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WO2025226912A1 - Administration spécifique de type cellulaire d'éditeurs géniques et procédés d'utilisation associés - Google Patents

Administration spécifique de type cellulaire d'éditeurs géniques et procédés d'utilisation associés

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Publication number
WO2025226912A1
WO2025226912A1 PCT/US2025/026135 US2025026135W WO2025226912A1 WO 2025226912 A1 WO2025226912 A1 WO 2025226912A1 US 2025026135 W US2025026135 W US 2025026135W WO 2025226912 A1 WO2025226912 A1 WO 2025226912A1
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WO
WIPO (PCT)
Prior art keywords
dope
peg
composition
cell
lnp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/026135
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English (en)
Inventor
Kevin James LUEBKE
Ibrahima YOUM
Allie ARMSTRONG
John Nicholas MARAFINO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SRI International Inc
Original Assignee
SRI International Inc
Stanford Research Institute
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Filing date
Publication date
Application filed by SRI International Inc, Stanford Research Institute filed Critical SRI International Inc
Publication of WO2025226912A1 publication Critical patent/WO2025226912A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered

Definitions

  • CRISPR-based editing comprises the largest segment of the projected genome editing market.
  • An important development in realizing the potential of genome editing was the FDA approval in December 2023 of exagamglogene autotemcel (exa-cel; CASGEVYTM), a CRISPR/Cas9 genome-edited cell therapy for the treatment of sickle cell disease in patients with recurrent vaso-occlusive crises.
  • This therapy relies on the ability to specifically edit a targeted cell-type by removing the target cells from the patient, editing them, and administering the edited cells to the patient. This strategy mitigates the risks of irreversible off-target editing, risks that are inherent in untargeted systemic administration of gene editors.
  • the invention relates to a composition for targeted delivery of a genome editing agent or transcriptional modulator, wherein the composition comprises a delivery vehicle comprising a targeting moiety specific for binding to a cell or tissue of interest.
  • the genome editing agent comprises a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA.
  • RNP ribonucleoprotein
  • the transcriptional modulator is selected from the group consisting of a methylase, a demethylase, a transcription factor, a transcriptional cofactor, and a transcriptional repressor.
  • the transcriptional modulator is a nucleic acid molecule encoding a methylase, a demethylase, a transcription factor, a transcriptional cofactor, or a transcriptional repressor.
  • the transcriptional modulator comprises a CRISPR Cas protein and a guide RNA.
  • the transcriptional modulator comprises a transcriptional modulator linked to a site-specific gene editing agent for directing the transcriptional modulator to a specific genomic locus.
  • the site-specific gene editing agent is a ribonucleoprotein comprising Cas9 and a guide RNA.
  • the site-specific gene editing agent is catalytically dead.
  • the site-specific gene editing agent is dCas9 and a guide RNA.
  • the composition comprises a targeted lipid nanoparticle (LNP) encapsulating the genome editing agent or transcriptional modulator.
  • LNP targeted lipid nanoparticle
  • the LNP comprises a PEG lipid conjugated to at least one molecular guidance system (MGS) peptide.
  • MGS molecular guidance system
  • the LNP comprises an ionizable cationic lipid, cholesterol, and at least one of DOPE, DOPE-PEG or DOPE-PEG-MGS peptide.
  • the LNP comprises Cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG- MGS peptide, wherein the Cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide are mixed in a molar ratio of 38.5 (Cholesterol):50 (D-Lin-MC3-DMA):10 (DOPE):1.5 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3- DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 46.5 (cholesterol):37 (D-Lin- MC3-DMA):16 (DOPE):0.8 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):6 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):11.5 (DOPE-PEG- MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 44 (cholesterol):37 (D-Lin-MC3- DMA):16 (DOPE):3 (DOPE-PEG-MGS peptide).
  • the LNP comprises an ionizable cationic lipid, cholesterol, at least one selected from the group consisting of DOPE, DOPE-PEG and DOPE-PEG-MGS peptide, and Cas9 ribonucleoprotein constituted with a sgRNA.
  • the LNP comprises wherein the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, DOPE-PEG-MGS peptide, sgRNA and Cas9, wherein the Cholesterol, D-Lin-MC3-DMA, DOPE, DOPE-PEG-MGS peptide or DOPE-Peg, sgRNA and Cas9 are mixed in a molar ratio of 44 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):3 (DOPE-PEG-MGS peptide or DOPE-Peg): 0.0069 (sgRNA): 0.0046 (Cas9).
  • the MGS peptide is SEQ ID NO:1-45, 47-56, or 58-62.
  • the target cell is a cancer cell, an immune cell, a stem cell, a fat cell, a dermal cell, an epithelial cell, a neuron, a glial cell, a cardiac cell, a hepatocyte, a lung cell, or a muscle cell.
  • the composition further comprises a nucleic acid molecule for insertion into the genome of the target cell.
  • the nucleic acid molecule for insertion into the genome of the target cell and the RNP are encapsulated within a single LNP.
  • the nucleic acid molecule for insertion into the genome of the target cell is encapsulated in a first LNP and the RNP is encapsulated in a second LNP, wherein each of the first and second LNP comprises a PEG lipid conjugated to at least one molecular guidance system (MGS) peptide.
  • MGS molecular guidance system
  • the MGS peptide of the first LNP encapsulating the nucleic acid molecule for insertion into the genome of the target cell and the MGS peptide of the second LNP encapsulating the RNP are the same MGS peptide.
  • the invention relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition for targeted delivery of a genome editing agent or transcriptional modulator, wherein the composition comprises a delivery vehicle comprising a targeting moiety specific for binding to a cell or tissue of interest to the subject.
  • the genome editing agent comprises a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA.
  • the disease or disorder is cancer, diabetes, a genetic defect, an autoimmune disease, a cardiac disease, a neurological disease, or an infectious disease.
  • the composition is administered by intravenous, intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.
  • the invention relates to a method of editing a target genomic locus of a target cell of a subject, the method comprising administering a composition for targeted delivery of a genome editing agent or transcriptional modulator, wherein the composition comprises a delivery vehicle comprising a targeting moiety specific for binding to a cell or tissue of interest to the subject.
  • the genome editing agent comprises a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA.
  • RNP ribonucleoprotein
  • the disease or disorder is cancer, diabetes, a genetic defect, an autoimmune disease, a cardiac disease, a neurological disease, or an infectious disease.
  • the composition is administered by intravenous, intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.
  • the invention relates to a method of modulating the transcriptional level of at least one gene in a target cell, the method comprising administering a composition for targeted delivery of a genome editing agent or transcriptional modulator, wherein the composition comprises a delivery vehicle comprising a targeting moiety specific for binding to a cell or tissue of interest to the subject.
  • the genome editing agent comprises a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA.
  • RNP ribonucleoprotein
  • the disease or disorder is cancer, diabetes, a genetic defect, an autoimmune disease, a cardiac disease, a neurological disease, or an infectious disease.
  • the composition is administered by intravenous, intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.
  • Figure 1 depicts a diagram showing that CRISPR nucleases allow precise, programmable gene editing.
