Compositions and methods for extrahepatic delivery of therapeutic substances targeting T cells

Abstract

This disclosure relates in part to lipid nanoparticle (LNP) compositions targeting immune cells, and to methods of using them for in vivo delivery of nucleic acid molecules and / or therapeutic substances to target cells. In certain embodiments, the target cells are located in the spleen of a subject. In certain embodiments, the nucleic acid molecules encode chimeric antigen receptors (CARs). In certain embodiments, the target cells are T cells. In certain embodiments, this disclosure relates to the use of the LNPs described herein for the treatment, prevention, and / or improvement of diseases and / or disorders, including but not limited to cancer. TIFF2026531625000043.tif64170

Description

[Technical Field]

[0001] Statement regarding federally funded research or development. This invention was made with government support under TR002776, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 581,876, filed on 11 September 2023 under 35 U.S.C. § 119(e), which is incorporated herein by reference in its entirety. [Background technology]

[0003] background Chimeric antigen receptor (CAR) T-cell therapy is reshaping the landscape of cancer immunotherapy, with six FDA-approved CAR T-cell immunotherapies for the treatment of relapsed or refractory acute lymphoblastic leukemia (ALL), B-cell lymphoma, and multiple myeloma, and additional cancer applications under investigation. Today, the production of these potent autologous cell therapies relies on the complex process of ex vivo cell engineering.

[0004] In short, patient T cells are isolated, modified with a virus to express transmembrane CAR constructs, and then reinjected into the patient. These CAR T cells then target and eliminate cancerous B cells, using the patient's own immune system to eradicate the cancer. However, because CAR expression is induced by a virus and is therefore persistent and potent, these CAR T cells also attack healthy B cells after eliminating cancerous cells. This can lead to adverse effects such as cytokine release syndrome, long-term B cell aplasia, and pancytopenia, exposing the patient to the risk of serious infections.

[0005] Accordingly, there is a need in the art for new approaches to engineering CAR-T cells that mitigate the severity of these off-target effects while providing potent cancer cell killing. The present disclosure addresses and meets this unmet need. Summary of the Invention

[0006] Summary In one aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising: (a) Formula (I): an ionizable lipid compound having the structure of TIFF2026531625000002.tif41147, or a salt thereof, wherein wherein A1, A2, L1, L2, L3, L4, L5, L6, R1, R2, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16a , R 16b , R 17 , R 18 , R 19 m, n, o, p, q, r, s, t, u, v, w, and x are as defined elsewhere herein, and the compound having the structure of Formula (I), or a salt thereof, constitutes from about 25 mol% to about 45 mol% of the LNP, the ionizable lipid compound or a salt thereof; (b) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which constitutes approximately 10 mol% to 20 mol% of LNP; (c) Cholesterol lipids, which make up approximately 40 mol% to 50 mol% of LNP; (d) Polyethylene glycol (PEG) conjugate lipids and / or modified derivatives thereof constituting approximately 0.5 mol% to approximately 5.0 mol% of the LNP; and (e) A cell targeting domain that is specific to binding to surface molecules of target cells, and is covalently conjugated to at least one component of an LNP.

[0007] In a particular embodiment, the ionizable lipid of formula (I) is 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azandiyl)bis(tetradecane-2-ol): Includes TIFF2026531625000003.tif52140.

[0008] In a particular embodiment, the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.5.

[0009] In another aspect, the Disclosure provides a pharmaceutical composition comprising at least one LNP of the Disclosure and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present disclosure provides a method for delivering at least one selected from the group consisting of nucleic acid molecules and therapeutic substances to target cells, the method comprising administering to the subject a therapeutically effective amount of at least one LNP of the present disclosure and / or a pharmaceutical composition of the present disclosure. [Brief explanation of the drawing]

[0011] The drawings generally illustrate various aspects of the present application by way of example, not by way of limitation. [Figure 1A] Schematic diagram of conventional CAR T cell engineering methods and the exemplary CAR T cell engineering method of the present disclosure. In conventional LNP-based T cell transfection methods, LNPs are combined with patient T cells ex vivo to generate CAR T cells outside the body. In the Ab-LNP-based T cell transfection of the present disclosure, Ab-LNP is administered to a patient such that CAR T cell therapy is generated in vivo, wherein the antibody promotes T cell targeting. [Figure 1B] Non-limiting schematic diagram of Ab-LNP formation. An ethanol phase containing LNP components and an aqueous phase containing mRNA cargo are combined via microfluidic mixing to produce mal-LNPs comprising mal-PEG on their surface. These are combined with cleaved and reduced antibody fragments, which conjugate to the mal-LNPs to form Ab-LNPs. [Figure 1C] Structures of the clinical standard MC3 lipid and the C14-4 ionizable lipid used to prepare certain exemplary LNPs of the present disclosure, as well as representative IVIS images of their respective in vivo performance demonstrating extrahepatic delivery using C14-4. [Figure 2-1]Figures 2A to 2E: Exemplary Ab-LNPs show increased size and efficacy compared to mal-LNPs. Figures 2A to 2C: A library of mal-LNPs was formulated by varying the mal-PEG:PEG ratio while keeping the ratio of excipient substances constant, allowing various amounts of anti-CD5 human antibody to be bound to the surface of mal-LNPs (Figure 2A). The library was then measured using DLS to observe changes in size before and after conjugation, showing a representative DLS curve for 1:5 mal-LNP (Figure 2B) and Ab-LNP, and summarizing the average diameter (average peak intensity) (n=3, error bar = standard deviation). Statistical analysis included one-way ANOVA with Bonferroni correction, and **p<0.001 compared with B10 (Figure 2C). Figures 2D to 2E: Screening of a library of mal-LNPs and Ab-LNPs was performed in Jurkat cells (CD5+) at a dose of 50 ng / 60,000 cells to measure delivery and cell viability of luciferase-encoding mRNA at the 24-hour time point (n=3 biological replicates, error bar = standard deviation). Results of luminescence measurements were normalized to B10 delivery, and viability was normalized to untreated cells. Statistical analysis included one-way ANOVA with Bonferroni correction, and **p<0.001 compared with B10. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3A]Figures 3A-3C: Dose-response and kinetics of Jurkat transfection using exemplary 1:5 LNP. Figures 3A-3B: Luciferase expression (Figure 3A) and viability (Figure 3B) of Jurkat cells treated with luciferase-encoding mRNA using 1:5 mal-LNP and Ab-LNP at a series of mRNA doses over 24 hours to confirm the potency and biocompatibility of Ab-LNP (n=3 biological repeats, error bars = standard deviation). Luminescence measurements were normalized to B10 delivery, and viability was normalized to untreated cells. For both measurements, results for each treatment group within each dose were compared using one-way ANOVA with Bonferroni correction, with *p<0.05 compared to B10. Figure 3C: Luciferase expression of Jurkat cells treated with 50 ng / 60,000 cells of 1:5 LNP over 0-24 hours (n=3 biological repeats, error bars = standard deviation). Statistical analysis included a two-way ANOVA with Bonferroni correction, and the p-value was *0.05 when compared with B10 at the same dose. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4A] Figures 4A-4C: The in vivo distribution of Ab-LNP is favorable for spleen delivery compared to standard LNP formulations. Figure 4A: Representative DLS curves of exemplary LNP-treated groups demonstrating size change after antibody conjugation. Figure 4B: Representative IVIS images of organs taken from mice 6 hours after intravenous injection of a specific exemplary LNP containing 0.6 mg / kg of luciferase-encoding mRNA. Figure 4C: Measured luminescence radiance of the liver, spleen, and lymph nodes (LNs) as target regions on IVIS images, and normalized to background (n=4 biological replicates, error bars = standard deviation). The normalized luminescence ratios for the spleen and liver of each mouse are also summarized. Statistical analysis included one-way ANOVA with Dunnett correction, with *p<0.05 and **p<0.001 compared to the MC3-treated group. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5-1] Figures 5A–5H: Ab-LNPs demonstrate improved delivery to T cells in vivo compared to non-targeting LNPs. Figures 5A–5D: Transfection rates in blood (Figure 5B), spleen (Figure 5C), and lymph nodes (Figure 5D) of B cells (CD19+), macrophages (CD11b+), and T cells (CD3+) 6 hours after intravenous injection of 0.6 mg / kg of LNPs containing GFP-encoding mRNA, measured using flow cytometry as depicted in a representative experimental scheme (Figure 5A) (biological replicates of n=4, error bars = standard deviation). Statistical analysis included two-way ANOVA with Dunnett correction, with *p<0.05 and **p<0.0001 in marked comparisons. Figures 5E-5H: Transfection rates in blood and spleen of T cells (Figure 5F), macrophages (Figure 5G), and B cells (Figure 5H) at various time points after intravenous injection of 0.6 mg / kg of LNP containing GFP-encoding mRNA, as depicted in the representative experimental scheme (Figure 5E) (n=4 biological replicates, error bars = standard deviation). Statistical analysis included two-way ANOVA with Tukey correction, with *p<0.05 and **p<0.001 compared to B10 at the same time point unless otherwise noted. [Figure 5-2] See the explanation in Figure 5-1. [Figure 5-3] See the explanation in Figure 5-1. [Figure 5-4] See the explanation in Figure 5-1. [Figure 5-5] See the explanation in Figure 5-1. [Figure 6-1]Figures 6A–6C: Exemplary increasing doses of CD3-LNPs show a decrease in T cell population and signs of toxicity; transfection rates of T cells in the blood (Figure 6A), normalized T cell counts (Figure 6B), and measured serum ALT and AST levels (Figure 6C) (n=3) 24 hours after intravenous injection of various doses of LNPs encapsulating GFP-encoding mRNA. T cell populations were measured as a percentage of single cell populations, and T cell counts, ALT, and AST were normalized relative to the PBS-treated group. Statistical analysis included two-way ANOVA with Dunnett correction, with *p<0.05 and **p<0.001 compared to PBS. [Figure 6-2] See the explanation in Figure 6-1. [Figure 7A] Figures 7A-7B: Ab-LNPs generate functional CAR T cells in vivo. Figure 7A: Transfection rates of T cells in the blood and mean fluorescence intensity (of CAR staining) at various time points after CAR mRNA delivery at low (0.5 mg / kg) and high (2 mg / kg) doses (n=4). Statistical analysis included a two-way ANOVA with Dunnett correction, with *p<0.05 and **p<0.0001 compared to PBS at the same time points. Figure 7B: In these same treatment groups, B cell depletion indicating CD19-specific CAR T cell activity was calculated as the percentage reduction in B cells present in a single cell population compared to mice treated with PBS. An additional group of LNPs containing luciferase-encoding mRNA was included as a negative control for CAR function (n=4). Statistical analysis included a two-way ANOVA with Tukey post-hoc correction, with *p<0.05 and **p<0.0001 compared to PBS at the same time points. [Figure 7B] See the explanation in Figure 7A. [Figure 8-1]Figures 8A-8C: Serum cytokine levels in CAR mRNA-treated mice. Serum concentrations of IL-6 (Figure 8A), GM-CSF (Figure 8B), and TNF-α (Figure 8C) at various time points after delivery of CAR mRNA at low (0.5 mg / kg) and high (2 mg / kg) doses, normalized to PBS-treated mice (n=4). Statistical analysis included a two-way ANOVA with Dunnett correction, with *p<0.05 and **p<0.0001. [Figure 8-2] See the explanation in Figure 8-1. [Modes for carrying out the invention]

[0012] Detailed description of the invention Hereafter, detailed references are made to certain aspects of the disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. The disclosed subject matter is described in conjunction with the enumerated claims, but it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0013] Throughout this specification, values ​​expressed in range form should be interpreted flexibly, including not only the numerical values ​​explicitly listed as limits to the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly listed. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The notation "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise indicated. Similarly, the notation "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise indicated.

