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WO2025184273A1 - Nano-compartiments bactériens basés sur des protéines - Google Patents

Nano-compartiments bactériens basés sur des protéines

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Publication number
WO2025184273A1
WO2025184273A1 PCT/US2025/017490 US2025017490W WO2025184273A1 WO 2025184273 A1 WO2025184273 A1 WO 2025184273A1 US 2025017490 W US2025017490 W US 2025017490W WO 2025184273 A1 WO2025184273 A1 WO 2025184273A1
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WO
WIPO (PCT)
Prior art keywords
rna
sequence
protein
nucleic acid
encapsulin
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Pending
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PCT/US2025/017490
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English (en)
Inventor
Tobias W. GIESSEN
Seokmu KWON
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University of Michigan System
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University of Michigan System
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Publication of WO2025184273A1 publication Critical patent/WO2025184273A1/fr
Pending legal-status Critical Current
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells are provided herein.
  • reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest.
  • Protein nanocages provide an engineering platform for diverse biotechnological and biomedical applications.
  • Synthetic encapsulation shells that self-assemble in vivo may comprise nucleic acid-binding peptides without disrupting native protein packaging mechanisms, and may be purified from bacterial cells.
  • Protein nanocompartment encapsulins are capable of size selective in vivo RNA packaging, may simultaneously load multiple functional RNAs, and support concurrent in vivo packaging of RNAs and proteins of use, for example, in codelivery of therapeutic and other RNAs with proteins to elicit synergistic effects.
  • a limitation of conventional encapsulins is the inability to provide simultaneous in vivo packaging of specific RNAs of interest, in contrast to non-specific loading together with specific proteins of interest.
  • compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells are provided herein.
  • reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest are described below.
  • the present invention provides a composition comprising a nanocompartment (e.g., an encapsulin) that contains a protein of interest and a specific RNA of interest (e.g., to the exclusion of other RNA molecules that are not of interest (e.g., with respect to RNA, consisting of or consisting essentially of the specific RNA of interest)).
  • a nanocompartment e.g., an encapsulin
  • the composition comprises an RNA binding protein fused to an encapsuling shell protein.
  • the composition comprises the specific RNA of interest fused to a target RNA sequence that binds to the RNA binding protein.
  • the nanocompartment composition is generated in vivo by expressing the two fusions so as to cause the specific RNA of interest to be selectively packaged into the nanocompartment. Also provided herein are methods for generating such composition.
  • the present invention provides a method of generating an encapsulin comprising a specific target RNA of interest, comprising: providing an encapsulin shell protein nucleic acid sequence; providing an RNA binding protein nucleic acid sequence; fusing the RNA binding protein nucleic acid sequence to the encapsulin shell protein nucleic acid sequence to generate an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; inserting the RNA binding protein encapsulin shell protein nucleic acid fusion sequence into a first vector under control of a first promoter to generate a first plasmid; providing an RNA sequence that binds the RNA binding protein; providing a specific target RNA sequence of interest; fusing the specific target RNA sequence of interest to the RNA sequence that binds the RNA binding protein to generate a specific target RNA sequence of interest RNA sequence that binds the RNA binding protein fusion sequence; inserting the specific target RNA sequence of interest_RNA sequence that binds the RNA binding protein fusion
  • the method comprises an encapsulin shell protein nucleic acid sequence that is a Myxococcus xanthus (MxT3), a Thermotaga maritima (TmTl) or a Quasibacillus thermotolerans (QtT4) encapsulin shell protein nucleic acid sequence.
  • MxT3 Myxococcus xanthus
  • TmTl Thermotaga maritima
  • QtT4 Quasibacillus thermotolerans
  • the RNA binding protein nucleic acid sequence is Escherichia A bacteriophage antiterminator protein N peptide (AN) nucleic acid sequence.
  • the first vector is a pETDuet vector.
  • the first promoter is an inducible T7 promoter.
  • the RNA binding sequence that binds the RNA binding protein is a BoxB RNA sequence.
  • the second vector is a pCDFDuet vector.
  • the second promoter is an inducible P70 promoter.
  • the bacterium is Escherichia coli BL21(DE3).
  • the cotransforming is electroporation co-transforming.
  • the method comprises isolating the encapsulin comprising said specific target RNA sequence of interest.
  • the method comprises purifying the isolated encapsulin comprising the specific target RNA sequence of interest.
