Targeted delivery of gene editing constructs and methods of use thereof

Abstract

The present invention relates to compositions for efficient delivery of gene editing agents to target cells, and methods of using the same to treat diseases or conditions.

Description

[0001] Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under grant AI045008 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Citation of Related Applications

[0004] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 384,692, filed on November 22, 2022, which is incorporated herein by reference in its entirety. Background Art

[0005] Altering the properties of regulatory immune cells by activating, inhibiting, or modifying them has emerged as a popular and highly demanded therapeutic class known as immunotherapy. Current immunotherapies largely rely on biologic protein-based reagents, which are costly and challenging to manufacture, or require ex vivo modification of immune cells. Some examples include antibodies or cytokines for regulating immune cell function, monoclonal antibodies for redirecting immune function, T cell gene editing for preventing viral infections, and chimeric antigen receptor (CAR) T cell therapy.

[0006] There remains a need in the art for a robust targeted mRNA delivery system for targeting multiple cell types to deliver functional RNA-based gene editing tools. The present invention addresses this need. Summary of the Invention

[0007] In one embodiment, the present invention relates to a composition for targeted delivery of a gene editing reagent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding a gene editing reagent and a delivery vehicle, wherein the delivery vehicle comprises a targeting moiety that specifically binds to the cell or particle of interest. In one embodiment, the reagent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

[0008] In one embodiment, it further comprises a guide RNA.

[0009] In one embodiment, the target cell or particle is a stem cell, an immune cell, an endothelial cell, a bacterial cell, a viral particle, a fungal cell, or a parasitic cell.

[0010] In one embodiment, the target cell is a hematopoietic stem cell. In one embodiment, the target cell or particle is a T cell.

[0011] In one embodiment, the composition comprises a combination of an mRNA molecule encoding a Cas9 protein and a guide RNA molecule for editing a gene in a target cell.

[0012] In one embodiment, the guide RNA molecule targets CCR5. In one embodiment, the targeting moiety specifically binds to CD4. In one embodiment, the targeting moiety specifically binds to CD34.

[0013] In one embodiment, at least one isolated nucleoside-modified RNA comprises pseudouridine or 1-methylpseudouridine.

[0014] In one embodiment, the delivery vehicle comprises lipid nanoparticles (LNPs). In one embodiment, at least one nucleoside-modified RNA is encapsulated within the LNPs.

[0015] In one embodiment, the present invention relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition for targeted delivery of a gene editing reagent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding a gene editing reagent and a delivery vehicle, wherein the delivery vehicle comprises a targeting moiety that specifically binds to the cell or particle of interest. In one embodiment, the reagent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

[0016] In one embodiment, the disease or disorder is selected from the group consisting of genetic defects and infectious diseases.

[0017] In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal or intramuscular delivery.

[0018] In one embodiment, the present invention relates to a method of treating HIV, the method comprising administering a composition comprising a therapeutic reagent and a delivery vehicle, wherein the delivery vehicle comprises a targeting moiety that specifically binds to CD34+ hematopoietic stem cells, and wherein the therapeutic reagent comprises an mRNA molecule encoding a Cas9 protein and a guide RNA specific for CCR5.

[0019] In one embodiment, the present invention relates to a method of treating HIV, the method comprising administering a composition comprising a therapeutic reagent and a delivery vehicle, wherein the delivery vehicle comprises a targeting moiety that specifically binds to CD4+ T cells, and wherein the therapeutic reagent comprises an mRNA molecule encoding a Cas9 protein and a guide RNA specific for CCR5. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0021] Figures 1A to 1C Depicts data showing the binding and functional activity of in vitro CD4-targeted particles. Figure 1A Depicts anti-human CD4 / 125 I-labeled mRNA-LNP specific in vitro binding to human CD4 + T cells after incubation at RT for 1 hour. Figure 1B Depicts the binding of anti-CD4 / mRNA-LNP and control IgG / mRNA-LNP to human CD4 + T cells with increasing mRNA-LNP dose, and their corresponding mean fluorescence intensity (MFI). Figure 1C Depicts the Luc activity measured in human CD4 + T cells treated with anti-human CD4 / mRNA-LNP or control IgG / mRNA-LNP.

[0022] Figure 2A and Figure 2B Depicts data showing Cre mRNA-mediated in vitro gene recombination. Figure 2A Depicts Cre mRNA-induced gene recombination and subsequent reporter gene expression, expressed as the % of ZsGreen1+ cells in CD3+CD8- cells. Spleen cells were harvested from Ai6 mice and incubated with Cre mRNA-LNP at a dose of 1, 3, 6, or 9 μg per 2 million cells. The % ZsGreen1+ cells after anti-CD4 / mRNA-LNP administration were compared with control IgG / mRNA-LNP and unconjugated mRNA-LNP administration (****P < 0.0001, two-way ANOVA with Bonferroni correction). Figure 2B Depicts the gating strategy for identifying ZsGreen1-positive cells in CD3+CD8- cells.

[0023] Figures 3A to 3D Depicts data showing mRNA-LNP targeting of CD4 + T cells in vivo. Figure 3A Depicts the biodistribution of 125 I-labeled anti-CD4 / and control IgG / poly(C)mRNA-LNP in mice at 0.5 h. Tissue uptake is expressed as mean ± SEM (****P < 0.0001). Figure 3B Depicts the localization ratio, calculated as the % ID / g of a given organ at 30 min post-injection compared to 125Ratio of %ID / g in blood of mice treated with I-labeled anti-CD4 / or control IgG / mRNA-LNP. Shown are mean ± SEM. After intravenous injection of mRNA-LNP, in vivo mRNA-LNP binding was quantified as the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs ( Figure 3C ) and the localization ratio in the spleen ( Figure 3D ). Group size was 3 animals. Statistical analysis was performed by two-way ANOVA with Bonferroni correction (****P < 0.0001).

[0024] Figures 4A to 4D Data depicting biodistribution showing in vivo-targeted mRNA-LNP expression are presented. Mice were IV injected with 8 μg mRNA-LNP. Luc activity in lysed tissues was measured by ( Figure 4A ) and the organ distribution of Luc mRNA expression was evaluated by luminescence imaging 5 h after administration of anti-CD4 / and control IgG / Luc mRNA-LNP by ( Figure 4B and Figure 4C ). Figure 4A Quantitative expression of Luc is depicted as light units (LU) / mg protein. Five minutes after administration of D-luciferin, a representative set of dissected mouse organ samples ( Figure 4B ) and whole carcasses after organ excision (showing luminescent lymph nodes) ( Figure 4C ) were analyzed. Figure 4D Quantitative expression of Luc in CD3+ cell preparations obtained from the spleens of mice injected with mRNA-LNP is depicted as LU / mg protein values. Figure 4A and Figure 4D : Error bars indicate SEM. Group size was 3 animals. Statistical analysis was performed by two-way ANOVA with Bonferroni correction (*P < 0.05, **P < 0.01, and ***P < 0.001).

[0025] Figure 5A To Figure 5E depict data showing Cre-mediated gene recombination after in vivo administration of CD4-targeted Cre mRNA-LNP. Figure 5A A schematic diagram is depicted that depicts anti-CD4 / mRNA-LNP targeted delivery for CD4 +Selective gene recombination in T cells, and the principle of the Ai6 reporter allele: Cre-mediated excision of the loxP-flanked STOP cassette allows robust expression of the fluorescent protein ZsGreen1. Ai6 mice received 3, 10, and 30 μg doses of Cre mRNA-LNP by IV administration. Spleens and lymph nodes were harvested 24 h after treatment, and flow cytometry was used to determine the percentage of ZsGreen1 + CD8 - cells in the CD3 + cell populations in the spleen (Figure 5B) and lymph nodes (Figure 5C). After IV injection of 10 μg mRNA-LNP, the change in the number of ZsGreen1-expressing CD4 + T cells over time was monitored in the spleen (Figure 5D) and lymph nodes (Figure 5E). In a total of three independent experiments, the group size was 8 or 9 animals (Figure 5B and Figure 5C) or 6 animals (Figure 5D and Figure 5E). Each symbol represents an animal, and the horizontal line shows the mean and SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction. The % ZsGreen1 #### cells were compared after injection of different doses of anti-CD4 / mRNA-LNP [*P<0.05, ****P<0.0001] and unconjugated mRNA-LNP + P<0.0001].

[0026] Figure 6A to Figure 6C depicts data showing in vivo uptake of Cre mRNA-LNP by different T cell subtypes. Spleens were harvested 24 h after treatment with 10 μg Cre mRNA-LNP, and flow cytometry was used to determine the percentage of ZsGreen1 + cells in the CD4 + T cell subsets (Figure 6A) and compared with the CD25 marker (Figure 6B). Naive CD4 + T cells were considered CD44 - CD62L - , central memory T cells were considered CD44 + CD62L + , and effector memory T cells were considered CD44 + CD62L - . The group size was 3 - 11 animals. Each symbol represents an animal, and the horizontal line shows the mean and SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction to compare T cell subtypes. Figure 6C depicts the gating strategy for identifying ZsGreen1-positive cells in different CD4 + T cell subtypes.

[0027] Figures 7A and 7B depict data showing the mRNA-LNP targeting efficiency using multiple administrations. Ai6 mice were injected daily via IV with 10 μg (0.4 mg / kg) anti-CD4 / , control IgG / , or unconjugated Cre mRNA-LNP for 3 or 5 days. Spleens and lymph nodes were harvested after three or five consecutive injections, and the percentage of ZsGreen1 + CD8 - cells in the CD3 + cell populations was determined in single cell suspensions of spleen (Figure 7A) and lymph nodes (Figure 7B). Group size was 9 animals. Each symbol represents one animal, and the horizontal line shows the mean. Error bars indicate SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction. The percentage of ZsGreen1 + cells was compared after different numbers of injections of anti-CD4 / mRNA-LNP [**P<0.01, ****P<0.0001].

[0028] Figure 8 Depicts the CCR5KO efficiency of CD5 / LNP / Cas9 mRNA / CCR5 gRNA in human T cells. Very promising KO efficiency was achieved by using CD5 / -targeted LNP encapsulating Cas9 mRNA / CCR5 gRNA as a proof of concept for RNA-based gene editing using targeted LNP-mRNA. Detailed Description

[0029] The present invention relates to a composition for the efficient delivery of a therapeutic agent, which comprises a delivery medium, wherein the delivery medium comprises at least one targeting domain or moiety for delivering the therapeutic agent to a specific target cell type, and wherein the therapeutic agent comprises at least one composition for gene editing.

[0030] In some embodiments, the composition for gene editing comprises a combination of an mRNA molecule encoding a Cas9 protein and a guide RNA.

[0031] In some embodiments, the LNPs comprise targeting domains that are specific for endothelial cells, immune cells, stem cells, or another cell type of particular interest. Target cells / tissues that can be targeted include, but are not limited to, T cells, hematopoietic stem cells (HSCs), endothelial cells, myeloid cells, and lung cells. In one embodiment, the targeted LNPs of the invention comprise a targeting domain that specifically binds an antigen on the target cell type of interest. For example, in one embodiment, the LNPs comprise a targeting domain that specifically binds an antigen expressed on immune cells. In one embodiment, the LNPs comprise a targeting domain that specifically binds an antigen expressed on tumor cells. In one embodiment, the LNPs comprise a targeting domain that specifically binds an antigen expressed on tumor cells. In one embodiment, the LNPs comprise a targeting domain that specifically binds an antigen expressed on a particular tissue type (e.g., a marker expressed on lung tissue).

[0032] The invention also relates to methods of targeted delivery of therapeutic agents using the compositions described herein and methods of treating a disease or disorder in a subject, the disease or disorder including, but not limited to, genetic defects and infectious diseases and disorders. In some embodiments, the genetic defect is a monogenic disease or disorder. Exemplary genetic disorders that can be treated using the compositions and methods of the invention include, but are not limited to, achondroplasia, α-1 antitrypsin deficiency, antiphospholipid syndrome, attention deficit hyperactivity disorder, autism, autosomal dominant polycystic kidney disease, breast cancer, Charcot-Marie-Tooth disease, colon cancer, cri du chat syndrome, Crohn's disease, cystic fibrosis, Duane syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, inborn errors of metabolism, Klinefelter syndrome, Marfan syndrome, methylmalonic aciduria, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, and Wilson disease.

[0033] In one embodiment, the disease or disorder is a non-malignant hematopoietic system disorder, a stem cell depletion disease or disorder, a stem cell proliferation disease or disorder, or any disease or disorder in which modulation of stem cells is beneficial.

[0034] In some embodiments, the infectious disease or disorder is a disease associated with a viral infection, a bacterial infection, a fungal infection, or a parasite. In some embodiments, the viral infection is a human immunodeficiency virus (HIV) infection.

[0035] Definition

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] As used herein, each of the following terms has the meaning associated with it in this section.

[0038] The articles “a” and “an” as used herein refer to one or more than one (i.e., at least one) grammatical object of the article. For example, “an element” refers to one element or more than one element.

[0039] As used herein, “about” when used in reference to a measured value (such as an amount, a time duration, etc.) is intended to cover variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0040] The term “adjuvant” as used herein refers to a reagent that modifies or enhances the intensity and duration of a desired therapeutic response, and / or broadens the therapeutic response to a concomitantly administered reagent.