[0014] In this specification, unless otherwise clearly determined by context, the terms “a,” “an,” or “the” are used to include one or more than one. The term “or” is used to refer to a non-exclusive “or” unless otherwise indicated. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.” In addition, it should be understood that any expressions or technical terms adopted herein and not otherwise defined are for illustrative purposes only and not for limitation. Any use of section headings is intended to aid the reading of this specification and should not be construed as limiting; information related to a section heading may appear within or outside that particular section. All publications, patents, and patent documents referenced herein are incorporated herein by reference in whole, as they are incorporated individually by reference.

[0015] In the methods described herein, the acts may be performed in any order unless a temporal or operational order is explicitly listed. Furthermore, the prescribed acts may be performed in parallel unless explicitly stated in the language of the claim that they are to be performed separately. For example, the claimed act of performing X and the claimed act of performing Y may be performed simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0016] explanation As described elsewhere in this specification, there is a need in the art for novel approaches to engineering CAR T cells that mitigate the severity of these off-target effects while providing potent cancer killing. mRNA-based cell engineering offers numerous advantages compared to viral transduction. From a production standpoint, mRNA avoids concerns commonly associated with viral vectors, such as cargo capacity limitations, insertional mutagenesis, and in vivo immunogenicity. Additionally, mRNA induces transient CAR expression, thereby mitigating the risks associated with long-term CAR T cell activity. Specifically, this transient CAR expression allows for temporal control of CAR T cell therapy to prevent the prolonged presence of CAR T cells in the absence of cancer cells. Overall, this potential has led to the evaluation of mRNA CAR T cell therapy in a variety of cancers, including melanoma, Hodgkin lymphoma, and ALL, demonstrating their ability to reduce short-term disease burden as effectively as virally engineered CAR T cells. However, because mRNA is rapidly degraded and does not easily pass through the cell membrane, an optimized delivery method is required to produce these mRNA CAR T cells.

[0017] One promising strategy for mRNA delivery is the use of nanoparticles, as they can mitigate cytotoxicity, stabilize mRNA cargoes, and enhance intracellular delivery. Specifically, lipid nanoparticles (LNPs) have been utilized as a platform for potent mRNA delivery to diverse cell types because their ionizable lipids become charged in acidic environments, promoting endosomal escape. Furthermore, their clinical applications have led to multiple FDA approvals, including Alnylam's Onpattro siRNA LNP therapy and Moderna and Pfizer-BioNTech's COVID-19 mRNA LNP vaccines, making them a potentially ideal platform for mRNA delivery to other immune cells, such as T cells. Previous studies have demonstrated potent T cell transfection using such LNPs, and optimized LNP platforms for ex vivo synthesis of mRNA-based CAR T cells have been established through excipient screening.

[0018] However, the costs and complexities associated with ex vivo CAR T cell engineering may hinder its widespread clinical implementation despite its therapeutic efficacy, necessitating the investigation of alternative production strategies such as in vivo CAR T cell engineering. Delivering CAR mRNA to T cells in vivo, thereby avoiding the processes of leukocyte apheresis and ex vivo T cell expansion, would streamline production and avoid the need for patient-specific manufacturing (Figure 1A). However, numerous barriers complicate in vivo T cell transfection, including the constant motility of circulating T cells, which can reduce the duration of LNP-T cell interactions, and hepatic clearance of LNPs, which can limit the bioavailability of LNPs.

[0019] To overcome this first barrier and improve LNP-T cell association, other nanoparticle platforms have utilized antibody conjugation to alter biodistribution, increase specificity and minimize off-target effects, and enhance delivery to target cell populations both in vitro and in vivo. Many of these benefits have been demonstrated for T cell applications using polymeric NPs or polymeric LNPs targeting various receptors including CD3, CD8, CD4, CD7, CD5, Nrp1, and β7. However, no study has yet directly compared the efficacy of these various antibodies against different T cell markers to establish the best candidates for targeted mRNA delivery in vivo.

[0020] Furthermore, most antibody-based targeting strategies do not utilize nanoparticle platforms that are also designed for delivery to extrahepatic tissues. Thus, targeted nanoparticle platforms face not only the challenge of reaching the desired cell population, but also the challenge of overcoming hepatic accumulation. As described herein, antibody conjugation targeting strategies have been applied to LNP platforms that achieve extrahepatic delivery to aid T cell targeting in vivo.

[0021] Therefore, in one aspect, this disclosure relates to the design, development, and evaluation of antibody-coated LNP platforms (Ab-LNPs) for T cell targeting and demonstrates their potential for in vivo CAR T cell engineering. To construct Ab-LNPs, a previously established B10 LNP platform containing C14-4 ionizable lipids was modified to include maleimide-functionalized PEG (mal-PEG). This is because previous studies have confirmed this strategy for antibody-nanoparticle conjugation (Figure 1B). To adapt this method for use with B10 LNPs, the optimized excipient molar ratio of mal-PEG was determined using in vitro screening in Jurkat cells, which demonstrated enhanced T cell transfection with minimal toxicity. These Ab-LNPs were then explored for in vivo T cell transfection.

[0022] Here, we investigated only pan-T cell markers, excluding T cell subpopulations (e.g., CD4+ or CD8+ T cells) that may be unfavorable for immunotherapeutic applications such as CAR T cell therapy. Specifically, we formulated Ab-LNPs targeting the pan-T cell markers CD3, CD5, and CD7, and compared their in vivo distribution with B10 LNPs and clinically relevant DLin-MC3-DMA LNPs. While many non-targeting LNPs have been reported to primarily migrate to the liver, thus demonstrating a bias that is counterproductive to delivery to desired immune cells, all C14-4 LNPs demonstrated a bias towards delivery to the spleen rather than the liver, and Ab-LNPs demonstrated low levels of hepatic delivery, indicating their ability to bypass this organ (Figure 1C). When the immune cell population was specifically observed, non-targeting LNPs failed to demonstrate in vivo T cell transfection, while only CD3-LNPs and higher doses of CD7-LNPs demonstrated significant delivery to T cells.

[0023] Next, we explored the in vivo delivery of non-limiting exemplary therapeutic cargo (i.e., mRNA encoding CD19 CAR) using these two Ab-LNPs. Both platforms resulted in a significant circulating CAR T cell population, with CD3-LNPs generating CAR T cells that persisted for up to 60 hours after administration. Furthermore, these Ab-LNPs led to significant depletion of circulating B cells, demonstrating their efficacy in eliminating B cell populations and thereby demonstrating their therapeutic potential for the treatment of B cell cancer. While many cytokines are associated with cytokine release syndrome observed in patients treated with CAR T cell therapy, we explored three of the cytokines considered relevant: IL-6, TFN-α, and GM-CSF. The levels of these cytokines showed transient, dose-dependent increases in serum, supporting the potential of Ab-LNPs to mitigate cytokine release and enable repeated dosing.

[0024] In summary, evaluation of three Ab-LNP platforms targeting pan-T cell markers for T cell transfection revealed that CD3-LNP and CD7-LNP are delivery platforms capable of generating functional CAR T cells in vivo, with dose-dependent effects on cytokine release. Thus, Ab-LNP is confirmed to be a platform for in vivo CAR T cell production.

[0025] Therefore, in one aspect, this disclosure provides an extrahepatic-targeted Ab-LNP platform for in vivo CAR T cell engineering. Exemplary Ab-LNPs were screened in Jurkat cells to determine optimized antibody densities by incorporating a series of mal-PEG to PEG ratios into the LNP formulations using antibodies against CD5. These Ab-LNPs were then produced using antibodies against the pan-T cell markers CD3, CD5, or CD7, thereby enabling comparison of these T cell targets and confirming the versatility of this platform. In vivo screening of these LNPs using different reporter cargoes revealed the ability of C14-4 LNPs to achieve a higher rate of spleen delivery compared to hepatic delivery than the clinical standard MC3, demonstrating Ab-LNPs' T cell transfection capabilities. Specifically, CD3-LNPs and CD7-LNPs were able to achieve significant transfection in circulating T cells with greater specificity compared to other immune cell types. Subsequently, these two Ab-LNP platforms were formulated using CAR-encoding mRNA, and they achieved significant CAR positivity in vivo, accompanied by potent B cell depletion. Therefore, the development of Ab-LNPs, including CD3-LNP and CD7-LNP, was confirmed as a means of producing functional CAR T cells in vivo. Thus, Ab-LNPs demonstrate promise as a platform for CAR T cell engineering and other applications in T cell engineering.

[0026] definition As used herein, the term “about” allows for some variability in the value or range, for example, within 10%, 5%, or 1% of the limit of the described value or range, and includes the described value or range itself.

[0027] As used herein, the term “adjuvant” is defined as any molecule that enhances an antigen-specific adaptive immune response.

[0028] As used herein, the term “alkenyl” refers to linear alkyl groups, branched alkyl groups, and cyclic alkyl groups as defined herein, except that they have at least one double bond between two carbon atoms. Thus, alkenyl groups have 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments, 2 to 8 carbon atoms. Examples include, but are not limited to, vinyl, -CH=C=CH2, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.

[0029] As used herein, the term “alkoxy” refers to an oxygen atom connected to an alkyl group, including cycloalkyl groups as defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group may contain about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to an oxygen atom, and may further contain double or triple bonds, and may also contain heteroatoms. For example, the allyloxy group or the methoxyethoxy group are also alkoxy groups in the sense of this specification, and the methylenedioxy group is also an alkoxy group in the context in which two adjacent atoms in the structure are substituted with methylenedioxy groups.

[0030] As used herein, the term “alkyl” refers to linear and branched alkyl groups, as well as cycloalkyl groups, having 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbon atoms, or in some embodiments, 1 to 8 carbon atoms. Examples of linear alkyl groups include those having 1 to 8 carbon atoms, such as the methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, and n-octyl group. Examples of branched alkyl groups include, but are not limited to, the isopropyl group, isobutyl group, sec-butyl group, t-butyl group, neopentyl group, isopentyl group, and 2,2-dimethylpropyl group. As used herein, the term “alkyl” encompasses n-alkyl groups, isoalkyl groups, and anteisoalkyl groups, as well as other branched forms of alkyl groups. Typical substituted alkyl groups are any of the groups listed herein, for example, amino groups, hydroxyl groups, cyano groups, carboxyl groups, nitro groups, thio groups, alkoxy groups, and halogen groups, which may be substituted once or multiple times.

[0031] As used herein, the term "alkynyl" refers to both linear and branched alkyl groups, except that they contain at least one triple bond between two carbon atoms. Thus, alkynyl groups have 2 to 40 carbon atoms, 2 to about 20 carbon atoms, 2 to 12 carbon atoms, or in some embodiments, 2 to 8 carbon atoms. Examples include, but are not limited to, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and CH2C≡C(CH2CH3).

[0032] As used herein, the term “amine” refers to primary, secondary, and tertiary amines having, for example, formula N(group)3, where each group can independently be H or non-H such as alkyl, aryl, and the like. Amines include, but are not limited to, R-NH2, e.g., alkylamines, arylamines, alkylarylamines; R2NH, where each R is independently selected, e.g., dialkylamines, diarylamines, aralkylamines, heterocyclylamines, and the like; and R3N, where each R is independently selected, e.g., trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. As used herein, the term “amine” also includes the ammonium ion.

[0033] As used herein, the term "amino group" refers to -NH2, -NHR, -NR2, -NR3, where each R is independently selected. + Substituents in the form of, and -NR3 which cannot be protonated + This refers to each protonation form excluding the one specified above. Therefore, any compound substituted with an amino group can be considered an amine. In this specification, "amino group" can be a primary, secondary, tertiary, or quaternary amino group. The "alkylamino" group includes monoalkylamino groups, dialkylamino groups, and trialkylamino groups.

[0034] 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 linked to neutral lipids.

[0035] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or they can be the immunoreactive moiety of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0036] The term "antibody fragment" refers to a portion of an intact antibody, and specifically to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0037] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structure.

[0038] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structures. κ-light chain and λ-light chain refer to the two main antibody light chain isotypes.