  • the method comprises detecting and/or monitoring at least one property of the encapsulin comprising the specific target RNA sequence of interest wherein the at least one property comprises at least one of nucleic acid sequencing, RNA sequencing, tissue sectioning, immunohistochemistry, optical detection, light intensity detection optical imaging, microscopy, photography and videography.
  • the present invention provides a composition comprising a cotransformed bacterium wherein said co-transformed bacterium is generated by a method comprising: providing an encapsulin shell protein nucleic acid sequence; providing an RNA binding protein nucleic acid sequence; fusing the RNA binding protein nucleic acid sequence to the encapsulin shell protein nucleic acid sequence to generate an RNA binding protein encapsulin shell protein nucleic acid fusion sequence; inserting the RNA binding protein encapsulin shell protein nucleic acid fusion sequence into a first vector under control of a first promoter to generate a first plasmid; providing an RNA sequence that binds the RNA binding protein; providing a specific target RNA sequence of interest; fusing the specific target RNA sequence of interest to the RNA sequence that binds the RNA binding protein to generate a specific target RNA sequence of interest RNA sequence that binds the RNA binding protein fusion sequence; inserting the specific target RNA sequence of interest RNA sequence that binds the RNA
  • the present invention provides a system, comprising a first plasmid comprising an RNA binding protein encapsulin shell protein nucleic acid fusion sequence in a first vector under control of a first promoter; a second plasmid comprising an RNA target sequence of interest_RNA binding sequence that binds an RNA binding protein fusion sequence in a second vector under control of a second promoter; and a bacterium comprising the first plasmid and the second plasmid.
  • the present invention provides a kit, comprising: an encapsulin shell protein nucleic acid sequence; an RNA binding protein nucleic acid sequence; an RNA sequence that binds the RNA binding protein; a first plasmid comprising a first vector under control of a first promoter; a second plasmid comprising a second vector under control of a second promoter; and a bacterium.
  • the present invention provides an encapsulin comprising a specific target RNA sequence of interest generated by a method of the present claims.
  • the present invention provides use of an encapsulin comprising a specific target RNA sequence of interest generated by a method of the present claims.
  • the present invention provides a cell comprising an encapsulin, the encapsulin comprising a protein/RNA complex that selectively encapsulates a specific RNA of interest into the encapsulin.
  • the protein/RNA complex comprises an Escherichia X bacteriophage antiterminator protein N peptide (XN) and a BoxB RNA sequence.
  • Figure 1 shows a schematic diagram of an engineered dual cargo-loaded protein nanocage comprising RNA and protein.
  • Figure 2A shows size exclusion chromatography of XN_MxT3_Brocolli (Superose 6).
  • Figure 2B shows size exclusion chromatography of XN_MxT3_BoxB-Brocolli (Superose 6).
  • Figure 2C shows SDS-PAGE analysis of purified ZN_MxT3_ BoxB Broccoli/Broccoli.
  • Figure 3 A shows negative stain electron microscopy of XN_MxT3_BoxB-Brocolli encapsulins. Scale bar: 50 nm.
  • Figure 3B shows negative stain electron microscopy of XN_MxT3_Brocolli. Scale bar: 50 nm.
  • Figures 4A and 4B show Broccoli fluorescence of cell lysate (Fig. 4A) and purified encapsulin (Fig. 4B).
  • FIG. 5 shows a TBE urea gel (6%) run with RNA extraction from purified XN_MxT3_ BoxB Broccoli/Broccoli. BoxB Broccoli: 123 bp (without T7 terminator: 76 bp) Broccoli: 104 bp (without T7 terminator: 57bp).
  • Figure 6 shows next generation sequencing of RNA extracted from purified XN_MxT3_ BoxB Broccoli/Broccoli
  • Figure 7 show next generation sequencing of RNA extracted from XN_MxT3_ Broccoli.
  • Figure 8 shows RNA-binding peptides/ aptamer combinations and RNA -sequences.
  • the term “or” is an inclusive “or” operator and is equivalent to the term “and/or” unless the context clearly dictates otherwise.
  • the term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise.
  • the meaning of “a,” “an,” and “the” include plural references.
  • the meaning of “in” includes “in” and “on.”
  • one or more refers to a number higher than one.
  • the term “one or more” encompasses any of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or an even greater number.
  • the higher number can be 10,000, 1,000, 100, 50, etc.
  • the higher number can be approximately 50 (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2).
  • nucleic acid or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA.
  • Nucleic acids include, without limitation, single- and double-stranded nucleic acids.
  • nucleic acid also includes DNA as described above that contains one or more modified bases.
