Fermentation-based method for double-stranded RNA production

Abstract

The present disclosure provides methods and tools that allow for the production of large quantities of double-stranded RNA (dsRNA) in bacterial cells, including plasmid vectors that can be used to transform Gram-positive or Gram-negative bacterial cells, such that the cells produce high yields of target dsRNA.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 424,778, filed on November 11, 2022, entitled “FERMENTATION BASED METHOD FOR DOUBLE STRANDED RNA PRODUCTION,” the contents of which are hereby incorporated by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] This application contains a sequence listing that has been submitted in .XML format by PatentCenter and is hereby incorporated by reference in its entirety. The WIPO sequence listing was created on November 10, 2023 and is named 066803-779123 Sequence Listing and is 121 kilobytes in size. Field of the Invention

[0005] The present disclosure generally relates to high-yield microbial plasmid vectors and their use in fermentation-based systems for the production of double-stranded RNA (dsRNA). Background of the Invention

[0007] RNAi biopesticides utilize a conserved eukaryotic gene regulatory mechanism, RNA interference, to disrupt the production of proteins essential for the survival of target pests. The active molecule of RNAi biopesticides, double-stranded RNA (dsRNA), can be delivered to plant pests through exogenous application. After application, dsRNA is ingested and absorbed by the cells of the target pest, where it engages the RNAi process to direct the sequence-specific degradation of the target mRNA transcript. Due to the selectivity of this mode of action and the favorable toxicity profile and instability of dsRNA, RNAi biopesticides present minimal risk to human health and the environment.

[0008] Despite progress in the identification of lethal RNAi gene targets and RNA delivery, the full potential of RNAi biopesticides for pest control remains unrealized due to the high cost of large-scale dsRNA production. Huge quantities of dsRNA are required to control agricultural pests. For a single spray application at a rate of 7 g / Ha, approximately 11,550 kg of dsRNA would be required to treat 0.1% of the total global crop area of ~1.65B hectares (Ha)15,16. Developing dsRNA production technologies that can be easily scaled up without specialized resources and that leverage existing production capacity to keep costs low will be necessary to allow widespread agricultural application of RNAi.

[0009] Existing RNA production methods include chemical synthesis, in vitro transcription, cell-free synthesis, and fermentation. Fermentation offers important advantages, including low raw material costs, scalability, and the availability of existing production infrastructure and capacity, but is not practical for commercial dsRNA production due to low yields.

[0010] Methods need to be developed to reduce the cost of dsRNA production to a level that allows the development of RNAi biopesticides that are cost-competitive with chemical pesticides. Microbial fermentation-based production systems could pave the way for cost-effective production of dsRNA, leveraging existing fermentation infrastructure and, unlike in vitro transcription, cell-free, and chemical synthesis methods, requiring no specialized equipment, materials, or conditions for dsRNA production.

[0011] Therefore, there is a need to develop high-yield plasmid constructs for bacterial cells for large-scale production of dsRNA in fermentation-based systems. SUMMARY OF THE INVENTION

[0013] In some aspects, the present disclosure provides a plasmid vector comprising a nucleic acid sequence comprising: an inducible bacterial promoter operably linked to a stem-loop sequence, the stem-loop sequence having a 3' end and a 5' end and comprising a target recombinant RNA sequence; a first pac site sequence at the 3' end of the stem-loop sequence and a second pac site sequence at the 5' end; an MS2 capsid protein (CP) expression cassette; and a pyrE coding sequence downstream of the MS2 CP expression cassette, which has a ribosome binding site (RBS) at its 5' end and a T1-T2 terminator at its 3' end.

[0014] In some aspects, the plasmid vector comprises an E. coli phage T7 promoter comprising the sequence of SEQ ID NO: 1.

[0015] In some aspects, the pyrE cassette of the plasmid vector comprises an RBS-pyrE cds-T1-T2 having SEQ ID NO: 2, which is driven by an upstream E. coli phage T7 promoter having SEQ ID NO: 1. In some aspects, expression of the pyrE coding sequence comprising the RBS-pyrE cds-T1-T2 is driven by a dedicated E. coli phage T7 promoter comprising the sequence of SEQ ID NO: 1. In some aspects, the pyrE coding sequence comprising the RBS-pyrE cds-T1-T2 having the sequence of SEQ ID NO: 2 is driven by a dedicated J23115 promoter comprising the sequence of SEQ ID NO: 33.

[0016] In some aspects, the target recombinant RNA in the plasmid vector is a dsRNA that specifically inhibits the expression of the target gene. In some aspects, the target recombinant RNA sequence is selected from dsRNA, siRNA, shRNA, hpRNA and miRNA.

[0017] In some aspects, the plasmid vector of the present disclosure comprises a first pac site sequence at the 3' end and a second pac site sequence at the 5' end of the stem-loop sequence. In some aspects, the first pac site sequence and the second pac site sequence each comprise the sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0018] In some aspects, the plasmid vector comprises a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 33, or any combination thereof. In further aspects, the disclosed vector does not comprise an antibiotic resistance Amp-r gene or a tetracycline resistance gene. In some aspects, the disclosed vector comprises a sequence of SEQ ID NO: 43.

[0019] In some respects, the plasmid vector of the present disclosure is expressed in gram-positive bacterial cells or gram-negative bacterial cells. In some respects, the bacterial cell expressing the disclosed plasmid vector is an Escherichia coli (E.coli) cell or Corynebacterium glutamicum (C.glutamicum) cell. In some respects, the bacterial cell expressing the disclosed plasmid vector is an Escherichia coli cell. In some respects, the Escherichia coli cell is an RNase III defective Escherichia coli strain. In some respects, the Escherichia coli cell is an RNase III defective Escherichia coli strain HT115 (DE3). In some respects, the Escherichia coli strain is a uracil auxotroph.

[0020] In some aspects, the present disclosure includes cultivating a bacterial cell colony transformed with a plasmid vector in a bioreactor. In some aspects, the bioreactor is selected from a fed-batch system, a semi-continuous system, and a continuous culture system. In some aspects, the bioreactor is a fed-batch system. In some aspects, the bacterial cells produce target dsRNA. In some aspects, the target dsRNA is produced in an amount of at least about 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 11g / L, or 12g / L.

[0021] In some aspects, the present disclosure includes a method for producing target recombinant RNA. The method includes maintaining a bacterial culture expressing the disclosed plasmid vector in a bioreactor, and the time and conditions for maintaining the method are enough to produce a target dsRNA of at least about 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 11g / L or 12g / L. In some aspects, the bioreactor is selected from a fed-batch system, a semi-continuous system and a continuous culture system. In some aspects, the bioreactor is a fed-batch system. In some aspects, the method further includes gathering in the crops target dsRNA. In some aspects, the target recombinant RNA is a dsRNA that specifically inhibits the expression of a target gene. In some aspects, the target recombinant RNA sequence is selected from dsRNA, siRNA, shRNA, hpRNA and miRNA.

[0022] Further provided herein is the use of a bacterial culture comprising a bacterial cell population disclosed herein for producing recombinant RNA without an antibiotic marker.In some aspects, the recombinant RNA is dsRNA, siRNA, shRNA, hpRNA, and miRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A is a schematic diagram of plasmid pAPSE10218.

[0025] Figure 1B is a schematic diagram of plasmid pAPSE10448.

[0026] Figure 1C is a schematic diagram of plasmid pAPSE10471.

[0027] Figure 1D is a schematic diagram of plasmid pAPSE10500.

[0028] Figure 1E is a schematic diagram of plasmid pAPSE10772.

[0029] Figure 1F is a schematic diagram of plasmid pAPSE10775.

[0030] Figure 1G is a schematic diagram of plasmid pAPSE10797.

[0031] Figure 1H is a schematic diagram of plasmid pAPSE10822.

[0032] Figure 1I is a schematic diagram of plasmid pAPSE10826.

[0033] Figure 1J is a schematic diagram of plasmid pAPSE10835.

[0034] Figure 1K is a schematic diagram of plasmid pAPSE10836.

[0035] Figure 2A Schematic diagram of the constructs used to study the effects of expressing MS2 CP or GFP on dsRNA accumulation in HT115(DE3) and JM109(DE3) cells.

[0036] Figure 2B is an image of a Coomassie-stained SDS-PAGE gel demonstrating the expression of MS2 CP and GFP in the disclosed strains.

[0037] Figure 2C is a bar graph showing shake flask dsRNA yields from different E. coli strains. The bars labeled a and b are significantly different.

[0038] Figure 2D is an image of an agarose gel used for quantification of dsRNA from different strains.

[0039] Figure 3 This image is an agarose gel image examining MS2 CP-mediated dsRNA accumulation in E. coli cells harboring a functional rnc gene. Lanes 1 to 3 contain RNA samples from three independent cultures of JM109(DE3) / pAPSE10218. Lanes 4 to 6 contain RNA samples from three independent cultures of JM109(DE3) / pAPSE10305.

[0040] Figure 4A is a schematic diagram illustrating the design of different plasmid constructs, which shows the location of the pac site on the hpRNA. The rectangles in the construct diagram represent the sense and antisense sequences of CPB β-actin, respectively.

[0041] Figure 4B is a bar graph showing shake flask dsRNA productivity data from HT115 (DE3) cells expressing different plasmid constructs. Bars labeled with the same letter are not significantly different.

[0042] Figure 5A Schematic diagram illustrating the construct used to produce CPB β-actin dsRNA as intramolecular hpRNA from a single transcript (pAPSE10218), and the construct to produce intermolecular dsRNA by bidirectional transcription (pAPSE10402).

[0043] Figure 5Bis a bar graph showing the dsRNA yields of CPB β-actin hpRNA and inter-molecular dsRNA from HT115 (DE3) cells expressing different plasmid constructs.

[0044] Figure 6A Schematic diagram showing constructs not expressing pyrE (pAPSE10218 and pAPSE10379) and overexpressing pyrE (pAPSE10775 and pAPSE10448).

[0045] Figure 6B Bar graph showing shake flask dsRNA yields of constructs pAPSE10379 and pAPSE10448 carrying the RIFA β-actin sequence in minimal medium.

[0046] Figure 6C Bar graph showing shake flask dsRNA yields of constructs pAPSE10218 and pAPSE10775 carrying the CPB β-actin sequence in minimal medium.

[0047] Figure 7 is a bar graph showing dsRNA yields from high cell density fed-batch fermentation cultures.

[0048] Figure 8 Line graph showing mortality of Colorado potato beetle (Leptinotarsa decemlineata (Say)) (CPB) larvae in a leaf disc bioassay when exposed to heat-killed E. coli HT115 (DE3) / pAPSE10218 cells containing β-actin dsRNA applied at three different concentrations or when fed on untreated leaf discs. Significant differences between treatments are indicated by different letters next to the corresponding lines.

[0049] Figure 9 Schematic diagram of HT115(DE3)-ΔpyrE surrounding micro-Tn10::rnc-era-reco.

[0050] The drawings do not limit the concepts herein to the specific aspects disclosed and described herein. The drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of certain aspects of the inventive concept. Detailed Description of the Invention

[0052] The present disclosure provides plasmid vectors and methods for improving the production of large quantities of non-encapsidated dsRNA in vivo. The disclosure is based on the following surprising discovery: the co-expression of sequences encoding the MS2 CP box, hpRNA, pac site, and pyrE in a plasmid vector can substantially improve the expression of dsRNA. These constructs can be expressed in bacterial cells and can be cultivated in scalable high-density fed-batch fermentation bioreactors to be used as a high-yield system for dsRNA production.

[0053] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications. The publications discussed herein are provided solely for their disclosure prior to the date of filing of this application. Nothing herein should be construed as an admission that the present disclosure is not entitled to preempt such publications by virtue of prior disclosure.

[0054] definition

[0055] When introducing elements of the present disclosure or preferred aspects thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Whenever the terms "comprising" or "including" are used, it is to be understood that the present disclosure also expressly contemplates and encompasses additional aspects "consisting of" the disclosed elements, which do not include additional elements other than the listed elements.

[0056] As used herein, the numerical range disclosed by numerical endpoints includes all numbers encompassed by the range (e.g., "1 to 5" includes but is not limited to 1, 1.25, 1.5, 1.75, 2, 2.3, 2.5, 2.8, 3, 3.1, 3.3, 3.8, 3.9, 4, 4.25, 4.5, 4.75, and 5). Unless otherwise indicated, all numbers used herein to express quantity, amount, size, measurement, etc. should be understood to encompass specific quantity, amount, size, measurement, etc., including those modified by the term "about". For example, the amount disclosed herein as "about 1 mg / mL" clearly includes the amount of 1 mg / mL, etc. Accordingly, unless otherwise indicated to the contrary, the numerical descriptions set forth herein may change while fully retaining within the teachings of the present disclosure. At least, each numerical value should be taken into account the number of significant figures and interpreted by applying conventional rounding techniques. As various changes could be made in the above cells and methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description and the examples given below shall be interpreted as illustrative and not in a limiting sense.

[0057] As used herein, the term "capsid protein" or "coat protein" refers to the coat protein of bacteriophage MS2, which is capable of binding to the cognate bacteriophage RNA pac site with high affinity and assembling into a complex hollow tertiary structure, wherein the bacteriophage RNA may be entirely encapsidated within the hollow tertiary structure. The term "capsid" refers to the hollow tertiary structure formed by the assembly of individual capsid proteins. An incomplete capsid is understood to mean a capsid that is not completely closed, such that a hollow tertiary structure is not formed.

[0058] As used herein, "dsRNA" refers to double-stranded RNA comprising substantially complementary RNA strands. dsRNA comprises RNA sequences having sufficient internal homology to form significant secondary structures, such as hairpins, due to internal complementary sequences hybridizing to each other via Watson-Crick base pairing of nucleotide bases within the complementary sequences.

[0059] As used interchangeably herein, the terms "RNA interference" and "RNAi" refer to the inhibition of mRNA degradation or protein synthesis by an endogenous pathway involving the DICER protein complex. DICER cuts longer double-stranded RNA (dsRNA) molecules into short fragments of approximately 21 nucleotides, called small interfering RNA (siRNA). siRNA unwinds into two single-stranded RNAs: a passenger strand and a guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Microribonucleic acids (miRNAs) are typically 22 nucleotides long and therefore very similar in size to siRNAs; however, miRNAs are cut from precursor molecules containing a polynucleotide "loop" connecting the hybridized passenger and guide strands, and they may also be similarly incorporated into RISC. Post-transcriptional gene silencing occurs when the guide strand specifically binds to a complementary mRNA molecule and induces cutting by the catalytic component of RISC, Argonaute. One possibility for generating siRNA in cells is to express a precursor RNA called hairpin RNA (hp-RNA). The transcription of hp-RNA is typically driven by an RNA polymerase III promoter such as the U6 or H1 promoter or the T7 RNA polymerase. RNA-mediated silencing using inverted repeats of a nucleic acid or portion thereof (in this case, a substantially contiguous stretch of nucleotides derived from the target gene, or any nucleic acid capable of encoding an ortholog, paralog, or homolog of a protein of interest) is preferably capable of forming a hairpin structure. The inverted repeats are cloned into an expression vector comprising a control sequence. Non-coding DNA nucleic acid sequences (spacers, such as matrix attachment region fragments (MARs), introns, polylinkers, etc.) are positioned between the two inverted nucleic acids that form the inverted repeats. After transcription of the inverted repeats, a chimeric RNA having a self-complementary structure (partial or complete) is formed. This double-stranded RNA structure is referred to as hairpin RNA (hpRNA). The hpRNA is processed by the insect into siRNA, which is incorporated into an RNA-induced silencing complex (RISC). The RISC then cleaves the target mRNA transcript, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide.