[0039] As used herein, the term “synthetic antibody” means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage. The term should also be interpreted as an antibody produced by the synthesis of an antibody-encoding DNA molecule, wherein the DNA molecule expresses an antibody protein or an amino acid sequence defining the antibody, and the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technology available and well known in the art. The term should also be interpreted as an antibody produced by the synthesis of an antibody-encoding RNA molecule, wherein the RNA molecule expresses an antibody protein or an amino acid sequence defining the antibody, and the RNA is obtained by the transcription of DNA (synthesized or cloned) or by other technology available and well known in the art.

[0040] As used herein, the terms “antigen” or “Ag” are defined as molecules that elicit an adaptive immune response. This immune response may involve either antibody production or activation of a given immunogenic eligible cell, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an adaptive immune response, therefore, encodes an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a full-length nucleotide sequence of a gene alone. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It is readily apparent that antigens can be produced and synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0041] As used herein, the term “aryl” refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in its ring. Thus, aryl groups include, but are not limited to, phenyl, azlenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenantrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbon atoms in the ring portion of the group. Aryl groups may be unsubstituted or substituted as defined herein. Typical substituted aryl groups may be monosubstituted or multiple substituted, such as a phenyl group substituted at one or more positions 2, 3, 4, 5, or 6 of the phenyl ring, or a naphthyl group substituted at one or more positions 2 through 8.

[0042] The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as a physiological pH (e.g., pH approximately 7.0). Cationic lipids containing alkyl chains with multiple unsaturated sites, e.g., at least two or three unsaturated sites, have been found to be particularly useful in forming lipid particles with increased membrane fluidity. Numerous cationic lipids and related analogues useful in this disclosure are described in U.S. Patent Applications Publications 20060083780 and 20060240554; U.S. Patents 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and International Publication No. 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids have a head group that is a protonable tertiary amine (e.g., pH titrable), C 18It contains an alkyl chain, an ether linkage between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0043] As used herein, the term "cycloalkyl" refers to cyclic alkyl groups, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups may have 3 to about 8 to 12 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups, including but not limited to norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and calenyl groups, as well as fused rings, including but not limited to dekalinyl and similar groups. Cycloalkyl groups also include rings substituted with linear or branched alkyl groups as defined herein. Typical substituted cycloalkyl groups are not limited to those substituted with amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups, such as 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl groups, or monosubstituted, disubstituted, or trisubstituted norbornyl or cycloheptyl groups, which can be monosubstituted or multiple times. The term "cycloalkenyl" alone or in combination refers to a cyclic alkenyl group.

[0044] A "disease" is a health condition in which an animal is unable to maintain homeostasis, and if the disease is not improved, the animal's health will continue to deteriorate. In contrast, a "disability" in animals is a health condition in which the animal is able to maintain homeostasis, but the animal's health is less desirable than if there were no disability. Even if left untreated, a disability does not necessarily lead to a further decline in the animal's health.

[0045] As used herein, the terms “effective dose,” “pharmaceutical effective dose,” and “therapeutic effective dose” refer to a non-toxic but sufficient amount of an active substance to produce a desired biological outcome. This outcome may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The appropriate therapeutic dose in any individual case can be determined by those skilled in the art using routine experiments.

[0046] In particular, in the case of mRNA, the “effective amount” or “therapeutic effective amount” of a therapeutic nucleic acid, as relating to mRNA, is an amount sufficient to produce the desired effect, for example, an amount sufficient to produce mRNA-directed expression of a protein that produces the desired biological effect in the organism in which the protein is expressed. For example, in some embodiments, the expressed protein is the active form of a protein normally expressed in a cell type in the body, and the therapeutic effective amount of mRNA is an amount that produces an amount of encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of protein normally expressed in a cell type in a healthy individual. For example, in some embodiments, the expressed protein is a protein normally expressed in a cell type in the body, and the therapeutic effective amount of mRNA is an amount that produces an expression level in an individual with abnormal protein expression (i.e., a protein-deficient individual) similar to that observed in a healthy individual. Suitable assays for measuring mRNA or protein expression include, but are not limited to, dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function assays, and phenotypic assays known to those skilled in the art.

[0047] As used herein, the term “encodes” refers to the products (e.g., proteins and RNA) defined by a given nucleotide sequence in nucleic acids (i.e., DNA and / or RNA), respectively, during the transcription or translation of DNA or RNA. In certain embodiments, the term “encodes” refers to the RNA sequence defined by the transcription of a DNA sequence. In certain embodiments, the term “encodes” refers to the amino acid sequence (e.g., polypeptides or proteins) defined by the translation of mRNA. In certain embodiments, the term “encodes” refers to the amino acid sequence defined by the transcription from DNA to mRNA and the subsequent translation of mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may include the direct transcript or translation product. In certain embodiments, the encoded product may include post-translational modifications that are understood or reasonably expected by those skilled in the art.

[0048] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the 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 known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), incorporating recombinant polynucleotides.

[0049] The term "fully encapsulated" indicates that the active or therapeutic substance in the lipid particles is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, in a treatment that would normally degrade 100% of the free active or therapeutic substance, preferably less than about 25% of the active or therapeutic substance in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5%. In the context of therapeutic substances that are nucleic acids, fully encapsulated can be determined by the OLIGREEN® assay. OLIGREEN® is an ultra-sensitive fluorescent nucleic acid dye for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). "Fully encapsulated" also indicates that the lipid particles are serum stable, i.e., that the lipid particles do not rapidly disintegrate into their constituent parts upon in vivo administration.

[0050] When used herein, the terms “halo” group, “halogen” group, or “halide” group mean, unless otherwise specified, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, either by themselves or as part of another substituent.

[0051] The term "haloalkyl" group, as used herein, includes monohaloalkyl groups, polyhaloalkyl groups in which all halo atoms may be the same or different, and perhaloalkyl groups in which all hydrogen atoms are replaced by halogen atoms such as fluoro. Examples of haloalkyl groups include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and similar groups.

[0052] As used herein, the term “helper lipid” refers to a lipid capable of increasing the effectiveness of delivery of lipid-based particles, such as cationic lipid-based particles, to a target, preferably into cells. Helper lipids can be neutral, positively charged, or negatively charged. In certain embodiments, helper lipids are neutral or negatively charged. Non-limiting examples of helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the health status of animals.

[0053] As used herein, the term “heteroaryl” refers to an aromatic ring compound containing five or more ring members, wherein one or more of the ring members are heteroatoms, such as but not limited to N, O, and S; for example, a heteroaryl ring can have five to about eight to twelve ring members. A heteroaryl group is a type of heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as C2-heteroaryl can be a five-ring with two carbon atoms and three heteroatoms, or a six-ring with two carbon atoms and four heteroatoms, and so on. Similarly, a C4-heteroaryl can be a five-ring with one heteroatom, or a six-ring with two heteroatoms, and so on. The sum of the number of carbon atoms and the number of heteroatoms is equal to the total number of ring atoms. Heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, prinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups may be unsubstituted or substituted with groups as discussed herein. Typical substituted heteroaryl groups may be substituted once or multiple times with groups such as those listed herein.

[0054] Examples of added aryl and heteroaryl groups include phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), and indenyl. Ryl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrrolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazole-1-yl, 1,2,3-triazole-2-yl) 1,2,3-Triazole-4-yl, 1,2,4-Triazole-3-yl), Oxazolyl (2-Oxazolyl, 4-Oxazolyl, 5-Oxazolyl), Thiazolyl (2-Thiazolyl, 4-Thiazolyl, 5-Thiazolyl), Pyridyl (2-Pyridyl, 3-Pyridyl, 4-Pyridyl), Pyrimidinyl (2-Pyrimidinyl, 4-Pyrimidinyl, 5-Pyrimidinyl, 6-Pyrimidinyl), Pyrazinyl, Pyridazinyl (3-Pyridazinyl, 4-Pyridazinyl, 5-Pyridazinyl), Quinolyl (2-Quinolyl, 3-Quinolyl, 4-Quinolyl, 5-Quinolyl, 6-Quinolyl, 7-Quinolyl, 8-Quinolyl), Isoquinolyl (1-Isoquinolyl, 3- Isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-Dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl) 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indazolyl, 2-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl) Zolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepine-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5 H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,This includes, but is not limited to, azepine-5-yl (f) and similar substances.

[0055] As used herein, the term “heterocycloalkyl” refers to aliphatic, partially unsaturated or fully saturated 3- to 14-membered ring systems, including monocyclic rings of 3 to 8 atoms, as well as bicyclic and tricyclic ring systems in which at least one of the carbon atoms of the ring is replaced by a heteroatom, such as but not limited to nitrogen, oxygen, sulfur, or phosphorus. Heterocycloalkyls can independently contain 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur, where the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may optionally be substituted. Representative heterocycloalkyl groups include, but are not limited to, the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0056] As used herein, the term “heterocyclyl” refers to aromatic and non-aromatic ring compounds containing three or more ring members, wherein one or more of the ring members are heteroatoms, such as but not limited to N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl or heteroaryl, or, in the case of a polycyclic compound, any combination thereof. In some embodiments, a heterocyclyl group contains 3 to about 20 ring members, while other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with 2 carbon atoms and 3 heteroatoms, or a 6-ring with 2 carbon atoms and 4 heteroatoms, and so on. Similarly, a C4-heterocyclyl can be a 5-ring with 1 heteroatom, or a 6-ring with 2 heteroatoms, and so on. The number of heteroatoms plus the number of carbon atoms equals the total number of ring atoms. A heterocyclyl ring can also contain one or more double bonds. A heteroaryl ring is one form of a heterocyclyl group. The term "heterocyclyl group" includes fused ring species, including those containing fused aromatic and non-aromatic groups. For example, the dioxolanyl ring and the benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups in the sense of this specification. This term also includes, but is not limited to, polycyclic ring systems containing heteroatoms, such as quinuclidyl. Heterocyclyl groups can be unsubstituted or substituted, as discussed herein.Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, prinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Typical substituted heterocyclyl groups include, but are not limited to, groups such as those listed herein, which may be monosubstituted or multiple substituted, such as piperidinyl or quinolinyl groups that are substituted at the 2nd, 3rd, 4th, 5th, or 6th position, or disubstituted.

[0057] As used herein, “homologous” refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. Two sequences being compared are homologous at a certain position if that position is occupied by the same base or amino acid monomer subunit, for example, if a certain position in each of two DNA molecules is occupied by adenine. The percentage of 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 being compared, and multiplied by 100. For example, if six out of ten positions in two sequences match or are homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when the two sequences are aligned to give the greatest homology.

[0058] As used herein, the term "ionizable lipid" refers to a lipid (e.g., a cationic lipid) having at least one protonable or deprotonable group such that the lipid is positively charged at a pH below the physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above the physiological pH. Those skilled in the art will understand that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of the dominant species, and do not require that all lipids exist in either a charged or neutral form. Generally, ionizable lipids have a pK of protonable groups in the range of about 4 to about 7. a It holds.

[0059] An "immunogen" refers to any substance introduced into the body to trigger an immune response. This substance can be a physical molecule such as a protein, or it can be encoded by a vector such as DNA, mRNA, or a virus.

[0060] When the term “immune cell” is used herein, it means any cell involved in the activation of an immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (e.g., dendritic cells and macrophages), monocytes, neutrophils, eosinophils, basophils, and the like.

[0061] "Isolated" means altered or removed from its natural context. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from its coexisting substances in its natural context is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0062] The term "lipids" refers to a group of organic compounds that include, but are not limited to, fatty acid esters, and are insoluble in water but soluble in many organic solvents. They are typically divided into at least three classes: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0063] As used herein, the term “conjugated lipid” refers to a lipid that is conjugated to one or more polymer groups and inhibits the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates (e.g., PEG coupled to dialkyloxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide (e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG may be conjugated directly to lipids or linked to lipids via a linker moisture. For example, any linker moisture suitable for coupling PEG to lipids can be used, including ester-free and ester-containing linker moistures. In a preferred embodiment, an ester-free linker moisture is used.