  • Figure 2 depicts a diagram showing ribonuclear particles (RNPs) encapsulated in lipid nanoparticles equipped with SRI molecular guidance system. The LNPs protect RNPs from degradation, diminish immunogenicity and allow for selective delivery to target cells.
  • Figure 3 depicts a diagram of a lipid nanoparticle.
  • Figure 4 depicts a TEM image demonstrating the structure formed using the nanoparticle formulation.
  • Figure 5 depicts data demonstrating conjugation of MGS1 dimer with PEG phospholipid for incorporation in LNP.
  • FIG. 6 depicts data demonstrating that nanoparticles targeted for cell selective uptake by a molecular guidance system (MGS) show specific uptake by the target cell type.
  • Figure 7 depicts a diagram demonstrating genomic cleavage by CRISPR gene editor creates insertions/deletions detectable with mismatch-dependent nuclease.
  • Figure 8 depicts data demonstrating that a functional CRISPR gene editor was delivered selectively to a targeted cell type by MGS1 LNP.
  • Cas9 with a nuclear localization signal and a single guide RNA (sgRNA) targeted to TGF ⁇ -3 coding sequence was delivered in an LNP with or without MGS1.
  • the Cas9-sgRNA complex was delivered with CRISPRMax as an untargeted commercial transfection agent as a positive control.
  • MGS1 LNP mediates uptake of the gene editor in the targeted cell type (H2009) but not in the untargeted cell type (H1299), demonstrating the cell-type selectivity of the targeted LNP.
  • the internalized gene editor accesses the nucleus.
  • Enough functional gene editor accesses the nucleus to effect efficient gene editing.
  • Figure 9 depicts data demonstrating cell-targeted genomic cleavage is characterized by formation of genomic insertions and deletions.
  • the present invention relates to compositions for efficient delivery of a genome editing complex or a transcriptional modulator to a target cell or locus comprising a delivery vehicle, wherein the delivery vehicle comprises at least one targeting domain or moiety for delivery of the genome editing complex or a transcriptional modulator to a specific cell type of interest.
  • the genome editing agent or complex is a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA for directing the CRISPR Cas protein to a specific target nucleotide sequence.
  • RNP ribonucleoprotein
  • the transcriptional modulator increases or decreases the level of transcription of at least one gene or genomic locus in a target cell.
  • the targeted LNP of the invention are targeted to one or more specific cell types.
  • the LNP comprises a targeting domain specific for binding to a surface receptor on a cancer cell, an immune cell, a stem cell, a tumor cell, a fat cell, a dermal cell, an epithelial cell, a neuron, a glial cell, a cardiac cell, a hepatocyte, a lung cell, a muscle cell, or another specific cell type of interest.
  • the targeted LNP comprises a targeting domain specific for directing the LNP to a target cell or locus of interest.
  • the targeted LNP comprises a targeting domain specific for binding to an antigen or receptor on a target cell type of interest.
  • the LNP comprises a targeting domain specific for binding to an antigen or receptor expressed on an immune cell.
  • the LNP comprises a targeting domain specific for binding to an antigen or receptor expressed on a tumor cell.
  • the LNP comprises a targeting domain specific for binding to an antigen or receptor expressed on a stem cell (e.g., a hematopoietic stem cell).
  • the LNP comprises a targeting domain specific for binding to an antigen or receptor expressed on a specific tissue type (e.g., an antigen or receptor expressed on lung tissue).
  • the LNP comprises a targeting domain specific for directing the LNP to the central nervous system (CNS).
  • the LNP comprises a targeting domain specific for directing the LNP across the blood brain barrier (BBB).
  • BBB blood brain barrier
  • the targeted LNP of the invention comprises a targeting domain that promotes uptake of the LNP by a target cell type of interest.
  • the targeted LNP of the invention comprises a targeting domain that promotes traversal of the LNP to a target locus of interest.
  • the LNP comprises a targeting domain specific that promotes uptake of the LNP by a tumor cell.
  • the LNP comprises a targeting domain that promotes uptake of the LNP by a stem cell (e.g., a hematopoietic stem cell).
  • the LNP comprises a targeting domain that promotes uptake of the LNP by an immune cell.
  • the LNP comprises a targeting domain that promotes uptake of the LNP by a glial cell.
  • the LNP comprises a targeting domain that promotes uptake of the LNP by a cardiac cell.
  • the LNP comprises a targeting domain that promotes uptake of the LNP by a specific tissue type (e.g., an antigen or receptor expressed on lung tissue). In one embodiment, the LNP comprises a targeting domain that promotes uptake of the LNP by lung fibroblasts. In one embodiment, the LNP comprises a targeting domain that promotes traversal of the LNP to the CNS. In one embodiment, the LNP comprises a targeting domain that promotes traversal of the LNP across the BBB. [0032] In one embodiment, the delivery vehicle is a lipid nanoparticle comprising at least one lipid conjugated to at least one molecular guidance system (MGS) peptide.
  • MMS molecular guidance system
  • Exemplary MGS peptides that can be conjugated to the LNP of the invention include but are not limited to the MGS peptides described in US Patent Publication No. US20220227823A1, US Patent No. US11738089B2, US Patent Publication No. US20220380764A1, US Patent Publication No. US20230174580, and International Patent Publication No. WO2023122632, each of which is incorporated herein by reference in its entirety.
  • the present invention also relates to methods of use of the compositions described herein for targeted delivery of at least one therapeutic agent as well as methods of treating diseases or disorders in subjects including, but not limited to, cancer including lung cancer, breast cancer, colorectal cancer, ovarian cancer, and pancreatic cancer, diabetes, immunodeficiency disorders, neurological and neurodegenerative diseases and genetic diseases or disorders.
  • cancer including lung cancer, breast cancer, colorectal cancer, ovarian cancer, and pancreatic cancer
  • diabetes immunodeficiency disorders
  • neurological and neurodegenerative diseases and genetic diseases or disorders are defined herein.
  • antibody refers to an immunoglobulin molecule, which specifically binds with an antigen or epitope. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
  • physiologically effective dosage refers to an amount of an agent that produces a measurable biologic or physiologic effect in the recipient subject that is related to the activity of the agent(s).
  • the physiologically effective dosage will vary depending on the compound, the age, weight, etc., of the subject being administered the agent, and the biologic or physiologic effect being measured.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • cosmids e.g., naked or contained in liposomes
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • percent (%) identity refers to or includes the level of nucleic acid or amino acid sequence identity when aligned with a wild type sequence or sequence of interest using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence.
  • Example levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., any of the MGS peptide sequences, as described herein.
  • Example computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet.
  • nucleotide identity between individual variant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • a “variant sequence” can be one with the specified identity to the parent or reference sequence (e.g., wild- type sequence) of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of the parent sequence.
  • a “variant sequence” can be a sequence that contains 1, 2, or 3, 4 nucleotide base changes as compared to the parent or reference sequence of the invention, and shares or improves biological function, specificity and/or activity of the parent sequence.
  • a “variant sequence” can be one with the specified identity to the parent sequence of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of the parent sequence.
