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WO2024235034A1 - Plate-forme de criblage à haut rendement de véhicules d'administration pour l'administration in vivo - Google Patents

Plate-forme de criblage à haut rendement de véhicules d'administration pour l'administration in vivo Download PDF

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Publication number
WO2024235034A1
WO2024235034A1 PCT/CN2024/091283 CN2024091283W WO2024235034A1 WO 2024235034 A1 WO2024235034 A1 WO 2024235034A1 CN 2024091283 W CN2024091283 W CN 2024091283W WO 2024235034 A1 WO2024235034 A1 WO 2024235034A1
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nucleic acid
delivery
composition
barcode
rna
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Bang Wang
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Genax Bio Ltd
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Genax Bio Ltd
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Priority to CN202480032192.6A priority Critical patent/CN121100187A/zh
Publication of WO2024235034A1 publication Critical patent/WO2024235034A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • 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
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • Nanoparticle libraries consisting of hundreds to thousands of LNPs, can be screened in vitro. This process is only efficient ifit predicts in vivo (in a living animal) delivery. In vivo mRNA delivery is affected by pulsatile blood flow, heterogenous vasculature, and clearance by the kidney, spleen, liver, lymphatics, and immune system. Barcoding technologies have quantified LNP biodistribution, which is necessary, but not sufficient, for cytoplasmic nucleic acid delivery. And the genes that alter whether the nanoparticle escapes into the endosome are likely to vary with each cell type. As a result, it is not possible to predict functional delivery of drug into the cytoplasm or nucleus by measuring biodistribution. Current technologies like gene editing require cargo to function at the genomic level, yet existing screening methods struggle to accurately reflect the optimal delivery strategy for the ultimate goal. Moreover, current methods impose significant restrictions on the types of delivery vehicles that can be screened, with many being incompatible with these methods.
  • a method is needed to characterize and screen delivery vehicles that exhibit the desired tropism and deliver functional cargo to specific cells or tissues, and this method should be applicable to a wide variety of delivery systems.
  • This disclosure provides a method for characterizing delivery vehicles for in vivo delivery of an agent comprising:
  • each different delivery vehicle comprises: a) one or more biologically active agents, and b) one or more identifiers that identify the composition of the delivery vehicle;
  • composition identifiers by counting the nanoparticle-associated barcode sequences
  • biologically active agents can edit a target nucleic acid by performing biological activity when delivered to the cells in vitro or delivered to the cell of the subject.
  • the target nucleic acid is in the genome. In some embodiments, the target nucleic acid is a DNA. In some embodiments, the target nucleic acid is an RNA.
  • the method further comprises isolating genomic DNA of tissues or cells and amplifying the genomic fragment before identifying the identifiers.
  • the method does not comprise any steps of sorting cells.
  • the count of vehicle-associated barcode sequences is determined by sequencing barcodes.
  • the multiple delivery vehicles are administered to the cells or to the subject simultaneously.
  • the biologically active agents comprise one or more nucleic acids.
  • the nucleic acids comprise RNAs, DNAs, or the combinations thereof.
  • the biologically active agents comprise one or more RNAs.
  • the RNA component comprises a modified RNA.
  • the RNA component comprises a sequence encoding RNA-guided DNA-binding agent.
  • the RNA component comprises mRNA.
  • the RNA component comprises a Cas nuclease mRNA.
  • the RNA component comprises a Class 2 Cas nuclease mRNA.
  • the RNA component comprises a mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k nuclease.
  • the RNA component comprises one or more gRNA nucleic acids.
  • the RNA component comprises one or more Cas nuclease mRNA and one or more gRNA.
  • the RNA component comprises a Cas nuclease mRNA and a gRNA.
  • the gRNA nucleic acid is or encodes a dual-guide RNA (dgRNA) .
  • the gRNA nucleic acid is or encodes a single-guide RNA (sgRNA) .
  • the gRNA is a modified gRNA.
  • the biologically active agents comprise component capable of altering (e.g. inserting) the target site by partial or complete sequence of the identifier.
  • the biologically active agents comprise nucleases, integrases, transposases or reverse transcriptases or nucleic acid encoding such nucleases, integrases, transposases or reverse transcriptases.
  • the nucleases herein include CRISPR/Cas, ZFN, Meganucleases, TALEN.
  • the biologically active agents comprise RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA-binding agents.
  • the RNA-guided DNA-binding agents comprise Cas nuclease, preferably, the Cas nuclease is SpCas9 or SaCas9.
  • the delivery vehicles comprise lipid nanoparticles, virus derived vehicles, exosomes, extracellular contractile injection systems, bacterial derived delivery systems, cell derived delivery systems or combinations thereof.
  • the delivery vehicles comprise LNP.
  • the delivery vehicles comprise AAV or LV.
  • the delivery vehicles comprise PLTS or eCIS.
  • the delivery vehicles comprise engineered erythrocytes.
  • the delivery vehicles comprise engineered Photorhabdus virulence cassette (PVC)
  • the delivery vehicles comprise endogenous virus protein particles.
  • the identifiers comprise one or more sequences of nucleic acid barcodes.
  • the nucleic acid barcodes are designed to alter (e.g. insert into) the target genes; In some embodiments, the nucleic acid barcodes are designed to insert into the target genes; In some embodiments, the nucleic acid barcodes comprise sequence to identify the composition of the delivery vehicle.
  • the nucleic acid barcodes are nucleic acid barcode oligonucleotides. In some embodiments, the nucleic acid barcodes are RNA barcode oligonucleotides. In some embodiments, the nucleic acid barcodes are single-stranded nucleic acids. In some embodiments, the nucleic acid barcodes are double-stranded nucleic acids.
  • delivery vehicles do not contain any components unrelated to gene alteration (e.g., insertion) .
  • delivery vehicles may contain components unrelated to gene alteration (e.g., insertion) , but do not solely rely on these components for characterizing or identifying.
  • the subject is non-human mammal.
  • the non-human mammal comprises rat, mice or non-human primate.
  • the method is a high-throughput method. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 different delivery vehicles are assayed in the method. In some embodiments, greater than 60 different delivery vehicles are assayed. In some embodiments, greater than 100 different delivery vehicles are assayed. In some embodiments, greater than 200 different delivery vehicles are assayed.
  • the tissues or cells herein are from liver, lung, spleen, heart, kidney, brain, bone marrow, lymph nodes, blood, T-cells, stem cells or ovary or testis and so on.