[0064] As used herein, “lipid-encapsulated” can mean lipid particles that provide complete, partial, or both complete encapsulation of an active or therapeutic substance, such as a nucleic acid (e.g., protein cargo). In a preferred embodiment, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles).

[0065] The term "lipid nanoparticles" refers to particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing one or more types of lipids and / or additional active substances.

[0066] The term “lipid particles” is used herein to refer to lipid formulations that can be used to deliver active or therapeutic substances, such as nucleic acids (e.g., mRNA), to a target site. In the lipid particles of this disclosure, which are typically formed from cationic lipids, non-cationic lipids, and conjugate lipids that prevent particle aggregation, the active or therapeutic substance may be encapsulated within the lipids, thereby protecting the active substance from enzymatic degradation.

[0067] In the context of the present invention, the following abbreviations are used for commonly existing nucleosides (nucleic acid bases linked to ribose sugar or deoxyribose sugar via N-glycoside linkages): "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0068] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term "nucleotide sequence encoding a protein or RNA" may also include introns, insofar as a nucleotide sequence encoding a protein may contain introns in some version.

[0069] The term "modulate," as used herein, means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the subject in the absence of treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. The term encompasses disrupting and / or influencing an innate signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0070] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns. In addition, nucleotide sequences may contain modified nucleosides that can be translated by the cell's translation mechanism. For example, mRNA may have all uridines replaced with pseudouridine, 1-methylpsudouridien, or another modified nucleoside.

[0071] The term "neutral lipids" refers to any of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at a given pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0072] The term "noncationic lipids" refers to any amphiphilic lipids and any other neutral or anionic lipids.

[0073] The term "functionally linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. As an example, when a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Generally, functionally linked DNA or RNA sequences are in close proximity and, if it is necessary to link two protein coding regions, they are within the same reading frame.

[0074] The terms “patient,” “subject,” “individual,” and similar terms are used interchangeably herein and refer to any animal, or its cells, whether in vitro or in situ, that are suitable for the methods described herein. In certain non-limiting aspects, patient, subject, or individual is human.

[0075] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxy-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, and similar molecules.

[0076] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR® and similar methods, as well as by synthetic means.

[0077] In certain specific examples, 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 that has been modified. For example, more than 100 different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0078] In certain aspects, "pseudolidine" is, in some aspects, m 1 a Mori 3 Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudolidine. In some embodiments, the term is m 1 This refers to Ψ(1-methylpseudolidine). In some embodiments, this term refers to Ψm(2'-O-methylpseudolidine). In some embodiments, this term refers to m 5 D(5-methyldihydrouridine) refers to the term m. In some embodiments, the term is m 3This refers to Ψ(3-methylpseudridine). In some embodiments, the term refers to unmodified pseudouridine moiety. In some embodiments, the term refers to any monophosphate, diphosphate, or triphosphate of the above pseudouridines. In some embodiments, the term refers to any other pseudouridine known in the art. Each possibility corresponds to a separate embodiment of the present invention.

[0079] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains commonly referred to in the art, for example, as peptides, oligopeptides, and oligomers, and longer chains commonly referred to in the art as proteins, of which there are many types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0080] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism that is necessary to initiate the specific transcription of a polynucleotide sequence. For example, a promoter may be recognized by bacteriophage RNA polymerase and used to produce mRNA by in vitro transcription.

[0081] As used herein with respect to antibodies, the term “specifically binding” means an antibody that recognizes a given antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such interspecies cross-reactivity does not in itself alter the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of that antigen. However, such cross-reactivity does not in itself alter the classification of the antibody as specific. In some practical applications, the terms “specifically binding” or “specifically binding” may be used with respect to interactions between an antibody, protein, or peptide and a second chemical species to mean that the interaction depends 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 given protein structure rather than the protein in general. If the antibody is specific to epitope "A", then 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 bound to the antibody.

[0082] As used herein in conjunction with the definition of a molecule or organic group, the term “substituted” refers to a situation in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. As used herein, the terms “functional group” or “substituent” refer to a group that can be substituted on or is substituted on a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogens (e.g., F, Cl, Br, and I); oxygen atoms in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxylic acids, carboxylates, and carboxyl groups including carboxyl groups; sulfur atoms in groups such as thiol groups, alkyl sulfide groups and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; nitrogen atoms in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines, and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to the substituted carbon (or other) atom include F, Cl, Br, I, OR, OCO(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O(oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OCO(O)R, C(O)N(R)2, OCO(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R included, where R can be a hydrogen or carbon-based molecule; for example, R is hydrogen, (C1~C 100 ) may be hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or two R groups bonded to one nitrogen atom or adjacent nitrogen atoms may together with the nitrogen atom(s) to form a heterocycline.

[0083] As used herein, the term “treatment” means procedure and / or prevention. A therapeutic effect is obtained by suppressing, reducing, relieving, or eradicating at least one sign or symptom of a disease or disorder.

[0084] The term "therapeutic dose" refers to the amount of a compound of interest that will elicit a biological or medical response in a tissue, system, or subject, as sought by researchers, veterinarians, physicians, or other clinicians. The term "therapeutic dose" includes the amount of a compound that, when administered, is sufficient to prevent the onset of, or to some extent alleviate, one or more signs or symptoms of the disorder or disease being treated. The therapeutic dose will vary depending on the compound, the disease and its severity, and the age, weight, and other factors of the subject being treated.

[0085] When the term “treatment” is used herein, “treatment” means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by the subject.

[0086] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary target cells and their offspring.

[0087] As used herein, the terms “transcriptionally controlled” or “functionally linked” mean that the promoter is in the correct location and orientation in relation to the polynucleotide in order to control transcription initiation by RNA polymerase and polynucleotide expression.

[0088] A “vector” is a composition of substances that contains isolated nucleic acids and can be used to deliver isolated nucleic acids into the interior of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0089] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the use of range form is solely for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges and the individual numerical values ​​within those ranges. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numerical values ​​within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0090] Lipids and lipid nanoparticles (LNPs) In one aspect, this disclosure provides lipid nanoparticle (LNP) compositions. In certain embodiments, the LNPs are LNPs that target immune cells.

[0091] In a particular embodiment, LNP is given by equation (I): It contains an ionizable lipid compound having the structure TIFF2026531625000004.tif39139 or a salt thereof, During the ceremony, A1 and A2 were independently selected from the group consisting of CH, N, and P; L1 and L6 are each independent of CR 19 Selected from the group consisting of and N; The occurrences of L2 and L5 are independently -CH2- and -CHR 19 -, -O-, -NH-, and -NR 19 -Selected from the group consisting of; L3 and L4 are independently -CH2- and -CHR 19 -, -O-, -NH-, and -NR 19 -Selected from the group consisting of; R1, R2, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16a , R 16b , R 17 , R 18 , and R 19 each occurrence is independently H, halogen, optionally substituted C1~C 28 alkyl, optionally substituted C3~C 12 cycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C3~C 12 cycloalkyl, optionally substituted C2~C 12 heterocycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C2~C 12 heterocycloalkyl), optionally substituted C2~C 28 alkenyl, optionally substituted C5~C 12 cycloalkenyl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C5~C 12 cycloalkenyl), optionally substituted C2~C 28 alkynyl, optionally substituted C6~C 12 cycloalkynyl, -Y(R 20 ) z` (R 21 ) z``-(C6~C may be substituted) 12 Cycloalkynyl) may be substituted C6-C 10 Ariel, -Y(R 20 ) z` (R 21 ) z`` -(C6~C may be substituted) 10 (aryl), may be substituted C2-C 12 Heteroaryl, -Y(R 20 ) z` (R 21 ) z`` -(C2~C may be substituted) 12 Heteroaryl), C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z` (R 21 ) z`` -ester, -Y(R 20 ) z` (R 21 ) z`` Selected from the group consisting of -NO2, -CN, and sulfoxy, Or, R 3a and R 3b , R 4a and R 4b , R 5a and R 5b , R 6a and R 6b , R 7a and R7b , R 8a and R 8b , R 9a and R 9b , R 10a and R 10b , R 11a and R 11b , R 12a and R 12b , R 13a and R 13b , R 14a and R 14b , or R 15a and R 15b Two more selected geminal substituents can combine with the carbon atom to which they are bonded to form a C=O group; Each occurrence of Y is independently selected from the group consisting of C, N, O, S, and P; R 20 and R 21 Each occurrence is independently of H, halogen, or possibly substituted C1-C 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, C6-C (may be substituted) 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Selected from the group consisting of alkoxy, carboxyl, carboxylate, ester, -NO2, -CN, and sulfoxy, Or, R 20 and R 21 These can combine with the Y atom to which they are bonded to form C=O; Each occurrence of z` and z`` is independently 0, 1, or 2; Each occurrence of m, n, o, p, q, r, s, t, u, v, w, and x is independently 0, 1, 2, 3, 4, or 5; and Here, compounds having the structure of formula (I) or their salts constitute approximately 25 mol% to 35 mol% of the LNP.

[0092] In certain embodiments, the LNP comprises at least one helper lipid.

[0093] In certain embodiments, LNPs include cholesterol lipids.

[0094] In certain embodiments, the LNP comprises polyethylene glycol (PEG) conjugated lipids and / or modified derivatives thereof.

[0095] In one particular embodiment, the LNP includes a cell-targeting domain that is specific for binding to surface molecules of target cells. In another embodiment, the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0096] In certain embodiments, the helper lipid is dioleoyl-phosphatidylethanolamine (DOPE). In certain embodiments, dioleoyl-phosphatidylethanolamine (DOPE) constitutes about 10 mol% to about 20 mol% of the LNP. In certain embodiments, dioleoyl-phosphatidylethanolamine (DOPE) constitutes less than about 10 mol% to about 20 mol% of the LNP. In certain embodiments, dioleoyl-phosphatidylethanolamine (DOPE) constitutes more than about 10 mol% to about 20 mol% of the LNP.

[0097] In certain embodiments, cholesterol constitutes approximately 40 mol% to approximately 50 mol% of LNP. In certain embodiments, cholesterol constitutes less than approximately 40 mol% to approximately 50 mol% of LNP. In certain embodiments, cholesterol constitutes more than approximately 40 mol% to approximately 50 mol% of LNP.

[0098] In certain embodiments, the conjugated lipid includes PEG-conjugated lipids. In certain embodiments, the PEG-conjugated lipid includes a mixture of PEG-conjugated lipids and maleimide-substituted PEG-conjugated lipids. In certain embodiments, the PEG-conjugated lipid and / or its modified derivatives constitute about 0.5 mol% to about 5.0 mol% of the LNP. In certain embodiments, the PEG-conjugated lipid and / or its modified derivatives constitute less than about 0.5 mol% to about 5.0 mol% of the LNP. In certain embodiments, the PEG-conjugated lipid and / or its modified derivatives constitute more than about 0.5 mol% to about 5.0 mol% of the LNP.

[0099] In a particular mode, R1, R2, R 17 , R 18 , and R 19These are, independently of each other, H, CH2CH(OH)(CH2)1CH3, CH2CH(OH)(CH2)2CH3, CH2CH(OH)(CH2)3CH3, CH2CH(OH)(CH2)4CH3, CH2CH(OH)(CH2)5CH3, CH2CH(OH)(CH2)6CH3, CH2CH(OH)(CH2)7CH3, CH2CH(OH)(CH2)8CH3, CH2CH(OH)(CH2)9CH3, CH2CH(OH)(CH2) 10 CH3, CH2CH(OH)(CH2) 11 CH3, CH2CH(OH)(CH2) 12 CH3, CH2CH(OH)(CH2) 13 CH3, CH2CH(OH)(CH2) 14 CH3, CH2CH(OH)(CH2) 15 CH3, CH2CH(OH)(CH2) 16 CH3, CH2CH(OH)(CH2) 17 CH3, CH2CH(OH)(CH2) 18 CH3, CH2CH(OH)(CH2) 19 CH3, CH2CH(OH)(CH2) 20 CH3, CH2CH(OH)(CH2) 21 CH3, CH2CH(OH)(CH2) 22 CH3, CH2CH(OH)(CH2) 23 CH3, CH2CH(OH)(CH2) 24 CH3 and CH2CH(OH)(CH2) 25 Selected from the group consisting of CH3.

