Targeted delivery of gene editing constructs and methods of use thereof

Abstract

The present invention relates to compositions for the effective delivery of gene editing agents to target cells and methods of use thereof for treating diseases or disorders.

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under award AI045008 from the National Institutes of Health (NIH). The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 384,692, filed November 22, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Modulation of immune cells by activating, inhibiting, or modifying them to change their properties has become a widespread and highly sought-after class of treatments known as immunotherapy. Today's immunotherapies rely primarily on biological protein-based drugs, which are expensive and difficult to manufacture or require ex vivo modification of immune cells. Some examples include antibodies or cytokines to modulate immune cell function, monoclonal antibodies to redirect immune function, gene editing of T cells to prevent viral infections, and chimeric antigen receptor (CAR) T-cell therapy.

[0004] There remains a need in the art for a robust targeted mRNA delivery system for delivering functional RNA-based gene editing tools to a variety of cell types. The present invention addresses this need. Summary of the Invention

[0005] In one embodiment, the present invention relates to a composition for targeted delivery of a gene editing agent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding the gene editing agent and a delivery vehicle, wherein the delivery vehicle comprises a specific targeting moiety for binding to the cell or particle of interest. In one embodiment, the gene editing agent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

[0006] In one embodiment, it further comprises a guide RNA.

[0007] 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.

[0008] In one embodiment, the target cell is a hematopoietic stem cell. In one embodiment, the target cell or particle is a T cell.

[0009] 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.

[0010] In one embodiment, the guide RNA molecule targets CCR5. In one embodiment, the targeting moiety is specific for binding to CD4. In one embodiment, the targeting moiety is specific for binding to CD34.

[0011] In one embodiment, the at least one isolated nucleoside modified RNA comprises pseudouridine or 1-methyl-pseudouridine.

[0012] In one embodiment, the delivery vehicle comprises a lipid nanoparticle (LNP). In one embodiment, at least one nucleoside-modified RNA is encapsulated within the LNP.

[0013] In one embodiment, the present invention relates to a method for treating a disease or disorder in a subject in need thereof, the method comprising administering a composition for targeted delivery of a gene editing agent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding the gene editing agent and a delivery vehicle, the delivery vehicle comprising a specific targeting moiety for binding to the cell or particle of interest. In one embodiment, the agent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

[0014] In one embodiment, the disease or disorder is selected from the group consisting of a genetic defect and an infectious disease.

[0015] In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.

[0016] In one embodiment, the present invention relates to a method of treating HIV, the method comprising administering a composition comprising a therapeutic agent and a delivery vehicle, the delivery vehicle comprising a CD34 + The therapeutic agent comprises a targeting moiety specific for binding to hematopoietic stem cells, and the therapeutic agent comprises an mRNA molecule comprising a Cas9 protein and a guide RNA specific for CCR5.

[0017] In one embodiment, the present invention relates to a method of treating HIV, the method comprising administering a composition comprising a therapeutic agent and a delivery vehicle, the delivery vehicle comprising a CD4 + It comprises a targeting moiety specific for binding to T cells, and the therapeutic comprises an mRNA molecule comprising a Cas9 protein and a guide RNA specific for CCR5.