[0041] The term “antibody” as used herein refers to an immunoglobulin molecule that specifically binds to an antigen or epitope. The antibody can be a full immunoglobulin from a natural source or from a recombinant source, and can be an immunoreactive portion of a full immunoglobulin. The term “antibody” as used herein includes “antibody fragments”. The term “antibody fragment” refers to a portion of a full antibody and refers to the antigen - specific - determining variable region of a full antibody. The antibodies or antibody fragments in the present invention can exist in various forms, including for example polyclonal antibodies, monoclonal antibodies, linear antibodies, Fv, Fab, F(ab)2, Fab' or F(ab')2 fragments, as well as single - chain antibodies, multispecific antibodies, and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879 - 5883; Bird et al., 1988, Science 242:423 - 426).

[0042] The “antibody heavy chain” as used herein refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0043] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. K and λ light chains refer to the two major antibody light chain isotypes.

[0044] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a phage. The term should also be construed to refer to an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art. The term should also be construed to refer to an antibody produced by synthesizing an RNA molecule encoding the antibody. The RNA molecule expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the RNA is obtained by transcribing DNA (synthetic or cloned) or other techniques available and well known in the art.

[0045] "Disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal refers to a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is not as good as it would be in the absence of the disorder. Without treatment, a disorder does not necessarily result in a further decline in the animal's state of health.

[0046] As used herein, "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0047] The term "physiologically effective dose" refers to the amount of a reagent that produces a measurable biological or physiological effect in a recipient subject that is related to the activity of the reagent. The physiologically effective dose will vary depending on the compound, the age, weight, etc. of the subject to which the reagent is administered, and the biological or physiological effect being measured.

[0048] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template in a biological process for synthesizing a polymer or macromolecule having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if transcription and translation of the mRNA corresponding to a certain gene produces a protein in a cell or other biological system, then that gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as the template for gene or cDNA transcription) can be said to encode the protein or other product of that gene or cDNA.

[0049] "Expression vector" means a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis - acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNAs, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno - associated viruses) incorporating the recombinant polynucleotide.

[0050] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the two molecules are homologous at that position. The percentage of homology between two sequences is determined by dividing the number of matching or homologous positions shared by the two sequences by the number of positions compared X 100. For example, if 6 out of 10 positions in two sequences match or are homologous, the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is made when the two sequences are aligned to provide maximum homology.

[0051] "Isolated" means altered or separated from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the co - existing materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non - native environment (such as, for example, a host cell).

[0052] In the context of the present invention, the following abbreviations for common nucleosides (nucleobases attached to ribose or deoxyribose via an N - glycosidic bond) are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0053] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein contains introns in some versions.

[0054] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of response in a subject as compared to the level of response in a subject in the absence of treatment or compound and / or as compared to the level of response in an otherwise identical untreated subject. The term encompasses disrupting and / or affecting the native signal or response such that a beneficial therapeutic response in the subject is mediated. In some embodiments, the subject is a human.

[0055] Unless otherwise indicated, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs can include introns. In addition, nucleotide sequences can contain modified nucleosides that are capable of being translated by the translation machinery in a cell. For example, all uridines in an mRNA are replaced with pseudouridine, 1-methylpseudouridine, or another modified nucleoside.

[0056] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence such that the expression of the latter is effected. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein-coding regions, are joined in the same reading frame.

[0057] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or its cells (whether in vitro or in situ) suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0058] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. It is common knowledge to those skilled in the art that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR TM etc. and by synthetic means.

[0059] In certain instances, the polynucleotides or nucleic acids of the invention are "nucleoside-modified nucleic acids", which refers to nucleic acids that contain at least one modified nucleoside. A "modified nucleoside" refers to a nucleoside having a modification. For example, over a hundred different nucleoside modifications have been identified in RNA (Rozenski et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27:196-197).

[0060] In certain embodiments, "pseudouridine" in another embodiment refers to m 1 acp 3 Y (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1 Y (1-methylpseudouridine). In another embodiment, the term refers to Ym(2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to m 3 Y (3-methylpseudouridine). In another embodiment, the term refers to an unmodified pseudouridine moiety. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0061] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein sequence or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined to each other by peptide bonds. As used herein, the term refers both to short chains, which are commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and to long chains, which are commonly referred to in the art as proteins, and there are many types of proteins. "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0062] The term "promoter" as used herein is defined as a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery and is required to initiate the specific transcription of a polynucleotide sequence. For example, a promoter recognized by a bacteriophage RNA polymerase and used to produce mRNA by in vitro transcription.

[0063] As used herein, the term "specifically binds" in reference to an affinity ligand (particularly an antibody) refers to an antibody that recognizes a specific antigen but essentially does not recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind antigens from one or more other species. However, this cross-species reactivity by itself does not change the classification of the antibody's specificity. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, this cross-reactivity by itself does not change the classification of the antibody's specificity. In some cases, the term "specific binding" or "specifically binds" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning the interaction depends on the presence of a specific structure (such as an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0064] As used herein, the term "treat" refers to treatment and / or prevention. A therapeutic effect is obtained by inhibiting, reducing, alleviating, or eliminating at least one sign or symptom of a disease or disorder.

[0065] The term "therapeutically effective amount" refers to the amount of a subject compound that will elicit a biological or medical response of a tissue, system, or subject that is sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of the treated disorder or disease or to alleviate to some extent one or more signs or symptoms of the treated disorder or disease. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0066] As used herein, the term "treating" a disease refers to reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0067] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0068] As used herein, the phrase "under transcriptional control" or "operably linked" means that a promoter is in the correct position and orientation relative to a polynucleotide to control transcription initiation by RNA polymerase and expression of the polynucleotide.

[0069] "Vector" refers to a substance composition that contains a separated nucleic acid and can be used to deliver the separated nucleic acid into the interior of a cell. A variety of vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0070] "Alkyl" refers to a straight-chain or branched-chain hydrocarbon chain group consisting only of carbon and hydrogen atoms, which may be saturated or unsaturated (i.e., contain one or more double bonds and / or triple bonds), having from one to twenty-four carbon atoms (C1-C 24 alkyl), from one to twelve carbon atoms (C1-C 12 alkyl), from one to eight carbon atoms (C1-C8 alkyl), or from one to six carbon atoms (C1-C6 alkyl), and is attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise explicitly stated, the alkyl group is optionally substituted.

[0071] "Alkylene" or "alkylene chain" refers to a straight-chain or branched-chain divalent hydrocarbon chain consisting only of carbon and hydrogen that connects the remainder of the molecule to a radical group, which is saturated or unsaturated (i.e., contains one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), and has, for example, from one to twenty-four carbon atoms (C1-C 24 alkylene), from one to fifteen carbon atoms (C1-C 15 alkylene), from one to twelve carbon atoms (C1-C 12 alkylene), from one to eight carbon atoms (C1-C8 alkylene), from one to six carbon atoms (C1-C6 alkylene), from two to four carbon atoms (C2-C4 alkylene), from one to two carbon atoms (C1-C2 alkylene), such as methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butylene, propynylene, n-butynylene, and the like. The alkylene chain is attached to the remainder of the molecule by a single bond or a double bond and is attached to the radical group by a single bond or a double bond. The attachment points of the alkylene chain to the remainder of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise explicitly stated in this specification, the alkylene chain can be optionally substituted.

[0072] "Cycloalkyl" or "carbocyclic ring" means a stable, non-aromatic, monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms and being saturated or unsaturated and attached to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethylbicyclo[2.2.1]heptyl, etc. Unless otherwise expressly stated, cycloalkyl is optionally substituted.

[0073] "Cycloalkylene" is a divalent cycloalkyl. Unless otherwise expressly stated in this specification, cycloalkylene may be optionally substituted.

[0074] "Heterocyclic radical" or "heterocyclic ring" means a stable 3- to 18-membered non-aromatic ring radical composed of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise expressly stated in the specification, the heterocyclic radical may be a monocyclic, bicyclic, tricyclic or tetracyclic system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclic radical may be optionally oxidized; the nitrogen atoms may be optionally quaternized; and the heterocyclic radical may be partially or fully saturated. Examples of such heterocyclic radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithialanyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidoneyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithialanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl. Unless otherwise expressly stated, the heterocyclic radical may be optionally substituted.

[0075] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, cycloalkyl or heterocyclic radical) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, the non-hydrogen atom being, for example but not limited to: a halogen atom, such as F, Cl, Br and I; an oxo group (=O); a hydroxy group (-OH); an alkoxy group (-OR a , where R a is C1-C 12 alkyl or cycloalkyl); a carboxyl group (-OC(=O)R a or -C(=O)OR a , where R a is H, C1-C 12 alkyl or cycloalkyl); an amino group (-NR a R b , where Ra and R b are each independently H, C1-C 12 alkyl or cycloalkyl); C1-C 12 alkyl; and cycloalkyl. In some embodiments, the substituent is C1-C 12 alkyl. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogen group, such as fluorine. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group.

[0076] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and the description includes both the substituted alkyl radical and the unsubstituted alkyl radical.

[0077] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges within that range as well as individual numerical values. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0078] Description

[0079] The present invention relates in part to compositions and methods for targeted delivery of gene editing reagents. In some embodiments, the present invention includes administering a therapeutic gene editing reagent to a subject in need thereof. In some embodiments, the therapeutic reagent comprises an mRNA molecule encoding a gene editing molecule.

[0080] Delivery medium

[0081] In some embodiments, the delivery vehicle is a colloidal dispersion system such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0082] Consider using a lipid formulation to introduce at least one reagent into a host cell (in vitro, ex vivo, or in vivo). In another aspect, at least one reagent can be associated with a lipid. The at least one reagent associated with a lipid can be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and an oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, included as a suspension in a lipid, included in or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or using a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include the naturally occurring fat droplets in the cytoplasm and the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0083] Suitable lipids for use are available from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Chol") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing various single and multi-layered lipid media formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure, with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components rearrange themselves before forming a closed structure and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having a structure different from the normal vesicular structure in solution are also encompassed. For example, the lipids may assume a micellar structure or simply exist as non-uniform aggregates of lipid molecules. Lipofectamine-reagent complexes are also considered.

[0084] In one embodiment, the delivery of at least one reagent includes any suitable delivery method, including the exemplary delivery methods described elsewhere herein. In certain embodiments, delivering at least one reagent to a subject includes mixing at least one reagent with a transfection reagent prior to the contacting step. In another embodiment, the methods of the invention further include co-administering at least one reagent with a transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0085] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is or In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0086] In another embodiment, the transfection reagent forms liposomes. In another embodiment, the liposomes increase intracellular stability, increase uptake efficiency and improve bioactivity. In another embodiment, the liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to those that make up cell membranes. In some embodiments, the liposomes contain an internal aqueous space for entrapping water-soluble compounds. In another embodiment, the liposomes can deliver at least one reagent to cells in an active form.

[0087] In one embodiment, the composition comprises lipid nanoparticles (LNPs) and at least one reagent.

[0088] The term "lipid nanoparticles" refers to particles having at least one dimension in the nanometer range (e.g., 1 - 1,000 nm) that comprise one or more lipids. In various embodiments, the particle comprises a lipid of formula (I), (II) or (III). In some embodiments, the lipid nanoparticles are included in a formulation comprising at least one reagent described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of formula (I), (II) or (III)) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., polyethylene glycolylated lipids such as the polyethylene glycolylated lipid of structure (IV), e.g., compound IVa). In some embodiments, at least one reagent is encapsulated within the lipid portion of the lipid nanoparticles or in an aqueous space enclosed by a partial or entire lipid portion of the lipid nanoparticles, thereby protecting it from enzymatic degradation or other adverse effects induced by the mechanisms of the host organism or cell (e.g., an adverse immune response).

[0089] In various embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In one embodiment, the lipid nanoparticles have an average diameter of about 83 nm. In one embodiment, the lipid nanoparticles have an average diameter of about 102 nm. In one embodiment, the lipid nanoparticles have an average diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, when present in the lipid nanoparticles, the at least one reagent resists degradation by intracellular or intercellular enzymes in an aqueous solution.

[0090] The LNP can comprise any lipid capable of forming a particle to which the at least one reagent is attached or in which the at least one reagent is encapsulated. The term "lipid" refers to a group of organic compounds that are fatty acid derivatives (e.g., esters), and which are generally characterized as being insoluble in water but soluble in a variety of organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids", which include fats and oils and waxes; (2) "compound lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0091] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and polyethylene glycolated lipids.

[0092] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH is lowered below the pK of the ionizable group of the lipid, but becomes increasingly more neutral at higher pH values. When the pH is below the pK, the lipid can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises an amphoteric lipid that exhibits a positive charge upon a decrease in pH.

[0093] In certain embodiments, the cationic lipid comprises any of a number of lipid species that bear a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In addition, commercial formulations of many cationic lipids are available and can be used in the present invention. These include, for example (commercially available cationic liposomes that contain DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); (commercially available cationic liposomes that contain N-(1-(2,3-dioleyloxy(propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and (commercially available cationic liposomes that contain dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol, from Promega Corp., Madison, Wis.). The following lipids are cationic and bear a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0094] In one embodiment, the cationic lipid is an amino lipid. Amino lipids suitable for use in the present invention include those described in WO2012 / 016184, which is hereby incorporated by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetyloxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinolenylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinolenylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinolenyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinolenyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0095] Suitable amino lipids include those having the following formula:

[0096]

[0097] wherein R1 and R2 are the same or different and independently are optionally substituted C 10 -C 24 alkyl, optionally substituted C 10 -C 24 alkenyl, optionally substituted C 10 -C 24 alkynyl or optionally substituted C 10 -C 24 acyl;

[0098] R3 and R4 are the same or different and independently are optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, or R3 and R4 may be joined to form an optionally substituted heterocyclic ring having 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;

[0099] R5 is absent or present, and when present is hydrogen or C1-C6 alkyl;

[0100] m, n, and p are the same or different and are independently 0 or 1, provided that m, n, and p are not simultaneously 0;

[0101] q is 0, 1, 2, 3, or 4; and

[0102] Y and Z are the same or different and are independently O, S, or NH.