  • the method further comprises one or more the following steps: (i) dividing the number of sequencing reads of one barcode delivered by a single vehicle by the total amount of reads from all barcodes delivered by all vehicle in a specific cell or tissue; (ii) dividing the number of sequencing reads of the same barcode (utilized in (i) ) by the total amount of reads from all barcodes of all vehicles in the non-injected vehicle pool; (iii) dividing the results from (i) by the results from (ii) .
  • this disclosure provides a composition
  • a composition comprising: one or more empty delivery vehicles, one or more nucleic acid barcodes, and one or more biologically active agents; wherein the biologically active agents can edit genome by performing a biological activity when delivered to the cells in vitro or delivered to the cell of the subject.
  • the delivery vehicle is a lipid nanoparticle.
  • the biologically active agents comprise one or more nucleic acids.
  • the nucleic acids comprise sequences encoding RNA-guided DNA-binding agent.
  • the nucleic acid comprises mRNA.
  • nucleic acid comprises a Cas nuclease mRNA.
  • the Cas nuclease mRNA comprises a Class 2 Cas nuclease mRNA.
  • the nucleic acid comprises a mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k nuclease.
  • the nucleic acid comprises a gRNA nucleic acid.
  • the nucleic acid comprises one or more Cas nuclease mRNAs and one or more gRNAs.
  • the nucleic acid comprises a Cas nuclease mRNA and a gRNA.
  • the gRNA nucleic acid is or encodes a dual-guide RNA (dgRNA) or a single-guide RNA (sgRNA) .
  • the gRNA is a modified gRNA.
  • the ionizable lipids, the molar amount of PEG lipids, the structure of PEG lipids, the molar amount of neutral lipids, the structure of neutral lipids and the molar amount of cholesterol, in the delivery vehicle are varied among the delivery vehicles.
  • this invention provides nucleic acid barcode according to the following formula: R1-R2-R3-R4-R5, wherein R1, R2, R3, R4 and R5 are independently selected from universal sequence, random nucleotide sequence, or barcode sequence.
  • R1 represents random nucleotide sequence
  • R2 represents universal sequence
  • R3 represents second random nucleotide sequence
  • R4 represents second universal sequence
  • R5 represents barcode sequence.
  • R1 represents universal sequence
  • R2 represents a random nucleotide sequence
  • R3 represents barcode sequence
  • R4 represents a second random nucleotide sequence
  • R5 represents a second universal sequence.
  • the nucleic acid barcodes are DNA barcode oligonucleotides. In some embodiments, the nucleic acid barcodes are RNA barcode oligonucleotides. In some embodiments, the nucleic acid barcodes are single-stranded nucleic acids. In some embodiments, the nucleic acid barcodes are double-stranded nucleic acids.
  • the nucleic acid barcodes are 5-100 nucleotides in length; In some embodiments, the nucleic acid barcodes are 10-70 nucleotides in length; In some embodiments, the nucleic acid barcodes are 20-60 nucleotides in length; In some embodiments, the nucleic acid barcodes are 20-50 nucleotides in length; In some embodiments, the nucleic acid barcodes are 30-50 nucleotides in length. In some embodiments, the nucleic acid barcodes are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the nucleic acid barcodes are 30, 31, 32, 33, 34, 35 nucleotides in length.
  • the random nucleotide sequence contains 0 to 20 nucleotides in length.
  • the nucleic acid barcode is modified.
  • the universal sequence is modified.
  • the random nucleotide sequence is modified.
  • the barcode sequence is modified.
  • the result of editing genome leads to the partial or complete sequence of nucleic acid barcodes being inserted into the target gene.
  • nucleic acid barcode comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%or 100%sequence identity to any one of SEQ ID NOs: 1-341.
  • the nucleic acid barcode comprises a sequence having at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that are identical to any one of the sequences selected from SEQ ID NOs: 1-341.
  • Figure 1 Nucleic acid barcoded nanoparticles for high throughput in vivo nanoparticle delivery.
  • A Lipid nanoparticles (LNPs) were formulated to carry Cas9 mRNA, sgRNA and nucleic acid barcodes.
  • B Stable LNPs were pooled together and administered to rodent or non-human primate. Cells or tissues were deep sequenced to quantify the relative delivery of all the LNPs simultaneously.
  • Figure 2 (Fig 2) : The indel frequency in HepG2 using different doses of Lipofectamine RNAiMAX and same doses of barcode.
  • Figure 3 (Fig 3) : The normalized barcode readouts in HepG2 using different doses of Lipofectamine RNAiMAX and same doses of barcode.
  • Figure 4 (Fig 4) : Correlation analysis between normalized barcode readouts and lipofectamine dose.
  • Figure 5 (Fig 5) : The indel frequency in HepG2 using same dose of Lipofectamine RNAiMAX and different doses of barcode.
  • Figure 6 (Fig 6) : The normalized barcode readouts in HepG2 using same dose of Lipofectamine RNAiMAX and different doses of barcode.
  • Figure 7 (Fig 7) : Correlation analysis between normalized barcode readouts and barcode dose.
  • Figure 8 Indel frequency biodistribution within the 5 tissues examined by normalized barcode readouts.
  • Figure 9 (Fig 9) : Heatmap of normalized delivery for all 68 LNPs in rat.
  • Figure 10 (Fig 10) : Typical representative at each level of LNP delivery efficiency (high, middle and low) to the liver, which were quantified by normalized barcode readouts from rats.
  • Figure 11 (Fig 11) : The barcode efficiency in HepG2 using same dose of Lipofectamine RNAiMAX and different barcodes.
  • Figure 12 (Fig 12) : The barcode efficiency in cynomolgus liver using different LNPs contained same RNA and different barcodes.
  • Figure 13 (Fig 13) : The barcode efficiency in cynomolgus liver using different LNPs contained same RNA and different lipids.
  • Figure 14 (Fig 14) : The indel frequency in cynomolgus liver using single LNP contained the lipid 32
  • Figure 15 The reduction in serum target protein concentration as a proportion of baseline in cynomolgus monkeys receiving intravenous administration of LNP100 at doses of 3.0 mg/kg (total RNA/body weight) .
  • Figure 16 The barcode efficiency in mice lung using different LNPs contained same RNA and different lipids.
  • Figure 17 (Fig 17) : The barcode efficiency in cynomolgus lung, spleen and heart using different LNPs contained same RNA and different lipids.
  • a method for characterizing delivery vehicles for delivery of an agent is disclosed in this invention. In vivo or in vitro delivery is all included in this disclosure.
  • the methods of the present invention may be used to characterize any factors that affect delivery or function of the delivery system or cargos; such as composition of the delivery system, amount, manufacturing company, composition of the delivered cargo, amount, etc.