  • nucleic acid DNA with a backbone modified for stability or for other reasons is a “nucleic acid.”
  • nucleic acid as it is used herein embraces such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells.
  • oligonucleotide or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
  • Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine.
  • Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.
  • the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor.
  • a functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained.
  • portion when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a “gene” may comprise fragments of the gene or the entire gene.
  • the term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full- length mRNA (e.g., comprising coding, regulatory, structural and other sequences).
  • the sequences that are located 5' of the coding region and that are present on the mRNA are referred to as 5' non-translated or untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' nontranslated or 3' untranslated sequences.
  • genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and polyadenylation.
  • primer refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method.
  • probe refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest.
  • a probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular nucleic acid sequences (e.g., a “capture probe”).
  • any probe used in the embodiments of the present disclosure may, in some embodiments, be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the various embodiments of the present disclosure be limited to any particular detection system or label.
  • kits refers to any delivery system for delivering materials.
  • delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • reaction reagents e.g., oligonucleotides, enzymes, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • fragment kit refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
  • fragment kit is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.”
  • a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
  • kit includes both fragmented and combined kits.
  • the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.).
  • the term “information related to a subject” refers to facts or data pertaining to a subject e.g., a human, plant, or animal).
  • the term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc.
  • compositions, methods, systems and kits for generation of protein nanocompartment encapsulins for concurrent RNA and protein encapsulation within bacterial cells are provided herein.
  • reagents and methodologies for generation and use of encapsulins providing simultaneous in vivo packaging of specific RNAs and specific proteins of interest are described below.
  • Protein nanocages are biocompatible with a shell-like structure that supports generation of multifunctional and atomically defined nano-devices by modifying both their inner and outer surfaces through genetic manipulation followed by expression, purification and scale-up.
  • encapsulins provide an engineering platform for applications in medicine, catalysis, and nanotechnology.
  • Encapsulins are self-assembling protein compartments composed of a type of shell protomer possessing the HK97 phage-like fold. They are able to assemble into Tl, T3 and T4 shells that are widely distributed throughout bacterial and archaeal domains.
  • a key feature of encapsulins is the capacity to selectively encapsulate dedicated cargo proteins in vivo.
  • Native cargo proteins may comprise N- or C-terminal domains or targeting peptides (TPs) for efficient cargo loading during shell self-assembly.
  • TPs targeting peptides
  • This feature may be used to package nonnative cargo proteins into the encapsulin shell via simple genetic fusion of TPs to proteins of interest.
  • Engineered encapsulins have utilites as nanoreactors, drug delivery systems, imaging agents and immunotherapies. Genetic and chemical shell modification supports small-molecule conjugation, peptide loop insertion, pore modification and fusion of protein domains to the N- and C-terminus of the encapsulin protomer.
  • Encapsulins capable of triggered reversible disassembly for in vitro cargo loading and stimulus-responsive cargo release have been generated.
  • Encapsulation of nucleic acids in vivo provides technologies for RNA regulation and cytosolic sampling, and RNA- and DNA-based therapeutics.
  • present technologies are limited by pharmacokinetic properties, difficulty in overcoming cell membranes, susceptibility to nucleases, inherent immunogenicity and rapid clearance from the body.
  • Due to desirable properties and engineerability encapsulins provide an alternate strategy for nucleic acid packaging and delivery.
  • nucleic acid encapsulation in encapsulins supports concurrent sequestration and colocalization of specific proteins and specific nucleic acids for codelivery of distinct types of functional macromolecules acting in either an orthogonal or a synergistic manner.
  • the present invention provides encapsulins as nanoencapsulation platforms for simultaneous specific RNA and specific protein packaging.
  • the present invention provides an encapsulin shell protein MxT3 genetically fused to the kN peptide resulting in kN being displayed on the interior of the assembled protein shell.
  • kN specifically binds a short RNA target sequence termed BoxB.
  • BoxB may be genetically linked to a target RNA of interest to generate efficient self-assembly of target RNA-loaded MxT3 upon co-expression in A. coli.
  • the present invention provides 2 types of specific therapeutic macromolecules ie., a specific RNA macromolecule and a specific protein macromolecule in a single nanocage.
  • codelivery of a specific RNA and aspecific protein may comprise any combination of therapeutic RNAs and proteins of interest to cells to target, for example, multiple intracellular target classes at the same time.
  • specific siRNA and antibodies are combined against the same target in a single nanocagebased delivery vehicle for improved suppression due to dual action at both the mRNA and protein level.