[0103] In one embodiment, both R1 and R2 are linoleyl, and the amino lipid is dilinoleyl amino lipid. In one embodiment, the amino lipid is dilinoleyl amino lipid.

[0104] Representative useful dilinoleyl amino lipids have the formula:

[0105]

[0106] where n is 0, 1, 2, 3, or 4.

[0107] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (the above DLin-K-DMA, where n is 2).

[0108] In one embodiment, the cationic lipid component of the LNP has the structure of formula (I):

[0109]

[0110] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, where:

[0111] L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond;

[0112] R 1a and R 1b are independently, each time they appear, (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached and together with the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0113] R 2a and R 2b are independently, each time they appear, (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1-C 12alkyl, and R 2b together with the carbon atom to which it is attached and with an adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0114] R 3a and R 3b are each independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached and with an adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0115] R 4a and R 4b are each independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached and with an adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0116] R 5 and R 6 are each independently methyl or cycloalkyl;

[0117] R 7 is, at each occurrence, independently H or C1-C 12 alkyl;

[0118] R 8 and R 9 are each independently C1-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring containing one nitrogen atom;

[0119] a and d are each independently an integer from 0 to 24;

[0120] b and c are each independently an integer from 1 to 24; and

[0121] e is 1 or 2.

[0122] In certain embodiments of formula (I), R 1a , R 2a , R 3a or R 4a at least one of which is C1-C 12 alkyl, or L1 or L 2 at least one of them is -O(C=O)- or -(C=O)O-. In other embodiments, R 1a and R 1b is not isopropyl when a is 6 or not n-butyl when a is 8.

[0123] In a further embodiment of formula (I), R 1a , R 2a , R 3a or R 4a at least one of them is C1-C 12 alkyl, or L 1 or L 2 at least one of them is -O(C=O)- or -(C=O)O-; and

[0124] R 1a and R 1b is not isopropyl when a is 6 or not n-butyl when a is 8.

[0125] In other embodiments of formula (I), R 8 and R 9 are each independently unsubstituted C1-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring containing one nitrogen atom;

[0126] In certain embodiments of formula (I), L 1 or L 2 either one of them can be -O(C=O)- or a carbon-carbon double bond. L 1 and L 2 can each be -O(C=O)- or can each be a carbon-carbon double bond.

[0127] In some embodiments of formula (I), L 1 or L 2 one of them is -O(C=O)-. In other embodiments, L 1 and L 2 are both -O(C=O)-.

[0128] In some embodiments of formula (I), L 1 or L 2 one of them is -(C=O)O-. In other embodiments, L 1 and L 2 are both -(C=O)O-.

[0129] In some other embodiments of formula (I), L 1 or L2 One of them is a carbon-carbon double bond. In other embodiments, L 1 and L 2 are both carbon-carbon double bonds.

[0130] In still other embodiments of formula (I), L 1 or L 2 One of them is -O(C=O)-, and the other of L 1 or L 2 is -(C=O)O-. In more embodiments, L 1 or L 2 One of them is -O(C=O)-, and the other of L 1 or L 2 is a carbon-carbon double bond. In still more embodiments, L 1 or L 2 One of them is -(C=O)O-, and the other of L 1 or L 2 is a carbon-carbon double bond.

[0131] It should be understood that the "carbon-carbon" double bond used throughout the specification refers to one of the following structures:

[0132]

[0133] wherein R a and R b are independently H or a substituent each time they appear. For example, in some embodiments, R a and R b are independently H, C1-C 12 alkyl or cycloalkyl each time they appear, such as H or C1-C 12 alkyl.

[0134] In other embodiments, the lipid compound of formula (I) has the following structure (Ia):

[0135]

[0136] In other embodiments, the lipid compound of formula (I) has the following structure (Ib):

[0137]

[0138] In still other embodiments, the lipid compound of formula (I) has the following structure (Ic):

[0139]

[0140] In certain embodiments of the lipid compound of formula (I), a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or an integer from 5 to 9. In some specific embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In still other embodiments, a is 16.

[0141] In some other embodiments of formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In still other embodiments, b is 16.

[0142] In some more embodiments of formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In still other embodiments, c is 16.

[0143] In some specific other embodiments of formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In still other embodiments, d is 16.

[0144] In some other various embodiments of formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same, and b and c are the same.

[0145] The sum of a and b and the sum of c and d in formula (I) are factors that can be varied to obtain a lipid of formula (I) with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In a further embodiment, the sum of a and b is the same as the sum of c and d. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer within the range of 14 to 24. In still more embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or greater.

[0146] In some embodiments of formula (I), e is 1. In other embodiments, e is 2.

[0147] R of formula (I) 1a 、R 2a 、R 3a and R 4a The substituents at are not particularly limited. In certain embodiments, R 1a 、R 2a 、R 3a and R 4a are each H upon each occurrence. In certain other embodiments, at least one of R 1a 、R 2a 、R 3a and R 4a is C1-C 12 alkyl. In certain other embodiments, R 1a 、R 2a 、R 3a and R 4aAt least one of them is a C1-C8 alkyl group. In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of them is a C1-C6 alkyl group. In some of the foregoing embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0148] In certain embodiments of formula (I), R 1a , R 1b , R 4a and R 4b Each occurrence is a C1-C 12 alkyl group.

[0149] In a further embodiment of formula (I), at least one of R 1b , R 2b , R 3b and R 4b is H, or R 1b , R 2b , R 3b and R 4b Each occurrence is H.

[0150] In certain embodiments of formula (I), R 1b together with the carbon atom to which it is attached and together with the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond. In other foregoing embodiments, R 4b together with the carbon atom to which it is attached and together with the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond.

[0151] In the foregoing embodiments, the substituents at R 5 and R 6 in formula (I) are not particularly limited. In certain embodiments, one or both of R 5 or R 6 are methyl. In certain other embodiments, one or both of R 5 or R 6 are cycloalkyl groups, such as cyclohexyl. In these embodiments, the cycloalkyl group can be substituted or unsubstituted. In certain other embodiments, the cycloalkyl group is substituted with a C1-C 12 alkyl group (such as tert-butyl).

[0152] In the foregoing embodiments of formula (I), the substituent at R 7 is not particularly limited. In certain embodiments, at least one R 7is H. In some other embodiments, R 7 is H each time it appears. In certain other embodiments, R 7 is C1-C 12 alkyl.

[0153] In certain other foregoing embodiments of formula (I), R 8 or R 9 is methyl. In other embodiments, R 8 and R 9 are both methyl.

[0154] In some different embodiments of formula (I), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, such as a pyrrolidinyl ring.

[0155] In various different embodiments, exemplary lipids of formula (I) may include

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] In some embodiments, the LNP comprises a lipid of formula (I), at least one reagent, and one or more excipients selected from neutral lipids, steroids, and polyethylene glycolated lipids. In some embodiments, the lipid of formula (I) is Compound I-5. In some embodiments, the lipid of formula (I) is Compound I-6.

[0165] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (II):

[0166]

[0167] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0168] L 1 and L 2 each independently is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)O-, or a direct bond;

[0169] G 1 is a C1-C2 alkylene group, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond;

[0170] G 2 is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a or a direct bond;

[0171] G 3 is a C1-C6 alkylene group;

[0172] R a is H or C1-C 12 alkyl;

[0173] R 1a and R 1b each occurrence is independently: (a) H or C1-C 12 alkyl; or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached and with the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0174] R 2a and R 2b each occurrence is independently: (a) H or C1-C 12 alkyl; or (b) R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached and with the adjacent R 2bTogether with the carbon atom to which it is attached, form a carbon-carbon double bond;

[0175] R 3a and R 3b Each occurrence independently is: (a) H or C1-C 12 alkyl; or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached and with the adjacent R 3b together with the carbon atom to which it is attached, form a carbon-carbon double bond;

[0176] R 4a and R 4b Each occurrence independently is: (a) H or C1-C 12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is attached and with the adjacent R 4b together with the carbon atom to which it is attached, form a carbon-carbon double bond;

[0177] R 5 and R 6 are each independently H or methyl;

[0178] R 7 is C4-C 20 alkyl;

[0179] R 8 and R 9 are each independently C1-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocyclic ring;

[0180] a, b, c and d are each independently an integer from 1 to 24; and

[0181] x is 0, 1 or 2.

[0182] In some embodiments of formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G 1 and G 2 are each independently -(C=O)- or a direct bond. In some different embodiments, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O- or a direct bond; and G 1 and G2 Each independently is -(C=O)- or a direct bond.

[0183] In some different embodiments of formula (II), L 1 and L 2 Each independently is -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR a S(O) x -, or -S(O) x NR a -.

[0184] In the foregoing embodiments of formula (II), the lipid compound has one of the following structures (IIA) or (IIB):

[0185]

[0186] In some embodiments of formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[0187] In any of the foregoing embodiments of formula (II), L 1 or L 2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each is -O(C=O)-.

[0188] In some different embodiments of formula (II), L 1 or L 2 One of them is -(C=O)O-. For example, in some embodiments, L 1 and L 2 Each is -(C=O)O-.

[0189] In different embodiments of formula (II), L 1 or L 2 One of them is a direct bond. As used herein, "direct bond" refers to a group (e.g., L1 or L 2 ) does not exist. For example, in some embodiments, L 1 and L 2 are each a direct bond.

[0190] In other different embodiments of formula (II), for at least one occurrence of R 1a and R 1b , R 1a is H or C1-C 12 alkyl, and R 1b , together with the carbon atom to which it is attached and together with the adjacent R 1b and the carbon atom to which it is attached, forms a carbon-carbon double bond.

[0191] In still other different embodiments of formula (II), for at least one occurrence of R 4a and R 4b , R 4a is H or C1-C 12 alkyl, and R 4b , together with the carbon atom to which it is attached and together with the adjacent R 4b and the carbon atom to which it is attached, forms a carbon-carbon double bond.

[0192] In more embodiments of formula (II), for at least one occurrence of R 2a and R 2b , R 2a is H or C1-C 12 alkyl, and R 2b , together with the carbon atom to which it is attached and together with the adjacent R 2b and the carbon atom to which it is attached, forms a carbon-carbon double bond.

[0193] In other different embodiments of formula (II), for at least one occurrence of R 3a and R 3b , R 3a is H or C1-C 12 alkyl, and R 3b , together with the carbon atom to which it is attached and together with the adjacent R 3b and the carbon atom to which it is attached, forms a carbon-carbon double bond.

[0194] In various other embodiments of formula (II), the lipid compound has one of the following structures (IIC) or (IID):

[0195]

[0196] wherein e, f, g, and h are each independently an integer from 1 to 12.

[0197] In some embodiments of formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0198] In various embodiments of structure (IIC) or (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0199] In certain embodiments of formula (II), a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or an integer from 5 to 9. In some specific embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In still other embodiments, a is 16.

[0200] In some embodiments of formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In still other embodiments, b is 16.

[0201] In some embodiments of formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In still other embodiments, c is 16.

[0202] In some specific embodiments of formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In still other embodiments, d is 16.

[0203] In some embodiments of formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In still other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In still other embodiments, e is 12.

[0204] In some embodiments of formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In still other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In still other embodiments, f is 12.

[0205] In some embodiments of formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In still other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In still other embodiments, g is 12.

[0206] In some embodiments of formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In still other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In still other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In still other embodiments, h is 12.

[0207] In some other various embodiments of formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same, and b and c are the same.

[0208] The sum of a and b and the sum of c and d in formula (II) are variable factors to obtain lipids with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In a further embodiment, the sum of a and b is the same as the sum of c and d. For example, in some embodiments, both the sum of a and b and the sum of c and d are the same integer that can be in the range of 14 to 24. In still more embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or greater.

[0209] R of formula (II) 1a , R 2a , R 3a and R 4a There are no particular restrictions on the substituents at. In some embodiments, at least one of R 1a , R 2a , R 3a and R 4a is H. In certain embodiments, each occurrence of R 1a , R 2a , R 3a and R 4a is H. In certain other embodiments, at least one of R 1a , R 2a , R 3a and R 4a is C1-C 12 alkyl. In certain other embodiments, at least one of R 1a , R 2a , R 3a and R 4a is C1-C8 alkyl. In certain other embodiments, at least one of R 1a , R 2a , R 3a and R4a At least one of them is a C1-C6 alkyl group. In some of the foregoing embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0210] In certain embodiments of formula (II), R 1a , R 1b , R 4a and R 4b are each a C1-C 12 alkyl group upon each occurrence.

[0211] In a further embodiment of formula (II), at least one of R 1b , R 2b , R 3b and R 4b is H, or R 1b , R 2b , R 3b and R 4b are each H upon each occurrence.

[0212] In certain embodiments of formula (II), R 1b together with the carbon atom to which it is attached and together with the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond. In other of the foregoing embodiments, R 4b together with the carbon atom to which it is attached and together with the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond.

[0213] In the foregoing embodiments, the substituents at R 5 and R 6 of formula (II) are not particularly limited. In certain embodiments, one of R 5 or R 6 is methyl. In other embodiments, each of R 5 or R 6 is methyl.

