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WO2024119101A1 - Plateforme d'ingénierie sans trace sensible aux stimuli pour distribution de charge utile intracellulaire - Google Patents

Plateforme d'ingénierie sans trace sensible aux stimuli pour distribution de charge utile intracellulaire Download PDF

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Publication number
WO2024119101A1
WO2024119101A1 PCT/US2023/082123 US2023082123W WO2024119101A1 WO 2024119101 A1 WO2024119101 A1 WO 2024119101A1 US 2023082123 W US2023082123 W US 2023082123W WO 2024119101 A1 WO2024119101 A1 WO 2024119101A1
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conjugate
disease
cells
inclusive
chemical moiety
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PCT/US2023/082123
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English (en)
Inventor
Jiangbing Zhou
Yong-hui JIANG
Ying Xie
Youmei BAO
Xiaona LU
Wendy SHEU
Jiang Yu
Gretchen LONG
Yuanyuan LUO
Wenzhe WANG
Kun-Yong Kim
Sung Eun Wang
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Yale University
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Publication of WO2024119101A1 publication Critical patent/WO2024119101A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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 RNAses, DNAses
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the invention is in the field of payload delivery, particularly the intracellular delivery of payloads using payload-membrane fusogenic molecule conjugates in which the payload and membrane fusogenic molecule are covalently linked preferably via a stimuli-responsive linker, and more preferably a redox-responsive, self-immolative linker.
  • conjugates that confer improved intracellular deliver of biologics. It is a further object of the invention to provide conjugates capable of passing through the cell membrane while retaining the biological activity of the biologics being delivered. It is another object of the invention to provide conjugates capable of delivering gene editing machinery into a cell. It is yet a further object of the invention to provide methods for using such conjugates.
  • SUMMARY OF THE INVENTION Described are conjugates and pharmaceutical compositions containing these conjugates for intracellular delivery of a payload.
  • the conjugates contain the payload, a chemical linker, and a cell membrane fusogenic molecule. Preferred payloads for delivery are gene editing machineries.
  • the cell membrane fusogenic molecule facilitates intracellular uptake of the conjugates.
  • the chemical linker contains a stimuli- responsive chemical moiety and a self-immolative chemical moiety.
  • the conjugate Upon entry into a cell, the conjugate is exposed to one or more stimuli, such as a reducing and/or an acidic environment, that cleave the stimuli-responsive chemical moiety, thereby activating self-immolation of the chemical linker.
  • the cleavage and self-immolation lead to the delivery of the payload in a stimuli-responsive traceless manner.
  • results described herein demonstrate that the platform can be used for the effective intracellular delivery of ribonucleoproteins (RNPs) composed of Cas9 protein sgRNA in in vitro cell cultures and in vivo in both reporter and diseased models.
  • RNPs ribonucleoproteins
  • STEP RNPs chemically modified RNPs, termed stimuli-responsive traceless engineering platform RNPs (STEP RNPs)
  • AST aspartate transaminase
  • ALT alanine transaminase
  • BUN blood urea nitrogen
  • STEP RNPs intracranial administration did not induce significant cellular damage to the brain based on microscopic imaging and by H&E staining. Storage of the STEP RNPs at - 20 o C over 2 months did not reduce their efficiency in genome editing.
  • the conjugate contains a structure: wherein: the dashed lines denote independently the presence or one or more covalent or non-covalent bonds, preferably the dashed lines denote the presence of one or more covalent bonds, P contains a protein, peptide, or nucleic acid; L is a linear or branched traceless or untraceless chemical linker; wherein when L is a linear or branched traceless chemical linker, L contains a stimuli-responsive chemical moiety and/or a self-immolative chemical moiety, wherein when L is a linear or branched untraceless chemical linker, L contains a substituted alkyl, unsubstituted alkyl, substituted alkylene, unsubstituted alkylene; M is a single-armed or multi-armed chemical moiety containing a cell membrane fusogenic molecule; and nl, nm, and nz are independently integers between 1 and 100, inclusive.
  • the values of nm and/or nz are selected such that the mole ratio of M to P ranges from 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the values of np, on the one hand, and nl, nm, and/or nz, on the other hand are selected such that the mole ratio of L-M to P ranges from 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the mole ratio can be determined using analytical methods (such as nuclear magnetic resonance spectroscopy) to analyze the final product.
  • the mole ratio can be a theoretical value based on the feed mole ratios of the reactants added to a reaction mixture to form the final product.
  • the ratio of the payload to membrane fusogenic moiety is between 1%:99% to 99%:1% by weight, 90%:10% to 97.5%:2.5% by weight, such as 50%:50% by weight or 95.6%:4.4% by weight. 45617673.1 3
  • the chemical linker is formed from: is In some forms, the conjugate contains a chemical linker formed from: is 24, kDa inserted between the cell membrane fusion moiety (e.g., the cholesterol moiety) and dibenzocyclooctyne (DBCO) group.
  • FIG.1 is a schematic of the stimuli-responsive traceless engineering (platform (STEP) for intracellular delivery of biologics payloads, such as protein payloads.
  • the schematic shows chemical modification of a payload with membrane fusogenic molecules through stimuli-responsive, self- immolative traceless linkers.
  • FIG.2A is a schematic diagram of the Ai9 loxP-flanked STOP reporter cassette.
  • FIGs.2B and 2C show the molecular structures of 11,12- didehydro- ⁇ -oxo-, 2-((2-(((4- nitrophenoxy)carbonyl)oxy)ethyl)disulfaneyl)ethyl ester (DBNPDEE) and cholesterol-PEG 24 -N 3 .
  • FIG.2D shows the non-limiting molecular structures of some selected STEP RNPs; n can be an integer from 1 to 10, with appropriate connectivities between the chemical moiety in the square brackets and the remainder of the STEP RNPs, as described in more detail below.
  • FIG.2E is a bar graph showing the characterization of the indicated chemically modified RNPs for genome editing.
  • FIG.3A shows the molecular structures of the indicated linkers.
  • FIG.3B is a bar graph showing the characterization of RNPs having surfaces modified with cholesterol through the indicated linkers for delivery of a genome editing machinery. RNPs were loaded with sgRNAs 276 and 280. Characterization was performed in Ai9 fibroblasts. Genome editing efficiency was determined based on the expression of tdTomato and expressed as percentage of that was achieved through DBNPDEE and cholesterol-PEG24-N3, which was defined as 100%.
  • FIGs.4A-4G show the molecular structures of the indicated membrane fusogenic molecules.
  • FIG.4H is a bar graph showing the characterization of RNPs having surfaces conjugated with the indicated 45617673.1 5 molecules through a linker, DBNPDEE, for delivery of a genome editing machinery in Ai9 fibroblasts. RNPs were loaded with sgRNAs 276 and 280. Genome editing efficiency was determined based on the expression of tdTomato and expressed as percentage of that was achieved through DBNPDEE and cholesterol-PEG 24 -N 3 , which was defined as 100%.
  • FIGs.5A-5G show the molecular structures of the indicated multi- arm, cholesterol-based fusogenic molecules.
  • FIG.5H is a bar graph showing the characterization of RNPs having surfaces conjugated with the indicated fusogenic molecules through DBNPDEE for delivery of a genome editing machinery in Ai9 fibroblasts.
  • RNPs were loaded with sgRNAs 276 and 280.
  • Genome editing efficiency was determined based on the expression of tdTomato and expressed as percentage of that was achieved through DBNPDEE and Cholesterol-PEG24-N3, which was defined as 100%.
  • FIGs.6A and 6B are bar graphs showing the size distribution of STEP RNPs determined by DLS analysis (FIG.6A), and quantification of the percentage of edited cells based on tdTomato expression (FIG.6B).
  • FIG.6B Ai9 fibroblasts were treated with STEP RNPs for 48 hours. Nuclei were stained with Hoechst33342. Edited cells showed tdTomato.
  • FIG.7A is a gel image of a Western blot analysis of STEP RNPs for AS treatment by observing Ube3a reactivation from paternal chromosome based on YFP expression in Ube3a-YFP reporter mice.
  • FIG.7B is a bar graph of semi-quantification of the data showing the reactivation persisted 90 days after STEP RNPs delivery in prefrontal cortex (PFC). Maternal Ube3a- YFP is a positive control.
  • FIGs.7C-7F are bar graphs (FIGs.7C-7E) and line graphs (FIGs.7F-7H) showing that delivery of STEP RNPs rescued abnormal behaviors of AS mouse model based on total travel distance in open field assay (FIGs.7C and 7H), center time in open field assay (FIG. 7D), percentage of new object recognition test (FIG.7E), and latency in rotarod test (FIGs.7F and 7G). ** p ⁇ 0.01,*** p ⁇ 0.001.
  • the order of the bars in FIG.7C corresponds with the order of those in FIGs.7D and 7E.
  • FIGs.8A and 8B are bar graphs showing the relative expression levels of Ube3a-ATS and Ube3a at different sections of the brain: prefrontal cortex (FIG.8A) and cerebrum (FIG.8B).
  • STEP RNPs were administered through either intracerebroventricular (ICV) or intrathecal (IT) 45617673.1 6 administration at a 40-ug dose in Ube3a mat-/pat+ mouse model pups.
  • ICV intracerebroventricular
  • IT intrathecal
  • the expression of Ube3a-ATS and Ube3a in different brain regions was examined 20-30 days post injection by qRT-PCR, quantitative immunoblot, and immunocytochemistry.
  • FIGs.9A-9D are a bar graph (FIG.9A) and line graphs (FIGs.9B- 9D) showing the effect of treatment with STEP RNPs loaded with an sgRNA (e.g., gRNA33) on rescuing neurological deficits in a Ube3a mat-/pat+ mouse model.
  • FIGs.9A and 9B show data for open field locomotor function and reduced anxiety tests;
  • FIGs.9C and 9D show data for the rotarod test. Closed circles: wild-type; closed squares: AS+gRNA33 treated; and closed triangles: AS+gRNA-control.
  • FIGs.10A and 10B show a schematic of a construct containing dCas9-TET4v (FIG.10A) and a gel image of the delivery of dCas9-TET4v via STEP (FIG.10B).
  • FIG.10B shows that delivery of dCas9-TET4v demethylated the histone modifications and turned on expression of Small Nuclear Ribonucleoprotein Polypeptide N (SNRPN).
  • FIGs.11A-11E are column graphs characterizing the toxicity (FIGs. 11A-11D) and stability (FIG.11E) of STEP RNPs. Solid bars: vehicle; unfilled bars: STEP RNPs.
  • FIGs.12A-12H are graphs showing the characterization of STEP RNPs H1-4 syndrome treatment.
  • FIG.12A is a line graph showing increased cellular prefoliation of H1-4 C-terminal frameshift tail (CFT) induced pluripotent stem cell (iPSC) and neural progenitor cells (NPCs).
  • FIG.12B is an image of mice showing growth retardation and lethality of H1-4 c.430G homozygous mice.
  • FIGs.12C and 12D are scatter plots showing that IT delivery of STEP RNPs rescued perinatal lethality in homozygotes of H1-4 C430G mice.
  • FIGs.12E-12G are gel images showing that H1-4 c430G-targeting ASO and RNPs effectively downregulated H1-4 in CFT.
  • FIG.12H is a line graph showing that in homozygous H1-4 430G mice, intrathecal administration of H1-4-targeting STEP RNPs rescued the perinatal lethality.
  • FIG.13A shows the molecular structures of cholesterol, F7- cholesterol and ⁇ -sitosterol.
  • FIGs.13B and 13C are column graphs quantifying the percentage of edited cells based on tdTomato expression. 45617673.1 7 Ai9 fibroblast cells were treated by traceless STEP Cas9/sgAi9 RNP at 2.5 ⁇ g/mL or 7.5 ⁇ g/mL. The cells were observed under fluorescence microscope at 48 h post treatment. The edited cells with tdTomato fluorescence (%) were quantified.
  • STEP Cas9/sgAi9 RNPs were prepared with STEP/Cas9 ratio of 10.
  • STEP Cas9/sgAi9 RNPs were prepared with STEP/Cas9 ratio of 20.