  • release of cargo from protein nanocages is promoted through inducing disassembly of the nanocage.
  • RNA for example specific tRNA
  • high cytosolic concentrations of specific RNA competes for Dps-N binding thereby liberating the delivered RNA cargo.
  • low pH in the late endosome promotes release of encapsulated nucleic acids.
  • copackaging of a specific RNA and a specific protein in a single nanocage provides codelivery to each target cell.
  • concurrent administration of the same therapeutics via separate delivery methods generates heterogeneous populations of singly and doubly targeted cells.
  • encapsulin-based protein cages i.e., Dps Encs
  • Dps Encs encapsulin-based protein cages
  • Dps Encs protect encapsulated specific RNAs from nuclease digestion.
  • the present invention comprises different sizes of Dps Encs, for example, MxTl (18 nm, luminal volume: ⁇ 905 nm 3 ), TmTl (24 nm, ⁇ 3054 nm 3 ), MxT3 (32 nm, ⁇ 9203 nm 3 ), and QtT4 (42 nm, ⁇ 24,429 nm 3 ) spanning over an order of magnitude in correlation with the specific RNA loading capacity per shell.
  • Dps_MxT3 encapsulins are capable of colocalizing and protecting 2 specific functional RNAs, e.g., the split aptamer Split Broccoli (SB).
  • the SB binding partner DFHBI-1T may access the shell interior. In some embodiments, access is via the 5-, 3-, or 2-fold pores natively present in MxT3. In some embodiments, Dps_MxT3 specifically sequesters a TP -tagged co-expressed cargo protein while simultaneously packaging a specific RNA.
  • the present invention provides encapsulin-based Dps Encs that co-package specific RNA and specific proteins in vivo in a single step.
  • in situ assembly of functional nanocages simplifies purification, and avoids nonphy si ologi cal in vitro conditions often required for disassembly and cargo loading of other protein nanocages.
  • the intrinsic specificity of encapsulins of the present invention provide methods for packaging coexpressed TP -tagged proteins that assemble into highly homogeneous cargo-loaded cages with minimal nonspecific loading.
  • after purification in vivo eGFP-loaded Dps_MxT3 of the present invention comprise minimal background of non-TP -tagged proteins.
  • encapsulin-based nanocage designs comprising Dps-N are replaced with RNA-binding peptides and/or domains that bind RNA in a sequence-specific manner.
  • functional specific RNAs are tagged with a packaging RNA sequence to generate sequence-selective in vivo specific RNA loading.
  • protein nanocages loaded in vivo find use as living therapeutics.
  • living therapeutics engineered bacteria are used as a drug delivery modality to reach a target site of interest. At the target, bioactive molecules are continuously produced locally by the bacteria to increase therapeutic effects with minimal systemic side effects.
  • nanocage systems that do not require in vitro assembly are locally assembled in vivo and released.
  • Dps Encs comprise cell targeting generated by genetic fusion of cell penetrating peptides or targeting systems to the encapsulin C-terminus exposed on the shell exterior.
  • the Dps Encs provide encapsulins for codelivery of specific therapeutic RNAs and specific proteins that provide homogeneous synergistic effects at a single cell level.
  • the present invention provides co-delivery of proteinaceous bio-PROTAC effectors and siRNA that simultaneously target one or more specific diseasecausing genes at the mRNA and protein level.
  • the target comprises antiviral therapy, anticancer therapy, and/or treatment of metabolic diseases.
  • the present invention comprises co-delivery of Argonaute 2 (Ago2) and siRNA to promote siRNA efficiency.
  • the present invention comprises codelivery of self-replicating RNA encoding a non-native immunogenic epitope for use as a cancer immunotherapy.
  • the present invention is combined with, or provided as, living therapeutics to target tumor cells.
  • the present invention comprises co-delivery of mRNA-based vaccines and protein-based adjuvants.
  • Broccoli is an RNA aptamer able to specifically bind the fluorogenic small molecule DFHBI-1T used as a read-out for specific RNA-loading.
  • the XN_MxT3 gene was inserted into the pETDuet vector under the control of an IPTG inducible T7 promoter.
  • For BoxB Broccoli/Broccoli transcription either BoxB Broccoli or Broccoli was inserted into the pCDFDuet vector under the control of the constitutive P70 promoter.
  • the resulting plasmids were used to cotransformed A. coll BL21(DE3).