[0214] In the foregoing embodiments, the substituent at R 7 of formula (II) is not particularly limited. In certain embodiments, R 7 is a C6-C 16 alkyl group. In some other embodiments, R 7 is a C6-C9 alkyl group. In some of these embodiments, R 7 is substituted with -(C=O)OR b , -O(C=O)R b , -C(=O)R b , -OR b , -S(O)x R b 、-S-SR b 、-C(=O)SR b 、-SC(=O)R b 、-NR a R b 、-NR a C(=O)R b 、-C(=O)NR a R b 、-NR a C(=O)NR a R b 、-OC(=O)NR a R b 、-NR a C(=O)OR b 、-NR a S(O) x NR a R b 、-NR a S(O) x R b or -S(O) x NR a R b Substitution, where: R a H or C1-C 12 Alkyl; R b C1-C 15 alkyl; and x is 0, 1 or 2. For example, in some embodiments, R 7 -(C=O)OR b or -O(C=O)R b replace.

[0215] In various embodiments of the aforementioned formula (II), R b For branched chain C1-C 15 For example, in some embodiments, R b Has one of the following structures:

[0216]

[0217] In certain other embodiments of the foregoing formula (II), R 8 or R 9 In other embodiments, R 8 and R 9 All are methyl.

[0218] In some different embodiments of formula (II), R 8 and R 9Together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocyclic ring. In some of the foregoing embodiments, R 8 and R 9 Together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, such as a pyrrolidinyl ring. In some different foregoing embodiments, R 8 and R 9 Together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, such as a piperazinyl ring.

[0219] In still other embodiments of the foregoing lipid of formula (II), G 3 is a C2-C4 alkylene group, such as a C3 alkylene group.

[0220] In various different embodiments, the lipid compound has one of the following structures:

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] In some embodiments, the LNP comprises a lipid of formula (II), at least one reagent, and one or more excipients selected from neutral lipids, steroids, and polyethylene glycolated lipids. In some embodiments, the lipid of formula (II) is compound II-9. In some embodiments, the lipid of formula (II) is compound II-10. In some embodiments, the lipid of formula (II) is compound II-11. In some embodiments, the lipid of formula (II) is compound II-12. In some embodiments, the lipid of formula (II) is compound II-32.

[0228] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (III):

[0229]

[0230] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0231] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, and L 1 or L 2 Another one of is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0232] G 1 and G 2 Each independently is unsubstituted C1-C 12 alkylene or C1-C 12 alkenylene;

[0233] G 3 is C1-C 24 alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0234] R a is H or C1-C 12 alkyl;

[0235] R 1 and R 2 Each independently is C6-C 24 alkyl or C6-C 24 alkenyl;

[0236] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0237] R 4 is C1-C 12 alkyl;

[0238] R 5 is H or a C1-C6 alkyl group; and

[0239] x is 0, 1 or 2.

[0240] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0241]

[0242] wherein:

[0243] A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;

[0244] R 6 is independently, in each occurrence, H, OH or a C1-C 24 alkyl group;

[0245] n is an integer in the range of 1 to 15.

[0246] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0247] In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID)

[0248]

[0249] wherein y and z are each independently an integer in the range of 1 to 12.

[0250] In any of the foregoing embodiments of formula (III), L 1 or L 2 is one of -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each -O(C=O)-. In some of any of the foregoing different embodiments, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each -(C=O)O-.

[0251] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[0252]

[0253] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

[0254]

[0255] In some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0256] In some other foregoing embodiments of formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

[0257] In some of the foregoing embodiments of formula (III), R 6 is H. In other foregoing embodiments, R 6 is C1-C 24 alkyl. In other embodiments, R 6 is OH.

[0258] In some embodiments of formula (III), G 3 is unsubstituted. In other embodiments, G 3 is substituted. In various embodiments, G 3 is a straight-chain C1-C 24 alkylene or a straight-chain C1-C 24 alkenylene.

[0259] In some other foregoing embodiments of formula (III), R 1 or R 2 or both are C6-C 24 alkenyl. For example, in some embodiments, R 1 and R 2 each independently have the following structure:

[0260]

[0261] wherein:

[0262] R 7a and R 7b are each independently H or C1-C 12 alkyl upon each occurrence; and

[0263] a is an integer from 2 to 12

[0264] wherein R 7a 、R 7b and a are each selected such that R 1 and R 2 each independently contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0265] In some of the foregoing embodiments of formula (III), at least one occurrence of R 7a is H. For example, in some embodiments, R 7a is H at each occurrence. In other different foregoing embodiments, at least one occurrence of R 7b is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0266] In different embodiments of formula (III), R 1 or R 2 、or both have one of the following structures:

[0267]

[0268] In some of the foregoing embodiments of formula (III), R 3 is OH, CN, -C(=O)OR 4 、-OC(=O)R 4 or -NHC(=O)R 4 . In some embodiments, R 4 is methyl or ethyl.

[0269] In various different embodiments, the cationic lipid of formula (III) has one of the following structures:

[0270]

[0271]

[0272]

[0273]

[0274]

[0275] In some embodiments, the LNP comprises a lipid of formula (III), at least one reagent, and one or more excipients selected from neutral lipids, steroids, and pegylated lipids. In some embodiments, the lipid of formula (III) is Compound III-3. In some embodiments, the lipid of formula (III) is Compound III-7.

[0276] In certain embodiments, the cationic lipid is present in the LNP in an amount of from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only the cationic lipid.

[0277] In certain embodiments, the LNP comprises one or more additional lipids that stabilize the formation of the particles during particle formation.

[0278] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0279] The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalins, and cerebrosides.

[0280] Exemplary neutral lipids include, for example, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1,2-ditrans oleoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0281] In some embodiments, the LNPs comprise neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid (e.g., the lipid of formula (I)) to neutral lipid ranges from about 2:1 to about 8:1.

[0282] In various embodiments, the LNPs further comprise a steroid or steroid analog. "Steroid" is a compound that contains the following carbon skeleton:

[0283]

[0284] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of cationic lipid (e.g., the lipid of formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0285] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaroyl phosphatidylethanolamine, lysyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylglycerol (POPG), and other anionic modifying groups conjugated to neutral lipids.

[0286] In certain embodiments, the LNPs comprise glycolipids (e.g., monosialoganglioside GM1). In certain embodiments, the LNPs comprise a sterol, such as cholesterol.

[0287] In some embodiments, the LNPs comprise polymer-conjugated lipids. The term "polymer-conjugated lipid" refers to a molecule that contains a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycolylated lipid. The term "polyethylene glycolylated lipid" refers to a molecule that contains a lipid moiety and a polyethylene glycol moiety. Polyethylene glycolylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-s-DMG), etc.

[0288] In certain embodiments, the LNP comprises an additional stabilizing lipid, which is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 ) carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNP comprises PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0289] In some embodiments, the LNP comprises a PEGylated lipid having the following structure (IV):

[0290]

[0291] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0292] R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

[0293] the average value of z ranges from 30 to 60.

[0294] In some of the foregoing embodiments of the PEGylated lipid (IV), when z is 42, R10 and R 11 are not both n-octadecyl. In some other embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 10 to 18 carbon atoms. In some embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R 10 and R 11 are each independently a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R 10 is a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 12 carbon atoms, and R 11 is a straight-chain or branched-chain, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0295] In various embodiments, the choice of the span of z is such that the PEG portion of (II) has an average molecular weight of from about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0296] In other embodiments, the pegylated lipid has one of the following structures:

[0297]

[0298] where n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g / mol.

[0299] In certain embodiments, the amount of the additional lipid present in the LNP is from about 1 to about 10 mole percent. In one embodiment, the amount of the additional lipid present in the LNP is from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP at about 1 mole percent or about 1.5 mole percent.

[0300] In some embodiments, the LNP comprises a lipid of formula (I), a nucleoside-modified RNA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (I) is Compound I-6. In various embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still other embodiments, the pegylated lipid is Compound IVa.

[0301] In certain embodiments, the LNP comprises one or more targeting moieties that target the LNP to a stem cell or population of stem cells. For example, in one embodiment, the targeting domain is a ligand that directs the LNP to a receptor found on the surface of the stem cell.

[0302] Exemplary LNPs and their manufacture have been described in the art, such as in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7):1357-1364; Basha et al., 2011, Mol Ther, 19(12):2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34):18440-18450; Lee et al., 2012, IntJ Cancer., 131(5):E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1:e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, MolTher Nucleic Acids.2, e139; Maier et al., 2013, Mol Ther., 21(8):1570-1578; and Tam et al., 2013, Nanomedicine, 9(5):665-74, each of which is incorporated by reference in its entirety.

[0303] The following reaction schemes illustrate methods for preparing lipids of formula (I), (II), or (III).

[0304] General Reaction Scheme 1

[0305]

[0306] Embodiments of the lipid of formula (I) (e.g., Compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), where R is a saturated or unsaturated C1-C24 An alkyl group or a saturated or unsaturated cycloalkyl group, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, the compound of structure A-1 can be purchased from a commercial source or prepared according to methods familiar to those of ordinary skill in the art. Treat a mixture of A-1, A-2 and DMAP with DCC to obtain bromide A-3. Heat a mixture of bromide A-3, a base (such as N,N-diisopropylethylamine) and N,N-dimethyldiamine A-4 at a certain temperature and for a certain time to produce A-5 after any necessary work-up and / or purification steps.

[0307] General Reaction Scheme 2

[0308]

[0309] Other embodiments of the compound of formula (I) (such as compound B-5) can be prepared according to General Reaction Scheme 2 ("Method B"), wherein R is a saturated or unsaturated C1-C 24 An alkyl group or a saturated or unsaturated cycloalkyl group, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, the compound of structure B-1 can be purchased from a commercial source or prepared according to methods familiar to those of ordinary skill in the art. Treat a solution of B-1 (1 equivalent) with an acid chloride B-2 (1 equivalent) and a base (such as triethylamine). Treat the crude product with an oxidizing agent (such as pyridinium chlorochromate) and recover the intermediate B-3. Then treat a solution of the crude B-3, an acid (such as acetic acid) and N,N-dimethylaminoamine B-4 with a reducing agent (such as sodium triacetoxyborohydride) to obtain B-5 after any necessary work-up and / or purification.

[0310] It should be noted that although the starting materials A-1 and B-1 depicted above only include saturated methylene carbons, starting materials including carbon-carbon double bonds can also be used to prepare compounds including carbon-carbon double bonds.

[0311] General Reaction Scheme 3

[0312]

[0313] Different embodiments of the lipid of formula (I) (such as compound C-7 or C9) can be prepared according to General Reaction Scheme 3 ("Method C"), wherein R is a saturated or unsaturated C1-C 24 An alkyl group or a saturated or unsaturated cycloalkyl group, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, the compound of structure C-1 can be purchased from a commercial source or prepared according to methods familiar to those of ordinary skill in the art.

[0314] General Reaction Scheme 4

[0315]

[0316] Embodiments of compounds of formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), where R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 3b 、R 4a 、R 4b 、R 5 、R 6 、R 8 、R 9 、L 1 、L 2 、G 1 、G 2 、G 3 、a, b, c, and d are as defined herein, and R 7’ represents R 7 or C3-C 19 alkyl. Referring to General Reaction Scheme 1, compounds of structures D-1 and D-2 can be purchased from commercial sources or prepared by methods familiar to those of ordinary skill in the art. Treating a solution of D-1 and D-2 with a reducing agent (e.g., sodium triacetoxyborohydride) gives D-3 after any necessary workup. Treating a solution of D-3 and a base (e.g., trimethylamine, DMAP) with an acyl chloride D-4 (or a carboxylic acid and DCC) gives D-5 after any necessary workup and / or purification. D-5 can be reduced with LiAlH4 to D-7 after any necessary workup and / or purification.

[0317] General Reaction Scheme 5

[0318]

[0319] Embodiments of lipids of formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), where R 1a 、R 1b 、R 2a 、R 2b 、R 3a 、R 3b 、R 4a 、R 4b 、R 5 、R 6 、R 7 、R 8 、R 9 、L 1 、L 2 、G 3, a, b, c, and d are as defined herein. Referring to General Reaction Scheme 2, the compounds of structures E-1 and E-2 can be purchased from commercial sources or prepared according to methods familiar to those of ordinary skill in the art. Heating a mixture of E-1 (in excess), E-2, and a base (such as potassium carbonate) gives E-3 after any necessary workup. Treating a solution of E-3 and a base (such as trimethylamine, DMAP) with an acyl chloride E-4 (or a carboxylic acid and DCC) gives E-5 after any necessary workup and / or purification.

[0320] General Reaction Scheme 6

[0321]

[0322] General Reaction Scheme 6 provides an exemplary method (Method F) for preparing the lipid of formula (III). G in General Reaction Scheme 6 1 , G 3 , R 1 , and R 3 are as defined herein for formula (III), and G1’ refers to a homolog of G1 that is one carbon shorter. The compound of structure F-1 is purchased or prepared according to methods known in the art. Reacting F-1 with the diol F-2 under suitable condensation conditions (such as DCC) gives the ester / alcohol F-3, which can then be oxidized (such as with PCC) to the aldehyde F-4. The reaction of F-4 with the amine F-5 under reductive amination conditions gives the lipid of formula (III).

[0323] It should be noted that those of ordinary skill in the art can employ various alternative strategies to prepare the lipid of formula (III). For example, other lipids of formula (III) in which L 1 and L 2 are not esters can be prepared using appropriate starting materials according to similar methods. Additionally, General Reaction Scheme 6 depicts the preparation of the lipid of formula (III) in which G 1 and G 2 are the same; however, this is not an essential aspect of the invention, and the above reaction scheme can be modified to produce compounds in which G 1 and G 2 are different.