  • the variant sequence can also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of a reference sequence (e.g., an MGS peptide sequence).
  • a reference sequence e.g., an MGS peptide sequence.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • nucleosides nucleobase bound to ribose or deoxyribose sugar via N- glycosidic linkage
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • Nucleotide sequences that encode proteins and RNA may include introns.
  • the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell.
  • the term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.”
  • the monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • a “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
  • the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • the term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.
  • affinity ligand in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • the term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.
  • the term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
  • the therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • the present invention relates to compositions and methods for targeted delivery of a genomic editing complex or transcriptional modulator.
  • the genomic editing complex or transcriptional modulator comprises a nuclear localization signal which promotes translocation to the nucleus.
  • the invention comprises administering a ribonucleoprotein (RNP) complex comprising CRISPR Cas protein and a guide RNA molecule specific for targeting the CRISPR Cas protein to a specific genomic locus in a target cell type.
  • the CRISPR Cas protein comprises a nuclear localization signal which promotes translocation of the RNP complex to the nucleus.
  • the invention comprises administering a transcriptional modulator to a subject in need thereof.
  • the transcriptional modulator comprises a transcription factor which increases transcription at one or more genomic locus.
  • the transcriptional modulator comprises a transcription repressor which decreases transcription at one or more genomic locus.
  • the transcriptional modulator comprises a protein which recruits a native transcription factor to at least one genomic locus which increases transcription at the locus. In some embodiments, the transcriptional modulator alters the methylation state of a chromatin region, which increases or decreases transcription at the locus. In some embodiments, the invention provides compositions and methods for activating or reactivating or de-repressing a heterochromatin gene or region. In some embodiments, the invention provides compositions and methods for modulating one or more epigenomic marker. Therefore, in some embodiments, transcriptional modulators include, but are not limited to, methylases, demethylases, transcription factors, transcriptional cofactors, and transcriptional repressors.
  • the transcriptional modulator comprises an artificial transcription factor. In some embodiments, the transcriptional modulator comprises an artificial transcription factor containing a DNA-binding component and a transcription modulating component. In some embodiments, the transcriptional modulator comprises a DNA- binding component that is a CRISPR Cas protein and a guide RNA. In some embodiments, the transcriptional modulator comprises a DNA-binding component that is a CRISPR Cas protein complexed with a guide RNA in which the CRISPR Cas protein has been modified to disable its endonuclease function.
  • the delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • lipid formulations are contemplated for the introduction of at least one genome editing complex or transcriptional modulator into a host cell (in vitro, ex vivo or in vivo).
  • An exemplary delivery vehicle for targeted delivery of a genome editing complex or transcriptional modulator in vitro and in vivo is a lipid nanoparticle (LNP) comprising a cationic ionizable lipid and at least one PEG lipid conjugated to a targeting moiety.
  • LNP lipid nanoparticle
  • the genome editing complex or transcriptional modulator may be associated with an LNP. In one embodiment, the genome editing complex or transcriptional modulator may be encapsulated in the interior of an LNP. In one embodiment, the genome editing complex may be a ribonucleoprotein (RNP) complex comprising a Cas9 protein and guide RNA. In one embodiment, the RNP is encapsulated in the interior of an LNP. In one embodiment, a nucleic acid molecule encoding a transcriptional modulator is encapsulated in the interior of an LNP. In one embodiment, a nucleic acid molecule encoding a site-specific endonuclease is encapsulated in the interior of the LNP.
  • RNP ribonucleoprotein
  • site-specific endonucleases include, but are not limited to, I-Sce I, I-Chu I, I-Cre I, I-Csm I, Pl- Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI- Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I,
  • a nucleic acid molecule encoding a site-specific endonuclease comprising a CRISPR Cas protein is encapsulated in the interior of the LNP.
  • a nucleic acid molecule encoding a site-specific endonuclease comprising a guide RNA is encapsulated in the interior of the LNP.
  • a transcriptional modulator is encapsulated in the interior of an LNP.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.
  • Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-agent complexes.
  • delivery of the genome editing complex or transcriptional modulator comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein.
  • delivery of the genome editing complex or transcriptional modulator to a subject comprises mixing the genome editing complex or transcriptional modulator with a transfection reagent prior to the step of contacting a cell with the genome editing complex.
  • a method of the present invention comprises administering the genome editing complex or transcriptional modulator together with a transfection reagent.
  • the transfection reagent is a lipid-based transfection reagent.
  • the transfection reagent is a protein-based transfection reagent.
  • the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art. [0077] In one embodiment, the transfection reagent forms a liposome. Liposomes, in some embodiments, increase intracellular stability, increase uptake efficiency and improve biological activity. In some embodiments, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane.
  • the liposomes comprise an internal aqueous space for entrapping water-soluble compounds.
  • liposomes can encapsulate the genome editing complex or transcriptional modulator and deliver the genome editing complex or transcriptional modulator to cells in an active form.
  • the genome editing complex or transcriptional modulator is operably linked to a nuclear localization signal (NLS) for translocation to the nucleus following uptake of the LNP by the target cell and release of the genome editing complex or transcriptional modulator from the LNP.
  • NLS nuclear localization signal
  • the delivery vehicle comprises a lipid nanoparticle (LNP).
  • lipid nanoparticle refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids.
  • lipid nanoparticles comprise a cationic lipid and at least one excipient.
  • excipients include, but are not limited to, neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid).
  • the at least one agent is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the LNP, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm
  • the lipid nanoparticles are substantially non-toxic.
  • the genome editing complex when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes.
  • the LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated.
  • lipid refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents.
  • Lipids are usually divided into at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • the LNP comprises a cationic lipid.
  • the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N- (1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DO
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).
  • LIPOFECTIN® commercially available cationic liposomes comprising
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), and N,N-dimethyl-2,3-bis(((9Z,12Z,15Z)-octadeca- 9,12,15-trien-1-yl)oxy)propan-1-amine (DLenDMA).
  • the cationic lipid is an amino lipid.
  • Representative amino lipids include, but are not limited to, (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (D-Lin-MC3-DMA), 1,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin- MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2- d
  • the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 37 mole percent. In one embodiment, D-Lin-MC3-DMA is present in the LNP in an amount of about 50 mole percent.
  • D-Lin-MC3-DMA is present in the LNP in an amount of about 37 mole percent.
  • the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation. Suitable stabilizing lipids include neutral lipids and anionic lipids.
  • neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.
  • Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DMPE),
  • the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).
  • the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the LNP comprises a neutral lipid in a mole percent of about 20 to about 5.
  • the LNP comprises a neutral lipid at a mole percent of about 16.
  • the LNP comprises a neutral lipid at a mole percent of about 10.
  • the LNP comprises a DOPE at a mole percent of about 16.
  • the LNP comprises DOPE at a mole percent of about 10.
  • the LNPs comprise a combination of a neutral lipid and a neutral lipid-PEG-MGS peptide conjugate.
  • the neutral lipid-PEG-MGS peptide conjugate comprises a DOPE-PEG-MGS peptide conjugate.