  • the methods of the present invention are used to characterize factors influencing the delivery system; in another embodiments, the methods of the present invention are used to characterize factors affecting the activity of the cargo.
  • the method comprising:
  • each different delivery vehicle comprises: i) one or more biologically active agents, and ii) one or more identifiers that identify the composition of the delivery vehicle;
  • biologically active agents can edit a target nucleic acid by performing biological activity when delivered to the cells in vitro or delivered to the cells of the subject.
  • the method further comprises isolating genomic DNA or RNA of tissues or cells, and amplifying the genomic fragment before identifying the identifiers.
  • tissue or cell that can be used to demonstrate tissue or cell-targeting specificity in a subject is included in the disclosure.
  • the tissues or cells are from liver, lung, spleen, heart, kidney, brain, bone marrow, lymph nodes, blood, T-cells, stem cells or ovary/tesis and so on.
  • counting is performed by sequencing.
  • Vehicle-associated barcode sequences refer to sequences used to characterize the corresponding "vehicle.
  • the biologically active agents comprise an RNA-guided DNA-binding proteins, such as a CRISPR/Cas effector, or the polynucleotides encoding such proteins.
  • the identifier is designed to be a nucleic acid sequence that is capble of altering (e.g. inserting into) the target site.
  • the target site in this disclosure can be a DNA or an RNA.
  • any biologically active agents (such as nucleases, integrases, transposases, reverse transcriptases, etc. ) capable of altering the target site with the identifier are included within the present disclosure.
  • the nucleases herein include CRISPR/Cas, ZFN, Meganucleases, TALEN, etc.
  • the biologically active agents are proteins (e.g. nucleases, integrases, transposases, reverse transcriptases herein) , or polynucleotides encoding such proteins.
  • the nucleic acid barcodes or the identifiers are (or are designed to be) capable of “altering the target site/sequence” , “altering the target gene” , “inserting into the target site/sequence” , “inserting into the target gene” , or to “alter the target site/sequence” , “alter the target gene” , “insert into the target site/sequence” , “insert into the target gene” , or other similar expressions, should be understood to alter or insert into or near the target site with partial or complete sequences of such nucleic acid barcodes or identifiers.
  • the biologically active agents can edit a target nucleic acid by performing a biological activity when delivered to the cells in vitro or delivered to the cell of the subject.
  • the target nucleic acid is in the genome.
  • the “genome” refers to the complete set of genes or genetic material present in a cell, tissue or organism.
  • the genome comprises a DNA.
  • the genome comprises an RNA (e.g., an mRNA) .
  • the method further comprises one or more the following steps: (d) dividing the number of sequencing reads of one barcode delivered by a single vehicle by the total amount of reads from all barcodes delivered by all vehicle in a specific cell or tissue; (e) dividing the number of sequencing reads of the same barcode (utilized in (d) ) by the total amount of reads from all barcodes of all vehicles in the non-injected vehicle pool; (f) dividing the results from (d) by the results from (e) .
  • this quantification method the delivery of different vehicles within the same organ can be compared, but the delivery of the same vehicles across different organs cannot be compared.
  • “vehicles” , “particles” , or similar terms are used interchangeably in the present disclosure. Delivery systems having any characteristics can be characterized by the methods described in the present disclosure. For example, such characteristics comprises composition, amount, preparation process, parameters (e.g., particle size, charge, PDI etc. ) .
  • any delivery vehicles capable of delivering the cargo into a cell is within the scope of this disclosure.
  • the “empty delivery vehicles” or similar terms means the composition of the vehicles and do not indicate any relationship with other components of the vehicles. Regardless of whether they are carrying cargo or not, as far as that part of the delivery system is concerned, it can be referred to as “empty delivery vehicles. " For instance, a vehicle wrapping cargo in this disclosure could be represented as an "empty delivery vehicle” and a “Cargo. " A vehicle with cargo adsorbed onto its surface can also be represented as an “empty delivery vehicle” and a “cargo. " A vehicle with cargo separated from it can also be represented as an “empty delivery vehicle” and a “cargo. " In some embodiments, the cargo delivered can edit a target nucleic acid by performing a biological activity.
  • the delivery vehicles may be provided as particles compositions.
  • the delivery vehicles comprise lipid nanoparticles, virus derived delivery system, virus like particles, virus-like protein derived delivery system, exosomes, extracellular contractile injection systems, bacterial derived delivery system, cell derived delivery system or combinations thereof.
  • the delivery vehicles comprise LNP.
  • the delivery vehicles comprise AAV or LV.
  • the delivery vehicles comprise PLTS or eCIS.
  • the delivery vehicles comprise engineered erythrocytes.
  • the delivery vehicles comprise engineered Photorhabdus virulence cassette (PVC) ,
  • the delivery vehicles comprise endogenous virus protein particles.
  • Suitable delivery vehicles or vectors include viral delivery vehicles or vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35: 25432549, 1994; Borras et al., Gene Ther 6: 515524, 1999; Li and Davidson, PNAS 92: 77007704, 1995; Sakamoto et al., H Gene Ther 5: 10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655) ; adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9: 81 86, 1998, Flannery et al., PNAS 94: 69166921, 1997; Bennett et al., Invest Opthalmol
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94: 1031923, 1997; Takahashi et al., J Virol 73: 78127816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, mye
  • a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector.
  • a recombinant expression vector of the present disclosure is a recombinant lentivirus vector.
  • a recombinant expression vector of the present disclosure is a recombinant retroviral vector.
  • the particles may be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes" -lamellar phase lipid bilayers that, in some embodiments are substantially spherical, and, in more particular embodiments can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules) , a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • microspheres including unilamellar and multilamellar vesicles, e.g., "liposomes" -lamellar phase lipid bilayers that, in some embodiments are substantially spherical, and, in more particular embodiments can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules) , a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • the particles may be comprised of a cationic lipid or ionizable lipid (such as compound described in prior art) , cholesterol, phospholipid (such as 1, 2-Distearoylsn-glycero-3-phosphocholine) , PEG derivative (such as DMG-PEG) ; at various mol: mol ratios.
  • a cationic lipid or ionizable lipid such as compound described in prior art
  • cholesterol such as 1, 2-Distearoylsn-glycero-3-phosphocholine
  • PEG derivative such as DMG-PEG
  • the composition is an LNP composition.
  • the lipid component comprises an ionizable lipid and a PEG lipid.
  • the non-cationic lipid is a neutral lipid.