  • STEP/Cas9 ratio refers to the feed molar ratios of compounds used to form L-M to Cas9).
  • FIG.14A shows the chemical structures of exemplary traceless and untraceless chemical linkers used in Example 3.
  • FIG.14B is a column graph showing quantification of the editing activity in Ai9 fibroblast cells of STEP Cas9/sgAi9 RNP or untraceless Cas9/sgAi9 RNP at 5 ⁇ g/mL or 10 ⁇ g/mL. Cholesterol was used as the cell membrane fusogenic molecule in each conjugate. The cells were observed under fluorescence microscope at 48 h post treatment. The edited cells with tdTomato fluorescence (%) were quantified.
  • FIG.15 is a gel image of a Western blot analysis of STEP RNPs for Cas9 by observing Cas9 antibodies are various time points.
  • FIG.16A is a schematic diagram showing dCas9-Tet1CD (upper panel) and dCas9-JMJD2a (lower panel) targeting methylated CpG and H3K9me2/3, respectively.
  • FIG.16B is another schematic showing the loci of sgRNA binding to Prader-Willi Syndrome (PWS)-imprinting center or around the region including CpG islands. Maternal imprinted/silenced gene also shown.
  • PWS Prader-Willi Syndrome
  • FIGs.16C-16G are bar graphs of RT-qPCR analysis showing the reactivation of maternal imprinted SNRPN gene in human fibroblasts derived from PWS (paternal deletion of 15q11-q13) by RNP including dCas9-Tet1CD with sgRNA (FIG.16C) and RT-qPCR analysis showing the reactivation of imprinted SNRPN, SNORD116, and 116HG genes by dCas9- JMJD2a with sgRNA#4 in human fibroblasts derived from PWS (FIGs. 16D-16G).
  • FIGs.17A and 17B are schematic diagrams showing a mouse model carrying maternal Snrpn-EGFP gene as a reporter (FIG.17A) and the sgRNA binding site on mouse chromosome 7C (FIG.17B). 45617673.1 8 DETAILED DESCRIPTION OF THE INVENTION I. Definitions The term "amino acid” refers to a molecule containing both an amino group and a carboxyl group. Amino acids include alpha-amino acids and beta-amino acids. In certain forms, an amino acid is an alpha-amino acid. Amino acids can be natural or synthetic.
  • Amino acids include, but are not limited to, the twenty standard or canonical amino acids: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • Common non-standard or non-canonical amino acids include, but are not limited to, selenocysteine, ornithine, pyrrolysine, and N- formylmethionine.
  • the term “natural amino acid” refers to both the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letter abbreviations)).
  • synthetic amino acid “non-natural amino acid” and “unnatural amino acid,” are used interchangeably, and refer to an organic compound that has an amino group and a carboxyl group, and is not one of the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides. Generally, it mimics the reactivity of a natural amino acid due to the presence of the amino and carboxyl groups. “Synthetic amino acid,” “non-natural amino acid,” or “unnatural amino acid” also refers to an amino acid that is not produced by an organism without genetic engineering.
  • the synthetic amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., reactivity towards a desired molecule) when the synthetic amino acid is either substituted for a natural amino acid or incorporated into a peptide.
  • “Synthetic amino acid,” “non-natural amino acid,” or “unnatural amino acid” can also refer to a natural amino acid whose side chain has been chemically modified to include a reactive group (e.g.
  • alkyne azide; alkene; triarylphosphine; aminooxy; carbonyl; hydrazide; 45617673.1 9 sulfonyl chloride; maleimide; aziridine; -CN; acryloyl; acrylamide; sulfone; vinyl sulfone; cyanate; thiocyanate; isocyanate; isothiocyanate; alkoxysilane; dialkyl dialkoxysilane; diaryl dialkoxysilane; trialkyl monoalkoxysilane; vinyl silane; acetohydrazide; acyl azide; acyl halides; epoxide; glycidyl; carbodiimides; thiol; amine; phosphoramidate; vinyl ether; substituted hydrazine; an alkylene glycol bis(diester), e.g.
  • thioester e.g., alkyl thioester, ⁇ -thiophenylester
  • allyl thioester e.g., allyl thioacetate, allyl thioproprionate
  • “Chemical moiety” refers to a part of a molecule, such as an organic molecule. “Conjugate,” “conjugation,” and related terms, refer to the covalent or non-covalent linkage of a molecule to another molecule, or one part of a molecule to a different part of the same molecule. The linkage can involve covalent or non-covalent linkage. Covalent linkages can be direct or indirect (i.e., mediated via a linker). “Covalent linkage”, refers to a bond or organic moiety that covalently links molecules or different parts of the same molecule.
  • Non-covalent linkage includes electrostatic interactions, hydrogen bonding interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, ⁇ -stacking interactions, van der Waals interactions, magnetic interactions, and dipole-dipole interactions.
  • the terms “genome editing,” “genome engineering” or “genome mutagenesis” refer to selective and specific changes to one or more targeted genes or DNA sequences within a recipient cell, for example, via delivery of CRISPR-Cas system to the cell.
  • the editing or changing of a targeted gene or genome can include one or more of a deletion, knock-in, point mutation, substitution mutation or any combination thereof in one or more genes of the recipient cell.
  • single guide RNA or “sgRNA” refer to the polynucleotide sequence comprising the guide sequence, tracr sequence and the tracr mate sequence.
  • Guide sequence refers to the around 20 base pair 45617673.1 10 (bp) sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer.”
  • Cas9 “Cas9 protein,” or “Cas9 nuclease” refer to a RNA-guided endonuclease that is a Cas9 protein that catalyzes the site- specific cleavage of double stranded DNA.
  • CRISPR-associated nuclease is an adaptive immune system found in bacteria that provides protection against mobile elements such as phage viruses and transposable elements. DNA binding and cleavage requires the Cas9 protein and two RNAs, a trans- encoded RNA (tracrRNA) and a CRISPR RNA (crRNA) in nature. Artificially, single-guided RNA or sgRNA can be engineered to incorporate aspects of both RNAs into a single species (Jinek, et al. Science, 337, 816- 821, doi: 10.1126/science.1225829 (2012)).
  • the CRISPR system has two components: the Cas9 nuclease and a single guide RNA (sgRNA) that provides DNA sequence-targeting accuracy.
  • the targeting of the Cas9- sgRNA complex is mediated by the protospacer adjacent motif (PAM) located at the DNA for Cas9 recognition and the homology between the ⁇ 20- nucleotide recognition sequence encoded in the sgRNA and the genomic DNA target.
  • PAM protospacer adjacent motif
  • the targeted gene can be knocked out after the Cas9-sgRNA complex finds and cleaves the exonic region of the gene to generate frameshift mutations.
  • Cas9 recognizes short motifs in CRISPR repeat sequences to help distinguish self from non-self.
  • Cas9 nuclease sequences and structures are known to those of skill in the art (Ferretti, et al. Proc Natl Acad Sci U.S.A, 98, 4658-4863, doi: 10.1073/pnas.071559398 (2001); Deltcheva, et al. Nature, 471, 602-607, doi: 10.1038/nature09886 (2011)).
  • Cas9 orthologs have been described in several species of bacteria, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus, Campylobacter jejuni and Neisseria meningitidis. (Slaymaker, et al.
  • “Pharmaceutically acceptable,” refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.
  • a “pharmaceutically acceptable carrier,” refers to all components of a pharmaceutical formulation which facilitate the delivery of the composition in vivo.
  • Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Protein refers of a chain of amino acids having a length of between two and 50 amino acids in length.
  • Protein refers to a chain of amino acids having a length of greater than 50 amino acids, such as greater than 50 amino acids and less than thirty- six thousand amino acids.
  • Small molecule refers to an organic molecule that is less than about 2500 g/mol in molecular weight, less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some forms, small molecules are non-polymeric and/or non-oligomeric.
  • treating means to ameliorate, reduce or otherwise stop a disease, disorder, or condition from occurring or progressing in an animal which may be predisposed to the disease, disorder, and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder, or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an 45617673.1 12 analgesic agent even though such agent does not treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully “treated” if one or more symptoms associated with a genetic neuropathy, a genetic based musculopathy, a genetic eye disease or disorder, a genetic lung disease or disorder, a genetic liver disease or disorder, or cancer are mitigated or eliminated, including, but are not limited to, reducing and/or inhibiting rate of progress of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.
  • Compositions Conjugates and pharmaceutical compositions containing these conjugates have been developed for intracellular delivery of a payload.
  • the conjugates contain the payload, a chemical linker, and a cell membrane fusogenic molecule.
  • the cell membrane fusogenic molecule facilitates intracellular uptake of the conjugates.
  • the conjugate enters the cell through a non-endocytic pathway.
  • the chemical linker can be a traceless chemical linker or an untraceless chemical linker.
  • the chemical linker is a traceless chemical linker, it contains a stimuli-responsive chemical moiety and/or a self-immolative chemical moiety.
  • the traceless chemical linker contains a stimuli-responsive chemical moiety and a self-immolative chemical moiety.
  • the conjugate Upon entry into a cell, the conjugate is exposed to one or more stimuli, such as a reducing and/or an acidic environment, that cleave the stimuli-responsive chemical moiety.
  • This cleavage event activates self-immolation of the chemical linker via an electronic cascade and/or cyclization elimination.
  • the linkers are cleaved and removed from the payload, such that the payload is delivered without any trace of the cell membrane fusogenic molecule and chemical linker on the payload.
  • the payload is delivered in a stimuli-responsive traceless manner, i.e., stimuli-responsive traceless engineering of conjugates.
  • the conjugates display higher efficiency in genome editing compared to similar 45617673.1 13 conjugates that are non-cleavable.
  • untraceless as relates to a chemical linker, describes is a chemical linker that is non-cleavable or a linker that is cleaved and leaves a chemical moiety thereof covalently bonded to the payload.
  • Untraceless chemical linkers can be used to form “non- cleavable” conjugates as a subset, which are those with linkers that form covalent bonds with the payloads, wherein the covalent bonds between the linkers and the payloads cannot be cleaved in the reductive or acidic microenvironments inside a cell within 24 hours or 48 hours after cell penetration.
  • RNP ribonucleoproteins
  • the conjugates can safely deliver payloads, such as STEP RNPs, given that intravenous administration of payloads, such as STEP RNPs, did not induce significant systemic toxicity to the liver and kidney based on AST, ALT, BUN, and creatine assays. Further, intracranial administration of STEP RNPs did not induce significant cellular damage to the brain based on microscopic imaging and by H&E staining. Storage of the STEP RNPs at temperatures below zero (such as -20 o C), over extended periods of time (such as 2 months) did not reduce their efficiency in genome editing.
  • the conjugate contains a structure: wherein: 45617673.1 14 the dashed lines denote independently the presence or one or more covalent or non-covalent bonds, preferably the dashed lines denote the presence of one or more covalent bonds, P contains a protein, peptide, or nucleic acid; L is a linear or branched chemical linker containing a stimuli- responsive chemical moiety and/or a self-immolative chemical moiety, M is a single-armed or multi-armed chemical moiety containing a cell membrane fusogenic molecule, and np, nl, nm, and nz are independently integers between 1 and 150, inclusive, between 1 and 100, inclusive, between 1 and 75, inclusive, between 1 and 50, inclusive, between 1 and 25, inclusive, between 1 and 15, inclusive, between 1 and 10, inclusive, between 1 and 7, inclusive, or between 1 and 5, inclusive.
  • the values of np, on the one hand, and nm and/or nz, on the other hand are selected such that the mole ratio of M to P ranges from 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the values of np, on the one hand, and nl, nm, and/or nz, on the other hand are selected such that the mole ratio of L-M to P ranges from 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the mole ratio can be determined using analytical methods (such as nuclear magnetic resonance spectroscopy) to analyze the final product.
  • the mole ratio can be a theoretical value based on the feed mole ratios of the reactants added to a reaction mixture to form the final product.