  • the LNP comprises a neutral lipid-PEG-MGS peptide conjugate in a mole percent of about 5 to about 1.
  • the LNP comprises a neutral lipid-PEG-MGS peptide conjugate in a mole percent of about 3.
  • the LNP comprises a neutral lipid-PEG- MGS peptide conjugate in a mole percent of about 1.5. In some embodiments, the LNP comprises a DOPE-PEG-MGS peptide conjugate at a mole percent of about 5 to about 1. In some embodiments, the LNP comprises a DOPE-PEG-MGS peptide conjugate at a mole percent of about 3. In some embodiments, the LNP comprises a DOPE-PEG-MGS peptide conjugate at a mole percent of about 1.5.
  • the LNP comprises Cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the Cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 38.5 (cholesterol):50 (D-Lin-MC3- DMA):10 (DOPE):1.5 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the Cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 44 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):3 (DOPE-PEG-MGS peptide).
  • the LNP comprises Cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG- MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 46.5 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):0.8 (DOPE- PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3- DMA):16 (DOPE):6 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):11.5 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 44 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):3 (DOPE-PEG- MGS peptide).
  • the LNPs further comprise a steroid or steroid analogue.
  • a “steroid” is a compound comprising the following carbon skeleton: .
  • In certain embodim d analogue is cholesterol.
  • the molar ratio o e ca o c p o cholesterol ranges from about 2:1 to 1:1.
  • the LNP comprises cholesterol at a mole percent of about 50 to about 30.
  • the LNP comprises cholesterol at a mole percent of about 38.5
  • the LNP comprises cholesterol at a mole percent of about 44.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH.
  • lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • the LNP comprises glycolipids (e.g., monosialoganglioside GM1).
  • the LNP comprises a sterol, such as cholesterol.
  • the LNPs comprise a polymer conjugated lipid.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.
  • the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid).
  • Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
  • the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).
  • the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-( ⁇ -methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ⁇ - methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(PEG-DA
  • the mole percent of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 4:1.
  • one or more additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent.
  • the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent.
  • the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.
  • the LNP comprises at least one PEG lipid conjugated to a targeting moiety that targets the LNP to a specific cell or cell population.
  • the targeting domain is a ligand which directs the LNP to a receptor found on a cell surface of a specific cell or cell population.
  • the LNP comprises an ionizable cationic lipid and cholesterol, and DOPE, DOPE-PEG, or DOPE-PEG-MGS, or any combination thereof.
  • the LNP comprises an ionizable cationic lipid and cholesterol, DOPE and DOPE- PEG-MGS.
  • the LNP comprises an ionizable cationic lipid, cholesterol, DOPE-PEG and DOPE-PEG-MGS.
  • the LNP comprises an ionizable cationic lipid, cholesterol, and DOPE-PEG-MGS.
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE- PEG-MGS peptide are mixed a mole percent of 38.5 (cholesterol):50 (D-Lin-MC3-DMA):10 (DOPE):1.5 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE- PEG-MGS peptide are mixed a mole percent of 44 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):3 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3- DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 46.5 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):0.8 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE-PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):6 (DOPE-PEG-MGS peptide).
  • the LNP comprises cholesterol, D-Lin-MC3-DMA, DOPE, and DOPE- PEG-MGS peptide, wherein the cholesterol, D-Lin-MC3-DMA, DOPE and DOPE-PEG-MGS peptide are mixed in a molar ratio of 41 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):11.5 (DOPE-PEG-MGS peptide).
  • the delivery vehicle comprises at least one gene editing agent.
  • the gene editing complex is a therapeutic agent.
  • the delivery vehicle may also include substances with biological activities which are not typically considered to be active ingredients, such as fragrances, sweeteners, flavorings and flavor enhancer agents, pH adjusting agents, effervescent agents, emollients, bulking agents, soluble organic salts, permeabilizing agents, anti-oxidants, colorants or coloring agents, and the like.
  • the LNP or the nanoparticle compositions of the invention further comprises a nucleic acid.
  • the nucleic acid is a gene editing component.
  • RNA for example, a guide RNA, a trans-activating CRISPR (tracr) RNA, guide RNA, an mRNA encoding an RNA-guided nuclease, a gene or base editing protein, a zinc-finger nuclease, a Talen, a CRISPR nuclease (such as Cas9), a DNA molecule to be inserted or serve as a template for repair, and the like, or a combination thereof.
  • the mRNA encodes a gene-editing or base-editing protein.
  • the nucleic acid is a guide RNA.
  • the LNP comprises both a gene-editing or base-editing protein and one or more guide RNAs.
  • CRISPR nucleases may have altered activity, for example, modifying the nuclease so that it is a nickase instead of making double-strand cuts or so that it binds the sequence specified by the guide RNA but has no enzymatic activity.
  • Base-editing proteins are often fusion proteins comprising a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease).
  • the LNP or nanoparticle comprises a ribonucleoprotein, that is a complex comprising a guide RNA bound to a RNA-guided nuclease.
  • the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene.
  • gRNA guide RNA
  • Cas CRISPR-associated peptide
  • the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA.
  • the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
  • the editing protein includes, but is not limited to, a CRISPR- associated (Cas) protein, a zinc finger nuclease (ZFN) protein, a transcription activator-like effector nuclease (TALEN), a sequence specific endonuclease, an artificial site-specific RNA endonuclease (ASRE) or a protein having an RNA binding domain.
  • Cas CRISPR-associated
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • ASRE artificial site-specific RNA endonuclease
  • Examples of gene editing proteins include, but are not limited to, I-Sce I, I-Chu I, I-Cre I, I-Csm I, Pl-Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-
  • the editing protein is a Cas protein.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2.
  • the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the Cas protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In one embodiment, the Cas protein is Cas9. In one embodiment, Cas protein is catalytically deficient (dCas).
  • dCas catalytically deficient
  • Cas protein is catalytically deficient Cas9 (dCas9).
  • the LNP further comprises an isolated nucleic acid molecule comprising a nucleotide sequence for insertion into the genome.
  • the isolated nucleic acid molecule is a DNA molecule.
  • the isolated nucleic acid molecule comprises a sequence to correct a genetic defect in a target cell.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the gene can include exons and introns.
  • the DNA to be administered with a genome editing complex is a human gene or a fragment thereof.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • the LNP comprises (i) an ionizable cationic lipid, (ii) cholesterol, (iii) DOPE, DOPE-PEG, or DOPE-PEG-MGS, or any combination thereof, (iv) sgRNA and (v) Cas9 or dCas9.
  • the LNP comprises i) an ionizable cationic lipid, (ii) cholesterol, (iii) DOPE, DOPE-PEG, or DOPE-PEG-MGS, or any combination thereof, (iv) sgRNA and (v) Cas9 or dCas9, wherein the i) an ionizable cationic lipid, (ii) cholesterol, (iii) DOPE, DOPE-PEG, or DOPE-PEG-MGS, or any combination thereof, (iv) sgRNA and (v) Cas9 or dCas9 are mixed a mole percent of 44 (Cholesterol):37 (ionizable cationic lipid):16 (DOPE):3 (DOPE-PEG-MGS peptide or DOPE-Peg): 0.0069 (sgRNA): 0.0046 (Cas9).
