Stimulus-reactive traceless engineered platform for intracellular payload delivery

Abstract

Disclosed are conjugates for intracellular delivery of a payload and pharmaceutical compositions containing these conjugates. The conjugate contains a gene editing machine or a protein as a payload; preferably, stimuli reactive, self-destructive chemical linkers and cell membrane fusion molecules. One or more stimuli within the cell, such as a reducing and / or acidic environment, cleaves the stimuli-reactive chemical moiety and activates the self-destructiveness of the chemical linker. In some forms, these processes result in release of the payload from the conjugate without the linker and components of the cell membrane fusion molecule being covalently linked to the gene editing machine or protein payload.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Application No. 63 / 385,632, filed December 1, 2022, the disclosure of which is incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant NS115597 from the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference Sequence Listing

[0006] Pursuant to 37 CFR 1.834(c)(1), the sequence listing submitted is hereby incorporated by reference in the text file entitled “YU_8549_PCT_ST26.xml” created on November 30, 2023, which is 6,961 bytes in size. Technical Field

[0007] The present invention relates to the field of payload delivery, in particular to the intracellular delivery of payloads using payload-membrane fusion molecule conjugates, wherein in the conjugates, the payload and the membrane fusion molecule are preferably covalently linked via a stimulus-responsive linker, more preferably via a redox-responsive self-destructive linker. Background Art

[0008] The clinical transformation of therapies based on biological agents (such as protein-based and gene-based therapies) has always been a major challenge because most biological agents (such as therapeutic agents based on genes and / or proteins) cannot effectively penetrate cells. Although significant efforts have been made in developing biological agents for clinical use, there are still major obstacles in developing payload delivery systems. These include penetrating the cell membrane, releasing the payload, effectively releasing the payload to maintain its biological activity, and / or releasing the payload in the absence of a carrier molecule covalently attached to the payload. Therefore, there is still a need for improved compositions and methods to effectively deliver biological agents in cells.

[0009] Therefore, it is an object of the present invention to provide conjugates that can improve the intracellular delivery of biologics.

[0010] Another object of the present invention is to provide conjugates that are able to cross cell membranes while maintaining the biological activity of the delivered biologic.

[0011] Another object of the present invention is to provide conjugates capable of delivering gene editing machinery into cells.

[0012] Another object of the present invention is to provide methods of using such conjugates. Summary of the Invention

[0013] The present invention describes conjugates for intracellular delivery of payloads and pharmaceutical compositions containing these conjugates. The conjugate comprises a payload, a chemical linker and a cell membrane fusion molecule. The payload preferably used for delivery is a gene editing machine. The cell membrane fusion molecule promotes the intracellular uptake of the conjugate. Preferably, the chemical linker comprises a stimulus-responsive chemical portion and a self-destructive chemical portion. After entering the cell, the conjugate is exposed to one or more stimuli, such as a reducing and / or acidic environment, which cleave the stimulus-responsive chemical portion, thereby activating the self-destructive property of the chemical linker. The cleavage and self-destructive property result in the payload being delivered in a stimulus-responsive, traceless manner.

[0014] The results described herein demonstrate that the platform can be used to effectively deliver ribonucleoproteins (RNPs) consisting of Cas9 protein sgRNA into cells in in vitro cell cultures and in vivo reporting and disease models. The results also show that chemically modified RNPs, called stimulus-responsive traceless engineering platform RNPs (STEP RNPs), are safe because intravenous administration of STEP RNPs does not cause significant systemic toxicity in the liver and kidneys based on aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatine measurements. In addition, based on microscopic imaging and H&E staining, intracranial administration of STEP RNPs does not cause significant cell damage in the brain. Storing STEP RNPs at -20°C for 2 months did not reduce the efficiency of their genome editing.

[0015] Preferably, the conjugate comprises the following structure:

[0016]

[0017] in:

[0018] Dashed lines independently represent the presence of one or more covalent bonds or non-covalent bonds, preferably, dashed lines represent the presence of one or more covalent bonds,

[0019] P comprises a protein, peptide, or nucleic acid;

[0020] L is a linear or branched traceless or trace chemical linker; wherein when L is a linear or branched traceless chemical linker, L comprises a stimulus-responsive chemical moiety and / or a self-destructive chemical moiety, wherein when L is a linear or branched trace chemical linker, L comprises a substituted alkyl, an unsubstituted alkyl, a substituted alkylene, or an unsubstituted alkylene;

[0021] M is a one-armed or multi-armed chemical moiety comprising a cell membrane fusion molecule; and

[0022] nl, nm and nz are independently an integer between 1 and 100, including 1 and 100. In some forms, the value of nm and / or nz is selected so that the molar ratio of M to P is in the range of 100: 1 to 1: 1, such as 100: 1, 50: 1, 25: 1, 20: 1, 10: 1, 5: 1 or 1: 1. In some forms, the value of np is selected on the one hand, and the value of nl, nm and / or nz is selected on the other hand so that the molar ratio of LM to P is in the range of 100: 1 to 1: 1, such as 100: 1, 50: 1, 25: 1, 20: 1, 10: 1, 5: 1 or 1: 1. In some forms, analytical methods (such as nuclear magnetic resonance spectroscopy) can be used to analyze the final product to determine the molar ratio. In some forms, the molar ratio can be a theoretical value based on the feed molar ratio of the reactants added to the reaction mixture to form the final product. In some forms, the ratio of payload to membrane fusion moiety is 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.

[0023] In a preferred form, the chemical linker is formed from:

[0024] And the cell membrane fusion molecule is formed by:

[0025]

[0026] Where n is 24.

[0027] In some forms, the conjugate comprises a chemical linker formed from: Cell membrane fusion molecules formed by:

[0028] wherein n is 24, and a poly(ethylene glycol) having a molecular weight of 200 Da to 10 kDa is inserted between a cell membrane fusogenic moiety (eg, a cholesterol moiety) and a dibenzocyclooctyne (DBCO) group.

[0029] Also described are in vitro and in vivo methods utilizing the conjugates and pharmaceutical compositions thereof in gene editing platforms, transfection platforms, and intracellular delivery of therapeutic proteins or peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the Stimuli-Responsive Traceless Engineering (STEP) platform for intracellular delivery of biologic payloads, such as protein payloads. The schematic shows chemical modification of the payload with a membrane fusion molecule via a stimuli-responsive, self-destructive, traceless linker.

[0031] Figure 2A Schematic diagram of the Ai9 loxP-flanked STOP reporter cassette. Figure 2B and Figure 2C Showing 11,12-didehydro-γ-oxo-, 2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ester (DBNPDEE) and cholesterol-PEG 24 -N3 molecular structure. Figure 2D Non-limiting molecular structures of some selected STEP RNPs are shown; n can be an integer from 1 to 10, and the chemical moiety in square brackets has appropriate connectivity to the rest of the STEP RNP, as described in more detail below. Figure 2E This is a bar graph showing the characteristics of the indicated chemically modified RNPs used for genome editing. Ai9 fibroblasts were treated with the indicated RNPs. 48 hours later, cells were imaged under a fluorescence microscope, and the percentage of cells expressing tdTomato was quantified.

[0032] Figure 3A Displays the molecular structure of the specified linker. Figure 3B Figure 2 is a bar graph showing the characteristics of RNPs with surfaces modified with cholesterol via the indicated linkers for delivery of genome editing machinery. The RNPs were loaded with sgRNAs 276 and 280. Characterization was performed in Ai9 fibroblasts. Genome editing efficiency was determined based on tdTomato expression and is expressed as the expression of tdTomato via DBNPDEE and cholesterol-PEG. 24 -Percentage of N3 achieved, defined as 100%.

[0033] Figures 4A-4G The molecular structures of the indicated membrane fusion molecules are shown. Figure 4H Figure 2 is a bar graph showing the characteristics of RNPs with surfaces coupled to the indicated molecules via the linker DBNPDEE for delivery of genome editing machinery in Ai9 fibroblasts. The RNPs were loaded with sgRNAs 276 and 280. Genome editing efficiency was determined based on tdTomato expression and is expressed as the expression of sgRNAs linked to DBNPDEE and cholesterol-PEG. 24 -Percentage of N3 achieved, defined as 100%.

[0034] Figures 5A-5G Molecular structures of the indicated multi-arm, cholesterol-based fusion molecules are shown. Figure 5HFigure 2 is a bar graph showing the characteristics of RNPs with surfaces coupled to the indicated fusion molecules via DBNPDEE for delivery of genome editing machinery in Ai9 fibroblasts. The RNPs were loaded with sgRNAs 276 and 280. Genome editing efficiency was determined based on tdTomato expression and is expressed as the expression of tdTomato by DBNPDEE and cholesterol-PEG. 24 -Percentage of N3 achieved, defined as 100%.

[0035] Figure 6A and Figure 6B is a bar graph showing the size distribution of STEP RNPs determined by DLS analysis ( Figure 6A ), and quantification of the percentage of edited cells based on tdTomato expression ( Figure 6B ).exist Figure 6B Ai9 fibroblasts were treated with STEPRNP for 48 hours. Cell nuclei were stained with Hoechst 33342. The edited cells expressed tdTomato.

[0036] Figure 7A Figure 2 is a gel image of Western blot analysis of STEP RNPs used in AS treatment, which observed reactivation of Ube3a from paternal chromosomes by YFP expression in Ube3a-YFP reporter mice. Figure 7B is a semiquantitative bar graph of data showing reactivation in the prefrontal cortex (PFC) that persists for 90 days after STEP RNP delivery. Maternal Ube3a-YFP is a positive control. Figures 7C-7F Is a bar chart ( Figures 7C-7E ) and line charts ( Figures 7F-7H ), showing the total distance traveled in the open field test ( Figure 7C and Figure 7H ), center time of open field test ( Figure 7D ), the percentage of novel object recognition trials ( Figure 7E ) and latency in the rotarod test ( Figure 7F and Figure 7G ), STEP RNP delivery rescued abnormal behaviors in the AS mouse model. **p<0.01, ***p<0.001. Figure 7C The order of columns in Figure 7D and Figure 7E The order of the columns in .

[0037] Figure 8A and Figure 8B is a bar graph showing the relative expression levels of Ube3a-ATS and Ube3a in different parts of the brain: prefrontal cortex ( Figure 8A ) and the brain ( Figure 8B ).mat- / pat+ In mouse model pups, STEP RNP was administered at a dose of 40 μg via intracerebroventricular (ICV) or intrathecal (IT) administration. Twenty to 30 days after injection, the expression of Ube3a-ATS and Ube3a in different brain regions was assessed by qRT-PCR, quantitative immunoblotting, and immunocytochemistry.

[0038] Figures 9A-9D Is a bar chart ( Figure 9A ) and line charts ( Figures 9B-9D ), showing that treatment with STEP RNP loaded with sgRNA (e.g. gRNA33) can rescue Ube3a mat- / pat+ Effects of neurological deficits in mouse models. Figure 9A and Figure 9B Data showing open-field motor function and anxiety reduction tests; Figure 9C and Figure 9D Data from the rotarod assay are shown. Closed circles: wild type; closed squares: AS+gRNA33 treatment; and closed triangles: AS+gRNA-control.

[0039] Figure 10A and Figure 10B Schematic diagram showing the construct containing dCas9-TET4v ( Figure 10A ) and gel images of dCas9-TEX4v delivered by STEP ( Figure 10B ). Figure 10B We show that delivery of dCas9-TET4v demethylates histone modifications and turns on expression of small nuclear ribonucleoprotein polypeptide N (SNRPN).

[0040] Figures 11A-11E is a characterization of the toxicity of STEP RNP ( Figures 11A-11D ) and stability ( Figure 11E ). Solid bars: vector; unfilled bars: STEP RNP.

[0041] Figures 12A-12H It is a diagram showing the treatment characteristics of STEP RNP H1-4 syndrome. Figure 12A is a line graph showing that H1-4 C-terminal frameshift tail (CFT)-induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs) increase cell preformation. Figure 12B These are images of mice showing growth retardation and lethality in H1-4 c.430G homozygous mice. Figure 12C and Figure 12D is a scatter plot showing that IT delivery of STEP RNP rescues perinatal lethality in H1-4 C430G homozygous mice. Figures 12E-12Gis a gel image showing that ASO and RNP targeting H1-4 c430G effectively downregulated H1-4 in CFT. Figure 12H is a line graph showing homozygous H1-4 430G Intrathecal administration of STEP RNP targeting H1-4 rescued perinatal lethality in mice.

[0042] Figure 13A shows the molecular structure of cholesterol, F7 cholesterol and β-sitosterol. Figures 13B and 13C are bar graphs based on tdTomato expression to quantify the percentage of edited cells. Ai9 fibroblasts were treated with 2.5 μg / mL or 7.5 μg / mL of traceless STEPCas9 / sgAi9 RNP. Cells were observed under a fluorescence microscope 48 hours after treatment. The edited cells were quantified using tdTomato fluorescence (%). In Figure 13B, STEPCas9 / sgAi9 RNP was prepared with a STEP / Cas9 ratio of 10. In Figure 13C, STEPCas9 / sgAi9 RNP was prepared with a STEP / Cas9 ratio of 20. With respect to Formula I (as described below) and Formula I' (as described above and below), the STEP / Cas9 ratio refers to the feed molar ratio of the compound used to form LM to Cas9.

[0043] Figure 14A shows the chemical structures of exemplary scarless and scar chemical linkers used in Example 3. Figure 14B is a bar graph showing the quantification of editing activity of STEP Cas9 / sgAi9 RNP or scarless Cas9 / sgAi9 RNP in Ai9 fibroblasts at 5 μg / mL or 10 μg / mL. Cholesterol was used as the cell membrane fusion molecule in each conjugate. 48 hours after treatment, cells were observed under a fluorescence microscope. Edited cells were quantified by tdTomato fluorescence (%).

[0044] Figure 15 This is a gel image of Western blot analysis of Cas9 in STEP RNP by observing Cas9 antibodies at different time points.

[0045] Figure 16A Schematic diagram showing dCas9-Tet1CD (upper panel) and dCas9-JMJD2a (lower panel) targeting methylated CpG and H3K9me2 / 3, respectively. Figure 16B Another schematic showing sgRNA binding to sites surrounding the center of a Prader-Willi syndrome (PWS) imprint or a region encompassing a CpG island. Maternally imprinted / silenced genes are also shown. Figures 16C-16Gis a bar graph showing RT-qPCR analysis of reactivation of the maternally imprinted SNRPN gene in human fibroblasts derived from PWS (paternal deletion of 15q11-q13) by RNP (including dCas9-Tet1CD and sgRNA) ( Figure 16C ), and bar graphs showing RT-qPCR analysis of reactivation of imprinted SNRPN, SNORD116, and 116HG genes in PWS-derived human fibroblasts by dCas9-JMJD2a with sgRNA#4 ( Figures 16D-16G ).

[0046] Figure 17A and Figure 17B This is a mouse model that shows the maternal Snrpn-EGFP gene as a reporter gene ( Figure 17A ) and the sgRNA binding site on mouse chromosome 7C ( Figure 17B ) schematic diagram. DETAILED DESCRIPTION

[0047] I. Definition

[0048] The term "amino acid" refers to a molecule that contains both an amino group and a carboxyl group. Amino acids include alpha amino acids and beta amino acids. In some 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.

[0049] The term "natural amino acids" refers to the D- and L-isomers of the common 20 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 (represented by single-letter abbreviations)).

[0050] The terms "synthetic amino acid," "non-natural amino acid," and "unnatural amino acid" are used interchangeably to refer to an organic compound having an amino group and a carboxyl group that is not one of the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides. Typically, due to the presence of the amino and carboxyl groups, it mimics the reactivity of natural amino acids. "Synthetic amino acid," "non-natural amino acid," or "unnatural amino acid" also refers to an amino acid that cannot be produced by an organism without genetic engineering. When a synthetic amino acid replaces a natural amino acid or is incorporated into a peptide, the synthetic amino acid, as defined herein, typically increases or enhances the properties of the peptide (e.g., reactivity toward a desired molecule). "Synthetic amino acid," "non-natural amino acid," or "unnatural amino acid" may also refer to amino acids whose side chains have been chemically modified to contain reactive groups (e.g., alkynes; azides; alkenes; triarylphosphines; aminooxy groups; carbonyl groups; hydrazides; sulfonyl chlorides; maleimides; aziridines; -CN; acryloyl groups; acrylamides; sulfones; vinylsulfones; cyanates; thiocyanates; isocyanates; isothiocyanates; alkoxysilanes; dialkyldialkoxysilanes; diaryldialkoxysilanes; trialkylmonoalkoxysilanes; vinylsilanes; acetohydrazides; acyl azides; acyl halides; epoxides; condensates) The invention also provides a method for preparing a natural amino acid containing the following: water, glycerol; carbodiimide; thiol; amine; phosphoramide; vinyl ether; substituted hydrazine; alkylene glycol bis (diester), such as ethylene glycol bis (succinate); thioester, such as alkyl thioester, α-thiophene ester, allyl thioester (such as allyl thioacetate, allyl thiopropionate); allyl ester (such as allyl acetate, allyl propionate); aryl acetate (such as benzoyl ester); orthoester; sulfonamide, such as 2-N-acylnitrobenzenesulfonamide; vinyl sulfide; or a combination thereof) such that the resulting amino acid is structurally different from any of the 20 typical naturally occurring amino acids.

[0051] "Chemical moiety" refers to a portion of a molecule, such as an organic molecule.

[0052] "Conjugation," "conjugate," and related terms refer to the covalent or non-covalent attachment of a molecule to another molecule or to a portion of a molecule to a different portion of the same molecule. Such attachment can involve covalent or non-covalent linkages. Covalent attachment can be direct or indirect (i.e., mediated by a linker). "Covalent attachment" refers to a bond or organic moiety that covalently links molecules or different portions of the same molecule. Non-covalent bonds include electrostatic interactions, hydrogen bonding interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, π-stacking interactions, van der Waals interactions, magnetic interactions, and dipole-dipole interactions.

[0053] The terms "genome editing," "genome engineering," or "genomic mutagenesis" refer to the selective and specific alteration of one or more target genes or DNA sequences within a recipient cell, for example, by delivering a CRISPR-Cas system to the cell. The editing or alteration of the target gene or genome can include one or more deletions, knock-ins, point mutations, substitution mutations, or any combination thereof, in one or more genes of the recipient cell.