[0060] As used herein, "plasmid" refers to any extrachromosomal episome that can replicate or stably maintain in a host cell. Particularly encompassed by this definition are plasmids such as pBR322, pCG1, and pACYC184, which represent the backbone of the plasmid. One of ordinary skill in the art will recognize that other plasmids or stably maintained viral episomes can provide the same desired functions of maintenance, expression, and selection, and may generate substitutes for the basic plasmid described herein from such other plasmids or stably maintained viral episomes without the need for excessive experimentation. A key feature of the present invention is the ability to express genes encoding dsRNA and capsid proteins, rather than specific replication patterns, expression, or selection markers found on episomes containing genes encoding dsRNA and capsid proteins. As used herein, "vector" comprises nucleic acid constructs or expression constructs. Vectors as described herein may be selected from any genetic element known in the art that can promote intercellular nucleic acid transfer, such as, but not limited to, plasmids, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, etc.

[0061] As used herein, "pyrE" refers to the pyrimidine biosynthetic pathway enzyme orotate phosphoribosyltransferase.

[0062] As used herein, "pac site" refers to the packaging site.

[0063] As used herein, "unencapsidated dsRNA" refers to double-stranded RNA that is not incorporated into a capsid and includes both dsRNA associated with an incomplete capsid and dsRNA that is completely unrelated to the bacteriophage coat protein. The dsRNA contemplated in the present invention comprises a single RNA having two complementary domains separated by a non-homologous recombination spacer / loop sequence capable of forming a hairpin structure. The dsRNA can be a hairpin RNA (hRNA) or a stem-loop RNA.

[0064] The polynucleotides described herein may comprise one or more nucleic acids each encoding a polypeptide, all of which are operably linked (i.e., in a functional relationship) to one or more regulatory sequences, such as a promoter. Such polynucleotides may alternatively be referred to herein as "nucleic acid constructs" or "constructs."

[0065] In a further preferred aspect, each nucleic acid sequence described herein by its percent identity or similarity to a given nucleic acid sequence has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or similarity to a given nucleotide sequence or amino acid sequence, respectively. The terms "homology", "sequence identity" and the like are used interchangeably in this article. Sequence identity is described as the relationship between two or more nucleic acid (polynucleotide) sequences in this article, as determined by comparing sequences. In a preferred aspect, sequence identity is calculated based on the full length (in terms of amino acids or nucleotides) of two given SEQ ID NOs or based on a part thereof. A part for a full-length sequence may be referred to as a fragment, and preferably means at least 50%, 60%, 70%, 80%, 90% or 100% of the length (in terms of nucleotides) of the reference sequence." identity " also refers to the sequence association degree between two nucleic acid sequences, as determined by the matching between the strings of this type of sequence. For example, two sequences can be compared using a computer program (such as a global or local alignment algorithm) that is commonly used for this purpose, to determine the sequence identity degree between two sequences. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, ClustalOmega, AlignMe, Praline, GAP, BESTFIT or another suitable method or algorithm. The Needleman and Wunsch global alignment algorithms can be used to align two sequences over their entire length or a portion thereof (which portion may mean at least 50%, 60%, 70%, 80% or 90% of the sequence length) to maximize the number of matches and minimize the number of gaps. Default settings can be used, and preferred programs are Needle for pairwise alignments (in one aspect, using EMBOSS Needle 6.6.0.0, Gap Open Penalty 10, Gap Extent Penalty: 0.5, End Gap Penalty: False, End Gap Open Penalty: 10, End Gap Extent Penalty: 0.5), and MAFFT for multiple sequence alignments.

[0066] An "expression construct" or "nucleic acid construct" carries a genome that is stable and remains episomal in a cell. In the context of the present invention, a cell may be intended to encompass a cell for preparing a construct or a cell to which the construct will be administered. Alternatively, the construct can be integrated into the genome of the cell, for example, by homologous recombination or otherwise. A "DNA construct" or "nucleic acid construct" prepared for introduction into a specific host may include a replication system recognized by the host, an expected DNA segment encoding the desired polypeptide, and transcription and translation start and stop regulatory sequences operably linked to the polypeptide coding segment. As a non-limiting example, if a promoter or enhancer stimulates transcription of a sequence, it is operably linked to a coding sequence. If the DNA for a signal sequence is expressed as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA encoding the polypeptide. Generally, operably linked DNA sequences are contiguous, and in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous to the coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites, or at adapters or linkers inserted in their place, or by gene synthesis.

[0067] As used herein, "insect" refers not only to insects but also to immature forms and larvae thereof.

[0068] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, including a pharmaceutical agent. As used herein, a pharmaceutical composition comprises one or more of the receptors, vectors, cells disclosed herein mixed with a suitable pharmaceutical carrier or excipient.

[0069] "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the intent to reverse, alleviate, ameliorate, inhibit, slow, or prevent the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease.

[0070] As used herein, the term "subject" refers to any organism to which the dsRNA described herein is administered in accordance with the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, humans, insects, worms, etc.). In one aspect, the subject is a human. In some aspects, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0071] The present disclosure encompasses plasmid vectors and methods for producing large amounts of dsRNA in vivo from microbial cells. In some aspects, the plasmid vector comprises a nucleic acid sequence comprising: an inducible bacterial promoter operably linked to a stem-loop sequence having a 3' end and a 5' end and comprising a target dsRNA sequence; a first pac site sequence at the 3' end of the stem-loop sequence and a second pac site sequence at the 5' end; an MS2 capsid protein (CP) expression cassette; and a pyrE coding sequence downstream of the MS2CP expression cassette having a ribosome binding site (RBS) at its 5' end and a T1-T2 terminator at its 3' end. Table 5 provides nucleic acid (DNA) sequences for the T7 promoter, MS2 capsid protein, pac site, the pyrE coding sequence having a ribosome binding site (RBS) at its 5' end, and T1-T2 terminators, T7 terminators, and expression cassettes.

[0072] In some aspects, the plasmid vector of the present disclosure is expressed in a host cell. The host cell can be a cell (e.g., bacteria, yeast cell, fungal cell, CHO, mammalian cell, etc.) that can be genetically altered, modified, or transformed using the methods and plasmids described herein. In some aspects, the host cell is a prokaryotic cell, a bacterial cell, or a eukaryotic cell. In some aspects, mammals, insects, plants, or yeast cells (e.g., Saccharomyces species (Saccharomyces sp.), Pichia species (Pichia sp.), or Schizosaccharomyces species (Schizosaccharomyces p.)) are considered for use in the methods disclosed herein.

[0073] In some respects, host cell is a bacterial cell. In some respects, bacterial cell is a gram-negative bacterial cell. In some respects, the limiting examples of gram-negative bacteria can include Escherichia species (Escherichiasp.), Salmonella species (Salmonella sp.), Shigella species (Shigella sp.), Agrobacterium (Agrobacterium), Campylobacter species (Campylobactersp.), Lactobacillus species (Lactobacillussp.), Neisseria species (Neisseria sp.), Legionella species (Legionella sp.) or Pseudomonas species (Pseudomonas sp.). In some respects, gram-negative bacterial cell is an Escherichia cell. In some respects, bacterial cell is an Escherichia coli cell. In some respects, Escherichia coli can be the bacterial strain of defect in one or more endogenous ribonucleases. For example, ribonuclease defect can be produced by disappearance, removal, knocking out, silence, suppression or otherwise lowering at least one endogenous ribonuclease. In some aspects, the E. coli cell is an RNase III-deficient E. coli strain. In some aspects, the E. coli is an RNase III-deficient E. coli strain HT115 (DE3).

[0074] In some respects, bacterial cell is a gram-positive bacterial cell. In some respects, the limiting examples of gram-positive bacteria can include bacillus species (Bacillus sp.), corynebacterium species (Corynebacterium sp.), lactobacillus species (Lactobacillus sp.), staphylococcus species (Staphylococcus sp.) or streptococcus species (Streptococcus sp.). In some respects, gram-positive bacterial cell is a corynebacterium cell. In some respects, bacterial cell is a Corynebacterium glutamicum cell.

[0075] In some aspects, the bacterial cell can be engineered to contain a promoter, which can be a constitutive promoter or a regulatable promoter. Non-limiting examples of inducible promoters and their subsequent inducers include lac (IPTG), lacUV5 (IPTG), tac (IPTG), trc (IPTG), P syn (IPTG), trp (tryptophan starvation), araBAD (1-arabinose), lpp a(IPTG), lpp-lac (IPTG), phoA (phosphate starvation), recA (nalidixic acid), proU (osmotic pressure), cst-1 (glucose starvation), teta (tetracycline), cada (pH), nar (anaerobic conditions), PL (thermal shift to 42°C), cspA (thermal shift to 20°C), T7 (heat induction), T7-lac operon (IPTG), T3-lac operon (IPTG), T5-lac operon (IPTG), T4 gene 32 (T4 infection), nprM-lac operon (IPTG), P syn (alkyl or halogenated benzoate), Pu (alkyl or halogenated toluene), Psal (salicylate), and VHb (oxygen).

[0076] In further aspects, bacterial cells disclosed herein are cells without antibiotic markers. In such aspects, bacterial cells lack the nucleic acid sequence encoding antibiotic selection markers. In such aspects, bacterial cells can grow or cultivate in the absence of antibiotics. In some aspects, antibiotic selection markers can be but are not limited to amphotericin B, bacitracin, carbapenem, cephalosporin, ethambutol, fluoroquinolone, isoniazid, cephalosporin, methicillin, oxacillin, vancomycin, streptomycin, quinoline, rifampicin (rifampin), rifampicin (rifampicin), sulfonamides, ampicillin, tetracycline, neomycin, ceftriaxone, erythromycin, streptomycin, kanamycin, gentamicin, penicillin and chloramphenicol resistance gene or other conventional non-auxotrophic selective markers. In some aspects, antibiotic selection markers can be chloramphenicol resistance genes. In some aspects, antibiotic selection markers can be tetracycline resistance genes.

[0077] In some aspects, bacterial cells disclosed herein can be transformed to induce auxotrophy. In some aspects, bacterial cells can be genetically modified to induce auxotrophy about at least one metabolite. Genetic modification can be one or more genes for encoding enzymes, and the enzyme operates in metabolic pathways, such as anabolic biosynthetic pathways or catabolism utilization pathways. Preferably, the host cell has all operator genes encoding a given biocatalytic activity of deletion or inactivation, so as to ensure the removal of biocatalytic activity. In some aspects, bacterial cells disclosed herein can be modified to uracil auxotrophy. In such aspects, bacterial cells can be maintained in a substratum comprising uracil.

[0078] In some aspects, the disclosure encompasses bacterial cells that do not contain antibiotic resistance marker genes. In some aspects, the bacterial cell is an Escherichia coli cell. In such aspects, provided herein are Escherichia coli cells that do not contain chloramphenicol resistance genes or tetracycline resistance genes.

[0079] In some aspects, the disclosure further encompasses transforming bacterial cells with plasmid vectors disclosed herein. In such aspects, bacterial cells are transformed to express recombinant RNA molecules related to RNAi. In some aspects, the recombinant RNA molecules related to RNAi can be double-stranded RNA (dsRNA), microribonucleic acid (miRNA), small interfering RNA (siRNA), hairpin RNA (hpRNA) or short hairpin RNA (shRNA). In some aspects, bacterial cells are transformed to express dsRNA.

[0080] In some aspects, the disclosure further includes cultivating a bacterial cell colony transformed with a plasmid vector in a bioreactor, wherein the bioreactor is selected from a fed-batch system, a semi-continuous system, and a continuous culture system. The method includes maintaining a bacterial culture expressing the disclosed plasmid vector in a bioreactor, the time and conditions of the maintenance being enough to produce a target recombinant RNA molecule with an amount of at least about 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 11g / L, or 12g / L. In some aspects, the bioreactor is a fed-batch system. In some aspects, the target recombinant RNA molecule is a dsRNA, siRNA, shRNA, hpRNA, or miRNA.

[0081] Construction of plasmid vector

[0082] Conventional microbial and molecular cloning methods and tools, including those for generating and purifying DNA, RNA and proteins, and for transforming host organisms and expressing recombinant proteins and nucleic acids as described herein, are well within the capabilities of those skilled in the art and are fully described in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Davis, et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., NY (1986); and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publ. Assoc., Wiley-Interscience, NY (1995). The disclosures of each of these references are incorporated herein by reference.

[0083] In some aspects, plasmid vectors are constructed to have the optimal expression of target dsRNA. Any plasmid vector or the stably maintained viral episome that can provide the desired function of maintenance, expression and selection can be used in the present disclosure. Alternatively, use a plasmid with the ability of expressing the gene encoding dsRNA and capsid protein, and not be restricted by specific replication mode, expression or the selection marker relevant to the gene encoding dsRNA and capsid protein. In some aspects, recombinant DNA constructs as herein described are based on the common plasmid vector series derived from plasmid pBR322. Alternatively, recombinant DNA constructs are generated by other plasmids or the stably maintained viral episome. Plasmid vectors can be assembled using any known method in the art. As non-limiting examples, the synthetic fragments of constructs can be obtained and amplified using appropriate primers. The amplified fragment can then be cloned into the respective restriction sites of the plasmid to obtain a plasmid vector comprising any or all disclosed construct sequences. Any sequence provided in Table 5 can be used to build a plasmid vector of the present disclosure.

[0084] In some aspects, the plasmid vector comprises one or more of the following: a nucleic acid sequence comprising a bacterial promoter operably linked to a stem-loop sequence having a 3' end and a 5' end, a target dsRNA sequence; a first pac site sequence at the 3' end of the stem-loop sequence and a second pac site sequence at the 5' end; an MS2 capsid protein (CP) expression cassette; a pyrE coding sequence, a ribosome binding site (RBS) at the 5' end of the pyrE coding sequence downstream of the MS2CP expression cassette and / or a T1-T2 terminator at its 3' end, or any combination thereof.

[0085] In some aspects, the bacteriophage capsid protein of the plasmid vector disclosed herein is encoded by a coat protein gene of a species of the leviviridae family. In some aspects, the coat protein gene encodes the capsid protein of the bacteriophage MS2. In some aspects, the plasmid vector comprises an MS2CP expression cassette.

[0086] In some aspects, the MS2 CP expression cassette can further comprise a promoter selected from a constitutive or inducible transcriptional promoter. In some aspects, non-limiting examples of promoters can include T7, T3, Sp6 RNA, or J23115 promoters. In some aspects, the promoter is an inducible promoter. In some aspects, the inducible promoter is a T7 promoter. In some aspects, the MS2 CP expression cassette comprises a terminator. In some aspects, the terminator is a T7 terminator. In some aspects, the MS2 CP cassette comprises a T7 promoter and a T7 terminator.