  • the LNP comprises i) D-Lin-MC3-DMA, (ii) cholesterol, (iii) DOPE, DOPE-PEG, or DOPE- PEG-MGS, or any combination thereof, (iv) sgRNA and (v) Cas9 or dCas9, wherein the i) D- Lin-MC3-DMA, (ii) cholesterol, (iii) DOPE, DOPE-PEG, or DOPE-PEG-MGS, or any combination thereof, (iv) sgRNA and (v) Cas9 or dCas9 are mixed a mole percent of 44 (Cholesterol):37 (ionizable cationic lipid):16 (DOPE):3 (DOPE-PEG-MGS peptide or DOPE- Peg): 0.0069 (sgRNA): 0.0046 (Cas9) Combinations [0114]
  • the composition of the present invention comprises a combination of agents described herein.
  • the invention provides a combination of a first LNP delivery vehicle comprising a genomic editing complex and a second LNP delivery vehicle comprising a nucleotide sequence to be inserted into a genome by way of recombination associated with repair of a nick or break in the DNA generated by the genome editing complex.
  • the first and second LNP delivery vehicle both are targeted using the same MGS peptide targeting molecule.
  • a composition comprising a combination of agents comprises individual agents in any suitable ratio.
  • the composition comprises a 1:1 ratio of two individual agents.
  • the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • the present invention relates to compositions and methods for modulating transcriptional activity of at least one gene or genomic locus in a subject.
  • the present invention provides a composition comprising a transcriptional modulator.
  • the present invention provides a composition comprising a nucleic acid molecule encoding a transcriptional modulator, wherein the transcriptional modulator increases or decreases the level of transcription of at least one gene or genomic locus.
  • the composition comprises a targeted LNP comprising a nucleic acid molecule encoding a transcriptional modulator.
  • the composition of the invention comprises in vitro transcribed (IVT) RNA molecule encoding a transcriptional modulator.
  • IVT in vitro transcribed
  • the composition of the invention comprises a targeted LNP encapsulating an IVT RNA which encodes a transcriptional modulator, wherein the transcriptional modulator increases or decreases the level of transcription of at least one gene or genomic locus.
  • the transcriptional modulator is at least one of a methylase, a demethylase, a transcription factor, a transcriptional cofactor, or a transcriptional repressor.
  • the transcriptional modulator comprises an artificial transcription factor.
  • the transcriptional modulator comprises an artificial transcription factor containing a DNA-binding component and a transcription modulating component.
  • the transcriptional modulator comprises a DNA-binding component that is a CRISPR Cas protein and a guide RNA.
  • the transcriptional modulator comprises a DNA-binding component that is a CRISPR Cas protein complexed with a guide RNA in which the CRISPR Cas protein has been modified to disable its endonuclease function.
  • the transcriptional modulator comprises a protein which recruits a native transcription factor to at least one genomic locus which increases transcription at the locus.
  • the transcriptional modulator comprises a non-specific transcriptional modulator tethered to a sequence specific DNA binding domain.
  • the transcriptional modulator comprises a methylase, a demethylase, a transcription factor, a transcriptional cofactor, or a transcriptional repressor tethered to a sequence specific DNA binding domain from a gene editor.
  • the transcriptional modulator comprises a methylase, a demethylase, a transcription factor, a transcriptional cofactor, or a transcriptional repressor tethered to a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN) protein, a transcription activator-like effector nuclease (TALEN), a sequence specific endonuclease, an artificial site-specific RNA endonuclease (ASRE) or a protein having an RNA binding domain.
  • Cas CRISPR-associated
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • ASRE artificial site-specific RNA endonuclease
  • the transcriptional modulator comprises a methylase, a demethylase, a transcription factor, a transcriptional cofactor, or a transcriptional repressor tethered to a catalytically dead gene editor, such as a dCas9-guide RNA.
  • the transcriptional modulator alters the methylation state of a chromatin region, which increases or decreases transcription at the locus.
  • the invention provides compositions and methods for activating or reactivating or de-repressing a heterochromatin gene or region.
  • the invention provides compositions and methods for modulating one or more epigenomic marker.
  • the nucleic acid of the present composition is a nucleoside-modified RNA.
  • the nucleoside that is modified in the nucleoside-modified RNA the present invention is uridine (U).
  • the modified nucleoside is cytidine (C).
  • the modified nucleoside is adenosine (A).
  • the modified nucleoside is guanosine (G).
  • Conjugation [0122]
  • the delivery vehicle comprises a PEG lipid conjugated to at least one MGS peptide targeting domain.
  • Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, hydrophobic, and Van der Waals interactions.
  • the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents.
  • the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.
  • the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting molecule.
  • activated polymer conjugated lipid refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group.
  • the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group.
  • the activated polymer conjugated lipid is an activated pegylated lipid.
  • the first coupling group is bound to the lipid portion of the pegylated lipid.
  • the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid.
  • the second functional group is covalently attached to the targeting domain.
  • the first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions.
  • the first coupling group or second coupling group are selected from the group consisting of maleimides, N- hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulf
  • the first coupling group or second coupling group is selected from the group consisting of free amines (–NH2), free sulfhydryl groups (–SH), free hydroxide groups (–OH), carboxylates, hydrazides, and alkoxyamines.
  • the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl.
  • the first coupling group is a maleimide.
  • the second coupling group is a sulfhydryl group.
  • the sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art.
  • the sulfhydryl group is present on a free cysteine residue.
  • the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine.
  • the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2- iminothilane or N-succinimidyl S-acetylthioacetate (SATA).
  • SATA N-succinimidyl S-acetylthioacetate
  • the polymer conjugated lipid and the targeting domain are functionalized with groups used in “click” chemistry.
  • Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles.
  • exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone.
  • the targeting molecule comprises at least one MGS peptide which specifically binds to a marker of a cell type of interest.
  • the targeting domain directs the vehicle to a cancer cell, an immune cell, a stem cell, a fat cell, a dermal cell, an epithelial cell, a neuron, a glial cell, a cardiac cell, a hepatocyte, a lung cell, a muscle cell, or another specific cell type of interest.
  • Cells and/or tissues that can be targeted include but are not limited to immune cells (e.g., dendritic cells, etc.), pancreatic cells (e.g., ⁇ - cells of the islets of Langerhans, etc.), hematopoietic stem cells (e.g., HSCs, etc.), lung fibroblasts, cardiovascular cells, microglial cells and tumor cells.
  • the targeted LNP of the invention comprises a PEG lipid conjugated to at least one MGS peptide specific for binding to an antigen on a target cell type of interest.
  • the targeting domain directs the vehicle to a target locus. In some embodiments, the targeting domain directs the vehicle to the CNS.
  • the targeting domain directs the vehicle across the BBB.
  • Exemplary MGS peptides that can be included in a targeting moiety of an LNP include, but are not limited to the MGS peptides of SEQ ID NO:1-45, 47-56, or 58-62 or a fragment or variant thereof.