[0054] The term "single guide RNA" or "sgRNA" refers to a polynucleotide sequence comprising a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to a 20-base pair (bp) sequence within a guide RNA that specifies a target site and is used interchangeably with "guide" or "spacer."

[0055] The term "Cas9", "Cas9 protein" or "Cas9 nuclease" refers to an RNA-guided endonuclease, which is a Cas9 protein that catalyzes site-specific cutting of double-stranded DNA. In addition, it is called "Cas nuclease" or "CRISPR-associated nuclease". In nature, the CRISPR system is an adaptive immune system found in bacteria that can provide protection against mobile elements such as phage viruses and transposable elements. In nature, DNA binding and cutting require Cas9 protein and two RNAs, a trans-encoding RNA (tracrRNA) and a CRISPR RNA (crRNA). Artificially, single guide RNA or sgRNA can be engineered to integrate various aspects of the two RNAs into a single species (Jinek et al., Science, 337, 816-821, doi: 10.1126 / science.1225829 (2012)). The CRISPR system has two components: 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 homology between the original spacer adjacent motif (PAM) for Cas9 recognition on DNA and the ~20 nucleotide recognition sequence encoded in the sgRNA and the genomic DNA target. After the Cas9-sgRNA complex finds and cuts the exon region of the gene to produce a frameshift mutation, the target gene can be knocked out. Cas9 recognizes short motifs in CRISPR repeat sequences to help distinguish between self and non-self. The Cas9 nuclease sequence and structure are known to those skilled in the art (Ferretti, et al. Proc Natl Acad Sci USA, 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 bacteria, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus, Campylobacter jejuni, and Neisseria meningitidis (Slaymaker, et al. Science, 351, 84-88 doi: 10.1126 / science.aad5227 (2016); Kleinstiver, et al. Nature, 529, 490-495, doi: 10.1038 / nature16526 (2016); Chen, et al. Nature, 550, 407-410, doi: 10.1038 / nature24268 (2017); Casini, et al. Nat Biotechnol, 6, 265-271, doi: 10.1038 / nbt.4066 (2018); Lee, et al. Nat Commun, 9, 3048, doi: 10.1038 / s41467-018-05477-x (2018); Vakulskas, et al. Nat Med, 24, 1216-1224, doi: 1.1038 / s41591-018-0137-0 (2018); Choi, et al. Nat Methods, 16, 722-730, doi: 10.1038 / s41592-019-0473-0 (2019); Kim, et al. Nat Commun, 8, 14500, doi: 10.1038 / ncomms14500 (2017); (Edraki, et al. Mol Cell, 73, 714-726, doi: (2019)).

[0056] "Pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, within the scope of sound medical judgment, according to the guidelines of the U.S. Food and Drug Administration and other agencies, without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. "Pharmaceutically acceptable carrier" refers to all ingredients in a pharmaceutical formulation that facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0057] "Peptide" refers to an amino acid chain between 2 and 50 amino acids in length.

[0058] "Protein" refers to an amino acid chain greater than 50 amino acids in length, eg, greater than 50 but less than 36,000 amino acids.

[0059] "Small molecule" refers to an organic molecule having a molecular weight of less than about 2500 g / mol, 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, a small molecule is a non-polymer and / or non-oligomer.

[0060] The terms "treat," "prevent," and related terms such as "treatment" mean ameliorating, reducing, or otherwise preventing the occurrence or progression of a disease, disorder, and / or condition in an animal that may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed as having the disease; inhibiting the disease, disorder, or condition, e.g., hindering its progression; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, e.g., treating pain in a subject by administering an analgesic, even if such drug does not treat the cause of the pain. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual would be successfully "treated" if one or more symptoms associated with an inherited neuropathy, an inherited myopathy, an inherited eye disease or condition, an inherited lung disease or condition, an inherited liver disease or condition, or cancer were alleviated or eliminated, including but not limited to reducing and / or inhibiting the rate of progression of the disease, improving the patient's quality of life, reducing the dose of other medications required to treat the disease, slowing the progression of the disease, and / or prolonging the patient's survival.

[0061] II. Composition

[0062] Conjugates for intracellular delivery of payloads and pharmaceutical compositions containing these conjugates have been developed. The conjugate comprises a payload, a chemical linker, and a cell membrane fusion molecule. The cell membrane fusion molecule promotes intracellular uptake of the conjugate. In some forms, the conjugate enters the cell via a non-endocytic pathway. The chemical linker can be a traceless chemical linker or a trace chemical linker. When the chemical linker is a traceless chemical linker, it comprises a stimulus-responsive chemical moiety and / or a self-destructive chemical moiety. Preferably, the traceless chemical linker comprises a stimulus-responsive chemical moiety and a self-destructive chemical moiety. After entering the cell, the conjugate is exposed to one or more stimuli, such as a reducing and / or acidic environment, which cleave the stimulus-responsive chemical moiety. This cleavage event activates the self-destructive property of the chemical linker through an electronic cascade and / or cyclization elimination. Ultimately, the linker is cleaved and removed from the payload, allowing the payload to be delivered without any trace of the cell membrane fusion molecule and chemical linker on the payload. Therefore, the payload is delivered in a stimulus-responsive and traceless manner, i.e., the stimulus-responsive traceless engineering of the conjugate is achieved. Preferably, when these conjugates include genome editing machinery, the conjugates show higher efficiency in genome editing compared to similar conjugates that cannot be cut. As used herein, "traceable" in relation to chemical linkers describes a chemical linker that cannot be cut or is cut and has its chemical part covalently bound to a joint on a payload. Traceable chemical linkers can be used to form "uncuttable" conjugates as a subset, and these conjugates have a joint that forms a covalent bond with a payload, wherein the covalent bond between the joint and the payload cannot be cut in a reducing or acidic microenvironment within 24 hours or 48 hours after cell infiltration. An example is poly (ethylene glycol) -N-hydroxysuccinimide, which reacts with a primary amino group on a payload such as an RNP surface.

[0063] This platform can be used for intracellular delivery of proteins, peptides or nucleic acids. In particular, the platform can be used for efficient intracellular delivery of genome editing systems. The results described in further detail below demonstrate that the platform can be used to effectively deliver ribonucleoproteins (RNPs) consisting of Cas9 protein and sgRNA into cells in in vitro cell cultures and in vivo reporting and disease models. The results also show that the conjugate can safely deliver payloads such as STEP RNP, because based on AST, ALT, BUN and creatine determinations, intravenous administration of payloads such as STEP RNP does not cause significant systemic toxicity in the liver and kidneys. In addition, based on microscopic imaging and H&E staining, intracranial administration of STEP RNP does not cause significant cell damage in the brain. Storing STEP RNP for a long time (such as 2 months) at subzero temperatures (such as -20°C) does not reduce the efficiency of its genome editing.

[0064] The conjugate contains the following structure:

[0065] Preferably

[0066] in:

[0067] Dashed lines independently represent the presence of one or more covalent bonds or non-covalent bonds, preferably, dashed lines represent the presence of one or more covalent bonds,

[0068] P comprises a protein, peptide, or nucleic acid;

[0069] L is a linear or branched chemical linker comprising a stimulus-responsive chemical moiety and / or a self-destructive chemical moiety,

[0070] M is a one-armed or multi-armed chemical moiety comprising a cell membrane fusion molecule, and

[0071] np, nl, nm and nz are independently an integer between 1 and 150, inclusive, an integer between 1 and 100, inclusive, an integer between 1 and 75, inclusive, an integer between 1 and 50, inclusive, an integer between 1 and 25, inclusive, an integer between 1 and 15, inclusive, an integer between 1 and 10, inclusive, an integer between 1 and 7, inclusive, or an integer between 1 and 5, inclusive.

[0072] In some forms, on the one hand, the value of np is selected, on the other hand, the value of nm and / or nz is selected so that the mol ratio of M to P is in the range of 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1 or 1:1. In some forms, on the one hand, the value of np is selected, on the other hand, the value of nl, nm and / or nz is selected so that the mol ratio of LM to P is in the range of 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1 or 1:1. In some forms, analytical methods (such as nuclear magnetic resonance spectroscopy) can be used to analyze the final product, so as to determine the mol ratio. In some forms, the mol ratio can be based on the theoretical value of the feed mole ratio of the reactant added to the reaction mixture to form the final product. Without being bound by theory, the mol ratio of the effective delivery payload can be determined by the balance of mol ratio / hydrophobicity and efficiency. For example, the ratio of M:P or LM:P is not always the higher the better. This is because most cell membrane fusion molecules are hydrophobic, and coupling too many cell membrane fusion molecules may increase cell penetration, but lead to toxicity and reduce the solubility of the conjugate (or precipitate payload). In some forms, the ratio of payload to membrane fusion part is expressed as weight percentage, i.e., the weight of the specified component is relative to the sum of the weight of payload and membrane fusion part. In some forms, the ratio of payload to membrane fusion part is 1% by weight: 99% to 99%: 1, by weight 90%: 10% to 97.5%: 2.5%, such as 50% by weight: 50% or by weight 95.6%: 4.4%.

[0073] In a preferred form, the conjugate comprises the gene editing machinery as a payload, a multi-arm chemical moiety comprising a five-ring or four-ring moiety of cholesterol, and the following moieties within the chemical linker:

[0074]

[0075] wherein Y is oxygen, W is -(CH2)2-, and X is sulfur.

[0076] Subsequent sections provide further details on the conjugates and their formulations.

[0077] (i) Payload to be delivered

[0078] The conjugate contains a payload, which can be one or more proteins; polypeptides; nucleic acids, such as mRNA, sgRNA or DNA; ribonucleoproteins; or combinations thereof. Preferably, the payload is covalently coupled to a chemical linker.

[0079] (a) Genome editing machine

[0080] In some embodiments, 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 catalytically impaired Cas proteins fused to DNA modification enzymes), containing catalytically impaired Cas proteins fused to engineered reverse transcriptases (such as Cas9 nickases - making DNA gaps instead of producing double-stranded breaks) of the Cas9 variants) leader (prime) editors, peptide nucleic acids (PNAs) and antisense oligonucleotides. In a preferred embodiment, the gene editing system is a CRISPR / Cas system. In some embodiments, gene editing technology is a donor oligonucleotide, which can be used alone to modify a gene. Strategies include, but are not limited to, small fragment homology replacement (e.g., small polynucleotide DNA fragments (SDFs)), single-stranded oligodeoxynucleotide-mediated gene modification (e.g., ssODN / SSO), and other strategies described in Sargent, Oligonucleotides, 21(2):55–75 (2011) and elsewhere. Other suitable gene editing technologies include, but are not limited to, intron-encoded meganucleases that are engineered to alter their target specificity. See, for example, Arnould, et al., Protein Eng. Des. Sel., 24(1-2):27-31 (2011).

[0081] In a preferred embodiment, the gene editing system is a protein-guided gene editing system, such as the CRISPR system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs).

[0082] (1) CRISPR / Cas

[0083] In some embodiments, the gene editing system that induces single-strand or double-strand breaks in the genome of the target cell is CRISPR / Cas, or a nucleic acid construct encoding a Cas nuclease.

[0084] CRISPR (clustered regularly interspaced short palindromic repeats) is an abbreviation for a DNA site that contains multiple short, direct base sequence repeats. Prokaryotic CRISPR / Cas systems have been modified for gene editing (silencing, enhancing, or changing specific genes) in eukaryotic organisms (see, for example, Cong, Science, 15: 339(6121): 819–823(2013) and Jinek, et al., Science, 337(6096): 816-21(2012)). By transfecting cells with the required elements (including cas genes and specially designed CRISPR), the genome of an organism can be cut and modified at any desired position. The method for preparing a composition for genome editing using the CRISPR / Cas system is described in detail in WO 2013 / 176772 and WO 2014 / 018423.

[0085] Typically, "CRISPR system" refers collectively to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") genes or direct their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. One or more tracr pairing sequences (e.g., direct repeat-spacer-direct repeat) operably linked to a guide sequence may also be referred to as pre-crRNA (pre-CRISPR RNA) before processing, or as crRNA after nuclease processing.

[0086] In some embodiments, tracrRNA and crRNA are connected to form a chimeric crRNA-tracrRNA hybrid, wherein the mature crRNA is fused to a portion of the 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 may also be referred to as a guide RNA or gRNA (or single guide RNA (sgRNA)). In sgRNA, the crRNA portion may be identified as a “target sequence” and the tracrRNA is often referred to as a “scaffold.”

[0087] Once the desired DNA target sequence is identified, there are many resources available to help practitioners identify suitable target sites. For example, a number of public resources, including a bioinformatics-generated list of approximately 190,000 potential sgRNAs targeting more than 40% of human exons, can be used to help practitioners select target sites and design relevant sgRNAs to affect nicks or double-strand breaks at the site. See also crispr.u-psud.fr / , a tool designed to help scientists find CRISPR target sites and generate appropriate crRNA sequences in various species.

[0088] In some embodiments, the payload to be delivered is one or more CRISPR-associated enzyme (Cas) nucleases. Exemplary Cas nucleases suitable as gene editing compositions include Cas9, CasX (also known as Cas12e), Cas7-11, CasFx, Cas12a, and Cas13.

[0089] In some embodiments, the payload to be delivered is one or more Cas nucleases, which are also complexed with one or more single guide RNAs (sgRNAs) to form CRISPR / Cas ribonucleoproteins (RNPs). In some embodiments, the Cas nuclease is optionally covalently coupled to one or more scarless linkers and one or more membrane fusion molecules via one or more linking moieties. In a preferred embodiment, the Cas nuclease is covalently coupled to one or more membrane fusion molecules via one or more scarless linkers. In one embodiment, one or more cholesterol molecules or PEGylated forms thereof (e.g., PEG n , n=1-30) were covalently coupled to the Cas9 nuclease via a scarless linker such as DBNPDEE.

[0090] In some embodiments, the payload is one or more Cas9 nucleases, preferably complexed with one or more single guide RNAs (sgRNAs) to form CRISPR / Cas ribonucleoproteins (RNPs). In a preferred embodiment, the Cas nuclease is covalently coupled to one or more traceless linkers and one or more membrane fusion molecules before or after the Cas9 is complexed with the sgRNA. In a preferred embodiment, the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease or a variant thereof.

[0091] In some forms, the payload is 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.

[0092] In some forms, the payload comprises a base editor. 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 describe base editors, the contents of which are incorporated herein by reference. Base editing is a genome editing technology based on CRISPR-Cas9 that allows point mutations to be introduced into DNA without producing double-stranded DNA breaks. An exemplary base editor is constructed by fusing Cas9 nickase (nCas9) with a base modification enzyme. Currently, there are three types of base editors: cytosine base editors (CBEs) that allow C>T conversion, adenine base editors (ABEs) that allow A>G conversion, and C- to -G base editors (CGBEs). In some forms, the payload includes a lead editor. Anzalone et al., Nature 2019, 576 (7785), 149-157 describe a lead editor, the contents of which are incorporated herein by reference. Similar to CRISPR, lead editing requires the presence of a Cas endonuclease and a single guide (sg) RNA. However, since the premise of lead editing is to edit the sequence without producing a double-strand break, both components are slightly modified. Unlike traditional Cas9, this method utilizes a variant of Cas9 nickase—Cas9 that makes a DNA gap instead of producing a double-strand break—fused to a reverse transcriptase. This Cas9 fusion is called a lead editor.

[0093] In some forms, the payload comprises a base editor and a prime editor.

[0094] In some embodiments, an inactivated Cas9 (dCas9), a nuclease-deficient mutant variant of the Cas9 protein (e.g., a point mutation that inactivates the DNA cleavage activity of the Cas9 protein (e.g., D10A, H840A)) is used as a payload. The CRISPR-Cas9 gene knockout system is also suitable for gene modulation technologies collectively referred to as CRISPR modulation (CRISPRmod), which include CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These technologies utilize nuclease-inactivated Cas9 (dCas9), which binds to the target genomic region with the same efficiency as Cas9, but cannot produce DSBs, but instead results in RNA-guided transcriptional control of the target region. CRISPRi utilizes dCas9 with or without a fused repressor domain and a guide RNA to target the promoter region for transcriptional inhibition or knockout of the gene. In contrast, CRISPRa uses dCas9 fused to a transcriptional activation domain, which can be guided to the promoter region by one or more guide RNAs that recruit additional effectors for transcriptional activation and increase the expression of the target gene. CRISPRi and CRISPRa technologies silence or activate transcription by attaching effector domains to dCas9, thereby generating artificial transcription factors. CRISPRi or CRISPRa require guide RNA design near the gene promoter region or transcription start site (TSS) to cause silencing or activation, respectively (L.A. Gilbert et al., Cell. 159, 647-661 (2014); S. Konermann et al., Nature. 517, 583–588 (2015)).

[0095] In a preferred embodiment, the payload is an epigenome editing machine based on CRISPR. For example, the catalytic domain of ten-eleven translocation (TET) 1 hydroxylase is fused to a catalytically inactive Cas9 nuclease (dCas9). A CRISPR-based epigenome editing machine has been described before (Nunez JK et al., Cell. 2021 Apr 29; 184 (9): 2503-2519). In some embodiments, the payload is an RNP based on dCas9-TETv4, resulting in effective epigenetic editing of the target region.

[0096] (2) Zinc finger nuclease

[0097] In some embodiments, the gene editing system that induces single-strand or double-strand breaks in the genome of the target cell is a zinc finger nuclease (ZFN) or a nucleic acid construct encoding a ZFN. ZFN is typically a fusion protein that includes a DNA-binding domain derived from a zinc finger protein connected to a cleavage domain.

[0098] The most common cleavage domain is the type IIS enzyme Fok1. Fok1 catalyzes double-stranded DNA cleavage, with one strand 9 nucleotides away from its recognition site and the other strand 13 nucleotides away from its recognition site. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al., Proc., Natl. Acad. Sci. USA 89 (1992): 4275-4279; Li et al., Proc. Natl. Acad. Sci. USA, 90: 2764-2768 (1993); Kim et al., Proc. Natl. Acad. Sci. USA. 91: 883-887 (1994a); Kim et al., J. Biol. Chem. 269: 31,978-31,982 (1994b). One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of a cleavage domain.