  • Neutral lipids suitable for use in a lipid composition of the invention include, for example, a variety of neutral, uncharged or zwitterionic lipids.
  • Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to: 5-heptadecylbenzene-1, 3-diol (resorcinol) , dipalmitoylphosphatidylcholine (DPPC) , distearoylphosphatidylcholine (DSPC) , phosphocholine (DOPC) , dimyristoylphosphatidylcholine (DMPC) , phosphatidylcholine (PLPC) , 1, 2-distearoyl-sn-glycero-3-phosphocholine (DAPC) , phosphatidylethanolamine (PE) , egg phosphatidylcholine (EPC) , dilauryloylphosphatidylcholine (DLPC) , dimyristoylphosphati
  • the PEG derivatives may be conjugated to one or more additional molecules, such as, a lipid.
  • the PEG derivative is selected from, but not limited to: PEG-DMG, 3-N- (-methoxy poly (ethylene glycol) 2000) carbamoyl-1, 2-dimyrisyl glycerol, PEG-CDMA, 3-N- (-methoxy poly ( ethylene glycol) 2000) carbamoyl-1, 2-dimyristyloxy-propylamine; PEG-CDSA, 3-N (-methoxy poly (ethylene glycol) 2000) carbamoyl-1, 2-distearyloxy-propylamine, DSPE-PEG, PEG-maleimide, DSPE-PEG-maleimide, or combinations thereof.
  • PEG-DMG 3-N- (-methoxy poly (ethylene glycol) 2000) carbamoyl-1, 2-dimyrisyl glycerol
  • PEG-CDMA 3-N- (-meth
  • the particles are nanoparticles.
  • the particles (including the nucleic acid encapsulated within) and the targeting moiety on the surface particles have a particle size (diameter) in the range of about 10 to about 500 nm.
  • the particles have a particle size (diameter) in the range of about 10 to about 350 nm.
  • the particles have a particle size (diameter) in the range of about 50 to about 250 nm.
  • the particles have a particle size (diameter) in the range of about 10 to about 200 nm.
  • the particles have a particle size (diameter) in the range of about 20 to about 200 nm.
  • the particles have a particle size (diameter) in the range of about 50 to about 200 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 75 to about 200 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 90 to about 200 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 100 to about 200 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 120 to about 200 nm.In some embodiments, the particles have a particle size (diameter) in the range of about 150 to about 200 nm.
  • the particles have a particle size (diameter) in the range of about 50 to about 150 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm. In some embodiments, the particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm.In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm.
  • the particles have a particle size (diameter) of over about 70 nm. In some embodiments, the particles have a particle size (diameter) of over about 80 nm. In some embodiments, the particles have a particle size (diameter) of over about 90 nm. In some embodiments, the particles have a particle size (diameter) of over about 100 nm. In some embodiments, the particles have a particle size (diameter) of over about 200 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 500 nm.
  • the particles (including the nucleic acid encapsulated within) have a particle size (diameter) in the range of about 5 to about 200 nm. In some embodiments, the particles (including the nucleic acid encapsulated within) have a particle size (diameter) in the range of about 50 to about 60 nm. In some embodiments, the particles (including the nucleic acid encapsulated within) have a particle size (diameter) in the range of about 55 to about 58 nm. In some embodiments, the size is a hydrodynamic diameter.
  • the “polydispersity index” or “PDI” is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution. In some embodiments, the polydispersity index may be less than 0.1. In some embodiments, the LNPs disclosed herein have a polydispersity index (PDI) that may range from about 0.005 to about 0.75. In some embodiments, the LNP have a PDI that may range from about 0.01 to about 0.5. In some embodiments, the LNP have a PDI that may range from about zero to about 0.4.
  • PDI polydispersity index
  • the LNP have a PDI that may range from about zero to about 0.35. In some embodiments, the LNP have a PDI that may range from about zero to about 0.35. In some embodiments, the LNP PDI may range from about zero to about 0.3. In some embodiments, the LNP have a PDI that may range from about zero to about 0.25. In some embodiments, the LNP PDI may range from about zero to about 0.2. In some embodiments, the LNP have a PDI that may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4.
  • LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution.
  • Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol.
  • the organic solvent may be 100%ethanol.
  • a pharmaceutically acceptable buffer e.g., for in vivo administration of LNPs, may be used.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0.
  • the composition has a pH ranging from about 7.2 to about 7.7.
  • the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6.
  • the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7.
  • the pH of a composition may be measured with a micro pH probe.
  • a cryoprotectant is included in the composition.
  • cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol.
  • compositions may include up to 10%cryoprotectant, such as, for example, sucrose.
  • the composition may comprise tris saline sucrose (TSS) .
  • the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%cryoprotectant.
  • the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%sucrose.
  • the LNP composition may include a buffer.
  • the buffer may comprise a phosphate buffer (PBS) , a Tris buffer, a citrate buffer, and mixtures thereof.
  • the buffer comprises NaCl.
  • the buffer lacks NaCl.
  • Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM.In some embodiments, the amount of NaCl is about 45 mM.
  • the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM.
  • the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP compositions contain 5%sucrose and 45 mM NaCl in Tris buffer.
  • compositions contain sucrose in an amount of about 5%w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5.
  • the salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall composition is maintained.
  • this invention provides one or more identifiers.
  • the identifiers are designed to alter the target genes.
  • the identifiers are designed to insert into the target genes.
  • the identifier comprises one or more sequences of nucleic acid barcodes.
  • the nucleic acid barcodes can be rationally designed to make the nucleic acid including the sequence of the barcodes successfully insert the genome.
  • the nucleic acid barcodes are designed to alter the target genes.
  • the nucleic acid barcodes are designed to insert into the target genes.
  • the insertion into the target genes is partial or complete sequence of the identifier.
  • nucleic acid barcode refers to an oligonucleotide having a nucleic acid sequence that contains a series of nucleotides ( “barcode sequence” ) unique to the barcode and optionally a series ofnucleotides common to other barcodes.
  • the common nucleotides can be used, for example, to isolate and sequence the barcode. Therefore, in some cases, the barcode sequence is flanked by upstream and/or downstream primer sites, such as, for example, universal primer sites.
  • the polynucleotide can include a DNA nucleotide, an RNA nucleotide, or a combination thereof.
  • Each delivery vehicle formulation is paired with its own unique nucleic acid barcode.
  • the unique nucleic acid barcode is paired to the chemical composition of the delivery vehicle formulation and by sequencing the nucleic acid barcode, one can identify the specific chemical composition used to produce that specific vehicle delivery formulation.