[0324] Those skilled in the art will appreciate that in the methods described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino, and guanidino include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto include -C(O)-R″ (where R″ is alkyl, aryl, or aralkyl), p-methoxybenzyl, trityl, etc. Suitable protecting groups for carboxylic acid include alkyl esters, aryl esters, or aralkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. Those skilled in the art will appreciate that the protecting group can also be a polymeric resin, such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0325] Reagent

[0326] In one embodiment, the delivery medium comprises at least one nucleic acid. In various embodiments, the nucleic acid is mRNA, self-replicating RNA, siRNA, miRNA, antisense oligonucleotide, DNA, DNA-RNA hybrid, gene editing component. Gene editing components include, but are not limited to, guide RNA, tracr RNA, sgRNA, mRNA encoding an RNA-guided nuclease, gene or base editing protein, zinc finger nuclease, Talen, CRISPR nuclease (such as Cas9), nucleic acid molecule to be inserted or used as a repair template, etc., or a combination thereof. In some embodiments, the mRNA encodes a gene-editing or base-editing protein. In some embodiments, the nucleic acid is a guide RNA.

[0327] In some embodiments, the LNP comprises a gene- or base-editing protein-encoding mRNA and one or more guide RNAs. The CRISPR nuclease may have altered activity, e.g., modifying the nuclease to be a nickase rather than making a double-stranded cut, or to bind to the sequence specified by the guide RNA but without enzymatic activity. Base-editing proteins are typically fusion proteins comprising a deaminase domain and a sequence-specific DNA-binding domain (such as an inactive CRISPR nuclease). In alternative embodiments, the LNP or nanoparticle does not comprise an mRNA encoding an RNA-guided nuclease and a guide RNA, but instead comprises a ribonucleoprotein, i.e., a complex comprising a guide RNA bound to an RNA-guided nuclease. In other embodiments, the nanoparticle comprises an RNA and a reverse transcriptase.

[0328] In some embodiments, the RNA molecule encodes a part of the TtAgo system. In eukaryotes, gene silencing is mediated by the Argonaute (Ago) protein family. In this paradigm, Ago binds to small (19-31 nt) RNAs. This protein-RNA silencing complex recognizes the target RNA through Watson-Crick base pairing between the small RNA and the target, and cleaves the target RNA by endonuclease. Ago-RNA-mediated DNA cleavage can be used to achieve various outcomes, including gene knockout using standard techniques in the art for exploiting DNA breaks.

[0329] In some embodiments, the RNA molecule encodes a cleavage domain that is operably linked to a DNA-binding domain to form a nuclease. For example, the ZFP DNA-binding domain has been fused to a nuclease domain to create ZFN—a functional entity that is capable of recognizing its intended nucleic acid target through its engineered (ZFP) DNA-binding domain and promoting DNA cleavage near the ZFP binding site through nuclease activity, including for genome modification in various organisms. Similarly, the TALE DNA-binding domain has been fused to a nuclease domain to construct TALEN.

[0330] As noted above, the cleavage domain can be heterologous to the DNA-binding domain, e.g., a zinc finger DNA-binding domain and a cleavage domain from a nuclease, or a TALEN DNA-binding domain and a cleavage domain, or a meganuclease DNA-binding domain and a cleavage domain from a different nuclease. The heterologous cleavage domain can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. Additional enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). One or more of these enzymes (or functional fragments thereof) can be used as a source of the cleavage domain and cleavage half-domains.

[0331] Similarly, the cleavage half-domains can be derived from any nuclease or part thereof as given above that requires dimerization to produce a cleavage activity. Generally, if the fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, an RNA molecule encoding a single protein containing two cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragment thereof). In addition, in some embodiments, the target sites of the two fusion proteins are positioned relative to each other such that binding of the two fusion proteins to their respective target sites positions the cleavage half-domains in a spatial orientation with respect to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerization. Thus, in certain embodiments, the proximal edges of the target sites are separated by 5-8 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or nucleotide pairs can intervene between the two target sites (e.g., 2 to 50 nucleotide pairs or more). Generally, the cleavage site is located between the target sites.

[0332] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at the recognition site) and cleaving the DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site distant from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes double-stranded DNA cleavage 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. Thus, in one embodiment, the RNA molecule of the invention can encode a fusion protein that comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, and the fusion protein can be or can not be engineered.

[0333] An exemplary type IIS restriction enzyme, whose cleavage domain can be separated from the binding domain, is FokI. This particular enzyme is active as a dimer. Thus, for the purposes of the present disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-strand cleavage and / or targeted replacement of cellular sequences using a zinc finger-FokI fusion, two fusion proteins (each containing a FokI cleavage half-domain) can be used to reconstitute the catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used.

[0334] The cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or the ability to oligomerize (e.g., dimerize) to form a functional cleavage domain. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure.

[0335] In some embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization.

[0336] In some embodiments, so-called "split-enzyme" technology can be used to assemble nucleases in vivo at a nucleic acid target site. The components of such split-enzymes can be encoded by separate RNA molecules. The components can be separate zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.

[0337] The Cas9-related CRISPR / Cas system comprises two non-coding RNA components: tracrRNA and a pre-crRNA array, which contains nuclease guide sequences (spacers) separated by identical direct repeats (DRs). For genome engineering using the CRISPR / Cas system, both functions of these RNAs must be present. In some embodiments, tracrRNA and pre-crRNA are supplied by separate expression constructs or as separate RNAs. In other embodiments, a chimeric RNA is constructed, where an engineered mature crRNA (conferring target specificity) is fused to tracrRNA (supplying interaction with Cas9) to form a chimeric cr-RNA-tracrRNA hybrid (also referred to as a single guide RNA).

[0338] As described in detail above, the DNA-binding domain can be engineered to bind any selected sequence. Compared to a naturally occurring DNA-binding domain, an engineered DNA-binding domain can have a new binding specificity.

[0339] In one aspect, the delivery vehicle can contain a vector that includes the nucleotide sequence or construct to be delivered. The choice of vector will depend on the host cell into which it is to be subsequently introduced. In certain embodiments, the vectors of the present invention are expression vectors. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a specific embodiment, the expression vectors are selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic and / or eukaryotic vector-based systems can be used in the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are commercially available and widely used.

[0340] For example, the vector into which the nucleic acid sequence is introduced can be a plasmid, which may or may not integrate into the genome of the host cell when introduced into the cell. Exemplary, non-limiting examples of vectors into which the nucleotide sequences of the present invention or the gene constructs of the present invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0341] Vectors can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0342] In one embodiment, the recombinant expression vector can also contain a nucleic acid molecule encoding a peptide or peptidomimetic.

[0343] The promoter can be a promoter naturally associated with the gene or polynucleotide sequence and can be obtained by isolating the 5' non-coding sequence located upstream of the coding region and / or exon. Such promoters can be referred to as "endogenous". Similarly, the enhancer can be an enhancer naturally associated with the polynucleotide sequence, located downstream or upstream of the sequence. Alternatively, certain advantages can be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the polynucleotide sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with the polynucleotide sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR TM ) can be used to generate the sequences (U.S. Patent 4,683,202, U.S. Patent 5,928,906). In addition, control sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. are contemplated.

[0344] Naturally, it will be important to employ promoters and / or enhancers that effectively direct the expression of DNA segments in the cell types, organelles, and organisms selected for expression. Those skilled in the art of molecular biology generally know how to use promoter, enhancer, and cell type combinations for protein expression, see, e.g., Sambrook et al. (2012). The promoters employed can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is beneficial for large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0345] The recombinant expression vector can also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes include genes encoding proteins conferring resistance to certain drugs (such as G418 and hygromycin), β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof (such as the Fc portion of an immunoglobulin, including but not limited to IgG). The selectable marker can be introduced on a vector different from the nucleic acid of interest.

[0346] Any polynucleotide can be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, adding flanking sequences at the 5' and / or 3' ends; using phosphorothioate or 2'-O-methyl in the backbone instead of phosphodiester bonds; and / or incorporating non-conventional bases, such as inosine, wybutosine, and queuosine, as well as acetyl-methyl-, thio-, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0347] In one embodiment of the invention, ribozymes are used as therapeutic agents to inhibit the expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule can be designed by incorporating the target sequence into a basic ribozyme structure, e.g., the basic ribozyme structure is complementary to the mRNA sequence encoding the target molecule. Ribozymes targeting a target molecule can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or can be expressed from DNA encoding them.

[0348] In one embodiment, the therapeutic agent can comprise one or more components of the CRISPR-Cas system, wherein a guide RNA (gRNA) targeting the gene encoding the target molecule and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0349] RNA molecules can be synthesized to include modifications that confer desired properties. For example, the modifications can improve stability, hybridization thermodynamics with target nucleic acids, targeting of specific tissues or cell types, or cellular permeability, e.g., by endocytosis-dependent or -independent mechanisms.

[0350] The modifications can also increase sequence specificity and thereby reduce off-targeting. Methods of synthesis and chemical modification are described in more detail below. If desired, the RNA molecules can be modified to stabilize the RNA against degradation, extend the half-life, or increase efficacy. For example, the desired modifications are described in U.S. Patent Publication Nos. 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, each of which is incorporated herein by reference in its entirety. To increase nuclease resistance and / or binding affinity for the target, the single-stranded oligonucleotide reagents described in this disclosure can include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acids), and certain nucleotide modifications can also increase binding affinity for the target. Inclusion of pyranosyl sugars in the oligonucleotide backbone can also reduce endonuclease cleavage. The oligonucleotide can be further modified by including a 3' cationic group or by inverting the nucleoside at the 3'-terminus with a 3-3' bond. In another alternative, the 3'-terminus can be blocked with an aminoalkyl. Other 3' conjugates can inhibit 3'-5' exonuclease cleavage. Although not bound by theory, the 3' can inhibit exonuclease cleavage by sterically blocking the exonuclease from binding to the 3'-end of the oligonucleotide. Even small alkyl chains, aryl or heterocyclic conjugates, or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonuclease.

[0351] In one embodiment, the RNA includes a 2'-modified oligonucleotide containing an oligodeoxynucleotide gap, wherein some or all of the internucleotide linkages are modified to phosphorothioates to achieve nuclease resistance. The presence of methylphosphonate modifications increases the oligonucleotide's affinity for its target RNA and thereby reduces the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It should be understood that the methods and reagents of this disclosure can be used in combination with any technology that may be developed to enhance the stability or efficacy of nucleic acid molecules.

[0352] RNA molecules include nucleotide oligomers containing modified backbones or unnatural internucleoside linkages. Oligomers with modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphonates, amino phosphates, phosphoramidates, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates. Also included are various salt, mixed salt, and free acid forms.

[0353] In some examples, the RNA composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80%, 50%, 30%, 20%, or 10% water). In another example, the RNA composition is in an aqueous phase, e.g., in a solution comprising water. The aqueous phase or crystalline composition can be incorporated into a delivery vehicle such as a liposome (especially for the aqueous phase) or a particle (e.g., a microparticle that can be suitable for the crystalline composition). Generally, the RNA composition is formulated in a manner compatible with the intended method of administration. The RNA composition can be formulated in combination with another reagent (e.g., another therapeutic reagent or a reagent that stabilizes an oligonucleotide reagent, such as a protein complexed with an oligonucleotide reagent). Other reagents include chelating agents, e.g., EDTA (e.g., for removing divalent cations such as Mg), salts, and RNase inhibitors (e.g., broad-spectrum specific RNase inhibitors). In one embodiment, the RNA composition includes two or more RNA molecules, e.g., a second sgRNA composition (e.g., a different sgRNA from the first). Other formulations can include at least three, five, ten, twenty, fifty, or one hundred or more different oligonucleotide species.

[0354] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of an sgRNA.

[0355] The oligonucleotides selected for inclusion in the compositions of the present invention can be of one of a variety of lengths. The length of such oligonucleotides can be from 7 to 100 linked nucleosides. For example, the length of an oligonucleotide sharing nucleobase identity with an miRNA can be from 7 to 30 linked nucleosides. The length of an oligonucleotide sharing identity with an miRNA precursor can be up to 100 linked nucleosides. In certain embodiments, the oligonucleotide comprises from 7 to 30 linked nucleosides. In certain embodiments, the oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, the oligonucleotide comprises from 19 to 23 linked nucleosides. In certain embodiments, the length of the oligonucleotide is from 40 to up to 50, 60, 70, 80, 90, or 100 linked nucleosides.

[0356] In vitro transcribed RNA

[0357] In one embodiment, the compositions of the present invention comprise in vitro transcribed (IVT) RNA. In one embodiment, the compositions of the present invention comprise in vitro transcribed (IVT) RNA encoding a gene editing molecule. In one embodiment, the compositions of the present invention comprise IVT RNA encoding a gene editing molecule and a guide RNA molecule.

[0358] In one embodiment, the IVT RNA can be incorporated into the delivery vehicle of the present invention. RNA is produced by in vitro transcription using a synthetically generated plasmid DNA template. DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis using appropriate primers and an RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source.

[0359] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence of an organism's genome. In one embodiment, the DNA is a full-length target gene that is part of a gene. The gene can include partial or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene that includes 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or symbiotic organism (including bacteria, viruses, parasites, and fungi). In another embodiment, the DNA to be used for PCR from a pathogenic or symbiotic organism (including bacteria, viruses, parasites, and fungi) includes 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a sequence containing gene segments ligated together to form an open reading frame encoding a fusion protein. The ligated DNA segments can be from a single organism, or from multiple organisms.