[0099] In principle, DNA-binding domains can be designed to target any genomic location of interest and can be tandem arrays of Cys2His2 zinc fingers, each of which typically 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. By linking multiple fingers together (the number varies: in published studies, three to six fingers per monomer have been used), ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotides long.

[0100] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. For example, rational design includes using a database comprising triplet (or quadruple) nucleotide sequences and single zinc finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a specific triplet or quadruple sequence. For example, see U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Published Application Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publication Nos. WO 98 / 53059 and WO 2003 / 016496.

[0101] (3) Transcription activator-like effector nuclease

[0102] In some embodiments, the gene editing system that induces single-strand or double-strand breaks in the genome of the target cell is a transcription activator-like effector nuclease (TALEN), or one or more nucleic acid constructs encoding TALEN. The overall structure of TALEN is similar to ZFN, with the main difference being that the DNA-binding domain comes from the TAL effector protein, a transcription factor from a plant pathogen. The DNA-binding domain of TALEN is a tandem array of amino acid repeats, each repeat being approximately 34 residues long. The repeats are very similar to each other; typically, they differ primarily at two positions (amino acids 12 and 13, called repeat variable diresidues or RVDs). Each RVD specifies a preference for binding to one of four possible nucleotides, meaning that each TALEN repeat binds to one base pair, although the NN RVD is known to bind to adenine in addition to guanine. Compared to zinc finger proteins, the mechanism of TAL effector DNA binding is less clear, but their seemingly simple code may be very beneficial for engineering nuclease design. TALENs can also cut as dimers with relatively long target sequences (the shortest target sequence reported to date binds 13 nucleotides per monomer) and appear to have less stringent requirements for the length of the spacer between binding sites than ZFNs. Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats.

[0103] Methods for engineering TALs to bind to specific nucleic acids are described in Cermak, et al., Nucl. Acids Res. 1-11 (2011). U.S. Publication No. 2011 / 0145940 discloses TAL effectors and methods of using them to modify DNA. Miller et al., Nature Biotechnol 29: 143 (2011) reported the preparation of TALENs for site-specific nuclease structures by linking TAL truncated variants to the catalytic domain of Fok1 nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. General design principles for TALE binding domains can be found, for example, in WO 2011 / 072246.

[0104] (b) Other biomolecular payloads

[0105] The payload may 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), bis-scFv, tri-scFv, diabodies, trivalent antibodies, tetravalent antibodies (teratbodies), disulfide-linked Fvs (sdFv), Fab', F(ab')2, Fv, single domain antibody fragments (sdAb).

[0106] (ii) Traceless stimuli-responsive linkers

[0107] (a) Stimuli-responsive chemical part

[0108] The above-mentioned stimulus-responsive chemical moiety may have one or more chemical groups or bonds that respond to external stimuli. The stimulus-responsive chemical moiety can be cut by a stimulus selected from pH (such as pH changes), redox (such as redox potential changes), reactive oxygen species (ROS), enzymes (such as overexpression of enzymes (such as proteases, esterases, etc.) due to a diseased state or condition), ionic strength (such as ionic strength changes) and hypoxia. Examples of stimulus-responsive chemical moieties that respond to ROS are described in Saravanakumar et al. Adv. Sci. 2017, 4, 1600124, the contents of which are incorporated herein by reference.

[0109] In some forms, the stimulus-responsive chemical moieties independently comprise disulfide bonds, amide bonds, orthoesters, hydrazones, hydrazides, hydrazines, imines (e.g., aldimines or ketimines), oximes, acetals, vinyl ethers, polyketals, methyl maleate, ester bonds, nitroaryls (e.g., nitrobenzyl), nitroheteroaryls (e.g., nitroimidazole), quinone groups, azoaryls (e.g., azophenyl), azoheteroaryls (e.g., azopyridyl), peroxyoxalates, aminoacrylates, alkyl thioethers or selenoethers (e.g., monoselenoether bonds, diselenoether bonds, etc.), thioketals, peroxyoxalates, or dimethyl maleate.

[0110] In some forms, the stimulus-responsive chemical moieties are independently disulfide bonds, amides, orthoesters, imines (such as aldimines or ketimines), hydrazones, hydrazides, hydrazines, imines, oximes, methyl maleate, ester bonds, dimethyl maleate, or combinations thereof.

[0111] In some forms, the stimulus-responsive chemical moieties independently comprise a disulfide bond, an amide, an orthoester, an imine (such as an aldimine or ketimine), or an ester bond.

[0112] In some forms, the orthoester contains

[0113]

[0114] For example

[0115]

[0116] and combinations thereof,

[0117] in:

[0118] Rx and Rx' are independently hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted C3-C 20 Cycloalkyl, substituted C3-C 20 Cycloalkyl, unsubstituted C1-C 20 Heterocyclic or substituted C3-C 20 Heterocyclic group.

[0119] Figure 3A Non-limiting examples of chemical structures that can be used to generate linkers containing stimulus-responsive chemical moieties are shown in .

[0120] (b) Self-destructive chemical part

[0121] The self-destructive chemical moiety is a chemical moiety that self-degrades through one or more elimination processes. Typically, self-degradation occurs through an electronic cascade process and / or cyclization elimination. Self-degradation can be initiated when a covalent bond is broken and / or by reduction of a chemical group (such as a nitro group) in the self-destructive chemical moiety. Preferably, this self-degradation is spontaneous and irreversible when a covalent bond is broken. Therefore, as used herein, a self-destructive chemical moiety is a moiety containing covalently linked atoms that is designed to spontaneously degrade in response to a stimulus. Degradation typically involves the breaking of one or more chemical bonds, preferably two or more chemical bonds.

[0122] In some forms, the self-destructive chemical moiety shares an atom or chemical group and a bond with the stimuli-responsive chemical moiety, such that breaking the bond causes the atom to become negatively charged or increase its ability to donate lone pairs of electrons. This atom or chemical group triggers self-destructive properties. The following schematic illustrates breakage-induced self-destructive properties, showing the sharing of atoms or chemical groups and bonds between the stimuli-responsive chemical moiety and the self-destructive chemical moiety.

[0123] Scheme 1: Non-limiting example of self-destruction by cyclization elimination

[0124]

[0125] In Scheme 1 above, M represents a single-arm or multi-arm chemical moiety comprising a cell membrane fusion molecule; the linker comprises a stimulus-responsive chemical moiety (SR) and a self-immolative chemical moiety (SE); and P represents a protein, peptide, or nucleic acid. Furthermore, when a black circle represents M, a gray circle represents P, and vice versa. As shown above, the stimulus-responsive chemical moiety and the self-immolative chemical moiety share a sulfur atom and a bond. Breaking this bond triggers a cascade of self-immolative reactions via cyclization. These reactions are typically driven by favorable entropy changes and the formation of stable products.

[0126] Self-destructive chemical moieties include p-aminobenzyl (e.g., p-NH-phenyl-CH2-), o-aminobenzyl (e.g., o-NH-phenyl-CH2-), p-oxybenzyl (e.g., p-O-phenyl-CH2-), o-aminobenzyl (e.g., o-O-phenyl-CH2-), p-thiobenzyl (e.g., p-S-phenyl-CH2-), o-thiobenzyl (e.g., o-S-phenyl-CH2-), cinnamyl ether, cyclization-driving moieties, Grob fragmentation moieties, etc. The cyclization-driving moieties include the following structures:

[0127]

[0128] wherein X can be O, NH, or S; Y can be O, NRs, substituted alkyl, or unsubstituted alkyl; W can be substituted alkyl or unsubstituted alkyl; wherein Rs is hydrogen, substituted alkyl, or unsubstituted alkyl. In some forms, Y is oxygen. In some forms, X is sulfur. In some forms, W is C2-C5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl, or C5 substituted alkyl. In some forms, Y is oxygen, W is C2-C5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl, or C5 substituted alkyl; and X is sulfur.

[0129] In some forms, the chemical linker is formed from:

[0130] L is formed using a structure selected from the following:

[0131]

[0132] or a combination thereof.

[0133] In a preferred form, the chemical linker is formed by:

[0134]

[0135] The Grob fragment contains the following structures:

[0136]

[0137] wherein X can be O, NH or S.

[0138] Chemical structures containing self-immolative chemical moieties that can be used to create suitable linkers are described in Ferhati, et al., Org. Lett. 2021, 23, 8580-8584 and Gavriel, et al., Poly. Chem. 2022, 13, 3188-3269, the contents of which are incorporated herein by reference.

[0139] (iii) Trace chemical joints

[0140] In some forms, the payload and the cell membrane fusion molecule are coupled to each other via a trace chemical linker. A trace chemical linker is a chemical linker that forms a covalent bond with the payload, wherein the covalent bond between the chemical linker and the payload cannot be cleaved in the reducing or acidic microenvironment within the cell within 24 hours or 48 hours after cell penetration, or a chemical linker that is cleaved and the chemical portion is covalently bound to the payload. Typically, the trace chemical linker does not contain a disulfide bond or is formed by a bifunctional molecule that does not contain a disulfide bond. In the features described herein, the trace chemical linker comprises the following structure:

[0141] -A-L1-B-

[0142] wherein A and B are independently selected from -NHC(O)-; -C(O)NH-; -C(O)O-; -OC(O)-; -O-; -NR-; -OC(O)NH-; -NHC(O)O-; -C(O)-; -OC(O)O-; -S(=O2)2-; -S(=O)- and -S-, wherein R is hydrogen, substituted alkyl or unsubstituted alkyl; and L1 is 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. Examples of bifunctional molecules that can be used to form trace chemical linkers include, but are not limited to, diols such as 1,2-ethanediol, 1,3-propylene glycol, 1,4-butanediol, 2,3-butanediol, 1,5-pentanediol, benzene-1,4-diol, 1,6-hexanediol, tetra(ethylene glycol) glycol), polyethylene glycol (PEG); dithiols such as 1,2-ethanedithiol, 1,3-propylenedithiol, 1,4-butanediol, 2,3- butanedithiol, 1,5-pentanedithiol, benzene-1,4-dithiol, 1,6-hexanedithiol, tetra(ethylene glycol)dithiol); dicarboxylic acids; aldehydes such as glyoxal, methylglyoxal, 2-formylmalondialdehyde, glutaraldehyde, adipaldehyde, heptanedial, octanedial; diglycidyl ethers; diamines such as ethylenediamine, propane-1,2-diamine, propane-1,3-diamine, N-methylethylenediamine, N,N'-dimethylethylenediamine, pentane-1, ,5-diamine, hexane-1,6-diamine, spermine and spermidine; divinyl adipate; divinyl sebacate; diamine-terminated PEG, diester PEG-N-hydroxysuccinimide; diisocyanate-terminated PEG, epichlorohydrin; S-acetylthioglycolate N-hydroxysuccinimide ester; 5-azido-2-nitrobenzoic acid N-hydroxysuccinimide ester; 4-azidobenzoyl bromide; bromoacetic acid N-hydroxysuccinimide Amine ester; N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; N-hydroxysuccinimide iodoacetate; N-hydroxysuccinimide 4-(N-maleimido)benzophenone 3-(2-pyridyldithio)propionate; N-hydroxysuccinimide 3-maleimidobenzoate; N,N'-cystamine bisacrylamide; N,N'-methylene bisacrylamide; and N,N'-ethylene bisacrylamide. In some forms, the trace chemical linker is formed by using a structure selected from the group consisting of:

[0143]

[0144] or a combination thereof.

[0145] (iv) Membrane fusion molecules (membrane fusion molecules)

[0146] In some forms, the membrane fusion molecule is a cell membrane fusion molecule. These cell membrane fusion molecules contain proteins, peptides, lipids and / or small molecules. Preferably, these cell membrane fusion molecules enhance the fusion between the conjugate and the cell membrane and / or promote the intracellular uptake of the conjugate.

[0147] Examples of peptides that can be used as cell membrane fusion molecules include cell penetrating peptides, also known as cell permeation peptides, protein transduction domains (PTDs), membrane translocation sequences (MTSs), and Trojan peptides. Cell penetrating peptides (CPPs) include, but are not limited to, virus-derived or mimetic polymers such as TAT, influenza fusion peptide, rabies virus glycoprotein fragment (RVG), neurofibromin, penetratin, and polyarginine. Anaspec has commercially available CPPs. Other examples of cell penetrating peptides and motifs are described in, for example, U.S. Patent Application Publication Nos. 2014016957, 20100061942, 20100061932, 20100048487, 20100022466, 20100016215, 20090280058, 20090186802, 20080234183, 20060014712, 20050260756, and 20030077289. Exemplary 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). Other examples of 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.

[0148] Examples of lipids that can be used as cell membrane fusion 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 phosphatidylethanolamines with two linked fatty acyl chains (preferably unsaturated fatty acyl chains); lysolipids, etc.

[0149] Examples of small molecule moieties that can be used as cell membrane fusion molecules include pentacyclic or tetracyclic moieties of cholesterol, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens, progestins, plant sterols (e.g., β-sitosterol); or hydrophobic amino acid residues (e.g., tyrosine, phenylamine, tryptophan, etc.).

[0150] In some forms, the cell membrane fusion molecule comprises a protein; and / or a peptide, such as a cell penetrating peptide. In some forms, the cell membrane fusion molecule comprises a small molecule portion, such as a five-ring or four-ring portion of the following substances: cholesterol, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens or progestogens, plant sterols (e.g., β-sitosterol). In some forms, the cell membrane fusion molecule comprises a small molecule portion, such as a five-ring or four-ring portion of cholesterol.

[0151] In some forms, the portion of the conjugate comprising the cell membrane fusion molecule is a one-arm chemical moiety. In some forms, the portion of the conjugate comprising the cell membrane fusion molecule is multi-armed, such that the portion comprises multiple cell membrane fusion molecules. In some forms, the portion of the conjugate comprising the cell membrane fusion molecule is a multi-arm chemical moiety containing 2 to 10, 2 to 9, 2 to 8, or 2 to 7 small molecule moieties, such as a tetracyclic or pentacyclic moiety of cholesterol.

[0152] In some forms, the portion of the conjugate comprising the cell membrane fusion molecule is a multi-arm chemical moiety comprising 2 to 7, preferably 2, small molecule moieties, such as a tetracyclic or pentacyclic moiety of cholesterol.

[0153] In some forms, the portion of the conjugate comprising the cell membrane fusion molecule is formed from:

[0154]

[0155] or Figure 5A Other molecules shown in

[0156]

[0157]

[0158] In some preferred forms, the conjugate comprises a hydrophilic polymer between L and M. In some forms, the hydrophilic polymer participates in the covalent bonding of L and M. In some forms, the hydrophilic polymer is directly bound to L. Preferably, the hydrophilic polymer is associated with M. Without wishing to be bound by theory, it is believed that the presence of such a 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 cellulose, alginates, glucosaminoglycans, and dextran; hydrophilic polypeptides and poly(amino acids), such as poly-L-glutamic acid, γ-polyglutamic acid, poly-L-aspartic acid, and poly-L-serine; poly(oxyethylated polyols); poly(olefin alcohols), such as poly(vinyl alcohol) and aminoacetated poly(vinyl alcohol); poly(N-vinylpyrrolidone); acrylic acid or acrylates, and alkylacrylic acid or alkylacrylate polymers, such as poly(acrylic acid), poly(methacrylic acid), poly(hydroxyethyl acrylate); poly(N,N-dimethylaminoethyl methacrylate), poly(hydroxyalkyl methacrylates), such as poly(hydroxyethyl methacrylate); acrylamide polymers such as poly(acrylamide), poly(hydroxyalkylmethacrylamides), such as poly(hydroxyethylmethacrylamide); and poly(4-vinylpyridine); and copolymers thereof. Preferably, the hydrophilic polymer segment comprises a neutral hydrophilic polymer, such as a neutral uncharged hydrophilic polymer. Examples of neutral uncharged hydrophilic polymers include, but are not limited to, polyalkylene glycols and polyalkylene oxides, such as polyethylene glycol (PEG); polysaccharides such as cellulose and dextran; hydrophilic polypeptides and poly(amino acids), such as poly-L-serine; poly(oxyethylated polyols); poly(olefin alcohols), such as poly(vinyl alcohol); poly(N-vinyl pyrrolidone); poly(hydroxyethyl acrylate); poly(hydroxyalkyl methacrylate), such as poly(hydroxyethyl methacrylate). Preferably, the hydrophilic polymer segment comprises a neutral uncharged hydrophilic polymer, such as polyalkylene glycol and polyalkylene oxide, such as polyethylene glycol. Preferably, the hydrophilic polymer has a molecular weight between 100 Da and 10 kDa, inclusive, or between 200 Da and 10 kDa, inclusive. In some forms, the hydrophilic polymer segment comprises 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.

[0159] In some forms, LM is formed by the reaction of two parts:

[0160]

[0161] wherein n is such that the molecular weight of the poly(ethylene glycol) is between 200 Da and 10 kDa. In the reaction, the alkyne group in the DBCO moiety reacts with the azide to form a triazole via a click reaction, thereby inserting the poly(ethylene glycol) having a molecular weight between 200 Da and 10 kDa between the cell membrane fusogenic moiety (e.g., the cholesterol moiety shown above) and the DBCO group. Preferably, the payload can react with the p-nitrophenyl carbonate (NPC) moiety to attach the payload to the rest of the conjugate. A non-limiting example of conjugation to LM in this configuration is shown below:

[0162]

[0163] In other forms, the conjugate is as described above, except that one or more of the above hydrophilic polymers are not present between L and M. This can be achieved when n is zero, and / or the oxygen atom in the moiety -(OCH2CH2)- is absent or is CR L R L’ Part, in CR L R L’ In, R L and R L’ are independently hydrogen, substituted alkyl or unsubstituted alkyl.

[0164] In some forms, conjugate is as described above, and difference is that the mol ratio of cell membrane fusion molecule and payload or the mol ratio of cell membrane fusion protein and chemical linker and payload on the one hand is 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1 or 1:1.In some forms, conjugate is as described above, and difference is that the mol ratio of cell membrane fusion molecule and joint and payload is 100:1 to 1:1, such as 100:1, 50:1, 25:1, 20:1, 10:1, 5:1 or 1:1.In some forms, analytical method (such as nuclear magnetic resonance spectroscopy) can be used to analyze final product, thus determine mol ratio.In some forms, mol ratio can be based on the theoretical value of the feed mole ratio of the reactant added to form final product in reaction mixture.

