Patent Application
20240368657
The technology described herein relates to methods for producing double-stranded RNA in bacterial cells.
The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said ST.26 copy, created on Jun. 11, 2024, is named SEQUENCE LISTING.xml and is 12,512 bytes in size.
RNA interference (RNAi) by double-stranded (ds) small interfering RNAs (siRNA) suppresses gene expression by inducing the degradation of mRNAs bearing complementary sequences. Transfection of synthetic siRNAs into eukaryotic cells to silence genes has become an indispensable tool to investigate gene function, and siRNA-based therapy is being developed to knockdown genes implicated in disease.
A novel siRNA design and production system has been invented by Dr. Linfeng Huang (Huang et al, Nat Biotechnology, 2013, 31(4):350-6; Huang & Lieberman, Nat Protocol, 2013, 8(12):2325-36). This system utilizes the unique function of the protein p19, which can bind to and stabilize 21-nt double stranded RNA species produced by endogenous RNase III in Escherichia coli (E. coli), producing a pool of siRNAs within a certain selected gene sequence. Those siRNAs produced in E. coli are double-stranded RNA fragments.
However, there are still needs for more efficient ways to produce double-stranded RNAs.
The technology described herein is directed to methods, insoluble complex and vector relating to the production of double-stranded RNA generated in vivo, e.g. in bacterial cells.
In an aspect, a vector includes a first nucleic acid sequence encoding a fusion protein which is siRNA-binding polypeptide comprising a p19 protein and a protein named as protein 1 (P1) and a second nucleic acid sequence substantially complementary to a target RNA.
In an embodiment, the vector further comprises a third nucleic acid sequence encoding an siRNA-generating enzyme, and the siRNA-generating enzyme is selected from RNase III and/or RNase III mutant at least 95% identical to the RNase III.
In an embodiment, the p19 protein is selected from following proteins: Tombusvirus (TBSV) p19 protein; TBSV p19 protein mutant at least 95% identical to the TBSV p19 protein; Carnation Italian ringspot virus (CIRV) p19 protein; or CIRV p19 protein mutant at least 95% identical to the CIRV p19 protein.
In an embodiment, wherein the nucleic acid sequence encodes for protein 1 as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:5.
In an embodiment, the siRNA-binding polypeptide comprises a purification tag.
In an embodiment, the vector comprises a promoter sequence.
In an embodiment, a composition for targeting a disease further comprises one or more pharmaceutical carriers, excipients, preservatives, colorants and/or diluents.
In an embodiment, the vector is a plasmid expressed by E. coli cell.
In an aspect, an inclusion body expressed by bacteria, wherein the inclusion body encodes by the vector disclosed herein.
In an aspect, an insoluble complex is generated by the interaction of a fusion protein of p19 and a double stranded RNA of target sequence.
In an embodiment, the fusion protein of p19 at least comprises p19 protein and P1 protein.
In an aspect, a method for double-stranded RNA producing comprises: transforming the vector disclosed herein to bacteria; culturing and inducing the bacteria expressed the inclusion body; extracting the inclusion body by the sonication or high-pressure lysis of the collected bacteria; resuspending the inclusion body to produce the released double-stranded RNA.
In an embodiment, the bacterium is an E. coli cell.
In an embodiment, the method further comprises purifying the released double-stranded RNA.
In an embodiment, the step of extracting the inclusion body further comprises: crushing the collected bacteria by sonication or high-pressure lysis method and extracting the inclusion body from the crushed bacteria by centrifugation.
In an aspect, a double-stranded RNA produced by the method described as below.
In an aspect, a method of RNAi comprising application of the double-stranded RNA produced by the method described as below to a subject in need of RNA interference, wherein the subject comprises eukaryotic cells and/or organisms.
One advance of the method for double-stranded RNA producing disclosed by the embodiment is that the method incorporates p19 protein with a new aggregation protein domain named “protein 1” to form an insoluble form known as the “inclusion body”, such that the yield and length diversity of the inclusion-body formulated double-stranded RNA was increased greatly compared with traditional double-stranded RNA producing methods.
Another advance of the method for double-stranded RNA producing disclosed by the embodiments are that protein 19 and protein 1 also empower those double-stranded RNA with potentially built-in delivery efficacy.
In order to more clearly illustrate the technical solutions of the embodiments of the present application, a brief description will be given below of the accompanying drawings which are required to be used in the embodiments. It is to be understood that the following drawings illustrate only some embodiments of the present application and are therefore not to be considered as limiting the scope. For ordinary technical personnel in this field, other relevant drawings can also be obtained according to these drawings without involving any inventive effort.