  • Table 1 MGS peptide sequences PEPTIDE SEQ ID NO: TARGET R GDLATLRQL 1 Carcinomas and Solid YAAWPASGAWT 2 Tumors KQYATPRVFWT 3 FHAVPQSFYTAP 4 F HAVPQSFYTA 5 F HAVPQSFYT 6 F HAVPQSFY 7 H AVPQSFYT 8 VSQTMRQTAVPLLWFWTGSL 9 Y AAWPASGAWTGTAPCSAGT 10 EAMNSAEQSAAVVQWEKRRI 11 ATEPRKQYATPRVFWTDAPG 12 MTVCNASQRQAHAQATAVSL 13 MRGQTGKLPTEHFTDTGVAF 14 MTGKAAAPHQEDRHANGLEQ 15 TNSCRGDWLCDAVPEKARV 16 EHPWFNMWSWATQVQE 17 YPGSPTQYPSSMHEYHSSSE 18 AHTIDDEWASYHMQQWNSPP 19 FEEFYSRQSNTIPYPQQ
  • the one or more MGS peptides have a sequence identity of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any of the sequences set forth in SEQ ID NOs:1-45, 47-56, or 58-62. In some embodiments, the one or more MGS peptides have 100% identity in the active portion of the peptide, wherein the active portion is the portion that retains its ability to target a specific cell. [0132] In some embodiments, MGS peptides can be modified. In some embodiments, modifying an MGS peptide comprises optimizing the peptide or stabilizing the peptide.
  • Optimized peptides can be obtained by applying modifications to the individual parental peptide sequences. Modification at the amino-terminus by acetylation (CH3CO-) and/or d-amino acids, such as d(Leu) can protect against degradation by peptidases.
  • the MGS peptide can have an N-terminal protection group.
  • the N-terminal protection group can be anything that prevents proteases from cleaving the amino acids from the N-terminus.
  • the MGS peptides disclosed herein can be modified by acetylation on the N-terminus.
  • the N-terminal protection group can be, but is not limited to, PEG, Formyl, CH3-(CH)n-CO, Fluorophore, Fatty acid, alkyl amine, aryl groups, carbohydrates, sulfonamide, or carbamate.
  • the targeting domain binds to a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell.
  • the targeting domain binds to a cell surface molecule that actively mediates internalization of bound molecules.
  • the composition comprises a delivery vehicle conjugated to a targeting domain that binds a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell.
  • the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a cancer cell.
  • the cancer is a solid tumor.
  • the targeting domain comprises at least one MGS peptide selected from SEQ ID NOs:1-43, or a fragment or variant thereof.
  • the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a lymphoma cancer cell.
  • the targeting domain comprises at least one MGS peptide selected from SEQ ID NOs:44-45, or a fragment or variant thereof.
  • the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a cardiovascular cell or cardiomyocyte. In some embodiments, the targeting domain comprises at least one MGS peptide comprising the sequence of SEQ ID NOs:47-49, or a fragment or variant thereof. [0137] In one embodiment, the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by an immune cell. In some embodiments, the targeting domain comprises at least one MGS peptide selected from SEQ ID NOs:50-54, or a fragment or variant thereof.
  • the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a pancreatic cell ( ⁇ -Cell of the islets of Langerhans). In some embodiments, the targeting domain comprises at least one MGS peptide selected from SEQ ID NOs:55-56, or a fragment or variant thereof. [0139] In one embodiment, the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a lung fibroblast. In some embodiments, the targeting domain comprises at least one MGS peptide comprising the sequence of SEQ ID NO:58, or a fragment or variant thereof.
  • the LNP comprises a targeting domain comprising at least one MGS peptide that promotes uptake by a neuronal cell.
  • the targeting domain comprises at least one MGS peptide comprising the sequence of SEQ ID NOs:59-62, or a fragment or variant thereof.
  • the present invention is not limited to vehicles directed to the specific cell types above. Rather, the present invention encompasses a delivery vehicle comprising a targeting domain that directs the vehicle to any specific target cell, as mediated by the binding of the targeting domain to a specific receptor on the surface of the target cell.
  • the vehicle is targeted to a specific treatment site in need.
  • the targeting domain can be directed specifically to target tumor cells or pathogens.
  • the targeting domain may be co-polymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle.
  • Peptides [0143] In one embodiment, the targeting domain of the invention comprises at least one MGS peptide. In one embodiment, the targeting domain of the invention comprises two MGS peptides. In certain embodiments, the MGS peptide targeting domain specifically binds to a receptor expressed on a cell type of interest. [0144] The peptide of the present invention may be made using chemical methods.
  • peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide.
  • the composition of a peptide may be confirmed by amino acid analysis, sequencing or mass spectrometry.
  • the variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
  • the fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation.
  • Therapeutic Methods [0148] In some embodiments, the invention provides methods for targeted delivery of a therapeutic agent to a target cell for the treatment of a disease or disorder in a subject. [0149] The present invention also provides methods of delivering at least one genome editing agent to a subject in need thereof.
  • the method is used to treat or prevent a disease or disorder in a subject.
  • diseases or disorders that can be treated using the methods of the invention include, but are not limited to, genetic defects, cancers, diabetes, autoimmune diseases, neurological diseases and disorders, cardiac diseases and disorders, and infectious diseases.
  • Exemplary genetic diseases or disorders that can be treated or prevented using the compositions and methods described herein include, but are not limited to, hemophilia, platelet disorders, sickle cell disease, thalassemia, thrombophilia, von Willebrand disease, a Trinucleotide Repeat disorder, Fragile X Syndrome, Parkinson’s Disease, Neuro transmission Disorders, Macular Degeneration, Aicardi-Goutieres Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi Syndrome, Alpers’ Disease, glioblastoma, Alzheimer's Disease, Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration, Fabry’s Disease, Gerstmann-Straussler-Scheinker Disease, Huntington’s Disease, Leigh's Disease, Lesch-Nyhan Syndrome, Menkes Disease, Mitochondrial Myopathies, Friedreich Ataxia (FRDA), Muscular Dystrophy, Maple Syrup Urine Disease, Peroxisome Bio
  • the invention relates to methods of treating or preventing neurological diseases or disorders in subjects in need thereof, the method comprising administering the LNP composition of the invention.
  • Exemplary neurological diseases or disorders that can be treated using the LNP compositions and methods of the invention include, but are not limited to, acute spinal cord injury, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, bell’s palsy, brain tumor, cerebral aneurysm, epilepsy, seizure, Guillain-Barré syndrome, headache, migraine, head injury, hydrocephalus, meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, Parkinson’s disease, stroke, cluster headache, tension headache, migraine headaches, encephalitis, or any combination thereof.
  • Infectious diseases that can be treated using the compositions and methods include, but are not limited to, bacterial infections, viral infections, parasitic infections and fungal infections.
  • the present invention also provides methods of delivering at least one genome editing agent or complex to a subject in need thereof in a cell-targeted manner.