[0087] In some aspects, the MS2 CP cassette comprises an MS CP having the nucleic acid sequence of SEQ ID NO: 5. In some aspects, the MS2 CP comprises a nucleic acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 5. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, the MS2 CP cassette comprises an MS2 CP having the amino acid sequence of SEQ ID NO: 9 (Table 6). In some aspects, the MS2 CP comprises an amino acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 9. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0088] In some aspects, the sequence of coding dsRNA may be relevant to inducible or constitutive transcription promoter and be expressed by it. In some aspects, the limiting examples of promotor can comprise T7, T3, Sp6 RNA or J23115 promotor. In some aspects, the sequence of coding dsRNA may be relevant to inducible promoter and be expressed by it. The inducible transcription promoter relevant to the expression of dsRNA may be the inducible transcription promoter identical with the transcription promoter relevant to the MS2 expression cassette or different inducible transcription promoters. In some aspects, the inducible transcription promoter relevant to the expression of dsRNA is a T7 promotor.

[0089] In some aspects, the plasmid vector comprises an E. coli phage T7 promoter having the sequence of SEQ ID NO: 1. In some aspects, the E. coli phage T7 promoter comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NO: 1. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0090] In some respects, the plasmid vector comprises a target dsRNA sequence. dsRNA can reduce, suppress or suppress the expression of the target gene by RNAi. In some respects, the sequence of the coding dsRNA can be transformed using methods known in the art. This type of sequence construct comprises a sense sequence and an antisense sequence, which is placed in the region of the lateral intron sequence with correct splicing orientation, along with donor and acceptor splicing sites. Alternatively, the self-complementary region of sequence in the construct can be separated by a spacer sequence of various lengths. In the processing of the gene construct transcript, the intron sequence can be spliced away, and allows the sense sequence and antisense sequence and splicing junction sequence to combine, forming dsRNA.

[0091] RNAi polynucleotide can be hybridized with the full-length mRNA encoded by the target gene, or hybridized with the fragment (target sequence) of target RNA or DNA.In some aspects, the length of the target sequence is 1 to 500 nucleotide.In some aspects, the length of the target sequence and / or dsRNA sequence is about 50 to 400 nucleotide.In some aspects, the length of the target sequence and / or dsRNA sequence is 100 to 300 nucleotide.In some aspects, the sequence of the dsRNA for RNAi has 100% identity or similarity with the target sequence of the target gene, but can have at least 70%, 80%, 90%, 95%, 98% or 99% or more similarity or identity with the target sequence. In some respects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some respects, use has the dsRNA greater than 90% or 95% sequence identity, makes it possible to tolerate the sequence variation that may be expected due to genetic mutation, bacterial strain polymorphism or evolution divergence.

[0092] In some aspects, the dsRNA targets specific essential genes to reduce, inhibit or suppress the expression of the gene. In some aspects, the dsRNA targets genes of insect pests or pathogens. In some aspects, the dsRNA targets genes of insect pests. In some aspects, the dsRNA comprises a nucleotide sequence complementary to at least a portion of the nucleotide sequence of the insect target gene. In some aspects, the target gene can be a midgut or non-midgut gene, a neurohormone gene, a pheromone biosynthesis activating neuropeptide (PBAN) / pyrokinin gene, tubulin, vATPase, acetylcholinesterase, chitin synthase gene (e.g., CHS1 and / or CHS2), a cytochrome P450 gene, Snf7, β-actin, a gene encoding an inhibitor of apoptosis (e.g., IAP), a ribosomal protein (e.g., CHD3, S4, or S9), or one of other conserved genes or insect-specific genes.

[0093] In some aspects, the plasmid vector may further comprise a pac site sequence. In some aspects, the pac site is an MS2 pac site. In some aspects, the plasmid vector comprises a first pac site sequence at the 3' end of the stem-loop sequence and a second pac site sequence at the 5' end. In some aspects, the plasmid vector comprises an MS2 pac site at each of the 5' and 3' ends of the dsRNA hairpin sequence. In some aspects, the plasmid vector is optimized to comprise a pac site at its 5' and 3' ends to maximize dsRNA productivity.

[0094] In some aspects, the first pac site sequence and the second pac site sequence can each comprise the nucleic acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some aspects, the pac site comprises a nucleic acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 3. In some aspects, the pac site comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NO: 4. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0095] In some aspects, the plasmid vector comprises the pyrE coding sequence. In some aspects, the pyrE coding sequence is included in the ribosome binding site at its 5' end and the T1-T2 terminator at its 3' end. In some aspects, the pyrE construct is inserted downstream of the MS2 CP expression cassette. In some aspects, the pyrE construct is transformed to allow the pyrE coding sequence to be transcribed through the read-through of the T7 promoter. In some aspects, the pyrE coding sequence comprising the RBS-pyrE cds-T1-T2 is a box and is driven by a dedicated promoter. The non-limiting examples of promoters can include T7, T3, Sp6RNA or J23115 promoters. In some aspects, the pyrE coding sequence or the construct comprising pyrE is transformed to be driven by the Escherichia coli phage T7 promoter. In some aspects, pyrE or the construct comprising pyrE is transformed to be driven by the J23115 promoter.

[0096] In some aspects, the pyrE construct of the disclosed plasmid vector comprises an RBS-pyrE cds-T1-T2 comprising the nucleic acid sequence of SEQ ID NO: 2. In some aspects, the RBS-pyrE cds-T1-T2 comprises a nucleic acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 2. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0097] In some aspects, pyrE or a construct comprising pyrE is driven by an upstream E. coli phage T7 promoter comprising the nucleic acid sequence of SEQ ID NO: 1. In some aspects, the E. coli phage T7 promoter comprises a nucleic acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 1. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0098] In some aspects, pyrE or a construct comprising pyrE is driven by a J23115 promoter comprising the nucleic acid sequence of SEQ ID NO: 33. In some aspects, the J23115 promoter comprises a nucleic acid sequence having at least 70% sequence identity or similarity to SEQ ID NO: 1. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0099] In some aspects, the pyrE coding sequence comprising the RBS-pyrE cds-T1-T2 is a cassette and is driven by a dedicated E. coli phage T7 promoter comprising the nucleic acid sequence of SEQ ID NO: 1. In some aspects, the dedicated E. coli phage T7 promoter comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NO: 1. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0100] In some aspects, the plasmid vector is plasmid pAPSE10775 engineered to express a target recombinant RNA molecule. In some aspects, the target recombinant RNA molecule is selected from dsRNA, siRNA, shRNA, hpRNA, and miRNA. In some aspects, the plasmid vector comprises one or more of a pac site, an MS2CP cassette, the RBS-pyrE cds-T1-T2 terminator of pAPSE10775, or any combination thereof, and the target dsRNA. In some aspects, the vector plasmid consists of or comprises the sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 33, or any combination thereof. In some aspects, the vector plasmid consists of or comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and / or 33. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0101] In some aspects, the plasmid vector is plasmid pAPSE10471 engineered to express a target recombinant RNA molecule. In some aspects, the target recombinant RNA molecule is selected from dsRNA, siRNA, shRNA, sshRNA, lshRNA, and miRNA. In some aspects, the plasmid vector comprises one or more of a pac site, an MS2 CP cassette, the T7-RBS-pyrE cds-T1-T2 of pAPSE10471, or any combination thereof, and the target dsRNA. In some aspects, the vector plasmid consists of or comprises the sequence of SEQ ID NO. 35. In some aspects, the vector plasmid consists of or comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NO. 35. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0102] In further aspect, plasmid vector disclosed herein is the plasmid without antibiotic marker. In this type of aspect, plasmid vector lacks the nucleotide sequence of encoding antibiotic selection marker. In some aspects, antibiotic selection marker can be but not limited to amphotericin B, bacitracin, carbapenem, cephalosporin, ethambutol, fluoroquinolone, isoniazid, cephalosporin, methicillin, oxacillin, vancomycin, streptomycin, quinoline, rifampicin (rifampin), rifampicin (rifampicin), sulfonamides, ampicillin, tetracycline, neomycin, ceftriaxone, erythromycin, streptomycin, kanamycin, gentamicin, penicillin and chloramphenicol resistance gene or other conventional non-auxotrophic selective marker. In some aspects, antibiotic selection marker can be Amp-r gene, for example bla, beta and / or beta-lactamase.

[0103] In some aspects, the plasmid vector is plasmid pAPSE10836 engineered to express a target recombinant RNA molecule. In some aspects, the target recombinant RNA molecule is selected from dsRNA, siRNA, shRNA, hpRNA, and miRNA. In some aspects, the plasmid vector comprises a pac site, an MS2CP cassette, and the P-J23115-pyrEcds-T1-T2 terminator of pAPSE10836, or any combination thereof, and one or more of the target dsRNA. In some aspects, the vector plasmid consists of or comprises the sequence of SEQ ID NO. 43. In some aspects, the vector plasmid consists of or comprises a sequence having at least 70% sequence identity or similarity to SEQ ID NO. 43. In some aspects, the identity or similarity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0104] Production of recombinant RNA

[0105] In some aspects, the disclosure further includes that the disclosed plasmid vector is transformed into a host cell. In some aspects, the disclosure comprises a cell line capable of expressing a plasmid vector and a target recombinant RNA molecule. In some aspects, the recombinant RNA molecule is dsRNA, siRNA, shRNA, hpRNA or miRNA. In some aspects, the target recombinant RNA molecule is dsRNA. In some aspects, the method comprises electroporating a plasmid vector as described herein into a bacterial cell. The bacterial cell can be any bacterium capable of transformation. In some aspects, the bacterial cell lacks double-stranded specific RNase III and / or contains an inducible T7 RNA polymerase gene. In some aspects, the bacterial cell is a gram-negative bacterial cell, such as an Escherichia coli strain, and its representative examples include K12 bacterial strains and derivatives thereof (such as MG1655, HT115 (DE3)) and B bacterial strains (such as BL21 (DE3), REL606). In some aspects, the bacterial cell is a gram-positive bacterial cell. In some aspects, the gram-positive bacterial cell is from Corynebacterium. In some aspects, the bacterial cell is Corynebacterium glutamicum. In some aspects, the disclosed bacterial cells may not contain antibiotic resistance marker genes. In some aspects, the bacterial cells are Escherichia coli cells that do not contain chloramphenicol resistance genes. In some aspects, the bacterial cells are Escherichia coli cells that do not contain tetracycline resistance genes. Any known method for transforming bacterial cells can be used in the art.

[0106] In some aspects, the use of a human having the genotype F -coli strain HT115 (DE3) containing the mcrA, mcrB, IN (rrnD-rrnE)1, rnc14::Tn10 (λDE3 lysogen: lacUV5 promoter-T7 polymerase). After transformation, as a non-limiting example, the resulting recombinant transformants were selected on LB agar plates containing tetracycline and / or ampicillin. Individual colonies were isolated, the presence of the intact plasmid was confirmed by restriction enzyme analysis, and confirmed transformed cells were saved for future use.

[0107] Further provided herein are bacterial cells comprising the vector plasmids disclosed herein. In such aspects, the bacterial cell comprises a plasmid comprising one or more of the pac site, MS2 CP cassette, and RBS-pyrE cds-T1-T2 terminator disclosed herein, a target recombinant RNA, or any combination thereof. In some aspects, the recombinant RNA molecule is a dsRNA, siRNA, shRNA, hpRNA, or miRNA. In some aspects, the disclosed bacterial cell may comprise the plasmid pAPSE10775, and one or more of the pac site, MS2 CP cassette, and RBS-pyrE cds-T1-T2 terminator of pAPSE10775. In some aspects, the disclosed bacterial cell may comprise the plasmid pAPSE10471, or one or more of the pac site, MS2 CP cassette, and T7-RBS-pyrE cds-T1-T2 of pAPSE10471. In some aspects, the disclosed bacterial cells can comprise plasmid pAPSE10836, or one or more of the pac site, MS2 CP cassette, and P-J23115-pyrE cds-T1-T2 terminator of pAPSE10836.

[0108] In some aspects, the bacterial cell is an Escherichia coli cell. In some aspects, the Escherichia coli cell is a cell that does not contain a chloramphenicol resistance gene. In some aspects, the Escherichia coli cell is a cell that does not contain a tetracycline resistance gene. In such aspects, the Escherichia coli cell comprises a plasmid comprising one or more of the pac site, MS2 CP cassette, and RBS-pyrE cds-T1-T2 terminator disclosed herein, a target recombinant RNA, or any combination thereof. In some aspects, the recombinant RNA molecule is a dsRNA, siRNA, shRNA, hpRNA, or miRNA. In some aspects, the disclosed Escherichia coli cell may comprise the plasmid pAPSE10775, and one or more of the pac site, MS2 CP cassette, and RBS-pyrE cds-T1-T2 terminator of pAPSE10775. In some aspects, the disclosed E. coli cells may comprise plasmid pAPSE10471, or one or more of the pac site, MS2 CP cassette, and T7-RBS-pyrE cds-T1-T2 of pAPSE10471. In some aspects, the disclosed E. coli cells may comprise plasmid pAPSE10836, or one or more of the pac site, MS2 CP cassette, and P-J23115-pyrE cds-T1-T2 terminator of pAPSE10836.

[0109] In some aspects, further provided is a method for producing a dsRNA comprising culturing a bacterial cell population transformed with a plasmid vector in a bioreactor, wherein the bioreactor is maintained for a sufficient time and under sufficient conditions to produce the target dsRNA.

[0110] In some aspects, the method includes cultivating the bacterial cells transformed in a bioreactor. The growth of the bacterial cells transformed with the plasmid vector comprising the target dsRNA may be carried out in a basic (mineral) culture medium or a rich culture medium. Such culture medium is well known to those of ordinary skill in the art. Method disclosed herein may be carried out using standard industrial microbiology techniques and standard fermentation procedures, as long as such methods are suitable for the requirements of specific plasmids and host cells, such as providing suitable selection markers to retain specific plasmid vectors, using suitable stimulants to induce transcription of specific promoters at the appropriate time, and maintaining the required temperature and respiratory conditions necessary for cell growth, each of which is within the working knowledge of those of ordinary skill in the art.

[0111] In some aspects, the culture method includes using a seed culture as an inoculum. In some aspects, the seed culture is used to inoculate a minimal culture medium. In some aspects, the minimal culture medium can include ammonium salts, potassium salts, sodium salts, magnesium salts, a carbon source, or any combination thereof. In some aspects, the ammonium salt can be (NH4)2SO4. In some aspects, the potassium salt in the culture medium can be KH2PO4. In some aspects, the sodium salt in the culture medium can be Na2HPO4. In some aspects, the magnesium salt in the culture medium can be MgSO4. In some aspects, the carbon source is glucose or lactose. In some aspects, the minimal culture medium can include (NH4)2SO4, KH2PO4, Na2HPO4, MgSO4, glucose, or any combination thereof. In some aspects, the minimal culture medium can include (NH4)2SO4, KH2PO4, Na2HPO4, MgSO4, glucose, lactose, or any combination thereof. In some aspects, the seed culture is grown for sufficient time under sufficient temperature conditions.