In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of this description.
Embodiments of the invention described herein are directed to methods, insoluble complex and vector relating to the production of double-stranded RNA in vivo, e.g. in bacterial cells (siRNAs produced according to the methods and compositions described herein are also referred to herein as “double-stranded RNA”). The technology described herein is derived from the inventors' discovery that the fusion of p19 protein and protein 1 forms an insoluble form known as the “inclusion body”. The yield and length diversity of the inclusion-body formulated double-stranded RNA was increased greatly compared with traditional double-stranded RNA producing methods. Beyond that, p19 protein and protein 1 also empower those double-stranded RNA (dsRNA) with potentially built-in delivery efficacy.
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, the terms “reduced”, “reduction”, “decrease”, or “inhibit” can mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or more or any decrease of at least 10% as compared to a reference level. In some embodiments, the terms can represent a 100% decrease, i.e., a non-detectable level as compared to a reference level. In the context of a marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
The term “proteins” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
The term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
The term operatively linked includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and, optionally, production of the desired polypeptide encoded by the polynucleotide sequence. In some examples, transcription of a nucleic acid modulatory compound is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the nucleic acid in a cell-type in which expression is intended. It will also be understood that the modulatory nucleic acid can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally occurring form of a protein.
The term “isolated” or “purified” as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered “isolated.”.
The term “complementary” or “complementary base pair” refers to A: T and G:C in DNA and A:U in RNA. Most DNA consists of sequences of nucleotide only four nitrogenous bases: base or base adenine (A), thymine (T), guanine (G), and cytosine (C). Together these bases form the genetic alphabet, and long ordered sequences of them contain, in coded form, much of the information present in genes. Most RNA also consists of sequences of only four bases. However, in RNA, thymine is replaced by uridine (U).
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean±1%.
The term “includes” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
The term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”.
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9). Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention can be performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
In the implementation of this application, applicant surprisingly find that a fusion protein of p19 and a double stranded RNAs of target sequence can interact and become an insoluble complex (i.e., inclusion body). Accordingly, the insoluble complex may use for purification of RNA product or their related application.
Specifically, the fusion protein of p19 may include p19 protein and protein 1. Alternatively, the p19 protein can be replaced by any suitable RNA-binding protein.
At least one advantage of the insoluble complex is that the RNAs of target sequence can be isolated from the protein and the cells according to the actual needs.
Furthermore, the bacteria cell lysates, insoluble particles (i.e., inclusion body) and the purified RNAs can be used for downstream applications.
Referring to
The term “vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. Many vectors useful for transferring exogenous genes into target cells are available, e.g. the vectors may be episomal, e.g., plasmids, virus derived vectors or may be integrated into the target cell genome, through homologous recombination or random integration. In some embodiments, a vector can be an expression vector. As used herein, the term “expression vector” refers to a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms. The nucleic acid incorporated into the vector can be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence. In some embodiments, the dsRNA and the nucleic acid encoding the siRNA-binding polypeptide can be within the same operon. In some embodiments, the dsRNA and the nucleic acid encoding the siRNA-binding polypeptide can be within separate operons.
As used herein, “p19 protein” can be also named as p19 polypeptides. “p19 protein” in particular but not limited to the p19 polypeptide such as tombusvirus p19 (NCBI Gene ID: 1493957) are able to bind to the siRNA and in particular include those as disclosed in US 2015/0337306A1 which are incorporated herein by reference. The p19 polypeptide as disclosed in US 2015/0337306A1, i.e. selected from a p19 polypeptide such as tombusvirus p19 polypeptide.
The term “protein 19” refers to a viral protein which binds specifically to dsRNAs and which suppresses RNAi-mediated host plant viral defenses. The sequences of p19 polypeptides from a number of species are known, e.g. tombusvirus p19 (NCBI Gene ID: 1493957). In some embodiments, referring to
Further preferred, the siRNA-binding polypeptide has a purification tag suitable for purification of the siRNA-binding polypeptide and siRNAs bound to the siRNA-binding polypeptide. The purification tag can bind to another moiety such as on a matrix or a resin with affinity for the purification tag such as Ni-NTA resin. Particular purification tags include histidine tags (“His-tagged”) such as disclosed in US 2015/0337306A1 which are incorporated herein by reference. The siRNA-binding polypeptide is in particular a His-tagged p19 polypeptide.
The vector is in particular able to express a siRNA-binding polypeptide, in particular a p19 polypeptide such as His-tagged p19 polypeptide, and an siRNA-generating enzyme like a RNase III or RNase III mutants, in particular p19 such as His-tagged p19, fused to a siRNA-generating enzyme like an E. coli RNase III. Such expression of siRNA-generating enzyme like an E. coli RNase III will enhance the siRNA production.