[0018] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings, it being understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0019] [Figure 1A]Figures 1A-1C show data demonstrating the binding and functional activity of CD4-targeted particles in vitro. Figure 1A shows the specific in vitro binding of anti-human CD4 / 125I-labeled mRNA-LNP to human CD4+ T cells after 1 hour of incubation at room temperature. Figure 1B shows the binding of anti-CD4 / mRNA-LNP and control IgG / mRNA-LNP to human CD4+ T cells with increasing doses of mRNA-LNP, as well as their corresponding mean fluorescence intensities (MFIs). Figure 1C shows the Luc activity measured in human CD4+ T cells treated with anti-human CD4 / mRNA-LNP or control IgG / mRNA-LNP. [Figure 1B] Figures 1A-1C show data demonstrating the binding and functional activity of CD4-targeted particles in vitro. Figure 1A shows the specific in vitro binding of anti-human CD4 / 125I-labeled mRNA-LNP to human CD4+ T cells after 1 hour of incubation at room temperature. Figure 1B shows the binding of anti-CD4 / mRNA-LNP and control IgG / mRNA-LNP to human CD4+ T cells with increasing doses of mRNA-LNP, as well as their corresponding mean fluorescence intensities (MFIs). Figure 1C shows the Luc activity measured in human CD4+ T cells treated with anti-human CD4 / mRNA-LNP or control IgG / mRNA-LNP. [Figure 1C] Figures 1A-1C show data demonstrating the binding and functional activity of CD4-targeted particles in vitro. Figure 1A shows the specific in vitro binding of anti-human CD4 / 125I-labeled mRNA-LNP to human CD4+ T cells after 1 hour of incubation at room temperature. Figure 1B shows the binding of anti-CD4 / mRNA-LNP and control IgG / mRNA-LNP to human CD4+ T cells with increasing doses of mRNA-LNP, as well as their corresponding mean fluorescence intensities (MFIs). Figure 1C shows the Luc activity measured in human CD4+ T cells treated with anti-human CD4 / mRNA-LNP or control IgG / mRNA-LNP. [Figure 2A]Figures 2A and 2B show data demonstrating Cre mRNA-mediated gene recombination in vitro. Figure 2A shows Cre mRNA-induced gene recombination and the resulting reporter gene expression (presented as the percentage of ZsGreen1+ cells among CD3+CD8- cells). Spleen cells were harvested from Ai6 mice and incubated with Cre mRNA-LNPs at doses of 1, 3, 6, or 9 μg per 2 million cells. The percentage of ZsGreen1+ cells following administration of anti-CD4 / mRNA-LNPs was compared with control IgG / mRNA-LNPs and non-binding mRNA-LNPs (****P<0.0001, two-way ANOVA with Bonferroni correction). Figure 2B shows the gating strategy used to identify ZsGreen1+ cells among CD3+CD8- cells. [Figure 2B] Figures 2A and 2B show data demonstrating Cre mRNA-mediated gene recombination in vitro. Figure 2A shows Cre mRNA-induced gene recombination and the resulting reporter gene expression (presented as the percentage of ZsGreen1+ cells among CD3+CD8- cells). Spleen cells were harvested from Ai6 mice and incubated with Cre mRNA-LNPs at doses of 1, 3, 6, or 9 μg per 2 million cells. The percentage of ZsGreen1+ cells following administration of anti-CD4 / mRNA-LNPs was compared with control IgG / mRNA-LNPs and non-binding mRNA-LNPs (****P<0.0001, two-way ANOVA with Bonferroni correction). Figure 2B shows the gating strategy used to identify ZsGreen1+ cells among CD3+CD8- cells. [Figure 2B-1]Figures 2A and 2B show data demonstrating Cre mRNA-mediated gene recombination in vitro. Figure 2A shows Cre mRNA-induced gene recombination and the resulting reporter gene expression (presented as the percentage of ZsGreen1+ cells among CD3+CD8- cells). Spleen cells were harvested from Ai6 mice and incubated with Cre mRNA-LNPs at doses of 1, 3, 6, or 9 μg per 2 million cells. The percentage of ZsGreen1+ cells following administration of anti-CD4 / mRNA-LNPs was compared with control IgG / mRNA-LNPs and non-binding mRNA-LNPs (****P<0.0001, two-way ANOVA with Bonferroni correction). Figure 2B shows the gating strategy used to identify ZsGreen1+ cells among CD3+CD8- cells. [Figure 2B-2] Figures 2A and 2B show data demonstrating Cre mRNA-mediated gene recombination in vitro. Figure 2A shows Cre mRNA-induced gene recombination and the resulting reporter gene expression (presented as the percentage of ZsGreen1+ cells among CD3+CD8- cells). Spleen cells were harvested from Ai6 mice and incubated with Cre mRNA-LNPs at doses of 1, 3, 6, or 9 μg per 2 million cells. The percentage of ZsGreen1+ cells following administration of anti-CD4 / mRNA-LNPs was compared with control IgG / mRNA-LNPs and non-binding mRNA-LNPs (****P<0.0001, two-way ANOVA with Bonferroni correction). Figure 2B shows the gating strategy used to identify ZsGreen1+ cells among CD3+CD8- cells. [Figures 3A-3D]Figures 3A-3D show data demonstrating mRNA-LNP targeting to CD4+ T cells in vivo. Figure 3A shows the biodistribution of 125I-labeled anti-CD4 / mRNA-LNP and control IgG / poly(C) mRNA-LNP in mice after 0.5 hours. Tissue uptake is shown as mean ± SEM (****P<0.0001). Figure 3B shows the localization ratio, calculated as the ratio of %ID / g in a given organ to %ID / g in blood, after 30 minutes in mice treated with 125I-labeled anti-CD4 / mRNA-LNP or control IgG / mRNA-LNP. Mean ± SEM is shown. In vivo mRNA-LNP binding capacity as a quantitative measure of the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs (Figure 3C) and the localization ratio in the spleen (Figure 3D) after intravenous administration of mRNA-LNP. Group size was 3 animals. Statistical analysis was performed by two-way analysis of variance with Bonferroni correction (****P<0.0001). [Figure 3A] Figures 3A-3D show data demonstrating mRNA-LNP targeting to CD4+ T cells in vivo. Figure 3A shows the biodistribution of 125I-labeled anti-CD4 / mRNA-LNP and control IgG / poly(C) mRNA-LNP in mice after 0.5 hours. Tissue uptake is shown as mean ± SEM (****P<0.0001). Figure 3B shows the localization ratio, calculated as the ratio of %ID / g in a given organ to %ID / g in blood, after 30 minutes in mice treated with 125I-labeled anti-CD4 / mRNA-LNP or control IgG / mRNA-LNP. Mean ± SEM is shown. In vivo mRNA-LNP binding capacity as a quantitative measure of the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs (Figure 3C) and the localization ratio in the spleen (Figure 3D) after intravenous administration of mRNA-LNP. Group size was 3 animals. Statistical analysis was performed by two-way analysis of variance with Bonferroni correction (****P<0.0001). [Figure 3B]Figures 3A-3D show data demonstrating mRNA-LNP targeting to CD4+ T cells in vivo. Figure 3A shows the biodistribution of 125I-labeled anti-CD4 / mRNA-LNP and control IgG / poly(C) mRNA-LNP in mice after 0.5 hours. Tissue uptake is shown as mean ± SEM (****P<0.0001). Figure 3B shows the localization ratio, calculated as the ratio of %ID / g in a given organ to %ID / g in blood, after 30 minutes in mice treated with 125I-labeled anti-CD4 / mRNA-LNP or control IgG / mRNA-LNP. Mean ± SEM is shown. In vivo mRNA-LNP binding capacity as a quantitative measure of the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs (Figure 3C) and the localization ratio in the spleen (Figure 3D) after intravenous administration of mRNA-LNP. Group size was 3 animals. Statistical analysis was performed by two-way analysis of variance with Bonferroni correction (****P<0.0001). [Figure 3C] Figures 3A-3D show data demonstrating mRNA-LNP targeting to CD4+ T cells in vivo. Figure 3A shows the biodistribution of 125I-labeled anti-CD4 / mRNA-LNP and control IgG / poly(C) mRNA-LNP in mice after 0.5 hours. Tissue uptake is shown as mean ± SEM (****P<0.0001). Figure 3B shows the localization ratio, calculated as the ratio of %ID / g in a given organ to %ID / g in blood, after 30 minutes in mice treated with 125I-labeled anti-CD4 / mRNA-LNP or control IgG / mRNA-LNP. Mean ± SEM is shown. In vivo mRNA-LNP binding capacity as a quantitative measure of the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs (Figure 3C) and the localization ratio in the spleen (Figure 3D) after intravenous administration of mRNA-LNP. Group size was 3 animals. Statistical analysis was performed by two-way analysis of variance with Bonferroni correction (****P<0.0001). [Figure 3D]Figures 3A-3D show data demonstrating mRNA-LNP targeting to CD4+ T cells in vivo. Figure 3A shows the biodistribution of 125I-labeled anti-CD4 / mRNA-LNP and control IgG / poly(C) mRNA-LNP in mice after 0.5 hours. Tissue uptake is shown as mean ± SEM (****P<0.0001). Figure 3B shows the localization ratio, calculated as the ratio of %ID / g in a given organ to %ID / g in blood, after 30 minutes in mice treated with 125I-labeled anti-CD4 / mRNA-LNP or control IgG / mRNA-LNP. Mean ± SEM is shown. In vivo mRNA-LNP binding capacity as a quantitative measure of the percentage of radiolabeled anti-CD4 / mRNA-LNP in selected organs (Figure 3C) and the localization ratio in the spleen (Figure 3D) after intravenous administration of mRNA-LNP. Group size was 3 animals. Statistical analysis was performed by two-way analysis of variance with Bonferroni correction (****P<0.0001). [Figure 4A] Figures 4A-4D show data demonstrating the biodistribution of targeted mRNA-LNP expression in vivo. Mice were injected IV with 8 μg of mRNA-LNP. Five hours after administration of anti-CD4 / Luc mRNA-LNP and control IgG / Luc mRNA-LNP, the organ distribution of Luc mRNA expression was assessed by measuring Luc activity in lysed tissue (Figure 4A) and luminescence imaging (Figures 4B and 4C). Figure 4A shows the quantitative expression of Luc as light units (LU) / mg protein. A representative sample set of dissected mouse organs (Figure 4B) and whole carcasses after organ removal (showing luminescent lymph nodes) (Figure 4C) were analyzed 5 minutes after D-luciferin administration. Figure 4D shows the quantitative expression of Luc as LU / mg protein in CD3+ cell preparations obtained from the spleens of mice injected with mRNA-LNP. 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, ***P<0.001). [Figure 4B]Figures 4A-4D show data demonstrating the biodistribution of targeted mRNA-LNP expression in vivo. Mice were injected IV with 8 μg of mRNA-LNP. Five hours after administration of anti-CD4 / Luc mRNA-LNP and control IgG / Luc mRNA-LNP, the organ distribution of Luc mRNA expression was assessed by measuring Luc activity in lysed tissue (Figure 4A) and luminescence imaging (Figures 4B and 4C). Figure 4A shows the quantitative expression of Luc as light units (LU) / mg protein. A representative sample set of dissected mouse organs (Figure 4B) and whole carcasses after organ removal (showing luminescent lymph nodes) (Figure 4C) were analyzed 5 minutes after D-luciferin administration. Figure 4D shows the quantitative expression of Luc as LU / mg protein in CD3+ cell preparations obtained from the spleens of mice injected with mRNA-LNP. 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, ***P<0.001). [Figure 4C] Figures 4A-4D show data demonstrating the biodistribution of targeted mRNA-LNP expression in vivo. Mice were injected IV with 8 μg of mRNA-LNP. Five hours after administration of anti-CD4 / Luc mRNA-LNP and control IgG / Luc mRNA-LNP, the organ distribution of Luc mRNA expression was assessed by measuring Luc activity in lysed tissue (Figure 4A) and luminescence imaging (Figures 4B and 4C). Figure 4A shows the quantitative expression of Luc as light units (LU) / mg protein. A representative sample set of dissected mouse organs (Figure 4B) and whole carcasses after organ removal (showing luminescent lymph nodes) (Figure 4C) were analyzed 5 minutes after D-luciferin administration. Figure 4D shows the quantitative expression of Luc as LU / mg protein in CD3+ cell preparations obtained from the spleens of mice injected with mRNA-LNP. 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, ***P<0.001). [Figure 4D]Figures 4A-4D show data demonstrating the biodistribution of targeted mRNA-LNP expression in vivo. Mice were injected IV with 8 μg of mRNA-LNP. Five hours after administration of anti-CD4 / Luc mRNA-LNP and control IgG / Luc mRNA-LNP, the organ distribution of Luc mRNA expression was assessed by measuring Luc activity in lysed tissue (Figure 4A) and luminescence imaging (Figures 4B and 4C). Figure 4A shows the quantitative expression of Luc as light units (LU) / mg protein. A representative sample set of dissected mouse organs (Figure 4B) and whole carcasses after organ removal (showing luminescent lymph nodes) (Figure 4C) were analyzed 5 minutes after D-luciferin administration. Figure 4D shows the quantitative expression of Luc as LU / mg protein in CD3+ cell preparations obtained from the spleens of mice injected with mRNA-LNP. 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, ***P<0.001). [Figure 5A]Figures 5A-5E show data demonstrating Cre-mediated gene recombination upon in vivo administration of CD4-targeted Cre mRNA-LNP. Figure 5A shows a schematic diagram illustrating the principle of targeted delivery of anti-CD4 / mRNA-LNP and the Ai6 reporter allele for selective gene recombination in CD4+ T cells. 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 of Cre mRNA-LNP intravenously. 24 hours after treatment, spleens and lymph nodes were harvested, and the percentage of ZsGreen1+ cells in the CD3+CD8- cell population was measured using flow cytometry in single-cell suspensions of the spleen (Figure 5B) and lymph nodes (Figure 5C). The time course of the number of ZsGreen1-expressing CD4+ T cells in the spleen (Figure 5D) and lymph nodes (Figure 5E) was monitored after IV injection of 10 μg of mRNA-LNP. In a total of three independent experiments, group sizes were 8 or 9 animals (Figures 5B and 5C) or 6 animals (Figures 5D and 5E). Each symbol represents one animal, and the horizontal line indicates the mean with standard error of the mean (SEM). Statistical analysis was performed by two-way ANOVA with Bonferroni correction. The percentage of ZsGreen1+ cells was compared after injection of different doses of anti-CD4 / mRNA-LNP [*P<0.05, ****P<0.0001] and unbound mRNA-LNP [****P<0.0001]. [Figures 5B-5E]Figures 5A-5E show data demonstrating Cre-mediated gene recombination upon in vivo administration of CD4-targeted Cre mRNA-LNP. Figure 5A shows a schematic diagram illustrating the principle of targeted delivery of anti-CD4 / mRNA-LNP and the Ai6 reporter allele for selective gene recombination in CD4+ T cells. 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 of Cre mRNA-LNP intravenously. 24 hours after treatment, spleens and lymph nodes were harvested, and the percentage of ZsGreen1+ cells in the CD3+CD8- cell population was measured using flow cytometry in single-cell suspensions of the spleen (Figure 5B) and lymph nodes (Figure 5C). The time course of the number of ZsGreen1-expressing CD4+ T cells in the spleen (Figure 5D) and lymph nodes (Figure 5E) was monitored after IV injection of 10 μg of mRNA-LNP. In a total of three independent experiments, group sizes were 8 or 9 animals (Figures 5B and 5C) or 6 animals (Figures 5D and 5E). Each symbol represents one animal, and the horizontal line indicates the mean with standard error of the mean (SEM). Statistical analysis was performed by two-way ANOVA with Bonferroni correction. The percentage of ZsGreen1+ cells was compared after injection of different doses of anti-CD4 / mRNA-LNP [*P<0.05, ****P<0.0001] and unbound mRNA-LNP [****P<0.0001]. [Figures 6A-6B]Figures 6A-6C show data demonstrating the in vivo uptake of Cre mRNA-LNP by different T cell subtypes. Spleens were collected 24 h after treatment with 10 μg of Cre mRNA-LNP, and flow cytometry was used to measure the percentage of ZsGreen1+ cells among CD4+ T cell subpopulations (Figure 6A) and the CD25 marker (Figure 6B). Naive CD4+ T cells are considered CD44-CD62L-, central memory T cells are considered CD44+CD62L+, and effector memory T cells are considered CD44+CD62L-. Group sizes ranged from 3 to 11 animals. Each symbol represents one animal, and horizontal lines indicate the mean with SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction to compare T cell subtypes. Figure 6C shows the gating strategy used to identify ZsGreen1+ cells among different CD4+ T cell subtypes. [Figure 6C] Figures 6A-6C show data demonstrating the in vivo uptake of Cre mRNA-LNP by different T cell subtypes. Spleens were collected 24 h after treatment with 10 μg of Cre mRNA-LNP, and flow cytometry was used to measure the percentage of ZsGreen1+ cells among CD4+ T cell subpopulations (Figure 6A) and the CD25 marker (Figure 6B). Naive CD4+ T cells are considered CD44-CD62L-, central memory T cells are considered CD44+CD62L+, and effector memory T cells are considered CD44+CD62L-. Group sizes ranged from 3 to 11 animals. Each symbol represents one animal, and horizontal lines indicate the mean with SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction to compare T cell subtypes. Figure 6C shows the gating strategy used to identify ZsGreen1+ cells among different CD4+ T cell subtypes. [Figure 6C-1]Figures 6A-6C show data demonstrating the in vivo uptake of Cre mRNA-LNP by different T cell subtypes. Spleens were collected 24 h after treatment with 10 μg of Cre mRNA-LNP, and flow cytometry was used to measure the percentage of ZsGreen1+ cells among CD4+ T cell subpopulations (Figure 6A) and the CD25 marker (Figure 6B). Naive CD4+ T cells are considered CD44-CD62L-, central memory T cells are considered CD44+CD62L+, and effector memory T cells are considered CD44+CD62L-. Group sizes ranged from 3 to 11 animals. Each symbol represents one animal, and horizontal lines indicate the mean with SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction to compare T cell subtypes. Figure 6C shows the gating strategy used to identify ZsGreen1+ cells among different CD4+ T cell subtypes. [Figures 7A-7B] Figures 7A and 7B show data demonstrating mRNA-LNP targeting efficiency using multiple doses. Ai6 mice received 10 μg (0.4 mg / kg) of anti-CD4 / mRNA-LNP, control IgG / mRNA-LNP, or unconjugated Cre mRNA-LNP intravenously as daily injections for 3 or 5 days. After three or five consecutive injections, spleens and lymph nodes were harvested, and the percentage of ZsGreen1+ cells in the CD3+CD8- cell population was measured using flow cytometry in single-cell suspensions of the spleen (Figure 7A) and lymph nodes (Figure 7B). Group sizes were 9 animals. Each symbol represents one animal, and horizontal lines indicate the mean. Error bars indicate the standard error of the mean (SEM). Statistical analysis was performed by two-way ANOVA with Bonferroni correction. The % of ZsGreen1+ cells (**P<0.01, ****P<0.0001) after different times of anti-CD4 / mRNA-LNP injection were compared. [Figure 8]Figure 8 shows the CCR5 knockout efficiency of CD5 / LNP / Cas9 mRNA / CCR5 gRNA in human T cells. By using CD5 / targeting LNP-encapsulated Cas9 mRNA / CCR5 gRNA, highly promising knockout efficiency was achieved as a proof-of-concept for RNA-based gene editing using targeted LNP-mRNA. [Figure 8-1] Figure 8 shows the CCR5 knockout efficiency of CD5 / LNP / Cas9 mRNA / CCR5 gRNA in human T cells. By using CD5 / targeting LNP-encapsulated Cas9 mRNA / CCR5 gRNA, highly promising knockout efficiency was achieved as a proof-of-concept for RNA-based gene editing using targeted LNP-mRNA. [Figure 8-2] Figure 8 shows the CCR5 knockout efficiency of CD5 / LNP / Cas9 mRNA / CCR5 gRNA in human T cells. By using CD5 / targeting LNP-encapsulated Cas9 mRNA / CCR5 gRNA, highly promising knockout efficiency was achieved as a proof-of-concept for RNA-based gene editing using targeted LNP-mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to a composition for efficiently delivering a therapeutic agent, comprising a delivery vehicle, wherein the delivery vehicle comprises at least one targeting domain or moiety for delivering the therapeutic agent to a specific cell type of interest, and the therapeutic agent comprises at least one composition for gene editing.

[0021] In some embodiments, the composition for gene editing comprises a combination of an mRNA molecule encoding a Cas9 protein and a guide RNA.

[0022] In some embodiments, the LNPs comprise a targeting domain specific for endothelial cells, immune cells, stem cells, or another particular cell type of interest. Target cells / tissues that can be targeted include, but are not limited to, T cells, hematopoietic stem cells (HSCs), endothelial cells, bone marrow cells, and lung cells. In one embodiment, the targeted LNPs of the present invention comprise a targeting domain specific for binding to an antigen on the target cell type of interest. For example, in one embodiment, the LNPs comprise a targeting domain specific for binding to an antigen expressed on immune cells. In one embodiment, the LNPs comprise a targeting domain specific for binding to an antigen expressed on tumor cells. In one embodiment, the LNPs comprise a targeting domain specific for binding to an antigen expressed on tumor cells. In one embodiment, the LNPs comprise a targeting domain specific for binding to an antigen expressed on a particular tissue type (e.g., a marker expressed on lung tissue).