[0112] In further aspects, inoculum is transferred to the fermentation vessel containing sufficient culture medium. In some aspects, culture medium can comprise minimal culture medium. In some aspects, minimal culture medium can comprise ammonium salt, potassium salt, sodium salt, magnesium salt, carbon source, trace metal or its any combination. In some aspects, ammonium salt can be (NH4)2SO4. In some aspects, the potassium salt in culture medium can be KH2PO4. In some aspects, the sodium salt in culture medium can be Na2HPO4. In some aspects, the magnesium salt in culture medium can be MgSO4. In some aspects, the carbon source is glucose. In some aspects, trace metal is commercially available trace metal solution (for example, Teknova, Hollister, CA). In some aspects, minimal culture medium can comprise (NH4)2SO4, KH2PO4, Na2HPO4, MgSO4, glucose and trace metal. In some aspects, culture is maintained by the supply of carbon source (for example, glucose feeding with the main salt of minimal culture medium).

[0113] In some aspects, the minimal medium disclosed herein comprises an ammonium salt at about 0.5 to about 100 mM, for example, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about In some aspects, the ammonium salt is (NH 4 ) 2 SO 4 and is present in the culture medium at about 20 mM to about 70 mM.

[0114] In some aspects, the minimal medium disclosed herein comprises a potassium salt in an amount of about 0.5 to about 50 mM, such as about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some aspects, the potassium salt is KH PO and is present in the culture medium at about 0.5 to about 20 mM.

[0115] In some aspects, the minimal medium disclosed herein comprises a sodium salt at about 0.5 to about 100 mM, for example, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about In some aspects, the sodium salt is Na2HPO4 and is present in the culture medium at about 0.5 mM to about 80 mM.

[0116] In some aspects, the minimal medium disclosed herein comprises a magnesium salt at about 0.01 mM to about 10 mM, such as about 0.01 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM. In some aspects, the magnesium salt is MgSO4 and is present in the culture medium at about 0.1 mM to about 10 mM.

[0117] In some aspects, the minimal medium disclosed herein comprises a carbon source at about 0.01% to about 80%, such as about 0.01%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%. In some aspects, the carbon source is glucose and is present in the culture medium at about 0.01% to about 80%. In some aspects, the carbon source is lactose and is present in the culture medium at about 0.01% to about 80%.

[0118] In some aspects, the minimal medium disclosed herein comprises a commercially available trace metal solution at about 0.01 ml / L to about 20 mL / L, such as about 0.01 ml / L, about 0.1 ml / L, about 0.5 ml / L, about 1 ml / L, about 1.5 ml / L, about 2 ml / L, about 2.5 mL / L, about 3 mL / L, about 3.5 ml / L, about 4 ml / L, about 4.5 ml / L, about 5 ml / L, about 5.5 ml / L, about 6 ml / L, about 6.5 ml / L, about 7 ml / L, about 7.5 ml / L, about 8 ml / L, about 8.5 ml / L, about 9 ml / L, about 9.5 ml / L, about 10 ml / L, or about 15 ml / L, or about 20 ml / L.

[0119] In a further aspect, biotin can be added to the culture medium used to culture the bacterial cells disclosed herein. In some cases, biotin is added at about 0.1 mg / L to about 100 mg / L. For example, the components may be added at concentrations of 0.1 mg / L, about 0.5 mg / L, about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 11 mg / L, about 12 mg / L, about 13 mg / L, about 14 mg / L, about 15 mg / L, about 16 mg / L, about 17 mg / L, about 18 mg / L, about 19 mg / L, about 20 mg / L, about 21 mg / L, about 22 mg / L, about 23 mg / L, about 24 mg / L, about 25 mg / L, about 26 mg / L, about 27 mg / L, about 28 mg / L, about 29 mg / L, about 30 mg / L, about 31 mg / L, about 32 mg / L, about 33 mg / L, about 34 mg / L, about 35 mg / L, about 36 mg / L, about 37 mg / L, about 38 mg / L, about 39 mg / L, about 40 mg / L, about 41 mg / L, about 42 mg / L, about 43 mg / L, about 44 mg / L, about 45 mg / L, about 46 mg / L, about 47 mg / L, about 48 mg / L, about 49 mg / L, about 50 mg / L, about 51 mg / L, about 52 mg / L, about 53 mg / L, about 54 mg / L About 17 mg / L, about 18 mg / L, about 19 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, about 50 mg / L, about 55 mg / L, about 60 mg / L, about 65 mg / L, about 70 mg / L, about 75 mg / L, about 80 mg / L, about 85 mg / L, about 90 mg / L, or about 100 mg / L.

[0120] In some aspects, the bacterial cells can be cultured at a temperature of about 25° C. to about 45° C. In some aspects, the culture is maintained at a temperature of about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., or about 45° C.

[0121] In some aspects, the bacterial cells can be maintained for about 30 minutes to about 48 hours or longer. In some aspects, the bacterial cells can be maintained for 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 hours or longer.

[0122] In some aspects, the culture is maintained at a pH of about 5 to about 8. In some aspects, the culture is maintained at a pH of about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8.

[0123] In some aspects, the culture is maintained at a desired level of dissolved oxygen (DO) of, for example, about 5% saturation to about 50% saturation. In some aspects, the DO is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% or about 50%.

[0124] In some aspects, after reaching required cell density, induced culture is used for dsRNA production. In some aspects, dsRNA production is induced with IPTG. In some aspects, IPTG is added with about 0.1mM to about 10mM. In some aspects, IPTG is added with about 0.01mM, about 0.1mM, about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM or about 10mM.

[0125] In some aspects, the culture is maintained to reach required cell density. In some aspects, culture growth and cell density are monitored by measuring OD600. In some aspects, the cell density of the substratum measured using OD600 is about 10 to about 90, such as about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 55, about 60, about 70, about 75, about 80, about 85 or about 90.

[0126] In further aspects, culture is maintained with controlled agitation of, for example, about 100 rpm to 2000 rpm. In some aspects, culture is maintained with controlled agitation of about 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1200 rpm, 1500 rpm, 1700 rpm, 1800 rpm or 2000 rpm.

[0127] In some aspects, the culture is performed on a large scale as a shaking culture or a bioreactor culture. In some aspects, a large scale culture can comprise a culture of about 10 liters up to about 200 ml. 3 , about 100 to 100m 3 In some aspects, the bacterial cells are cultured in a culture vessel of about 100 to 1000 liters. In some aspects, the culture vessel can comprise a bioreactor (e.g., a stirred tank), or a simple (disposable) container, such as a plastic bag of up to several cubic meters, for example a 500 liter plastic bag (e.g., a WAVE bioreactor).

[0128] In some aspects, the culture can be a batch culture comprising a discontinuous process, wherein a sterile growth medium with all required substrates is initially inoculated with a pure culture of bacterial cells, and no additional growth medium is added during the operating process. In some aspects, the batch process is a partially closed system, wherein the only materials added and removed during the operating process are air / gas exchangers, defoamers, and pH control agents. In some aspects, the batch culture is continuously shaken or stirred to maintain the required uniformity of substrate and cells and to ensure high as possible oxygen transfer for aerobic cultures.

[0129] In some aspects, the culture can be a fed-batch culture comprising a process in which an amount of fresh growth-limiting substrate is continuously added to the culture medium to provide a low concentration thereof in the culture medium and obtain control of bacterial cell growth.

[0130] In some aspects, the cultures disclosed herein are high cell density cultures, which include cultures that produce a large number of bacterial cells within a defined culture period. The high cell density value depends on the microbial cell and can be defined as the cell density value achieved by the gradual addition of a growth-limiting substrate (fed-batch) without poisoning the bacterial cells, i.e., about 3×10 for E. coli. 9 cells / ml, or higher than about 1×10 10 The viable cell concentration was 1 cell / ml.

[0131] In some aspects, the bioreactor is selected from a fed-batch system, a semi-continuous system, and a continuous culture system. In some aspects, the bioreactor is a fed-batch system. In some aspects, the fed-batch system is a high cell density fed-batch system. In some aspects, the fed-batch system is a high cell density fed-batch fermentation system. The disclosed target dsRNA production method can also be modified to adapt to any existing fermentation and / or culture system infrastructure available to the user.

[0132] In some aspects, the target dsRNA is produced in an amount of at least about 4 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 5 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 6 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 7 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 8 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 9 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 10 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 11 g / L. In some aspects, the target dsRNA is produced in an amount of at least about 12 g / L.

[0133] In a further aspect, methods for producing target dsRNAs that are performed in an antibiotic-free environment are provided herein. In such aspects, problems that may accompany the use of antibiotics for selection of plasmids, such as regulatory agency concerns, product safety, final product analysis (exhaustion of antibiotics in the product), and the risks and costs associated therewith, can be avoided. By methods according to the present disclosure, selection pressure during the fermentation process is maintained, and there is no need to use antibiotics in the fermentation medium.

[0134] In some aspects, the method further comprises purifying the dsRNA from the bacterial cell culture.The method comprises lysing the cells to produce a lysate, and purifying the dsRNA from cellular components within the lysate before processing the purified dsRNA for use.

[0135] In some aspects, the dsRNA is treated prior to application. Such treatments may include, but are not limited to, mixing with excipients, binders, or fillers to improve physical handling properties, mixing with stabilizers to reduce degradation, or mixing with other active agents such as chemical pesticides, fungicides, defoliants, or other RNAi molecules to broaden the target range of application, and may include granulating, spray drying, or dissolving the material into a liquid carrier.

[0136] In some aspects, dsRNA is not purified from the lysate, but is directly processed for use. In some aspects, bacterial cells are not cracked, but are directly processed for use, and the dsRNA remains unpurified in the cells processed. In some aspects, the bacterial cells in the culture comprising the target dsRNA are killed or inactivated before use. The bacterial cells can be killed or inactivated by heating or other known methods in the art, which do not interfere with the activity of the target dsRNA.

[0137] In some aspects, the methods disclosed herein can be further modified. As used herein, "modifying a method" can include modifying or changing one or more features or aspects of one or more steps of a disclosed method. In one aspect, a method can be modified, for example, by changing the amount of salts used in the culture medium used in the disclosed method, or changing the duration of the culture, or replacing one or more of the disclosed components used in the culture medium with similar or equivalent components and / or reagents.

[0138] Applications of dsRNA

[0139] In some aspects, the target dsRNA produced by the disclosed constructs and methods is used for non-pharmaceutical applications. In some aspects, the target dsRNA produced by the disclosed constructs and methods is used for pharmaceutical applications. In some aspects, the target dsRNA is used to prevent and treat pests, bacteria or viruses that infest plants, animals and / or humans. In some aspects, the target dsRNA is used to treat diseases or conditions associated with plants, animals and / or humans. It is within the working knowledge of those of ordinary skill in the art to use or adjust the plasmid vectors and methods for producing the disclosed target dsRNA for any dsRNA of interest.

[0140] This article further provides disclosed plasmids and / or bacterial cells comprising the plasmids for use in the production of target recombinant RNA without antibiotic markers. In such aspects, the recombinant RNA is dsRNA, siRNA, shRNA, hpRNA or miRNA. In some aspects, the bacterial cell is an Escherichia coli cell. In some aspects, the Escherichia coli cell is a cell that does not contain a chloramphenicol resistance gene. In some aspects, the Escherichia coli is a cell that does not contain a tetracycline resistance gene.

[0141] I. dsRNA for non-pharmaceutical applications

[0142] The present disclosure encompasses methods and compositions of dsRNA that can be used for the agricultural control of plant viruses, parasites, insects, nematodes, fungal infections, or any other organism that affects plant growth or development.

[0143] In some aspects, the delivery of dsRNA is used to control insect pests. In some aspects, the delivery of dsRNA induces a lethal phenotype in insect pests.

[0144] Suitable genes to be targeted by dsRNA in insect pests include midgut genes or non-midgut genes. Representative, non-limiting examples of suitable insect genes that can be targeted by dsRNA include tubulin, vATPase, acetylcholinesterase, chitin synthase gene A, β-actin, and genes encoding inhibitors of apoptosis (e.g., IAPs). Targets can also be genes disclosed in US 2009 / 0285784A1, the disclosure of which is incorporated herein by reference.

[0145] In some aspects, as used herein, controlling insects also encompasses inhibiting the viability, growth, development, or reproduction of insects, or reducing the pathogenicity or infectivity of insects. In some aspects, controlling insects can inhibit biological activity in insects, resulting in one or more of the following attributes: by a reduction in insect feeding, a reduction in insect viability, insect death, inhibition of insect differentiation and development, by the absence or reduction of insect sexual reproduction, muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, apoptosis, and any component of the eukaryotic cell cytoskeletal structure, such as actin and tubulin.

[0146] In some aspects, the insect pests to be controlled are selected from the order Acari, Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera, In some aspects, the insect pest to be controlled is a member of the order Coleoptera or Lepidoptera.

[0147] In some aspects, the insect pest to be controlled is a member of the order Lepidoptera. Lepidoptera larvae and adults include, but are not limited to, armyworms, cutworms, loopers, and heliothines. Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, geometrids, and heliothines of the family Noctuidae, Spodoptera frugiperda JESmith (fall armyworm); S. exigua Hübner (beet armyworm) / S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (berthaarmyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A.subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leafworm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvet bean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough-skinned cutworm); Euxoa messona Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H.zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, sheath moths, webworms, coneworms and skeletonizers from the family Pyralidae: Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagastakuehniella Zeller (Mediterranean flour moth); Cadracautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocismedinalis Guenee (rice leafroller); Desmiafuneralis Hübner (grape borer); leaffolder); Diaphania hyalinata Linnaeus (melon borer); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer); D.saccharalis Fabricius (sugarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth), Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sodwebworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia gnsella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beetwebworm); Orthagathyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellowstem borer); Udea rubigalis Guenee (celery leaftier); and leaf rollers, aphids, seed worms, and fruit worms in the family Tortricidae, Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leafroller); A.rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischervon Rosslerstamm (summer fruit tortrixmoth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermiiller (European grapevine moth); Spilonota ocellana Denis & Schiffermiiller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salubncola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruitmoth); Suleima helianthana Riley (sunflower budmoth); Argyrotaenia spp.; Choristoneura spp.), Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peachtwigborer); Anisotasenatoria J.E. Smith (orange striped oakworm); Antheraeapernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyxmori Linnaeus (silkworm); Cotton miner (. Busck (cottonleaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm looper); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall caterpillar); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria l. ugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth). moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); Manduca hornworm (M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter looper moth); Paleacritavernata Peck (spring looperworm); Papilio cresphontes Cramer (giant walltail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white cabbage butterfly); butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizuraconcinna JESmith (red humped caterpillar); Sitotrogacerealella Olivier (Angoumois grain moth); Thaumetopoeapityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (clothes moth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (apple erminemoth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp. Tenebhonidae.

[0148] In some aspects, the insect pest to be controlled is a member of the order Coleoptera. Larvae and adults of the order Coleoptera include weevils from the families Anthribidae, Bruchidae, and Curculionidae (including, but not limited to, Anthonomus grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (cloverleaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus (yellow claw weevil); and Cylindrocopturus adspersus LeConte (sunflower stem weevil). LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize bill bug); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leaf miners in the family Chrysomelidae (including but not limited to Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith & Lawrence (northern corn rootworm); D.undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae (including but not limited to: Epilachna v. vestis Mulsant (Mexican bean beetle)); beetle); scarabs and other beetles from the family Scarabaeidae (including but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer); Phyllophagacrinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot beetle); beetle), carpet beetle from the family Dermestidae; from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.); species of the genus Ctenicera; nematodes of the genus Aeolus; bark beetles from the subfamily Scolytidae and beetles from the family Tenebrionidae.