  • the mole ratio for effective delivery of payload can be determined by a balance of mole ratios/hydrophobicity and efficiency. For instance, it is not always the case that the higher the M:P or L-M:P ratio the better. This is because most cell membrane fusogenic molecules are hydrophobic, and conjugation of too many cell membrane fusogenic molecules may increase cell penetration but cause toxicity and reduce conjugate solubility (or precipitate payloads).
  • the ratio of the payload to the membrane fusogenic moiety is expressed as a weight percent, i.e., weight of the indicated component to the sum of the weights of the payload and membrane fusogenic moiety. In some forms, the ratio of the payload to membrane fusogenic moiety is between 1%:99% to 45617673.1 15 99%:1% by weight, 90%:10% to 97.5%:2.5% by weight, such as 50%:50% by weight or 95.6%:4.4% by weight.
  • the conjugates contain a gene editing machinery as the payload, a multi-arm chemical moiety containing pentacyclic or tetracyclic moieties of cholesterol, and the following moiety within the chemical linker: wherein Y is oxygen, W is -(CH2)2-, and X is sulfur. Further details on the conjugates and formulations thereof are provided in the ensuing sections.
  • Payloads to be delivered The conjugates contain payloads that can be one or more proteins; peptides; nucleic acids such as mRNAs, sgRNAs, or DNAs; ribonucleoproteins; or a combination thereof.
  • the payloads are covalently conjugated to the chemical linker.
  • the payload to be delivered is one or more gene editing systems, or at least one or more components thereof.
  • Exemplary gene editing systems include, but are not limited to, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), meganucleases (MNs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, base editors (containing a catalytically impaired Cas protein fused to a DNA modifying enzyme), prime editors containing a catalytically impaired Cas protein (e.g., Cas9 nickase - a variant of Cas9 that nicks the DNA rather than generating double-strand breaks) fused to an engineered reverse transcriptase, peptide nucleic acids (PNAs), and anti-sense oligonucleotides.
  • ZFNs Zinc Finger Nucleases
  • TALENs Transcription Activator-Like Effector Nucleases
  • the gene editing system is the CRISPR/Cas system.
  • the gene editing technology is the donor oligonucleotide, which can be used be used alone to modify genes.
  • Strategies include, but are not limited to, small fragment homologous replacement (e.g., polynucleotide small DNA fragments (SDFs)), single-stranded oligodeoxynucleotide-mediated gene modification 45617673.1 16 (e.g., ssODN/SSOs) and other described in Sargent, Oligonucleotides, 21(2): 55–75 (2011)), and elsewhere.
  • SDFs polynucleotide small DNA fragments
  • ssODN/SSOs single-stranded oligodeoxynucleotide-mediated gene modification 45617673.1 16
  • Other suitable gene editing technologies include, but are not limited to, intron encoded meganucleases that are engineered to change their target specificity.
  • the gene editing system is a protein- guided gene editing system such as a CRISPR system, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN).
  • CRISPR/Cas the gene editing system that induces a single or a double strand break in the target cell’s genome is CRISPR/Cas, or a nucleic acid construct encoding the Cas nuclease.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing, or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819–823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • gene editing stress, enhancing, or changing specific genes
  • eukaryotes see, for example, Cong, Science, 15:339(6121):819–823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012).
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • tracr trans-activating CRISPR
  • tracrRNA or an active partial tracrRNA e.g., tracrRNA or an active partial tracrRNA
  • a tracr-mate sequence encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogen
  • One or more tracr mate sequences operably linked to a guide sequence can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.
  • pre-crRNA pre-CRISPR RNA
  • a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819– 823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • a single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)).
  • gRNA guide RNA
  • sgRNA single-guide RNA
  • the crRNA portion can be identified as the “target sequence” and the tracrRNA is often referred to as the “scaffold.”
  • the payload to be delivered is to one or more CRISPR-Associated Enzyme (Cas) nucleases.
  • Cas nucleases suitable as a gene editing composition include Cas9, CasX (also referred as Cas12e), Cas7-11, CasFx, Cas12a, and Cas13.
  • the payload to be delivered is one or more Cas nucleases, which are further complexed with one or more single guide RNA (sgRNA) to form CRISPR/Cas ribonucleoproteins (RNPs).
  • sgRNA single guide RNA
  • RNPs CRISPR/Cas ribonucleoproteins
  • a Cas nuclease is covalently conjugated to one or more traceless linkers, and one or more membrane fusogenic molecules, optionally via one or more linking moieties.
  • a Cas nuclease is covalently conjugated to one or more membrane fusogenic molecules via one or more traceless linkers.
  • one or more cholesterol molecules or pegylated forms are covalently conjugated to a Cas9 nuclease via traceless linkers such as DBNPDEE. 45617673.1 18
  • the payloads are one or more Cas9 nucleases, preferably complexed with one or more single guide RNA (sgRNA) to form CRISPR/Cas ribonucleoproteins (RNPs).
  • sgRNA single guide RNA
  • RNPs CRISPR/Cas ribonucleoproteins
  • a Cas nuclease is covalently conjugated to one or more traceless linkers, and one or more membrane fusogenic molecules, prior to or subsequent to complexing of Cas9 with sgRNA.
  • the Cas9 nuclease is Streptococcus pyogenes Cas9 nuclease, or variants thereof.
  • the payloads are one or more CRISPR–Cas-derived genome editing agents. Exemplary classes of CRISPR–Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases, and prime editors—are currently available for modifying genomes.
  • the payload contains a base editor.
  • Base editors are described in Komor, et al., Nature 2016, 533, 420-424; Gaudelli, et al., Nature 2017, 551, 464-471, Mok, et al., Nature 2020, 583, 631-637, and Koblan, et al., Nature 2021, 589 (7843), 608-614, the contents of which are hereby incorporated by reference.
  • Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating double-stranded DNA breaks.
  • Exemplary base editors are constructed by fusing a Cas9 nickase (nCas9) with a base- modifying enzyme.
  • CBEs cytosine base editors
  • ABEs adenine base editors
  • CGBEs C-to-G base editors
  • the payload contains a prime editor.
  • Prime editors are described in Anzalone, et al., Nature 2019, 576 (7785), 149-157, the contents of which are hereby incorporated by reference. Similar to CRISPR, prime editing requires the presence of a Cas endonuclease and a single guide (sg) RNA. However, as the premise of prime editing is to edit sequences without generating a double-stranded break, both components are slightly modified.
  • this method utilizes Cas9 nickase—a variant of Cas9 that nicks the DNA rather than generating double-strand breaks—fused to a reverse transcriptase.
  • This Cas9 fusion is referred to as a prime editor.
  • the payload contains both a base editor and a prime editor. 45617673.1 19
  • a deactivated Cas9 dCas9
  • a nuclease- deficient mutant variant of the Cas9 protein for example, point mutations (e.g., D10A, H840A) that inactivate the DNA cleavage activity of the Cas9 protein
  • point mutations e.g., D10A, H840A
  • CRISPR-Cas9 gene knockout system has also been adapted into gene modulation technologies collectively known as CRISPR modulation (CRISPRmod), which includes CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa).
  • CRISPR modulation CRISPRmod
  • CRISPRi CRISPR interference
  • CRISPRa CRISPR activation
  • CRISPRi nuclease-deactivated Cas9
  • CRISPRi nuclease-deactivated Cas9
  • CRISPRi utilizes dCas9 with or without fused repressor domains along with a guide RNA to target the promoter regions for transcriptional repression, or knockdown, of a gene.
  • CRISPRa employs dCas9 fused to transcriptional activation domains, which can be directed to promoter regions by one or more guide RNA(s) that recruit additional effectors for transcriptional activation and increased expression of the target gene.
  • CRISPRi and CRISPRa technologies generate artificial transcription factors by attaching an effector domain to dCas9 to silence or activate transcription.
  • CRISPRi or CRISPRa requires guide RNA designs in proximity to the gene’s promoter region or the transcriptional start site (TSS) to result in silencing or activation, respectively (L. A. Gilbert et al., Cell.159, 647–661 (2014); S. Konermann et al., Nature.517, 583–588 (2015)).
  • the payloads are CRISPR-based epigenome editing machinery.
  • An exemplary CRISPR-based epigenome editing machinery has been previously described (Nunez JK, et al., Cell.2021 Apr 29;184(9):2503- 2519).
  • the payloads are dCas9-TETv4 based RNPs, leading to efficient epigenetic editing of the target region(s).
  • the gene editing system that induces a single or a double strand break in the target cell’s genome is zinc finger nuclease (ZFN), or a nucleic acid construct encoding ZFN.
  • ZFNs are typically fusion 45617673.1 20 proteins that include a DNA-binding domain derived from a zinc-finger protein linked to a cleavage domain.
  • the most common cleavage domain is the Type IIS enzyme Fokl.
  • Fok1 catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat.
  • the DNA-binding domain which can, in principle, be designed to target any genomic location of interest, can be a tandem array of Cys2His2 zinc fingers, each of which generally recognizes three to four nucleotides in the target DNA sequence.
  • the Cys2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)- Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His.
  • Rational design includes, for example, using databases including triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos.6, 140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S.
  • the gene editing system that induces a single or a double strand break in the target cell’s genome is a transcription activator-like effector nuclease (TALEN), or a nucleic acid construct or constructs encoding TALEN.
  • TALENs have an overall architecture similar to that of ZFNs, with the main difference that the DNA-binding domain comes from TAL effector proteins, transcription factors from plant pathogenic bacteria.
  • the DNA-binding domain of a TALEN is a tandem array of amino acid repeats, each about 34 residues long.
  • the repeats are very similar to each other; typically, they differ principally at two positions (amino acids 12 and 13, called the repeat variable diresidue, or RVD).
  • RVD specifies preferential binding to one of the four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, though the NN RVD is known to bind adenines in addition to guanine.
  • TAL effector DNA binding is mechanistically less well understood than that of zinc-finger proteins, but their seemingly simpler code could prove very beneficial for engineered-nuclease design.
  • TALENs also cleave as dimers, have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and appear to have less stringent requirements than ZFNs for the length of the spacer between binding sites.
  • Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats. Methods of engineering TAL to bind to specific nucleic acids are described in Cermak, et al, Nucl. Acids Res.1-11 (2011). U.S. Published Application No.2011/0145940, which discloses TAL effectors and methods of using them to modify DNA. Miller et al.
  • TALENs for site-specific nuclease architecture by linking TAL truncation variants to the catalytic domain of Fokl nuclease.
  • the resulting TALENs were shown to induce gene modification in immortalized human cells.
  • General design principles for TALE binding domains can be found in, for example, WO 2011/072246. 45617673.1 22 (b)
  • Other biomolecular payloads can also include molecules such as therapeutic, diagnostic, and/or prophylactic proteins or peptides.
  • Proteins or peptides include, but are not limited to transcription factors, enzymes, peptide nucleic acids, antibodies and fragments thereof such as monoclonal and polyclonal antibodies, single chain antibodies, affibodies, single chain variable fragments (scFv), di-scFv, tri-scFv, diabody, triabody, teratbody, disulfide- linked Fvs (sdFv), Fab', F(ab')2, Fv, single domain antibody fragments (sdAb).
  • sdFv single chain variable fragments
  • sdFv single chain variable fragments
  • Fab' F(ab')2, Fv, single domain antibody fragments
  • sdAb single domain antibody fragments
  • the stimuli-responsive chemical moieties can be cleaved by a stimulus selected from pH (such as change in pH), redox (such as change in redox potential), reactive oxygen species (ROS), enzyme (such as over-expression of an enzyme (e.g., a protease, esterase, etc.) due to a diseased state or disorder), ionic strength (such as a change in ionic strength), and hypoxia.
  • a stimulus selected from pH (such as change in pH), redox (such as change in redox potential), reactive oxygen species (ROS), enzyme (such as over-expression of an enzyme (e.g., a protease, esterase, etc.) due to a diseased state or disorder), ionic strength (such as a change in ionic strength), and hypoxia.