[0100] In a particular manner, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16a , and R 16b Each of these is independently selected from the group consisting of H and OCH2CH3.

[0101] In certain configurations, m is 0. In certain configurations, m is 1. In certain configurations, m is 2. In certain configurations, m is 3. In certain configurations, m is 4. In certain configurations, m is 5. In certain configurations, n is 0. In certain configurations, n is 1. In certain configurations, n is 2. In certain configurations, n is 3. In certain configurations, n is 4. In certain configurations, n is 5. In certain configurations, o is 0. In certain configurations, o is 1. In certain configurations, o is 2. In certain configurations, o is 3. In certain configurations, o is 4. In certain configurations, o is 5. In certain configurations, p is 0. In certain configurations, p is 1. In certain configurations, p is 2. In certain configurations, p is 3. In certain configurations, p is 4. In certain configurations, p is 5. In certain configurations, q is 0. In certain configurations, q is 1. In certain configurations, q is 2. In certain configurations, q is 3. In certain configurations, q is 4. In certain configurations, q is 5. In certain configurations, r is 0. In certain configurations, r is 1. In certain configurations, r is 2. In certain configurations, r is 3. In certain configurations, r is 4. In certain configurations, r is 5. In certain configurations, s is 0. In certain configurations, s is 1. In certain configurations, s is 2. In certain configurations, s is 3. In certain configurations, s is 4. In certain configurations, s is 5. In certain configurations, t is 0. In certain configurations, t is 1. In certain configurations, t is 2. In certain configurations, t is 3. In certain configurations, t is 4. In certain configurations, t is 5. In certain configurations, u is 0. In certain configurations, u is 1. In certain configurations, u is 2. In certain configurations, u is 3. In certain configurations, u is 4. In certain configurations, u is 5. In certain configurations, v is 0. In certain configurations, v is 1. In certain configurations, v is 2. In certain configurations, v is 3.In a certain mode, v is 4. In a certain mode, v is 5. In a certain mode, w is 0. In a certain mode, w is 1. In a certain mode, w is 2. In a certain mode, w is 3. In a certain mode, w is 4. In a certain mode, w is 5. In a certain mode, x is 0. In a certain mode, x is 1. In a certain mode, x is 2. In a certain mode, x is 3. In a certain mode, x is 4. In a certain mode, x is 5.

[0102] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000005.tif48140.

[0103] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000006.tif47128.

[0104] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000007.tif48128.

[0105] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000008.tif48128.

[0106] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000009.tif47128.

[0107] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000010.tif46128.

[0108] In certain embodiments, the following definitions apply independently to compounds of formulas (II), (III), (IV), (V), (VI), and (VII): R1, R2, R3, R4, R5, R6, and R7 are each independently H, halogen, or C1-C which may be substituted. 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, may be substituted C6~C 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; b 1 , b 2 , b 3 , b 4 , and b 5Each of these is independently 0, 1, 2, 3, 4, or 5; c 1 and c 2 Each of these is independently 0, 1, 2, 3, 4, or 5; and d 1 d 2 d 3 , and d 4 Each of these is independently 0, 1, 2, 3, 4, or 5.

[0109] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000011.tif51144.

[0110] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000012.tif43148.

[0111] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000013.tif50128.

[0112] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000014.tif50144.

[0113] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000015.tif39147.

[0114] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000016.tif47128.

[0115] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000017.tif48128.

[0116] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000018.tif48128.

[0117] In a particular embodiment, the compound of formula (I) The filename is TIFF2026531625000019.tif47128.

[0118] In certain embodiments, the following definitions apply independently to compounds of formulas (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), and (XVI): R1, R2, R3, R4, and R5 are each independently H, halogen, or C1-C which may be substituted. 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, C6-C (may be substituted) 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; and a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0119] In a particular embodiment, R1, R2, R3, R4, and R5 are each independently selected from the group consisting of H, methyl, ethyl, isopropyl, n-propyl, n-butyl, t-butyl, isobutyl, and sec-butyl.

[0120] In a particular manner, a 1 is 0. In a particular manner, a 1 is 1. In a particular manner, a 1 It is 2. In a particular manner, a 1 It is 3. In a particular manner, a 1 It is 4. In a particular manner, a 1 It is 5. In a particular manner, a 1 It is 6. In a particular manner, a 1 It is 7. In a particular manner, a 1 It is 8. In a particular manner, a 1 It is 9. In a particular manner, a 1 It is 10. In a particular manner, a 1 is 11. In a particular manner, a 1 It is 12. In a particular manner, a 1 It is 13. In a particular manner, a 1 It is 14. In a particular manner, a 1 It is 15. In a particular manner, a 1 It is 16. In a particular manner, a 1 It is 17. In a particular manner, a 1 It is 18. In a particular manner, a 1It is 19. In a particular manner, a 1 It is 20. In a particular manner, a 1 It is 21. In a particular manner, a 1 It is 22. In a particular manner, a 1 It is 23. In a particular manner, a 1 It is 24. In a particular manner, a 1 It is 25. In a particular manner, a 2 is 0. In a particular manner, a 2 is 1. In a particular manner, a 2 It is 2. In a particular manner, a 2 It is 3. In a particular manner, a 2 It is 4. In a particular manner, a 2 It is 5. In a particular manner, a 2 It is 6. In a particular manner, a 2 It is 7. In a particular manner, a 2 It is 8. In a particular manner, a 2 It is 9. In a particular manner, a 2 It is 10. In a particular manner, a 2 is 11. In a particular manner, a 2 It is 12. In a particular manner, a 2 It is 13. In a particular manner, a 2 It is 14. In a particular manner, a 2 It is 15. In a particular manner, a 2 It is 16. In a particular manner, a 2 It is 17. In a particular manner, a 2 It is 18. In a particular manner, a 2 It is 19. In a particular manner, a 2 It is 20. In a particular manner, a 2 It is 21. In a particular manner, a 2 It is 22. In a particular manner, a 2 It is 23. In a particular manner, a 2 It is 24. In a particular manner, a 2 It is 25. In a particular manner, a3 is 0. In a particular manner, a 3 is 1. In a particular manner, a 3 It is 2. In a particular manner, a 3 It is 3. In a particular manner, a 3 It is 4. In a particular manner, a 3 It is 5. In a particular manner, a 3 It is 6. In a particular manner, a 3 It is 7. In a particular manner, a 3 It is 8. In a particular manner, a 3 It is 9. In a particular manner, a 3 It is 10. In a particular manner, a 3 is 11. In a particular manner, a 3 It is 12. In a particular manner, a 3 It is 13. In a particular manner, a 3 It is 14. In a particular manner, a 3 It is 15. In a particular manner, a 3 It is 16. In a particular manner, a 3 It is 17. In a particular manner, a 3 It is 18. In a particular manner, a 3 It is 19. In a particular manner, a 3 It is 20. In a particular manner, a 3 It is 21. In a particular manner, a 3 It is 22. In a particular manner, a 3 It is 23. In a particular manner, a 3 It is 24. In a particular manner, a 3 It is 25. In a particular manner, a 4 is 0. In a particular manner, a 4 is 1. In a particular manner, a 4 It is 2. In a particular manner, a 4 It is 3. In a particular manner, a 4 It is 4. In a particular manner, a 4 It is 5. In a particular manner, a 4It is 6. In a particular manner, a 4 It is 7. In a particular manner, a 4 It is 8. In a particular manner, a 4 It is 9. In a particular manner, a 4 It is 10. In a particular manner, a 4 is 11. In a particular manner, a 4 It is 12. In a particular manner, a 4 It is 13. In a particular manner, a 4 It is 14. In a particular manner, a 4 It is 15. In a particular manner, a 4 It is 16. In a particular manner, a 4 It is 17. In a particular manner, a 4 It is 18. In a particular manner, a 4 It is 19. In a particular manner, a 4 It is 20. In a particular manner, a 4 It is 21. In a particular manner, a 4 It is 22. In a particular manner, a 4 It is 23. In a particular manner, a 4 It is 24. In a particular manner, a 4 It is 25. In a particular manner, a 5 is 0. In a particular manner, a 5 is 1. In a particular manner, a 5 It is 2. In a particular manner, a 5 It is 3. In a particular manner, a 5 It is 4. In a particular manner, a 5 It is 5. In a particular manner, a 5 It is 6. In a particular manner, a 5 It is 7. In a particular manner, a 5 It is 8. In a particular manner, a 5 It is 9. In a particular manner, a 5 It is 10. In a particular manner, a 5 is 11. In a particular manner, a 5 It is 12. In a particular manner, a5 It is 13. In a particular manner, a 5 It is 14. In a particular manner, a 5 It is 15. In a particular manner, a 5 It is 16. In a particular manner, a 5 It is 17. In a particular manner, a 5 It is 18. In a particular manner, a 5 It is 19. In a particular manner, a 5 It is 20. In a particular manner, a 5 It is 21. In a particular manner, a 5 It is 22. In a particular manner, a 5 It is 23. In a particular manner, a 5 It is 24. In a particular manner, a 5 It is 25.

[0121] In a particular manner, b 1 is 0. In a particular manner, b 1 is 1. In a particular manner, b 1 It is 2. In a particular manner, b 1 It is 3. In a particular manner, b 1 It is 4. In a particular manner, b 1 It is 5. In a particular manner, b 2 is 0. In a particular manner, b 2 is 1. In a particular manner, b 2 It is 2. In a particular manner, b 2 It is 3. In a particular manner, b 2 It is 4. In a particular manner, b 2 It is 5. In a particular manner, b 3 is 0. In a particular manner, b 3 is 1. In a particular manner, b 3 It is 2. In a particular manner, b 3 It is 3. In a particular manner, b 3 It is 4. In a particular manner, b 3 It is 5. In a particular manner, b 4is 0. In a particular manner, b 4 is 1. In a particular manner, b 4 It is 2. In a particular manner, b 4 It is 3. In a particular manner, b 4 It is 4. In a particular manner, b 4 It is 5. In a particular manner, b 5 is 0. In a particular manner, b 5 is 1. In a particular manner, b 5 It is 2. In a particular manner, b 5 It is 3. In a particular manner, b 5 It is 4. In a particular manner, b 5 It is 5.

[0122] In a particular manner, c 1 is 0. In a particular manner, c 1 is 1. In a particular manner, c 1 It is 2. In a particular manner, c 1 It is 3. In a particular manner, c 1 It is 4. In a particular manner, c 1 It is 5. In a particular manner, c 2 is 0. In a particular manner, c 2 is 1. In a particular manner, c 2 It is 2. In a particular manner, c 2 It is 3. In a particular manner, c 2 It is 4. In a particular manner, c 2 It is 5.

[0123] In a particular manner, d 1 is 0. In a particular manner, d 1 is 1. In a certain manner, d 1 is 2. In a particular manner, d 1 It is 3. In a certain manner, d 1 It is 4. In a certain manner, d 1 It is 5. In a certain manner, d 2 is 0. In a particular manner, d2 is 1. In a certain manner, d 2 is 2. In a particular manner, d 2 It is 3. In a certain manner, d 2 It is 4. In a certain manner, d 2 It is 5. In a certain manner, d 3 is 0. In a particular manner, d 3 is 1. In a certain manner, d 3 is 2. In a particular manner, d 3 It is 3. In a certain manner, d 3 It is 4. In a certain manner, d 3 It is 5. In a certain manner, d 4 is 0. In a particular manner, d 4 is 1. In a certain manner, d 4 is 2. In a particular manner, d 4 It is 3. In a certain manner, d 4 It is 4. In a certain manner, d 4 It is 5.