[0360] In various embodiments, plasmids are used to generate templates for in vitro transcription of RNA, which is incorporated into LNPs or delivery vehicles.

[0361] Chemical structures having the ability to promote stability and / or translation efficiency can also be used. In some embodiments, the RNA has 5' and 3' UTRs. In one embodiment, the length of the 5' UTR is between 0 and 3000 nucleotides. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this method, one of ordinary skill in the art can modify the desired 5' and 3' UTR lengths to achieve optimal translation efficiency of the transcribed RNA.

[0362] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs for the target gene. Alternatively, UTR sequences that are non-endogenous to the target gene can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are non-endogenous to the target gene can be useful for modifying the stability and / or translation efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to reduce RNA stability. Thus, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0363] In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when adding a 5'UTR that is not endogenous to the gene of interest as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but it does not seem that all RNAs require it for efficient translation. Many RNAs are known in the art to require the Kozak sequence. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonucleolytic degradation of the RNA.

[0364] To be able to synthesize RNA from a DNA template without gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When the sequence to be used as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 RNA polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0365] In one embodiment, the RNA has a cap at the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and stability of the mRNA in cells. On circular DNA templates such as plasmid DNA, RNA polymerase produces long concatemer products that are not suitable for expression in eukaryotic cells. Transcription of linearized plasmid DNA at the 3'UTR end produces normal-sized RNA, which is effective in eukaryotic transfection when polyadenylated after transcription.

[0366] On a linear DNA template, bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0367] The conventional method for integrating a polyA / T stretch into a DNA template is molecular cloning. However, the polyA / T sequence integrated into plasmid DNA may cause plasmid instability, which can be improved by using recombination-deficient bacterial cells for plasmid propagation.

[0368] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase such as Escherichia coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 to 400 nucleotides results in approximately a two-fold increase in the translation efficiency of the RNA. In addition, attaching different chemical groups at the 3' end can increase RNA stability. Such attachments can contain modified / artificial nucleotides, aptamers, and other compounds. For example, an ATP analogue can be incorporated into the poly(A) tail using a poly(A) polymerase. The ATP analogue can further increase the stability of the RNA.

[0369] The 5' cap also provides stability to the RNA molecule. In one embodiment, the RNA produced by this method includes a 5' cap1 structure. Such cap1 structures can be generated using vaccinia capping enzymes and 2'-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, techniques known in the art and described herein are used to provide the 5' cap (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0370] Nucleoside-modified RNA

[0371] In one embodiment, the compositions of the present invention comprise nucleoside-modified nucleic acids. In one embodiment, the compositions of the present invention comprise nucleoside-modified RNA, guide RNA, or a combination thereof encoding a gene editing protein.

[0372] For example, in one embodiment, the composition comprises nucleoside-modified RNA. In one embodiment, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA has particular advantages over unmodified mRNA, including, for example, increased stability, lower or no innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, which is incorporated herein by reference in its entirety.

[0373] In certain embodiments, nucleoside-modified mRNAs do not activate any pathophysiological pathways, are translated very efficiently and almost immediately after delivery, and serve as templates for continuous protein production in vivo for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is less, and this makes it suitable for human therapy.

[0374] In some cases, expressing a protein by delivering the encoding mRNA has many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA-based vectors and virus-based vectors, mRNA does not carry the risk of being integrated into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein are measured within 15 to 30 minutes after in vivo injection of the encoding mRNA. In certain embodiments, using mRNA instead of protein also has many advantages. The half-life of proteins in circulation is usually short, so protein therapy requires frequent dosing, while mRNA provides a template for protein production for several days. The purification of proteins is problematic, and they may contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0375] In certain embodiments, the nucleoside-modified RNA contains the naturally occurring modified nucleoside pseudouridine. In certain embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, NucleicAcids Research39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0376] It has been demonstrated that the presence of modified nucleosides (including pseudouridine) in RNA inhibits their innate immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Also, protein-coding, in vitro transcribed RNA containing pseudouridine can be translated more efficiently compared to RNA lacking or containing other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it was shown that the presence of pseudouridine improved the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and attenuated the activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established, which is crucial for obtaining pseudouridine-containing RNA with excellent translation potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified, pseudourine-containing RNA encoding erythropoietin to mice and macaques resulted in a significant increase in serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0377] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or modified nucleosides. In certain embodiments, the composition comprises an isolated nucleic acid, wherein the nucleic acid comprises pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises a vector that comprises an isolated nucleic acid, wherein the nucleic acid comprises pseudouridine or a modified nucleoside.

[0378] In one embodiment, the nucleoside-modified RNA of the present invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0379] In one embodiment, the modified nucleoside is m 1 acp 3 Ψ (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the modified nucleoside is m 1Ψ(1-methylpseudouridine). In another embodiment, the modified nucleoside is Ψm(2'-O-methylpseudouridine). In another embodiment, the modified nucleoside is m 5 D(5-methyldihydrouridine). In another embodiment, the modified nucleoside is m 3 Ψ(3-methylpseudouridine). In another embodiment, the modified nucleoside is an unmodified pseudouridine moiety. In another embodiment, the modified nucleoside is a monophosphate, diphosphate or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0380] In another embodiment, the modified nucleoside in the nucleoside-modified RNA of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0381] In another embodiment, the modified nucleoside of the present invention is m 5 C(5-methylcytidine). In another embodiment, the modified nucleoside is m 5 U(5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 -methyladenosine). In another embodiment, the modified nucleoside is s 2 U(2-thiouridine). In another embodiment, the modified nucleoside is Ψ(pseudouridine). In another embodiment, the modified nucleoside is Um(2'-O-methyluridine).

[0382] In other embodiments, the modified nucleoside is m 1 A(1-methyladenosine); m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N 6 -methyladenosine); i 6 A(N 6 -isopentenyladenosine); ms 2 i6A(2-methylthio-N 6 -isopentenyladenosine); io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine); g 6A(N 6 -(glycinylcarbamoyl)adenosine); t 6 A(N 6 -(threonylcarbamoyl)adenosine); ms 2 t 6 A(2-methylthio-N 6 -(threonylcarbamoyl)adenosine); m 6 t 6 A(N 6 -methyl-N 6 -(threonylcarbamoyl)adenosine); hn 6 A(N 6 -(hydroxy-norvalylcarbamoyl)adenosine); ms 2 hn 6 A(2-methylthio-N 6 -(hydroxy-norvalylcarbamoyl)adenosine); Ar(p)(2'-O-ribosyladenosine(phosphate)); I(inosine); m 1 I(1-methylinosine); m 1 Im(1,2'-O-dimethylinosine); m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine); ac 4 C(N 4 -acetylcytidine); f 5 C(5-formylcytidine); m 5 Cm(5,2'-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine); k 2 C(lysidine); m 1 G(1-methylguanosine); m 2 G(N 2 -methylguanosine); m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine); m 2 Gm(N 2 ,2'-O-dimethylguanosine); m 2 2Gm(N 2 ,N 2, 2'-O-trimethylguanosine); Gr(p)(2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (under-modified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0 (7-cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G + (archiguanosine); D (dihydrouridine); m 5 Um (5,2'-O-dimethyluridine); s 4 U (4-thiouridine); m 5 s 2 U (5-methyl-2-thiouridine); s 2 Um (2-thio-2'-O-methyluridine); acp 3 U (3-(3-amino-3-carboxypropyl)uridine); ho 5 U (5-hydroxyuridine); mo 5 U (5-methoxyuridine); cmo 5 U (uridine 5-oxyacetic acid); mcmo 5 U (uridine 5-oxyacetic acid methyl ester); chm 5 U (5-(carboxyhydroxymethyl)uridine)); mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U (5-methoxycarbonylmethyluridine); mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U (5-methoxycarbonylmethyl-2-thiouridine); nm 5 s 2 U (5-aminomethyl-2-thiouridine); mnm 5 U (5-methylaminomethyluridine); mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U (5-methylaminomethyl-2-selenouridine); ncm 5 U (5-carbamoylmethyluridine); ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm 5 U (5-carboxymethylaminomethyluridine); cmnm 5Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine); m 6 2A(N 6 ,N 6 -dimethyladenosine); Im (2'-O-methylinosine); m 4 C(N 4 -methylcytidine); m 4 Cm(N 4 ,2'-O-dimethylcytidine); hm 5 C (5-hydroxymethylcytidine); m 3 U (3-methyluridine); cm 5 U (5-carboxymethyluridine); m 6 Am(N 6 ,2'-O-dimethyladenosine); m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine); m 2,7 G(N 2 ,7-dimethylguanosine); m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine); m 3 Um (3,2'-O-dimethyluridine); m 5 D (5-methyldihydrouridine); f 5 Cm (5-formyl-2'-O-methylcytidine); m 1 Gm (1,2'-O-dimethylguanosine); m 1 Am (1,2'-O-dimethyladenosine); τm 5 U (5-tauromethyluridine); τm 5 s 2 U (5-tauromethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac 6 A(N 6 -acetyladenosine).

[0383] In another embodiment, the nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above-mentioned modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above-mentioned modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above-mentioned modifications.

[0384] In another embodiment, 0.1% to 100% of the residues in the nucleoside-modified RNA of the present invention are modified (e.g., by the presence of pseudouridine or a modified nucleobase). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0385] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0386] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of the given nucleotide that is modified is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0387] In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0388] In another embodiment, the nucleoside-modified RNA of the present invention is translated more efficiently in cells than an unmodified RNA molecule having the same sequence. In another embodiment, the nucleoside-modified RNA exhibits an enhanced ability to be translated by target cells. In another embodiment, the translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, the translation is enhanced by a factor of 3-fold. In another embodiment, the translation is enhanced by a factor of 5-fold. In another embodiment, the translation is enhanced by a factor of 7-fold. In another embodiment, the translation is enhanced by a factor of 10-fold. In another embodiment, the translation is enhanced by a factor of 15-fold. In another embodiment, the translation is enhanced by a factor of 20-fold. In another embodiment, the translation is enhanced by a factor of 50-fold. In another embodiment, the translation is enhanced by a factor of 100-fold. In another embodiment, the translation is enhanced by a factor of 200-fold. In another embodiment, the translation is enhanced by a factor of 500-fold. In another embodiment, the translation is enhanced by a factor of 1000-fold. In another embodiment, the translation is enhanced by a factor of 2000-fold. In another embodiment, the factor is 10 - 1000-fold. In another embodiment, the factor is 10 - 100-fold. In another embodiment, the factor is 10 - 200-fold. In another embodiment, the factor is 10 - 300-fold. In another embodiment, the factor is 10 - 500-fold. In another embodiment, the factor is 20 - 1000-fold. In another embodiment, the factor is 30 - 1000-fold. In another embodiment, the factor is 50 - 1000-fold. In another embodiment, the factor is 100 - 1000-fold. In another embodiment, the factor is 200 - 1000-fold. In another embodiment, the translation is enhanced by any other significant amount or range of amounts.

[0389] Combination

[0390] In one embodiment, the composition of the present invention comprises a combination of the reagents described herein. In certain embodiments, the compositions comprising the combinations of reagents described herein cooperate to produce an effect (e.g., the Cas9 protein and the guide RNA cooperate to effect gene editing).

[0391] Compositions comprising a combination of reagents comprise the individual reagents in any suitable ratio. For example, in one embodiment, the composition comprises a 1:1 ratio of two individual reagents. However, the combination is not limited to any particular ratio. Instead, any ratio that is shown to be effective is encompassed.

[0392] Conjugation

[0393] In various embodiments of the present invention, the delivery medium is conjugated to a targeting domain. In some embodiments, the delivery medium is conjugated to a CD34 or CD4 targeting domain. Exemplary conjugation methods can include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic ("van der Waals") interactions. In one embodiment, the conjugation is a reversible conjugation such that upon exposure to certain conditions or chemical reagents, the delivery medium can dissociate from the targeting domain. In another embodiment, the conjugation is an irreversible conjugation such that under normal conditions, the delivery medium does not dissociate the targeting domain.

[0394] In some embodiments, the conjugation comprises a covalent bond between an activated polymer-conjugated lipid and the targeting domain. The term "activated polymer-conjugated lipid" refers to a molecule comprising a lipid moiety and a polymer moiety that has been activated by functionalizing the polymer-conjugated lipid with a first coupling group. In one embodiment, the activated polymer-conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer-conjugated lipid is an activated polyethylene glycolylated lipid. In one embodiment, the first coupling group is bound to the lipid moiety of the polyethylene glycolylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol moiety of the polyethylene glycolylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0395] The first coupling group and the second coupling group can be any functional groups known to those skilled in the art to form a covalent bond together, for example, under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of maleimide, N-hydroxysuccinimide (NHS) ester, carbodiimide, hydrazide, pentafluorophenyl (PFP) ester, phosphine, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, vinyl sulfone, α-haloacetyl, aryl azide, acyl azide, alkyl azide, diazirine, benzophenone, epoxide, carbonate, anhydride, sulfonyl chloride, cyclooctyne, aldehyde, and thiol. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of free amine (-NH2), free thiol (-SH), free hydroxyl (-OH), carboxylate ester, hydrazide, and alkoxyamine. In some embodiments, the first coupling group is a functional group reactive with a thiol, such as maleimide, pyridyl disulfide, or haloacetyl. In one embodiment, the first coupling group is maleimide.