[0149] In certain aspects, the insect pest to be controlled is a member of the order Hymenoptera, such as an ant, sawfly, wasp, or bee. In some aspects, the insect pest to be controlled is an ant (Formicoidea) selected from the group consisting of Tapinoma sessile (stink ants), Solenopsis spp. (e.g., Solenopsis invicta (fire ants)), Monomorium spp. (e.g., Monomoriumpharaonis (Pharaoh ants)), Camponotus spp. (e.g., Camponotus spp. (carpenter ants)), Lasius spp. (e.g., Lasius niger (small black ants)), Tetramorium spp. (e.g., Tetramorium caespitum (pavement ants)), Myrmica spp. (e.g., Myrmica rubra (red ants)), Formica spp. (e.g., spp. (carpenter ants), Crematogaster spp. (e.g., Crematogaster lineolata (Acrobat Ant)), Iridomyrmex spp. (e.g., Iridomyrmex humilis (Argentine Ant)), Pheidole spp. (big-headed ants), and Dasymutilla spp. (e.g., Dasymutilla occidentalis (velvet ants)).

[0150] In certain aspects, the insect pest to be controlled is a termite (Isoptera and / or Termitidae) selected from the group consisting of Amitermes spp. (e.g., Amitermes floridensis (Florida dark-winged subterraneantermite)), Reticulitermes spp. (e.g., Reticulitermes flavipes (Eastern subterranean termite), Reticulitermes hesperus (Western subterranean termite)), Coptotermes spp. (e.g., Coptotermes formosanus (Foreign subterranean termite)), Incisitermes spp. (e.g., Incisitermes minor (Western drywood termite)), Neotermes spp. (e.g., Neotermes connexus (forest termites).

[0151] In certain aspects, the insect pest to be controlled is a member of the order Diptera, such as a mosquito or a fly, for example, A. gambiae (malaria mosquito) or Ae. aegypti (yellow fever mosquito).

[0152] In some aspects, the insect pest to be controlled is a member of the subclass Acari (e.g., a tick or mite) selected from the families Argasidae, Dermanyssidae, Ixodidae, Psoroptidae, or Sarcoptidae, and representatives of the following species: Amblyommas spp., Anocentor spp., Argas spp., Boophilus spp., Cheyletiella spp., Chorioptes spp., Demodex spp., Dermacentor spp., Denmanyssus spp., Haemophysalis spp. spp.), Hyalomma spp., Ixodes spp., Lynxacarus spp., Mesostigma spp., Notoedres spp., Ornithodoros spp., Ornithonyssus spp., Otobius spp., Otodectes spp., Pneumonyssus spp., Psoroptes spp., Rhipicephalus spp., Sarcoptes spp., or Trombicula spp.; Anoplura (sucking and biting lice) selected from the following species: Bovicola spp. spp.), Haematopinus spp., Linognathus spp., Menopon spp., Pediculus spp., Pemphigus spp., Phylloxera spp. or Solenopotes spp.; of the order Diptera (flies), for example representatives of the following species: Aedes spp., Anopheles spp., Calliphora spp., Chrysomyia spp.), Chrysops spp., Cochliomyia spp., Culex spp., Culicoides spp., Cuterebra spp., Dermatobia spp., Gastrophilus spp., Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp., Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrus spp., Phaenicia spp.), Phlebotomus spp., Phormia spp., Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp., or Tipula spp.; a Trichophaga (biting lice) selected from the group consisting of Damalina spp., Felicola spp., Heterodoxus spp., or Trichodectes spp.; or a Siphonaptera (wingless insects) selected from the group consisting of Ceratophyllus spp., Pulex spp., or Xenopsylla spp.); Cimicidae (truebugs) selected from the group consisting of Cimex spp., Tritominae spp., Rhodinius spp., or Triatoma spp.; Blattodea (cockroaches) such as Blatella spp. (e.g., Blatella germanica (German cockroach)), Periplaneta spp.) (e.g., Periplaneta americana (American cockroach) and Periplaneta australiasiae (Australian cockroach)), Blatta spp. (e.g., Blatta orientalis (Oriental cockroach)) and Supella spp. (e.g., Supella longipalpa (Brown-banded cockroach)); as well as other insects such as Dermoptera (earwigs), Hemiptera (e.g., bed bugs), Siphonaptera (fleas), Sternorrhyncha (aphids), or Zygentoma (silverfish), crickets, silverfish, booklice, and beetles.

[0153] In some aspects, the dsRNA targets a plant virus or viroid. Exemplary viruses and viroids include viruses or viroids from the following families: Alphaflexiviridae (Potato Virus X (PVX)); Bromoviridae (Alfalfa Mosaic Virus (AMV), Cucumber Mosaic Virus, and Brome Mosaic Virus (BMV)); Bunyaviridae (Tomato Spotted Wilt virus); Caulimoviridae (Cauliflower Mosaic Virus (CaMV) and Rice Tungro Bacilliform Virus); Closteroviridae (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus (Citrus Tristeza Virus Virus); Geminiviridae (Mungbean Yellow Mosaic India Virus, African Cassava Mosaic Virus, Tomato Yellow Leaf Curl Sardinia Virus, Tomato Yellow Leaf Curl Virus, and African Cassava Mosaic Virus); Luteoviridae (Barley Yellow Dwarf Virus and Potato Leafroll Virus); Pospiviroidae (Potato Spindle Tuber Viroid);Potyviridae (Potato Virus Y (PVY), Tobacco Etch Virus (TEV), Papaya Ringspot Virus W (PRSV-W), Plum Pox Virus (PPV), Sugarcane Mosaic Virus, Bean Common Mosaic Virus, and Cassava Brown Streak Virus); Sequiviridae (Rice Tungro Spherical Virus); Tombusviridae (Maize Chlorotic Mottle Virus and Tomato Bushy Stunt Virus); and Virgaviridae (Tobacco Mosaic Virus). Virus (TMV), Tomato Mosaic Virus, Pepper Mild Mottle Virus (PMMoV), Cucumber Green Mottle Mosaic Virus, and Benyvirus (Beet Necrotic Yellow Vein Virus).

[0154] In some aspects, the dsRNA controls plant fungi. Non-limiting examples of fungi include Magnaporthe species (Magnaporrhe oryzae), Botryhs species (particularly Borrytis cinema), Puccinia species, Fusarium species (Fusarium graminearum and Fusarium oxysporum), Blumeria species (Blumeria graminis f.sp), Mycosphaerella species (Mycosphaerella graminicola), Colletotrichum species, Ustilago species (Ustilago maydis), Melampsora species (Melampsora spp.), and lini), Phakopsora species (Phakopsora pachyrhizi), Rhizoctonia species (Rhizoctonia solani), and Aspergillus species.

[0155] In some aspects, the dsRNA controls oomycetes.Non-limiting examples of oomycetes include Phytophthora species, Phytophthora infestans, Hyaloperonospora arabidopsidis, Phytophthora ramorum, Ramorum disease, Phytophthora sojae, Phytophthora capsica, Plasmoparaviticola, Phytophthora cinnamomic, Phytophthora parasitica, Pythium ultimum, Albugo candida, Aphanomyces euteiches, Albugo laibachii, Bremia lactucae, Phytophthora rapalmivora, Pseudoperonospora cubensis), Plasmopara halstedii, Peronophythora litchi, Peronosclerospora sorghi, Peronospora belbahrii, Phytophthora alni, Phytophthora brassicae, Phytophthora cactorum, Phytophthora meadii, Phytophthora phaseoli, Phytophthora pulrivora (formerly P. citricola), Plasmopara obducens, Pythium aphanidermatum, Pythium oligandrum, Sclerophthora raysiae, Hyaloperonospora brassicae, Saprolegnia parasitica (fish parasite), Lagenidium giganteum (mosquito parasite), Pythium insidiosum (mammalian parasite).

[0156] The dsRNAs disclosed herein can be engineered to control nematodes (e.g., root-knot nematodes) or parasitic weeds (e.g., Striga asiatica L). One skilled in the art can construct dsRNAs based on any organism for which protection is sought, and the appropriate sequence can be readily selected by the skilled artisan.

[0157] In some respects, the bacterial cells comprising dsRNA or the dsRNA of purification or partial purification may be prepared with suitable carriers or excipients or diluents. Preparation may be any physical form suitable for application, such as solid forms such as powders, pills or baits, liquid forms such as sprays, or gels, coatings or paste forms. In some respects, preparation comprises components that act on stable dsRNA and / or prevent dsRNA from degrading during storage. In some respects, preparation comprises components that enhance or promote dsRNA to be absorbed by insects, such as generally promote RNA to be absorbed into intracellular chemical reagents (such as lipofectamine). In some respects, composition may also include one or more other active ingredients or provide a composition of benefit to plant. Such active ingredient may be, for example, insecticide, pesticide, fungicide, antibiotic, anthelmintic, antiparasitic agent, antiviral agent or nematicide.

[0158] II. dsRNA for pharmaceutical applications

[0159] In some respects, the delivery of dsRNA is used for pharmaceutical applications. In some respects, the delivery of dsRNA treats the situation, disease or illness in a subject, wherein the dsRNA targeting gene is relevant to the situation, disease or illness in the subject. Target gene is selected from oncogenes, cytokine genes, DNA binding and inhibitor of cell differentiation (Id) protein genes, genes related to growth and prion genes. In some respects, target gene is expressed in pathogenic organisms, such as viruses, viroids, bacterium, fungi or plasmodium. In some respects, dsRNA is used to treat diseases controlled by heredity, such as cancer, viral diseases or Alzheimer's disease. In some respects, dsRNA can be used for targeting specific sites or organs such as the gene in the liver, and the site or organ are responsible for some metabolic diseases or illness such as high cholesterol levels or HIV infected cells. In some respects, dsRNA is used in vaccines to prevent or protect the subject from infectious diseases or situations.

[0160] In some aspects, the disease to be treated is cancer. The disclosed dsRNA can be used to target for treatment or development of treatments for any type of cancer, including solid tumors and leukemias, including: apudoma, bud tumor, branchial proto-tumor, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker carcinoma, basal cell carcinoma, basosquamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, carcinoma in situ, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma), histiocytic disorders, leukemias (e.g., B-cell leukemia, mixed cell leukemia, null cell leukemia, T-cell leukemia, chronic T-cell leukemia, HTLV-II-associated leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia), malignant histiocytosis, Hodgkin's disease, immunoproliferative disorders small), non-Hodgkin's lymphoma, plasmacytoma, reticuloendothelioma, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymal stem cell tumor Follicular tumor, mesonephroma, myosoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastoma, adenocarcinoma, adenoma, cholangiocarcinoma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynecoma, hepatocellular carcinoma, sweat adenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myoma , rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioma, glioma, medulloblastoma, meningioma, neurilemoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-pheochromocytic paraganglioma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing angioma, angiomatosis, glomus tumor, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, angiosarcoma, leiomyosarcoma, leukemic sarcoma (leukosarcoma), liposarcoma, lymphangiosarcoma, sarcoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcomas (e.g., Ewing's sarcoma, experimental sarcoma, Kaposi's sarcoma, and mast cell sarcoma), tumors (e.g.,bone tumors, breast tumors, digestive system tumors, colorectal tumors, liver tumors, pancreatic tumors, pituitary tumors, testicular tumors, orbital tumors, head and neck tumors, central nervous system tumors, acoustic nerve tumors, pelvic tumors, respiratory tract tumors, and genitourinary system tumors), neurofibromatosis, and cervical dysplasia.

[0161] Non-limiting examples of possible target genes for dsRNA include developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, winged helix family members, members), Hox family members, cytokines / lymphokines and their receptors, growth / differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RBI, TP53, and WT1); and enzymes (e.g., ACC, WT ... Enzymes and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophosphorylase, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidase, granule-bound starch synthase, GTPases, helicases, hemicellulases, integrases, inulinase, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteases and peptidases, pullanase, recombinases, reverse transcriptases, RUBISCO, topoisomerases, apolipoprotein B, PCSK9, and xylanases.

[0162] The dsRNA disclosed herein can be used to treat or prevent viral infections. Non-limiting examples include human retroviral infections, including HIV, members of the herpes virus family, such as herpes simplex and herpes zoster; cytomegalovirus or CMV (sometimes classified as herpes-type viruses); localized skin cancers with and without significant etiology, susceptible sexually transmitted viral conditions, and sexually transmitted infections, including chlamydia, and where the causative viral agent is primary or secondary to another sexually transmitted infection.

[0163] Viral pathogens can include, but are not limited to, Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV, HTLV-1 and HTLV-II, Herpesviruses, such as EBV; CMV or herpes simplex virus; Lentiviruses, such as HIV-1 and HIV-2; Rhabdoviruses, such as rabies virus; Picornoviruses, such as poliovirus; Poxviruses, such as vaccinia virus; Rotavirus; Parvoviruses, such as adeno-associated virus 1 and Coronavirues, such as alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. Other viruses that may be targeted by the present disclosure include, but are not limited to, influenza, coxsackieviruses, herpes simplex virus type I and II, St. Louis encephalitis virus, Epstein-Barr virus, myxovirus, JC virus, coxsackievirus B, togaviruses, measles virus, paramyxovirus, echovirus, bunyavirus, cytomegalovirus, varicella-zoster virus, mumps virus, equine encephalitis virus, lymphocytic choriomeningitis virus, rhabdoviruses including rabies virus, simian virus 40, human polyomavirus, parvovirus, papillomavirus, primate adenovirus, coronavirus, retrovirus, dengue virus, yellow fever virus, Japanese encephalitis virus, and / or BK virus, or any virus of the following species / families: Astoviridae, Togaviridae, Flaviviridae, Paramyxoviridae ramyxoviridae), arteriviruses, Rhabdoviridae, Filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, Birnaviridae, circoviridae, adenoviridae, Iridoviridae, retroviruses, herpesviruses, hepadenoviruses, papillomaviruses, and papovaviruses.

[0164] Some non-limiting examples of viral targets include, but are not limited to, human immunodeficiency virus Nef, Gag, Env, Tat, mutant derivatives of Tat such as Tat-Δ31-45, and Pol and T cell epitopes and B cell epitopes of gp120, chimeric derivatives of HIV-1 Env and gp120 such as, but not limited to, fusions between gp120 and CD4; truncated or modified derivatives of HIV-1 env such as, but not limited to, gp140 or HIV-1 Env and / or derivatives of its gp140, hepatitis B virus targets, rotaviruses such as VP4 and VP7, influenza virus hemagglutinin or nucleoprotein, and herpes simplex virus thymidine kinase.