[0165] III. Preparation Methods and Reagents

[0166] The conjugate can be synthesized using various methods known to those skilled in the art, including but not limited to chemical synthesis, semi-synthesis, or a combination thereof. Preferably, the conjugate is prepared by chemical synthesis. In a non-limiting example, RNP is assembled by incubating Cas9 protein with sgRNA (molar ratio, for example, 1:3). Next, DSC-cholesterol (72) can be added (molar ratio, for example, 3:1 with RNP). After removing unreacted DSC-cholesterol (2) and sgRNA, cls-RNP can be obtained. For the synthesis of DSC-cholesterol (2), a precursor linker molecule such as DBCO-DSC DBNPDEE can be mixed with an azide-functionalized Tris derivative terminated with two cholesterols. The alkynyl group in the DBCO moiety can react with an azide to form a triazole through a click reaction.

[0167] IV. How to use

[0168] Methods of using the disclosed conjugates and compositions are described. In some embodiments, the compositions are capable of editing in vitro, ex vivo, and in vivo in the context of prokaryotic and eukaryotic cells. In further embodiments, the compositions are capable of editing genes in agricultural contexts, such as in plants.

[0169] (i) Intracellular delivery methods of gene editing platforms

[0170] The composition can be used for ex vivo or in vivo gene editing. The method generally comprises contacting a cell with an effective amount of the disclosed composition to modify the genome of the cell. As discussed in more detail below, contact can occur in vitro or in vivo. In a preferred embodiment, the method comprises contacting a target cell population with an effective amount of a gene editing composition to modify the genome of a sufficient number of cells to achieve a desired outcome, such as a therapeutic outcome or a modified trait.

[0171] For example, an effective amount or therapeutically effective amount can be a dose sufficient to treat, inhibit or alleviate one or more symptoms of a disease or condition, or otherwise provide a desired pharmacological and / or physiological effect, such as reducing, inhibiting or reversing one or more potential pathophysiological mechanisms behind a disease or condition. The effective amount can include a dose 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), 0.1 mg and 50 mg (inclusive), 0.1 mg and 60 mg (inclusive), 0.1 mg and 7 ... and 40mg), between 5mg and 70mg (including 5mg and 70mg), between 5mg and 60mg (including 5mg and 60mg), between 5mg and 50mg (including 5mg and 50mg), between 5mg and 40mg (including 5mg and 40mg), between 10mg and 70mg (including 10mg and 70mg), between 10mg and 60mg (including 10mg and 60mg), between 10mg and 50mg (including 10mg and 50mg), between 10mg and 40mg (including 10mg and 40mg), 1 Between 5mg and 70mg (including 15mg and 70mg), between 15mg and 60mg (including 15mg and 60mg), between 15mg and 50mg (including 15mg and 50mg), between 15mg and 40mg (including 15mg and 40mg), between 20mg and 70mg (including 20mg and 70mg), between 20mg and 60mg (including 20mg and 60mg), between 20mg and 50mg (including 20mg and 50mg), between 20mg and 40mg (including 20mg and 40mg), 2 Between 5mg and 70mg (including 25mg and 70mg), between 25mg and 60mg (including 25mg and 60mg), between 25mg and 50mg (including 25mg and 50mg), between 25mg and 40mg (including 25mg and 40mg), between 30mg and 70mg (including 30mg and 70mg), between 30mg and 60mg (including 30mg and 60mg), between 30mg and 50mg (including 30mg and 50mg), or between 30mg and 40mg (including 30mg and 40mg).In preferred forms, the effective amount includes a dose of the conjugate between 20 mg and 50 mg (inclusive), between 25 mg and 40 mg (inclusive), between 30 mg and 45 mg (inclusive), or between 30 mg and 40 mg (inclusive).

[0172] Preparations are prepared to suit the mode of administration. The pharmaceutically acceptable carrier depends in part on the specific composition being administered and the specific method for administering the composition. Thus, there are a variety of suitable formulations for pharmaceutical compositions containing nucleic acids. The precise dosage will vary according to various factors, such as subject-dependent variables (e.g., age, immune system health, clinical symptoms, etc.). Exemplary symptoms, pharmacological and physiological effects will be discussed in more detail below.

[0173] The composition can be administered or otherwise contacted with the target cells once, twice, or three times daily; once, twice, three times, four times, five times, six times, seven times weekly; or once, twice, three times, four times, five times, six times, seven times, or eight times monthly. For example, in some embodiments, the composition is administered once every two or three days, or an average of about 2 to about 4 times per week.

[0174] In a preferred embodiment, the composition is administered in an amount effective to induce genetic modification of at least one target allele in a target cell at a 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%. In some embodiments, particularly in ex vivo applications, the frequency of genetic modification in at least one target allele is about 0.1-25%, or 0.5-25%, or 1-25%, or 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 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%.

[0175] In some embodiments, particularly for in vivo applications, the frequency of genetic modification in at least one target allele is from about 0.1% to about 10%, or from about 0.2% to about 10%, or from about 0.3% to about 10%, or from about 0.4% to about 10%, or from about 0.5% to about 10%, or from about 0.6% to about 10%, or from about 0.7% to about 10%, or from about 0.8% to about 10%, or from about 0.9% to about 10%, or from about 1.0% to about 10%, or from about 1.1% to about 10%, or from about 1.1% to about 10%. 0%, or about 1.2% to 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%.

[0176] In some embodiments, the genetic modification occurs with low off-target effects. In some embodiments, the off-target modification is not detectable using conventional analysis. In some embodiments, the frequency of off-target events is 0-1%, or 0-0.1%, or 0-0.01%, or 0-0.001%, or 0-000.1%, or 0-0.00001%. In some embodiments, the frequency of off-target modifications is about 10% lower than that of the on-target site. 2 , 10 3 , 10 4 or 10 5 times.

[0177] In some embodiments, the method includes the step of selecting a subject who may benefit from treatment with a disclosed gene editing composition.

[0178] (1) In vitro gene therapy

[0179] In some embodiments, ex vivo gene therapy of cells is used to treat a genetic disease in a subject. For ex vivo gene therapy, cells are isolated from the subject and contacted with a composition ex vivo to produce cells containing a mutation in or near a gene. In a preferred embodiment, the cells are isolated from the subject to be treated or a syngeneic host. Prior to contact with the gene editing composition, the target cells are removed from the subject. These cells can be hematopoietic progenitor cells or stem cells. In a preferred embodiment, the target cells are CD34 +Hematopoietic stem cells. Hematopoietic stem cells (HSCs), such as CD34+ cells, are multipotent stem cells that give rise to all blood cell types, including red blood cells. Therefore, CD34+ cells can be isolated from patients with, for example, thalassemia, sickle cell disease, or lysosomal storage diseases, and the mutated gene can be altered or repaired ex vivo using compositions and methods, and the cells can be reintroduced into the patient as a treatment or cure.

[0180] Stem cells can be isolated and enriched by those skilled in the art. + Such separation and enrichment methods for hematopoietic progenitor cells and other cells are known in the art, for example, as disclosed in U.S. Patent Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136; 5,716,827; 5,750,397 and 5,759,793. As used herein in the context of enrichment of a composition in hematopoietic progenitor cells and stem cells, "enrichment" means that the proportion of the desired element (e.g., hematopoietic progenitor cells and stem cells) is greater than the proportion in the natural source of the cell. The cell composition can be enriched in at least one order of magnitude, preferably two or three orders of magnitude, more preferably 10, 100, 200 or 1000 orders of magnitude over the natural source of the cell.

[0181] In humans, CD34 + Cells can be recovered from umbilical cord blood, bone marrow, or blood after cytokine mobilization by subcutaneous or intravenous injection of hematopoietic growth factors (such as granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF)) in an amount sufficient to move hematopoietic stem cells from the bone marrow space to the peripheral circulation. Initially, bone marrow cells can be obtained from any suitable bone marrow source, such as tibia, femur, spine, and other bone cavities. In order to separate the bone marrow, the bones can be flushed with an appropriate solution, which will be a balanced salt solution, conveniently supplemented with fetal bovine serum or other naturally occurring factors, and combined with a low concentration of an acceptable buffer, typically about 5 to 25 mM. Convenient buffers include Hepes, phosphate buffer, lactate buffer, etc.

[0182] Cells can be selected by positive and negative selection techniques. Cells can be selected using commercially available antibodies that bind to hematopoietic progenitor or stem cell surface antigens (such as CD34) using methods known to those skilled in the art. For example, the antibody can be coupled to magnetic beads and an immunogenicity program can be used to recover the desired cell type. Other techniques include the use of fluorescence activated cell sorting (FACS). The CD34 antigen is found on progenitor cells in the hematopoietic system of non-leukemic individuals and is expressed on a population of cells recognized by the monoclonal antibody My-10 (i.e., expressing the CD34 antigen), which can be used to isolate stem cells for bone marrow transplantation. My-10, deposited as HB-8483 at the American Type Culture Collection (Rockville, Md.), is commercially available as anti-HPCA 1. In addition, negative selection of differentiated and "specialized" cells in human bone marrow can be used to select essentially any desired cell marker. For example, progenitor or stem cells, most preferably CD34 + cells, which can be characterized as CD3 - 、CD7 - 、CD8 - 、CD10 - 、CD14 - 、CD15 - 、CD19 - 、CD20 - 、CD33 - , HLA class II + and Thy-1 + Any of the above.

[0183] Once isolated progenitor cells or stem cells, they can be bred by growing in any suitable culture medium. For example, progenitor cells or stem cells can be grown in a conditioned medium from stromal cells (such as those stromal cells associated with factor secretion that can be obtained from bone marrow or liver) or in a culture medium comprising cell surface factors that support stem cell proliferation. Use appropriate monoclonal antibodies to remove unwanted cells, and the hematopoietic cells in the stromal cells can be removed.

[0184] The separated cells are contacted in vitro with the disclosed gene editing composition in an amount that effectively causes the desired mutation in or near the gene that needs to be repaired or changed (such as human β-globin or α-L-arabinuronidase gene). Methods for transfecting 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 cells in the S-phase to further increase the frequency of gene correction. Methods for synchronizing cultured cells, such as by double thymidine blocking, are known in the art.

[0185] 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. In particular, CD34 + The maintenance conditions have been well studied, and several suitable methods are available. A common method for in vitro multipotent hematopoietic cell expansion is to culture purified progenitor cells or stem cells in the presence of early acting cytokines such as interleukin-3. It has also been shown that the addition of thrombopoietin (TPO), stem cell factor (SCF) and flt3 ligand (Flt-3L; i.e., a ligand of the flt3 gene product) to the nutrient medium for in vitro maintenance of hematopoietic progenitor cells helps to expand primitive (i.e., relatively undifferentiated) human hematopoietic progenitor cells in vitro, and these cells can be implanted in SCID-hu mice (Luens et al., 1998, Blood 91: 1206-1215). In other known methods, cells can be maintained in vitro (e.g., for minutes, hours, or 3, 6, 9, 13 days or longer) in nutrient media, including murine prolactin-like protein E (mPLP-E) or murine prolactin-like protein F (mPIP-F; collectively referred to as mPLP-E / IF) (U.S. Patent No. 6,261,841). It should be understood that other suitable cell culture and expansion methods may also be used according to the present invention. Cells can also be grown in serum-free medium, as described in U.S. Patent No. 5,945,337.

[0186] In another embodiment, the modified hematopoietic stem cells are differentiated ex vivo into CD4 T cells using a specific combination of interleukins and growth factors prior to administration to a subject using methods well known in the art. + Cell Cultures. The cells can be expanded ex vivo in greater quantities than the originally isolated hematopoietic stem cell population, preferably at least 5-fold, more preferably at least 10-fold, and even more preferably at least 20-fold.

[0187] In another embodiment, the cells used for ex vivo gene therapy can be dedifferentiated somatic cells. Somatic cells can be reprogrammed into pluripotent stem-like cells, which can be induced to become hematopoietic progenitor cells. Then, the above-disclosed CD34 + The gene editing composition of the cell is used to treat hematopoietic progenitor cells to produce recombinant cells with one or more modified genes. Representative somatic cells that can be reprogrammed include but are not limited to fibroblasts, adipocytes, and muscle cells. Hematopoietic progenitor cells from induced stem-like cells have been successfully developed in mice (Hanna, J. et al., Science, 318: 1920-1923 (2007)).

[0188] In order to produce hematopoietic progenitor cells from induced stem-like cells, somatic cells are harvested from the host. In a preferred embodiment, 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. The transduced ES cells are cultured and induced to produce induced stem-like cells. The cells are then screened for CD41 and c-kit markers (early hematopoietic progenitor cell markers) as well as myeloid and erythroid differentiation markers.

[0189] The modified hematopoietic stem cells or modified induced hematopoietic progenitor cells are then introduced into the subject. Delivery of the cells can be effected using a variety of methods, most preferably including intravenous administration by infusion and direct depot injection into the periosteum, bone marrow, and / or subcutaneous sites.

[0190] Subjects receiving the modified cells may undergo myeloconditioning therapy to enhance engraftment. Prior to administration of the cells, the recipient may be treated with radiation or chemotherapy to enhance engraftment. Following administration, it typically takes some time for the cells to engraft. Significant engraftment of hematopoietic stem or progenitor cells typically takes weeks to months.

[0191] It is not expected that a high percentage of modified hematopoietic stem cells will be required to achieve a significant prophylactic or therapeutic effect. It is expected that the implanted cells will expand over time after implantation to increase the percentage of modified cells. It is expected that only a small number or a small percentage of modified hematopoietic stem cells will be required to provide a prophylactic or therapeutic effect.

[0192] In preferred embodiments, the cells to be administered to a subject will be autologous, eg, derived from the subject, or syngeneic.

[0193] In a preferred embodiment, the guide RNA can be used for specific gene editing of ex vivo cell therapies, including CAR-T, CAR-NK, and CAR-macrophage editing. In a further embodiment, the guide RNA can knock out and knock in one or more genes or gene fragments in iPSCs, ESCs, mesenchymal stem cells, and stem-derived cell lines.

[0194] (2) In vivo gene therapy

[0195] The composition can be directly administered to a subject for in vivo gene therapy. The composition and method are suitable for treating one or more diseases and conditions in the brain, neurons, eyes, lungs, heart, kidneys, liver, etc.

[0196] (ii) Conditions and diseases to be treated

[0197] Although these strategies can also be used to treat non-genetic diseases such as HIV in the context of ex vivo and in vivo cell modification, gene therapy using gene editing compositions is evident when studied in the context of human genetic diseases (e.g., hereditary neuropathies, hereditary myopathies, hemophilia, gammopathy, such as sickle cell anemia and beta thalassemia, xeroderma pigmentosum, and lysosomal storage diseases). The composition is particularly suitable for treating genetic defects, disorders, and diseases caused by single gene mutations, for example, correcting genetic defects, disorders, and diseases caused by point mutations. If the target gene contains a mutation that causes a genetic disease, the composition can be used for mutagenic repair to 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 a DNA sequence that regulates the expression of the target gene, including a promoter or enhancer sequence.

[0198] In some embodiments, the compositions are particularly useful for treating monogenic and polygenic diseases, wherein one or more sgRNA constructs are delivered together in the same or separate compositions.

[0199] If the target gene is an oncogene that causes uncontrolled proliferation, such as in cancer cells, oligonucleotides can be used to cause mutations, inactivate the gene and terminate or reduce the uncontrolled proliferation of cells. Oligonucleotides are also useful anticancer agents for activating suppressor genes that have lost the ability to inhibit proliferation. The target gene can also be a gene encoding an immunomodulatory factor, such as programmed cell death protein 1 (PD-1), to enhance the host's immune response to cancer. Gene modification technology can be designed to reduce or prevent the expression of PD-1 and administered in an effective amount to do so. Therefore, in some embodiments, the composition is used to treat cancer.

[0200] For example, when designed to modify specific portions of the viral genome that are necessary for normal viral proliferation or function, these compositions can be used as antiviral agents.

[0201] In some embodiments, the subject to be treated is a human. In some embodiments, the subject to be treated is a child or infant. All methods can include the steps of identifying and selecting a subject in need of treatment or a subject who will benefit from the administration of the composition.

[0202] (1) Nervous and muscular diseases or disorders

[0203] In some forms, the disease or condition to be treated is a hereditary neuropathy. Hereditary neuropathy can be degenerative or non-degenerative. Examples include, but are not limited to, 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 ataxia, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, and Jakob-Creutzfeldt disease.

[0204] In some forms, the disease or condition to be treated is a genetically based myopathy. Myopathies can be degenerative or non-degenerative. Examples include, but are not limited to, dystonia, amyotrophic lateral sclerosis, muscular dystrophy, muscular dystrophy (e.g., Duchenne, Becker, facioscapulohumeral, spastic, congenital, distal, Emery-Dreifuss, oculopharyngeal, and limb-girdle), congenital myopathies (e.g., central nucleus, myotubes, rods, Ullrich / Bethlem, and RyR1), or metabolic disorders (e.g., Pompe disease).

[0205] Martier and Konstantinova, Front. Neurosci. 2020, 14: 580-179 and Xie, et al., Journal of Molecular Medicine (2022) 100: 385-394 reviewed hereditary neuropathies, myopathies and their gene therapy, the contents of which are incorporated herein by reference.

[0206] (2) Hereditary eye, lung and liver diseases or conditions

[0207] In some forms, the gene-editing machinery can be used to treat inherited eye diseases or conditions, including but not limited to retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber congenital amaurosis.

[0208] In some forms, gene editing machines can be used to treat inherited lung diseases or conditions. These can be diseases or conditions that primarily affect the lungs or other organs including the lungs. These include, but are not limited to, cystic fibrosis, primary ciliary dyskinesia, alpha-1-antitrypsin deficiency, surfactant dysmetabolism types 1-4, familial pulmonary fibrosis, alveolar microlithiasis, dyskeratosis congenita, neurofibromatosis type 1, tuberous sclerosis / LAM, Birt-Hogg-Dubé syndrome, hyper-IgE syndrome, Hermansky-Pudlak syndrome, Gaucher disease type 1, Niemann-Pick disease type B, and lysinuric protein intolerance.

[0209] In some forms, the gene-editing machinery could be used to treat inherited liver diseases or disorders, including but not limited to hemochromatosis, Wilson's disease, and alpha-1-antitrypsin deficiency.