  • RNAs BoxB Broccoli/Broccoli
  • XN_MxT3 expression 250 mL of fresh ZYM-5052 auto-inducing medium containing appropriate antibiotics was inoculated 1 : 1000 using a 5 mL overnight culture and grown at 30°C for 24 hours. Cells were harvested via centrifugation (5,000 g, 12 min, 4°C).
  • cell pellets were suspended in 5 mL/g (wet cell mass) of Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.5). Lysis components [lysozyme (0.5 mg/mL), Benzonase nuclease (25 units/mL), MgCh (1.5 mM), and SIGMAFAST EDTA-free protease inhibitor cocktail (one tablet per 100 mL)] were added, and cells were incubated on ice for 15 min. Samples were then sonicated at 60% amplitude and a pulse time of 10 s on and 20 s off for 5 min total (Model 120 Sonic Dismembrator, Fisher Scientific).
  • samples were clarified by centrifugation (10,000 g, 15 min, 4 °C). To the supernatant, NaCl and PEG-8000 were added to a final concentration of 0.5 M and 10%, respectively, and incubated on ice for 50 min, followed by centrifugation (8,000 g, 10 min, 4 °C). The supernatant was removed, and the pellet was resuspended in 3 mL of Tris buffer (pH 7.5) and filtered using a 0.2 pm syringe filter. The filtered sample was subjected to SEC using a Sephacryl S-500 16/60 column and Tris buffer (pH 7.5) at a flow rate of 1 mL/min.
  • Broccoli fluorescence was measured using purified ZN_MxT3_BoxB_Broccoli and XN_MxT3_Broccoli.
  • 3 pL of DFHBI-1T final concentration: 300 pM
  • 75 pL of Tris buffer pH 7.5
  • Phenol:chloroform:isoamyl alcohol 25:24: 1, pH 8) was used for phenol-chloroform extraction, and after ethanol precipitation, the desalted nucleic acid extracts were dissolved in TEN buffer (Tris 10 mM, EDTA 1 mM, pH 8) and stored at -80 °C. Quantification of RNA was carried out using a Nanodrop Spectrophotometer from ThermoFisher Scientific, Inc. (USA).
  • Dps Encs Dps-N-fused encapsulins
  • IDT Integrated DNA Technologies
  • SB Split Broccoli
  • Top and Bottom of SB separated with a 270 bp spacer were inserted into a single pCDFDuet-1 vector.
  • E. coli BL21 (DE3) cells were transformed with the assembled plasmids via electroporation and were confirmed through Sanger sequencing (Eurofins Scientific).
  • LB lysogeny broth
  • pETDuet-1 100 mg/mL ampicillin
  • pCDFDuet-1 50 mg/mL spectinomycin
  • 500 mL of fresh LB medium was inoculated 1 : 100 using a 5 mL overnight culture, grown at 37 °C to OD600 of 0.4-0.5, and then induced with 0.1 mM IPTG. After induction, cultures were grown at 30 °C overnight for ca. 18 h and harvested via centrifugation (8000 g, 10 min, 4 °C). The resulting cell pellets were frozen and stored at -20 °C until further use.
  • Frozen cell pellets were resuspended in 5 mL/g (wet cell mass) of Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.5). Lysis components [lysozyme (0.5 mg/mL), Benzonase nuclease (25 units/mL), MgCh (1.5 mM), and SIGMAFAST EDTA-free protease inhibitor cocktail (one tablet per 100 mL)] were added, and cells were incubated on ice for 15 min. Samples were then sonicated at 55% amplitude and a pulse time of 10 s on and 20 s off for 5 min total (Model 120 Sonic Dismembrator, Fisher Scientific).
  • samples were clarified by centrifugation (10,000 g, 15 min, 4 °C). NaCl and PEG-8000 were added to the supernatant to a final concentration of 0.5 M and 10%, respectively, and incubated on ice for 40 min, followed by centrifugation (8000 g, 10 min, 4 °C). The supernatant was removed, and the pellet was resuspended in 3 mL of Tris buffer (pH 7.5) and filtered using a 0.2 pm syringe filter.
  • the filtered sample was subjected to SEC using a Sephacryl S-500 16/60 column and Tris buffer (pH 7.5) at a flow rate of 1 mL/min. Fractions were evaluated using SDS-PAGE and encapsulin-containing fractions were combined, concentrated, and dialyzed using Amicon filter units (100 kDa MWCO) and Tris buffer without NaCl (20 mM Tris, pH 7.5). The low salt sample was then loaded on a HiPrep DEAE FF 16/10 Ion Exchange column at a flow rate of 3 mL/min to remove nucleic acid contamination.