[0165] Infection by other pathogens may also be treated or prevented using the methods of the present disclosure, including protozoan, bacterial, yeast, and fungal infections. Non-limiting examples of bacterial pathogens include Mycobacterium species, Helicobacter pylori, Salmonella species, Shigella species, Escherichia coli, Rickettsia species, Listeria species, Legionella pneumoniae, Pseudomonas species, Vibrio species, and Borellia burgdorferi. dsRNA targets for bacterial infections can be from enterotoxigenic Escherichia coli, such as CFA / I fimbriae antigens and the non-toxic B subunit of heat-labile toxin; pertactin from Bordetella pertussis, adenylate cyclase hemolysin from Bordetella pertussis, fragment C of tetanus toxin from Clostridium tetani, OspA from Borrelia burgdorferi, protective quasi-crystalline surface proteins from Rickettsia prowazekii and Rickettsia typhi, listeriolysin (also known as "Llo" and "Hly") and / or superoxide dismutase (also known as "SOD" and "p60") from Listeria monocytogenes, urease from Helicobacter pylori, and urease from Bacillus anthracis. anthrax) receptor binding domain and / or protective antigens of the lethal toxin.

[0166] The dsRNAs of the present disclosure can target parasitic pathogens, not limited to Plasmodium spp., such as Plasmodium falciparum; Trypanosome spp., such as Trypanosoma cruzi; Giardia spp., such as Giardia intestinalis; Boophilus spp., Babesia spp., such as Babesia microti; Entamoeba spp., such as Entamoeba histolytica; Eimeria spp., such as Eimeria maxima; Leishmania spp.; Schistosoma spp. spp.), Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp. For example, for intracellular parasites such as Plasmodium, pathogen-specific dsRNAs effective against the pathogen can be delivered to cells susceptible to infection.

[0167] In some aspects, dsRNA is used as a formulation. In some aspects, bacterial cells comprising unpurified or partially purified dsRNA may be formulated with suitable carriers, excipients, or diluents. In some aspects, the present disclosure encompasses direct administration of dsRNA.

[0168] dsRNA can be prepared and administered by several different means. For example, the composition can generally be administered parenterally, intraperitoneally, intravascularly, transdermally, subcutaneously or intrapulmonaryly in a dosage unit formulation containing conventional non-toxic pharmaceutically acceptable adjuvants, carriers, excipients and vehicles as needed. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intrathecal or intrasternal injection or infusion techniques. The preparation of the pharmaceutical composition is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H.A. and Lachman, L., editors, Pharmaceutical Dosage Forms, Marcel Decker, New York, NY (1980).

[0169] Pharmaceutical formulations as disclosed herein comprise one or more pharmaceutically acceptable excipients. Non-limiting examples of excipients include chemical enhancers, humectants, pressure-sensitive adhesives, antioxidants, solubilizers, thickeners, plasticizers, adjuvants, carriers, excipients, vehicles, coatings, and any combination thereof. One or more excipients can be selected for oral, transdermal, parenteral, intraperitoneal, intravascular, subcutaneous, by inhalation spray, rectal, or intrapulmonary administration.

[0170] Kits for dsRNA production

[0171] In some aspects, the present invention comprises a kit for dsRNA production. The kit comprises a plasmid vector for transiently expressing a target dsRNA in preselected cells and / or genetically modified cells that can be cultured and maintained for the production of the target dsRNA. The kit may further comprise the necessary chemicals, equipment, and instructions for setting up target dsRNA production and / or administering the dsRNA for the desired purpose. Example

[0172] Unless otherwise stated, the reagents used in the examples are commercially available or can be prepared using commercially available instruments, methods or reagents known in the art. The embodiments illustrate various aspects of the present invention and the practice of the method of the present invention. The embodiments are not intended to provide a detailed description of many different aspects of the present invention. Therefore, although the present invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily appreciated by those skilled in the art that various changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0173] It should be understood that the embodiments and aspects described herein are for illustrative purposes only and that various modifications or changes thereto will occur to those skilled in the art and are included within the spirit and scope of this application and the appended claims.

[0174] Example 1

[0175] Bacterial strains.

[0176] Escherichia coli 5-α (NEB, Ipswich, MA) was used in all cloning experiments. Escherichia coli HT115 (DE3) with genotype F-, mcrA, mcrB, IN (rrnD-rrnE) 1, rnc14:: Tn10 (DE3) lysogen: lacUV5 promoter-T7 polymerase) was obtained from the University of Minnesota Caenorhabditis Genetics Center (Minneapolis, MN) and used for dsRNA production in shake flasks and fed-batch fermentations. Escherichia coli JM109 (DE3) (Promega, Madison, WI) was used to study the effect of ribonuclease III (RNase III) on the accumulation of dsRNA inside the E. coli cells mediated by MS2 CP. All E. coli strains were maintained on LB medium with appropriate antibiotics.

[0177] Construction of plasmid vector

[0178] Vector pBR322 was used to generate dsRNA production vectors. A custom synthetic DNA fragment consisting of a T7 promoter sequence driving transcription of a single copy of the bacteriophage MS2 capsid gene, followed by a T7 terminator, was obtained from GenScript (Piscataway, NJ). This fragment was amplified using primer pair P1 and P2 (Table 1) and cloned into the BamHI / SphI restriction sites of pBR322 to generate plasmid pAPSE10118. A second synthetic sequence consisting of a T7 promoter sequence followed by an MS2 pac site sequence, a multiple cloning site, and, in 5' to 3' order, the following: AsiSI-PmeI-AscI-RsrII-NotI-PacI restriction sites, a second high-affinity variant MS2 pac-type sequence (C-pac), a T7 terminator, and a SphI restriction site was obtained from GenScript. This fragment was PCR amplified with primers P3 and P4 and cloned into the EcoR1 / EcoRV sites of pAPSE10118, generating plasmid pAPSE10136. To prepare the Colorado potato beetle (CPB) (Say) β-actin (NCBI accession number: KJ577616) hairpin construct, a synthetic 294-bp long sequence starting 49 bp upstream of the first codon was obtained, and primer pairs P5 / P6 and P7 / P8 were used to amplify the sense and antisense sequences, respectively, along with a loop region for cloning into pAPSE10136 via Gibson assembly. The pAPSE10136 plasmid backbone was PCR amplified with primers P9 / P10, and the CPB β-actin sense and antisense fragments were assembled to generate vector pAPSE10218.

[0179] To construct plasmids containing hairpin constructs containing zero pac sites (pAPSE10279), one 5' pac site (pAPSE10338), or one 3' pac site (pAPSE10219), a series of synthetic T7 promoter-T7 terminator sequences containing zero, one 5' pac site, or one 3' pac site were obtained. Each was PCR amplified with primers P3 / P4 and cloned into the EcoR1 / EcoRV sites of plasmid pAPSE10118, followed by Gibson assembly cloning of the CPB β-actin hairpin construct as described above for the generation of pAPSE10218. To add a third pac site to the hairpin loop region, the reverse sense primer and forward antisense primer were modified to incorporate a pac site into the loop region, generating plasmid pAPSE10777.

[0180] To generate the invasive fire ant (RIFA) (Solenopsis invicta) β-actin hairpin construct, a synthetic sequence containing a 300-bp region of the RIFA β-actin (NCBI Accession No. XM_011175337) was used as a template to amplify the sense and antisense sequences, along with the loop region, using primer pairs P11 / P12 and P13 / P14, respectively. The construct was then introduced into pAPSE10136 via Gibson assembly, generating plasmid pAPSE10379. To clone the pyrE expression cassette, the rrnBT1-T2 terminator sequence was amplified from synthetic DNA using forward primer P15, which carries SalI and AvrII sites, and reverse primer P16, which carries an NruI site, and cloned into the SalI / NruI sites of pAPSE10379, generating plasmid pAPSE10424. Then, primers P17 / P18 were used to isolate the The pyrE coding sequence was amplified from 5-α genomic DNA and cloned into the SalI / AvrII sites of pAPSE10424, generating plasmid pAPSE10448. Plasmid pAPSE10775 was generated by excising the CPB β-actin hairpin cassette (T7 promoter-CPB β-actin hairpin-T7 terminator) as an EcoRI / NotI fragment from pAPSE10218 and cloning into the EcoRI / NotI sites of pAPSE10448.

[0181] To generate vector pAPSE10402, which contains a cassette for producing intermolecular dsRNA via bidirectional transcription, a synthetic DNA fragment consisting of the following: T7 terminator-T7 promoter-MS2 pac site-294 bp CPB β-actin sense strand (Genscript, Piscataway, NJ) was amplified with primers P3 / P19 and cloned into the EcoRI / PacI sites of vector pAPSE10218, generating vector pAPSE10400. A second synthetic DNA sequence consisting of the following: MS2 pac site-T7 promoter-T7 terminator was amplified with primers P20 / P21 and cloned into the PacI / EcoRV sites of vector pAPSE10400, generating plasmid pAPSE10402. Plasmid pAPSE10776 was generated by excising the CPB β-actin hairpin and MS2 CP cassette as an EcoRI / SalI fragment and cloning into the same sites of pUC19. To construct vector pAPSE10773, a synthetic DNA fragment consisting of a T7 promoter driving the eGFP coding sequence followed by a T7 terminator was amplified using primer pair P22 / P23 and cloned into the BglII / SalI sites of vector pAPSE10218. Vector pAPSE10305 was constructed by excising the MS2 CP expression cassette from pAPSE10218 via EcoRV / NruI digestion and religating the pAPSE10218 backbone.

[0182] The primers used for cloning are listed together with the primer sequences in Table 1. The plasmid vectors are summarized in Table 2. All constructs were confirmed by Sanger sequencing.

[0183] Table 1: Primers used in the study. Restriction site sequences are underlined.

[0184]

[0185]

[0186]

[0187] Table 2: Summary of plasmids used in the experiments

[0188]

[0189]

[0190] dsRNA production in shake flasks

[0191] dsRNA production in shake flasks is performed using minimal medium. A carbon source (e.g., glucose and / or α-lactose) is added for autoinduction of dsRNA production. A single colony of the appropriate culture is transferred to 2 ml of liquid LB medium supplemented with appropriate antibiotics to initiate seed culture. After 5 to 6 hours of incubation at 37°C, 100 μl of the seed culture is used to inoculate a shake flask containing 25 ml of 25% Super Broth. The shake flask is incubated overnight at 37°C in a shaking incubator at 250 rpm. After 18 to 19 hours of incubation, the culture OD is recorded. 600 , and culture aliquots were collected and stored at -80°C. 1-ml aliquots of the culture were saved for TRIzol TM RNA extraction.

[0192] Minimal medium was used for shake flask RNA production. Seed cultures were grown as described above. Cell pellets obtained from the seed cultures were resuspended in 1 ml of minimal medium and used to inoculate shake flasks containing 25 ml of minimal medium. The shake flasks were incubated at 37°C in a shaking incubator at 250 rpm for 24 hours. After 24 hours of incubation, the culture OD was recorded. 600 , and culture aliquots were collected and stored at -8° C. 3-ml aliquots of the culture were saved for RNA extraction.

[0193] RNA extraction

[0194] Cell mass from the aliquots collected from shake flask culture or from the 250-μ l aliquots of fermentation liquid is processed as follows for RNA extraction.Cell mass is resuspended in ice-cold 20mM Tris-HCl pH 7.0, so that volume reaches 500 μ l.Resuspended cells are transferred to ice-cold 2-ml bead grinding tube (bead beater tube) (Sigma, St.louis, MO), and by bead grinding (bead beating) 2 minutes in Biospec micro-bead mill 16 (Biospec Products, Bartlesville, OK) and cracked.After bead grinding, lysate was incubated on ice for 5 minutes after centrifugation for 2 minutes with 14000rcf.The 100-μ l aliquots of centrifugal lysate are transferred to ice-cold 20mMTris-HCl, pH 7 and 150 μ lTRIzol containing 300-μ l. TM The RNA pellet was resuspended in 200 μl of 20 mM Tris-HCl, pH 7.

[0195] RNA quantification

[0196] Total RNA concentration is measured using nanodrop 200 ℃ of spectrophotometers (ThermoFisher Scientific). 5-μg aliquots of the total RNA from the sample were subjected to RNAse A (NEB, Ipswich, MA) digestion for 2 hours, followed by Proteinase K (NEB, Ipswich, MA) digestion for 1 hour. The total RNA of 100ng (shake flask sample) or 50ng (fermentation sample) RNAse A / Protease K digestion, together with 100bp quantitative DNA ladder (Lambda Biotech, St.Louis, MO), was run on 1.5% agarose gel to dsRNA content of quantitative sample. Gel images were captured with Bio-Rad molecular imager gel doc XR+ gel imaging system, and the manufacturer's guide was followed to use Image Lab software 6.1 (Bio-rad, Hercules, CA) to quantitatively analyze the RNA bands on the gel.

[0197] Protein analysis

[0198] Cell lysates generated by bead beating were used for recombinant protein analysis by SDS-PAGE accompanied by Coomassie staining. 600 Cell lysate samples containing approximately equal numbers of cells were used for analysis. Each cell lysate sample (1.5 to 6 μl) was mixed with 2.5 μl NuPAGE 4X LDS sample buffer (Invitrogen, Waltham, MA), 1 μl NuPAGE sample reducing agent (Invitrogen, Waltham, MA), and the total sample volume was made up to 10 μl with 20 mM Tris HCl pH 7. The prepared protein samples were incubated at 75°C for 10 minutes, followed by incubation on ice until gel loading. Protein samples were stained on NuPAGE 12% PAGE gels (Invitrogen) together with SeeBlue TM Plus2 prestained protein standards (Invitrogen, Waltham, MA) were run together. After the gel was run, the gel was removed from the casting tray, rinsed with Milli-Q water, and subjected to Coomassie blue staining.

[0199] Example 2

[0200] High levels of extracapsid dsRNA in Δrnc Escherichia coli cells co-expressing the bacteriophage MS2 coat protein accumulation

[0201] Studies were conducted to determine whether dsRNA could be produced at high levels and packaged into MS2 virus-like particles (VLPs). To produce MS2 VLPs containing dsRNA, the vector pAPSE10218 (Figure 1) was introduced into the Δrnc Escherichia coli strain HT115 (DE3). The E. coli rnc gene encodes a dsRNA-specific RNase (RNase III), and inactivation of rnc is required for accumulation of dsRNA in E. coli. The vector pAPSE10218 co-expresses the bacteriophage MS2 (MS2) CP cassette with an 893-base hairpin RNA (hpRNA) derived from the β-actin gene of the Colorado potato beetle (Leptinotarsa decemlineata (Say)) (CPB), and contains 19-nucleotide RNA stem-loop sequences called MS2 packaging (pac) sites at the 5' and 3' ends of the hairpin to promote capsid self-assembly and packaging of dsRNA ( Figure 2A MS2 VLP-encapsidated dsRNA was recovered from E. coli HT115(DE3) / pAPSE10218. The average dsRNA yield of isolated encapsidated dsRNA in shake flask cultures was 5 mg / L (data not shown). In addition, a significant amount of unencapsidated ('exocapsid') dsRNA was also recovered from E. coli HT115(DE3) / pAPSE10218. The total shake flask dsRNA yield, including both encapsidated and exocapsid dsRNA, ranged from 200 to 500 mg / L.