[0210] (3) Cancer

[0211] In some embodiments, the target cell is a cancer cell. In these embodiments, the disclosed gene editing composition is administered to a subject with a proliferative disease (such as a benign or malignant tumor). In some embodiments, the subject to be treated has been diagnosed with stage I, II, III, or IV cancer.

[0212] Compositions that can be delivered to cancer cells include, but are not limited to, constructs for expressing one or more pro-apoptotic factors, immunogenic factors, or tumor suppressors; gene editing compositions; and inhibitory nucleic acids targeting oncogenes. In some embodiments, the composition is a construct encoding a pro-apoptotic factor or immunogenic factor that increases the immune response to the cell. In other embodiments, the composition is a construct that disrupts the expression of an oncogene or other cancer-causing transcript.

[0213] In mature animals, a balance is maintained between cell renewal and cell death in most organs and tissues. Each type of mature cell in the body has a finite lifespan; as these cells die, new cells are generated by the proliferation and differentiation of various stem cell types. Under normal circumstances, the production of new cells is tightly regulated, so that the number of cells of any particular type remains constant. However, cells occasionally emerge that no longer respond to normal growth control mechanisms. These cells give rise to cell clones that can expand to considerable size, forming tumors or neoplasms. Tumors that do not grow indefinitely and do not extensively invade surrounding healthy tissue are benign. Tumors that continue to grow and gradually invade are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors can also exhibit metastasis. In this process, small clusters of cancer cells detach from the tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way, a primary tumor in one location can give rise to secondary tumors in another.

[0214] The compositions and methods described herein can be used to treat a subject having a benign or malignant tumor by delaying or inhibiting tumor growth, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth in the subject.

[0215] Treatable malignant tumors are categorized herein based on the embryonic origin of the tissue from which the tumor originates. Cancers are tumors arising from endoderm or ectoderm tissues, such as the skin or the epithelial linings of internal organs and glands. The disclosed compositions are particularly effective in treating cancers. Sarcomas are less common and arise from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of hematopoietic cells in the bone marrow. Leukemias proliferate as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can arise in many organs or tissues of the body, forming cancers.

[0216] Cancer types that can be treated using the provided compositions and methods include, but are not limited to, cancers such as vascular cancers, such as multiple myeloma, adenocarcinomas and sarcomas of the bone, bladder, brain, breast, cervix, colorectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors at multiple sites.

[0217] The disclosed conjugates and pharmaceutical compositions thereof can be used in the context of preparing lymphocytes expressing immune receptors, particularly chimeric immune receptors (CIRs), such as chimeric antigen receptors (CARs). Artificial immune receptors (also referred to herein as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs), and chimeric immune receptors (CIRs)) are engineered receptors that graft selected specificities onto cells.

[0218] In some forms, mRNA or DNA encoding chimeric antigen receptor payload can be delivered to immune cells, such as lymphocytes. Payload can be delivered to immune cells in vivo, in vitro or in vitro. In some forms, payload is mRNA. In some forms, immune cells (such as T cells) are harvested from subjects who need CAR T cell therapy, and conjugates disclosed herein and pharmaceutical compositions thereof are used to deliver the mRNA encoding one or more CAR T cell constructs to the harvested cells, and then the cells are returned to the subject. In some forms, the process from initially harvesting cells to returning them to the subject requires 1 week or less, such as 1, 2, 3, 4, 5, 6 or 7 days. In some forms, the process from initially harvesting cells to returning them to the subject is carried out in 1 or 2 days or less than 1 day, such as 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.

[0219] Strategies for the design and development of chimeric antigen receptors are reviewed in Dotti, et al., Immunol Rev. 2014 January; 257(1):doi:10.1111 / imr.12131 (35 pages) (the entire contents of which are specifically incorporated herein by reference), as well as Dotti, Molecular Therapy, 22(5):899-890 (2014), Karlsson, et al., Cancer Gene Therapy, 20:386-93 (2013), Charo, et al., Cancer Res., 65(5):2001-8 (2005), Jensen, et al., Immunol Rev., 257(1):127–144 (2014), Eaton, et al., Gene Therapy, 9:527-35 (2002), Barrett, et al., Annu Rev. Med., 65:333–347 (2014), Cartellieri, et al., Journal of Biomedicine and Biotechnology, Volume 2010, Article ID 956304, Page 13, doi: 10.1155 / 2010 / 956304; and U.S. Published Application Nos. 2015 / 0017120, 2015 / 0283178, 2015 / 0290244, 2014 / 0050709, and 2013 / 0071414.

[0220] CAR combines the antigen binding properties of monoclonal antibodies with the lytic ability and self-renewal of T cells, and has 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). Therefore, target cell recognition is not affected by some mechanisms by which tumors escape MHC-restricted T-cell recognition (such as downregulation of human leukocyte antigen (HLA) class I molecules and antigen processing defects).

[0221] Chimeric immunoreceptors were first developed in the 1980s and initially included the variable (antigen binding) region of a monoclonal antibody and the constant region of the T cell receptor (TCR) α and β chains (Kuwana, et al., Biochem Biophys Res Commun., 149:960–968 (1987)). In 1993, the design was modified to include an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain being a single-chain variable fragment (scFv) from the heavy and light chain antigen binding regions of a monoclonal antibody, and the intracellular domain having a signaling domain derived from CD3-ζ. Later CARs generally follow a similar structural design, with a costimulatory signaling intracellular domain. Therefore, a CAR construct useful in a conjugate described herein or a pharmaceutical composition thereof may include an antigen binding domain or an extracellular domain, a hinge domain, a transmembrane domain, an intracellular domain, and a combination thereof.

[0222] In some forms, the extracellular domain is scFv. The affinity of scFv predicts CAR function (Hudecek, et al., Clin Cancer Res., 19 (12): 3153-64 (2013), Chmielewski, et al., J Immunol., 173: 7647–7653 (2004)). Antigen binding and subsequent activation can also be modified by adding a flexible linker sequence to CAR, which allows the expression of two different scFvs that can recognize two different antigens (Grada, et al., Mol Ther Nucleic Acids, 2: e105 (2013)) (referred to as tandem CAR (TanCAR)). Tandem CAR can more effectively kill cancers that express low levels of each antigen alone, and can also reduce the risk of tumor immune escape due to single antigen loss variants. Other ectodomains include IL13Rα2 (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-ligands and CD70 receptors, peptide ligands (e.g., T1E peptide ligands), and so-called “universal ectodomains” (e.g., anti-biotin ectodomains designed to recognize targets that have been contacted with biotinylated monoclonal antibodies, or FITC-specific scFvs designed to recognize targets that have been contacted with FITC-labeled monoclonal antibodies (Zhang, et al., Blood, 106:1544–1551 (2005), Barber, et al., Exp. Hematol., 36:1318–1328 (2008), Shaffer, et al., Blood, 117:4304–4314 (2011), Davies, et al., Mol Med., 18:565–576 (2012), Urbanska, et al., Cancer Res., 72:1844–1852 (2012), Tamada, et al., Clin Cancer Res., 18:6436–6445 (2012)).

[0223] In some forms, CAR includes a hinge region. Although the extracellular domain is important for CAR specificity, the sequence connecting the extracellular domain and the transmembrane domain (hinge region) can also affect CAR-T cell function by producing differences in CAR length and flexibility. The hinge can include, for example, a CH2CH3 hinge or a fragment thereof, which is derived from an immunoglobulin such as IgG1. For example, Hudecek et al. (Hudecek, et al., Clin Cancer Res., 19(12):3153-64 (2013)) compared the effects of a CH2-CH3 hinge [229 amino acids (AA)], a CH3 hinge (119AA), and a short hinge (12AA) on the effector function of T cells expressing a third-generation ROR1-specific CAR and found that T cells expressing the “short hinge” CAR had excellent anti-tumor activity, whereas other researchers found that the CH2-CH3 hinge impaired epitope recognition of a first-generation CD30-specific CAR (Hombach, et al., Gene Ther., 7:1067–1075 (2000)).

[0224] There is usually a transmembrane domain between the hinge (or extracellular domain if there is no hinge domain) and the signaling intracellular domain, most typically from CD3-ζ, CD4, CD8, or CD28 molecules. Like the hinge, the transmembrane domain can also affect the function of CAR-T-cell effectors.

[0225] After antigen recognition, CAR intracellular domain transmits activation and costimulatory signals to T cells. T cell activation depends on the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) present in the cytoplasmic domain to the cytoplasmic CD3-ζ domain of the TCR complex (Irving, et al., Cell, 64:891–901 (1991)). Although most CAR intracellular domains contain activation domains derived from CD3-ζ, others may include domains containing ITAM, such as Fc receptors of IgE-γ domains (Haynes, et al., J Immunol., 166:182–187 (2001)).

[0226] The target specificity of the cell expressing CAR is determined by the antigen recognized by the antibody / extracellular domain. The disclosed conjugate and pharmaceutical composition can be used to construct a construct targeting any antigen and a cell expressing the construct. In the context of immunotherapy, particularly cancer immunotherapy, many antigens and suitable extracellular domains for targeting them are well known. Unlike natural TCR, most scFv-based CARs recognize target antigens expressed on the cell surface, rather than internal antigens processed and presented by cell MHC, however, CAR has an advantage over classical TCR in 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)), thereby increasing the potential target antigen library. Preferred targets include antigens that are expressed only on cancer cells or in their surrounding stroma (Cheever, et al., Clin Cancer Res., 15:5323–5337 (2009)), such as a splice variant of EGFR (EGFRvIII), which is specific for glioma cells (Sampson, et al., Semin Immunol., 20(5):267-75 (2008)). However, human antigens meet this requirement, as most target antigens are expressed at low levels on normal cells (e.g., GD2, CAIX, HER2) and / or in a lineage-restricted manner (e.g., CD19, CD20).

[0227] Preferred targets and CARs targeting them are known in the art (see, e.g., Dotti, et al., Immunol Rev. 2014 January; 257(1):.doi:10.1111 / imr.12131 (page 35)). For example, CAR targets for hematological malignancies include, but are not limited to, CD 19 (e.g., B cells) (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, et al., N Engl J Med., 365:725-733 (2011); Kalos, et al., Sci Transl Med., 3:95ra73 (2011); Brentjens, et al., Sci Transl Med., 5:177ra38 (2013); Grupp, et al., N Engl J Med (2013)); CD20 (e.g., B cells) (Jensen et al., Biol Blood Marrow Transplant (2010); Till, et al., Blood, 112:2261-2271 (2008); Wang, et al., Hum Gene Ther., 18:712-725 (2007); Wang, et al., Mol Ther., 9:577-586 (2004); Jensen, et al., Biol Blood Marrow Transplant (2010); Till, et al., Blood, 112:2261-2271 (2008); Wang, et al., Hum Gene Ther., 18:712-725 (2007); Wang, et al., Mol Ther., 9:577-586 (2004); Jensen, et al., Biol Blood Marrow Transplant, 4:75-83 (1998)); CD22 (e.g., B cells) (Haso et al., Blood, 121:1165-1174 (2013)); CD30 (e.g., B cells) (Di Stasi, et al., Blood, 113:6392-6402 (2009); Savoldo, et al., Blood, 110:2620-2630 (2007); Hombach, et al., Cancer Res., 58:1116-1119 (1998)); CD33 (e.g., myeloid) (Finney et al., J Immunol., 161:2791-2797 (1998)); CD70 (e.g., B cells / T cells) (Shaffer et al., Blood, 117:4304-4314 (2011)); CD123 (e.g., myeloid) (Tettamanti et al., Br J Haematol., 161:389-401 (2013)); Kappa (e.g., B cells) (Vera et al., Blood, 108:3890-3897 (2006)); Lewis Y (e.g., myeloid) (Peinnert et al., Gene Ther., 17:678-686 (2010); Ritchie et al., Mol. Biol., 20:116-1119 (1998)); CD33 (e.g., myeloid) (Finney et al., J Immunol., 161:2791-2797 (1998)); CD70 (e.g., B cells / T cells) (Shaffer et al., Blood, 117:4304-4314 (2011)); CD123 (e.g., myeloid) (Tettamanti et al., Br J Haematol., 161:389-401 (2013)); Kappa (e.g., B cells) (Vera et al., Blood, 108:3890-3897 (2006)); Lewis Y (e.g., myeloid) Ther. (2013)); NKG2D ligands (e.g., myeloid) (Barber et al., Exp Hematol., 36:1318-1328 (2008); Lehner, et al., PLoS One., 7:e31210 (2012); Song, et al., Hum Gene Ther., 24:295-305 (2013); Spear, et al., J Immunol. 188:6389-6398 (2012)); ROR1 (e.g., B cells) (Hudecek et al., Clin Cancer Res. (2013)).

[0228] 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. (2006); Weijtens, et al., Int J Cancer, 77:181-187 (1998)); CD44 v6 / v7 (e.g., cervix) (Hekele et al., Int J Cancer, 68:232-238 (1996); Dall, et al., Cancer Immunol Immunother, 54:51-60 (2005)); CD171 (e.g., neuroblastoma) (Park et al., Mol Ther., 15:825-833 (2007)); CEA (e.g., colon) (Nolan et al., Clin Cancer Res., 5:3928-3941 (1999)); EGFRvIII (e.g., glioma) (Bullain et al., J Neurooncol. (2009); Morgan, et al., Hum Gene Ther., 23:1043-1053 (2012)); EGP2 (e.g., cancer) (Meier et al., Magn Reson Med., 65:756-763 (2011); Ren-Heidenreich, et al., Cancer Immunol Immunother., 51:417-423 (2002)); EGP40 (e.g., colon) (Daly et al., Cancer Gene Ther., 7:284-291 (2000)); EphA2 (e.g., glioma, lung) (Chow et al., Mol. Biol., 2003; Ther., 21:629-637 (2013)); ErbB2 (HER2) (e.g., breast, lung, prostate, glioma) (Zhao et al., J Immunol., 183:5563-5574 (2009); Morgan, et al., Mol Ther., 18:843-851 (2010); Pinthus, et al., 114:1774-1781 (2004); Teng, et al., Hum Gene Ther., 15:699-708 (2004); Stancovski, et al., J Immunol., 151:6577-6582 (1993); Ahmed, et al., Mol Ther., 17:1779-1787 (2009); Ahmed, et al., Clin Cancer Res., 16:474-485 (2010); Moritz, et al., Proc Natl Acad Sci USA, 91:4318-4322 (1994)); ErbB receptor family (e.g., breast, lung, prostate, glioma) (Davies et al., Mol Med., 18:565-576 (2012)); ErbB3 / 4 (e.g., breast, ovary) (Muniappan et al., Cancer Gene Ther., 7:128-134 (2000); Altenschmidt, et al., Clin Cancer Res., 2:1001-1008 (1996)); HLA-A1 / MAGE1 (e.g., melanoma) (Willemsen et al., Gene Ther., 8:1601-1608 (2001); Willemsen, et al., J Immunol., 174:7853-7858 (2005)); HLA-A2 / NY-ESO-1 (e.g., sarcoma, melanoma) (Schuberth et al., Gene Ther., 20:386-395 (2013)); FR-α (e.g., ovary) (Hwu et al., J Exp Med., 178:361-366 (1993); Kershaw, et al., Nat Biotechnol., 20:1221-1227 (2002); Kershaw, et al., Clin Cancer Res., 12:6106-6115 (2006); Hwu, et al., Cancer Res., 55:3369-3373 (1995)); FAP (e.g., cancer-associated fibroblasts) (Kakarla et al., Mol Biol., 20:1147-1151 (2006)); Ther. (2013)); FAR (e.g., rhabdomyosarcoma) (Gattenlohner et al., Cancer Res., 66:24-28 (2006)); GD2 (e.g., neuroblastoma, sarcoma, melanoma) (Pule et al., Nat Med., 14:1264-1270 (2008); Louis, et al., Blood, 118:6050-6056 (2011); Rossig, et al., Int J Cancer., 94:228-236 (2001)); GD3 (e.g., melanoma, lung cancer) (Yun et al., Neoplasia., 2:449-459 (2000)); HMW-MAA (e.g., melanoma) (Burns et al., Cancer Res., 70:3027-3033 (2010)); IL11Rɑ (e.g. osteosarcoma) (Huang et al., Cancer Res., 72:271-281 (2012)); IL13Rα2 (e.g., glioma) (Kahlon et al., Cancer Res., 64:9160-9166 (2004); Brown, et al., Clin Cancer Res. (2012); Kong, et al., Clin Cancer Res., 18:5949-5960 (2012); Yaghoubi, et al., Nat Clin Pract Oncol., 6:53-58 (2009)); Lewis Y (e.g., breast / ovary / pancreas) (Peinert et al., Gene Ther., 17:678-686 (2010); Westwood, et al., Proc Natl Acad Sci USA, 102:19051-19056 (2005); Mezzanzanica, et al., Cancer Gene Ther., 5:401-407 (1998)); mesothelin (e.g., mesothelioma, breast, pancreas) (Lanitis et al., Mol Ther., 20:633-643 (2012); Moon, et al., Clin Cancer Res., 17:4719-4730 (2011)); Muel (e.g., ovary, breast, prostate) (Wilkie et al., J Immunol., 180:4901-4909 (2008)); NCAM (e.g., neuroblastoma, colorectal) (Gilham et al., J Immunother., 25:139-151 (2002)); NKG2D ligands (e.g., ovary, sarcoma) (Barber et al., Exp Hematol., 36:1318-1328 (2008); Lehner, et al., PLoS One, 7:e31210 (2012); Song, et al., Gene Ther., 24:295-305 (2013); Spear, et al., J Immunol., 188:6389-6398 (2012)); PSCA (e.g., prostate, pancreas) (Morgenroth et al., Prostate, 67:1121-1131 (2007); Katari, et al., HPB, 13:643-650 (2011)); PSMA (e.g., prostate) (Maher et al., Nat Biotechnol., 20:70-75 (2002); Gong, et al., Neoplasia., 1: 123-127 (1999)); TAG72 (e.g., colon) (Hombach et al., Gastroenterology, 113: 1163-1170 (1997); McGuinness, et al., Hum Gene Ther., 10: 165-173 (1999)); VEGFR-2 (e.g., tumor vasculature) (J Clin Invest., 120: 3953-3968 (2010); Niederman, et al., Proc Natl Acad Sci USA, 99: 7009-7014 (2002)).