[0023] The present invention also relates to methods of using the compositions described herein for targeted delivery of therapeutic agents and methods of treating diseases or disorders, including, but not limited to, genetic defects and infectious diseases and disorders in a subject, hi 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 present invention include achondroplasia, achondroplasia, alpha 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 thrombosis, 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 acidemia, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, and others. These include, but are not limited to, 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's disease.

[0024] In one embodiment, the disease or disorder is a non-malignant hematological 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.

[0025] In some embodiments, the infection or disorder is a viral infection, a bacterial infection, a fungal infection, or a parasitic disease, hi some embodiments, the viral infection is a human immunodeficiency virus (HIV) infection.

[0026] definition Unless defined otherwise, 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.

[0027] As used herein, each of the following terms has the meaning associated with it in this section.

[0028] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0029] As used herein, "about," when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0030] As used herein, the term "adjunctive agent" refers to an agent that alters or enhances the intensity and duration of a desired therapeutic response and / or extends the therapeutic response to a co-administered agent.

[0031] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen or epitope. An antibody can be an intact immunoglobulin from natural sources or from recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin. As used herein, the term "antibody" includes "antibody fragments." The term "antibody fragment" refers to a portion of an intact antibody, and refers to the variable region that determines the antigen specificity of the intact antibody. The antibodies or antibody fragments of the present invention may exist in a variety of 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).

[0032] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0033] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa light chain and lambda light chain refer to the two major antibody light chain isotypes.

[0034] 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 bacteriophage. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses the antibody protein or amino acid sequence specifying the antibody, where 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 taken to mean an antibody produced by synthesis of an RNA molecule encoding the antibody, where the RNA molecule expresses the antibody protein or amino acid sequence specifying the antibody, where the RNA is obtained by transcription of DNA (synthetic or clonal) or other techniques available and well known in the art.

[0035] "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 health is less favorable than it would be in the absence of the disorder. A disorder, if left untreated, does not necessarily result in further deterioration of the animal's health.

[0036] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0037] The term "physiologically effective dose" refers to an amount of a drug that produces a measurable biological or physiological effect in a recipient subject that is related to the activity of the drug. A physiologically effective dose varies depending on the compound, the age, weight, etc. of the subject receiving the drug, and the biological or physiological effect being measured.

[0038] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, mRNA) or a defined amino acid sequence and biological properties derived therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0039] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids incorporating the recombinant polynucleotide, plasmids (e.g., naked plasmids or those encapsulated in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0040] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both compared sequences 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, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0041] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal would not be "isolated," but the same nucleic acid or peptide would be "isolated" if it was partially or completely separated from the coexisting materials of its natural state. An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as a host cell.

[0042] In the context of the present invention, the following abbreviations are used for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond): "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0043] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that the nucleotide sequence encoding the protein may contain introns in some form.

[0044] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of the treatment or compound and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject. In some embodiments, the subject is a human.

[0045] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs may contain introns. Furthermore, the nucleotide sequence may contain modified nucleosides that can be translated by the translational machinery in a cell. For example, an mRNA in which all uridines are replaced with pseudouridine, 1-methylpseudouridine, or another modified nucleoside.

[0046] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to link two protein-coding regions, in the same reading frame.

[0047] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof, in vitro or in situ, to which the methods described herein are applicable. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0048] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art generally know that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is 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 cellular genomes using conventional cloning techniques, PCR™, etc.) and synthetic means.

[0049] In certain cases, the polynucleotide or nucleic acid of the present invention is a "nucleoside-modified nucleic acid," which refers to a nucleic acid containing at least one modified nucleoside. A "modified nucleoside" refers to a nucleoside having a modification. For example, more than 100 different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucleic Acids Res 27:196-197).

[0050] In certain embodiments, "pseudouridine" refers to, in another embodiment, m 1 acp 3 Y(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1 In another embodiment, the term refers to Y(1-methylpseudouridine). In another embodiment, the term refers to Ym(2'-O-methylpseudouridine). 5 In another embodiment, the term refers to m 3Y (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the pseudouridines listed above. In another embodiment, the term refers to other pseudouridines known in the art. Each alternative represents a separate embodiment of the present invention.

[0051] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting 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 may comprise a protein or peptide sequence. Polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins (of which there are many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0052] The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate specific transcription of a polynucleotide sequence, e.g., the promoter recognized by bacteriophage RNA polymerase and used to generate mRNA by in vitro transcription.

[0053] The term "specifically binds" as used herein with respect to affinity ligands, particularly antibodies, refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. However, such cross-species reactivity, in and of itself, does not alter the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity, in and of itself, does not alter the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" are used in reference to the interaction of an antibody, protein, or peptide with a second chemical species and can mean that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A," in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0054] As used herein, the term "treatment" refers to treatment and / or prophylaxis. A therapeutic benefit is obtained by suppressing, reducing, ameliorating, or eradicating at least one sign or symptom of a disease or disorder.

[0055] The term "therapeutically effective amount" refers to an amount of a compound of interest that elicits the biological or medical response in a tissue, system, or subject that is desired 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 onset of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0056] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0057] As used herein, the terms "transfected," "transformed," or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected," "transformed," or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0058] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0059] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0060] "Alkyl" means an alkyl group consisting solely of carbon and hydrogen atoms, saturated or unsaturated (i.e., containing one or more double and / or triple bonds), and having 1 to 24 carbon atoms (C1 to C 24alkyl), 1 to 12 carbon atoms (C1 to C 12 "(C-C alkyl)" refers to a straight or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C-C alkyl), or 1 to 6 carbon atoms (C-C alkyl), attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, propynyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise specified, alkyl groups are optionally substituted.

[0061] An "alkylene" or "alkylene chain" is an alkylene group consisting of only carbon and hydrogen, saturated or unsaturated (i.e., containing one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), and having, for example, 1 to 24 carbon atoms (C1 to C 24 Alkylene, 1 to 15 carbon atoms (C1 to C 15 Alkylene, 1 to 12 carbon atoms (C1 to C 12 "C-C alkylene" refers to a linear or branched divalent hydrocarbon chain having 1 to 8 carbon atoms (C-C alkylene), 1 to 6 carbon atoms (C-C alkylene), 2 to 4 carbon atoms (C-C alkylene), or 1 to 2 carbon atoms (C-C alkylene), which connects the rest of the molecule to a radical group, such as methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, and n-butynylene. The alkylene chain is connected to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon atom or any two carbon atoms within the chain. Unless otherwise specifically stated herein, the alkylene chain may be optionally substituted.

[0062] "Cycloalkyl" or "carbocycle" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having 3 to 15 carbon atoms, saturated or unsaturated, and attached to the rest 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, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, cycloalkyl groups may be optionally substituted.

[0063] "Cycloalkylene" refers to a divalent cycloalkyl group. Unless stated otherwise specifically in the specification, a cycloalkylene group may be optionally substituted.

[0064] "Heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical, which consists of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atoms can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl. Unless otherwise specifically stated, heterocyclyl groups may be optionally substituted.

[0065] As used herein, the term "substituted" refers to at least one hydrogen atom being replaced with a halogen atom, such as, but not limited to, F, Cl, Br, and I, an oxo group (=O), a hydroxyl group (-OH), an alkoxy group (-OR), or a substituted or unsubstituted alkyl group. a , where R a is C1~C 12 alkyl or cycloalkyl), carboxyl group (-OC(=O)R a or -C(=O)OR a , where R a H, C1~C 12 alkyl or cycloalkyl), an amine group (—NR a R b , where R a and Rb are each independently H, C1 to C 12 alkyl or cycloalkyl), C1-C 12 It refers to any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) replaced by a bond to a non-hydrogen atom, such as alkyl groups and cycloalkyl groups. In some embodiments, the substituent is C1-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group. In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group.

[0066] "Optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and the description includes instances when the event or circumstance occurs and instances when the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may be substituted or unsubstituted, and that the description includes both substituted and unsubstituted alkyl groups.

[0067] Ranges: 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 an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range of 1 to 6 should be considered to have specifically disclosed subranges 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 numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the broadness of the range.

[0068] definition The present invention relates, in part, to compositions and methods for targeted delivery of gene editing agents. In some embodiments, the invention comprises administering a therapeutic gene editing agent to a subject in need thereof. In some embodiments, the therapeutic agent comprises an mRNA molecule encoding the gene editing molecule.

[0069] Delivery Vehicle In some embodiments, the delivery vehicle is a colloidal dispersion system such as a macromolecule complex, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0070] The use of lipid formulations is contemplated for introducing at least one agent into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / nucleic acid, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure such as a micelle, or in a "collapsed" structure. They may also simply be dispersed in solution, potentially forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0071] Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY), cholesterol ("Chol") is available from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure consisting of a phospholipid bilayer and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess amount of aqueous solution. The lipid components undergo self-rearrangement to form closed structures, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-drug complexes are also contemplated.

[0072] In one embodiment, the delivery of at least one agent comprises any suitable delivery method, including the exemplary delivery method described elsewhere herein.In certain embodiments, the delivery of at least one agent to a subject comprises mixing at least one agent with a transfection reagent before the contacting step.In another embodiment, the method of the present invention further comprises administering at least one agent together with a transfection reagent.In another embodiment, the transfection reagent is a cationic lipid reagent.

[0073] 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 Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0074] In another embodiment, the transfection reagent forms a liposome. In another embodiment, the liposome has enhanced intracellular stability, enhanced uptake efficiency, and improved biological activity. In another embodiment, the liposome is a hollow spherical vesicle composed of lipids arranged in a manner similar to that of the lipids that make up the cell membrane. In some embodiments, the liposome contains an internal aqueous space for encapsulating water-soluble compounds. In another embodiment, the liposome can deliver at least one drug to cells in an active form.

[0075] In one embodiment, the composition comprises a lipid nanoparticle (LNP) and at least one agent.

[0076] The term "lipid nanoparticle" refers to a particle comprising one or more lipids and having at least one dimension on the nanometer order (e.g., 1-1,000 nm). In various embodiments, the particle comprises a lipid of Formula (I), (II), or (III). In some embodiments, the lipid nanoparticle is included in a formulation comprising at least one agent 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., a PEGylated lipid, e.g., a PEGylated lipid of structure (IV), e.g., compound IVa). In some embodiments, at least one agent is encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting the agent from enzymatic degradation or other undesirable effects elicited by mechanisms of the host organism or cells, such as a harmful immune response.

[0077] 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 nm 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, the at least one drug, when present in the lipid nanoparticles, is resistant to degradation by intracellular or extracellular enzymes in aqueous solution.

[0078] LNPs may include any lipid capable of forming particles having at least one drug bound thereto or encapsulated therein. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically classified into at least three classes: (1) "simple lipids," including oils and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0079] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and PEGylated lipids.

[0080] In one embodiment, LNP comprises 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 gradually neutral at higher pH values.Then, at a pH value lower than the pK, the lipid can associate with negatively charged nucleic acid.In certain embodiments, cationic lipids include zwitterionic lipids, which become positively charged when the pH is lowered.

[0081] In certain embodiments, the cationic lipid comprises any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Such lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 ... Cationic lipids include, but are not limited to, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(2-(dimethylamino)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercially available cationic lipid preparations are available that can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), manufactured by GIBCO / BRL, Grand Island, NY), LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), manufactured by GIBCO / BRL), and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, manufactured by Promega Corp., Madison, WI).The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0082] In one embodiment, cationic lipid is amino lipid.Suitable amino lipids useful for the present invention include those described in WO2012 / 016184 (which is incorporated herein by reference in its entirety).Representative amino lipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0083] Suitable amino lipids include those having the formula: [ka] In the formula, R 1 and R 2are the same or different and independently represent 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 It is acyl, R 3 and R 4 are the same or different and independently optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, or R 3 and R 4 may be joined to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R 5 is absent or present, and if present, is hydrogen or C1-C6 alkyl; m, n, and p are the same or different and independently 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different and independently O, S, or NH.

[0084] In one embodiment, R 1 and R 2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid. Representative useful dilinoleylamino lipids have the formula: [ka] wherein n is 0, 1, 2, 3, or 4.

[0085] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA as described above, where n is 2).

[0086] In one embodiment, the cationic lipid component of the LNP has the structure of formula (I): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 and L 2 are each independently —O(C═O)—, —(C═O)O—, or a carbon-carbon double bond; R 1a and R 1b is, in each occurrence, 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, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 2a and R 2b is, in each occurrence, 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, form an adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 3a and R 3b is, in each occurrence, independently: (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, form an adjacent R 3band together with the carbon atom to which it is attached form a carbon-carbon double bond, R 4a and R 4b is, in each occurrence, independently: (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, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is, in each occurrence, independently H or C1-C 12 is alkyl, R 8 and R 9 are each independently C1 to 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; a and d each independently represent an integer of 0 to 24, b and c each independently represent an integer of 1 to 24; e is 1 or 2.