[0396] In one embodiment, the second coupling group is a thiol group. Any method known to those skilled in the art can be used to attach the thiol group to the targeting domain. In one embodiment, the thiol group is present on a free cysteine residue. In one embodiment, the thiol group is revealed by reducing a disulfide bond on the targeting domain, for example, by reacting with 2-mercaptoethylamine. In one embodiment, the thiol group is attached by a chemical reaction, such as the reaction between a free amine and 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA).

[0397] In some embodiments, the polymer-conjugated lipid and the targeting domain are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves the reaction between a functional group with a 1,3-dipole (such as azide, nitrile oxide, nitrone, isocyanide, and alkyne) and an alkene or alkyne dipolarophile. Exemplary dipolarophiles include any strained cycloalkene and cycloalkyne known to those skilled in the art, including but not limited to cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, and bisarylazacyclooctynone.

[0398] Targeting domain

[0399] In one embodiment, the targeting domain specifically binds to a marker of the target cell type of interest. For example, in one embodiment, the targeting domain directs the agent to endothelial cells, immune cells, stem cells, or another specific target cell type of interest. Target cells / tissues that can be targeted include but are not limited to T cells, hematopoietic stem cells (HSCs), myeloid cells, and lung cells. In one embodiment, the targeted LNP of the present invention comprises a targeting domain that specifically binds to an antigen on the target cell type of interest.

[0400] Any cell can be modified using the compositions and methods of the present invention, including but not limited to prokaryotic or eukaryotic cells, such as bacterial cells, insect cells, yeast cells, fish cells, mammalian (including non-human mammalian) cells, and plant cells. In certain embodiments, the cell is an immune cell, such as a T-cell (e.g., CD4+, CD3+, CD8+, etc.), dendritic cell, B cell, etc. In other embodiments, the cell is a pluripotent, totipotent, or multipotent stem cell, such as an induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC, e.g., CD34+), embryonic stem cell, etc.

[0401] In some embodiments, the methods and compositions of the present invention provide materials useful for in situ editing of cells in a subject in need thereof. In some embodiments, the method includes directly delivering a composition (e.g., a gene editing molecule) to a tissue. In certain aspects, the tissue is a secretory tissue, such as the liver, while in other aspects, the tissue has a specific function, such as bone marrow or lung.

[0402] For example, in one embodiment, the LNP comprises a targeting domain that specifically binds to an antigen expressed on an immune cell. In one embodiment, the LNP comprises a targeting domain that specifically binds to an antigen expressed on a tumor cell. In one embodiment, the LNP comprises a targeting domain that specifically binds to an antigen expressed on a specific tissue type (e.g., a marker expressed on lung tissue).

[0403] In certain embodiments, the targeting domain binds to a cell surface molecule of a target cell of interest, thereby guiding the composition to the target cell. In one embodiment, the composition comprises a delivery vehicle conjugated to a targeting domain that binds to a cell surface molecule of a target cell of interest, thereby guiding the composition to the target cell.

[0404] For example, in various embodiments, for a composition targeting endothelial cells, the targeting domain binds to a molecule selected from the group consisting of, but not limited to, the following molecules: (ICAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, angiotensin-converting enzyme (ACE), aminopeptidase P (APP), plasma membrane vesicle protein-1 (PV1), P-selectin, VE-cadherin, cytokine receptors, plasma proteins, and microorganisms.

[0405] In some embodiments, the targeted delivery vehicle of the present invention comprises a targeting moiety that binds to a surface molecule of an immune cell, including but not limited to T cells (including cytotoxic T cells, helper T cells, regulatory T cells, and γδ T cells), natural killer (NK) cells, antigen-presenting cells, dendritic cells, B cells, or Langerhans cells. In some embodiments, the targeted delivery vehicle comprises a targeting moiety that binds to a T cell surface molecule. Exemplary targeting moieties useful for targeting T cells with the compositions of the present invention include, but are not limited to, anti-CD4, anti-CD-8, anti-CD5, anti-CD3, or anti-CD25 targeting ligands. Exemplary compositions and methods for in vivo targeting of T cells are described in WO2022 / 081694, WO2022 / 081699, and WO2022 / 081702, which are hereby incorporated by reference in their entirety.

[0406] In some embodiments, the targeted delivery vehicle of the present invention comprises a targeting moiety that binds to surface molecules of stem cells, including but not limited to adult stem cells, mesenchymal stem cells, or hematopoietic stem cells. Exemplary surface molecules of stem cells include but are not limited to CD34, CD117, CD90, CD133, CD105, ABCG2, bone morphogenetic protein receptor (BMPR), CD44, Sca-1, Thy-1, CD133, alkaline phosphatase, alpha-fetoprotein, CD70, CD105, CD73, Stro-1, SSEA-4, CD271, CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1, CD56, CD200, PODXL, CD13, CD29, CD44, and CD10. Exemplary compositions and methods for in vivo targeting of stem cells are described in International Patent Application No. PCT / US22 / 26933 and International Patent Application No. PCT / US22 / 26981, which are hereby incorporated by reference in their entirety.

[0407] However, the present invention is not limited to vehicles targeting endothelial cells, immune cells, or stem cells. Instead, the present invention encompasses a delivery vehicle that comprises a targeting domain that directs the vehicle to any specific target cell, such as mediated by binding of the targeting domain to a specific marker. In some embodiments, the vehicle targets a specific therapeutic site in need. For example, the targeting domain can specifically target an inflammatory site, or can target tumor cells or pathogens.

[0408] The targeting domain can comprise nucleic acids, peptides, antibodies, small molecules, organic molecules, inorganic molecules, glycans, sugars, hormones, etc., which target the particle to a site that particularly requires a therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein.

[0409] In some embodiments, the targeting domain can be copolymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain can be covalently attached to the composition comprising the delivery vehicle, such as by a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. The targeting domains of the present invention include but are not limited to antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0410] In one embodiment, the composition comprises a targeting domain that directs the delivery vehicle to CD34. In some embodiments, the targeting domain is an affinity ligand that specifically binds CD34.

[0411] In one embodiment, the composition comprises a targeting domain that directs the delivery vehicle to CD4. In some embodiments, the targeting domain is an affinity ligand that specifically binds to CD4.

[0412] Peptide

[0413] In one embodiment, the targeting domain of the invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a marker of a target cell type. In one embodiment, the targeting domain directs the vehicle to endothelial cells, immune cells, stem cells or another specific target cell type. For example, in one embodiment, the targeting domain directs the vehicle to CD34+-expressing stem cells. In one embodiment, the targeting domain directs the vehicle to CD4+-expressing T cells.

[0414] The peptides of the invention can be prepared by chemical methods. For example, the peptides can be synthesized by solid-phase techniques (Roberge J Y et al. (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. For example, automated synthesis can be accomplished using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0415] Alternatively, the peptides can also be prepared recombinantly or by cleavage from a longer polypeptide. The composition of the peptides can be confirmed by amino acid analysis or sequencing.

[0416] Variants of the peptides according to the invention can be (i) variants in which one or more of the amino acid residues are replaced with conservative or non-conservative amino acid residues and such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code, (ii) variants in which there are one or more modified amino acid residues (e.g., residues modified by attachment of substituents), (iii) variants in which the peptide is an alternative splicing variant of the peptide of the invention, (iv) fragments of the peptide and / or (v) variants in which the peptide is fused to another peptide such as a leader sequence or a secretion sequence or a sequence for purification (e.g., His-tag) or a sequence for detection (e.g., Sv5 epitope tag). Fragments include peptides produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The variants can be post-translationally modified or chemically modified. Such variants are considered to be within the scope of those skilled in the art according to the teachings herein.

[0417] As is known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conservative amino acid substitutions with the sequence of a second peptide. Variants are defined as including peptide sequences that differ from the original sequence. In some embodiments, the variant has less than 40% residue differences from the original sequence in each target segment and retains the function of the original sequence at the same time. In some embodiments, the variant has less than 25% residue differences from the original sequence in each target segment and retains the function of the original sequence at the same time. In some embodiments, the variant has less than 10% residue differences from the original sequence in each target segment and retains the function of the original sequence at the same time. In some embodiments, the variant differs from the original sequence by only a few residues in each target segment and retains the function of the original sequence at the same time. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. Computer algorithms and methods well known to those skilled in the art are used to determine the degree of identity between two peptides. In some embodiments, the BLASTP algorithm is used to determine the identity between two amino acid sequences [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0418] The peptides of the present invention can be post-translationally modified. For example, post-translational modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, prenylation, proteolysis, myristoylation, protein folding, and proteolytic processing, etc. Some modification or processing events require the introduction of additional biomachinery. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding canine microsomal membranes or Xenopus laevis oocyte extracts (U.S. Patent No. 6,103,489) to a standard translation reaction.

[0419] The peptides of the present invention can include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0420] Nucleic acid

[0421] In one embodiment, the targeting domain of the present invention comprises isolated nucleic acids, including, for example, DNA oligonucleotides and RNA oligonucleotides. In certain embodiments, the nucleic acid targeting domain specifically binds to CD34. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to CD4.

[0422] The nucleotide sequence of the nucleic acid targeting domain may alternatively comprise sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid is capable of performing the original function and specifically binding to the target cell.

[0423] For the purposes used in this specification, a nucleotide sequence is "substantially homologous" to any nucleotide sequence described herein when the nucleotide sequence has a degree of identity of at least 60%, at least 70%, at least 85%, or at least 95% relative to any nucleotide sequence described herein. Examples of other possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides at either end of the sequence, or the deletion of one or more nucleotides at either end or within the sequence. Computer algorithms and methods well known to those skilled in the art are used to determine the degree of identity between two polynucleotides. In some embodiments, the identity between two nucleotide sequences is determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0424] Antibody

[0425] In one embodiment, the targeting domain of the invention comprises an antibody or an antibody fragment. In certain embodiments, the antibody targeting domain specifically binds to CD34 or CD4. Such antibodies include polyclonal antibodies, monoclonal antibodies, their Fab and single-chain Fv (scFv) fragments, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0426] The antibody can be a complete monoclonal or polyclonal antibody, as well as immunoreactive fragments (such as Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778), or chimeric antibodies, such as antibodies that contain the binding specificity of a murine antibody but the remainder of which is of human origin. Methods known to those skilled in the art can be used to prepare antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras.

[0427] Such antibodies can be produced in a variety of ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. The choice of manufacturing method depends on several factors, including the desired antibody structure, the importance of the carbohydrate moiety to the antibody, the ease of culture and purification, and cost. Many different antibody structures can be generated using standard expression techniques, including full-length antibodies, antibody fragments (such as Fab and Fv fragments), and chimeric antibodies containing components from different species. Small-sized antibody fragments, such as Fab and Fv fragments, may be produced in bacterial expression systems with no effector function and limited pharmacokinetic activity. Single-chain Fv fragments exhibit lower immunogenicity.

[0428] Therapeutic method

[0429] In some embodiments, the present invention provides methods for the cell-targeted delivery of a therapeutic agent to treat a disease or disorder in a subject.

[0430] In some embodiments, the present invention provides methods for the targeted delivery of a gene editing agent to treat a disease or disorder in a subject. In certain embodiments, the method is used to treat or prevent a disease or disorder in a subject. Exemplary diseases or disorders that can be treated using the methods of the present invention include, but are not limited to, blood monogenic disorders, genetic defects, bone marrow genetic defects, cancer, autoimmune diseases, and infectious diseases.

[0431] Exemplary genetic disorders that can be treated using the compositions and methods of the present invention include, but are not limited to, achondroplasia, α-1 antitrypsin deficiency, antiphospholipid syndrome, attention deficit hyperactivity disorder, autism, autosomal dominant polycystic kidney disease, breast cancer, Charcot-Marie-Tooth disease, colon cancer, cri du chat syndrome, Crohn's disease, cystic fibrosis, Duane syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, inborn errors of metabolism, Klinefelter syndrome, Marfan syndrome, methylmalonic aciduria, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, velo-cardio-facial syndrome, and Wilson disease.

[0432] Infectious diseases that can be treated using the compositions and methods include, but are not limited to, bacterial infections, viral infections, parasitic infections, and fungal infections.

[0433] Bacterial infections

[0434] In one embodiment, the infectious disease or disorder is associated with bacteria. In some embodiments, the bacteria can be from any of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Syntrophobacterales, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0435] The bacteria can be Gram-positive bacteria or Gram-negative bacteria. The bacteria can be aerobic bacteria or anaerobic bacteria. The bacteria can be autotrophic bacteria or heterotrophic bacteria. The bacteria can be mesophilic, neutrophilic, polarophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or osmophilic.

[0436] The bacteria can be Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. The bacteria can be Mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0437] Viral infection

[0438] In one embodiment, the infectious disease or disorder is associated with a virus. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen can be from human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomavirus (e.g., human papillomavirus (HPV)), poliovirus, hepatitis virus (e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV)), smallpox virus (variola major and variola minor), vaccinia virus, influenza virus, rhinovirus, equine encephalitis virus, rubella virus, yellow fever virus, norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), varicella zoster (chickenpox - shingles, also known as varicella), cytomegalovirus (CMV) (e.g., human CMV), Epstein - Barr virus (EBV), flavivirus, foot - and - mouth disease virus, Lassa virus, arenavirus, severe acute respiratory syndrome - related coronavirus (SARS), Middle East respiratory syndrome - related coronavirus (MERS), severe acute respiratory syndrome - related coronavirus 2 (SARS CoV 2), or oncogenic virus.