[0229] (iii) Delivery of other payloads

[0230] The payload may also include therapeutic, diagnostic and / or preventive proteins, peptides or nucleic acids. In particular, proteins such as antibodies and fragments thereof may be utilized due to their target specificity. The use of antibodies in therapeutic settings directed against intracellular targets is hindered by the low membrane-penetrating properties of their cell membranes. The data described below demonstrate effective antibody delivery into cells. Therefore, in some forms, the conjugates described herein contain antibodies and / or fragments thereof. Some examples of intracellular antigens that can be targeted include, but are not limited to, melanoma-associated antigens, pan-cancer antigens, 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, and the like.

[0231] V. Preparation

[0232] Pharmaceutical compositions of the disclosed conjugates can be prepared using a pharmaceutically acceptable "carrier," which is composed of materials considered safe and effective and can be administered to an individual without causing undesirable biological side effects or unwanted interactions. A "carrier" includes all components of a pharmaceutical formulation other than the active ingredient. The term "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, and coating compositions. In some forms, a "carrier" includes components other than water.

[0233] In some forms, the conjugate is administered as an aqueous solution thereof. The formulation may also be in the form of a suspension, emulsion, lyophilized powder, or powder in a tablet. Typically, a pharmaceutical composition is provided comprising an effective amount of a peptide or polypeptide and optionally comprising a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and optional additives such as detergents and solubilizers (e.g., 20. TWEEN (polysorbate 80), antioxidants (such as ascorbic acid, sodium metabisulfite) and preservatives (such as thiomycin, benzyl alcohol) and bulking agents (such as lactose, mannitol). Examples of non-aqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), gelatin and injectable organic esters (such as ethyl oleate). The formulation can be lyophilized and re-dissolved / re-suspended immediately before use. The formulation can be sterilized by, for example, filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0234] The preferred route of administering the conjugate or its pharmaceutical composition is parenteral administration. Suitable parenteral routes include, but are not limited to, intracranial, intracerebral, intraventricular, intrathecal, intravenous, ocular, subretinal, intravitreal, intranasal, intrapleural, or intratracheal. Among them, a more preferred approach is intracerebral, intraventricular, intrathecal, intravenous, subretinal, and intravitreal. In some forms, administering the conjugate or its pharmaceutical composition relates to convection-enhanced delivery (CED). CED involves infusing the conjugate into the brain through a catheter under a positive pressure gradient (Bobo et al., Proc Natl Acad Sci USA 91, 2076-2080 (1994)). CED has been shown to be clinically safe and feasible (Kunwar et al., Neuro Oncol 12, 871-881 (2010); Sampson, et al., Neuro Oncol 10, 320-329 (2008); Jacobs, et al., Lancet 358, 727-729 (2001)). By generating a large amount of fluid movement in the brain interstitium, the distribution volume of the conjugate injected by CED can be much larger than the volume achieved by diffusion (Morrison et al., American Journal of Physiology 266, R292-R305 (1994)).

[0235] The disclosed compositions and methods can be further understood through the following enumerated paragraphs or embodiments.

[0236] 1. A conjugate comprising the following structure:

[0237] Preferably

[0238] in:

[0239] Dashed lines independently represent the presence of one or more covalent bonds or non-covalent bonds, preferably, dashed lines represent the presence of one or more covalent bonds,

[0240] P includes proteins, peptides, or nucleic acids;

[0241] L is a linear or branched traceless or traceable chemical linker;

[0242] M is a one-armed or multi-armed chemical moiety comprising a cell membrane fusion molecule; and

[0243] np, nl, nm and nz are independently an integer between 1 and 150, inclusive, an integer between 1 and 100, inclusive, an integer between 1 and 75, inclusive, an integer between 1 and 50, inclusive, an integer between 1 and 25, inclusive, an integer between 1 and 15, inclusive, an integer between 1 and 10, inclusive, an integer between 1 and 7, inclusive, or an integer between 1 and 5, inclusive.

[0244] 2. The conjugate according to paragraph 1, wherein L is a linear or branched traceless chemical linker comprising a stimulus-responsive chemical moiety and / or a self-destructive chemical moiety.

[0245] 3. The conjugate according to paragraph 1, wherein L is a linear or branched trace chemical linker comprising 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.

[0246] 4. The conjugate according to any one of paragraphs 1 to 3, wherein P comprises a protein or a peptide.

[0247] 5. The conjugate according to any one of paragraphs 1 to 3, wherein P comprises a nucleic acid.

[0248] 6. The conjugate according to any one of paragraphs 1 to 5, wherein P comprises genome editing machinery.

[0249] 7. The conjugate according to paragraph 6, wherein the genome editing machinery comprises one or more of a CRISPR / Cas system, a zinc finger nuclease, a transcription activator-like effector nuclease, a meganuclease, a peptide nucleic acid, a base editor, a lead editor or an antisense oligonucleotide.

[0250] 8. A conjugate according to paragraph 6 or 7, wherein the genome editing machinery comprises a CRISPR / Cas system.

[0251] 9. The conjugate according to paragraph 7 or 8, wherein the CRISPR / Cas system comprises a Cas nuclease and a single guide RNA (sgRNA).

[0252] 10. The conjugate according to paragraph 9, wherein the Cas nuclease is selected from Cas9, CasX, Cas7-11, CasFx, Cas12a and Cas13.

[0253] 11. The conjugate according to paragraph 9 or 10, wherein the Cas nuclease is a Cas9 nuclease.

[0254] 12. The conjugate according to any one of paragraphs 9 to 11, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease.

[0255] 13. The conjugate according to any one of paragraphs 9 to 12, wherein the Cas9 nuclease is a nuclease-deficient mutant variant of the Cas9 protein (dCas9).

[0256] 14. The conjugate according to paragraph 13, wherein dCas9 is further bound to or conjugated to an effector domain for dCas9 to silence or activate transcription.

[0257] 15. The conjugate according to paragraph 13, wherein dCas9 is further bound to or conjugated to the catalytic domain of ten-eleven translocation (TET) 1 hydroxylase.

[0258] 16. The conjugate according to any one of paragraphs 2 or 4 to 15, wherein the stimulus-responsive chemical moiety is responsive to a stimulus selected from the group consisting of pH (e.g., a change in pH), redox (e.g., a change in redox potential), reactive oxygen species (ROS), enzymes (e.g., overexpression of enzymes (e.g., proteases, esterases, etc.) due to a disease state or condition), ionic strength (e.g., a change in ionic strength), hypoxia, and combinations thereof.

[0259] 17. The conjugate according to any one of paragraphs 2 or 4 to 16, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide bond, an orthoester, a hydrazone, a hydrazide, a hydrazine, an imine (such as an aldimine or ketimine), an oxime, an acetal group, a vinyl ether, a polyketal, methyl maleate, an ester bond, a nitroaryl group (such as nitrobenzyl), a nitroheteroaryl group (such as nitroimidazole), a quinone group, an azoaryl group (such as azophenyl), an azoheteroaryl group (such as azopyridyl), a peroxyoxalate, an aminoacrylate, an alkyl thioether or a selenoether (such as a monoselenoether bond, a diselenoether bond, etc.), a thioketal, a peroxyoxalate, or dimethyl maleate.

[0260] 18. The conjugate according to any one of paragraphs 2 or 4 to 17, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide, an orthoester, an imine (such as an aldimine or ketimine), a hydrazone, a hydrazide, a hydrazine, an imine, an oxime, methyl maleate, an ester bond, dimethyl maleate, or a combination thereof.

[0261] 19. The conjugate according to any one of paragraphs 2 or 4 to 18, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide, an orthoester, an imine (such as an aldimine or ketimine), or an ester bond.

[0262] 20. The conjugate according to any one of paragraphs 2 or 4 to 19, wherein the self-destructive chemical moiety degrades by electronic cascade, cycloelimination, or both.

[0263] 21. The conjugate according to any one of paragraphs 2 or 4 to 20, wherein the self-destructive chemical moiety comprises p-aminobenzyl (e.g., p-NH-phenyl-CH2-), o-aminobenzyl (e.g., o-NH-phenyl-CH2-), p-oxybenzyl (e.g., p-O-phenyl-CH2-), o-aminobenzyl (e.g., o-O-phenyl-CH2-), p-thiobenzyl (e.g., p-S-phenyl-CH2-), o-thiobenzyl (e.g., o-S-phenyl-CH2-), cinnamyl ether, a cyclization-driving moiety, a Grob fragmentation moiety, or a combination thereof.

[0264] 22. The conjugate according to any one of paragraphs 2 or 4 to 21, wherein the self-destructive chemical moiety comprises the following structure:

[0265]

[0266] in:

[0267] X is oxygen (O), NH or sulfur (S),

[0268] Y is O, NRs, substituted alkyl or unsubstituted alkyl,

[0269] W is a substituted alkyl group or an unsubstituted alkyl group, and

[0270] Rs is hydrogen, substituted alkyl or unsubstituted alkyl.

[0271] 23. The conjugate according to paragraph 22, wherein Y is oxygen.

[0272] 24. The conjugate according to paragraph 22 or 23, wherein X is sulfur.

[0273] 25. The conjugate according to any one of paragraphs 22 to 24, wherein W is C2-C5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl or C5 substituted alkyl.

[0274] 26. The conjugate according to any one of paragraphs 1, 2 or 4 to 23, wherein L is formed using a structure selected from the group consisting of:

[0275]

[0276]

[0277] or a combination thereof.

[0278] 27. The conjugate according to any one of paragraphs 1 to 24, wherein L is formed using:

[0279]

[0280] 11,12-Didehydro-γ-oxo-, 2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ester (DBNPDEE).

[0281] 28. The conjugate according to any one of paragraphs 2 or 4 to 19, wherein the self-destructive chemical moiety comprises the following structure:

[0282]

[0283] in:

[0284] X is oxygen (O), NH or sulfur (S).

[0285] 29. The conjugate according to any one of paragraphs 1 to 28, wherein the cell membrane fusion molecule comprises a protein; a peptide, such as a cell penetrating peptide; a lipid (e.g., 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 phosphatidylethanolamines with two linked fatty acyl chains (preferably unsaturated fatty acyl chains); lysolipids, etc.); a small molecule moiety, such as a pentacyclic or tetracyclic moiety of the following substances: cholesterol, a steroid hormone, a glucocorticoid, a mineralocorticoid, an androgen, an estrogen, a progestin, a plant sterol (e.g., β-sitosterol); or a hydrophobic amino acid residue (e.g., tyrosine, aniline, tryptophan, etc.).

[0286] 30. The conjugate according to any one of paragraphs 1 to 29, wherein the cell membrane fusion molecule comprises a protein; a peptide, such as a cell penetrating peptide.

[0287] 31. The conjugate according to any one of paragraphs 1 to 29, wherein the cell membrane fusion molecule comprises a small molecule portion, such as a pentacyclic or tetracyclic portion of the following substances: cholesterol, a steroid hormone, a glucocorticoid, a mineralocorticoid, an androgen, an estrogen or a progestogen, a plant sterol (e.g. β-sitosterol).

[0288] 32. The conjugate according to any one of paragraphs 1 to 29, wherein the cell membrane fusion molecule comprises a small molecule portion, such as a tetracyclic or pentacyclic portion of cholesterol.

[0289] 33. The conjugate according to any one of paragraphs 1 to 32, which comprises a hydrophilic polymer between the moieties in L and M, preferably a neutral uncharged hydrophilic polymer such as polyalkylene glycols and polyalkylene oxides such as poly(ethylene glycol); polysaccharides such as cellulose and dextran; hydrophilic polypeptides and poly(amino acids), such as poly-L-serine; poly(oxyethylated polyols); poly(olefin alcohols), such as poly(vinyl alcohol); poly(N-vinylpyrrolidone); poly(hydroxyethyl acrylate); poly(hydroxyalkyl methacrylate), for example poly(hydroxyethyl methacrylate), preferably polyalkylene glycols and polyalkylene oxides, such as poly(ethylene glycol), preferably wherein the molecular weight of the hydrophilic polymer is between 100 Da and 10 kDa, inclusive, or between 200 Da and 10 kDa, inclusive.

[0290] 34. The conjugate according to any one of paragraphs 1 to 33, wherein M is a one-armed chemical moiety comprising a cell membrane fusion molecule.

[0291] 35. The conjugate according to any one of paragraphs 1 to 34, wherein M is

[0292] Where n is between 2 and 300, inclusive, such as 24.

[0293] 36. The conjugate according to any one of paragraphs 1 to 33, wherein M is a multi-armed chemical moiety comprising multiple cell membrane fusion molecules.

[0294] 37. The conjugate according to any one of paragraphs 1 to 33 or 36, wherein M is a multi-arm chemical moiety comprising 2 to 10, 2 to 9, 2 to 8 or 2 to 7 small molecule moieties, such as a tetracyclic or pentacyclic moiety of cholesterol.

[0295] 38. The conjugate according to any one of paragraphs 1 to 33, 36 or 37, wherein M is a multi-arm chemical moiety comprising 2 to 7, preferably 2, small molecule moieties, such as a tetracyclic or pentacyclic moiety of cholesterol.

[0296] 39. The conjugate according to any one of paragraphs 1 to 33 or 36 to 38, wherein M is

[0297]

[0298] 40. The conjugate according to any one of paragraphs 1 to 39, wherein the molar ratio of M to P or ML 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.

[0299] 41. A pharmaceutical composition comprising the conjugate of any one of paragraphs 1 to 40 and a pharmaceutically acceptable carrier.

[0300] 42. A method of delivering a protein, peptide, or nucleic acid to a subject in need thereof, the method comprising administering the conjugate of any one of paragraphs 1 to 40 or the pharmaceutical composition of paragraph 36.

[0301] 43. The method according to paragraph 42, wherein the administration is parenteral, such as intracranial, intracerebral, intracerebroventricular, intrathecal, intravenous, intraocular, subretinal, intravitreal, intranasal, intrapleural or intratracheal.

[0302] 44. The method according to paragraph 42 or 43, wherein administering comprises convection-enhanced delivery.

[0303] 45. A method of editing the genome of a subject in need thereof, the method comprising administering the conjugate of any one of paragraphs 1 to 40 or the pharmaceutical composition of paragraph 41.

[0304] 46. ​​The method according to paragraph 45, comprising administering the conjugate or pharmaceutical formulation to one or more cells, tissues or organs of the subject.

[0305] 47. The method according to paragraph 45 or 46, wherein the administering is performed in vitro or in vivo.

[0306] 48. The method according to any one of paragraphs 42 to 47, wherein the subject exhibits one or more signs or symptoms associated with an inherited neuropathy, an inherited myopathy, an inherited eye disease or disorder, an inherited lung disease or disorder, an inherited liver disease or disorder, or cancer.

[0307] 49. The method according to any one of paragraphs 42 to 48, wherein the subject exhibits a condition consistent 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 ataxia, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Jakob-Creutzfeldt disease, dystonia, amyotrophic lateral sclerosis, muscular dystrophy, muscular dystrophy (such as Duchenne, Becker, facioscapulohumeral, ankylosing, congenital, distal, Emery-Dreifuss, oculopharyngeal and limb-girdle), congenital myopathies (such as central nucleus, myonephritis, One or more signs or symptoms associated with certain disorders (e.g., retinal dysplasia, fibrosis, pulmonary fibrosis, alveolar microlithiasis, dyskeratosis congenita, neurofibromatosis type 1, tuberous sclerosis / LAM, Birt-Hogg-Dubé syndrome, hyper-IgE syndrome, Hermansky-Pudlak syndrome, Gaucher disease type 1, Niemann-Pick disease type B, lysinuria, protein intolerance, hemochromatosis, Wilson disease, alpha-1-antitrypsin deficiency, or cancer).

[0308] 50. The method according to any one of paragraphs 42 to 49, 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 ataxia, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, or Jakob-Creutzfeldt disease.

[0309] 51. The method according to any one of paragraphs 42 to 50, wherein the subject exhibits one or more signs or symptoms associated with Angelman syndrome, HIST1H1E(H1-4) syndrome, or Prader-Willi syndrome.

[0310] Example

[0311] Example 1: Stimuli-responsive, traceless engineering platform (STEP) for intracellular protein delivery

[0312] The clinical translation of biologics-based therapies (e.g., protein-based therapies) has been a major challenge because most biologics (e.g., protein therapeutics) cannot effectively penetrate cells. This non-limiting example describes a stimulus-responsive traceless engineering platform (STEP) that can be used to effectively deliver biologics, such as proteins, into cells without compromising the biological activity of the biologics. In this non-limiting example, STEP is achieved by chemically coupling membrane fusion molecules to amino or sulfhydryl groups on the surface of the protein payload through stimulus-responsive linkers that can be cleaved and completely removed by intracellular stimuli (e.g., reducing and / or acidic environments). As a result, the payload (e.g., protein) is released without any trace molecules, thereby fully restoring its biological function after cell penetration ( Figure 1 The data showed that this technology can be used to effectively deliver ribonucleoproteins (RNPs) made from sgRNAs for the Cas9 protein in vitro in cell culture and in vivo in reporter and disease models. The data further showed that this technology can be used to deliver other payloads, such as protein payloads including green fluorescent protein (GFP), Cas9 / dCas9 fusions, and antibodies.

[0313] Materials, methods, and results

[0314] Ai9 mice were purchased from The Jackson Laboratory. Ai9 fibroblasts were provided by the NIH Common Fund's Somatic Cell Genome Editing (SCGE) program. Chemically synthesized starting molecules can be obtained from commercial suppliers such as Sigma and Santa Cruz Biotechnology. Cas9 protein was purchased from Integrated DNA Technologies (IDT) or Couragene. Guide RNA was synthesized by Synthego.