[0087] In certain embodiments of Formula (I), R 1a , R 2a , R 3a , or R 4a At least one of C1~C 12 alkyl or L 1 Or L 2 At least one of R is -O(C=O)- or -(C=O)O-. 1a and R 1b is not isopropyl when a is 6 and is not n-butyl when a is 8. In yet another embodiment of Formula (I), R 1a , R2a , R 3a , or R 4a At least one of C1~C 12 alkyl or L 1 Or L 2 at least one of is -O(C=O)- or -(C=O)O-, and R 1a and R 1b is not an isopropyl group when a is 6, and is not an n-butyl group when a is 8.

[0088] In other embodiments of Formula (I), R 8 and R 9 are each independently an unsubstituted C1 to C 12 is an alkyl group, 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.

[0089] In certain embodiments of Formula (I), L 1 or L 2 Any one of L may be -O(C=O)- or a carbon-carbon double bond. 1 and L 2 may each be —O(C═O)— or each be a carbon-carbon double bond.

[0090] In some embodiments of Formula (I), L 1 or L 2 In another embodiment, any one of L 1 and L 2 Both are -O(C=O)-.

[0091] In some embodiments of Formula (I), L 1 or L 2 In another embodiment, any one of L 1 and L 2 Both are —(C═O)O—.

[0092] In some embodiments of Formula (I), L1 or L 2 In another embodiment, any one of L 1 and L 2 Both of these are carbon-carbon double bonds.

[0093] In still other embodiments of Formula (I), L 1 or L 2 is -O(C=O)-, and L 1 or L 2 and the other is —(C═O)O—. 1 or L 2 is -O(C=O)-, and L 1 or L 2 and the other is a carbon-carbon double bond. 1 or L 2 is -(C=O)O-, and L 1 or L 2 The other is a carbon-carbon double bond.

[0094] As used throughout this specification, a "carbon-carbon" double bond is understood to refer to either of the following structures: [ka] In the formula, R a and R b is, at each occurrence, independently H or a substituent. For example, in some embodiments, R a and R b is, in each occurrence, independently H, C1 to C 12 alkyl, or cycloalkyl, for example, H or C1-C 12 It is alkyl.

[0095] In another embodiment, the lipid compound of formula (I) has the following structure (Ia): [ka]

[0096] In another embodiment, the lipid compound of formula (I) has the following structure (Ib): [ka]

[0097] In yet another embodiment, the lipid compound of formula (I) has the following structure (Ic): [ka]

[0098] In particular embodiments of lipid compounds of Formula (I), a, b, c, and d are each independently an integer between 2 and 12 or an integer between 4 and 12. In other embodiments, a, b, c, and d are each independently an integer between 8 and 12 or between 5 and 9. In some particular embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0099] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0100] In some further embodiments of Formula (I), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0101] In more particular embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0102] In various other 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.

[0103] The sum of a and b and the sum of c and d in Formula (I) are factors that may be varied to obtain a lipid of Formula (I) with desired properties. In one embodiment, a and b are selected so that their sum is an integer ranging from 14 to 24. In another embodiment, c and d are selected so that their sum is an integer ranging from 14 to 24. In a further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which may vary in the range of 14 to 24. In yet a further embodiment, a, b, c, and d are selected so that the sum of a and b and the sum of c and d is 12 or greater.

[0104] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0105] R in formula (I) 1a , R 2a , R 3a , and R 4aThe substituents in R are not particularly limited. 1a , R 2a , R 3a , and R 4a is H in each occurrence. In other particular embodiments, R 1a , R 2a , R 3a , and R 4a At least one of C1~C 12 In other particular embodiments, R 1a , R 2a , R 3a , and R 4a At least one of R is C1-C8 alkyl. 1a , R 2a , R 3a , and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0106] In certain embodiments of Formula (I), R 1a , R 1b , R 4a , and R 4b In each case, C1 to C 12 It is alkyl.

[0107] In a further embodiment of formula (I), R 1b , R 2b , R 3b , and R 4b At least one of is H or R 1b , R 2b , R 3b , and R 4b is H in each case.

[0108] In certain embodiments of Formula (I), R 1b together with the carbon atom to which it is attached, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond.4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0109] In the above embodiment, R 5 and R 6 The substituents in R are not particularly limited. 5 or R 6 In certain other embodiments, one or both of R 5 or R 6 One or both of is cycloalkyl, e.g., cyclohexyl. In these embodiments, the cycloalkyl may be substituted or unsubstituted. In other particular embodiments, the cycloalkyl is C1-C 12 It is substituted with alkyl, for example, tert-butyl.

[0110] In the above embodiment of formula (I), R 7 The substituents in are not particularly limited. In certain embodiments, at least one R 7 is H. In some other embodiments, R 7 is H in each occurrence. In other particular embodiments, R 7 is C1~C 12 It is alkyl.

[0111] In certain other of the above embodiments of formula (I), R 8 or R 9 One of R is methyl. 8 and R 9 are both methyl.

[0112] 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. 8 and R 9taken together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[0113] In various different embodiments, exemplary lipids of formula (I) can include: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0114] In some embodiments, the LNPs comprise a lipid of Formula (I), at least one drug, and one or more excipients selected from a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of Formula (I) is compound I-5. In some embodiments, the lipid of Formula (I) is compound I-6.

[0115] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (II): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 and L 2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, or -S(O) x -, -SS-, -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, G 1 -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond, G 2 are -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a or by direct bond, G 3 is a C1-C6 alkylene, R a is H or C1~C 12 is alkyl, R 1a and R 1b is, in each occurrence, 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, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 2a and R 2b is, in each occurrence, 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, form an adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 3a and R 3bis, in each occurrence, independently: (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, form an adjacent R 3b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 4a and R 4b is, in each occurrence, independently: (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, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond, R 5 and R 6 are each independently H or methyl; R 7 is C4~C 20 is alkyl, R 8 and R 9 are each independently C1 to 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; a, b, c, and d each independently represent an integer of 1 to 24; x is 0, 1, or 2.

[0116] In some embodiments of Formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond. 1 and G 2 are each independently —(C═O)— or a direct bond. 1 and L 2are each independently -O(C=O)-, -(C=O)O-, or a direct bond; G 1 and G 2 are each independently —(C═O)— or a direct bond.

[0117] In some different embodiments of Formula (II), L 1 and L 2 are each independently -C(=O)-, -S(O) x -, -SS-, -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 -It is.

[0118] In other of the above embodiments of formula (II), the lipid compound has one of the following structures (IIA) or (IIB): [ka]

[0119] In some embodiments of formula (II), the lipid compound has the structure (IIA): In other embodiments, the lipid compound has the structure (IIB):

[0120] In any of the above embodiments of formula (II), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —O(C═O)—.

[0121] In some different embodiments of Formula (II), L 1 or L 2 One of L is -(C=O)O-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0122] In some different embodiments of Formula (II), L 1 or L 2 As used herein, a "direct bond" refers to a bond that is bonded to a group (e.g., L 1 or L 2 ) is absent. For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0123] In another different embodiment of formula (II), R 1a and R 1b For at least one of 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0124] In yet another different embodiment of formula (II), R 4a and R 4b For at least one of 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0125] In a further embodiment of formula (II), R 2a and R 2b For at least one of 2a is H or C1~C 12 alkyl, and R 2btogether with the carbon atom to which it is attached, form an adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0126] In another different embodiment of formula (II), R 3a and R 3b For at least one of 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, form an adjacent R 3b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0127] In various other embodiments of formula (II), the lipid compound has one of the following structures (IIC) or (IID): [ka] In the formula, e, f, g, and h each independently represent an integer of 1 to 12.

[0128] In some embodiments of formula (II), the lipid compound has the structure (IIC): In other embodiments, the lipid compound has the structure (IID):

[0129] In various embodiments of structure (IIC) or (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0130] 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 particular embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0131] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0132] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0133] In some particular embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0134] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In still other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In still other embodiments, e is 12.

[0135] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In still other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In still other embodiments, f is 12.

[0136] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In still other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In still other embodiments, g is 12.

[0137] 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.

[0138] In various other 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.

[0139] The sum of a and b and the sum of c and d in Formula (II) are factors that may be varied to obtain lipids with desired properties. In one embodiment, a and b are selected so that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected so that their sum is an integer in the range of 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which may be in the range of 14 to 24. In still other embodiments, a, b, c, and d are selected so that the sum of a and b and the sum of c and d are 12 or greater.

[0140] R in formula (II) 1a , R 2a , R 3a , and R 4a The substituents in R are not particularly limited. 1a , R 2a , R 3a , and R 4a At least one of R is H. In certain embodiments, 1a , R 2a , R 3a , and R 4a is H in each occurrence. In other particular embodiments, R 1a , R 2a , R 3a , and R 4a At least one of C1~C 12 In other particular embodiments, R 1a , R 2a , R 3a , and R 4a At least one of R is C1-C8 alkyl. 1a , R 2a , R 3a , and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0141] In certain embodiments of Formula (II), R 1a , R 1b , R 4a , and R 4b In each case, C1 to C 12 It is alkyl.

[0142] In a further embodiment of formula (II), R 1b , R 2b , R 3b , and R 4b At least one of is H or R 1b , R 2b , R 3b , and R 4b is H in each case.

[0143] In certain embodiments of Formula (II), R 1b together with the carbon atom to which it is attached, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0144] In the above embodiment, R 5 and R 6 The substituents in R are not particularly limited. 5 or R 6 In other embodiments, one of R 5 or R 6 Each of is methyl.

[0145] In the above embodiment, R 7 The substituents in R are not particularly limited. 7 is C6~C 16 In some other embodiments, R 7 is C6-C9 alkyl. In some other embodiments, R 7 -(C=O)ORb , -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 wherein R a is H or C1~C 12 alkyl, and R b is 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 is replaced by .

[0146] In the above various embodiments of formula (II), R b is molecular C1~C 15 For example, in some embodiments, R b has one of the following structures: [ka]

[0147] In this other particular embodiment of formula (II), R 8 or R 9 In other embodiments, one of R 8 and R 9 are both methyl.

[0148] In some different embodiments of Formula (II), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring. 8 and R 9 are taken together with the nitrogen atom to which they are attached to form a 5-membered heterocyclic ring, e.g., a pyrrolidinyl ring. In several different embodiments of the above, R 8 and R 9 taken together with the nitrogen atom to which they are attached form a six-membered heterocyclic ring, for example a piperazinyl ring.

[0149] In yet another embodiment of the lipid of formula (II) above, G 3 is a C2 to C4 alkylene, for example, a C3 alkylene.

[0150] In various different embodiments, the lipid compound has one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0151] In some embodiments, the LNP comprises a lipid of Formula (II), at least one drug, and one or more excipients selected from a neutral lipid, a steroid, and a pegylated lipid. 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.

[0152] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -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 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -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, G 1 and G 2 are each independently an unsubstituted C1 to C 12 Alkylene or C1-C 12 is alkenylene, G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1~C 12 is alkyl, R 1 and R 2 are each independently C6 to C 24 Alkyl or C6-C 24 It is alkenyl. R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 is C1~C 12 is alkyl, R 5 is H or C1-C6 alkyl, x is 0, 1, or 2.

[0153] In some of the above embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB): [ka] During the ceremony, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is, in each occurrence, independently H, OH, or C1-C 24 is alkyl, n is an integer from 1 to 15.

[0154] In some of the above embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0155] In another embodiment of formula (III), the lipid has one of the following structures (IIIC) or (IIID): [ka] In the formula, y and z are each independently an integer ranging from 1 to 12.

[0156] In any of the above embodiments of formula (III), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of the following is —O(C═O)—. In any of the above 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 Each of is —(C═O)O—.

[0157] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF): [ka]

[0158] In some of the above embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ). [ka]

[0159] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 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.

[0160] In some other embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0161] In some of the above embodiments of formula (III), R 6 is H. In other embodiments, R 6 is C1~C 24 In another embodiment, R 6 is OH.