As used herein, “protein 1” refers to a new aggregation protein domain. The fusion of p19 protein and protein 1 forms inclusion body to achieve significant yield improvement of the double-stranded RNAs.
In some embodiments, referring to
The parameters of fusion protein and their dsRNA products can be shown in
As shown in
As shown in
As shown in
As shown in
As used herein, “substantially complementary” refers to a first nucleotide sequence having at least 90% complementarity over the entire length of the sequence with a second nucleotide sequence, e.g. 90% complementary, 95% complementary, 98% complementary, 99% complementary, or 100% complementary. Two nucleotide sequences can be substantially complementary even if less than 100% of the bases are complementary, e.g. the sequences can be mismatched at certain base.
As used herein, the term “target RNA” refers to an RNA present in a cell (i.e. the “target cell”). The target RNA comprises a target sequence to which one strand of a siRNA according to the methods and compositions described herein binds, thereby causing RNAi silencing of the target RNA. The target cell can be the bacterial cell comprising a siRNA-binding polypeptide or another cell, either prokaryotic or eukaryotic. The target sequence can be an RNA that can be translated (i.e. it can encode a polypeptide, e.g. mRNA) or it can be an RNA that is not translated (i.e. a non-coding RNA). In some embodiments, the target sequence can be any portion of an mRNA. In some embodiments, the target sequence can be a sequence endogenous to the cell. In some embodiments, the target sequence can be a sequence exogenous to the cell. In some embodiments, the target sequence can be sequence from an organism that is pathogenic to the target cell, e.g. the target sequence can be sequence from a viral, bacterial, fungal, and/or parasitic origin. In some embodiments, the target sequence is a viral nucleotide sequence.
In an embodiment, the siRNAs can be generated from the dsRNA comprising a nucleic acid sequence substantially complementary to a target RNA. As used herein, the term “siRNA” refers to a nucleic acid that forms an RNA molecule comprising two individual strands of RNA which are substantially complementary to each other. Typically, the siRNA is at least about 15-40 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-40 nucleotides in length, and the double stranded siRNA is about 15-40 base pairs in length, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In some embodiments, a siRNA can be blunt-ended. In some embodiments, a siRNA can comprise a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. The siRNA molecules can also comprise a 3′hydroxyl group. In some embodiments, the siRNA can comprise a 5′phosphate group. A siRNA has the ability to reduce or inhibit expression of a gene or target RNA when the siRNA is present or expressed in the same cell as the target gene, e.g. the target RNA. siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target RNA molecule at a site guided by the siRNA.
Preferably, the vector further comprises a “P19-P1 fusion protein expression cassette” including a promoter and a sequence encoding a P19-P1 fusion as described below and optionally a siRNA-generating enzyme, in particular a ribonuclease like an Escherichia coli (E. coli) RNase III. One of skill in the art will understand that the method of the present invention could also use sequences encoding a siRNA-generating enzyme such as a RNase III from any other bacterial species. In preferred embodiments of the present invention, the vector is a plasmid further comprising a siRNA-binding polypeptide expression cassette including a promoter, a sequence encoding a siRNA-binding polypeptide and a sequence encoding a siRNA-generating enzyme, wherein the siRNA-binding polypeptide is a P19-P1 fusion protein and the siRNA-generating enzyme is an E. coli RNase III.
As show in
Referring to
Firstly, the vector used herein, such as plasmid, is transformed to bacteria such as Escherichia coli cell (E. coli). Alternatively, the bacterial cell can be from any species although E. coli cells are preferred.
Secondly, culturing and inducing the bacteria either through IPTG induction method or lactose auto-induction method to express the vector.
Thirdly, extracting the inclusion body by the lysis of the bacteria.
The term “extracting” as used herein means separating the siRNA from other components such as from the bacterial cells and other DNA or RNA sequences or polypeptides that are present resulting from the materials used and conditions applied for producing the double-stranded RNAs. In particular, isolating the double-stranded RNAs comprises extracting and purifying the double-stranded RNAs.
In some embodiments, the inclusion body can be resuspended and the released double-stranded RNAs may continually purify to generate the purified double-stranded RNAs.
In some embodiments, the bacteria cell lysates, released or purified ds RNAs and the inclusion body can also be used together for any suitable RNAi applications, according to the actual needs.
Referring to
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/116876 | Sep 2022 | WO | international |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/116412 | Sep 2023 | WO |
| Child | 18748282 | US |