  • ROS reactive oxygen species
  • Examples of stimuli-responsive chemical moieties that are responsive to ROS are described in Saravanakumar, et al., Adv. Sci.2017, 4, 1600124, the
  • the stimuli-responsive chemical moieties independently contain a disulfide bond, an amide bond, an orthoester, a hydrazone, a hydrazide, a hydrazine, an imine (such as aldimine or ketoimine), an oxime, an acetal group, a vinyl ether, a polyketal, a methyl maleate, an ester bond, a nitroaryl group (e.g., nitrobenzyl), a nitroheteroaryl (e.g, nitroimidazole), a quinone group, an azoaryl group (e.g., azophenyl), an azoheteroaryl group (e.g., azopyridinyl), peroxalate ester, aminoacrylate, alkyl thioether or selenide (e.g., monoselenide bond, diselenide bond, etc.), thioketal, peroxalate ester, or a di
  • the stimuli-responsive chemical moieties are independently a disulfide bond, an amide, an orthoester, an imine (such as aldimine or ketoimine), a hydrazone, a hydrazide, a hydrazine, an imine, an 45617673.1 23 oxime, a methyl maleate, an ester bond, a dimethyl maleate, or a combination thereof.
  • the stimuli-responsive chemical moieties independently contain a disulfide bond, an amide, an orthoester, an imine (such as aldimine or ketoimine), or an ester bond.
  • the orthoester contains O O O Rx' O O Rx and Rx’ are independently hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted C 3 -C 20 cyclyl, substituted C3-C20 cyclyl, unsubstituted C1-C20 heterocyclyl, or substituted C 3 -C 20 heterocyclyl.
  • Non-limiting examples of chemical structures that can be used to generate linkers containing stimuli-responsive chemical moieties are shown in FIG.3A.
  • (b) Self-immolative chemical moieties The self-immolative chemical moieties described above are chemical moieties that self-degrade through one or more elimination processes.
  • self-degradation occurs via an electronic cascade process and/or cyclization elimination.
  • the self-degradation can be triggered upon cleavage of a covalent bond and/or by reduction of a chemical group (e.g., nitro-) in the self-immolative chemical moiety.
  • a chemical group e.g., nitro-
  • self-degradation is spontaneous and irreversible upon cleavage of a covalent bond. Therefore, as used herein a self-immolative chemical moiety is a moiety that contains 45617673.1 24 covalently linked atoms designed to degrade spontaneously in response to a stimulus. The degradation typically involves cleavage of one or more chemical bonds, preferably two or more chemical bonds.
  • the self-immolative chemical moiety shares an atom or chemical group and a bond with the stimuli-responsive chemical moiety such that cleavage of the bond results in the atom having a negative charge or increased ability to donate a lone pair of electrons.
  • This atom or chemical group triggers self-immolation.
  • the following schematic illustrates a cleavage-triggered self-immolation showing the sharing of an atom or chemical group and a bond between a stimuli-responsive chemical moiety and a self-immolative chemical moiety.
  • Scheme 1 A non-limiting example of self-immolation via cyclization elimination
  • M represents a single-armed or multi-armed chemical moiety containing a cell membrane fusogenic molecule
  • the linker contains a stimuli-responsive chemical moiety (SR) and a self-immolative chemical moiety (SE)
  • P represents a protein, peptide, or nucleic acid.
  • the black circle is M
  • the grey circle is P, and vice versa.
  • the stimuli-responsive chemical moiety and the self-immolative chemical moiety share a sulfur atom and a bond. The cleavage of this bond 45617673.1 25 triggers a cascade of self-immolation via cyclization elimination.
  • Self-immolative chemical moieties contain p-aminobenzyl groups (e.g., -para-NH-phenyl-CH 2 -), o-aminobenzyl groups (e.g., -ortho-NH- phenyl-CH2-), p-oxybenzyl groups (e.g., -para-O-phenyl-CH2-), o- aminobenzyl groups (e.g., -ortho-O-phenyl-CH 2 -), p-thiobenzyl groups (e.g., -para-S-phenyl-CH2-), o-thiobenzyl groups (e.g., -ortho-S-phenyl-CH2-), cinnamyl ethers, cyclization-driven moieties, Grob fragmentation moieties, etc.
  • p-aminobenzyl groups e.g., -para-NH-phenyl-CH 2 -
  • the cyclization-driven moieties contain the structure: wherein X can be O, NH, or S; Y can be O, NRs, substituted alkyl, or unsubstituted alkyl; and W can be substituted alkyl or unsubstituted alkyl; wherein Rs is hydrogen, substituted alkyl, or unsubstituted alkyl.
  • Y is oxygen.
  • X is sulfur.
  • W is C 2 -C 5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl, or C 5 substituted alkyl.
  • Y is oxygen
  • W is C2-C5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl, or C 5 substituted alkyl
  • X is sulfur.
  • the chemical linker is formed from: L is formed using a structure selected from: , 45617673.1 26 , , , , or a combination thereof.
  • the chemical linker is formed from: . Chemical structures containing self-immolative chemical moieties that can be used to generate suitable linkers are described in Ferhati, et al., Org. Lett.2021, 23, 8580-8584 and Gavriel, et al., Poly.
  • Untraceless chemical linkers In some forms, the payload and the cell membrane fusogenic molecule are conjugated to each other through an untraceless chemical 45617673.1 27 linker. Untraceless chemical linkers are chemical linkers that form covalent bonds with the payloads, wherein the covalent bonds between the chemical linkers and the payloads cannot be cleaved in the reductive or acidic microenvironments inside a cell within 24 hours or 48 hours after cell penetration, or chemical linkers that are cleaved and leave a chemical moiety thereof covalently bonded to the payload.
  • untraceless chemical linkers do not contain a disulfide bond or are formed from bifunctional molecules that do not contain a disulfide bond.
  • the untraceless chemical linker is formed from using a structure selected from: 4 5617673.1 29 (iv) Membrane fusion molecules (membrane fusogenic molecules)
  • the membrane fusogenic molecules are cell membrane fusogenic molecules. These cell membrane fusogenic molecules contain proteins, peptides, lipids, and/or small molecules. Preferably, these cell membrane fusogenic molecules enhance fusion between the conjugate and a cell membrane and/or facilitate intracellular uptake of the conjugate.
  • cell-penetrating peptides also known as cell permeable peptides, protein transduction domains (PTDs), membrane translocating sequences (MTSs) and Trojan peptides.
  • Cell penetrating peptides include, but are not limited to, virus-derived or mimicking polymers such as TAT, influenza fusion peptide, rabies virus glycoprotein fragment (RVG), neuropilin, penetratin, and polyarginines. Anaspec has commercially available CPPs. Further examples of cell penetrating peptides and motifs are described in, for example, U.S. Patent Application Publication Nos.
  • CPPs include: CRGDKGPD (SEQ ID NO:1), YGRKKRRQRRR (SEQ ID NO:2), AAVALLPAVLLALLAP (SEQ ID NO:3), PIEVCMYREP (SEQ ID NO:4), RQIKIWFQNRRMKWKK (SEQ ID NO:5), LLIILRRRIRKQAHAHSK (SEQ ID NO:6), AGYLLGKINLKALAALAKKIL (SEQ ID NO:7).
  • Examples of additional CPPs are described in Qin, et al., Mol. Pharmacol. 2017, 92, 219-231, and Chang, et al., J. Drug Target.2016, 24(6), 475-491.
  • Examples of lipids that can be utilized as cell membrane fusogenic molecules include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; phosphatidylethanolamine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16-PC); 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (18-PC); 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or other related phosphatidylethanolamine with two attached fatty acyl chains, preferably unsaturated fatty acyl chains; lysolipids, etc.
  • DOPE 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine
  • the cell membrane fusogenic molecule contains proteins; and/or peptides such as cell-penetrating peptides.
  • the cell membrane fusogenic molecule contains small molecule moieties such as pentacyclic or tetracyclic moieties of cholesterol, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens, or progestogens, phytosterols (e.g., ⁇ -sitosterol).
  • the cell membrane fusogenic molecule contains small molecule moieties such as pentacyclic or tetracyclic moieties of cholesterol.
  • the portion of the conjugate containing the cell membrane fusogenic molecule is a single-armed chemical moiety.
  • the portion of the conjugate containing the cell membrane fusogenic molecule is multi-armed, such that that portion contains a plurality of the cell membrane fusogenic molecules.
  • the portion of the conjugate containing the cell membrane fusogenic molecule is a multi-armed chemical moiety containing 2 to 10, 2 to 9, 2 to 8, or 2 to 7 small molecule moieties such as tetracyclic or pentacyclic moieties of cholesterol.
  • the portion of the conjugate containing the cell membrane fusogenic molecule is a multi-armed chemical moiety comprising 2 to 7, preferably 2, small molecule moieties such as tetracyclic or pentacyclic moieties of cholesterol.
  • the portion of the conjugate containing the cell membrane fusion molecule is formed from: H N O H H or 45617673.1 31 N H H N3 O some a between L and M.
  • the hydrophilic polymer is involved in covalent bonding between L and M.
  • the hydrophilic polymer is bonded directly to L.
  • the hydrophilic polymer is associated with M. Without wishing to be bound by theory, it is believed that the presence of this hydrophilic polymer improves the solubility of the conjugate.
  • Suitable hydrophilic polymers that can be included in the hydrophilic polymer segment include, but are not limited to, polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG); polysaccharides such as celluloses, alginates, glucosaminoglycans, and dextrans; hydrophilic polypeptides and poly(amino acids) such as poly-L- glutamic acid, gamma-polyglutamic acid, poly-L-aspartic acid, and poly-L- serine; poly(oxyethylated polyol); poly(olefinic alcohol) such as poly(vinyl alcohol) and aminoacetalized poly(vinyl alcohol); poly(N-vinylpyrrolidone); acrylic or acrylate, and alkacrylic or alkacrylate polymers such as 4 5617673.1 32 poly(acrylic acid), poly(methacrylic acid), poly(hydroxyethyl acrylate); poly(N,N-dimethyla
  • the hydrophilic polymer segment contains a neutral hydrophilic polymer, such as a neutral uncharged hydrophilic polymer.
  • neutral uncharged hydrophilic polymers include, but are not limited to, polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG); polysaccharides such as celluloses and dextrans; hydrophilic polypeptides and poly(amino acids) such as poly-L-serine; poly(oxyethylated polyol); poly(olefinic alcohol) such as poly(vinyl alcohol); poly(N-vinylpyrrolidone); poly(hydroxyethyl acrylate); poly(hydroxyalkyl methacrylate), e.g., poly(hydroxyethyl methacrylate).
  • PEG polyethylene glycol
  • polysaccharides such as celluloses and dextrans
  • hydrophilic polypeptides and poly(amino acids) such as poly-L-serine
  • poly(oxyethylated polyol) such as poly(oxyethylated polyol
  • poly(olefinic alcohol) such as poly(
  • the hydrophilic polymer segment contains a neutral uncharged hydrophilic polymer, such as polyalkylene glycols and polyalkylene oxides such as polyethylene glycol.
  • the hydrophilic polymer has a molecular weight between preferably wherein the hydrophilic polymer has a molecular weight between 100 Da and 10 kDa, inclusive, or between 200 Da and 10 kDa, inclusive.
  • the hydrophilic polymer segment contains a neutral uncharged hydrophilic polymer, such as polyalkylene glycols and polyalkylene oxides such as polyethylene glycol, having a molecular weight between 100 Da and 10 kDa, inclusive, or between 200 Da and 10 kDa, inclusive.
  • L-M is formed from reacting the following two moieties: 45617673.1 33 and , where n is such that the poly(ethylene glycol) has a molecular weight of 200 Da to 10 kDa.
  • the alkyne group in the DBCO moiety reacts with the azide to form a triazole through a click reaction, such that the poly(ethylene glycol) with the molecular weight of 200 Da to 10 kDa is inserted between the cell membrane fusion moiety (e.g., the cholesterol moiety shown above) and DBCO group.
  • a payload can be reacted with the p- nitrophenyl carbonate (NPC) moiety to attach the payload to the rest of the conjugate.
  • NPC p- nitrophenyl carbonate
  • a non-limiting example of a conjugate with L-M in this configuration is shown below: . or more of the hydrophilic polymers described above are absent between L and M. These can be achieved in instances where n is zero and/or the oxygen atom in the moiety -(OCH2CH2)- is absent or a CRLRL’ moiety, CRLRL’ where RL and RL’ are independently hydrogen, substituted alkyl, or unsubstituted alkyl.