  • the genome editing agent or complex is a therapeutic agent for the treatment of a disease or disorder.
  • the disease or disorder is a genetic defect.
  • the methods comprise administering at least one agent for genetic editing to a target cell for the treatment of a genetic defect.
  • At least one agent for genetic editing is a RNP comprising a CRISPR Cas protein and a guide RNA molecule for targeting the CRISPR Cas protein to a specific genomic locus.
  • the present invention includes a method for preventing diseases or disorders, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of diseases or disorders, thereby preventing diseases or disorders.
  • a composition as discussed previously elsewhere herein, can be administered to a subject prior to the onset of diseases or disorders, thereby preventing diseases or disorders.
  • the prevention of a disease or disorder encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder.
  • the compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other.
  • the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result.
  • any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with diseases or disorders.
  • the invention includes a method comprising administering a combination of compositions described herein.
  • the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor.
  • the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.
  • the method comprises administering a combination of compositions in any suitable ratio.
  • the method comprises administering two individual compositions at a 1:1 ratio.
  • the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • Pharmaceutical Compositions [0160] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • Such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
  • compositions of the invention include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • a pharmaceutical composition of the invention may further comprise one or more additional adjuvants.
  • Exemplary adjuvants include, but are not limited to, aluminum-based adjuvant and monophosphoryl lipid A.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In some embodiments, the diameter is in the range from about 1 to about 6 nanometers.
  • compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure.
  • the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent.
  • the propellant has a particle size of the same order as particles comprising the active ingredient.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • Example 1 Targeted Delivery of Gene Editors is an Important Frontier Technology
  • Gene editing involves site-specific cleavage of genomic DNA and subsequent repair of the site-specific DNA break.
  • the break can be repaired by non-homologous end joining with the creation of insertions and deletions (indels) at the cut site.
  • Indels insertions and deletions
  • a donor DNA sequence with flanking sequences that match the DNA sequence flanking the cut site can be inserted into the break by homology-directed repair, resulting in insertion of the donor sequence into the genome at the cut site.
  • CRISPR-based editing accomplishes site-specific cleavage with a CRISPR endonuclease, composed of a guide RNA used to locate (bind) the target DNA to be edited and a CRISPR-associated protein such as Cas9, which bears the enzymatic capability to cleave the DNA at the location identified by guide RNA ( Figure 1).
  • Accomplishing gene editing by systemic administration of an editing agent requires delivery of the gene editing agent to the cells to be edited.
  • an editing agent e.g., CAS9 ribonucleoprotein
  • the editor must be protected from degradation by nucleases and proteases that they encounter en route to the targeted cells.
  • CRISPR-based gene editors originated in bacteria, they are immunogenic in mammalian hosts, so they must also be protected from immune surveillance. Editors must further be internalized by the cells in which they are to act. Encapsulation of gene editors in lipid nanoparticles can protect them from degradation and sequester them from recognition by the immune system. Immunogenicity of the nanoparticle is further suppressed by the presence of polyethylene glycol (PEG) chains on its surface.
  • PEG polyethylene glycol
  • lipid nanoparticle formulations that efficiently encapsulate ribonucleoprotein gene editors. These formulations comprise a cationic lipid, cholesterol, and a helper lipid, such as 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), a fraction of which is conjugated to PEG ( Figure 3).
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • Nanoparticles with these formulations were produced by precipitation from an aqueous ethanol mixture with rapid mixing followed by centrifugal filtration. This procedure produces nanoparticles in the size range of 100 nm. They provide high encapsulation efficiency of ribonucleoprotein complexes and protect the encapsulated RNA component from degradation by ribonucleases. Transmission electron microscopy of the resulting nanoparticles is consistent with their anticipated structure (Figure 4). [0181] The LNP particles have been optimized for ribonucleoprotein payload (Figure 3) and show ⁇ 90% Encapsulation efficiency for CRISPR/PGM payload. They are about 100-200 nm in diameter as measured by dynamic light scattering and TEM ( Figure 4).
  • the targeting agent was conjugated to the PEG moiety on the helper lipid.
  • a dimeric form of the targeting peptide MGS1 (SEQ ID NO:1) was used. This MGS is internalized by certain cancer cell types. It was conjugated with DOPE-PEG by reaction of a sulfhydryl from a cysteine in the peptide with a maleimide on the end of the PEG chain, forming the conjugate shown.
  • the MGS1 nanoparticles were loaded with fluorophore AF647 and applied to cells that internalize MGS1 (lung tumor cell line H2009) or cells that do not internalize MGS1 (lung tumor cell line H1299).
  • Red fluorescence (AF647) in the fluorescence micrograph of H2009 (upper left) treated with the nanoparticles shows uptake of the fluorophore-loaded particles. Red-fluorescence is not apparent in the treated H1299 cells (lower left), indicating the nanoparticles were not internalized by these cells. Fluorophore internalization was quantified by flow cytometry (right).
  • Nanoparticles formulated with MGS1 delivered 10,000 AF647 molecules per cell to the targeted cell type (H2009) in one hour of incubation but only 500 molecules per cell to the non-targeted cell type under the same conditions, a 20-fold selectivity for the targeted cell type.
  • MGS1 was omitted from the lipid nanoparticles, uptake of fluorophore was poor in both cell lines (1000 molecules per cell for H2009, 120 molecules per cell for H1299) ( Figure 6).
  • a genomic cleavage assay enables assessment of site-specific DNA double- stranded breaks produced by Cas9 endonuclease.
  • Double stranded breakage of the genome at the selected site, followed by cell-mediated repair of the breaks results in a mixture of sequence insertions and deletions (indels) at the cleavage site after cell-mediated repair of the breaks.
  • Indels sequence insertions and deletions
  • Isolation of the genomic DNA and amplification of the region surrounding the break site by the Polymerase Chain Reaction (PCR) produces DNA that recapitulates the distribution of indel sequences and un-modified DNA within the amplified region of the genome. Denaturation and renaturation of the DNA scrambles the pairing of the different sequences, resulting in mismatched or bulged segments in the annealed duplexes.
  • FIG. 7 A single-strand specific endonuclease, T7 endonuclease, cleaves the annealed duplexes at sites of earlier genomic cleavage by the Cas9. This cleavage can be observed from the sizes of the resulting fragments using agarose gel electrophoresis ( Figure 7).
  • Figure 8 provides data demonstrating cell-type specific delivery and function of a Cas9 gene editor directed to a site in the coding sequence for the growth factor TGFbeta-3.
  • a Cas9-guide RNA ribonucleoprotein complex was generated in which the guide RNA directs cleavage at a site in the coding sequence of TGFbeta-3, and the resulting editor complex was encapsulated in lipid nanoparticles with and without MGS1. These nanoparticles were applied to MGS1-targeted cells (H2009) and MGS1-non-targeted cells (H1299) and after a 24 hour incubation, genomic DNA was isolated from the treated cells. In control experiments, the CRISPR complex was delivered to both cell types using the commercial transfection agent CRISPRMax and the resulting genomic DNA was isolated.