[0162] In some embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G 3 is substituted. In various different embodiments, G 3 is a linear C1~C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0163] In some other embodiments of Formula (III), R 1 or R 2 Or both are C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the following structure: [ka] During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C1-C 12 is alkyl, a is an integer from 2 to 12, R 7a , R 7b , and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0164] In some of the above embodiments of formula (III), R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0165] In different embodiments of formula (III), R 1 or R 2 or both have any one of the following structures: [ka]

[0166] In some of the above embodiments of formula (III), R 3 OH, OH, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0167] In various different embodiments, the cationic lipid of formula (III) has one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0168] In some embodiments, the LNPs comprise a lipid of formula (III), at least one drug, and one or more excipients selected from a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the lipid of formula (III) is compound III-7.

[0169] In certain embodiments, the cationic lipid is present in the LNP in an amount of about 30 to about 95 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mol %. In one embodiment, the LNP comprises only cationic lipid.

[0170] In certain embodiments, the LNP comprises one or more additional lipids that stabilize the particle during its formation.

[0171] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0172] The term "neutral lipid" refers to any one of a number of lipid species that exist in uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0173] Examples of neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N- Maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0174] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid (e.g., a lipid of Formula (I)) to neutral lipid ranges from about 2:1 to about 8:1.

[0175] In various embodiments, the LNP further comprises a steroid or steroid analog. A "steroid" is a compound that contains the following carbon skeleton: [ka]

[0176] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of cationic lipid (e.g., a lipid of Formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0177] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0178] In certain embodiments, the LNPs comprise a glycolipid (e.g., monosialoganglioside GM1). In certain embodiments, the LNPs comprise a sterol, such as cholesterol.

[0179] In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. Examples of polymer-conjugated lipids include pegylated lipids. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEGs-DMG), etc.

[0180] In certain embodiments, the LNPs comprise an additional stabilizing lipid that 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, and PEG-modified dialkylglycerol. Exemplary 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 )carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol lipid is PEG-c-DOMG. In other embodiments, the LNP is a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (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)butanediol). PEG-3 hydroxybenzoates (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyl)propyl)carbamate or 2,3-di(tetradecanoyl)propyl-N-(β-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.

[0181] In some embodiments, the LNP comprises a PEGylated lipid having the following structure (IV): [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0182] R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds; z has an average value in the range of 30 to 60.

[0183] In some of the above embodiments of PEGylated lipid (IV), when z is 42, R 10 and R 11 and R are not both n-octadecyl. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 18 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. 10 is a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms, and R 11 is a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0184] In various embodiments, z ranges from a range selected such that the PEG moiety of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0185] In other embodiments, the PEGylated lipid has one of the following structures: [ka] where n is an integer selected so that the average molecular weight of the PEGylated lipid is about 2500 g / mol.

[0186] In certain embodiments, the additional lipid is present in the LNP in an amount of about 1 to about 10 mol %. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 to about 5 mol %. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mol % or about 1.5 mol %.

[0187] 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 a different embodiment, the neutral lipid is DSPC. In another embodiment, the steroid is cholesterol. In yet a different embodiment, the PEGylated lipid is compound IVa.

[0188] In certain embodiments, LNPs comprise one or more targeting moieties that target the LNP to stem cells or stem cell populations. For example, in one embodiment, the targeting domain is a ligand that directs the LNP to a receptor found on the surface of stem cells.

[0189] Exemplary LNPs and methods for their production are described in the art, e.g., in U.S. Patent Application Publication No. 2012 / 0276209; 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, Int J Cancer., 131(5):E781-90; Belliveau et al., 2012, Mol Ther Nucleic Acids, 1:e37; Jayaraman et al. al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, Mol Ther 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 herein by reference in its entirety.

[0190] The following reaction schemes illustrate methods for preparing lipids of formula (I), (II), or (III).

[0191] General Reaction Scheme 1 [ka]

[0192] Lipid embodiments 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-C 24A-1 is alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods known to those skilled in the art. A mixture of A-1, A-2, and DMAP is treated with DCC to provide bromide A-3. Heating a mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine), and N,N-dimethyldiamine A-4 at a sufficient temperature and for a sufficient time, after any necessary workup and / or purification steps, produces A-5.

[0193] General Reaction Scheme 2 [ka]

[0194] Other embodiments of compounds of Formula (I) (e.g., Compound B-5) can be prepared according to General Reaction Scheme 2 ("Method B"), wherein R is a saturated or unsaturated C1-C 24 where m is alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-1 can be purchased from commercial sources or prepared according to methods known to those skilled in the art. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinium chlorochromate) to recover intermediate product B-3. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to afford B-5, after any necessary workup and / or purification.

[0195] It should be noted that although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbons, starting materials containing carbon-carbon double bonds may also be used to prepare compounds containing carbon-carbon double bonds.

[0196] General Reaction Scheme 3 [ka]

[0197] Different embodiments of lipids of formula (I) (e.g., compounds C-7 or C-9) can be prepared according to General Reaction Scheme 3 ("Method C"), where R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art.

[0198] General Reaction Scheme 4 [ka]

[0199] 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; R 7 ' is R 7 Or C3~C 19represents alkyl. Referring to General Reaction Scheme 1, compounds of structures D-1 and D-2 can be purchased from commercial sources or prepared according to methods known to those skilled in the art. A solution of D-1 and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to provide D-3 after any necessary workup. A solution of D-3 and a base (e.g., trimethylamine, DMAP) is treated with an acyl chloride D-4 (or a carboxylic acid and DCC) to provide D-5 after any necessary workup and / or purification. D-5 can be reduced with LiAlH4 D-6 to provide D-7 after any necessary workup and / or purification.

[0200] General Reaction Scheme 5 [ka]

[0201] 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, compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. Heating a mixture of E-1 (in excess), E-2, and a base (e.g., potassium carbonate) affords E-3 after any necessary workup. Treating a solution of E-3 and a base (e.g., trimethylamine, DMAP) with an acyl chloride E-4 (or a carboxylic acid and DCC) affords E-5 after any necessary workup and / or purification.

[0202] General Reaction Scheme 6 [ka]

[0203] General Reaction Scheme 6 provides an exemplary method (Method F) for preparing lipids of formula (III). 1 , G 3 , R 1 , and R 3 is as defined herein for formula (III), and G 1 ' is one carbon shorter than G 1 The term "F-1" refers to a homologue of the formula (III). Compounds of structure F-1 can be purchased or prepared according to methods known in the art. Reaction of F-1 with diol F-2 under appropriate condensation conditions (e.g., DCC) provides ester / alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions provides lipids of formula (III).

[0204] It should be noted that various alternative strategies for preparing lipids of formula (III) are available to those skilled in the art. For example, L 1 and L 2 Other lipids of formula (III) where G is other than an ester can be prepared according to similar methods using appropriate starting materials. 1 and G 2 The preparation of lipids of formula (III) in which G is the same is shown, but this is not a required aspect of the present invention, and the above reaction scheme can be modified to prepare lipids of formula (III) in which G is the same as G 1 and G 2 It is also possible to obtain compounds that differ from the above.

[0205] It will be appreciated by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Hutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one skilled in the art will appreciate, the protecting group may also be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0206] Drugs In one embodiment, the delivery vehicle 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, tracrRNA, sgRNA, mRNA encoding an RNA-guided nuclease, gene editing or base editing protein, zinc finger nuclease, Talen, CRISPR nuclease such as Cas9, nucleic acid molecule to be inserted or function as a repair template, etc., or combinations thereof. In some embodiments, the mRNA encodes the gene editing or base editing protein. In some embodiments, the nucleic acid is a guide RNA.

[0207] In some embodiments, the LNP contains both an mRNA encoding a gene-editing or base-editing protein and one or more guide RNAs. The CRISPR nuclease may have altered activity, for example, the nuclease is modified so that it is a nickase instead of making a double-strand cleavage, or so that it binds to the sequence specified by the guide RNA but does not have enzymatic activity. The base-editing protein is often a fusion protein containing 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 contain an mRNA encoding an RNA-guided nuclease and a guide RNA, but rather contains a ribonucleoprotein that is a complex containing a guide RNA bound to an RNA-guided nuclease. In other embodiments, the nanoparticle contains RNA and a reverse transcriptase.

[0208] In some embodiments, the RNA molecule encodes part of the TtAgo system. In eukaryotes, gene silencing is mediated by the Argonaute (Ago) family of proteins. In this paradigm, Ago binds to small (19-31 base) RNAs. This protein-RNA silencing complex recognizes the target RNA through Watson-Crick base pairing between the small RNA and the target RNA and endonucleolytically cleaves the target RNA. Ago-RNA-mediated DNA cleavage can be used to affect a variety of outcomes, including gene knockout, using standard techniques in the art for utilizing DNA cleavage.

[0209] In some embodiments, RNA molecule encodes cleavage domain that is operably linked with DNA binding domain to form nuclease.For example, ZFP DNA binding domain is fused with nuclease domain to create ZFN, that is, functional entity that can recognize intended nucleic acid target through engineered (ZFP) DNA binding domain and cause DNA cleavage near ZFP binding site by nuclease activity, including being used for genome modification in various organisms.Similarly, TALE DNA binding domain is fused with nuclease domain to create TALEN.

[0210] 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 derived from a nuclease, or a TALEN DNA-binding domain and a cleavage domain derived from a meganuclease, or a cleavage domain derived from a different nuclease. Heterologous cleavage domains can be derived from any endonuclease or exonuclease. Exemplary endonucleases from which cleavage domains 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 cleavage domains and cleavage half-domains.

[0211] Similarly, the cleavage half-domain can be derived from any of the nucleases or portions thereof described above that require dimerization for cleavage activity. Generally, when a 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 fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). Furthermore, 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 places the cleavage half-domains in a spatial orientation that allows them to form a functional cleavage domain, e.g., by dimerization. Thus, in certain embodiments, the proximal ends of the target sites are separated by 5-8 nucleotides or 15-18 nucleotides. However, any integral number of nucleotides or nucleotide pairs (e.g., 2-50 nucleotide pairs or more) can be present between the two target sites. Generally, a cleavage site is located between the target sites.

[0212] Restriction endonucleases (restriction enzymes) exist in many species and can bind to DNA in a sequence-specific manner (at their recognition site) and cleave 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 Fok I catalyzes double-stranded cleavage of DNA on one strand at 9 nucleotides from the recognition site and on the other strand at 13 nucleotides from the recognition site. Thus, in one embodiment, an RNA molecule of the invention can encode a fusion protein comprising a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains (which may or may not be engineered).

[0213] An exemplary Type IIS restriction enzyme in which the cleavage domain is separate from the binding domain includes FokI. This particular enzyme is active as a dimer. Therefore, for 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-stranded cleavage and / or targeted replacement of cellular sequences using zinc finger-FokI fusion proteins, two fusion proteins, each containing a FokI cleavage half-domain, can be reconstituted using a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can be used.

[0214] A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain. Additional restriction enzymes also contain separable binding and cleavage domains and are contemplated by this disclosure.

[0215] 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.

[0216] In some embodiments, nucleases may be assembled in vivo at nucleic acid target sites using the so-called "split enzyme" technique. The components of such split enzymes may be encoded by separate RNA molecules. The components may be individual zinc finger binding domains or domains of meganuclease nucleic acid binding domains.

[0217] The Cas9-associated CRISPR / Cas system comprises two non-coding RNA components: a tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). To achieve genome engineering using the CRISPR / Cas system, both of these RNAs must function. In some embodiments, the tracrRNA and pre-crRNA are provided via separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (which confers target specificity) is fused with a tracrRNA (which confers interaction with Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also called a single guide RNA).

[0218] As detailed above, DNA-binding domains can be engineered to bind to any selected sequence, and engineered DNA-binding domains can have novel binding specificities compared to naturally occurring DNA-binding domains.

[0219] In one aspect, the delivery vehicle may comprise a vector containing the nucleotide sequence or construct to be delivered. The choice of vector depends on the host cell into which the vector is subsequently introduced. In certain embodiments, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be used for use in the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0220] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not integrate into the genome of the host cell when introduced into the cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of the invention or the genetic construct of the invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0221] The vector may 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 transformation of animal cells.

[0222] In one embodiment, the recombinant expression vector may also include a nucleic acid molecule encoding the peptide or peptidomimetic.

[0223] A promoter may be one naturally associated with a gene or polynucleotide sequence or may be obtained by isolating 5' non-coding sequences located upstream of the coding region and / or exons. Such promoters may be referred to as "endogenous." Similarly, an enhancer may be located downstream or upstream of a polynucleotide sequence and naturally associated with the polynucleotide sequence. Alternatively, certain advantages may be obtained by placing a coding polynucleotide region under the control of a recombinant or heterologous promoter (recombinant or heterologous promoter refers to a promoter not normally associated with a polynucleotide sequence in its natural environment). Recombinant or heterologous enhancer also refers to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes and promoters or enhancers isolated from other prokaryotes, viruses, or eukaryotic cells, as well as "non-naturally occurring" promoters or enhancers, i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or expression-altering mutations. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR™, in connection with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202 and 5,928,906). It is further contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria and chloroplasts, may also be used.