[0315] i. Cholesterol conjugation via a scarless linker facilitates RNP cell penetration for efficient genome editing

[0316] Experiments were performed to determine whether the coupling of cholesterol to the RNP surface facilitates RNP penetration into cells to achieve genome editing. RNPs were assembled by incubating Cas9 protein with sgRNAs 276 and 280 targeting loxP-flanked STOP cassettes in Ai9 cells ( Figure 2ACholesterol was chemically coupled to the surface of RNPs via the redox-reactive, self-destructive linker dibenzo[b,f]azepin-5(6H)-butyric acid, 11,12-didehydro-γ-oxo-, 2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ester (DBNPDEE). Figure 2B ), and then incubated with azide-conjugated cholesterol ( Figure 2C ). This coupling chemistry was chosen so that upon entry into the cytosol, the reducing microenvironment in the cytosol can trigger disulfide cleavage and subsequent self-destruction of the chemical linker, thereby releasing the RNP without any trace molecules on the surface. Due to its ability to release the payload without trace molecules, this linker is called a traceless linker. Control RNPs were synthesized by incubating pre-synthesized cholesterol-PEG-NHS, which reacts with primary amino groups on the surface of the RNP. Unlike DBNPDEE, this coupling method results in the covalent attachment of cholesterol molecules to the surface of the RNP, which cannot be cleaved inside the cell. The genome editing capacity of the resulting chemically modified RNPs was evaluated in primary fibroblasts isolated from Ai9 mice, which was determined based on the expression of tdTomato. The results showed that coupling of cholesterol via DBNPDEE facilitated efficient cell penetration and genome editing, resulting in 61.0% of the cells expressing tdTomato. For comparison, the extent of this editing efficiency was defined as 100% "relative genome editing activity". In contrast, coupling via a non-cleavable linker resulted in a significantly lower efficiency ( Figure 2E These findings suggest that cholesterol conjugation facilitates RNP delivery into cells for genome editing and that the use of scarless linkers significantly improves delivery and / or editing efficiency.

[0317] ii. Screening for traceless joints

[0318] Figure 2EThe data in show that the use of scarless linkers leads to efficient delivery of RNPs for genome editing. To identify traceless linkers that may be more beneficial for RNP delivery than DBNPDEE, a library of stimuli-responsive traceless linkers was synthesized and screened, including the acid-responsive linker dibenzo[b,f]azepin-5(6H)-butyric acid, 11,12-didehydro-γ-oxo-, 4-n-phenyl ester (DBFPE); the redox-responsive, self-immolative linker 2-((2-(((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl acrylate (NPDEA); and the redox-responsive, self-immolative linker dibenzo[b,f]azepin-5(6H)-butyric acid, 11,12-didehydro-γ-oxo-, 2-((2-(((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl acrylate (D BNPPDEE); dibenzo[b,f]azepine-5(6H)-butyric acid 8,24-dioxo-11,14,17,20-tetraoxa-3,4-dithia-7,23-diazacosanoyl (4-nitrophenyl) carbonate (DBTDC); 2,5-dioxo-1-pyrrolidinyl 5-[[3-(11,12-didehydrodibenzo[b,f]azepine-5 [(2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ester (DBPDEE)] Figure 3A ). All of these linkers were designed to be cleaved and completely removed by intracellular stimuli. DBNPDEE was included as a baseline control for comparison. Through these linkers, cholesterol was coupled to the surface of RNPs loaded with sgRNAs 276 and 280. The resulting RNPs were evaluated in Ai9 fibroblasts. It was found that the structure of the chemical linker significantly affected the efficiency of RNP delivery, and among all the linkers tested, DBNPPDEE and DBNPDEE proved to be the most efficient ( Figure 3B ).

[0319] iii. Screening of membrane fusion molecules

[0320] In addition to the scarless linker, the use of membrane fusion molecules also plays a key role in determining the intracellular delivery activity of RNPs ( Figures 2A-2E ). A fusion molecule library was screened, including lipids DSPE, PE, 16-PC, and 18-PC; cholesterol; cholesterol analogs OA and β-sitosterol; and multi-arm tyrosine ( Figure 4ACandidate small molecules were coupled to RNPs via DBNPDEE. The resulting RNPs were tested in Ai9 fibroblasts. Although all tested molecules demonstrated varying degrees of ability to deliver RNPs, cholesterol remained the most effective. Therefore, cholesterol was selected for the following studies.

[0321] iv. Characteristics of cholesterol-based multi-arm fusion molecules

[0322] Experiments were performed to determine whether the presentation of cholesterol at a local high density would further improve the delivery of RNPs for genome editing through multivalency effects. A series of fusion molecules presenting 2-7 cholesterol groups via multi-arm PEG or sugar backbones were synthesized and conjugated to RNPs via DBNPDEE ( Figures 5A-5G The resulting RNPs were evaluated in Ai9 fibroblasts. Of all the fusion molecules tested, the one presenting cholesterol via 2-arm PEG (Cholesterol x 2) demonstrated the highest efficiency in delivering RNPs for genome editing ( Figure 5H Based on this finding, an RNP with 2-arm PEG (cholesterol x 2) coupled to its surface via DBNPDEE was selected for further study and this formulation was designated as a non-limiting example of STEP RNP.

[0323] v. Characteristics of STEP RNP

[0324] Dynamic light scattering (DLS) analysis showed that the diameter of STEP RNP was 12 nm ( Figure 6A When used to deliver sgRNA 276 and sgRNA 280 targeting the Ai9 cassette, the RNP efficiently edited Ai9 fibroblasts, resulting in 72.4% of cells expressing tdTomato after two days of treatment ( Figure 6B ).

[0325] vi. Local administration of STEP RNPs effectively edits the brain

[0326] Using the Ai9 transgenic mouse model, STEP RNP was characterized to deliver the genome editing machinery to the brain. STEP RNP loaded with sgRNA276 and sgRNA280 was administered to the brain parenchyma by convection-enhanced delivery (CED). One week later, the mice were euthanized. Analysis of the brain found that CED of STEP RNP effectively edited the genome of brain cells, as evidenced by the expression of tdTomato in the diffuse zone (mainly in neuronal cells). STEP RNP was characterized in Ai9 mice by intracerebroventricular and intrathecal administration. These treatments resulted in the editing of approximately 76% of NeuN+ neurons in the prefrontal cortex.

[0327] vii. STEP RNP for the treatment of neurogenetic diseases

[0328] Angelman syndrome (AS) is a devastating neurological genetic disorder caused by a defect in the maternal UBE3A gene in the human chromosome 15q11-q13 region. Due to brain-specific imprinting of the UBE3A gene, there is an opportunity to reactivate / unsilence the paternal allele, expressing the existing 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). In all AS cases, the structure of UBE3A is intact in the paternal chromosome but is transcriptionally repressed in neurons by a mechanism mediated by UBE3A noncoding antisense RNA (UBE3A-ATS) (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). Inhibition of UBE3A-ATS by antisense oligonucleotides (ASOs), small molecules, and CRISPR in human cells and in vivo mouse models can reactivate UBE3A expression and rescue the phenotype (Meng et al., Nature 2015; 518(7539):409-12; Wolter et al., Nature 2020; 587(7833):281-4; Schmid et al., The Journal of Clinical Investigation 2021. Epub 2021 / 01 / 08.doi:10.1172 / JCI142574; Huang et al., Nature 2011; 481(7380):185-9). Like Alzheimer's disease, AS affects multiple regions of the brain, and treating AS requires genome editing throughout the brain. Here, we evaluated whether delivery of RNPs targeting noncoding antisense RNA could turn on UBE3A expression and rescue neurological deficits.

[0329] First, using Ube3a-YFP transgenic mice, we determined whether the delivery of STEP RNPs could lead to editing of neuronal cells in the brain. In these specific mice, the expression of paternal Ube3a-YFP (pUbe3a-YFP) is imprinted and silenced in neurons in the mouse brain. In mice without intervention, pUbe3a-YFP is not expressed. Therefore, the expression of pUbe3a-YFP can be used as an indicator to quantify the efficiency of STEP RNP-mediated genome editing. STEP RNPs loaded with sgRNA targeting non-coding antisense RNA were synthesized and administered to pups on the first or second day of life via intracerebroventricular injection (ICV) at a dose of 40 μg. One month later (30 days in this case), the mice were euthanized and their brains analyzed for YFP expression. YFP was efficiently expressed throughout the brain, indicating that ICV administration of STEP RNPs can induce genome editing throughout the brain. In Ai9 mice, the neuronal editing efficiency and whole-brain genome editing scale of STEP RNP were characterized by ICV and intrathecal administration (IT). A single administration effectively activated the expression of UBE3A-YFP throughout the brain and edited approximately 76% of NeuN+ neurons in selected cortical areas such as the prefrontal cortex. These findings were replicated in AS mouse models of >10 cohorts and three different families. Western blot analysis showed that 90 days after a single IT at P1-2, the reactivation of UBE3A was still significant ( Figure 7A and Figure 7B Compared with untreated AS mice (AS-NT) and AS mice treated with STEP RNP-scratching sgRNA control, treatment with STEP RNP targeting UBE3A-ATS by a single IT administration significantly improved or almost completely rescued motor function (total distance traveled) in the open field test, reduced anxiety (reduced center time), improved short-term and long-term memory, and improved performance in the rotarod test ( Figures 7C-7H ) and rescued the impaired hippocampal long-term potentiation. It was also found that IT administration of cRNPs-sgRNA33 on D21 achieved a therapeutic benefit comparable to that observed in these mice treated on D1-2, and that two administrations of cRNPs-sgRNA33 on D1-2 and D21 did not provide additional therapeutic benefit ( Figure 7C and Figure 7D ). We also investigated whether treatment could be given at later time points. The data showed that administration of cRNPs-sgRNA33 at D42 was still highly effective ( Figures 7C-7E ). Figure 7ETwo entries containing D21 twice in the training, short-term memory, and long-term memory datasets are shown. One dataset was IDT Cas9 and the other was Couragene Cas9. In preparation for clinical translation, 15 guide sequences were screened that target human UBE3A in neurons differentiated from iPSCs derived from AS patients. A candidate guide was found that could be delivered using STEPRNP to reactivate tau expression in almost 100% of neurons. The selected guide was characterized by 300X whole genome sequencing (WGS) and targeted amplicon sequencing, and no significant off-target effects were identified. Delivery of this candidate guide was completed in a single treatment.

[0330] Next, use Ube3a mat- / pat+ Mouse models were used to evaluate STEP RNP for genome editing therapy for AS treatment. STEP RNP was administered by ICV or intrathecal (IT) at a dose of 40 μg. 20-30 days after injection, the expression of Ube3a-ATS and Ube3a in different brain regions was detected by qRT-PCR, quantitative immunoblotting, and immunocytochemistry. Delivery of STEP RNP loaded with four different gRNAs by ICV and IT injection in P1-2 mouse pups reduced the expression of Ube3a-ATS and reactivated the expression of Ube3a 20-30 days after IT or ICV injection ( Figure 8A and Figure 8B There was no significant difference in whole-brain editing efficiency between ICV and IT administration routes.

[0331] Finally, the effect of STEP RNP treatment loaded with sgRNA (such as gRNA33) on rescuing neurological deficits was investigated. Behavioral analysis also showed that RNP-treated mice had significantly improved or almost completely rescued motor function (total distance traveled) and reduced anxiety (reduced center time) in the open field test compared to untreated AS mice and AS mice treated with chemically modified RNPs carrying a scrambled sgRNA control (STEPRNP). Similarly, the treatment significantly improved performance in the rotating rod test and improved the novel object recognition test, such as Figures 9A-9D shown.

[0332] viii. STEP RNP for delivering genome editing machinery to the eye

[0333] STEP RNPs were evaluated for delivering the genome editing machinery to the eye. STEP RNPs loaded with sgRNA 276 and sgRNA 280 were administered via subretinal injection. One week later, mice were euthanized. Imaging analysis using DAPI demonstrated that subretinal administration of STEP RNPs successfully enabled genome editing.

[0334] ix. STEP RNP for delivering genome editing machinery to the lungs

[0335] Intranasal delivery of STEP RNPs loaded with sgRNA 276 and sgRNA 280 was evaluated for their ability to deliver the genome editing machinery to the lungs. One week after treatment, mice were euthanized. Lung imaging analysis using DAPI demonstrated that intranasal delivery of STEP RNPs resulted in efficient genome editing in the lungs.

[0336] x. STEP for intracellular delivery of CRISPR epigenetic editing machinery

[0337] Using dCas9-TETv4-based RNPs as an example, we characterized the STEP for delivering the CRISPR epigenetic editing machinery. Data showed that STEP facilitated the delivery of dCas9-TETv4-based RNPs, leading to efficient epigenetic editing and re-expression of the silenced SNRPN gene in the Prader-Willie syndrome candidate region. Figure 10A and Figure 10B TETv4 is the catalytic subunit of a ten-eleven translocation (TET) enzyme that oxidizes 5-methylcytosine and promotes site-specific reversal of DNA methylation. dCas9 is the inactive form of the spCas9 nuclease.

[0338] xi. STEP for intracellular delivery of other protein payloads

[0339] Experiments were conducted to determine whether STEP could be used to deliver protein payloads other than RNP, including GFP (28kDa), Cas9-EGFP fusion protein (194kDa), and AF568-labeled IgG (150kDa). Both proteins were surface-coupled to cholesterol via DBNPDEE and 2-arm PEG (cholesterol x 2). The resulting STEP Cas9-EGFP and IgG were added to U87 cells. After two, four, or six hours, the cells were imaged. STEP effectively delivered GFP, Cas9-EGFP, and IgG into cells, which was evident by observing strong fluorescence in the cytoplasm. These findings suggest that STEP technology can be used to effectively deliver various protein payloads into cells.

[0340] xii. Toxicity and stability

[0341] Results from the immediate study suggest that STEP RNP is potentially safe, as intravenous administration of STEP RNP did not cause significant systemic toxicity in the liver and kidneys based on AST, ALT, BUN, and creatine measurements ( Figures 11A-11D). In addition, based on microscopic imaging and H&E staining, intracranial administration of STEP RNPs did not cause significant cellular damage in the brain. In addition, after IT administration, H&E staining did not detect significant brain toxicity or cellular damage, and measurements of AST, ALT, BUN, and creatinine levels did not indicate significant hepatotoxicity or nephrotoxicity. Finally, storage of STEP RNPs at -20°C for 2 months did not reduce their efficiency in genome editing ( Figure 11E ).

[0342] xiii. Pharmacokinetics

[0343] Given that no significant differences were observed between IT and ICV injections in terms of whole-brain editing, IT was chosen for pharmacokinetic (PK) studies. The PK of CRISPR-Cas9 ribonucleoprotein (cRNP) was characterized. Mice were euthanized 1, 3, 6, and 12 hours after IT injection. Immunostaining analysis found that cRNP permeated the entire brain at the 6-hour time point and was almost undetectable at 12 hours. Some cRNP leaked into the circulation, as detected by positive staining in peripheral organs.

[0344] xiv. STEP RNP-based genome editing therapy for HIST1H1E (H1-4) syndrome

[0345] STE RNPs have been characterized for the treatment of H1-4 syndrome, a neurological disorder characterized primarily by autism spectrum disorder (ASD) and intellectual disability (ID) (Burkardt and Tatton Brown, HIST1H1E syndrome. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, eds. Gene Reviews ((R)). Seattle (WA) 1993); Duffney, et al., Am J Med Genet B Neuropsychiatr Genet. 2018; 177(4):426-33). Most mutations in H1-4 syndrome are frameshift mutations and are clustered at the C-terminus, resulting in a protein with an abnormal C-terminal frameshift tail (CFT) of approximately 40 new amino acids (Duffney et al., Am J Med Genet B Neuropsychiatr Genet. 2018; 177(4):426-33; Tatton-Brown, et al., Am J Hum Genet. 2017; 100(5):725-36; Burkardt, et al., Am J Med Genet A. 2019; 179(10):2049-55; Flex, et al., Am J Hum Genet. 2019; 105(3):493-508; Tremblay, et al., Hum Mol Genet. 2021. Epub 2021 / 11 / 18.doi:10.1093 / hmg / ddab321). Importantly, gene-disrupting mutations at the human H1-4 N-terminus do not have any obvious clinical phenotypes (Tremblay et al., Hum Mol Genet. 2021. Epub 2021 / 11 / 18. doi: 10.1093 / hmg / ddab321), and homozygous H1-4 knockout mice do not show any abnormal phenotypes (Fan et al., Mol Cell Biol. 2001; 21(23): 7933-43). These observations support a gain-of-function mechanism for H1-4 syndrome and suggest that knocking out H1-4 CFTs may provide therapeutic benefits. H1-4 CFT patient-derived iPSCs were found to exhibit increased cell proliferation, abnormalities in nuclear ultrastructure and transcriptome, and abnormal H3K27me3 distribution ( Figure 12A). These cellular and molecular phenotypes were used to evaluate the efficacy of STEP RNP loaded with gRNA targeting H1-4. The most common and recurrent mutation in H1-4 syndrome is c.430dupG (430G). However, unlike humans, the same frameshift mutation in the mouse H1-4 protein does not lead to CFT (Tremblay et al., Hum Mol Genet. 2021. Epub 2021 / 11 / 18. doi: 10.1093 / hmg / ddab321). Therefore, humanized H1-4 mutant mice carrying 430G were generated ( Figure 12B As a control, humanized H1-4 WT mice were generated. 430G Mice exhibit highly penetrant perinatal lethality and growth retardation ( Figure 12B ). Heterozygous H1-4 430G The mice survived but had mild early growth retardation and behavioral impairments in multiple areas ( Figure 12C and Figure 12D ). H1-4 CFT iPSC and H1-4 430G Humanized mice were treated with ASO and STEP RNP targeting the 430G mutation. In iPSC-derived neural progenitor cells, a highly efficient reduction of H1-4 CFT mediated by STEP RNP in human neurons and mice was observed ( Figures 12E-12G ). In homozygous H1-4 430G In mice, IT administration of STEP RNP targeting H1-4 rescued perinatal lethality ( Figure 12H ).

[0346] Example 2: Using STEP for intracellular protein delivery using a low STEP / Cas9 ratio

[0347] Materials and methods

[0348] A library of fusion molecules was created according to the protocol discussed in Example 1, which includes cholesterol analogs and bile acid analogs. STEP Cas9 / sgAi9 RNP was prepared according to the protocol discussed in Example 1, and these molecules are in the library. Ai9 fibroblasts were treated with 2.5 μg / mL or 7.5 μg / mL of scarless STEP Cas9 / sgAi9 RNP. The membrane fusion molecules used were cholesterol, F7-cholesterol, and β-sitosterol. 48 hours after treatment, the cells were observed under a fluorescence microscope. The edited cells were quantified using tdTomato fluorescence (%). In this example, the STEP / Cas9 ratios were 10 and 20, respectively.