[0202] To determine whether MS2 CP is required for dsRNA accumulation, the MS2 CP expression cassette was deleted from vector pAPSE10218, generating vector pAPSE10305 ( Figure 2A Another vector, pAPSE10773, was constructed by replacing the MS2CP coding sequence in pAPSE10218 with the green fluorescent protein (GFP) coding sequence ( Figure 2A ) was produced to test whether co-expression of another protein also leads to dsRNA accumulation via certain non-specific interactions with dsRNA. The newly generated vector was introduced into E. coli strain HT115 (DE3) and the shake flask dsRNA yield was measured. MS2 CP and GFP expression were confirmed by SDS-PAGE and Coomassie staining ( Figure 2B , lanes 1-6). The recombinant protein band was not visible in the HT115(DE3) / pAPSE10305 cell lysate ( Figure 2BStrong bands of GFP were visible in HT115(DE3) / pAPSE10773 cell lysates, as well as strong bands of MS2CP in HT115(DE3) / pAPSE10218 cell lysates. The average dsRNA yield of HT115(DE3) / pAPSE10305 cultures lacking MS2 CP was 4 mg / L, while HT115(DE3) / pAPSE10773 cultures co-expressing GFP and dsRNA produced an average of 7 mg / L ( Figure 2C and Figure 2D , lanes 1-6). The average dsRNA yield of HT115(DE3) / pAPSE10218 cultures co-expressing MS2 CP and dsRNA was significantly higher, at 501 mg / L ( Figure 2C ), confirming that co-expression of MS2CP is necessary for dsRNA accumulation in E. coli cells.

[0203] To confirm whether MS2 CP promotes dsRNA accumulation by protecting dsRNA from cellular nucleases, vectors pAPSE10218 and pAPSE10305 were introduced into E. coli strain JM109(DE3) containing a functional rnc gene. The strain JM109(DE3) / pAPSE10218 co-expressing MS2 CP and dsRNA accumulated dsRNA at low levels (~15 mg / L). Figure 3 No dsRNA accumulation was detected in the absence of MS2 CP expression in strain JM109(DE3) / pAPSE10305 ( Figure 3 The dsRNA yield of the JM109(DE3) / pAPSE10218 strain carrying a functional rnc gene was 15 mg / L, significantly lower than the 501 mg / L obtained with HT115(DE3) / pAPSE10218 ( Figure 2C and Figure 2D , lanes 3-4 and 7-8). SDS-PAGE with Coomassie staining ( Figure 2B , lanes 3-4, 7-8) show that JM109(DE3) / pAPSE10218 and HT115(DE3) / pAPSE10218 cultures expressed similar levels of MS2 CP. This result from this study demonstrates that in the presence of dsRNA-specific ribonucleases, MS2 CP is not sufficient to protect dsRNA to support its high accumulation.

[0204] Example 3

[0205] The presence and number of MS2pac sites affect MS2CP-mediated dsRNA accumulation.

[0206] To further explore whether the interaction between the dsRNA pac site and the MS2 CP promotes the accumulation of dsRNA, the vector pAPSE10218 ( Figure 2A To test whether the presence and / or number of MS2 pac sites affects dsRNA yield, four modifications of pAPSE10218 were generated: 1) removal of both dsRNA pac sites (pAPSE10279), 2) removal of the 3' dsRNA pac site (pAPSE10338), 3) removal of the 5' dsRNA pac site (pAPSE10219), and 4) addition of a third pac site to the hairpin loop (pAPSE10777) ( Figure 4A Each vector was introduced into Escherichia coli HT115 (DE3).

[0207] It was found that E. coli harboring the vector pAPSE10279 (which produces dsRNA lacking the pac site) accumulated a relatively high level of dsRNA (121 mg / L) in the presence of MS2 CP ( Figure 4B ), and the addition of 1-2 pac sites to hpRNA improved dsRNA accumulation. E. coli carrying vectors pAPSE10338 or pAPSE10219 (each of which produces dsRNA with a single pac site) produced 142 mg / L and 186 mg / L dsRNA, respectively. Of the constructs tested, E. coli carrying pAPSE10218 (which produces hpRNA containing a pac site at both its 5' and 3' ends) was the best design for maximizing cumulative dsRNA yield. This strain produced 478 mg / L dsRNA, more than double the amount produced by any other strain in the experiment. HT115(DE3) / pAPSE10777, which contains a third pac site in the hairpin loop, produced dsRNA at similar levels to HT115(DE3) / pAPSE10219 ( Figure 4B ).

[0208] Example 4

[0209] Intramolecular hairpin RNA (hpRNA) accumulates to higher levels than intermolecular dsRNA.

[0210] To examine the effect of transcript design on dsRNA accumulation, vector pAPSE10402 was generated by modifying pAPSE10218 to produce an intermolecular dsRNA consisting of a 294-bp sequence from the CPB β-actin gene flanked at each end by MS2pac sites ( Figure 5AWhen compared to HT115(DE3) / pAPSE10402 producing intermolecular dsRNA, which yielded 109 mg / L, the average shake flask yield of hpRNA-producing E. coli HT115(DE3) / pAPSE10218 was almost 2.5 times higher at 251 mg / L ( Figure 5B ).

[0211] Example 5

[0212] Complementary expression of the pyrE gene improves the growth of Escherichia coli K-12 derivatives grown in minimal medium. dsRNA yield per bottle

[0213] To determine whether increasing pyrE expression increases dsRNA yield, pAPSE10218 was modified with a pyrE coding sequence that includes a ribosome binding site at its 5' end and a T1-T2 terminator at its 3' end. The pyrE construct was inserted downstream of the MS2 CP expression cassette to allow read-through transcription of the pyrE coding sequence via the T7 promoter. The resulting vector, pAPSE10775 ( Figure 6A ) were introduced into E. coli HT115(DE3), and dsRNA production was evaluated in minimal medium.

[0214] On average, E. coli HT115(DE3) / pAPSE10775 produced 45% more dsRNA than E. coli HT115(DE3) / pAPSE10218 ( Figure 6B ). To confirm the yield improvement with the second dsRNA sequence, the CPB β-actin hairpin sequence in vectors pAPSE10218 and pAPSE10775 was replaced with the Invasive Fire Ant (RIFA) (Red Imported Fire Ant) β-actin hairpin sequence, generating plasmids pAPSE10379 and pAPSE10448, respectively. Each strain used for shake flask dsRNA production was grown in triplicate. Data from shake flask experiments involving more than two strains were subjected to one-way analysis of variance (ANOVA), and data from experiments involving two strains were subjected to t-test analysis. Following the ANOVA analysis, a post hoc test with Bonferroni correction was used to identify constructs that differed significantly in dsRNA yield. Results from shake flask experiments using minimal medium showed that E. coli HT115(DE3) / pAPSE10448 produced 30% more dsRNA ( ) relative to E. coli HT115(DE3) / pAPSE10379. Figure 6C ).

[0215] Example 6

[0216] A plasmid vector modified with a stem-loop sequence, 3' and 5' pac sites, an MS2CP cassette and pyrE complement expression Fed-batch fermentation of Enterobacter HT115(DE3) to produce large amounts of dsRNA

[0217] To evaluate the maximum productivity potential of the disclosed dsRNA production system in a benchtop high cell density fed-batch bioreactor, the pyrE-deficient strain E. coli HT115 (DE3) / pAPSE10218 and the pyrE-overexpressing strain E. coli HT115 (DE3) / pAPSE10775 were each evaluated in three independent fermentation runs. pAPSE10775 contains a T7 promoter operably linked to a CPB β-actin hairpin with a pac site at each of the 3' and 5' ends, an MS2 CP cassette, and an RBS-pyrE cds-T1-T2 terminator downstream of the MS2 CP cassette.

[0218] Fed-batch fermentation was carried out in a 3-L vessel using a New Brunswick BioFlo 115 (Eppendorf, Enfield, CT) benchtop bioreactor with a working volume of 2L. Seed cultures for fermentation inoculum were grown in 2ml LB medium with appropriate antibiotics. After 8 hours of growth, the seed culture was used to inoculate 50ml minimal medium. Before the 50ml inoculum was transferred to a fermentation vessel containing 1.2L minimal medium, the inoculum OD600 was recorded. The glucose feed consisted of 50% glucose and the main salts of minimal medium. In order to support the initial growth of the culture, 5ml glucose feed was added to the vessel before inoculating the fermentation vessel with the inoculum. The pH was maintained at 7.0 by adding 30% (v / v) NH4OH. Dissolved oxygen (DO) was set to 30% saturation and controlled by agitation-DO cascade during the first 16 hours of the fermentation run. After 16 hours, agitation was set to 1000rpm, and DO was used to control the glucose feed. Culture growth was monitored by measuring OD600. Cultures were induced with 1 mM IPTG for dsRNA production at an OD600 between 65 and 70. Fermentation samples were collected at the time of induction and every hour up to 5 hours after induction for evaluation of dsRNA production. Three different fermentation runs were performed for each strain, and dsRNA data were subjected to t-test analysis.

[0219] The dsRNA yields of three HT115(DE3) / pAPSE10218 fermentation runs ranged from 4 g / L to 4.8 g / L and averaged 4.69 g / L ( Figure 7The highest dsRNA yield achieved with strain HT115(DE3) / pAPSE10775 was 11.26 g / L, with an average yield of 10.12 g / L over three fermentation runs. The yield obtained was 115.78% higher than that of HT115(DE3) / pAPSE10218 ( Figure 7 The results demonstrated that supplemental expression of pyrE compensated for the OPRT enzyme deficiency of K-12-derived E. coli HT115(DE3) and significantly increased dsRNA productivity in cultures grown in minimal medium in high cell density fed-batch bioreactors.

[0220] Example 7

[0221] dsRNA produced by fed-batch fermentation of Escherichia coli transformed with pAPSE10775 in leaf disc bioassays High efficacy against CPB larvae

[0222] dsRNA produced by fed-batch fermentation of E. coli transformed with pAPSE10775 was evaluated for its efficacy against Colorado potato beetle (CPB) larvae in a leaf disc bioassay.

[0223] Colorado potato beetle (CPB) larval bioassays were performed by AgMetrics Group (Albion, MI). First-instar Colorado potato beetle larvae of mixed sex and similar size used in these bioassays were obtained from colonies maintained under laboratory conditions. Potato plants (Solanum tuberosum cv. Kennebec) used in the bioassays were grown in a greenhouse. To kill E. coli cells containing β-actin dsRNA prior to use in the bioassays, the cells were incubated at 55°C for 15 minutes. dsRNA was quantified in heat-inactivated samples, and three different dsRNA solutions (2.5 ng / μl, 10 ng / μl, and 40 ng / μl) were prepared by diluting the samples with Milli-Q water.

[0224] A 10-μl volume of each dsRNA solution including 0.1% Tween-20 was pipetted onto a leaf disc (D = 1 cm 2 ) and smeared over the entire leaf surface using a glass rod to ensure even coverage. The leaf discs were allowed to air dry, and each disc was then transferred to a 100-mm petri plate lined with filter paper, and a single larva was placed on each treated disc. Untreated leaf discs were provided in the negative control treatment group. Each treatment group included 20 biological replicates.

[0225] After 24 hours, the residual leaf material was removed from each culture dish and replaced with a freshly cut and treated leaf disc. This process was repeated for the first four days of the bioassay. On the fifth day, the residual leaf material was removed from each culture dish and replaced with a fresh, untreated leaf disc. This process was repeated until the tenth day, at which time the bioassay was terminated. The number of dead larvae in each treatment group was recorded daily during the assay. Larvae that did not respond to the probe were considered dead. The data were analyzed by one-way ANOVA followed by Fisher's least significant difference test to identify significant differences among the groups (p<0.05). The results indicated that when delivered at a concentration of 10 ng / μl, the dsRNA was found to cause 100% mortality seven days after treatment ( Figure 8 ).

[0226] Example 8

[0227] E. coli transformed with a plasmid vector harboring the MS2CP cassette and pyrE under the control of a dedicated T7 promoter Fed-batch fermentation of HT115(DE3)

[0228] To evaluate the productivity potential of a plasmid vector containing an MS2 CP cassette and pyrE under the control of a dedicated promoter, dsRNA production was evaluated in E. coli HT115 (DE3) / pAPSE10471 in a benchtop, high-cell-density fed-batch bioreactor. pAPSE10471 was engineered to contain a T7 promoter operably linked to RIFA B-actin with a pac site at each of the 3' and 5' ends, an MS2 CP cassette, and a T7-RBS-pyrE cds-T1-T2 terminator downstream of the MS2 CP cassette, with pyrE under the control of a dedicated T7 promoter. Fed-batch fermentation and dsRNA production were performed using the methods described in Example 6. The dsRNA yield from the E. coli HT115 (DE3) / pAPSE10471 fermentation run was 5.5 g / L (Table 3). This result suggests that higher dsRNA production can be obtained by engineering the plasmid vector with the MS2 CP cassette and having pyrE expression under the control of its own promoter.

[0229] Table 3: dsRNA yield in E. coli HT115 (DE3) transformed with plasmid vectors harboring pyrE under the control of a dedicated T7 promoter

[0230]

[0231] Example 9

[0232] Fed-batch fermentation of Corynebacterium glutamicum transformed with plasmid vector

[0233] Evaluated the dsRNA produced by the fed-batch fermentation of Corynebacterium glutamicum transformed with a plasmid vector. Use New Brunswick BioFlo115 (Eppendorf, Enfield, CT) bench-top bioreactor with a working volume of 2L to carry out fed-batch fermentation in a 3-L vessel. The seed culture used for the fermentation inoculum is grown in 5ml brain heart infusion sorbitol (BHIS) culture medium with appropriate antibiotics. After overnight growth at 30°C, the seed culture is used to inoculate 50ml BHIS culture medium and grows 6 to 8 hours at 30°C. Before the 50-ml inoculum is transferred to the fermentation vessel containing 1.2L minimal medium, record the inoculum OD600. The glucose feed is composed of the main salts and 20mg / L biotin of 50% glucose and minimal medium. In order to support the initial growth of the culture, before inoculating the fermentation vessel with the inoculum, 5ml glucose feed is added to the vessel. pH is maintained at 7.2 by adding 30% (v / v) NH4OH. Dissolved oxygen (DO) was set to 30% saturation and controlled by agitation-DO cascade during the fermentation run. Culture growth was monitored by measuring OD600. Cultures were induced with 0.1 mM IPTG for dsRNA production at an OD600 between 75 and 80. Fermentation samples were collected at the time of induction and every hour up to 5 hours after induction for evaluation of dsRNA production.

[0234] To evaluate the productivity potential of plasmid vectors with an MS2 CP cassette and pyrE, dsRNA production was evaluated in C. glutamicum transformed with pAPSE10772, pAPSE10797, or pAPSE10500 (SEQ ID NO: 36) in a benchtop high cell density fed-batch bioreactor. pAPSE10772 (SEQ ID NO: 38) was engineered to have a T7 promoter operably linked to RIFA β-actin with a pac site at each of the 3' and 5' ends, an MS2 CP cassette, and an RBS-pyrE cds-T1-T2 terminator downstream of the MS2 CP cassette. pAPSE10797 (SEQ ID NO: 39) has a T7 promoter operably linked to CPB β-actin with a pac site at each of the 3' and 5' ends, an MS2 CP cassette, and an RBS-pyrE cds-T1-T2 terminator downstream of the MS2 CP cassette, while pAPSE10500 has an MS2 CP cassette without the pyrE construct. Table 4 summarizes the results of dsRNA yields produced by C. glutamicum / pAPSE10772 and C. glutamicum / pAPSE10797, as well as the predicted yield for C. glutamicum / pAPSE10500. dsRNA yields from fermentation runs of C. glutamicum / pAPSE10772 reached 1.1 g / L (Table 4), while yields from C. glutamicum / pAPSE10797 were approximately 1 g / L (Table 4). The predicted yield for C. glutamicum / pAPSE10500 lacking the pyrE construct was approximately 0.8 g / L (Table 4). These results suggest that higher dsRNA production can be achieved by engineering a plasmid vector that co-expresses the MS2 CP cassette and the pyrE construct in C. glutamicum.