  • Encapsulin-containing fractions were concentrated, centrifuged (10,000 g, 10 min, 4 °C), and then subjected to SEC using a Superose 6 10/300 GL column and Tris buffer (pH 7.5) at a flow rate of 0.5 mL/min. Purified proteins were stored in Tris buffer (pH 7.5) at 4 °C until further use.
  • Encapsulin samples for negative-stain TEM were diluted to 0.15 mg/mL in Tris buffer (pH 7.5).
  • Gold grids 200-mesh coated with a Formvar-carbon film, EMS) were made hydrophilic by glow discharge at 5 mA for 60 s (easiGlow, PELCO). 4 pL of sample was added to the grid and incubated for 1 min, wicked with filter paper, and washed with 0.75% uranyl formate before staining with 0.75% uranyl formate for 1 min. Stain was removed using filter paper, and the grid was dried for at least 20 min before imaging.
  • TEM micrographs were captured using a Morgagni transmission electron microscope at 100 keV at the University of Michigan Life Sciences Institute.
  • the desalted nucleic acid extracts were dissolved in TEN buffer (Tris 10 mM, EDTA 1 mM, pH 8) and stored at -80 °C.
  • E. coli total RNA was purchased from ThermoFisher Scientific (AM7940). Quantification of RNA was conducted using a Nanodrop Spectrophotometer from ThermoFisher Scientific, Inc. (USA).
  • DNase ThermoFisher Scientific, EN0521
  • RNase ThermoFisher Scientific, EN0531
  • Benzonase Sigma Aldrich, E8263
  • 1 pL (1.5 pL) of DNase, RNase, and Benzonase was added to 9 pL (13.5 pL) of extracted RNA (RNA-loaded Dps_Encs) samples (final concentration: 10, 5, and 25 U/mL, respectively), followed by 30 min incubation at 37 °C.
  • 3% native agarose gels were used to determine the nucleic acid encapsulation capacity of Dps Encs and to demonstrate the nuclease resistance of Dps Encs shell.
  • U TAE buffer was used to make agarose gels. The amount of Dps Enc loaded per lane was adjusted for each Dps Enc encapsulin so as to visualize nucleic acid signal after GelRed staining, while corresponding Nat Encs were loaded at equal amounts for direct comparison.
  • Per lane 15 pL of sample was loaded with an additional 2 pL of 70% (v/v) aqueous glycerol. Gel electrophoresis was carried out using 1 x TAE buffer at a constant voltage of 90 V for 35 min.
  • 2% native agarose gels were used for nucleic acid extracted from purified Dps Encs. The extracted nucleic acid was incubated with nucleases and loaded on the gels along with undigested nucleic acid for comparison. Per lane, 10 pL of sample was loaded with an additional 10 pL of 2 RNA loading buffer. Gel electrophoresis was carried out in 1 x TAE buffer at a constant voltage of 125 V for 25-30 min. The gel was stained with GelRed to visualize nucleic acids. Native Polyacrylamide Gel Electrophoresis
  • Native PAGE gels were run at a constant voltage of 150 V for 1 h, followed by an additional 1 h run at 250 V at 4 °C. Gels were then stained, first with GelRed for nucleic acid visualization and then with Coomassie blue for protein detection. For eGFP_MxTP_Dps_MxT3, the gel was first exposed to UV light for eGFP visualization before staining with GelRed and Coomassie blue.
  • SB fluorescence was also measured using purified Nat_MxT3, Dps_MxT3, SB + Nat_MxT3, and SB + Dps_MxT3 samples.
  • 2 pL of DFHBI-1T final concentration: 200 pM was added to 75 pL of each sample containing 5 pmol of capsid, and incubated at 37 °C for 40 min, followed by fluorescence analysis as above.
  • 75 pL of Tris buffer (pH 7.5) was used and subtracted from the fluorescence signal of each sample.
  • In-gel digestion with trypsin was performed using a robot (ProGest, DigiLab) with the following protocol: (a) washed with 25 mM ammonium bicarbonate, followed by acetonitrile, (b) reduced with 10 mM dithiothreitol at 60 °C, (c) alkylated with 50 mM iodoacetamide at RT, (d) digested with sequencing grade trypsin (Promega) at 37 °C for 4 h, and (e) quenched with formic acid, and the supernatant was analyzed directly without further processing.
