01-08 Schering Corporation Anti-igf1r
US20100136024A1 (en) * 2008-11-14 2010-06-03 Bartels Stephen P Selective Inhibition of Histone Deacetylase 6 for Ocular Neuroprotection or for Treatment or Control of Glaucoma
WO2010124009A2 (en) * 2009-04-21 2010-10-28 Schering Corporation Fully human anti-vegf antibodies and methods of using
WO2015189816A1 (en) * 2014-06-13 2015-12-17 Friedrich Miescher Institute For Biomedical Research New treatment against influenza virus
WO2016060520A1 (ko) * 2014-10-16 2016-04-21 차의과학대학교 산학협력단 줄기 세포의 신경 전구 세포로의 분화용 조성물 및 이를 이용한 방법
CN104404630A (zh) * 2014-12-11 2015-03-11 东南大学 一种天然的双峰驼源噬菌体展示纳米抗体文库、构建方法及用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANG RENREN ET AL.: "Application and the Research Progress of Nanobodies", CHEMISTRY OF LIFE, vol. 33, no. 3, 31 December 2013 (2013-12-31), pages 307 - 315, XP009507680 *
ZHOU TAORAN ET AL.: "Current status of nanobody technology, Application in disease diagnosis and therapy", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 43, no. 10, 31 December 2016 (2016-12-31), pages 936 - 945 *

Also Published As

Publication number Publication date
US20200024365A1 (en) 2020-01-23
CN106929513A (zh) 2017-07-07

Similar Documents

Publication Publication Date Title
Wang et al. AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells
Borst et al. The human cytomegalovirus UL51 protein is essential for viral genome cleavage-packaging and interacts with the terminase subunits pUL56 and pUL89
Eystathioy et al. A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles
WO2018184267A1 (zh) mRNA编码的纳米抗体及其应用
JP2024059823A (ja) 操作された細菌ユビキチンリガーゼ模倣物を用いる、幅広い範囲にわたるプロテオーム編集
JP2016028604A (ja) ＩＬ−４Ｒαに結合する涙液リポカリンムテイン
WO2022247943A1 (en) Constructs and methods for preparing circular rnas and use thereof
JP2008200041A (ja) モジュラートランスフェクション系
CN112004932B (zh) 一种CRISPR/Cas效应蛋白及系统
Whitley et al. Encapsulating Cas9 into extracellular vesicles by protein myristoylation
WO2014135998A1 (en) In vitro and in vivo delivery of genes and proteins using the bacteriophage t4 dna packaging machine
WO2015089881A1 (zh) 全人源抗cd26抗体及其应用
CN115885039A (zh) 蛋白质降解
WO2018167070A1 (en) Virus-like particles and uses thereof
Chan et al. Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma
Kershberg et al. Protein composition of axonal dopamine release sites in the striatum
CN109207518B (zh) 用于基因转录激活的药物诱导型CRISPR/Cas9系统
CN111410695B (zh) 基于自噬机制介导Tau蛋白降解的嵌合分子及其应用
CN113249408A (zh) 一种靶向激活体液免疫和细胞免疫的核酸疫苗载体构建及应用
EP2554672B1 (en) Nucleic acid structure, method for producing complex using same, and screening method
WO2023212584A2 (en) Rna-binding by transcription factors
US20220281931A1 (en) Chemically inducible polypeptide polymerization
Zhao et al. Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan
CN110893240B (zh) Nme2基因在抑制禽呼肠病毒复制中的应用
CN113880924A (zh) 一种SARS-CoV-2假病毒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17904730

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17904730

Country of ref document: EP

Kind code of ref document: A1