[0124] In a particular embodiment, the ionizable lipid of formula (I) is 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azandiyl)bis(tetradecane-2-ol): Includes TIFF2026531625000020.tif50133.

[0125] In a particular embodiment, the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.5.

[0126] In certain embodiments, PEG conjugate lipids are C14-PEG: Includes TIFF2026531625000021.tif24141.

[0127] In a particular embodiment, the modified derivative of the PEG conjugate lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000(ammonium salt)(mPEG): Includes TIFF2026531625000022.tif19145.

[0128] In certain embodiments, the total PEG comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio greater than approximately 1:1 to approximately 1:10 (mPEG:PEG).

[0129] In a particular embodiment, total PEG comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio selected from the group consisting of 1:3, 1:5, 1:7, and 1:10 (mPEG:PEG).

[0130] In certain embodiments, maleimide PEG is covalently conjugated to a cell-targeting domain specific for binding to surface molecules of target cells. In certain embodiments, the covalent conjugation involves a covalent bond between the α-carbon of the maleimide carbonyl and the thiol moiety of the cell-targeting domain. In certain embodiments, the covalent conjugation occurs via a [1,4]-conjugation (i.e., Michael addition) between the maleimide of maleimide PEG and the thiol of the cysteine ​​residue of the cell-targeting domain.

[0131] In certain embodiments, LNPs are delivered to the spleen at a greater rate than to the liver.

[0132] In a particular embodiment, the target cells are selected from a group consisting of stem cells, peripheral blood mononuclear cells, and immune cells.

[0133] In certain embodiments, the LNP further comprises at least one selected from the group consisting of nucleic acid molecules and therapeutic substances.

[0134] In a particular embodiment, the LNP further comprises at least one active agent selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

[0135] In certain embodiments, LNPs include nucleic acid molecules.

[0136] In certain embodiments, nucleic acid molecules are DNA molecules or RNA molecules.

[0137] In a particular embodiment, the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagonists, antisense molecules, and targeted nucleic acids, or any combination thereof.

[0138] In certain embodiments, nucleic acid molecules encode chimeric antigen receptors (CARs).

[0139] In certain embodiments, CARs are specific to binding to surface antigens of pathogenic or tumor cells.

[0140] In a particular embodiment, a cell targeting domain specific to the binding surface molecules of target cells is an immune cell targeting domain specific to binding to T cells.

[0141] In a particular embodiment, the surface molecules of the target cell are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD At least one selected from the group consisting of 183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7.

[0142] As used herein, the term "cationic lipid" refers to a lipid that is cationic, or a lipid that becomes cationic (protonated) as the pH decreases below the pK of the lipid's ionizable group, but gradually becomes more neutral at higher pH values. Below the pK, the lipid can then associate with negatively charged nucleic acids. In certain embodiments, cationic lipids include zwitterionic lipids that become positively charged as the pH decreases.

[0143] In some embodiments, cationic lipids include one of a number of lipid species that carry a net positive charge at a selective 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-(2,3-dioleoyl) This includes, but is not limited to, xy)propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamideglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In addition, numerous commercially available preparations of cationic lipids that can be used in the present invention are available. These include, for example, LIPOFECTIN® (a commercially available cationic liposome from GIBCO / BRL, Grand Island, NY, containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE)); LIPOFECTAMINE® (a commercially available cationic liposome from GIBCO / BRL, containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE)); and TRANSFECTAM® (a commercially available cationic lipid from Promega Corp., Madison, Wis., containing dioctadecylamideglycylcarboxyspermine (DOGS) in ethanol).The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0144] In certain embodiments, cationic lipids are aminolipids. Suitable aminolipids useful in the present invention include those described in International Publication No. 2012 / 016184, which is incorporated herein by reference in whole. Representative aminolipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), and 1,2-dilinoleyl-3 This includes, but is not limited to, 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).

[0145] In certain embodiments, the lipids are PEGylated lipids, including but not limited to DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxy-NHS, DOPE-PEG-carboxylic acid, and DSPE-PEG-carboxylic acid.

[0146] The term "neutral lipid" refers to one of many lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0147] Examples of neutral lipids include, for example, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine. This includes luamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylethanolamine (DSPE)-maleimide-PEG, distearoylphosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleoylphosphatidiethanolamine (SOPE), stearoyloleoylphosphatidylcholine (SOPC), and 1,2-dieryloyl-sn-glycero-3-foethanolamine (transDOPE). In certain embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0148] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0149] "Steroids" are defined as having the following carbon skeleton: This is a compound containing TIFF2026531625000023.tif25128.

[0150] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of cationic lipids.

[0151] 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 linked to neutral lipids.

[0152] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid and a polyethylene glycol portion. PEGylated lipids are known in the art and include polyethylene glycol (PEG), maleimide PEG (mPEG), DSPE-PEG-DBCO, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxy-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, and similar molecules.

[0153] In certain embodiments, the LNP includes additional stabilizing lipids, which are polyethylene glycol-lipids (PEGylated lipids). 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. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In certain embodiments, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 The substance is )carbamyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In certain embodiments, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP includes PEG-DAGs such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEG-PEs, PEG succinate diacylglycerols such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediol (PEG-S-DMG), PEG-S-DAGs, PEG-ceramides, or PEG-dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl) carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl) carbamate.

[0154] In certain embodiments, the additional lipids are present in the LNP in an amount of approximately 1 mol% to approximately 10 mol%. In certain embodiments, the additional lipids are present in the LNP in an amount of approximately 1 mol% to approximately 5 mol%. In certain embodiments, the additional lipids are present in the LNP in an amount of approximately 1 mol% or approximately 2.5 mol%.

[0155] The term "lipid nanoparticles" refers to particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing one or more types of lipids, such as lipids of formulas (I) to (XVI).

[0156] In various embodiments, lipid nanoparticles have an average diameter of approximately 30 nm to 150 nm, approximately 40 nm to 150 nm, approximately 50 nm to 150 nm, approximately 60 nm to 130 nm, approximately 70 nm to 110 nm, approximately 70 nm to 100 nm, approximately 80 nm to 100 nm, approximately 90 nm to 100 nm, approximately 70 nm to 90 nm, approximately 80 nm to 90 nm, approximately 70 nm to 80 nm, or approximately 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.

[0157] In various embodiments, the lipids or LNPs of the present invention are substantially nontoxic.

[0158] In various embodiments, the lipids or LNPs described herein are formulated for stability against in vivo immune cell targeting.

[0159] In some embodiments, the LNP formulated for stability against in vivo immune cell targeting contains C14-4 in a concentration range of approximately 10 mol% to approximately 45 mol%. In some embodiments, C14-4 is present at a molar ratio of approximately 40%.

[0160] In some embodiments, LNPs formulated for stability against in vivo immune cell targeting contain phospholipids in a concentration range of approximately 10 mol% to approximately 45 mol%. In certain embodiments, the phospholipid is dioleoyl-phosphatidylethanolamine (DOPE), and DOPE is present in a molar ratio of approximately 25 or approximately 25% molar percentage.

[0161] In some embodiments, LNPs formulated for stability against in vivo immune cell targeting contain cholesterol lipids in a concentration range of approximately 5 mol% to approximately 50 mol%. In certain embodiments, cholesterol is present at a molar ratio of approximately 30 or a molar percentage of approximately 30%.

[0162] In some embodiments, LNPs formulated for stability against in vivo immune cell targeting contain total PEG in a concentration range of approximately 0.5 mol% to approximately 12.5 mol%. In certain embodiments, total PEG is present in a molar ratio of approximately 2.5 or a molar percentage of approximately 2.5%.

[0163] In a particular embodiment, the LNP formulated for stability against in vivo immune cell targeting comprises the ionizable lipid C14-4, DOPE, cholesterol, and total PEG, where C14-4:DOPE:cholesterol:total PEG are present in a molar ratio of approximately 40:25:30:2.5, or approximately 40%:25%:30%:2.5% molar percentages.

[0164] In some embodiments, total PEG contains maleimide PEG (mPEG) and PEG in molar ratios of approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or greater than 1:15, or any molar ratio between these. In certain embodiments, LNP contains total PEG in a molar ratio of approximately 2.5, where total PEG contains mPEG and PEG in a molar ratio of 1:3. In certain embodiments, LNP contains total PEG in a molar ratio of approximately 2.5, where total PEG contains PEG and mPEG in a molar ratio of 1:5. In certain embodiments, LNP contains total PEG in a molar ratio of approximately 2.5, where total PEG contains PEG and mPEG in a molar ratio of 1:7. In a particular embodiment, the LNP contains total PEG in a molar ratio of about 2.5, and the total PEG contains PEG and mPEG in a molar ratio of 1:10.

[0165] Small molecule therapeutic substances In various embodiments, the active substance is a therapeutic substance. In various embodiments, the therapeutic substance is a small molecule. When the therapeutic substance is a small molecule, it can be obtained using standard methods known to those skilled in the art. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis, and in vitro translation systems using methods well known in the art. In certain embodiments, therapeutic substances that are small molecules include organic molecules, inorganic molecules, biomolecules, synthetic molecules, and the like.

[0166] Combinatorial libraries of molecularly diverse chemical compounds, potentially useful in the treatment of a wide range of diseases and conditions, are well known in the art, as are methods for creating such libraries. These methods may employ a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrangements, deconvolution strategies, tagging techniques, and the creation of unbiased molecular landscapes for lead discovery and biased structures for lead development. In some aspects of the present invention, therapeutic substances are synthesized and / or identified using combinatorial techniques.

[0167] In a common method for synthesizing small libraries, an activated core molecule is condensed with numerous building blocks to produce a combinatorial library of covalently linked core-building block assemblies. The shape and stiffness of the core determine the orientation of the building blocks in shape space. The library can be biased ("focused library") by altering the core, linkages, or building blocks to target a characterized biological structure, or it can be synthesized with less structural bias using a flexible core. In some aspects of the present invention, therapeutic substances are synthesized via small library synthesis.

[0168] Small molecules and small molecule compounds described herein may exist as salts even if salts are not described, and as is well understood by those skilled in the art, the present invention is understood to encompass all salts and solvates of the therapeutic substances described herein, as well as the unsalted and unsolvated forms of the therapeutic substances. In some embodiments, the salts of the therapeutic substances of the present invention are pharmaceutically acceptable salts.

[0169] Where tautomers may exist for any of the therapeutic substances described herein, each and all tautomers are intended to be included in the present invention, even if only one or some of the tautomers are explicitly described. For example, when 2-hydroxypyridylmoyety is described, the corresponding 2-pyridone tautomer is also intended.

[0170] The present invention also includes any or all stereochemical forms of the described therapeutic substance, including any enantiomer or diastereoisomer. In this specification, the listing of structures or names is intended to encompass all possible stereoisomers of the described therapeutic substance. All forms of the therapeutic substance, such as crystalline or amorphous therapeutic substances, are also encompassed by the present invention. Compositions containing the therapeutic substance of the present invention, for example, a substantially pure therapeutic substance composition including a predetermined stereochemical form thereof, or a composition containing a mixture of the therapeutic substance of the present invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture, are also intended.

[0171] The present invention also includes any or all active analogs or derivatives, e.g., prodrugs, of any therapeutic substance described herein. In certain embodiments, the therapeutic substance is a prodrug. In certain embodiments, the small molecules described herein are candidates for derivatization. Thus, in certain examples, analogs of the small molecules described herein, with modulated potency, selectivity, and solubility, are included herein and provide useful leads for drug discovery and drug development. Thus, in certain examples, during optimization, the new analogs are designed with consideration to drug delivery, metabolism, novelty, and safety.