[0235] Table 4: dsRNA yield in Corynebacterium glutamicum transformed with plasmid vectors

[0236]

[0237] *Predicted yield

[0238] Example 10

[0239] Construction of antibiotic marker-free plasmid backbones and modified E. coli strains to allow for the generation of Proliferation and dsRNA expression

[0240] Further development of antibiotic-marker-free plasmids and host systems for dsRNA expression was conducted. The pyrE gene was selected as a selectable marker for plasmid selection and maintenance. PyrE knockout strains of E. coli and related microorganisms are uracil auxotrophs, but remain fully viable and 'healthy' if supplemented with uracil in the growth medium or if complemented (in trans) with a functional pyrE gene on the plasmid.

[0241] A previously developed suicide plasmid system (Link et al., 1997) was selected to knock out the pyrE gene in the E. coli genome. Plasmid pAPSE10822 (SEQ ID NO: 40), a chloramphenicol resistance plasmid containing a temperature-sensitive replication origin, and a Bacillus SacB gene for reverse selection in the presence of sucrose, was assembled. It contained an E. coli K-12 genomic DNA sequence of ~0.7 kb upstream of pyrE and a DNA sequence of ~0.7 kb downstream of pyrE (extracted from the genome of E. coli K-12 strain W3110, GenBank CP017979). This design deleted the pyrE coding sequence from amino acids 1 to 175, making it a functionally inactive truncated peptide. The knockout vector was integrated into the genome of HT115 (DE3) cells by homologous recombination and excised after selection for sucrose resistance. Sucrose-resistant, chloramphenicol-sensitive E. coli colonies were screened using PCR with primer pairs that identify the pyrE gene deletion. Colonies with relevant PCR patterns suggestive of gene deletion were selected and tested for uracil auxotrophy by examining growth on M9-glucose minimal agar versus minimal agar supplemented with 50 μg / ml uracil. The new strain was confirmed to be genetically deleted in pyrE and uracil auxotrophic. This resulted in strain HT115(DE3)-ΔpyrE.

[0242] Bacterial strain HT115 (DE3) -ΔpyrE is a recA + bacterial strain, which is therefore not suitable for cloning or direct transformation with a ligation mixture to produce a new construct. By cloning the E. coli recA coding region into the backbone of pAPSE10822, a new suicide plasmid pAPSE10826 (SEQ ID NO: 41) was assembled. Unlike plasmid pAPSE10822, pAPSE10826 can be integrated into the genome of E. coli strain NEB-5 α and subsequently excised therefrom, and the NEB-5 α is a recA - bacterial strain commonly used for gene cloning and stable plasmid maintenance. Basically following the same steps outlined above for HT115 (DE3), bacterial strain NEB-5 α -ΔpyrE was produced. The genomic deletion and uracil auxotrophy of pyrE were confirmed. By losing the suicide plasmid backbone, the bacterial strain was once again recA -, which was confirmed by its high sensitivity to UV cell killing.

[0243] Plasmid pAPSE10775, which contains a promoterless pyrE gene, was unable to complement strain NEB5-ΔpyrE to allow growth on M9-glucose minimal agar plates, likely due to insufficient pyrE expression. To make pyrE a suitable selectable marker (i.e., allowing plasmid selection by uracil prototrophy), pAPSE10835 (SEQ ID NO: 42) was constructed by modifying the pyrE gene in pAPSE10775 with the addition of the constitutive J23115 promoter driving pyrE expression. pAPSE10835 transformants in both HT115(DE3)-ΔpyrE and NEB5-ΔpyrE could be selected by growth on M9-glucose minimal medium without added uracil. pAPSE10835 also confers ampicillin resistance.

[0244] Plasmid pAPSE10836 (SEQ ID NO: 43) was prepared by deleting the Amp-r gene (bla, β-lactamase) from the backbone of pAPSE10835 via BspHI restriction digestion and religation, transformation into competent NEB5-ΔpyrE cells, and selection for growth on M9-glucose (without antibiotics). For growth of the plasmid preparation, transformants could be grown in minimal medium and in LB without plasmid loss. The nucleotide sequence of pAPSE10836 was confirmed by whole plasmid sequencing.

[0245] Plasmid pAPSE10836 was transformed into competent HT115(DE3)-ΔpyrE cells and selected for growth in minimal medium as described for NEB5-ΔpyrE. Recombinant HT115(DE3)-ΔpyrE cells harboring pAPSE10836 were first tested for dsRNA production in shake flasks. Shake flask dsRNA yields from the three HT115(DE3)-ΔpyrE / pAPSE10836 clones were similar to those from HT115(DE3) / pAPSE10775. The HT115(DE3)-ΔpyrE / pAPSE10836 clone with the highest shake flask dsRNA yield was evaluated for dsRNA production in fermentors. The fermentation dsRNA yield of HT115(DE3)-ΔpyrE / pAPSE10836 was approximately 5.4 g / L, which fell within the range of the fermentation dsRNA yield of HT115(DE3) / pAPSE10775.

[0246] Example 11

[0247] Removal of antibiotic marker genes from the genome of Escherichia coli dsRNA-producing strain HT115(DE3)-ΔpyrE Tetracycline resistance

[0248] The rnc / RNase III knockout mutation in E. coli HT115 results from an insertion event of the mini-transposon Tn10 close to the 5'- / N-terminus of rnc within the E. coli rnc-era-recO operon. This transposon functionally knocks out rnc while allowing expression of the downstream era gene (an essential gene), making HT115 and its derivatives suitable for dsRNA production. However, the presence of mini-Tn10 places a tetracycline resistance gene in the HT115 genome and its derivatives, such as HT115(DE3)-ΔpyrE.

[0249] SEQ ID NO:44 comprises the genomic sequence of HT115(DE3)-ΔpyrE, Figure 9 The complete sequence of the mini-Tn10 transposon and its insertion site within the rnc-era-recO operon are provided in [ 15 ]. Transposon insertion positions the regulatory gene TetR(B) in the same orientation as Drnc-era-recO. The tetracycline resistance gene, Tet(B), is placed upstream of TetR(B) and oriented in the opposite direction.

[0250] Using a similar approach to that used to generate the ΔpyrE deletion using plasmid APSE10822, a ΔTet(B) deletion template can be assembled in a suicide plasmid backbone containing approximately 0.7-1 kb of sequence upstream and downstream of the Tet(B) coding sequence, where Tet(B) is deleted. Alternatively, a 'deletion plus insertion' template can be assembled, deleting the Tet(B) coding sequence and replacing it with the MS2 coat protein coding sequence, placing MS2 CP expression under the control of the medium-strength constitutive Tet(B) promoter.

[0251] Insertion and subsequent excision of the 'knockout' and 'knockout plus knockin' constructs into the mini-Tn10 locus of HT115(DE3)-ΔpyrE by homologous recombination will generate sucrose-resistant colonies that can be screened for sensitivity to growth in medium with 12.5 μg / ml tetracycline. Tetracycline-sensitive colonies will be screened by PCR using primer pairs and sequencing, which will confirm the correct ΔTet(B) or ΔTet(B)::MS2 genomic structure.

[0252] The modified strain will completely lack Tet(B) and, when paired with plasmid pAPSE10836 (or variants), will allow for dsRNA production by fermentation without antibiotics and without antibiotic resistance genes in the vector or host. Strain HT115(DE3)-ΔpyrE-ΔTet(B)::MS2 additionally has the ability to provide MS2 from a chromosomal copy, allowing for further modification of dsRNA expression vectors by removing the P-T7-MS2 expression cassette from the backbone.

[0253] Summary of the embodiments

[0254] The results from the study confirmed that MS2 CP is essential for dsRNA accumulation in Escherichia coli HT115 (DE3), an RNase III-deficient strain widely used in microbial dsRNA production. The addition of the pac site further improved dsRNA productivity, wherein a pac site flanking the transcribed RNA is optimal for dsRNA production (Fig. 2). The data indicate that vector constructs with pac sites are desirable for obtaining higher dsRNA accumulation. Further, using the MS2 CP-dsRNA production system, hpRNA accumulates to a level higher than that of intermolecular dsRNA. The sense and antisense fragments of hpRNA are connected via a loop sequence that promotes intramolecular RNA folding and the formation of dsRNA molecules immediately after transcript synthesis. Supplementary expression of pyrE from the dsRNA production plasmid has increased the shake flask and high cell density fed-batch fermentation dsRNA productivity of CPB β-actin and RIFA β-actin dsRNA. The highest dsRNA yields in high cell density fed-batch fermentations were achieved with the pAPSE10775 construct, which contains a T7 promoter operably linked to a CPB β-actin hairpin with a pac site at each of the 3' and 5' ends, an MS2 CP cassette, and an RBS-pyrE cds-T1-T2 terminator downstream of the MS2 CP cassette construct. Fermentation runs in E. coli transformed with a plasmid vector harboring the MS2 CP cassette, combined with a pyrE construct engineered with a dedicated T7 promoter, demonstrated high dsRNA yields, suggesting alternative configurations of the pyrE construct that can be engineered to generate high-yield dsRNA plasmid vectors. Additionally, examination of dsRNA production in C. glutamicum transformed with a plasmid vector harboring the MS2CP cassette and the pyrE construct revealed high yield levels, confirming the feasibility of the disclosed high-yield plasmid vector in a fed-batch fermentation system using C. glutamicum.

[0255] The high dsRNA yield produced by the disclosed system paves the way for reducing the cost of dsRNA production and helps develop RNAi biopesticides that are cost-competitive with chemical pesticides. This is a production system based on microbial fermentation that can utilize existing fermentation infrastructure and, unlike in vitro transcription, cell-free, and chemical synthesis methods, does not require specialized equipment, materials, or conditions for dsRNA production. Since the inducible expression system based on T7 RNA polymerase has been evaluated in various microorganisms, this system can be extended to other industrial microorganisms - including species designated as "Generally Recognized as Safe" (GRAS) by the FDA, such as Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae. The dsRNA produced using this system has been demonstrated in laboratory bioassays for use in agricultural pest control ( Figure 8 ).

[0256] Table 5: Nucleic acid (DNA) sequences used in plasmid vectors.

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] *Bold: RBS; Italic: pyrE CDS; Underline: T1 terminator; Bold and italic: T2 terminator

[0355] Table 6: Amino acid sequence of MS2 CP

[0356]

Claims

1. A plasmid vector comprising a nucleic acid sequence, the nucleic acid sequence comprising: an inducible bacterial promoter operably linked to a stem-loop sequence, the stem-loop sequence having a 3' end and a 5' end and comprising a target recombinant RNA sequence; a first pac site sequence at the 3' end and a second pac site sequence at the 5' end of the stem-loop sequence; an MS2 capsid protein (CP) expression cassette; and a pyrE coding sequence downstream of the MS2 CP expression cassette, the pyrE coding sequence having a ribosome binding site (RBS) at its 5' end and a T1-T2 terminator at its 3' end. 2 . The vector according to claim 1 , wherein the promoter comprises an Escherichia coli phage T7 promoter having a sequence of SEQ ID NO:

1.

3. The vector of claim 1, wherein expression of the pyrE coding sequence comprising RBS-pyrE cds-T1-T2 having the sequence of SEQ ID NO: 2 is driven by an upstream Escherichia coli phage T7 promoter comprising the sequence of SEQ ID NO:

1.

4. The vector of claim 1, wherein expression of the pyrE coding sequence comprising RBS-pyrE cds-T1-T2 having the sequence of SEQ ID NO: 2 is driven by a dedicated E. coli phage T7 promoter comprising the sequence of SEQ ID NO:

1.

5. The vector of claim 1, wherein expression of the pyrE coding sequence comprising RBS-pyrE cds-T1-T2 having the sequence of SEQ ID NO: 2 is driven by a dedicated J23115 promoter comprising the sequence of SEQ ID NO:

33. The vector according to claim 1 , wherein the target recombinant RNA is a dsRNA that specifically inhibits the expression of a target gene.

7. The vector according to claim 1, wherein the target recombinant RNA is selected from the group consisting of dsRNA, siRNA, shRNA, hpRNA and miRNA.

8. The vector of claim 1, wherein the first pac site sequence and the second pac site sequence each comprise the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

9. The vector according to claim 1, comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 33 or any combination thereof.

10. The vector according to claim 1, wherein the vector does not comprise an antibiotic resistance Amp-r gene or a tetracycline resistance gene. The vector according to claim 1 , comprising the sequence of SEQ ID NO:

43.

12. A Gram-positive or Gram-negative bacterial cell comprising a vector according to any preceding claim. 13 . The bacterial cell according to claim 12 , wherein the bacterial cell is an Escherichia coli cell or a Corynebacterium glutamicum cell.

14. An Escherichia coli cell comprising the vector according to any one of claims 1 to 11.

15. The E. coli cell according to claim 14, wherein the E. coli cell is an RNase III-deficient E. coli strain.

16. The E. coli cell according to claim 14, wherein the RNase III-deficient E. coli strain is HT115 (DE3).

17. The E. coli cell of claim 15, wherein the E. coli strain is auxotrophic for uracil.

18. A bacterial culture comprising a population of bacterial cells according to any one of claims 12 to 17 in a bioreactor.

19. The bacterial culture of claim 18, comprising the target dsRNA in an amount of at least about 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L.

20. The bacterial culture of claim 18, wherein the bioreactor is selected from the group consisting of a fed-batch system, a semi-continuous system, and a continuous culture system.

21. The bacterial culture of claim 20, wherein the bioreactor is a fed-batch system.

22. A method for producing a target recombinant RNA, comprising maintaining the bacterial culture of claim 18 in a bioreactor for a time and under conditions sufficient to produce the target recombinant RNA in an amount of at least about 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L.

23. The method of claim 22, wherein the bioreactor is selected from the group consisting of a fed-batch system, a semi-continuous system, and a continuous culture system.

24. The method of claim 22, wherein the bioreactor is a fed-batch system.

25. The method of any one of claims 22-24, further comprising harvesting the target recombinant RNA.

26. The method according to any one of claims 22 to 24, wherein the target recombinant RNA is a dsRNA that specifically inhibits the expression of a target gene.

27. The method of any one of claims 22-24, wherein the target recombinant RNA is selected from the group consisting of dsRNA, siRNA, shRNA, hpRNA, and miRNA.

28. Use of a bacterial culture comprising a population of bacterial cells according to any one of claims 12 to 17 for producing antibiotic marker-free recombinant RNA.

29. The use according to claim 27, wherein the recombinant RNA is dsRNA, siRNA, shRNA, hpRNA and miRNA.

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