  • the nucleic acid barcode can contain 5 to 250 nucleotides in length, about 5 to about 200 nucleotides in length, about 5 to about 150 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides.
  • the nucleic acid barcodes are 5-100 nucleotides in length; In some embodiments, the nucleic acid barcodes are 10-70 nucleotides in length; In some embodiments, the nucleic acid barcodes are 20-60 nucleotides in length; In some embodiments, the nucleic acid barcodes are 20-50 nucleotides in length; In some embodiments, the nucleic acid barcodes are 30-50 nucleotides in length. In some embodiments, the nucleic acid barcodes are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the nucleic acid barcodes are 30, 31, 32, 33, 34, 35 nucleotides in length.
  • the nucleic acid barcodes can be covalently or non-covalently attached to the disclosed delivery vehicle. In some embodiments, the nucleic acid barcode is encapsulated by the delivery vehicle.
  • the random nucleotide sequence contains 0 to 20 nucleotides in length. In some embodiments, the random nucleotide sequence contains 5 to15 nucleotides in length. In some embodiments, the random nucleotide sequence contains 8 to12 nucleotides in length. In some embodiments, the random nucleotide sequence contains 10 nucleotides in length.
  • nucleic acid sequences of the barcodes are modified, In certain embodiments.
  • Modified nucleosides, nucleotides or oligonucleotide can be present in the RNA or DNA, for example the nucleic acid barcodes comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA or DNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, T, and U residues.
  • a modified nucleic acid synthesized with a non-canonical nucleoside or nucleotide here called “modified. ”
  • the both the double strands are modified. In some embodiments, only one strand of the double strands is modified.
  • Modified nucleosides or nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification) ; (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′hydroxyl on the ribose sugar (an exemplary sugar modification) ; (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification) ; (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification) ; (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification) ; (vi) modification of the 3′ end or 5′ end of the
  • the sequence comprises phosphorylation at the 5' end (5'-phospht or 5'-phosp) ; In some embodiments, the sequence comprises phosphorylation at the 3' end (3'-phospht or 3'-phosp) ; In some embodiments, the sequence comprises phosphorothioate linkage between the nucleosides or nucleotides of the sequences. In some embodiments, the sequence comprises phosphorothioate linkage at 3' end. In some embodiment, the sequence comprises phosphorothioate linkage between the last 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides or nucleotides of the sequences.
  • the sequence comprises phosphorothioate linkage between the first 1-20 nucleosides or nucleotides of the sequences; In some embodiments, the sequence comprises phosphorothioate linkage between the first 2-10 nucleosides or nucleotides of the sequences; In some embodiments, the sequence comprises phosphorothioate linkage between the first 3-8 nucleosides or nucleotides of the sequences; In some embodiments, the sequence comprises phosphorothioate linkage between the last 1-20 nucleosides or nucleotides of the sequences; In some embodiments, the sequence comprises phosphorothioate linkage between the last 2-10 nucleosides or nucleotides of the sequences; In some embodiments, the sequence comprises phosphorothioate linkage between the last 3-8 nucleosides or nucleotides of the sequences; Certain embodiments comprise phosphorylation at 5' end and linkage between the nucleosides or nucleot
  • Certain embodiments comprise phosphorylation at 5' end and 1-5 phosphorothioate linkage of the sequences. In some embodiments, the sequence comprises phosphorylation at 5' end and three phosphorothioate linkages of the sequences. In some embodiments, the sequence comprises phosphorylation at 5' end and three phosphorothioate linkages of the last four nucleotides.
  • the phosphorylation at the 5’ end (5’-phospht) or the phosphorylation at the 3’ end (3’-phospht) , can be described as followings:
  • phosphorylation is indicated by ′/phosp/′, and the position of ′/phosp/′ denotes the site of phosphorylation.
  • ′/5phosp/CGTCCAGTTACGACGTAGGTCGACGAGTCGC′ represents a phosphorylated 5′-end.
  • ′*′ represents a phosphorothioate linkage, as defined elsewhere in this disclosure, such as ′/phosp/CGTCCAGTTACGACGTAGGTCGACGAGT*C*G*C′ indicating both a 5′-end phosphorylation and phosphorothioate linkages between the last four nucleotides.
  • the modifications in this invention comprise modified phosphate groups include, phosphorylation, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp) .
  • the backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside) , with nitrogen (bridged phosphoroamidates) , sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates) .
  • the replacement can occur at either linking oxygen or at both of the linking oxygens.
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • the nucleic acid barcode in the compositions and methods disclosed herein may be used to alter (e.g., insert) a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease.
  • the methods comprise introducing a template to the cell.
  • single-stranded nucleic acid (s) or may be provided;
  • double-stranded nucleic acid (s) may be provided.
  • the nucleic acid sequence (s) to be altered or inserted is/are the target nucleic acid sequence of the genome.
  • two or more nucleic acid (s) may be provided such that editing may occur at two or more target sites.
  • different nucleic acid (s) may be provided to edit a single gene in a cell, or two different genes in a cell.
  • the nucleic acid barcode (s) may be used in homologous recombination. In some embodiments, the homologous recombination may result in the integration of the nucleic acid barcode (s) sequence or a portion of the nucleic acid (s) sequence into the target nucleic acid molecule. In other embodiments, the nucleic acid (s) may be used in homology-directed repair, which involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some embodiments, the homology-directed repair may result in including the nucleic acid (s) sequence in the edited target nucleic acid molecule.
  • the nucleic acid (s) may be used in gene editing mediated by non-homologous end joining.
  • the nucleic acid (s) sequence has no similarity to the nucleic acid sequence near the cleavage site.
  • the nucleic acid (s) or a portion of the nucleic acid (s) sequence is incorporated.
  • the nucleic acid (s) includes flanking inverted terminal repeat (ITR) sequences.
  • the barcodes may be single-strand or double-strand types. If the double-stranded types are used, the double-stranded nucleic acids consist of one strand of the barcodes and the complementary strand. If the single-strand types are used, the nucleic acids only have one strand of the barcode.
  • the examples of one strand of the barcodes used in this invention are shown in the following table 1:
  • the dosage of the identifiers is optimized to improve editing efficiency.
  • the mass ratio of the dosage of identifier to that of total RNA is 1: 5-1: 200.