  • Dps-N consists of the 13 N-terminal residues of Dps and includes 3 positively charged lysines and is able to bind to both DNA and RNA.
  • binding arise from the electrostatic interaction of the positively charged lysine residues with the negatively charged DNA/RNA phosphate backbone.
  • Dps-N fusion constructs provide broad specificity in use as a nucleic acid-binding peptide.
  • the N-termini of all protomers are pointed toward the shell interior.
  • engineered Dps Encs 3 additional positive charges per protomer were introduced to the encapsulin lumen resulting in overall charge increases of +180 (Tl), +540 (T3), and +720 (T4) fusion constructs. This increased positive charge of the shell interior serves to five the encapsulation of RNA during shell self-assembly.
  • Dps Encs and unmodified native Tm, Mx, and Qt controls were produced in E. coli and purified through a combination of polyethylene glycol (PEG) precipitation, ion exchange chromatography (IEC), and size-exclusion chromatography (SEC). SDS-PAGE analysis of purified Dps Encs and Nat Encs was used to confirm sample homogeneity. Further analyses using negative-stain transmission electron microscopy (TEM), dynamic light scattering (DLS), and analytical SEC indicated that all Dps Encs formed stable shells with similar size and appearance compared to the corresponding Nat Encs.
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • analytical SEC indicated that all Dps Encs formed stable shells with similar size and appearance compared to the corresponding Nat Encs.
  • Dps MxTl and Dps_MxT3 have the highest relative nucleic acid packaging capacity with 20- and 11 -fold increases in signal when compared to their native forms.
  • Dps TmTl and Dps_QtT4 yield moderate signal increases of 2.4- and 4-fold, respectively.
  • Encapsulin shells possess small pores at the 5-, 3-, and 2-fold symmetry axes with diameters ranging from 2 to 7 A that is too small to allow nuclease access to the shell interior. Once formed encapsulin shells are stable may only be disassembled under harsh nonphy si ologi cal conditions, thus making them suitablecontainers for protecting labile nucleic acids.
  • Dps Encs encapsulate specific RNAs in a size- selective manner within a relevant size range for, for example, delivery of specific siRNAs between 20 and 25 nt in length. Specific RNA packaging capacity per shell increases with shell diameter. Larger Dps Encs provide larger volumes for RNA packaging and contain more Dps-N-fused protomers that result in an increased number of positive luminal charges.
  • RNAs are copackaged at the same time and protected from nucleases.
  • the split fluorogenic aptamer Split Broccoli (SB) was used to coexpress its two RNAs— Top (97 nt) and Bottom (153 nt)— together with Dps_MxT3 Dps_MxT3 was used due to its a high upper size limit for RNA, low background, and high loading capacity.
  • Benzonase was added to cleared cell lysates from cells expressing SB alone, SB + Nat_MxT3, or SB + Dps_MxT3 to remove free SB, to test the protective role of encapsulin shells, and to allow the detection of encapsulated SB via addition of the smallmolecule SB binding partner DFHBI-1T, thereby yielding a fluorescence readout.
  • the highest SB fluorescence signal was observed for SB + Dps_MxT3, indicating that Dps_MxT3 packages both SB RNAs, protected them from nuclease digestion, and allows access of the small-molecule DFHBI-1T to the shell interior.
  • Nat_MxT3 and Dps_MxT3 were purified alone or from cells coexpressing SB, followed by incubation with DFHBI-1T to generate higher SB fluorescence signals for Dps_MxT3, confirming our initial results.
  • Dps Encs to encapsulate RNA was combined with encapsulins’ native capacity for specific protein encapsulation.
  • the Mx targeting peptide (MxTP, PEKRLTVGSLRR) with a flexible six-residue linker (GGSGGS) was genetically fused to the C -terminus of eGFP and cloned immediately upstream of the Dps_MxT3 gene for coexpression SDS-PAGE analysis of purified Dps_MxT3 confirmed the successful in vivo loading and copurification of MxTP -tagged eGFP. Protein cargo loading for Dps_MxT3 was comparable with Nat_MxT3.
  • Negative-stain TEM analysis confirmed that eGFP-loaded Dps_MxT3 particles form homogeneous shells similar in size and appearance to Nat_MxT3.
  • Concurrent loading of both eGFP and RNA with native PAGE analysis on purified eGFP- loaded Dps_MxT3 shells confirms that Nat_MxT3, eGFP-loaded Dps_MxT3 exhibit higher RNA signal intensity and eGFP fluorescence in the high-molecular-weight encapsulin band.