[0224] Naturally, it is important to use a promoter and / or enhancer that effectively induces expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al. (2012)). The promoter used may be constitutive, tissue-specific, inducible, and / or useful for inducing high-level expression of the introduced DNA segment under appropriate conditions, for example, advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0225] The recombinant expression vector may also contain a selectable marker gene that facilitates selection of host cells. Suitable selectable marker genes include proteins that confer resistance to certain drugs, such as G418 and hygromycin, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes encoding immunoglobulins or portions thereof, such as the Fc region of immunoglobulins, including, but not limited to, IgG. The selectable marker may be introduced into a vector separate from the nucleic acid of interest.

[0226] Any polynucleotide can be further modified to enhance its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends, the use of phosphorothioate or 2'-O-methyl (rather than phosphodiester) linkages in the backbone, and / or the inclusion of non-traditional bases such as inosine, queosine, and vabutosine, and acetyl, methyl, thio, and other modifications of adenine, cytidine, guanine, thymine, and uridine.

[0227] In one embodiment of the present invention, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes useful for inhibiting the expression of target molecules can be designed, for example, by incorporating a target sequence complementary to the mRNA sequence encoding the target molecule into the basic structure of the ribozyme. Ribozymes that target target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or can be genetically expressed from DNA that encodes them.

[0228] In one embodiment, the therapeutic agent can comprise one or more components of a CRISPR-Cas system in which a guide RNA (gRNA) that targets a gene encoding a target molecule and a CRISPR-associated (Cas) peptide form a complex to induce a mutation in the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding the gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding the Cas peptide.

[0229] RNA molecules can be synthesized to contain modifications that confer desired properties, such as improved stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, e.g., by endocytosis-dependent or -independent mechanisms.

[0230] Modifications can also enhance sequence specificity, resulting in reduced off-site targeting. Synthesis and chemical modification methods are described in more detail below. If desired, RNA molecules can be modified to stabilize the RNA against degradation, extend half-life, or otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Publication Nos. 2007 / 0213292, 2006 / 0287260, 2006 / 0035254, 2006 / 0008822, and 2005 / 028824, each of which is incorporated herein by reference in its entirety. To enhance nuclease resistance and / or binding affinity to the target, the single-stranded oligonucleotide agents featured in the present disclosure can include 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Target binding affinity can also be enhanced by including locked nucleic acids (LNAs), ethylene nucleic acids (ENAs), e.g., 2'-4'-ethylene-bridged nucleic acids, and specific nucleotide modifications. Endonucleolytic cleavage can also be reduced by including pyranose sugars in the oligonucleotide backbone. Oligonucleotides can be further modified by including a 3' cationic group or by inverting the nucleoside at the 3' end with a 3-3' linkage. In another alternative, the 3' end can be blocked with an aminoalkyl group. Other 3' linkages can inhibit 3'-5' exonucleolytic cleavage. Without being bound by theory, the 3' may inhibit exonucleolytic cleavage by sterically blocking the oligonucleotide from binding to the 3' end of the exonuclease. Even small alkyl chains, aryl groups, or heterocyclic conjugates, or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

[0231] In one embodiment, the RNA comprises a 2'-modified oligonucleotide containing an oligodeoxynucleotide gap, in which some or all internucleotide linkages are modified to phosphorothioate for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for the target RNA, thereby lowering the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in combination with any technology that can be developed to enhance the stability or efficacy of nucleic acid molecules.

[0232] RNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers with modified backbones include oligomers with and without a phosphorus atom in the backbone. For purposes of this disclosure, modified oligonucleotides without a phosphorus atom in the internucleoside backbone are also considered nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts, and free acid forms are also included.

[0233] 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., a solution comprising water. The aqueous phase or crystalline composition can be incorporated into a delivery vehicle, e.g., a liposome (particularly in the case of an aqueous phase), or a particle (e.g., a microparticle, which may be appropriate for a 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 agent, e.g., another therapeutic agent, or an agent that stabilizes the oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., broad-specificity RNAse inhibitors). In one embodiment, the RNA composition comprises two or more RNA molecules, e.g., a second sgRNA composition (e.g., a different sgRNA from the first sgRNA). Still other preparations can include at least 3, 5, 10, 20, 50, or 100 or more different oligonucleotide species.

[0234] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of an sgRNA.

[0235] Oligonucleotides selected for inclusion in the compositions of the invention may be any of a number of lengths. Such oligonucleotides may be 7 to 100 linked nucleosides in length. For example, oligonucleotides having nucleobase identity to an miRNA may be 7 to 30 linked nucleosides in length. Oligonucleotides having identity to an miRNA precursor may be up to 100 linked nucleosides in length. In certain embodiments, oligonucleotides contain 7 to 30 linked nucleosides. In certain embodiments, oligonucleotides contain 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, oligonucleotides contain 19 to 23 linked nucleosides. In certain embodiments, oligonucleotides are 40 up to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0236] In vitro transcribed RNA In one embodiment, a composition of the invention comprises an in vitro transcribed (IVT) RNA. In one embodiment, a composition of the invention comprises an in vitro transcribed (IVT) RNA encoding a gene editing molecule. In one embodiment, a composition of the invention comprises an IVT RNA encoding a gene editing molecule and a guide RNA molecule.

[0237] In one embodiment, IVT RNA can be incorporated into the delivery vehicle of the present invention. RNA is produced by in vitro transcription using a synthetically produced plasmid DNA template. DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source.

[0238] In one embodiment, the DNA used for PCR contains an open reading frame. The DNA can be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest, a portion of the gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene including 5' and 3' UTRs. In another embodiment, the DNA used for PCR is a gene from a pathogenic or symbiotic organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA used for PCR is a gene from a pathogenic or symbiotic organism, including bacteria, viruses, parasites, and fungi, including 5' and 3' UTRs. Alternatively, the DNA can be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains gene portions joined together to form an open reading frame encoding a fusion protein. The gene portions joined together can be from a single organism or from two or more organisms.

[0239] In various embodiments, a plasmid is used to generate a template for in vitro transcription of RNA to be incorporated into the LNP or delivery vehicle.

[0240] Chemical structures capable of promoting stability and / or translation efficiency may be used. In some embodiments, the RNA has a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 0 to 3,000 nucleotides in length. The lengths of the 5'UTR and 3'UTR sequences added to the coding region can be varied by different methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can vary the lengths of the 5'UTR and 3'UTR necessary to achieve optimal translation efficiency of the transcribed RNA.

[0241] The 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR of the gene of interest. Alternatively, a UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence into the forward primer and reverse primer, or by any other modification of the template. The use of a UTR sequence that is not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence can reduce RNA stability. Therefore, based on the characteristics of UTRs known in the art, 3'UTR can be selected or designed to increase the stability of transcribed RNA.

[0242] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, if a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Skozak sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to enable efficient translation. It is known in the art that many RNAs require a Skozak sequence. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' UTR or 5' UTR to prevent exonuclease degradation of the RNA.

[0243] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be added to the DNA template upstream of the sequence to be transcribed. If a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be 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 described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0244] In one embodiment, the RNA has both a 5'-end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase generates long concatameric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end generates RNA of a normal size that, when polyadenylated after transcription, is effective for eukaryotic transfection.

[0245] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the final 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)).

[0246] The traditional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which can be ameliorated by using recombinant non-competent bacterial cells for plasmid propagation.

[0247] The poly(A) tail of an RNA can be further extended after in vitro transcription with a poly(A) polymerase, such as Escherichia coli poly(A) polymerase (E-PAP) or yeast poly(A) polymerase. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the translation efficiency of the RNA by approximately two-fold. Furthermore, the stability of the RNA can be enhanced by adding different chemical groups to the 3' end. Such additions can include modified / artificial nucleotides, aptamers, and other compounds. For example, poly(A) polymerase can be used to incorporate ATP analogs into the poly(A) tail. The ATP analogs can further enhance the stability of the RNA.

[0248] The 5' cap also confers stability to the RNA molecule. In one embodiment, the RNA produced by the method includes a 5' Cap 1 structure. Such Cap 1 structures can be generated using vaccinia capping enzyme and 2'-O-methyltransferase enzyme (CellScript, Madison, Wisconsin). Alternatively, the 5' cap can be provided using techniques known in the art and described herein (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)).

[0249] Nucleoside-modified RNA In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid. In one embodiment, a composition of the invention comprises a nucleoside-modified RNA encoding a gene-editing protein, a guide RNA, or a combination thereof.

[0250] For example, in one embodiment, the composition comprises nucleoside-modified RNA. In one embodiment, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA has certain advantages over unmodified mRNA, such as increased stability, reduced or absent natural immunogenicity, and improved 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.

[0251] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiological pathways, is translated very efficiently almost immediately after delivery, and serves as a template for continuous protein production in vivo, lasting for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). Only small amounts of mRNA are required to exert a physiological effect, making it applicable to human therapy.

[0252] In certain cases, expressing proteins by delivering encoding mRNA offers numerous advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence for the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with the plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, mRNA does not risk integration into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15–30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, the use of mRNA rather than protein also offers numerous advantages. Proteins often have a short half-life in circulation, thus necessitating frequent administration of protein therapeutics, whereas mRNA provides a template for continuous protein production over several days. Purifying proteins is difficult and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0253] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified nucleoside pseudouridine. In certain embodiments, the inclusion of pseudouridine makes mRNA more stable, non-immunogenic, and enhances translation (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, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0254] The presence of pseudouridine-containing modified nucleosides in RNA has been shown to suppress its natural immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed RNA encoding proteins containing pseudouridine can be translated more efficiently than RNA without the modified nucleoside or RNA containing other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Furthermore, the presence of pseudouridine has been shown to increase RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and attenuate both PKR activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established to obtain pseudouridine-containing RNA with excellent translational activity and no natural immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified pseudouridine-containing erythropoietin-encoding RNA to mice and macaques significantly increased serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), confirming that pseudouridine-containing mRNA is suitable for in vitro protein therapy.

[0255] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules that contain pseudouridine or modified nucleosides. In certain embodiments, a composition comprises an isolated nucleic acid, wherein the nucleic acid comprises pseudouridine or modified nucleosides. In certain embodiments, a composition comprises a vector that contains the isolated nucleic acid, wherein the nucleic acid comprises pseudouridine or modified nucleosides.

[0256] In one embodiment, the nucleoside-modified RNA of the present invention is the IVT RNA 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.

[0257] 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 1 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 a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the pseudouridines described above. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0258] 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).

[0259] In another embodiment, the modified nucleoside of the present invention is m 5 C(5-methylcytidine). In another embodiment, the modified nucleoside is m 5U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 -methyladenosine). In another embodiment, the modified nucleoside is s 2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0260] 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 -isopentenyl adenosine), 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 6 A(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 -hydroxynorvalylcarbamoyl adenosine), ms 2 hn 6 A(2-methylthio-N 6-hydroxynorvalylcarbamoyl 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 (lycidin), 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 * (undermodified hydroxywybutosine), imG (wybutosine), mimG (methylwybutosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQ0 (7-cyano-7-deazaguanosine), preQ1 (7-aminomethyl-7-deazaguanosine), G + (Archiosin), D (Dihydrouridine), m 5 Um (5,2'-O-dimethyluridine), s4 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-hydroxyacetic 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 5 Um (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-taurinomethyluridine), τm 5 s 2 U (5-taurinomethyl-2-thiouridine), imG-14 (4-demethylwiosin), imG 2 (Isowyosin), or ac 6 A(N 6 -acetyladenosine).

[0261] In another embodiment, the nucleoside-modified RNA of the present invention comprises a combination of two or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of three or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of four or more of the above modifications.