[0349] result

[0350] As shown in Figures 13B and 13C, the cholesterol analog F7-cholesterol showed similar gene editing activity to cholesterol when the STEP / Cas9 ratio was 10 and 20. It should be noted that in Figures 13B and 13C, β-sitosterol showed better cell editing efficiency than cholesterol when the STEP / Cas9 ratio was 10. In addition, when the STEP / Cas9 ratio was 20, the cell editing efficiency of β-sitosterol was also slightly higher than that of cholesterol. Without being bound by theory, in some cases (lower STEP / Cas feed ratio, lower Cas9 concentration), β-sitosterol may have better editing efficiency than cholesterol. However, the cytotoxicity of cholesterol may be lower than that of β-sitosterol. Therefore, for safety reasons, cholesterol can be selected. Nevertheless, this does not rule out the possibility that β-sitosterol can be used in some aspects of the present disclosure.

[0351] Example 3: Trace chemical linkers can also effectively deliver RNPs

[0352] Materials and methods

[0353] The editing efficiency of STEP Cas9 / sgAi9 RNP and scar Cas9 / sgAi9 RNP in Ai9 fibroblasts was compared. In this example, cholesterol was used as a cell membrane fusion molecule. Ai9 fibroblasts were treated with these conjugates at 5 μg / mL or 10 μg / mL. Cells were observed under a fluorescence microscope 48 hours after treatment. Edited cells were quantified using tdTomato fluorescence (%). The conjugates are shown below:

[0354]

[0355] wherein n is 24. The corresponding conjugate containing a trace chemical linker differs in that it does not contain the disulfide bond shown above.

[0356] result

[0357] As shown in Figure 14B, at an RNP concentration of 10 μg / mL, scar delivery using a scarless chemical linker effectively edited Ai9 cells with an efficiency of up to 57%. Importantly, at 5 μg / mL and 10 μg / mL, scarless delivery using a scarless chemical linker exhibited significantly higher editing efficiency than scar delivery. Scarless STEP delivery achieved an editing efficiency of 82% at a concentration of 10 μg / mL, 25% higher than scar delivery. These findings indicate that RNPs can also be effectively delivered using a scar chemical linker, and the use of a scarless chemical linker further significantly improves delivery and / or editing efficiency.

[0358] Example 4: STEP delivers Cas9-RNP into cells via a cytosolic delivery mechanism

[0359] Materials and methods

[0360] Endosomal capture during endocytosis is a major delivery limitation for genetic medicine, including Cas9 gene editing machines (Tong et al., Nature Reviews Materials 4.11 (2019): 726-737). The intracellular delivery mechanism of STEP-engineered RNP was evaluated. STEP-engineered GFP-fused Cas9 RNP was added to Ai9 fibroblasts and observed with a fluorescence microscope 2 hours and 6 hours after incubation. Endosomes were also stained with LysoTtracker Red.

[0361] result

[0362] GFP-Cas9 fluorescence did not fuse with endosomal fluorescence at 2 hours and was further completely distributed in the cytoplasm at 6 hours. These observations indicate that STEP-engineered RNP can deliver payloads into cells via a direct cytoplasmic delivery mechanism, thereby effectively avoiding endocytosis capture. GFP-Cas9 fluorescence was also observed in the nucleus (data not shown), confirming the effective nuclear delivery of payloads by STEP.

[0363] Example 5: Degradation of STEP RNP in cells

[0364] Materials and methods

[0365] To investigate the degradation of STEP RNP in cells, Ai9 fibroblasts were treated with STEP RNP for 3 hours. After 3 hours, the RNP was removed with fresh cell culture medium. Protein was harvested from cells at different time points and analyzed by Western blot using a Cas9 antibody.

[0366] result

[0367] like Figure 15 As shown, most Cas9 is degraded within 24 hours. After 48 hours, Cas9 is undetectable. This rapid degradation of Cas9 will significantly reduce the safety concerns of Cas9 gene editing therapies.

[0368] Example 6: STEP RNP treatment of Prader-Willi syndrome

[0369] Materials and methods

[0370] dCas9 is fused to the catalytic domain of a specific enzyme and binds to the target DNA sequence to modify methylation on CpG islands or histones without genome editing, thereby performing epigenetic regulation. Figure 16ASchematic diagram of methylated CpG on the target region targeted by dCas9-TET1 with sgRNA demethylase and histone H3K9me2 / 3 on the target region targeted by dCas9-JMJD2a with sgRNA demethylase are shown. To rescue the defects of paternally expressed genes, several sgRNAs were designed that bind to the PWS imprinting center (IC) to reactivate the silenced genes on maternal chromosome 15 ( Figure 16B In addition, this technology was applied to an in vivo mouse model in which EGFP was fused to the end of exon 3 of Snrpn, one of the imprinted genes on the maternal chromosome ( Figure 17A ). We designed an sgRNA that binds to the conserved PWS-IC on mouse chromosome 7C ( Figure 17B In addition, sgRNAs that bind to the upstream region of PWS-IC (which harbors H3K9me3, a target of JMJD2a) were also tested. Two IV injections were performed in mSnrpn-EGFP / p+ mice.

[0371] result

[0372] dCas9-TET1 was treated with sgRNA packaged by STEP into fibroblasts from a patient with Prader-Willi syndrome who has a large deletion on paternal chromosome 15q11-q13. The ribonucleoprotein reactivated the maternally imprinted / silenced gene SNPRN ( Figure 16C Compared with CPP (cell penetrating peptide), dCas9-JMJD2a with sgRNA packaged by STEP showed higher efficacy for reactivation of imprinted genes in PWS fibroblasts ( Figure 16D 、 16E and 16F). dCas9-JMJD2a with sgRNA can reactivate SNRPN, SNORD116, 116HG (host genes) from maternal chromosomes ( Figure 16D 、 16E and 16F). However, IPW located far from PWS-IC was not reactivated by dCas9-JMJD2a with sgRNA ( Figure 16G In vivo experiments demonstrated that dCas9-JMJD2a with sgRNA bound to PWS-ICs, reactivating the imprinted maternal EGFP gene via STEP delivery. Reactivated gene expression of the silenced gene (Snrpn-EGFP) was detected in the liver and blood of mice (Figure 17C). Expression levels approached those of wild-type mice, indicating that STEP effectively delivered RNPs via IV injection.

[0373] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A conjugate comprising the following structure: Preferably in: The dashed lines independently represent the presence of one or more covalent bonds or non-covalent bonds, preferably, the dashed lines represent the presence of one or more covalent bonds, P includes proteins, peptides, or nucleic acids; L is a linear or branched traceless or traceable chemical linker; M is a one-armed or multi-armed chemical moiety comprising a cell membrane fusion molecule; and np, nl, nm and nz are independently an integer between 1 and 150, inclusive, an integer between 1 and 100, inclusive, an integer between 1 and 75, inclusive, an integer between 1 and 50, inclusive, an integer between 1 and 25, inclusive, an integer between 1 and 15, inclusive, an integer between 1 and 10, inclusive, an integer between 1 and 7, inclusive, or an integer between 1 and 5, inclusive.

2. The conjugate according to claim 1, wherein L is a linear or branched traceless chemical linker comprising a stimulus-responsive chemical moiety and / or a self-destructive chemical moiety.

3. The conjugate of claim 1, wherein L is a linear or branched trace chemical linker comprising 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.

4. The conjugate according to any one of claims 1 to 3, wherein P comprises a protein or a peptide.

5. The conjugate according to any one of claims 1 to 3, wherein P comprises a nucleic acid.

6. The conjugate of any one of claims 1 to 5, wherein P comprises genome editing machinery.

7. The conjugate of claim 6, wherein the genome editing machinery comprises one or more of a CRISPR / Cas system, a zinc finger nuclease, a transcription activator-like effector nuclease, a meganuclease, a peptide nucleic acid, a base editor, a lead editor, or an antisense oligonucleotide.

8. The conjugate of claim 6 or 7, wherein the genome editing machinery comprises a CRISPR / Cas system.

9. The conjugate of claim 7 or 8, wherein the CRISPR / Cas system comprises a Cas nuclease and a single guide RNA (sgRNA).

10. The conjugate of claim 9, wherein the Cas nuclease is selected from Cas9, CasX, Cas7-11, CasFx, Cas12a, and Cas13.

11. The conjugate of claim 9 or 10, wherein the Cas nuclease is a Cas9 nuclease.

12. The conjugate according to any one of claims 9 to 11, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease.

13. The conjugate of any one of claims 9 to 12, wherein the Cas9 nuclease is a nuclease-deficient mutant variant of the Cas9 protein (dCas9).

14. The conjugate of claim 13, wherein the dCas9 is further bound to or coupled to an effector domain for dCas9 to silence or activate transcription.

15. The conjugate of claim 13, wherein the dCas9 is further bound to or conjugated to the catalytic domain of ten-eleven translocation (TET) 1 hydroxylase.

16. The conjugate of any one of claims 2 or 4 to 15, wherein the stimulus-responsive chemical moiety is responsive to a stimulus selected from the group consisting of pH (e.g., a change in pH), redox (e.g., a change in redox potential), reactive oxygen species (ROS), enzymes (e.g., overexpression of enzymes (e.g., proteases, esterases, etc.) due to a disease state or condition), ionic strength (e.g., a change in ionic strength), hypoxia, and combinations thereof.

17. The conjugate of any one of claims 2 or 4 to 16, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide bond, an orthoester, a hydrazone, a hydrazide, a hydrazine, an imine (such as an aldimine or ketimine), an oxime, an acetal group, a vinyl ether, a polyketal, methyl maleate, an ester bond, a nitroaryl group (such as nitrobenzyl), a nitroheteroaryl group (such as nitroimidazole), a quinone group, an azoaryl group (such as azophenyl), an azoheteroaryl group (such as azopyridyl), a peroxyoxalate, an aminoacrylate, an alkyl thioether or a selenoether (such as a monoselenoether bond, a diselenoether bond, etc.), a thioketal, a peroxyoxalate, or dimethyl maleate.

18. The conjugate of any one of claims 2 or 4 to 17, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide, an orthoester, an imine (such as an aldimine or ketimine), a hydrazone, a hydrazide, a hydrazine, an imine, an oxime, methyl maleate, an ester bond, dimethyl maleate, or a combination thereof.

19. The conjugate according to any one of claims 2 or 4 to 18, wherein the stimulus-responsive chemical moiety comprises a disulfide bond, an amide, an orthoester, an imine (such as an aldimine or ketimine), or an ester bond.

20. The conjugate of any one of claims 2 or 4 to 19, wherein the self-destructive chemical moiety degrades by electronic cascade, cycloelimination, or both.

21. The conjugate of any one of claims 2 or 4 to 20, wherein the self-destructive chemical moiety comprises p-aminobenzyl (e.g., p-NH-phenyl-CH2-), o-aminobenzyl (e.g., o-NH-phenyl-CH2-), p-oxybenzyl (e.g., p-O-phenyl-CH2-), o-aminobenzyl (e.g., o-O-phenyl-CH2-), p-thiobenzyl (e.g., p-S-phenyl-CH2-), o-thiobenzyl (e.g., o-S-phenyl-CH2-), cinnamyl ether, a cyclization-driving moiety, a Grob fragmentation moiety, or a combination thereof.

22. The conjugate according to any one of claims 2 or 4 to 21, wherein the self-destructive chemical moiety comprises the following structure: in: X is oxygen (O), NH or sulfur (S), Y is O, NRs, substituted alkyl or unsubstituted alkyl, W is a substituted alkyl group or an unsubstituted alkyl group, and Rs is hydrogen, substituted alkyl or unsubstituted alkyl.

23. The conjugate according to claim 22, wherein Y is oxygen.

24. The conjugate according to claim 22 or 23, wherein X is sulfur.

25. The conjugate according to any one of claims 22 to 24, wherein W is C2-C5 substituted alkyl, such as C2 substituted alkyl, C3 substituted alkyl, C4 substituted alkyl or C5 substituted alkyl.

26. The conjugate according to any one of claims 1, 2 or 4 to 23, wherein L is formed using a structure selected from the group consisting of: or a combination thereof.

27. The conjugate according to any one of claims 1 to 24, wherein L is formed using: 11,12-Didehydro-γ-oxo-, 2-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ester (DBNPDEE).

28. The conjugate according to any one of claims 2 or 4 to 19, wherein the self-destructive chemical moiety comprises the following structure: in: X is oxygen (O), NH or sulfur (S).

29. according to the conjugate described in any one of claims 1 to 28, wherein said cell membrane fusion molecule comprises protein; Peptide, such as cell penetrating peptide; Lipid (for example, 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 phosphatidylethanolamines with two connected fatty acyl chains (preferably unsaturated fatty acyl chains); Lysolipid etc.); Small molecule part, such as the pentacyclic or tetracyclic part of following substance: cholesterol, steroid hormone, glucocorticoid, mineralocorticoid, androgen, estrogen, progestogen, phytosterol (for example, β-sitosterol); Or hydrophobic amino acid residue (for example, tyrosine, aniline, tryptophan etc.).

30. The conjugate according to any one of claims 1 to 29, wherein the cell membrane fusion molecule comprises a protein; a peptide, such as a cell penetrating peptide.

31. according to the conjugate described in any one in claim 1 to 29, wherein said cell membrane fusion molecule comprises small molecule part, as the pentacyclic or tetracyclic part of following substance: cholesterol, steroid hormone, glucocorticoid, mineralocorticoid, androgen, estrogen or progestogen, phytosterol (for example β-sitosterol).

32. The conjugate according to any one of claims 1 to 29, wherein the cell membrane fusion molecule comprises a small molecule portion, such as a tetracyclic or pentacyclic portion of cholesterol.

33. The conjugate of any one of claims 1 to 32, comprising a hydrophilic polymer between the moieties in L and M, preferably a neutral, uncharged hydrophilic polymer such as polyalkylene glycols and polyalkylene oxides such as poly(ethylene glycol); polysaccharides such as cellulose and dextran; hydrophilic polypeptides and poly(amino acids), such as poly-L-serine; poly(oxyethylated polyols); poly(olefin alcohols), such as poly(vinyl alcohol); poly(N-vinyl pyrrolidone); poly(hydroxyethyl acrylate); poly(hydroxyalkyl methacrylate), for example poly(hydroxyethyl methacrylate), preferably polyalkylene glycols and polyalkylene oxides, such as poly(ethylene glycol), preferably wherein the hydrophilic polymer has a molecular weight between 100 Da and 10 kDa, inclusive, or between 200 Da and 10 kDa, inclusive.

34. according to any one of claims 1 to 33 conjugates, wherein M is a one-arm chemical part comprising the cell membrane fusion molecule.

35. according to any one of claims 1 to 34, wherein M is Where n is between 2 and 300, inclusive, such as 24.

36. according to any one of claims 1 to 33 conjugates, wherein M is the multi-arm chemical part that comprises a plurality of described cell membrane fusion molecules.

37. according to any one of claims 1 to 33 or 36, wherein M is a multi-arm chemical part, it comprises 2 to 10, 2 to 9, 2 to 8 or 2 to 7 small molecule parts, such as the tetracyclic or pentacyclic part of cholesterol.

38. The conjugate of any one of claims 1 to 33, 36 or 37, wherein M is a multi-arm chemical moiety comprising 2 to 7, preferably 2, small molecule moieties such as the tetracyclic or pentacyclic moieties of cholesterol.

39. according to any one of claims 1 to 33 or 36 to 38, wherein M is 40. The conjugate according to any one of claims 1 to 39, wherein the molar ratio of M to P or ML 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.

41. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 40 and a pharmaceutically acceptable carrier.

42. A method of delivering a protein, peptide or nucleic acid to a subject in need thereof, the method comprising administering the conjugate of any one of claims 1 to 40 or the pharmaceutical composition of claim 36.

43. The method of claim 42, wherein the administration is parenteral, such as intracranial, intracerebral, intracerebroventricular, intrathecal, intravenous, intraocular, subretinal, intravitreal, intranasal, intrapleural, or intratracheal.

44. The method of claim 42 or 43, wherein administering comprises convection-enhanced delivery.

45. A method of editing the genome of a subject in need thereof, the method comprising administering the conjugate of any one of claims 1 to 40 or the pharmaceutical composition of claim 41.

46. ​​The method of claim 45, comprising administering the conjugate or pharmaceutical formulation to one or more cells, tissues or organs of the subject.

47. The method of claim 45 or 46, wherein administration is performed in vitro or in vivo.

48. The method of any one of claims 42 to 47, wherein the subject exhibits one or more signs or symptoms associated with an inherited neuropathy, an inherited myopathy, an inherited eye disease or condition, an inherited lung disease or condition, an inherited liver disease or condition, or cancer.

49. The method of any one of claims 42 to 48, wherein the subject exhibits a condition consistent 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 ataxia, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Jakob-Creutzfeldt disease, dystonia, amyotrophic lateral sclerosis, muscular dystrophy, muscular dystrophy (e.g., Duchenne, Becker, facioscapulohumeral, ankylosing, congenital, distal, Emery-Dreifuss, oculopharyngeal, and limb-girdle), congenital myopathies (e.g., central One or more signs or symptoms associated with certain disorders (e.g., nuclei, myotubes, rods, Ullrich / Bethlem, and RyR1), metabolic disorders (e.g., Pompe disease), retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, cystic fibrosis, primary ciliary dyskinesia, alpha-1-antitrypsin deficiency, surfactant metabolism disorders types 1-4, familial pulmonary fibrosis, pulmonary alveolar microlithiasis, dyskeratosis congenita, neurofibromatosis type I, tuberous sclerosis / LAM, Birt-Hogg-Dubé syndrome, hyper-IgE syndrome, Hermansky-Pudlak syndrome, Gaucher disease type I, Niemann-Pick disease type B, lysinuria protein intolerance, hemochromatosis, Wilson disease, alpha-1-antitrypsin deficiency, or cancer.

50. The method of any one of claims 42 to 49, 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 ataxia, Pick's disease, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, or Jakob-Creutzfeldt disease.

51. The method of any one of claims 42 to 50, wherein the subject exhibits one or more signs or symptoms associated with Angelman syndrome, HIST1H1E (H1-4) syndrome, or Prader-Willi syndrome.

Images