  • the conjugate is as described above, except that the mole ratio of the cell membrane fusion molecule to the payload, or the cell membrane fusion protein, on the one hand, to the chemical linker and payload, on the other hand, ranges from 100:1 to 1:1, such as 100:1, 50:1, 4 5617673.1 34 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the conjugate is as described above, except that the mole ratio of the cell membrane fusion molecule and linker to the payload ranges from 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1, or 1:1.
  • the mole ratio can be determined using analytical methods (such as nuclear magnetic resonance spectroscopy) to analyze the final product.
  • the mole ratio can be a theoretical value based on the feed mole ratios of the reactants added to a reaction mixture to form the final product.
  • the conjugates can be synthesized using a variety of methods known to those of skill art including, but not limited to, chemical synthesis, semisynthesis, or a combination thereof. Preferably, the conjugates are produced via chemical synthesis.
  • RNPs are assembled through incubation of Cas9 protein with sgRNA (at a mole ratio such as 1:3). Next, DSC-cholesterols(72) can be added ((at a mole ratio such as 3:1 to RNPs).
  • cls-RNPs After removal of unreacted DSC-cholesterols(2) and sgRNA, cls-RNPs can be obtained.
  • a precursor linker molecule such as DBCO-DSC DBNPDEE can be mixed together with an azide-functionalized Tris derivative terminated with two cholesterols.
  • the alkyne group in the DBCO moiety can react with the azide to form a triazole through a click reaction.
  • Methods of Use Methods of using the disclosed conjugates and compositions are described.
  • the compositions can enable editing in the context of prokaryotic and eukaryotic cells, in vitro, ex vivo, and in vivo.
  • the compositions can enable gene editing in agricultural contexts, such as in plants.
  • Methods of Intracellular Delivery of Gene Editing Platforms The compositions can be used to ex vivo or in vivo gene editing.
  • the methods typically include contacting a cell with an effective amount of the disclosed composition to modify the cell’s genome. As discussed in more detail below, the contacting can occur ex vivo or in vivo.
  • the method includes contacting a population of target cells 45617673.1 35 with an effective amount of gene editing composition to modify the genomes of a sufficient number of cells to achieve a desired result e.g., therapeutic outcome or modified traits.
  • the effective amount or therapeutically effective amount can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder.
  • the effective amount can include a dosage of the conjugate between 0.1 mg and 100 mg, inclusive, 0.1 mg and 90 mg, inclusive, 0.1 mg and 80 mg, inclusive, 0.1 mg and 70 mg, inclusive, 0.1 mg and 60 mg, inclusive, 0.1 mg and 50 mg, inclusive, 0.1 mg and 40 mg, inclusive, 5 mg and 70 mg, inclusive, 5 mg and 60 mg, inclusive, 5 mg and 50 mg, inclusive, 5 mg and 40 mg, inclusive, 10 mg and 70 mg, inclusive, 10 mg and 60 mg, inclusive, 10 mg and 50 mg, inclusive, 10 mg and 40 mg, inclusive, 15 mg and 70 mg, inclusive, 15 mg and 60 mg, inclusive, 15 mg and 50 mg, inclusive, 15 mg and 40 mg, inclusive, 20 mg and 70 mg, inclusive, 20 mg and 60 mg, inclusive, 20 mg and 50 mg, inclusive, 20 mg and 40 mg, inclusive, 25 mg and 70 mg, inclusive, 25 mg and 60 mg, inclusive, 25 mg and 50 mg, inclusive, 25 mg and 40 mg, inclusive, 30 mg and 70 mg, inclusive, 30 mg and 60 mg, inclusive, 30 mg
  • the effective amount includes a dosage of the conjugate between 20 mg and 50 mg, inclusive, 25 mg and 40 mg, inclusive, 30 mg and 45 mg, inclusive, or 30 mg and 40 mg, inclusive.
  • Formulation is made to suit the mode of administration.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions containing the nucleic acids. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, clinical symptoms etc.). Exemplary symptoms, pharmacologic, and physiologic effects are discussed in more detail below.
  • compositions can be administered or otherwise contacted with target cells once, twice, or three times daily; one, two, three, four, five, six, seven times a week, one, two, three, four, five, six, seven or eight times a month.
  • the composition is administered every two or three days, or on average about 2 to about 4 times a week.
  • the compositions are administered in an amount effective to induce gene modification in at least one target allele to occur at frequency of at least 0.1, 0.2.0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of target cells.
  • gene modification occurs in at least one target allele at a frequency of about 0.1-25%, or 0.5-25%, or 1-25% 2-25%, or 3-25%, or 4-25% or 5-25% or 6- 25%, or 7-25%, or 8-25%, or 9-25%, or 10-25%, 11-25%, or 12-25%, or 13%-25% or 14%-25% or 15-25%, or 2-20%, or 3-20%, or 4-20% or 5-20% or 6-20%, or 7-20%, or 8-20%, or 9-20%, or 10-20%, 11-20%, or 12-20%, or 13%-20% or 14%-20% or 15-20%, 2-15%, or 3-15%, or 4-15% or 5-15% or 6-15%, or 7-15%, or 8-15%, or 9-15%, or 10-15%, 11-15%, or 12-15%, or 13%-15% or 14%-15%.
  • gene modification occurs in at least one target allele at a frequency of about 0.1% to about 10%, or about 0.2% to about 10%, or about 0.3% to about 10%, or about 0.4% to about 10%, or about 0.5% to about 10%, or about 0.6% to about 10%, or about 0.7% to about 10%, or about 0.8% to about 10%, or about 0.9% to about 10%, or about 1.0% to about 10% , or about 1.1% to about 10%, or about 1.1% to about 10%, 1.2% to about 10%, or about 1.3% to about 10%, or about 1.4% to about 10%, or about 1.5% to about 10%, or about 1.6% to about 10%, or about 1.7% to about 10%, or about 1.8% to about 10%, or about 1.9% to about 10%, or about 2.0% to about 10%, or about 2.5% to about 10% , or about 3.0% to about 10%, or about 3.5% to about 10%, or about 4.0% to about 10%, or about 4.5% to about 10%, or about 5.0% to about 10%.
  • gene modification occurs with low off-target effects.
  • off-target modification is undetectable using routine analysis.
  • off-target incidents occur at a 45617673.1 37 frequency of 0-1%, or 0-0.1%, or 0-0.01%, or 0-0.001%, or 0-0.0001%, or 0- 0000.1%, or 0-0.000001%.
  • off-target modification occurs at a frequency that is about 10 2 , 10 3 , 10 4 , or 10 5 -fold lower than at the target site.
  • the methods include a step of selecting a subject who is likely to benefit from treatment with the disclosed gene editing compositions.
  • ex vivo gene therapy of cells is used for the treatment of a genetic disorder in a subject.
  • cells are isolated from a subject and contacted ex vivo with the compositions to produce cells containing mutations in or adjacent to genes.
  • the cells are isolated from the subject to be treated or from a syngeneic host.
  • Target cells are removed from a subject prior to contacting with a gene editing composition.
  • the cells can be hematopoietic progenitor or stem cells.
  • the target cells are CD34 + hematopoietic stem cells.
  • HSCs Hematopoietic stem cells
  • CD34+ cells are multipotent stem cells that give rise to all the blood cell types including erythrocytes. Therefore, CD34+ cells can be isolated from a patient with, for example, thalassemia, sickle cell disease, or a lysosomal storage disease, the mutant gene altered or repaired ex-vivo using the compositions and methods, and the cells reintroduced back into the patient as a treatment or a cure.
  • Stem cells can be isolated and enriched by one of skill in the art. Methods for such isolation and enrichment of CD34 + and other cells are known in the art and disclosed, for example, in U.S.
  • compositions enriched in hematopoietic progenitor and stem cells “enriched” indicates a proportion of a desirable element (e.g., hematopoietic progenitor and stem cells) which is higher than that found in the natural source of the cells.
  • a composition of cells may be enriched over a natural source of the cells by at least one order of magnitude, preferably two or three orders, and more preferably 10, 100, 200 or 1000 orders of magnitude.
  • CD34 + cells can be recovered from cord blood, bone marrow or from blood after cytokine mobilization effected by injecting the donor with hematopoietic growth factors such as granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) subcutaneously or intravenously in amounts sufficient to cause movement of hematopoietic stem cells from the bone marrow space into the peripheral circulation.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte-monocyte colony stimulating factor
  • SCF stem cell factor
  • bone marrow cells may be obtained from any suitable source of bone marrow, e.g., tibiae, femora, spine, and other bone cavities.
  • an appropriate solution may be used to flush the bone, which solution will be a balanced salt solution, conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5 to 25 mM.
  • Convenient buffers include Hepes, phosphate buffers, lactate buffers, etc.
  • Cells can be selected by positive and negative selection techniques. Cells can be selected using commercially available antibodies which bind to hematopoietic progenitor or stem cell surface antigens, e.g., CD34, using methods known to those of skill in the art. For example, the antibodies may be conjugated to magnetic beads and immunogenic procedures utilized to recover the desired cell type.
  • FACS fluorescence activated cell sorting
  • progenitor or stem cells can be characterized as being any of CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CD19-, CD20-, CD33-, Class II HLA + and Thy-1 + .
  • progenitor or stem cells may be propagated by growing in any suitable medium.
  • progenitor or 45617673.1 39 stem cells can be grown in conditioned medium from stromal cells, such as those that can be obtained from bone marrow or liver associated with the secretion of factors, or in medium including cell surface factors supporting the proliferation of stem cells.
  • Stromal cells may be freed of hematopoietic cells employing appropriate monoclonal antibodies for removal of the undesired cells.
  • the isolated cells are contacted ex vivo with the disclosed gene editing compositions in amounts effective to cause the desired mutations in or adjacent to genes in need of repair or alteration, for example the human beta-globin or ⁇ -L-iduronidase gene.
  • Methods for transfection of cells with oligonucleotides and peptide nucleic acids are well known in the art (Koppelhus, et al., Adv. Drug Deliv. Rev., 55(2): 267-280 (2003)). It may be desirable to synchronize the cells in S-phase to further increase the frequency of gene correction.
  • the modified cells can be maintained or expanded in culture prior to administration to a subject.
  • Culture conditions are generally known in the art depending on the cell type. Conditions for the maintenance of CD34 + in particular have been well studied, and several suitable methods are available.
  • a common approach to ex vivo multi-potential hematopoietic cell expansion is to culture purified progenitor or stem cells in the presence of early-acting cytokines such as interleukin-3.
  • TPO thrombopoietin
  • SCF stem cell factor
  • Flt-3L flt3 ligand
  • cells can be maintained ex vivo in a nutritive medium (e.g., for minutes, hours, or 3, 6, 9, 13, or more days) including murine prolactin-like protein E (mPLP-E) or murine prolactin-like protein F (mPIP-F; collectively mPLP- E/IF) (U.S. Patent No.6,261,841).
  • a nutritive medium e.g., for minutes, hours, or 3, 6, 9, 13, or more days
  • mPLP-E murine prolactin-like protein E
  • mPIP-F murine prolactin-like protein F
  • the modified hematopoietic stem cells are differentiated ex vivo into CD4 + cells culture using specific combinations of interleukins and growth factors prior to administration to a subject using methods well known in the art.
  • the cells may be expanded ex vivo in large numbers, preferably at least a 5-fold, more preferably at least a 10-fold and even more preferably at least a 20-fold expansion of cells compared to the original population of isolated hematopoietic stem cells.
  • cells for ex vivo gene therapy the cells to be used can be dedifferentiated somatic cells. Somatic cells can be reprogrammed to become pluripotent stem-like cells that can be induced to become hematopoietic progenitor cells.
  • the hematopoietic progenitor cells can then be treated with the disclosed gene editing compositions above with respect to CD34 + cells to produce recombinant cells having one or more modified genes.
  • Representative somatic cells that can be reprogrammed include, but are not limited to, fibroblasts, adipocytes, and muscles cells.