[0172] In some examples, the small molecules of therapeutic substances described herein are derivatives or analogues of known therapeutic substances, as is well known in the art of combinatorial chemistry and medicinal chemistry. Analogues or derivatives can be prepared by adding and / or substituting functional groups at various locations. Thus, the small molecules described herein can be converted into derivatives / analogues using well known chemical synthesis procedures. For example, new analogues can be created by selectively modifying all hydrogen atoms or substituents. Linking atoms or linking groups can also be modified into longer or shorter linkers with a carbon skeleton or heteroatoms. Furthermore, ring groups can be altered to have different numbers of atoms in the ring and / or to contain heteroatoms. Moreover, aromatics can be converted into cyclic rings and vice versa. For example, a ring can have 5 to 7 atoms and can be carbocyclic or heterocyclic.

[0173] As used herein, the terms “analog,” “derivative,” and “synonym” mean a chemical compound or molecule produced from a parent compound or parent molecule by one or more chemical reactions. Therefore, an analog may have a structure similar to, or based on, the structure of, a small molecule therapeutic substance described herein, but differing in certain components or structural configurations, and may have metabolically similar or opposing effects. Any analog or derivative of any small molecule inhibitor according to the present invention can be used to treat a disease or disorder.

[0174] In certain embodiments, therapeutic substances, which are small molecules described herein, can be independently derivatized by modifying hydrogen groups with other substituents independently of each other, or analogues can be prepared from such active substances. That is, each atom on each molecule can be independently modified with respect to other atoms on the same molecule. Any conventional modification can be used to produce derivatives / analogs. For example, atoms and substituents can be independently composed of hydrogen, alkyl, aliphatic, linear aliphatic, aliphatic with chain heteroatoms, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic with one or more heteroatoms, aromatic, heteroaromatic, polycyclic aromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo-substituted aliphatic, and the like. Additionally, any ring group on a compound can be derivatized to increase and / or decrease the ring size, and to replace skeletal atoms with carbon atoms or heteroatoms.

[0175] Nucleic acid therapeutic substances In certain embodiments, the composition of the present invention comprises an in vitro transcribed (IVT) RNA molecule. For example, in certain embodiments, the composition of the present invention comprises an IVT RNA molecule encoding an active agent. In certain embodiments, the IVT RNA molecule of the composition of the present invention is a nucleoside-modified mRNA molecule. In certain embodiments, the active agent is for targeting immune cells to a target pathogen or tumor cell. In certain embodiments, the IVT RNA molecule encodes a chimeric antigen receptor (CAR).

[0176] In some embodiments, the CAR is specific to binding to one or more antigens. In some embodiments, the antigens include at least one viral antigen, bacterial antigen, fungal antigen, parasitic antigen, influenza antigen, tumor-associated antigen, tumor-specific antigen, or any combination thereof.

[0177] However, the present invention is not limited to any specific active substance or combination of active substances. In certain embodiments, the composition comprises an adjuvant. In certain embodiments, the composition comprises a nucleic acid molecule encoding the adjuvant. In certain embodiments, the composition comprises a nucleoside-modified RNA encoding the adjuvant.

[0178] In certain embodiments, the composition comprises at least one RNA molecule encoding a combination of at least two active substances. In certain embodiments, the composition comprises a combination of two or more RNA molecules encoding a combination of two or more active substances.

[0179] In certain embodiments, the present invention provides a method for inducing an immune response in a subject. For example, the method can be used to provide immunity in a subject to viruses, bacteria, fungi, parasites, cancer, or similar. In some embodiments, the method includes administering to a subject a composition comprising one or more LNP molecules formulated for in vivo targeting of immune cells, comprising one or more RNAs encoding at least one antigen, an adjuvant, or a combination thereof.

[0180] In certain embodiments, the present invention provides a method for gene editing of target immune cells. For example, the method can be used to deliver one or more components of a gene editing system (e.g., components of a CRISPR system) to target immune cells. In some embodiments, the method includes administering to a target a composition comprising one or more ionizable LNP molecules formulated for T cell target delivery, which comprises one or more nucleoside-modified RNA molecules for gene editing.

[0181] In certain embodiments, the method includes administering the composition to a subject. In certain embodiments, the method includes administering multiple doses to a subject. In some embodiments, the method includes administering a single dose of the composition, which is effective for delivering the therapeutic substance of interest.

[0182] In other relevant aspects, the therapeutic substance is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of either a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA, or miRNA molecule. In certain embodiments, the isolated nucleic acid molecule encodes therapeutic peptides, such as antithrombotic proteins including thrombomodulin, endothelial protein C receptor (EPCR), plasminogen activator and their variants, catalase, superoxide dismutase (SOD), and antioxidant proteins including iron-scavenging proteins. In some embodiments, the therapeutic substance is an siRNA, miRNA, shRNA, or antisense molecule that inhibits a targeted nucleic acid, including one that encodes a protein involved in the exacerbation of a pathological process.

[0183] In certain embodiments, the nucleic acid includes a promoter / regulatory sequence such that the nucleic acid can direct the expression of the nucleic acid. Thus, the present invention encompasses expression vectors and methods for introducing exogenous nucleic acids into cells, including, for example, those described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as well as those otherwise described herein, with the contingent expression of the exogenous nucleic acid in the cell.

[0184] In certain applications, siRNA is used to reduce the levels of a targeted protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a wide range of organisms and cell types leads to the degradation of complementary mRNA. In cells, long dsRNA is cleaved by ribonucleases, known as dicers, into short, small interfering RNAs of 21-25 nucleotides, namely siRNA. Subsequently, the siRNA is assembled with protein components into an RNA-induced silencing complex (RISC), which unravels in the process. The activated RISC then binds to the complementary transcript via base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and as a result of sequence-specific degradation of the mRNA, gene silencing occurs. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications to siRNA that facilitate intravenous systemic delivery. siRNA optimization requires consideration of the overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' overhang. See Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216 for examples.Therefore, the present invention also includes a method for reducing PTPN22 levels using RNAi technology.

[0185] In one aspect, the present invention comprises a vector containing an siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide can inhibit the expression of a target polypeptide. The incorporation of desired polynucleotides into vectors and the selection of vectors are well known in the art, for example, as described in Sambrook et al. (2012) and Ausubel et al. (1997), and elsewhere herein.

[0186] In certain embodiments, the expression vectors described herein encode a therapeutic substance that is a short hairpin RNA (shRNA). shRNA molecules are well known in the art and are directed toward a target mRNA, thereby reducing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell and subsequently processed into siRNA. For example, in certain instances, cells possess an innate enzyme (e.g., dicer) that cleaves shRNA to form siRNA.

[0187] Expression vectors introduced into cells to assess the expression of siRNA, shRNA, or antisense polynucleotides may also contain either or both a selection marker gene or a reporter gene to facilitate the identification of expressing cells from a cell population to be transfected or infected using the delivery vehicle of the present invention. In other embodiments, the selection marker may be supported on a separate DNA fragment and may also be contained within the delivery vehicle. Both the selection marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers are known in the art and include, for example, antibiotic resistance genes such as neomycin resistance and similar genes.

[0188] Therefore, in one aspect, the delivery vehicle may contain a vector comprising the nucleotide sequence or construct to be delivered. The selection of the vector will depend on the host cell into which it will subsequently be introduced. In certain embodiments, the vector of the present invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic vectors and / or eukaryotic vector-based systems can be employed for use in the present invention to produce polynucleotides or their corresponding polypeptides. Many such systems are commercially available and widely accessible.

[0189] As an example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not be integrated into the host cell's genome when introduced into the cell. Exemplary, non-limiting examples of vectors into which the nucleotide sequence or gene construct of the present invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0190] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is useful for transforming animal cells.

[0191] In certain embodiments, recombinant expression vectors may also contain nucleic acid molecules encoding peptides or peptide mimetic molecules.

[0192] A promoter can be a promoter naturally associated with a gene or polynucleotide sequence, such as one that can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters may be referred to as “endogenous.” Similarly, an enhancer can be naturally associated with a polynucleotide sequence and located either downstream or upstream of that sequence. Alternatively, certain advantages are gained by positioning a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a polynucleotide sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic cell, viral cell, or eukaryotic cell, as well as promoters or enhancers that are not “naturally present,” i.e., contain different elements of different transcriptional regulatory regions and / or contain mutations that alter expression. In addition to synthetically generating promoter and enhancer nucleic acid sequences, sequences may also be generated using nucleic acid amplification techniques, including recombinant cloning and / or PCR®, in relation to the compositions disclosed herein (U.S. Patents 4,683,202 and 5,928,906). Furthermore, it is intended that regulatory sequences directing the transcription and / or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and similar structures may also be employed.

[0193] Naturally, it is crucial to employ promoters and / or enhancers that effectively direct the expression of the DNA segment in the selected cell type, organelle, and organism. Those skilled in the field of molecular biology are generally familiar with how to use combinations of promoters, enhancers, and cell types for protein expression; see, for example, Sambrook et al. (2012). The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under conditions suitable for directing high-level expression of the introduced DNA segment, which is advantageous, for example, in the large-scale production of recombinant proteins and / or peptides. Promoter may be heterogeneous or endogenous.

[0194] Recombinant expression vectors may also contain selection marker genes to facilitate the selection of host cells. Suitable selection marker genes are genes encoding proteins, such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or their portions, such as the Fc portion of immunoglobulins, preferably IgG. The selection marker may be introduced into a vector separate from the nucleic acid of interest.

[0195] After the preparation of an siRNA polynucleotide, those skilled in the art will understand that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, siRNA polynucleotides can be further designed to withstand degradation by modifying them to include phosphorothioates or other linkages, methylphosphonates, sulfones, sulfates, ketyls, phosphorodithioates, phosphoramidates, phosphate esters, and the like (e.g., Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117). (See 1989).

[0196] Any polynucleotide may be further modified to increase 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 phosphorothioates or 2' O-methyl groups instead of phosphodiester linkages in the backbone; and / or the use of unconventional bases such as inosine, queuosine, and weibutosine, and similar groups, as well as acetyl-modified, methyl-modified, thio-modified, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0197] In certain embodiments of the present invention, an antisense nucleic acid sequence expressed by a plasmid vector is used as a therapeutic substance to inhibit the expression of a target protein. The antisense expression vector is used to transfect mammalian cells or the mammal itself, thereby causing a reduction in the endogenous expression of the target protein.

[0198] Antisense molecules and their use to inhibit gene expression are well known in the art (see, for example, Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a given mRNA molecule, as the term is defined elsewhere herein (Weintraub, 1990, Scientific American 262:40). In cells, antisense nucleic acids hybridize to the corresponding mRNA to form a double-stranded molecule, thereby inhibiting gene translation.

[0199] The use of antisense methods to inhibit gene translation is known in the art and is described, for example, by Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be delivered to cells via genetic expression using DNA encoding the antisense molecule, as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0200] Alternatively, the antisense molecules of the present invention may be synthesized and then supplied to cells. Antisense oligomers of about 10 to about 30 nucleotides, more preferably about 15 nucleotides, are preferred because they are easily synthesized and introduced into target cells. The synthetic antisense molecules intended by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0201] In certain aspects of the present invention, ribozymes are used as therapeutic substances to inhibit the expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule can be designed by incorporating a target sequence, for example, complementary to the mRNA sequence encoding the target molecule, into a basic ribozyme structure. Ribozymes targeting a target molecule can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or they can be genetically expressed from the encoding DNA.

[0202] In certain embodiments, the therapeutic substance may contain one or more components of the 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 that induces a mutation in the targeted gene. In certain embodiments, the therapeutic substance contains gRNA or a nucleic acid molecule encoding gRNA. In certain embodiments, the therapeutic substance contains Cas peptide or a nucleic acid molecule encoding Cas peptide.

[0203] In certain embodiments, the active substance includes miRNA or a miRNA mimic. In certain embodiments, the active substance includes a nucleic acid molecule encoding miRNA or a miRNA mimic.