  • a 470 bp region of the TGFbeta-3 coding sequence containing the anticipated Cas9 cleavage site was amplified, denatured, and the amplified product was re-annealed.
  • the resulting DNA pool was digested with T7 endonuclease, and the resulting fragments were analyzed by agarose gel electrophoresis. Cleavage products of the 470 bp amplicon consistent with the anticipated lengths of 313 and 137 bp are apparent in the DNA from both cell types when the editor complex was delivered with CRISPRMax (top(H2009) and bottom(H1299) gels, lane 7).
  • a donor DNA will be co- delivered with the editor.
  • the donor DNA may include flanking sequences homologous to the genomic DNA flanking the editor cleavage site to promote homologous end joining at the cleavage site.
  • the donor DNA may be co-encapsulated with the editor or encapsulated separately. If encapsulated separately, it may be targeted for delivery with the same MGS as the editor. [0187] Co-delivery of a donor DNA with a targeted lipid nanoparticle to achieve targeted site-specific insertion of DNA into the genome extends the utility of the cell-targeted editing and facilitate in vivo studies by allowing modification of targeted cells with a reporter gene for visualization of successful editing.
  • CRISPR/Cas9-loaded LNPs Guide RNA/Cas9-loaded lipid nanoparticles (CRISPR/Cas9-loaded LNPs) were synthesized using a nanoprecipitation/filtration method that provides a scalable, robust, reproducible and procedurally simple alternative to lipid nanoparticle formation using detergent dialysis, particle extrusion, and fluidic mixing methods commonly used (Lloyd B Jeffs, Lorne R Palmer, Ellen G Ambegia, Cory Giesbrecht, Shannon Ewanick, Ian MacLachlan.
  • Lipid stock solutions in ethanol containing cholesterol, ionizable/cationic lipid (e.g., D-Lin-MC3-DMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and pegylated DOPE (DOPE-PEG or DOPE-PEG-MGS peptide conjugate) were mixed.
  • lipids were mixed at a molar ratio of 38.5 (Cholesterol):50 (D-Lin-MC3-DMA):10 (DOPE):1.5 (DOPE-PEG-MGS peptide).
  • PBS phosphate buffered saline
  • the CRISPR guide RNA/cas9 ribonucleoprotein complex was formed by mixing equimolar amounts of Cas9 from Streptococcus pyogenes bearing two copies of a nuclear localization signal sequence (NLS-Cas9-NLS, Novateinbio, PR-137211) and the single guide RNA (sgRNA) in 25 ⁇ l of Opti-MEM medium (Invitrogen, Waltham, MA) and incubating the mixture at room temperature for 5 minutes (Xin Yu, Xiquan Liang, Huimin Xie, Shantanu Kumar, Namritha Ravinder, Jason Potter, Xavier de Mollerat du Jeu, Jonathan D. Chesnut.
  • Solution components not incorporated into nanoparticles or forming particles below a desirable size threshold were removed by diluting the suspension with two volumes of PBS and filtering with a sterile ultra-centrifugal filter (AmiconTM Ultra-15 Centrifugal Filter Units, MWCO:100kDa, MilliporeSigma, St-Louis, MO) at 3,000 rpm for 10 min at 4°C (Allegra X-14R centrifuge, Beckman Coulter, Brea, CA). The retentate contained the purified nanoparticles, which were stored at -80°C until use. [0190] Dye-loaded and empty LNPs made of similar lipid components were prepared using the same method.
  • Example 2 Cell-Targeted Genomic Cleavage Characterized by Formation of Genomic Insertions and Deletions. [0192] Production of sequence insertions and deletions (InDels) was used as a measure of site-specific genomic cleavage by Cas9-sgRNA RNP ( Figure 9).
  • Cas9 ribonucleoprotein constituted with a sgRNA targeted to a sequence (5’-CCCTCCGGGCTGCGGCTGCT-3’ (SEQ ID NO: 76)) in the human gene for transforming growth factor beta-1 (TGFB-1) was encapsulated in LNP bearing DOPE-PEG-MGS1 peptide conjugate (MGS1) or in LNP formulated with DOPE-PEG (PEG) in place of the DOPE-PEG-MGS1 conjugate.
  • Human lung cancer cell lines H1299 and H2009 in culture were treated with the RNP-loaded nanoparticles or with the RNP complexed with Lipofectamine CRISPRMAX Cas9 Transfection Reagent (Thermo).
  • the components of the lipid nanoparticle were mixed at a molar ratio of 44 (cholesterol):37 (D-Lin-MC3-DMA):16 (DOPE):3 (DOPE-PEG-MGS peptide):0.0069 (sgRNA): 0.0046 (Cas9).
  • the Cas9 sequence included a nuclear localization signal.
  • H1299 and H2009 cells were plated in 24-well plates at 35,000 cells per well in 500 ⁇ l of growth medium at 37°C so that the cells reached 30–70 % confluence at the time of transfection. 24 hours after plating, cells were treated with 400 ⁇ g of RNP-loaded LNPs in each well containing 500 ⁇ l of complete cell culture medium for 24h. After 24h, cells were harvested, and the genomic DNA extracted using PureLinkTM Genomic DNA Mini Kit following vendor protocol (Thermo Fisher Scientific). The 280 base pair sequence to be analyzed was amplified by polymerase chain reaction and subjected to Illumina paired-end sequencing by a commercial sequencing service (Azenta).

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Abstract

La présente invention concerne des compositions pour l'administration ciblée efficace d'un agent d'édition génique ou d'un modulateur transcriptionnel à une cellule cible, ainsi que des procédés d'utilisation de celles-ci pour le traitement de maladies comprenant le cancer et des maladies génétiques. Dans certains modes de réalisation, l'invention concerne une composition pour l'administration ciblée d'un agent d'édition génomique ou d'un modulateur transcriptionnel, la composition comprenant un véhicule d'administration comprenant une fraction de ciblage spécifique de la liaison à une cellule ou un tissu d'intérêt. Dans certains modes de réalisation, l'agent d'édition génomique comprend un complexe ribonucléoprotéique (RNP) comprenant une protéine CRISPR Cas et un ARN guide.
PCT/US2025/026135 2024-04-26 2025-04-24 Administration spécifique de type cellulaire d'éditeurs géniques et procédés d'utilisation associés Pending WO2025226912A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220267807A1 (en) * 2012-05-25 2022-08-25 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US20230374549A1 (en) * 2020-09-29 2023-11-23 Flagship Pioneering Innovations V, Inc. Compositions and methods for inhibiting the expression of multiple genes
US20230407282A1 (en) * 2022-06-21 2023-12-21 Novocure Gmbh Systems and methods for treating conditions and diseases using alternating electric fields and crispr-cas system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220267807A1 (en) * 2012-05-25 2022-08-25 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US20230374549A1 (en) * 2020-09-29 2023-11-23 Flagship Pioneering Innovations V, Inc. Compositions and methods for inhibiting the expression of multiple genes
US20230407282A1 (en) * 2022-06-21 2023-12-21 Novocure Gmbh Systems and methods for treating conditions and diseases using alternating electric fields and crispr-cas system

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