  • Hematopoietic progenitor cells from induced stem-like cells have been successfully developed in the mouse (Hanna, J. et al. Science, 318:1920- 1923 (2007)).
  • somatic cells are harvested from a host.
  • the somatic cells are autologous fibroblasts.
  • the cells are cultured and transduced with vectors encoding Oct4, Sox2, Klf4, and c-Myc transcription factors.
  • the transduced cells are cultured and screened for embryonic stem cell (ES) morphology and ES cell markers including, but not limited to,AP, SSEA1, and Nanog.
  • ES embryonic stem cell
  • the transduced ES cells are cultured and induced to produce induced stem-like cells.
  • Cells are then screened for CD41 and c-kit markers (early hematopoietic progenitor markers) as well as markers for myeloid and erythroid differentiation.
  • the modified hematopoietic stem cells or modified induced hematopoietic progenitor cells are then introduced into a subject.
  • Delivery of the cells may be affected using various methods and includes most 45617673.1 41 preferably intravenous administration by infusion as well as direct depot injection into periosteal, bone marrow and/or subcutaneous sites.
  • the subject receiving the modified cells may be treated for bone marrow conditioning to enhance engraftment of the cells.
  • the recipient may be treated to enhance engraftment, using a radiation or chemotherapeutic treatment prior to the administration of the cells.
  • the cells Upon administration, the cells will generally require a period of time to engraft. Achieving significant engraftment of hematopoietic stem or progenitor cells typically takes weeks to months.
  • the cells to be administered to a subject will be autologous, e.g., derived from the subject, or syngeneic.
  • the guide RNA enables specific gene editing for ex vivo cell therapies, including CAR-T, CAR-NK, and CAR- macrophage editing.
  • the guide RNA enables knockout and knock-in of one of more genes or gene segments in iPSC, ESC, mesenchymal stem cell, and stem-derived cell lines.
  • the compositions can be administered directly to a subject for in vivo gene therapy.
  • the compositions and methods are suitable for treatment of one or more diseases and conditions in the brain, neurons, eye, lung, heart, kidneys, liver, etc.
  • the compositions can be used for mutagenic repair that may restore the DNA sequence of the target gene to normal.
  • the target sequence can be within the coding DNA sequence of the gene or within an intron.
  • the target sequence can also be within DNA sequences that regulate expression of the target gene, including promoter or enhancer sequences.
  • the compositions are especially useful to treat monogenic diseases and polygenic diseases, where one or more sgRNA constructs are delivered together in the same or separate compositions.
  • the oligonucleotide is useful for causing a mutation that inactivates the gene and terminates or reduces the uncontrolled proliferation of the cell.
  • the oligonucleotide is also a useful anti-cancer agent for activating a repressor gene that has lost its ability to repress proliferation.
  • the target gene can also be a gene that encodes an immune regulatory factor, such as Programmed cell death protein 1 (PD-1), in order to enhance the host’s immune response to a cancer.
  • PD-1 Programmed cell death protein 1
  • the gene modification technology can be designed to reduce or prevent expression of PD-1, and administered in an effective amount to do so. Therefore, in some embodiments, compositions are used to treat cancer.
  • compositions can be used as antiviral agents, for example, when designed to modify a specific a portion of a viral genome necessary for proper proliferation or function of the virus.
  • the subject to be treated is a human.
  • the subject to be treated is a child, or an infant. All the methods can include the step of identifying and selecting a subject in need of treatment, or a subject who would benefit from administration with the described compositions.
  • Neurological and muscular diseases or 45617673.1 43 disorders In some forms, the disease or disorder to be treated is a genetic neuropathy.
  • the genetic neuropathy can be degenerative or non- degenerative.
  • the disease or disorder to be treated is a genetic based musculopathy.
  • the musculopathy can be degenerative or non-degenerative.
  • Examples include, but are not limited to, dystonia, amyotrophic lateral sclerosis, muscular atrophies, muscular dystrophies (such as Duchenne, Becker, facioscapulohumeral, myotonic, congenital, distal, Emery-Dreifuss, oculopharyngeal, and limb girdle), congenital myopathies (such as central core, Myotubular, Nemaline, Ullrich/Bethlem, and RyR1), or metabolic disorders (such as Pompe’s disease). Genetic neuropathies, musculopathies, and gene therapies thereof are reviewed in Martier and Konstantinova, Front.
  • the gene editing machineries can be used for the treatment of genetic eye diseases or disorders. These include, but are not limited to Retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber congenital amaurosis. In some forms, the gene editing machineries can be used for the treatment of genetic lung diseases or disorders. These can be diseases or disorders that affect primarily the lungs or other organs including the lungs.
  • cystic fibrosis include, but are not limited to cystic fibrosis, primary ciliary dyskinesia, alpha-1-antitrypsin deficiency, surfactant metabolism dysfunction 1-4, familial pulmonary fibrosis, pulmonary alveolar microlithiasis, Dyskeratosis congenita, neurofibromatosis type I, tuberous 45617673.1 44 sclerosis/LAM, Birt-Hogg-Dubé Syndrome, Hyper IgE syndrome, Hermansky-Pudlak syndrome, Gaucher disease type I, Niemann-Pick disease type B, and Lysinuric protein intolerance.
  • the gene editing machineries can be used for the treatment of genetic liver diseases or disorders.
  • the target cells are cancer cells.
  • compositions of the disclosed gene editing compositions are administered to a subject having a proliferative disease, such as a benign or malignant tumor.
  • the subjects to be treated have been diagnosed with stage I, stage II, stage III, or stage IV cancer.
  • Composition that may be delivered to cancer cells include, but are not limited to, constructs for the expression of one or more pro-apoptotic factors, immunogenic factors, or tumor suppressors; gene editing compositions, inhibitory nucleic acids that target oncogenes.
  • the compositions are constructs that encode a pro-apoptotic factor, or immunogenic factor that increases and immune response against the cells.
  • the compositions are constructs the disrupt expression of an oncogene or other cancer-causing transcript.
  • a balance usually is maintained between cell renewal and cell death in most organs and tissues.
  • the various types of mature cells in the body have a given life span; as these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is so regulated that the numbers of any particular type of cell remain constant. Occasionally, though, cells arise that are no longer responsive to normal growth-control mechanisms.
  • cancer refers specifically to a malignant tumor.
  • 45617673.1 45 malignant tumors exhibit metastasis.
  • small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site.
  • compositions and methods described herein may be useful for treating subjects having benign or malignant tumors by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.
  • Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands.
  • the disclosed compositions are particularly effective in treating carcinomas.
  • Sarcomas which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
  • the types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas, and sarcomas, of bone, bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine.
  • the disclosed compositions are used to treat multiple cancer types concurrently.
  • the compositions can also be used to treat metastases or tumors at multiple locations.
  • the disclosed conjugates and pharmaceutical compositions thereof can be used in the context of preparing lymphocytes expressing immune receptors, particularly chimeric immune receptors (CIR) such as chimeric antigen receptors (CAR).
  • CIR chimeric immune receptors
  • Artificial immune receptors also known and referred to herein, as chimeric T cell receptors, chimeric immunoreceptors, 45617673.1 46 chimeric antigen receptors (CARs), and chimeric immune receptors (CIR) are engineered receptors, which graft a selected specificity onto a cell.
  • mRNA or DNA encoding a chimeric antigen receptor payload can be delivered to immune cells, such as lymphocytes.
  • the payload can be delivered to immune cells in vivo, ex vivo, or in vitro.
  • the payload is mRNA.
  • immune cells e.g., T cells
  • the conjugates and pharmaceutical compositions thereof disclosed herein are used to deliver mRNA encoding one or more CAR T cell constructs into the harvested cells, and the cells are returned to the subject.
  • the process from initially harvesting the cells to returning them to the subject, takes 1 week or less, for example, 1, 2, 3, 4, 5, 6, or 7 days.
  • the process from initially harvesting the cells to returning them to subject is carried in out in 1 or 2 days, or in less than 1 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • 1 or 2 days or in less than 1 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • CARs combine the antigen-binding property of monoclonal antibodies with the lytic capacity and self-renewal of T cells and have several advantages over conventional T cells (Ramos and Dotti, Expert Opin Biol Ther., 11:855–873 (2011), Curran, et al., J Gene Med., 14:405–415 (2012), Maher, ISRN Oncol.2012:278093 (2012)).
  • CAR-T cells recognize and kill cancer cells independently of the major histocompatibility complex (MHC).
  • target cell recognition is unaffected by some of the 45617673.1 47 mechanisms by which tumors evade MHC-restricted T-cell recognition, for example downregulation of human leukocyte antigen (HLA) class I molecules and defective antigen processing.
  • Chimeric immune receptors were initially developed in the 1980s and originally included the variable (antigen binding) regions of a monoclonal antibody and the constant regions of the T-cell receptor (TCR) ⁇ and ⁇ chains (Kuwana, et al., Biochem Biophys Res Commun., 149:960–968 (1987)).
  • the design was modified to include an ectodomain, from a single chain variable fragment (scFv) from the antigen binding regions of both heavy and light chains of a monoclonal antibody, a transmembrane domain, and an endodomain with a signaling domain derived from CD3- ⁇ .
  • scFv single chain variable fragment
  • the CAR constructs that can be utilized in the conjugates or pharmaceutical compositions thereof described herein can include an antigen binding domain or ectodomain, a hinge domain, a transmembrane domain, an endodomain, and combinations thereof.
  • the ectodomain is an scFv.
  • Tandem CARS may be more effective in killing cancers expressing low levels of each antigen individually and may also reduce the risk of tumor immune escape due by single antigen loss variants.
  • Other ectodomains include IL13R ⁇ 2 (Kahlon, et al., Cancer Res., 64:9160–9166 (2004), Brown, et al., Clin Cancer Res., 18(8):2199-209 (2012), Kong, et al., Clin Cancer Res., 18:5949–5960 (2012), NKG2D-ligand and CD70 receptor, peptide ligands (e.g., T1E peptide ligand), and so-called “universal ectodomains” (e.g., avidin ectodomain designed to recognize targets that have been contacted with biotinylated monoclonal antibodies, or FITC-specific scFv designed to recognize targets that have been contacted with FITC-labeled 45617673.1 48 monoclonal antibodies (Zhang,
  • the CAR includes a hinge region. While the ectodomain is important for CAR specificity, the sequence connecting the ectodomain to the transmembrane domain (the hinge region) can also influence CAR-T-cell function by producing differences in the length and flexibility of the CAR.
  • Hinges can include, for example, a CH2CH3 hinge, or a fragment thereof, derived from an immunoglobulin such as IgG1. For example, Hudecek et al.
  • transmembrane domain typically derived from CD3- ⁇ , CD4, CD8, or CD28 molecules.
  • the transmembrane domain can also influence CAR-T-cell effector function.
  • CAR endodomains Upon antigen recognition, CAR endodomains transmit activation and costimulatory signals to T cells.
  • T-cell activation relies on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain to the cytoplasmic CD3- ⁇ domain of the TCR complex (Irving, et al., Cell, 64:891–901 (1991)).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • CAR endomains contain an activation domain derived from CD3- ⁇ , others can include ITAM-containing domains such as the Fc receptor for IgE- ⁇ domain (Haynes, et al., J Immunol., 166:182–187 (2001)).
  • ITAM-containing domains such as the Fc receptor for IgE- ⁇ domain (Haynes, et al., J Immunol., 166:182–187 (2001)).
  • the target specificity of the cell expressing a CAR is determined by the antigen recognized by the antibody/ectodomain.
  • the disclosed 45617673.1 49 conjugates and pharmaceutical compositions can be used to create constructs, and cells expressing the constructs, that target any antigen.
  • numerous antigens, and suitable ectodomains for targeting them are well known.
  • CARs Unlike the native TCR, the majority of scFv-based CARs recognize target antigens expressed on the cell surface rather than internal antigens that are processed and presented by the cells’ MHC, however, CARs have the advantage over the classical TCR that they can recognize structures other than protein epitopes, including carbohydrates and glycolipids Dotti, et al., Immunol Rev.2014 January ; 257(1): . doi:10.1111/imr.12131 (35 pages) thus increasing the pool of potential target antigens.