[0262] In another embodiment, 0.1% to 100% of the residues in a modified nucleoside of the invention are modified (e.g., by the presence of pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the percentage of modified residues is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0263] In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0264] 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 percentage of a given nucleotide that is modified is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0265] In another embodiment, the percentage of a given nucleotide that is modified is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0266] 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 improved ability to be translated by a target cell. In another embodiment, translation is improved 2-fold compared to its unmodified counterpart. In another embodiment, translation is improved 3-fold. In another embodiment, translation is improved 5-fold. In another embodiment, translation is improved 7-fold. In another embodiment, translation is improved 10-fold. In another embodiment, translation is improved 15-fold. In another embodiment, translation is improved 20-fold. In another embodiment, translation is improved 50-fold. In another embodiment, translation is improved 100-fold. In another embodiment, translation is improved 200-fold. In another embodiment, translation is improved 500-fold. In another embodiment, translation is improved 1000-fold. In another embodiment, translation is improved 2000-fold. In another embodiment, the ratio is 10-1000-fold. In another embodiment, the ratio is 10-100-fold. In another embodiment, the ratio is 10-200-fold. In another embodiment, the ratio is 10-300 fold. In another embodiment, the ratio is 10-500 fold. In another embodiment, the ratio is 20-1000 fold. In another embodiment, the ratio is 30-1000 fold. In another embodiment, the ratio is 50-1000 fold. In another embodiment, the ratio is 100-1000 fold. In another embodiment, the ratio is 200-1000 fold. In another embodiment, translation is improved by any other significant amount or range of amounts.

[0267] combination In one embodiment, a composition of the invention comprises a combination of agents described herein. In certain embodiments, compositions comprising a combination of agents described herein act together to have an effect (e.g., the Cas9 protein and guide RNA function together to edit genes).

[0268] Compositions that contain a combination of drugs can contain the individual drugs in any suitable ratio. For example, in one embodiment, the composition contains two individual drugs in a 1:1 ratio. However, the combination is not limited to any specific ratio. Rather, any ratio that has been shown to be effective is encompassed.

[0269] join In various embodiments of the present invention, the delivery vehicle is conjugated to a targeting domain. In some embodiments, the delivery vehicle is conjugated to a CD34 or CD4 targeting domain. Exemplary methods of conjugation include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic ("van der Waals") interactions. In one embodiment, the conjugation is reversible, such that the delivery vehicle can dissociate from the targeting domain upon exposure to certain conditions or chemicals. In another embodiment, the conjugation is irreversible, such that the delivery vehicle does not dissociate from the targeting domain under normal conditions.

[0270] In some embodiments, the bond comprises a covalent bond between the activated polymer-bound lipid and the targeting domain. The term "activated polymer-bound lipid" refers to a molecule comprising a lipid portion and a polymer portion that has been activated by functionalizing the polymer-bound lipid with a first linking group. In one embodiment, the activated polymer-bound lipid comprises a first linking group that can react with a second linking group. In one embodiment, the activated polymer-bound lipid is an activated pegylated lipid. In one embodiment, the first linking group is attached to the lipid portion of the pegylated lipid. In another embodiment, the first linking group is attached to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0271] The first and second linking groups may 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 or second linking 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 sulfhydryl group. In some embodiments, the first or second coupling group is selected from the group consisting of free amine (-NH), free sulfhydryl group (-SH), free hydroxide group (-OH), carboxylate, hydrazide, and alkoxyamine. In some embodiments, the first linking group is a functional group reactive with sulfhydryl groups, such as maleimide, pyridyl disulfide, or haloacetyl. In one embodiment, the first linking group is maleimide.

[0272] In one embodiment, the second linking group is a sulfhydryl group. Any method known to those skilled in the art can be used to introduce a sulfhydryl group onto the targeting domain. In one embodiment, the sulfhydryl group is present on a free cysteine ​​residue. In one embodiment, the sulfhydryl group is achieved by reducing a disulfide on the targeting domain, for example, by reaction with 2-mercaptoethylamine. In one embodiment, the sulfhydryl group is introduced by a chemical reaction, such as the reaction of a free amine with 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA).

[0273] In some embodiments, the polymer-bound lipid and targeting domain are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves the reaction of 1,3-dipole functional groups, such as azides, nitrile oxides, nitrones, and isocyanides, with alkene or alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those skilled in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorocyclooctynes, difluorocyclooctynes, and biarylazacyclooctynones.

[0274] Targeted Domains In one embodiment, the targeting domain specifically binds to a marker of a cell type of interest. For example, in one embodiment, the targeting domain directs the vehicle to endothelial cells, immune cells, stem cells, or another specific cell type of interest. Target cells / tissues that can be targeted include, but are not limited to, T cells, hematopoietic stem cells (HSCs), bone marrow cells, and lung cells. In one embodiment, the targeted LNPs of the present invention comprise a targeting domain that is specific for binding to an antigen on the target cell type of interest.

[0275] 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, insect, yeast, fish, mammalian (including non-human mammalian), and plant cells. In certain embodiments, the cell is an immune cell, e.g., a T cell (e.g., a CD4 + , CD3 + , CD8 + In other embodiments, the cells are pluripotent, totipotent, or multipotent stem cells, such as induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs, e.g., CD34 + ), embryonic stem cells or the like.

[0276] In some embodiments, the methods and compositions of the present invention provide materials useful for editing cells in situ in a subject in need thereof. In some embodiments, the methods involve delivering the composition (e.g., a gene-editing molecule) directly to a tissue. In some aspects, the tissue is a secretory tissue such as the liver, and in other aspects, the tissue has a specific function, such as bone marrow or lung.

[0277] For example, in one embodiment, the LNP comprises a targeting domain specific for binding to an antigen expressed on immune cells. In one embodiment, the LNP comprises a targeting domain specific for binding to an antigen expressed on tumor cells. In one embodiment, the LNP comprises a targeting domain specific for binding to an antigen expressed on a particular tissue type (e.g., a marker expressed on lung tissue).

[0278] In certain embodiments, the targeting domain binds to a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell. In one embodiment, the composition comprises a delivery vehicle attached to a targeting domain that binds to a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell.

[0279] For example, in various embodiments, in the case of compositions targeting endothelial cells, the targeting domain binds to a molecule selected from the group including, but not limited to, 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), plasmalemma vesicle protein-1 (PV1), P-selectin, VE-cadherin, cytokines, plasma proteins, and microbial receptors.

[0280] 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, a T cell (including killer T cells, helper T cells, regulatory T cells, and gamma delta T cells), a natural killer (NK) cell, an antigen-presenting cell, a dendritic cell, a B cell, or a Langerhans cell. In some embodiments, the targeted delivery vehicle comprises a targeting moiety that binds to a surface molecule of a T cell. Exemplary targeting moieties that can be used to target the compositions of the present invention to T cells include, but are not limited to, anti-CD4, anti-CD8, anti-CD5, anti-CD3, or anti-CD25 targeting ligands. Exemplary compositions and methods for targeting T cells in vivo are described in WO 2022 / 081694, WO 2022 / 081699, and WO 2022 / 081702, which are incorporated by reference in their entireties.

[0281] In some embodiments, the targeted delivery vehicle of the present invention comprises a targeting moiety that binds to a surface molecule of stem cells, including, but not limited to, somatic stem cells, mesenchymal stem cells, or hematopoietic stem cells. Exemplary stem cell surface molecules 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 targeting stem cells in vivo are described in International Application Nos. PCT / US22 / 26933 and PCT / US22 / 26981, which are incorporated herein by reference in their entireties.

[0282] However, the present invention is not limited to vehicles that target endothelial cells, immune cells, or stem cells. Rather, the present invention encompasses delivery vehicles that include a targeting domain that targets the vehicle to any specific target cell, mediated by the targeting domain binding to a specific marker. In some embodiments, the vehicle is targeted to a specific therapeutic site where treatment is required. For example, the targeting domain can be specifically targeted to an inflammatory site or can target tumor cells or pathogens.

[0283] The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, etc. that targets the particle to a site specifically requiring the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, meaning the particle comprises multiple targeting mechanisms as described herein.

[0284] In some embodiments, the targeting domain may be copolymerized with a composition comprising a delivery vehicle. In some embodiments, the targeting domain may be covalently attached to a composition comprising a 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. Targeting domains of the present invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0285] In one embodiment, the composition comprises a targeting domain that targets the delivery vehicle to CD34, hi some embodiments, the targeting domain is an affinity ligand that specifically binds to CD34.

[0286] In one embodiment, the composition comprises a targeting domain that targets the delivery vehicle to CD4, hi some embodiments, the targeting domain is an affinity ligand that specifically binds to CD4.

[0287] peptide In one embodiment, the targeting domain of the present invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a marker of a cell type of interest. In one embodiment, the targeting domain directs the vehicle to an endothelial cell, an immune cell, a stem cell, or other specific cell type of interest. For example, in one embodiment, the targeting domain directs the vehicle to CD34 + In one embodiment, the targeting domain targets the vehicle to CD4 + Induce T cells to express

[0288] The peptides of the present invention may be produced using chemical methods. For example, the peptides can be synthesized by solid-phase techniques (Roberge JY et al. (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be performed, for example, using an ABI431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0289] Alternatively, the peptide may be produced by recombinant means or by cleavage from a longer polypeptide. The composition of the peptide may be confirmed by amino acid analysis or sequencing.

[0290] Variants of the peptides of the present invention may include (i) those in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (such substituted amino acid residues may or may not be those encoded by the genetic code); (ii) those in which one or more modified amino acid residues (e.g., residues modified by the addition of a substituent group); (iii) those in which the peptide is an alternative splice variant of a peptide of the present invention; (iv) fragments of the peptide; and / or (v) those in which the peptide is fused to another peptide, such as a leader or secretory sequence or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multiple proteolysis) of the original sequence. Variants may also be post-translationally or chemically modified. Such variants are considered to be within the purview of those skilled in the art from the teachings herein.

[0291] As known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conserved amino acid substitutes with the sequence of another peptide. A variant is defined as comprising a peptide sequence that differs from the original sequence. In some embodiments, the variant differs from the original sequence in fewer than 40% of the residues per segment of interest while retaining the functionality of the original sequence. In some embodiments, the variant differs from the original sequence in fewer than 25% of the residues per segment of interest while retaining the functionality of the original sequence. In some embodiments, the variant differs from the original sequence in fewer than 10% of the residues per segment of interest while retaining the functionality of the original sequence. In some embodiments, the variant differs from the original sequence in several residues per segment of interest while retaining the functionality of the original sequence. The present invention encompasses 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. The degree of identity between two peptides can be determined using computer algorithms and methods well known to those skilled in the art. In some embodiments, identity between two amino acid sequences is determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0292] The peptides of the present invention may be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding dog microsomal membranes or Xenopus egg extract (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0293] The peptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0294] nucleic acid In one embodiment, the targeting domain of the present invention comprises an isolated nucleic acid, 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.

[0295] Alternatively, the nucleotide sequence of the nucleic acid targeting domain can include sequence changes relative to the original nucleotide sequence, e.g., substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid functions similarly to the original nucleic acid and specifically binds to the target cell.

[0296] As used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein if the nucleotide sequence has at least 60%, at least 70%, at least 85%, or at least 95% identity with respect to the nucleotide sequence. Other examples of 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 internally. The degree of identity between two polynucleotides is determined using computer algorithms and methods well known to those skilled in the art. In some embodiments, the identity between two nucleotide sequences is determined 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)].

[0297] antibody In one embodiment, a targeting domain of the invention comprises an antibody or antibody fragment. In certain embodiments, the antibody targeting domain specifically binds to CD34 or CD4. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single-chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0298] The antibody may be a complete monoclonal or polyclonal antibody, an immunologically active fragment (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, a humanized antibody, a genetically engineered single-chain Fv molecule (Ladner et al., U.S. Pat. No. 4,946,778), or a chimeric antibody (e.g., an antibody that contains the binding specificity of a murine antibody, but whose remaining portions are human). Antibodies, including monoclonal antibodies, polyclonal antibodies, fragments, and chimeras, may be prepared using methods known to those skilled in the art.

[0299] 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 production method depends on several factors, including the desired antibody structure, the importance of carbohydrate moieties on the antibody, ease of culture and purification, and cost. Using standard expression techniques, many different antibody structures can be produced, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies containing components from different species. Small antibody fragments, such as Fab and Fv fragments, which lack effector function and have limited pharmacokinetic activity, may be produced in bacterial expression systems. Single-chain Fv fragments exhibit low immunogenicity.

[0300] treatment method In some embodiments, the present invention provides methods for delivering a therapeutic agent to a cellular target for the treatment of a disease or disorder in a subject.

[0301] In some embodiments, the present invention provides a method for targeted delivery of a gene editing agent for the treatment of 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, monogenic blood disorders controlled by a pair of genes, genetic defects, bone marrow genetic defects, cancer, autoimmune diseases, and infectious diseases.