  • the mass ratio of the dosage of identifier to that of total RNA is 1: 5, 1: 10, 1: 15, 1: 20, 1: 25, 1: 30, 1: 35, 1: 40, 1: 45, 1: 50, 1: 55, 1: 60, 1: 65, 1: 70, 1: 75, 1: 80, 1: 85, 1: 90, 1: 95, 1: 100, 1: 105, 1: 110, 1: 115, 1: 120, 1: 125, 1: 130, 1: 135, 1: 140, 1: 145, 1: 150, 1: 155, 1: 160, 1: 165, 1: 170, 1: 175, 1: 180, 1: 185, 1: 190, 1: 195 or 1: 200.
  • biologically active agents is used to refer to compounds or entities that alter, inhibit, activate, or otherwise affect biological or chemical events.
  • biologically active agents may be chemical entities or biological products that have therapeutic or diagnostic activity when delivered to a cell in vitro or delivered to the cell of the subject.
  • the chemical entity or biological product can be an organic or inorganic molecule.
  • the biologically active agents are modified or unmodified polynucleotide.
  • the biologically active agents are peptides or peptidomimetics.
  • the one or more biologically active agents comprise proteins.
  • the one or more biologically active agents comprise nucleic acids (e.g. mRNA, gRNA or the combinations) .
  • one or more biologically active agents comprise vector comprising a nucleic acid encoding a therapeutic or diagnostic nucleic acids or proteins.
  • the biologically active agent comprises a polypeptide, optionally in combination with a nucleic acid.
  • the biologically active agent comprises one or more nucleic acid, such as RNA (e.g. : siRNA, miRNA, shRNA, anti-sense RNA, mRNA, gRNA and the combinations or the like) .
  • the nucleic acid component comprises DNA.
  • the component, such as an RNA component may comprise an mRNA, such as an mRNA encoding an RNA-guided DNA-binding agent.
  • the RNA-guided DNA-binding agent is a Cas nuclease.
  • the biologically active agent may comprise an mRNA that encodes Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, cas12k or cas13.
  • the biologically active agent may comprise a gRNA.
  • the biologically active agent comprising an mRNA encoding an RNA-guided DNA-binding agent further comprises a gRNA nucleic acid, such as a gRNA.
  • the biologically active agent comprises an RNA-guided DNA-binding agent and a gRNA.
  • the biologically active agent comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the biologically active agent comprises a Class 2 Cas nuclease mRNA and a gRNA. In some embodiments, the biologically active agent comprises a SpCas9 or/and an SaCas9, or a nucleic acid encoding such SpCas9 or/and an SaCas9; In some embodiments, the biologically active agent further comprises a gRNA.
  • mRNA refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs) .
  • mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2'-methoxy ribose residues.
  • the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxy ribose residues, or a combination thereof.
  • mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%thymidine content) .
  • an mRNA can contain modified uridines at some or all of its uridine positions.
  • the mRNA comprises pseudouridine modifications at some or all of its uridine positions;
  • the mRNA comprises N1-methyl-pseudouridine modifications at some or all of its uridine positions.
  • unmodified nucleotides are replaced with modified nucleotides, such as using pseudouridine or N1-methyl-pseudouridine instead of uridine.
  • Cas nuclease comprises DNA-binding agents (e.g. Cas cleavases, Cas nickases, and dCas) .
  • Cas cleavases/nickases and dCas DNA-binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm 1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.
  • a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA-binding activity.
  • Class 2 Cas nucleases include Class 2 Cas cleavases/nickases (e.g., H840A, D10A, or N863A variants) , which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA-binding agents, in which cleavase/nickase activity is inactivated.
  • Class 2 Cas cleavases/nickases e.g., H840A, D10A, or N863A variants
  • Class 2 dCas DNA-binding agents in which cleavase/nickase activity is inactivated.
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants) , HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants) , eSPCas9 (1.0) (e.g, K810A, K1003A, R1060A variants) , and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof.
  • Cas9 is SpCas9 or SaCas9.
  • Guide RNAs can include modified RNAs as described herein.
  • a gRNA may be either a crRNA (also known as CRISPR RNA) , or the combination of a crRNA and a trRNA (also known as tracrRNA) .
  • the crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA) .
  • guide RNA or “gRNA” refers to each type.
  • the trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.
  • the sgRNA is a “Cas9 sgRNA” capable of mediating RNA guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a “Cpf1 sgRNA” capable of mediating RNA-guided DNA cleavage by a Cpfl protein. In certain embodiments, the gRNA comprises a crRNA and a tracrRNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient for forming an active complex with a Cpf1 protein and mediating RNA-guided DNA cleavage.
  • the delivery vehicles or the compositions, such as LNP comprise modified nucleic acids, including modified RNAs.
  • Modified nucleosides or nucleotides can be present in an RNA, for example a gRNA or mRNA.
  • a gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a modified RNA is synthesized with a non-canonical nucleoside or nucleotide, here called “modified” or “modification. ”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification) ; (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification) ; (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification) ; (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification) ; (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification) ; (vi) modification of the 3′ end or 5′ end of
  • Certain embodiments comprise a 5′ end modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a 3′ end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5′ end and 3′ end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, an mRNA includes at least one modified residue.
  • the modifications in this invention comprise modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp) .
  • the backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside) , with nitrogen (bridged phosphoroamidates) , sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates) .
  • the replacement can occur at either linking oxygen or at both of the linking oxygens.
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • modified sugars are believed to control the puckering ofnucleotide sugar rings, a physical property that influences oligonucleotide binding affinity for complementary strands, duplex formation, and interaction with nucleases. Substitutions on sugar rings can therefore alter the conformation and puckering of these sugars. For example, 2'-O-methyl (2'-OMe) modifications can increase binding affinity and nuclease stability of oligonucleotides.
  • mA mA
  • mC mU
  • mG mG
  • a “*” may be used to depict a phosphorothioate (PS) modification.
  • PS phosphorothioate
  • the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS bond.
  • mX mX
  • mN mN
  • mA mC*
  • mU* mU*
  • mG* a nucleotide that has been substituted with 2'-OMe or/and that is linked to the next (e.g., 3') nucleotide with a PS linkage, which may sometimes be referred to as a “PS bond” .
  • fX fX
  • fN fN
  • fA fC*
  • fU* fU*
  • fG* a nucleotide that has been substituted with 2'-F or/and that is linked to the next (e.g., 3') nucleotide with a PS linkage, which may sometimes be referred to as a “PS bond” .
  • pX pX
  • pA pA
  • pC* pU*
  • pG* a nucleotide that has been substituted with 2'-phosphate or/and that is linked to the next (e.g., 3') nucleotide with a PS linkage, which may sometimes be referred to as a “PS bond” .