  • Preferred targets include antigens that are only expressed on cancer cells or their surrounding stroma (Cheever, et al., Clin Cancer Res.,15:5323–5337 (2009)), such as the splice variant of EGFR (EGFRvIII), which is specific to glioma cells (Sampson, et al., Semin Immunol., 20(5):267-75 (2008)).
  • EGFRvIII the splice variant of EGFR
  • human antigens meet this requirement, and the majority of target antigens are expressed either at low levels on normal cells (e.g. GD2, CAIX, HER2) and/or in a lineage restricted fashion (e.g. CD19, CD20).
  • CAR targets for hematological malignancies include, but are not limited to, CD 19 (e.g., B- cell) (Savoldo, et al., J Clin Invest., 121:1822-1826 (2011), Cooper, et al., Blood, 105:1622-1631 (2005); Jensen, et al., Biol Blood Marrow Transplant (2010), Kochenderfer, et al., Blood, 119:2709-2720 (2012), Brentjens, et al., Molecular Therapy, 17:S157 (2009), Brentjens, et al., Nat Med., 9:279-286 (2003), Brentjens, et al., Blood, 118:4817-4828 (2011), Porter
  • NKG2D ligands e.g., Myeloid
  • NKG2D ligands e.g., Myeloid
  • ROR1 e.g., B-cell
  • CAR targets for solid tumors include, but are not limited to, B7H3 (e.g., sarcoma, glioma) (Cheung, et al., Hybrid Hybridomics, 22:209–218 (2003)); CAIX (e.g., kidney) (Lamers, et al., J Clin Oncol., 24:e20–e22.
  • B7H3 e.g., sarcoma, glioma
  • CAIX e.g., kidney
  • CD44 v6/v7 e.g., cervical
  • CD171 e.g., neuroblastoma
  • CEA e.g., colon
  • EGFRvIII e.g., glioma
  • EGP2 e.g., carcinomas
  • EGP40 e.g., colon
  • EphA2 e.g., glioma, lung
  • ErbB2(HER2) e.g., breast, lung, prostate, glioma
  • FAR e.g., rhabdomyosarcoma
  • GD2 e.g., neuroblastoma, sarcoma, melanoma
  • GD3 e.g., melanoma, lung cancer
  • HMW-MAA e.g., melanoma
  • IL11R ⁇ e.g., osteosarcoma
  • the payload can also include therapeutic, diagnostic, and/or prophylactic proteins, peptides, or nucleic acids.
  • proteins, such as antibodies and fragments thereof can be utilized due to their target specificity.
  • the use of antibodies in therapeutic settings directed towards intracellular targets is hampered by their low membrane-crossing characteristics. cellular membrane.
  • the data described below show effective antibody delivery into a cell. Therefore, in some forms, the conjugates described herein contain antibodies and/or fragments thereof.
  • antigens that can be targeted intracellular include, but are not limited to melanoma-associated antigen, pan-carcinoma antigen, human B- cell lymphoma, HIV-1 Gag, Bcl-2, Akt, HIV-1 TAT-protein, nuclear pore complex, Hepatitis B virus X protein, transcription factors, reporter molecules, etc.
  • a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • the “carrier” includes all components present in the pharmaceutical formulation other than the active ingredient or ingredients.
  • carrier includes, but is not limited, to diluents, binders, lubricants, disintegrators, fillers, and coating compositions. In some forms, the “carrier” includes other components besides water. In some forms, the conjugates are administered in an aqueous solution.
  • the formulation may also be in the form of a suspension, emulsion, lyophilized powder, or powder in tablets.
  • pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include sterile water, buffered saline (e.g., Tris-HCl, 45617673.1 53 acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN 80® (polysorbate 80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
  • buffered saline e.g., Tris-HCl, 45617673.1 53 acetate, phosphate
  • pH and ionic strength e.g., Tris-HCl, 45617673.1 53 acetate, phosphate
  • additives e.g., Tris-HCl, 45617673.1 53 acetate, phosphate
  • additives
  • non-aqueous solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • the formulations may be lyophilized and redissolved/resuspended immediately before use.
  • the formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
  • a preferred route for administering the conjugate or pharmaceutical composition thereof is parenteral administration.
  • Suitable parental routes include, but are not limited to, intracranial, intracerebral, intracerebroventricular, intrathecal, intravenous, ocular, subretinal, intravitreal, intranasal, intrapleural, or intratracheal.
  • more preferred routes are intracerebral, intracerebroventricular, intrathecal, intravenous, subretinal, and intravitreal.
  • administering the conjugate or pharmaceutical composition thereof involves convection enhanced delivery (CED).
  • CED involves infusion of the conjugates into the brain through a catheter under a positive pressure gradient, (Bobo, et al., Proc Natl Acad Sci U S A 91, 2076-2080 (1994)).
  • a conjugate containing a structure: 45617673.1 54 preferably Formula I Formula I’ wherein: the dashed lines denote independently the presence of one or more covalent or non-covalent bonds, preferably the dashed lines denote the presence of one or more covalent bonds, P comprises a protein, peptide, or nucleic acid; L is a linear or branched traceless or untraceless chemical linker; M is a single-armed or multi-armed chemical moiety containing a cell membrane fusogenic molecule; and np, nl, nm, and nz are independently integers between 1 and 150, inclusive, between 1 and 100, inclusive, between 1 and 75, inclusive, between 1 and 50, inclusive, between 1 and 25, inclusive, between 1 and 15, inclusive, between 1 and 10, inclusive, between 1 and 7, inclusive, or between 1 and 5, inclusive.
  • L is a linear or branched traceless chemical linker comprising a containing a stimuli- responsive chemical moiety and/or a self-immolative chemical moiety.
  • L is a linear or branched untraceless chemical linker containing a substituted alkyl, unsubstituted alkyl, substituted alkylene, unsubstituted alkylene, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted heterocyclyl, or unsubstituted heterocyclyl.
  • the Cas nuclease is selected from the group composed of Cas9, CasX, Cas7-11, CasFx, Cas12a, and Cas13.
  • the Cas nuclease is Cas9 nuclease.
  • a stimulus selected from pH (such as change in pH), redox (such as change in redox potential), reactive oxygen species (ROS), enzyme (such as over-expression of an enzyme (e.g., a protease, esterase, etc.) due to a diseased state or disorder), ionic strength (such as a change in ionic strength), hypoxia, and combinations thereof. 17.
  • a stimulus selected from pH (such as change in pH), redox (such as change in redox potential), reactive oxygen species (ROS), enzyme (such
  • the stimuli-responsive chemical moiety contains a disulfide bond, an amide 45617673.1 56 bond, an orthoester, a hydrazone, a hydrazide, a hydrazine, an imine (such as aldimine or ketoimine), an oxime, an acetal group, a vinyl ether, a polyketal, a methyl maleate, an ester bond, a nitroaryl group (e.g., nitrobenzyl), a nitroheteroaryl (e.g, nitroimidazole), a quinone group, an azoaryl group (e.g., azophenyl), an azoheteroaryl group (e.g., azopyridinyl), peroxalate ester, aminoacrylate, alkyl thioether or selenide (e.g., monoselenide bond, diselenide bond, etc.),
  • the stimuli-responsive chemical moiety comprises a disulfide bond, an amide, an orthoester, an imine (such as aldimine or ketoimine), a hydrazone, a hydrazide, a hydrazine, an imine, an oxime, a methyl maleate, an ester bond, a dimethyl maleate, or a combination thereof.
  • the stimuli-responsive chemical moiety contains a disulfide bond, an amide, an orthoester, an imine (such as aldimine or ketoimine), or an ester bond.
  • a hydrophilic polymer between L and a moiety in M preferably a neutral uncharged hydrophilic polymer such as polyalkylene glycols and polyalkylene oxides such as poly(ethylene glycol); polysaccharides such as celluloses and dextrans; hydrophilic polypeptides and poly(amino acids) such as poly-L-serine; poly(oxyethylated polyol); poly(olefinic alcohol) such as poly(vinyl alcohol); poly(N-vinylpyrrolidone); poly(hydroxyethyl acrylate); poly(hydroxyalkyl methacrylate), e.g., poly(hydroxyethyl methacrylate), preferably polyalkylene glycols and polyalkylene glycols and polyalkylene glycols and polyalky
  • administration is parenterally, such as intracranially, intracerebrally, intracerebroventricularly, intrathecally, intravenously, ocularly, subretinally, intravitreally, intranasally, intrapleurally, or intratracheally.
  • administration comprises convection enhanced delivery.
  • any one of paragraphs 42 to 48 wherein the subject exhibits one or more signs or symptoms associated with Angelman syndrome, HIST1H1E (H1-4) syndrome, Prader-Willi syndrome, Alzheimer’s disease, Huntington’s disease, Parkinson's Disease (PD), Multiple Sclerosis (MS), Cerebral Palsy (CP), Spinocerebellar Ataxias, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Jakob-Creutzfeldt disease, dystonia, amyotrophic lateral sclerosis, muscular atrophies, muscular dystrophies (such as Duchenne, Becker, facioscapulohumeral, myotonic, congenital, distal, Emery-Dreifuss, oculopharyngeal, and limb girdle), congenital myopathies (such as central core, Myotubular, Nemaline, Ullrich/Bethlem, and RyR1),
  • Example 1 Stimuli-responsive traceless engineering platform (STEP) for intracellular protein delivery
  • STEP Stimuli-responsive traceless engineering platform
  • biologics-based therapies such as protein- based therapies
  • This non-limiting example describes a stimuli-responsive traceless engineering platform (STEP) that can be utilized for efficient delivery of biologics, such as proteins, into cells without compromising the biological activities the biologics.
  • STEP is achieved through chemical conjugation of membrane fusogenic molecules to amino- or thiol- groups on the surface of protein payloads via stimuli-responsive linkers that can be cleaved and completely removed by intracellular stimuli, such as a reducing and/or an acidic environment.
  • the payloads e.g., protein
  • the payloads are released without any trace molecules and thus fully recover their biological functions after cell penetration (FIG.1).
  • RNPs ribonucleoproteins 45617673.1 63
  • the data further show that the technology can be utilized for the delivery of other payloads, for instance protein payloads including green fluorescent protein (GFP), Cas9/dCas9 fusions, and antibodies.
  • GFP green fluorescent protein
  • Cas9/dCas9 fusions include antibodies.
  • Materials, methods, and results Ai9 mice were purchased from the Jackson Laboratory.
  • Ai9 fibroblasts were provided by the NIH Common Fund's Somatic Cell Genome Editing (SCGE) program. Starting molecules for chemical synthesis were obtained from commercial vendors, such as Sigma and Santa Cruz Biotechnology.
  • Cas9 proteins were obtained from Integrated DNA Technologies IDT or Couragene. Guide RNA was synthesized by Synthego Corporation. i.
  • Conjugation of cholesterol through a traceless linker facilitates RNPs to penetrate cells to achieve efficient genome editing
  • Experiments were performed to determine whether conjugation of cholesterol to the surface of RNPs facilitates penetration of the RNPs into cells to achieve genome editing.
  • RNPs were assembled through incubation of Cas9 protein with sgRNAs 276 and 280 that target the loxP-flanked STOP cassette in Ai9 cells (FIG.2A).
  • Cholesterol was chemically conjugated to the surface of RNPs via a redox-responsive, self-immolative linker dibenz[b, f]azocine-5(6H)-butanoic acid, 11,12-didehydro- ⁇ -oxo-, 2-((2-(((4- nitrophenoxy)carbonyl)oxy)ethyl)disulfaneyl)ethyl ester (DBNPDEE) (FIG. 2B), followed by incubation with azide-conjugated cholesterol (FIG.2C).
  • DNPDEE 2-((2-(((4- nitrophenoxy)carbonyl)oxy)ethyl)disulfaneyl)ethyl ester
  • This conjugation chemistry was selected so that, after getting into the cytosol, the reducing microenvironment in the cytosol can initiate disulfide cleavage and subsequent self-immolation of the chemical linker, resulting in release of RNPs without any trace molecules on the surface. Because of the capacity of release of payloads without trace molecules, such linkers are termed traceless linkers.