[0302] Exemplary genetic disorders that can be treated using the compositions and methods of the present invention include achondroplasia, alpha 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 thrombosis, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, and cystic fibrosis. These include, but are not limited to, rheumatoid arthritis, Huntington's disease, inborn errors of metabolism, Klinefelter's syndrome, Marfan's syndrome, methylmalonic acidemia, myotonic dystrophy, neurofibromatosis, Noonan's 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's syndrome, velocardiofacial syndrome, and Wilson's disease.

[0303] Infectious diseases that can be treated using the compositions and methods include, but are not limited to, bacterial, viral, parasitic, and fungal infections.

[0304] bacterial infection In one embodiment, the infection or disorder is associated with bacteria. In some embodiments, the bacteria can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusim icrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0305] The bacterium can be a gram-positive or gram-negative bacterium. The bacterium can be aerobic or anaerobic. The bacterium can be autotrophic or heterotrophic. The bacterium can be mesophilic, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or osmophilic.

[0306] The bacteria can be anthrax, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or Clostridium tetanus. The bacteria can be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthraci, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0307] Viral infections In one embodiment, the infection or disorder is associated with a virus, hi some embodiments, the virus is from any one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae. Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae.Viral antigens include human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue fever virus, papillomaviruses such as human papillomavirus (HPV), poliovirus, hepatitis viruses such as 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 leukemia virus (CVE-201002266), and the like. The virus may be derived from an influenza virus, such as encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex type 1 (oral herpes), herpes simplex type 2 (genital herpes), shingles (varicella, also known as chickenpox), cytomegalovirus (CMV), e.g., human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, Lassa fever virus, arenavirus, severe acute respiratory syndrome-related coronavirus (SARS), Middle East respiratory syndrome-related coronavirus (MERS), severe acute respiratory syndrome-related coronavirus 2 (SARSCoV2), or a cancer-causing virus.

[0308] Parasitic infections In one embodiment, the infection or disorder is associated with a parasite. In some embodiments, the parasite can be a protozoan, a helminth, or an ectoparasite. The helminth (i.e., worm) can be a flatworm (e.g., flukes and tapeworms), an anchovy, or a roundworm (e.g., pinworm). The ectoparasite can be a lice, flea, tick, or mite.

[0309] The parasite can be any parasite that causes any one of the following diseases: acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, Baylisscariasis, Chagas disease, clonorchiasis, myiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, tumefacilitatoriasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0310] Parasites include Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chigger, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, and Loa loa, Paragonimus (lung fluke), Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Tick, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.

[0311] fungal infection In one embodiment, the infection or disorder is associated with a fungus. In some embodiments, the fungus is selected from the group consisting of Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, and the like. schenckii), Exserohilum, or Cladosporium.

[0312] The present invention encompasses the delivery of a delivery vehicle comprising at least one gene editing agent to edit at least one disease-associated gene in a subject in need thereof. In one embodiment, the gene is associated with intracellular entry of a bacterial, viral, fungal, or parasitic pathogen. In some embodiments, the gene is associated with a genetic disease or disorder.

[0313] In one embodiment, the delivery vehicle further comprises at least one targeting domain. To practice the methods of the present invention, those skilled in the art will understand how to formulate an appropriate composition and administer it to a subject based on the disclosure provided herein. The present invention is not limited to any particular administration method or treatment regimen.

[0314] The present invention also provides methods for cell-specific delivery of at least one agent to a subject in need thereof. In some embodiments, the agent is a therapeutic agent for the treatment of a disease or disorder. In some embodiments, the disease or disorder is a genetic defect. In some embodiments, the method comprises administering at least one gene editing agent to a target cell (e.g., a stem cell) for the treatment of the genetic defect. In some embodiments, the method comprises administering at least one gene editing agent to a target cell (e.g., a stem cell) for the treatment or prevention of a disease or disorder associated with an infectious agent.

[0315] In some embodiments, the method comprises administering at least one gene editing agent to T cells, hematopoietic stem cells, or a combination thereof for the treatment or prevention of HIV or a disease or disorder associated with HIV infection (e.g., AIDS). In one embodiment, the at least one gene editing agent is Cas9 mRNA, a guide RNA, or a combination thereof. In one embodiment, the guide RNA is specific for binding to CCR5.

[0316] Armed with the present disclosure, including the methods detailed herein, those skilled in the art will understand that the present invention is not limited to the treatment of established diseases or disorders. In particular, the disease or disorder need not be present to the extent that it is detrimental to the subject, and in fact the disease or disorder need not be detected in the subject before treatment is administered. That is, noticeable signs or symptoms of the disease or disorder need not be present before the present invention can provide benefit. Thus, the present invention includes methods for preventing a disease or disorder, in that the compositions already discussed elsewhere herein can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder.

[0317] Those skilled in the art, once armed with the disclosure herein, will understand that preventing a disease or disorder includes administering a composition to a subject as a prophylactic measure against the onset or progression of the disease or disorder.

[0318] Those skilled in the art will understand that the compositions of the present invention can be administered alone or in any combination. Furthermore, the compositions of the present invention can be administered alone or in any combination in a temporal sense, in that they may be administered simultaneously or before and / or after each other. Those skilled in the art will understand, based on the disclosure herein, 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 affect the outcome of treatment. 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 a disease or disorder.

[0319] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In certain embodiments, the method has an additive effect, where 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, where the overall effect of administering the combination of compositions is greater than the sum of the effects of administering each individual composition.

[0320] The method includes administering the combination of compositions in any suitable ratio. For example, in one embodiment, the method includes administering two individual compositions in a 1:1 ratio. However, the method is not limited to a particular ratio. Rather, any ratio shown to be effective is included.

[0321] In some embodiments, the present invention includes methods of preparing a therapeutic composition for delivering at least one agent to endothelial cells present in the lumen of a blood vessel.

[0322] Pharmaceutical Composition The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desirable, distributing or packaging the product in a desired single- or multi-dose unit.

[0323] While the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and veterinary pharmacologists of ordinary skill can design and implement such modifications with routine skill and, if necessary, experimentation. Subjects to which administration of the pharmaceutical compositions of the present invention is contemplated include, but are not limited to, humans and mammals, including other primates, non-human primates, and commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs.

[0324] Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenicity-based formulations.

[0325] The pharmaceutical composition of the present invention may be prepared, packaged, or sold in bulk as a single unit dose or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, for example, half or one-third of such a dosage.

[0326] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route of administration of the composition. For example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0327] The pharmaceutical compositions of the present invention may further comprise, in addition to the active ingredient, one or more additional pharmaceutically active agents.

[0328] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional adjuvants. Exemplary adjuvants include, but are not limited to, aluminum-based adjuvants and monophosphoryl lipid A.

[0329] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention may be prepared using conventional techniques.

[0330] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically puncturing a target tissue and administering the pharmaceutical composition through the puncture. Thus, parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and kidney dialysis infusion techniques.

[0331] Pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending 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 granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0332] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-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 diglycerides. Other useful parenteral formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0333] Pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising 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 are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir capable of directing a propellant stream to disperse the powder, or using a self-propelling solvent / powder dispensing container, e.g., a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some embodiments, such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles (by number) have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles (by number) have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0334] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F (about 20°C) at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components such as a liquid nonionic surfactant or solid diluent or a solid anionic surfactant or solid diluent. In some embodiments, the diluent has a particle size similar to that of the particles comprising the active ingredient.

[0335] Pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending 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 granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0336] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-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 diglycerides. Other useful parenteral formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0337] 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, sweeteners, flavoring agents, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may 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. [Example]

[0338] The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the present invention unless otherwise specified. Therefore, the present invention is not limited to the following examples in any way, but should be construed to encompass any and all variations that become apparent as a result of the disclosure provided herein.

[0339] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed methods. Thus, the following examples are not to be construed as limiting in any way the remainder of the disclosure.

[0340] Example 1: Targeted LNPs for in vivo delivery of RNA-based gene editing tools To date, several mRNA delivery technologies have been developed to target various cell types in vivo, including endothelial cells, hematopoietic stem cells (HSCs), T cells, and other immune cells (Figures 1-7). This disclosure builds on existing targeted delivery platforms to provide a novel therapeutic application for delivering functional mRNA-based gene editing tools to T cells and stem cells for the treatment of HIV infection. Initial in vitro studies have shown that targeted delivery of Cas9 mRNA / CCR5 gRNA to T cells effectively knocked down CCR5 expression on T cells, thereby preventing or attenuating HIV infection (Figure 8). CCR5 is a cell surface chemokine receptor that promotes HIV entry into immune cells and is a valid target for HIV drug development. Targeted knockdown or knockout of CCR5 in both T cells and stem cells can confer durable resistance to HIV infection to the immune system. The long-term goal is to combine this technology with additional gene-editing tools or therapies to interrupt HIV infection and reinfection pathways, and to inactivate or eliminate proviral HIV DNA in the latent HIV reservoir to achieve an HIV cure.

[0341] Compared to the early stages of anti-HIV drug development, today's antiretrovirals with lower toxicity have been developed, and when administered in combination, antiretroviral therapy (ART) drug regimens have significantly reduced mortality and enabled the management of HIV / AIDS as a chronic infection. However, ART has yet to eliminate persistent / latent HIV cell reservoirs. These persistent HIV cell reservoirs remain a challenging challenge to achieve an HIV cure due to their frequency, slow elimination rate, and the lack of an efficient targeted delivery system. One potential cure for HIV involves inactivating proviral DNA in the latent HIV reservoir and targeting CD34. + modifying the CCR5 coreceptor on HSCs (or differentiated CD4 T cells) to resist future HIV infection, respectively. +The key to success may be to promote the expression of mRNA-based gene editing tools in T cells and HSCs. Therefore, the development of mRNA-LNP therapeutics, such as mRNA-based gene editing, has the potential to change current HIV treatment methods. CD4-targeted nucleoside-modified mRNA-LNPs have demonstrated efficient and specific delivery in vitro and in vivo. Comprehensive radioactivity- or luminescence-based biodistribution analysis of CD4-targeted mRNA-LNPs revealed the CD4 expression in lymphoid organs. + Significant targeting of T cells was demonstrated. The CD4-targeted mRNA-LNP platform induces potent and specific gene editing in vivo using a Cre / loxP reporter system. Preliminary results also demonstrated effective CCR5 knockout in T cells (Figure 8).

[0342] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. 1. A composition for targeted delivery of a gene editing agent to a target cell or particle of interest, the composition comprising at least one RNA molecule comprising or encoding the gene editing agent and a delivery vehicle, the delivery vehicle comprising a specific targeting moiety for binding to the cell or particle of interest.

2. 10. The composition of claim 1, wherein the agent comprises at least one isolated nucleoside-modified RNA molecule encoding a Cas9 protein.

3. The composition of claim 2, wherein the composition further comprises a guide RNA.

4. 4. The composition of claim 1, wherein the target cell or particle is selected from the group consisting of a stem cell, an immune cell, an endothelial cell, a bacterial cell, a viral particle, a fungal cell, and a parasitic cell.

5. The composition of claim 1 , wherein the target cells are hematopoietic stem cells.

6. The composition of claim 1 , wherein the target cell or particle is a T cell.

7. 7. The composition of any one of claims 1 to 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 of claim 7, wherein the guide RNA molecule targets CCR5.

9. 9. The composition of claim 1, wherein the targeting moiety is specific for binding to CD4.

10. 9. The composition of claim 1, wherein the targeting moiety is specific for binding to CD34.

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

12. 12. The composition of claim 1, wherein the delivery vehicle comprises a lipid nanoparticle (LNP).

13. The composition of claim 12, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

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

15. 15. The method of claim 14, wherein the disease or disorder is selected from the group consisting of a genetic defect and an infectious disease.

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

17. 1. A method of treating HIV, the method comprising administering a composition comprising a therapeutic agent and a delivery vehicle, the delivery vehicle comprising a CD34 ＋ The method, wherein the therapeutic agent comprises a specific targeting moiety for binding to hematopoietic stem cells, and the therapeutic agent comprises an mRNA molecule comprising a Cas9 protein and a guide RNA specific for CCR5.

18. 1. A method of treating HIV, said method comprising administering a composition comprising a therapeutic agent and a delivery vehicle, said delivery vehicle comprising a CD4 ＋ The method, wherein the therapeutic agent comprises a specific targeting moiety for binding to T cells, and the therapeutic agent comprises an mRNA molecule comprising a Cas9 protein and a guide RNA specific for CCR5.

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