[0439] Parasitic infection

[0440] In one embodiment, the infectious disease or disorder is associated with a parasite. In some embodiments, the parasite can be a protozoan, a worm, or an ectoparasite. Worms (i.e., helminths) can be flatworms (e.g., flukes and tapeworms), acanthocephalans, or roundworms (e.g., pinworms). Ectoparasites can be lice, fleas, ticks, and mites.

[0441] The parasite can be any parasite that causes any of the following diseases: Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiasis, baylisascariasis, Chagas disease, clonorchiasis, screw-worm fly disease, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, katayama fever, leishmaniasis, Lyme disease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0442] The parasite can be Acanthamoeba, Anisakis, Ascaris, botfly, Balantidium coli, bedbug, cestode (tapeworm), chigger, screw-worm fly (Cochliomyia hominivorax), Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatula serrata, liver fluke, Loa loa, Paragonimus - lung fluke, pinworm, Plasmodium falciparum, schistosome, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, trypanosome, whipworm, or Wuchereria bancrofti.

[0443] Fungal infection

[0444] In one embodiment, the infectious disease or condition is associated with a fungus. In some embodiments, the fungus can be an Aspergillus species, Blastomyces dermatitidis, Candida yeast (e.g., Candida albicans), Coccidioides immitis, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0445] The present invention encompasses the delivery of a delivery medium that comprises at least one gene editing reagent for editing at least one disease-related gene in a subject in need thereof. In one embodiment, the gene is related to cellular entry of a bacterium, virus, fungus, or parasitic pathogen. In some embodiments, the gene is related to a genetic disease or condition.

[0446] In one embodiment, the delivery medium further comprises at least one targeting domain. To practice the methods of the present invention; based on the disclosure provided herein, those skilled in the art will know how to formulate an appropriate composition and administer it to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0447] The present invention also provides a method for delivering at least one reagent to a subject in need thereof in a cell-specific manner. In some embodiments, the reagent is a therapeutic reagent for treating a disease or disorder. In some embodiments, the disease or disorder is a genetic defect. In some embodiments, the method comprises administering to a target cell (e.g., a stem cell) at least one reagent for gene editing to treat a genetic defect. In some embodiments, the method comprises administering to a target cell (e.g., a stem cell) at least one reagent for gene editing to treat or prevent a disease or disorder associated with an infectious agent.

[0448] In some embodiments, the method comprises administering to a T cell, a hematopoietic stem cell, or a combination thereof at least one reagent for gene editing to treat or prevent HIV or a disease or disorder associated with HIV infection (e.g., AIDS). In one embodiment, the at least one reagent for gene editing is Cas9 mRNA, a guide RNA, or a combination thereof. In one embodiment, the guide RNA specifically binds to CCR5.

[0449] Those skilled in the art will understand, upon learning of the present disclosure (including the methods detailed herein), that the present invention is not limited to treating a disease or disorder that has been identified. In particular, the disease or disorder need not have reached a point where it is causing harm to the subject; in fact, it is not necessary to detect the disease or disorder in the subject prior to treatment. That is, the overt signs or symptoms of the disease or disorder need not be present before the present invention can provide a benefit. Accordingly, the present invention includes a method for preventing a disease or disorder, which comprises administering to a subject a composition as discussed elsewhere hereinbefore, prior to the onset of the disease or disorder, thereby preventing the disease or disorder.

[0450] Those skilled in the art will understand, upon learning of the present disclosure herein, that preventing a disease or disorder encompasses administering a composition to a subject as a prophylactic measure against the development or progression of the disease or disorder.

[0451] Those skilled in the art will understand that the compositions of the present invention may be administered alone or in any combination. Further, the compositions of the present invention may be administered alone or in any combination in the sense of time, in that they may be administered simultaneously with each other, or before, and / or after each other. Based on the disclosure provided herein, those skilled in the art will understand that the compositions of the present invention can be used to prevent or treat a disease or disorder, and that the compositions can be used alone or in any combination with another composition to achieve a therapeutic effect. In various embodiments, any of the compositions of the present invention described herein can be administered alone or in combination with other modulators of other molecules associated with the disease or disorder.

[0452] In one embodiment, the present invention includes a method that comprises administering a combination of the compositions described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of administering the combination of compositions is approximately equal to the sum of the effects of administering each individual composition. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering the combination of compositions is greater than the sum of the effects of administering each individual composition.

[0453] The method comprises administering the combination of compositions at any suitable ratio. For example, in one embodiment, the method comprises administering two individual compositions at a ratio of 1:1. However, the method is not limited to any particular ratio. Instead, any ratio that is shown to be effective is encompassed.

[0454] In some embodiments, the present invention includes a method of preparing a therapeutic composition for delivering at least one agent to endothelial cells lining a blood vessel lumen.

[0455] Pharmaceutical composition

[0456] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparative methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose unit.

[0457] Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to a variety of animals. Modifications of such compositions to render them suitable for administration to various animals are well known, and the ordinary veterinary pharmacologist can design and perform such modifications with only ordinary (if any) experimentation. Subjects contemplated for administration of the pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0458] The pharmaceutical compositions useful in the methods of the present invention can be prepared, packaged, or sold in a formulation suitable for ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intraventricular, intradermal, intramuscular, or other routes of administration. Other contemplated formulations include injectable nanoparticles, liposomal formulations, resealed red blood cells containing the active ingredient, and immunogenic matrix formulations.

[0459] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk, as a single unit dose, or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is usually equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as, for example, half or one-third of such a dose.

[0460] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject to be treated, and further depending on the route by which the composition is to be administered. By way of example, the composition can contain from 0.1% to 100% (w / w) of the active ingredient.

[0461] In addition to the active ingredient, the pharmaceutical compositions of the present invention can further comprise one or more additional pharmaceutically active agents.

[0462] In addition to the active ingredient, the pharmaceutical compositions of the present invention can further comprise one or more additional adjuvants. Exemplary adjuvants include, but are not limited to, aluminum-based adjuvants and monophosphoryl lipid A.

[0463] Conventional techniques can be used to prepare controlled-release or sustained-release formulations of the pharmaceutical compositions of the present invention.

[0464] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically disrupting the tissue of a subject and administering the pharmaceutical composition through the breach in the tissue. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by surgical incision, by non-surgical tissue-penetrating wound, etc. In particular, parenteral administration is contemplated to include, but is not limited to, intravitreal, intraperitoneal, intramuscular, intradermal, intracardiac, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0465] Formulations of pharmaceutical compositions suitable for parenteral administration comprise an active ingredient and a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in an ampoule or in a multi-dose container containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and implantable sustained release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable medium (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0466] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Such suspension or solution may be formulated according to known techniques and, in addition to the active ingredient, may contain additional ingredients such as dispersing agents, wetting agents, or suspending agents as described herein. For example, such sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides of fatty acids. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, in liposomal formulations, or as components of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0467] The pharmaceutical composition of the present invention can be prepared, packaged or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations can contain dry particles containing the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers. In some embodiments, the diameter is in the range of about 1 to about 6 nanometers. Such compositions can conveniently be in the form of a dry powder for administration using a device comprising a dry powder reservoir (into which a stream of propellant can be introduced to disperse the powder) or a self-propelling solvent / powder-dispensing container (such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container). In some embodiments, such powders contain particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nanometers and at least 95% by number of the particles have a diameter less than 7 nanometers. In some embodiments, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter less than 6 nanometers. In some embodiments, the dry powder composition includes a solid fine powder diluent (such as sugar) and is conveniently provided in unit dose form.

[0468] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant can account for 50 to 99.9% (w / w) of the composition, and the active ingredient can account for 0.1 to 20% (w / w) of the composition. The propellant can further contain additional ingredients, such as liquid nonionic or solid anionic surfactants or solid diluents. In some embodiments, the diluent has a particle size of the same order of magnitude as the particles containing the active ingredient.

[0469] Formulations of pharmaceutical compositions suitable for parenteral administration contain the active ingredient and a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable formulations can be prepared, packaged or sold in unit dosage forms, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and implantable sustained-release or biodegradable formulations. Such formulations can further contain one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable medium (such as sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0470] A pharmaceutical composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Such suspension or solution can be formulated according to known techniques and, in addition to the active ingredient, can include additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. For example, such sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides of fatty acids. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, in liposomal formulations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation can include pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0471] As used herein, "additional ingredients" includes, but is not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous media and solvents; oily media and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffering agents; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric materials or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0472] Experimental Examples

[0473] The present invention will be further described in detail by reference to the following experimental examples. These examples are for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations that become apparent from the teachings provided herein.

[0474] Without further description, it is believed that one of ordinary skill in the art can make and utilize the present invention and practice the claimed methods using the foregoing description and the following illustrative examples. Accordingly, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0475] Example 1: Targeted LNP for in vivo delivery of RNA-based gene editing tools

[0476] In the past, a variety of mRNA delivery technologies have been developed for multiple cell types such as endothelial cells, hematopoietic stem cells (HSCs), T cells, and other immune cells in vivo (Figures 1-7). In the present disclosure, a new therapeutic application is proposed based on existing targeted delivery platforms to deliver functional mRNA-based gene editing tools to T cells and stem cells for the treatment of HIV infection. Preliminary in vitro studies have shown that T cell-targeted Cas9 mRNA / CCR5 gRNA delivery can effectively knockdown CCR5 expression on T cells, thereby enabling the prevention or reduction of HIV infection ( Figure 8 ). CCR5 is a cell surface chemokine receptor that facilitates HIV entry into immune cells and is a validated target for HIV drug development. With T cell and stem cell-targeted knockdown or knockout of CCR5, the immune system can acquire permanent resistance to HIV infection. The long-term goal is to combine this technology with additional gene editing tools or therapies to block the HIV infection and reinfection pathways and also inactivate or eliminate proviral HIV DNA in the latent HIV reservoir to achieve an HIV cure.

[0477] Compared to the early stages of anti-HIV drug development, less toxic antiretroviral drugs have now been developed, which can significantly reduce mortality and control HIV / AIDS as a chronic infection when administered in combination with antiretroviral therapy (ART) drug regimens. However, ART still cannot eliminate the persistent / latent HIV cell reservoir. Due to the frequency of these persistent cell HIV reservoirs, slow decay rate, and lack of an effective targeted delivery system, they remain a formidable challenge for achieving an HIV cure. A potential HIV cure could be to drive the expression of mRNA-based gene editing tools in CD4+ T cells and HSCs respectively to inactivate proviral DNA in the latent HIV reservoir and modify the CCR5 co-receptor in CD34+ HSCs (or differentiated CD4 T cells) to resist future HIV infection. Therefore, the development of mRNA-LNP therapies such as mRNA-based gene editing has the potential to change the current way of treating HIV. CD4-targeted nucleoside-modified mRNA-LNPs exhibit efficient and specific in vitro and in vivo delivery. Comprehensive radio- or luminescence-based biodistribution analysis of CD4-targeted mRNA-LNPs has shown significant targeting of CD4+ T cells in lymphoid organs. The CD4-targeted mRNA-LNP platform uses the Cre / loxP reporter system to induce potent and specific gene editing in vivo. Preliminary results have also shown effective CCR5 knockout in T cells ( Figure 8 ).

[0478] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention may be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A composition for targeted delivery of a gene editing reagent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding a gene editing reagent and a delivery medium, wherein the delivery medium comprises a targeting moiety that specifically binds to the cell or particle of interest.

2. The composition according to claim 1, wherein The reagent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

3. The composition according to claim 2, wherein, The composition further comprises a guide RNA.

4. The composition according to any one of claims 1-3, wherein, The target cell or particle is selected from the group consisting of stem cells, immune cells, endothelial cells, bacterial cells, viral particles, fungal cells, and parasite cells.

5. The composition according to any one of claims 1-4, wherein, The target cell is a hematopoietic stem cell.

6. The composition according to any one of claims 1-4, wherein, The target cell or particle is a T cell.

7. The composition according to any one of claims 1-6, wherein, The composition comprises a combination of an mRNA molecule encoding a Cas9 protein and a guide RNA molecule for editing a gene in the target cell.

8. The composition according to claim 7, wherein, The guide RNA molecule targets CCR5.

9. The composition according to any one of claims 1-8, wherein, The targeting moiety specifically binds to CD4.

10. The composition according to any one of claims 1-8, wherein, The targeting moiety specifically binds to CD34.

11. The composition according to any one of claims 1-8, wherein, At least one isolated nucleoside-modified RNA comprises at least one selected from the group consisting of pseudouridine and 1-methylpseudouridine.

12. The composition according to any one of claims 1-11, wherein, The delivery medium comprises lipid nanoparticles (LNPs).

13. The composition according to claim 12, wherein, At least one nucleoside-modified RNA is encapsulated within the LNP.

14. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1-13.

15. The method according to claim 14, wherein The disease or disorder is selected from the group consisting of genetic defects and infectious diseases.

16. The method according to claim 14, wherein, The composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

17. A method of treating HIV, the method comprising administering a composition comprising a therapeutic reagent and a delivery medium, wherein the delivery medium comprises a targeting moiety that specifically binds to CD34+ hematopoietic stem cells, and wherein the therapeutic reagent comprises an mRNA molecule encoding a Cas9 protein and a guide RNA specific for CCR5.

18. A method of treating HIV, the method comprising administering a composition comprising a therapeutic reagent and a delivery medium, wherein the delivery medium comprises a targeting moiety that specifically binds to CD4+ T cells, and wherein the therapeutic reagent comprises an mRNA molecule encoding a Cas9 protein and a guide RNA specific for CCR5.

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