  • psX psA
  • psC* psU*
  • psG* a nucleotide that has been substituted with 2'-phosphorothioate or/and that is linked to the next (e.g., 3') nucleotide with a PS linkage, which may sometimes be referred to as a “PS bond” .
  • PS bond a nucleotide that has been substituted with 2'-phosphorothioate or/and that is linked to the next (e.g., 3') nucleotide with a PS linkage.
  • PS bond e.g., PS bond
  • a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent, such as a Cas nuclease, or Class 2 Cas nuclease as described herein.
  • an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease is provided, used, or administered.
  • An mRNA may comprise one or more of a 5′ cap, a 5′ untranslated region (UTR) , a 3′ UTRs, and a polyadenine tail.
  • the mRNA may comprise a modified open reading frame, for example to encode a nuclear localization sequence or to use alternate codons to encode the protein.
  • the mRNA in the disclosed LNP compositions may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide or other protein of interest that is normally secreted.
  • the mRNA may optionally have chemical or biological modifications which, for example, improve the stability and/or half life of such mRNA or which improve or otherwise facilitate protein production.
  • suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the mRNA.
  • Cs cytidines
  • U′s uridines
  • RNA devoid of C and U residues have been found to be stable to most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994) ) .
  • the number of C and/or U residues in an mRNA sequence is reduced.
  • the number of C and/or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid.
  • Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridines.
  • the incorporation of pseudouridines into the mRNA nucleic acids of the present invention may enhance stability and translational capacity, as well as diminishing immunogenicity in vivo. See, e.g., Kariko, K., et al., Molecular Therapy 16 (11) : 1833-1840 (2008) .
  • Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one or ordinary skill in the art.
  • modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both the 3′ and 5′ ends of an mRNA molecule encoding a functional secreted protein or enzyme) .
  • Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail) , the alteration of the 3′ UTR or the 5′ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule) , and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures) .
  • bases e.g., the inclusion of a poly A tail or a longer poly A tail
  • an agent e.g., a protein or a complementary nucleic acid molecule
  • inclusion of elements which change the structure of an mRNA molecule e.g., which form secondary structures
  • the poly A tail is thought to stabilize natural messengers. Therefore, in one embodiment a long poly A tail can be added to an mRNA molecule thus rendering the mRNA more stable.
  • Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed mRNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256) .
  • a transcription vector can also encode long poly A tails.
  • poly A tails can be added by transcription directly from PCR products. In one embodiment, the length of the poly A tail is at least about 90, 200, 300, 400 at least 500 nucleotides.
  • the length of the poly A tail is adjusted to control the stability of a modified mRNA molecule of the invention and, thus, the transcription of protein.
  • the length of the poly A tail can influence the half-life of an mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of protein expression in a cell.
  • the stabilized mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases) , such that they may be delivered to the target cell without a transfer vehicle.
  • an mRNA can be modified by the incorporation 3′ and/or 5′ untranslated (UTR) sequences which are not naturally found in the wild-type mRNA.
  • 3′ and/or 5′ flanking sequence which naturally flanks an mRNA and encodes a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein in order to modify it.
  • 3′ or 5′ sequences from mRNA molecules which are stable can be incorporated into the 3′ and/or 5′ region of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule.
  • stable e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes
  • More detailed descriptions of the mRNA modifications can be found in US2017/0210698A1, at pages 57-68, the contents of which are incorporated herein.
  • delivering means providing an entity to a destination.
  • delivering a therapeutic and/or prophylactic vehicle to a subject may involve administering a nanoparticle composition including the therapeutic and/or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route) .
  • Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.
  • contacting means establishing a physical connection between two or more entities.
  • contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and a nanoparticle are made to share a physical connection.
  • Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts.
  • contacting a nanoparticle composition and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of nanoparticle compositions.
  • routes of administration e.g., intravenous, intramuscular, intradermal, and subcutaneous
  • more than one mammalian cell may be contacted by a nanoparticle composition.
  • polynucleotide refers to a polymer of nucleotides.
  • polynucleotide refers to a polymer of nucleotides.
  • polynucleotide refers to a polymer of nucleotides.
  • oligonucleotide refers to a polymer of nucleotides.
  • a polynucleotide comprises at least two nucleotides.
  • DNAs and RNAs are polynucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) , nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, and 2-thiocytidine) , chemically modified
  • Nucleic acids also include nucleic acid-based therapeutic agents, for example, nucleic acid ligands, siRNA, short hairpin RNA, antisense oligonucleotides, ribozymes, aptamers, and SPIEGELMERSTM, oligonucleotide ligands described in Wlotzka, et al., Proc. Natl. Acad. Sci. USA, 2002, 99 (13) : 8898, the entire contents of which are incorporated herein by reference.
  • nucleic acid ligands for example, nucleic acid ligands, siRNA, short hairpin RNA, antisense oligonucleotides, ribozymes, aptamers, and SPIEGELMERSTM, oligonucleotide ligands described in Wlotzka, et al., Proc. Natl. Acad. Sci. USA, 2002, 99 (13) : 8898, the entire contents of which are incorporated herein by reference
  • Nucleic acids can also include nucleotide analogs (e.g., BrdU) , and non-phosphodiester intemucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages) .
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal and particularly a human.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • nucleic acid barcodes were prepared by modified oligonucleotides which designed rationally with several characteristics. Nucleic acid barcodes were 20-200 nucleotides in length, with or without the modifications described above. The barcode portion comprised 4-50 nucleotides in the center of the oligonucleotide.
  • LNPs were formulated by mixing an aqueous phase containing nucleic acid with an ethanol phase containing lipids and excipients using a microfluidic chip device as previously described (Chen D, et al. (2012) Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc 134 (16) : 6948-6951) .
  • the ethanol phase contained a mixture of an ionizable lipid (e.g., 9, 12-Octadecadienoic acid (9Z, 12Z) -, 3- [4, 4-bis (octyloxy) -1-oxobutoxy] -2- [ [ [ [3- (diethylamino) propoxy] carbonyl] oxy] methyl] propyl ester, LP01, in this disclosure Lipid 40) , 1, 2-Dioctadecanoyl-sn-glycero-3-phophocholine (DSPC) , cholesterol, and 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) .
  • an ionizable lipid e.g., 9, 12-Octadecadienoic acid (9Z, 12Z) -, 3- [4, 4-bis (octyloxy) -1-oxobutoxy] -2- [ [ [ [
  • the ethanol phase was prepared by solubilizing a mixture.
  • the aqueous phase was prepared in 25 mM citrate buffer with nucleic acid barcode and RNA (gRNA and mRNA, Table 2-3) .