  • Control RNPs were synthesized through incubation of pre-synthesized cholesterol-PEG-NHS, which reacts with primary amino groups on the surface of RNPs. Unlike DBNPDEE, this conjugation method leads to covalent attachment of cholesterol molecules to the surface of RNPs, 45617673.1 64 which could not be cleaved within cells.
  • the resulting chemically modified RNPs were evaluated in primary fibroblasts isolated from Ai9 mice for their capacity for genome editing, which was determined based on the expression of tdTomato.
  • the results show that conjugation of cholesterol through DBNPDEE facilitates efficient cell penetration and genome editing, resulting in 61.0% of cells expressing tdTomato. For purposes of comparison, this degree of editing efficiency is defined as 100% “relative genome editing activity”.
  • conjugation through the non-cleavable linker led to significantly lower efficiency (FIG.2E).
  • FIGs.2A-2E Screening of membrane fusogenic molecules Besides the traceless linker, the use of membrane fusogenic molecules also plays a key role in determining the intracellular delivery activity of RNPs (FIGs.2A-2E).
  • a library of fusogenic molecules was screened, including lipids DSPE, PE, 16-PC, and 18-PC; cholesterol; cholesterol analogues OA and ⁇ -sitosterol; and multi-arm tyrosine (FIG. 4A).
  • Candidate small molecules were conjugated to RNPs through DBNPDEE. The resulting RNPs were tested in Ai9 fibroblasts. Although all the tested molecules demonstrated various degrees of capacity to deliver RNPs, cholesterol remained the most efficient one. Therefore, cholesterol was selected in the following studies. iv.
  • STEP RNPs were characterized in terms of delivery of a genome editing machinery to the brain.
  • STEP RNPs loaded with sgRNA 276 and sgRNA 280 were administered to the brain parenchyma through convection enhanced delivery (CED).
  • CED convection enhanced delivery
  • the mice were euthanized.
  • Analysis of the brain found that CED of the STEP RNPs efficiently edited the genome of brain cells, evident by the expression of tdTomato in the diffusion region, mostly in neuronal cells.
  • STEP RNPs were characterized in Ai9 mice through both intracerebroventricular and intrathecal administration. The treatments gave rise to the editing of ⁇ 76% NeuN + neurons in the prefrontal cortex.
  • STEP RNPs for treatment of neurogenetic diseases Angelman Syndrome (AS) is a devastating neurogenetic disease caused by the deficiency of the maternal UBE3A gene in human chromosome 15q11-q13 region. Due to brain specific imprinting of the UBE3A gene, there is an opportunity to reactivate/unsilenced the paternal allele, expressing a present and functional copy (Albrecht, et al., Nature genetics 1997;17(1):75-8; Judson, et al., The Journal of Comparative Neurology 2014;522(8):1874-96).
  • UBE3A The structure of UBE3A is intact in the paternal chromosome in all AS cases, but is transcriptionally repressed by a non- coding antisense RNA of UBE3A in neurons (UBE3A-ATS) mediated mechanism (Meng, et al., Hum Mol Genet.2012;21(13):3001-12; Meng, et al., PLoS Genet.2013;9(12):e1004039; Meng, et al., Nature. 2015;518(7539):409-12).
  • AS Like Alzheimer’s disease, AS affects multiple regions of the brain and treatment of AS requires delivery of genome editing 45617673.1 67 throughout the brain.
  • RNPs targeting the non-coding antisense RNA could turn on expression of UBE3A and rescue neurological deficits.
  • Ube3a-YFP transgenic mice it was determined whether delivery of STEP RNPs could lead to the editing of neuronal cells in the brain.
  • expression of paternal Ube3a-YFP pUbe3a- YFP
  • mice without interventions there is no expression of pUbe3a- YFP.
  • STEP RNPs loaded with sgRNA targeting the non-coding antisense RNA were synthesized and administered at 40 ug dose intracerebroventricular (ICV) to post-natal day1-2 pups. One month later (in this case 30 days later), the mice were euthanized, and their brains were analyzed for expression of YFP. YFP was expressed throughout the brain in high efficiency, suggesting that ICV administration of STEP RNPs can induce genome editing throughout the brain.
  • STEP RNPs were characterized in Ai9 mice for their neuronal editing efficiency and the scale of brain-wide genome editing through both ICV and intrathecal administration (IT).
  • FIG.7E shows two entries containing D21 twice in the training, short term memory, and long term memory data sets. One date set is IDT Cas9 and the other is Couragene Cas9.
  • STEP for intracellular delivery of CRISPR epigenetic editing machinery STEPs were characterized for delivery of CRISPR epigenetic editing machinery by using dCas9-TETv4 based RNPs as an example.
  • the data showed that STEP facilitated delivery of dCas9-TETv4 based RNPs, leading to efficient epigenetic editing and re-expression of silenced SNRPN gene in the Prader-Willie syndrome candidate region FIGs.10A and 10B.
  • TETv4 is the catalytic subunit of a ten elven translocation (TET) enzyme that oxidizes 5-methylcytosines and promote locus-specific reversal of DNA methylation.
  • TET ten elven translocation
  • dCas9 is an inactive form of spCas9 nuclease.
  • STEP for intracellular delivery of other protein payloads Experiments were performed to determine whether the STEP can be employed for the delivery of protein payloads other than RNPs, including GFP (28 kDa), Cas9-EGFP fusion protein (194 kDa), and AF568-labeled IgG (150 kDa). Both proteins were surface conjugated with cholesterol through DBNPDEE and 2 arm PEG (cholesterol x 2). The resulting STEP Cas9-EGFP and IgG were added to U87 cells. Two, four, or six hours later, the cells were imaged.
  • STEP efficiently delivered GFP, Cas9-EGFP, and IgG into cells, evident by the observation of strong fluorescence in the 45617673.1 70 cytoplasm.
  • HIST1H1E H1-4
  • ID intellectual disability
  • H1-4 CFT patient derived iPSCs display increased cell proliferation, abnormal nuclear ultrastructure and transcriptome, and aberrant distribution of H3K27me3 (FIG.12A). These cellular and molecular phenotypes were used to assess the efficacy of STEP RNPs loaded with H1-4-targeting gRNA.
  • the most common and recurrent mutation found in H1-4 syndrome is c.430dupG (430G).
  • the same frameshift mutation does not result in a CFT in mouse H1-4 protein, unlike in human (Tremblay, et al., Hum Mol Genet.2021. Epub 2021/11/18.
  • H1-4 mutant mouse carrying 430G was generated (FIG.12B).
  • humanized H1-4 WT mice were generated.
  • Homozygous H1-4 430G mice display high penetrant perinatal lethality and growth retardation (FIG.12B).
  • Heterozygous H1-4 430G mice are viable and have mild early growth retardation and behavioral impairments in multiple domains (FIGs.12C and 12D).
  • H1-4 CFT iPSCs and H1-4 430G humanized mice were treated with both ASO and STEP RNPs targeting the 430G mutation.
  • Ai9 fibroblast cells were treated by traceless STEP Cas9/sgAi9 RNP at 2.5 ⁇ g/mL or 7.5 ⁇ g/mL.
  • the membrane fusogenic molecules employed were cholesterol, F7- cholesterol, and ⁇ -sitosterol.
  • the cells were observed under fluorescence microscope at 48 h post treatment.
  • the edited cells with tdTomato fluorescence (%) were quantified.
  • the ratios of STEP/Cas9 were 10 and 20. Results
  • cholesterol analogs F7- cholesterol showed similar gene editing activity with cholesterol at both STEP/Cas9 ratio of 10 and 20.
  • ⁇ -sitosterol showed better cell editing efficiency than cholesterol at STEP/Cas9 ratio of 10. Further, ⁇ -sitosterol also showed slightly better cell editing efficiency than cholesterol at STEP/Cas9 ratio 20.
  • ⁇ -sitosterol may have better editing efficiency than cholesterol. But cholesterol may have lower cytotoxicity than ⁇ -sitosterol. Thus, cholesterol may be selected for the safety. Nonetheless, this does not exclude the possibility that ⁇ -sitosterol may be used in some aspects of this disclosure.
  • Example 3 An untraceless chemical linker can also effectively deliver RNP Materials and methods
  • the editing efficiencies of STEP Cas9/sgAi9 RNP and untraceless Cas9/sgAi9 RNP in Ai9 fibroblast cells were compared.
  • cholesterol was used as the cell membrane fusogenic molecule.
  • Ai9 fibroblast cells were treated with these conjugates at 5 ⁇ g/mL or 10 ⁇ g/mL. The cells were observed under fluorescence microscope at 48 h post 45617673.1 73 treatment. The edited cells with tdTomato fluorescence (%) were quantified.
  • the conjugate is shown below: , where n is chemical above.
  • Example 4 STEP delivers Cas9 RNP into cells through a cytosolic delivery mechanism
  • Endosome entrapment upon intracellular endocytosis is a major delivery limitation for genetic medicine including Cas9 gene editing machinery (Tong, et al., Nature Reviews Materials 4.11 (2019): 726-737).
  • the intracellular delivery mechanism of STEP engineered RNP was evaluated.
  • GFP fusion Cas9 RNP engineered by STEP was added to Ai9 fibroblast cells and observed with fluorescence microscope at 2 h and 6 h post incubation. Endosome was also stained with LysoTtracker Red.
  • Example 6 STEP RNPs for treating Prader-Willi Syndrome Materials and methods dCas9 fused with the catalytic domain of a specific enzyme binds to target DNA sequence without genome editing, to modify methylation on CpG island or histone for epigenetic regulation.
  • FIG.16A Schematics of dCas9-TET1 with sgRNA demethylase methylated CpGs on target region and dCass9- JMJD2a with sgRNA demethylase Histone H3K9me2/3 on target region are shown in FIG.16A.
  • IC PWS-imprinting center
  • sgRNA binding to PWS-IC on mouse chromosome 7C which has conserved was designed (FIG.17B). Additionally, sgRNA binding to upstream region of PWS-IC which has 45617673.1 75 H3K9me3, a target of JMJD2a, was tested. I.V. injection was performed twice in mSnrpn-EGFP/p+ mice. Results dCas9-TET1 was treated with sgRNA packaged by STEP to fibroblasts derived from patient with Prader-Willi Syndrome which has a large deletion on paternal chromosome 15q11-q13. This ribonucleoprotein reactivated maternal imprinted/silenced gene, SNPRN (FIG.16C).
  • dCas9- JMJD2a with sgRNA packaged by STEP showed more efficacy on reactivation of imprinted genes in PWS fibroblasts compared to CPP (cell penetration peptide) (FIG.16D, 16E, and 16F).
  • dCas9-JMJD2a with sgRNA could reactivate SNRPN, SNORD116, 116HG (host gene) from the maternal chromosome (FIG.16D, 16E, and 16F).
  • IPW located far from PWS-IC was not reactivated by dCas9-JMJD2a with sgRNA (FIG.16G).

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Abstract

Des conjugués et des compositions pharmaceutiques contenant ces conjugués pour la distribution intracellulaire d'une charge utile sont divulgués. Les conjugués contiennent une machinerie ou une protéine d'édition de gènes en tant que charge utile ; de préférence un lieur chimique auto-immolable sensible aux stimuli, et une molécule fusogène de membrane cellulaire. Un ou plusieurs stimuli à l'intérieur d'une cellule, tels qu'un environnement réducteur et/ou acide, clivent la fraction chimique sensible aux stimuli et activent l'auto-immolation du lieur chimique. Dans certaines formes, ces procédés conduisent à la libération de la charge utile à partir du conjugué sans composants du lieur et de la molécule fusogène de membrane cellulaire liée de manière covalente à la machinerie d'édition de gène ou à la charge utile de protéine.
PCT/US2023/082123 2022-12-01 2023-12-01 Plateforme d'ingénierie sans trace sensible aux stimuli pour distribution de charge utile intracellulaire WO2024119101A1 (fr)

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