  • Coelution of RNA and eGFP signals confirmsl copackaging of RNA and a specific heterologously expressed protein in vivo.
  • Encapsulin-containing fractions of the present invention were concentrated, centrifuged (10,000 g, 10 min, 4 °C), and then subjected to size exclusion chromatography (SEC) using a Superose 6 10/300 GL column and Tris buffer (pH 7.5) at a flow rate of 0.5 mL/min.
  • SEC size exclusion chromatography
  • XN_MxT3_Brocolli Superose 6
  • Figure 3 shows negative stain electron microscopy of XN_MxT3_BoxB-Brocolli encapsulins (3 A) and XN_MxT3_Brocolli (3B).
  • Encapsulin samples for negative-stain TEM were diluted to 0.15 mg/mL in Tris buffer (pH 7.5).
  • Gold grids 200-mesh coated with a Formvar-carbon film, EMS) were made hydrophilic by glow discharge at 5 mA for 60 s. 4 pL of sample was added to the grid and incubated for 1 min, wicked with filter paper, and washed with 0.75% uranyl formate before staining with 0.75% uranyl formate for 1 min.
  • FIG. 3 A) shows BoxB Broccoli-loaded XN_MxT3 shells highlighting their homogeneity and structural integrity.
  • Figure 3B) shows XN_MxT3 shells without loaded Broccoli.
  • Figure 4A shows Broccoli fluorescence of cell lysate. More fluorescence based on DFHBI-IT-Broccoli binding is observed for XN_MxT3_BoxB_Broccoli-expressing cells after nuclease treatment. These results indicate that Broccoli is more protected from digestion in this sample due to more efficient loading arising from the presence of BoxB.
  • Figure 4B shows Broccoli fluorescence of purified encapsuling. Greater fluorescence based on DFHBI- IT-Broccoli binding is observed for the XN_MxT3_BoxB_Broccoli sample. These results indicate that more Broccoli co-purifies with the sample due to the improved specific loading of ZN_MxT3 with the co-transcribed BoxB Broccoli RNA.
  • Figure 5 shows a TBE urea gel (6%) with RNA extraction from purified ZN_MxT3_ BoxB Broccoli/Broccoli.
  • RNA extracted from purified ZN_MxT3_BoxB_Broccoli encapsulins contains more target RNA compared to the negative control with BoxB fused to the target RNA.
  • Figure 7 shows next generation sequencing of RNA extracted from +N_MxT3_ Broccoli. No target RNA was detected in the negative control lacking BoxB.
  • Figure 8 shows alternate RNA-binding peptides/aptamer combinations and RNA- sequences in some embodiments of the present invention.

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Abstract

L'invention concerne des compositions, des méthodes, des systèmes et des kits destinés à la génération d'encapsulines de nano-compartiment protéique pour une encapsulation d'ARN et de protéine simultanées dans des cellules bactériennes. En particulier, l'invention concerne des réactifs et des méthodologies destinés à la génération et à l'utilisation d'encapsulines fournissant une encapsidation in vivo simultanée d'ARN spécifiques et de protéines d'intérêt spécifiques.
PCT/US2025/017490 2024-02-28 2025-02-27 Nano-compartiments bactériens basés sur des protéines Pending WO2025184273A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210213139A1 (en) * 2016-10-03 2021-07-15 President And Fellows Of Harvard College Delivery of therapeutic rnas via arrdc1-mediated microvesicles
US20220213467A1 (en) * 2019-09-25 2022-07-07 Yeda Research And Development Co. Ltd. Assembly of protein complexes on a chip
US20220411785A1 (en) * 2019-11-15 2022-12-29 The University Of Tokyo Library of barcoded extracellular vesicles

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210213139A1 (en) * 2016-10-03 2021-07-15 President And Fellows Of Harvard College Delivery of therapeutic rnas via arrdc1-mediated microvesicles
US20220213467A1 (en) * 2019-09-25 2022-07-07 Yeda Research And Development Co. Ltd. Assembly of protein complexes on a chip
US20220411785A1 (en) * 2019-11-15 2022-12-29 The University Of Tokyo Library of barcoded extracellular vesicles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KWON ET AL.: "Engineered Protein Nanocages for Concurrent RNA and Protein Packaging In Vivo", ACS SYNTHETIC BIOLOGY, vol. 11, no. 10, 21 October 2022 (2022-10-21), pages 3504 - 3515, XP093230286, DOI: 10.1021/acssynbio.2c00391 *

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