  • Each LNP was formulated to carry its own unique nucleic acid barcode.
  • the resulting LNPs were dialyzed against TSS buffer contained 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) in a 30,000 MWCO cassette at room temperature for 2 hours and then extruded through a 0.22 ⁇ m sterile filter.
  • TSS pH 7.5
  • the final LNP was stored at -80 °C until further use.
  • a series of ionizable lipids and formulations were used.
  • the ionizable lipids used in the examples included the commercially available LP01, ALC-0315, MC3, 306-O12B, ALC-0366, as well as our proprietary ionizable lipids (Cationic lipids) , which have been documented in PCT/CN2024/074415, PCT/CN2023/108217, PCT/CN2023/108216, PCT/CN2023/120619 and PCT/CN2023/137046.
  • Nanoparticles with different compositions and with different barcodes were prepared by the similar method. All LNP contained 8 molar ratios of the four lipids, and each ratio is prepared by four different processes and 2 mass ratios between gRNA (e.g., sgRNA) and mRNA, any CRISPR-system could be used in performing the examples, for example, sgRNA targeting TTR and mRNA encoding SaCas9 or SpCas9 (which can be found in our previous work or description herein) are introduced in the examples.
  • the mRNA used in the examples is N1-methyl-pseudouridine modified of the all the uracil.
  • Nanoparticle diameter and polydispersity were measured using dynamic light scattering (DLS) .
  • Nanoparticles were diluted to ⁇ 0.001 mg/mL nucleic acid in PBS and analyzed at room temperature.
  • encapsulation was >80%, particle size was ⁇ 150 nm, and PDI was ⁇ 0.2.
  • the LNP compositions are listed in Table 10.
  • the human hepatocellular carcinoma cell line HepG2 (American Type Culture Collection, Cat. HB-8065) was cultured at 37°C and 5%CO 2 in DMEM media supplemented with 10%fetal bovine serum. Cells were seeded in 48-well plates at a density of 20,000 cells per well. After 24 hours, cells were treated with Lipofectamine RNAiMAX encapsulating different barcodes at same concentration (6.25 nM) . After 72 hours transfection, cells were washed with PBS and genomic DNA was extracting using QuickExtract DNA Extraction Solution (Epicentre, QE09050) following the manufacturer′s recommended protocol.
  • mice All animals were purchased from Charles River Labs.
  • 8-week-old male CD-1 mice were injected intravenously via the tail vein with a pool of different barcoded LNPs at a dose of 2 mg/kg.
  • All animals were euthanized for necropsy.
  • Assessment of clinical signs, body weight, serum chemistry, organ weights and histopathology was performed.
  • the cynomolgus monkeys were pretreated with dexamethasone at least 1 hour before Cyn-LNP infusion to mimic planned clinical pretreatment.
  • the cynomolgus monkeys received intravenous administration of t LNP at doses of 3.0 mg/kg (total RNA/body weight) .
  • Transthyretin (TTR) gene editing in liver was evaluated using next-generation sequencing on day 30 and 43. Serum target protein concentrations were assayed by ELISA. Liver or lung were cut into small slices, and genomic DNA was extracting using TIANamp Genomic DNA kit following the manufacturer′s recommended protocol.
  • PCR samples were purified by AMPure XP beads. Final library QC was conducted using the Agilent Bioanalyzer 2100. Illumina deep sequencing was conducted on an Illumina MiniSeqTM. Primers were designed based on Nextera XT adapter sequences.
  • Sequencing results were processed using a custom python-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized with an R script prior to further analysis. The barcode counts were then normalized to 100%. Correlation analyses were run assuming a Gaussian distribution in order to obtain Pearson correlation coefficients. R2 values (0-1) were computed by squaring Pearson correlation coefficients.
  • LNP delivery of a specific barcode to cells or liver was calculated according to the following 3 steps: (i) dividing the number of sequencing reads of one barcode delivered by a single LNP formulation by the total amount of reads from all barcodes delivered by all LNPs in a specific tissue; (ii) dividing the number of sequencing reads of the same barcode (utilized in (i) ) by the total amount of reads from all barcodes of all LNPs in the non-injected LNP pool. (iii) dividing the results from (i) by the results from (ii) . By using this quantification method, the delivery of different LNP formulations within the same organ can be compared.
  • LNPs lipid nanoparticles
  • Fig 1A RNA and nucleic acid barcode 1
  • LNP-N RNA and nucleic acid barcode N.
  • Fig 1B RNA and nucleic acid barcode N.
  • nucleic acid barcoded screening system using lipofectamine in vitro.
  • Different doses of lipofectamine carried same dose RNA and nucleic acid barcodes were used to simulate different delivery efficiency of LNPs.
  • the indel frequency which is positively correlated with delivery efficiency increased with the increasing lipofectamine dose (Fig 2) .
  • the detected nucleic acid barcode counts were also positively correlated with the lipofectamine dose (Fig 3, 4) .
  • the nucleic acid barcode dose can also affect indel frequency and barcode counts. Excessive nucleic acid barcode dose brings negative effect to indel frequency possible due to toxicity (Fig 5) . Within certain limits, nucleic acid barcode dose and counts are also positively correlated (Fig 6, 7) .
  • LNP information was given in Table 5.
  • Twenty-eight days after LNP administration, which is a sufficiently long time for functional cargo delivery to generate Cas9/sgRNA ribonucleoprotein complexes (RNPs) we isolated liver and sequenced the barcodes using NGS. Consistent with the in vitro results, Barcodes that are 31 bases (LNP1-12) are more efficient than 33 bases (LNP 13-20) (Fig 12) .
  • lipid 32 lipid 32
  • LNP information was given in Table 7.
  • Thirty and forty-three days after LNP administration, which is a sufficiently long time for functional cargo delivery to generate Cas9/sgRNA ribonucleoprotein complexes (RNPs) we isolated liver and sequenced the barcodes using NGS.
  • the lipid 32 performed very well, the indel frequency is extremely high (Fig 14) .
  • Reductions in serum TTR protein concentration from baseline were observed by day14 and deepened by day 28 ( Figure 15) . At day 28 and beyond, TTR reductions more than 90%.

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Abstract

L'invention concerne un procédé de caractérisation de véhicules d'administration pour l'administration in vivo d'un agent. Les procédés peuvent être utilisés pour caractériser diverses propriétés d'une grande variété de véhicules d'administration.
PCT/CN2024/091283 2023-05-12 2024-05-06 Plate-forme de criblage à haut rendement de véhicules d'administration pour l'administration in vivo Pending WO2024235034A1 (fr)

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