[0204] miRNAs are small, non-coding RNA molecules capable of inducing post-transcriptional silencing of a given gene in a cell by inhibiting the translation of a targeted mRNA or by its degradation. miRNAs can be perfectly complementary to the target nucleic acid, or they can have regions that are incompatible with the target nucleic acid, resulting in a "bulge" in the incompatible region. miRNAs can inhibit gene expression by suppressing translation when the miRNA is not perfectly complementary to the target nucleic acid, or by inducing degradation of the target RNA, which is thought to occur only when the miRNA binds to its target in perfect complementarity. This disclosure may also include double-stranded precursors of miRNAs. miRNAs or pri-miRNAs can be 18–100 nucleotides long, or 18–80 nucleotides long. Mature miRNAs can be 19–30 nucleotides long, or 21–25 nucleotides long, particularly 21, 22, 23, 24, or 25 nucleotides long. miRNA precursors typically have a length of approximately 70–100 nucleotides and possess a hairpin three-dimensional structure. miRNAs are produced in vivo from pre-miRNAs by enzyme dicers and drothers that specifically process long pre-miRNAs into functional miRNAs. The hairpin or mature microRNAs, or pri-microRNA activators, discussed herein can be synthesized in vivo by cell-based systems or in vitro by chemosynthesis.

[0205] In various embodiments, the active ingredient comprises an oligonucleotide containing the nucleotide sequence of a disease-related miRNA. In certain embodiments, the oligonucleotide contains the nucleotide sequence of the disease-related miRNA in pre-microRNA form, mature form, or hairpin form. In other embodiments, a combination of oligonucleotides is envisioned, containing the sequences of one or more disease-related miRNAs, any pre-miRNA, any fragment, or any combination thereof.

[0206] miRNAs can be synthesized to include modifications that confer desired characteristics. For example, modifications can enhance stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, for instance, through endocytosis-dependent or independent mechanisms.

[0207] Modifications can also increase sequence specificity and, as a result, reduce off-site targeting. Methods of synthesis and chemical modification are described in more detail below. If desired, miRNA molecules may be modified to stabilize them against degradation, to extend their half-life, or to otherwise improve their efficacy. Desired modifications are described, for example, in U.S. Patent Applications Publications 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, each of which is incorporated herein by reference in whole. To increase nuclease resistance and / or binding affinity to targets, the single-stranded oligonucleotide activators discussed herein may include 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Binding affinity to targets can also be increased by including locked nucleic acids (LNAs), ethylene nucleic acids (ENAs), e.g., 2'-4'-ethylene-bridged nucleic acids, and certain nucleotide modifications. Endonucleases may 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' terminus with a 3'-3' linkage. Another alternative example is the 3' terminus being blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' cleavage by exonucleases. While not theoretically bound, the 3'-5'-exonuclease can inhibit exonuclease cleavage by sterically blocking the exonuclease from binding to the 3' end of the oligonucleotide. Small alkyl chains, aryl groups, heterocyclic conjugates, or even modified sugars (such as D-ribose, deoxyribose, and glucose) can block 3'-5'-exonucleases.

[0208] In certain embodiments, the miRNA comprises a 2'-modified oligonucleotide containing an oligodeoxynucleotide gap in which some or all internucleotide links are modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modification increases the affinity of the oligonucleotide to its target RNA and thus reduces its IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents disclosed in this invention may be used in conjunction with any techniques that may be developed to enhance the stability or efficacy of inhibitory nucleic acid molecules.

[0209] miRNA molecules include nucleotide oligomers containing a modified skeleton or unnatural internucleoside linkages. Oligomers with a modified skeleton include those that retain a phosphorus atom in the skeleton and those that do not. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside skeleton are also considered nucleotide oligomers. Nucleotide oligomers with a modified oligonucleotide skeleton include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriester-L, and boranophosphates. Various salts, mixed salts, and free acid forms are also included.

[0210] The miRNAs described herein, which may be in mature or hairpin form, may be provided as naked oligonucleotides. In some cases, it may be desirable to use formulations that assist in the delivery of miRNAs or other nucleotide oligomers to cells (see, for example, U.S. Patents 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each incorporated herein by reference).

[0211] In some cases, the miRNA composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In other cases, the miRNA composition is present in an aqueous phase, for example, in a solution containing water. The aqueous or crystalline composition can be incorporated into a delivery vehicle, e.g., liposomes (especially in the case of the aqueous phase), or particles (e.g., microparticles that may be suitable for crystalline compositions). Generally, the miRNA composition is formulated in a manner compatible with the intended method of administration. The miRNA composition can be formulated in combination with another active agent, e.g., another therapeutic agent, or an active agent that stabilizes the oligonucleotide active agent, e.g., a protein that complexes with the oligonucleotide active agent. Other active agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., broadly specific RNAse inhibitors). In certain embodiments, the miRNA composition includes another miRNA, for example, a second miRNA composition (e.g., a microRNA distinct from the first one). Other preparations may contain at least 3, 5, 10, 20, 50, or 100, or more than 100, different oligonucleotide species.

[0212] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of miRNA. In certain embodiments, the composition comprises an oligonucleotide having nucleic acid base identity to the nucleic acid base sequence of miRNA, and is therefore designed to mimic the activity of miRNA. In certain embodiments, the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin or a processed miRNA double helix.

[0213] In certain embodiments, the oligonucleotide shares identity with the nucleic acid base sequence of an endogenous miRNA or miRNA precursor. The oligonucleotide selected for inclusion in the composition of the present invention may be one of a number of lengths. Such an oligonucleotide may be 7 to 100 ligated nucleosides long. For example, an oligonucleotide sharing nucleic acid base identity with miRNA may be 7 to 30 ligated nucleosides long. An oligonucleotide sharing identity with a miRNA precursor may be up to 100 ligated nucleosides long. In certain embodiments, the oligonucleotide contains 7 to 30 ligated nucleosides. In certain embodiments, the oligonucleotide contains 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 ligated nucleotides. In certain embodiments, the oligonucleotide contains 19 to 23 ligated nucleosides. In certain embodiments, the oligonucleotide is the length of 40 to a maximum of 50, 60, 70, 80, 90, or 100 linked nucleosides.

[0214] In certain embodiments, oligonucleotides have sequences that have a particular identity with respect to miRNA or its precursor. The mature miRNA nucleic acid sequences and their corresponding stem-loop sequences described herein are sequences found in miRBase, an online searchable database of miRNA sequences and annotations. Entries in the miRBase sequence database represent the predicted hairpin portion (stem-loop) of a miRNA transcript, along with information about the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and in some instances may include pre-miRNAs and some adjacent sequences from the predicted primary transcript. The miRNA nucleic acid sequences described herein encompass any version of miRNA, including sequences listed in miRBase sequence database release 10.0 and any earlier releases of the miRBase sequence database. Releases of the sequence database may result in the renaming of certain miRNAs. Releases of the sequence database may result in variations of mature miRNA sequences. The compositions of the present invention include oligomeric compounds comprising oligonucleotides having a certain identity with respect to any nucleic acid sequence version of the miRNA described herein.

[0215] In certain embodiments, the oligonucleotide has a nucleic acid base sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to that of the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases. Thus, in certain embodiments, the nucleic acid base sequence of the oligonucleotide may have one or more non-identical nucleic acid bases with respect to the miRNA.

[0216] In certain embodiments, the composition comprises nucleic acid molecules encoding miRNA, its precursor, its mimic, or its fragments. For example, the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing miRNA, its precursor, its mimic, or its fragments in desired mammalian cells or tissues.

[0217] vaccine In certain embodiments, the present invention provides immunogenic compositions for inducing or activating an immune response in a subject. For example, in certain embodiments, the immunogenic composition is a vaccine. As used herein, “immunogenic composition” may include LNPs comprising an antigen (e.g., a peptide or polypeptide), an antibody or antibody fragment (e.g., an antigen-binding molecule), a nucleic acid encoding an antigen or antigen-binding molecule, a cell expressing or presenting an antigen or antigen-binding molecule, or a combination thereof. In certain embodiments, the composition comprises or encodes all or part of any peptide antigen or antigen-binding molecule, or an immunogenically functional equivalent thereof. In other embodiments, the composition comprises a mixture of mRNA molecules encoding one or more additional immunostimulants. Immunostimulants include, but are not limited to, additional antigens or antigen-binding molecules, immunomodulators, or adjuvants. In the context of the present invention, the term “vaccine” refers to a substance that induces immunity upon inoculation into an animal.

[0218] The vaccine of the present invention may have a variety of nucleic acid component compositions. In non-limiting examples, nucleic acids encoding an antigen or antigen-binding molecule may also be formulated together with an adjuvant. Naturally, it will be understood that the various compositions described herein may further contain additional components. The vaccine of the present invention and its various components may be prepared and / or administered by any method disclosed herein or known to those skilled in the art in light of the disclosure of the present invention.

[0219] In some embodiments, the therapeutic compounds or compositions of the present invention may be administered prophylactically (i.e., to prevent disease or disorder) or therapeutically (i.e., to treat disease or disorder) to subjects who are suffering from or at risk of developing (or are susceptible to) a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration is performed before the manifestation of obvious clinical symptoms of a disease, so that the disease or disorder may be prevented or its progression may be delayed. In the context of the pharmaceutical field, the term “prevent” encompasses any activity that reduces the burden of death or pathological conditions due to the disease. Prevention can be carried out at the primary, secondary, and tertiary levels. Primary prevention aims to avoid the onset of the disease, while secondary and tertiary levels of prevention encompass activities aimed at preventing the progression and manifestation of symptoms of the disease, as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0220] nucleic acid In certain embodiments, the present invention comprises an ionizable LNP molecule formulated for in vivo T cell target delivery, containing or encapsulating one or more nucleic acid molecules. In certain embodiments, the nucleic acid molecule is an mRNA molecule. In certain embodiments, the mRNA molecule encodes a CAR. In certain embodiments, the nucleoside-modified mRNA molecule encodes a CAR. In certain embodiments, the present invention comprises a nucleoside-modified mRNA molecule encoding an adjuvant.

[0221] The nucleotide sequences encoding CARs described herein may, on the condition that the resulting polynucleotide encodes the polypeptide according to the present invention, alternatively include sequence variations of the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides. Accordingly, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences listed herein and that encode the antigen or antigen-binding molecule or adjuvant of interest.

[0222] Furthermore, the scope of the present invention includes nucleotide sequences that are substantially homologous to the amino acid sequences listed herein and that maintain the immunogenicity of the original amino acid sequences.

[0223] As used herein, an amino acid sequence is "substantially homologous" to any of the amino acid sequences described herein if the amino acid sequence has a degree of identity of at least 60%, preferably at least 70%, more preferably at least 85%, and more preferably at least 95% with respect to the described amino acid sequences. The identity between two amino acid sequences is preferably determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0224] In certain embodiments, the present invention relates to a construct comprising a nucleotide sequence encoding a CAR. In certain embodiments, the construct comprises a plurality of nucleotide sequences encoding a plurality of antigens. For example, in certain embodiments, the construct encodes one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more antigens. In certain embodiments, the present invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In certain embodiments, the construct comprises a first nucleotide sequence encoding a CAR and a second nucleotide sequence encoding an adjuvant.

[0225] In certain embodiments, the composition comprises a plurality of constructs, each construct encoding one or more antigens. In certain embodiments, the composition comprises one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more constructs. In certain embodiments, the composition comprises a first construct comprising a nucleotide sequence encoding a CAR; and a second construct comprising a nucleotide sequence encoding an adjuvant.

[0226] In another specific embodiment, the construct is functionally bound to a translational regulatory element. The construct can incorporate a functionally bound regulatory sequence for expressing the nucleotide sequence of the present invention, thereby forming an expression cassette.

[0227] vector The nucleic acid sequences encapsulated in the immune cell-targeting LNP molecules of the present invention can be obtained using recombinant methods known in the art, such as by screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the target nucleic acid molecules can be synthesized.

[0228] Nucleic acids can be cloned into numerous types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe-generating vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0229] In certain embodiments, the composition of the present invention comprises in vitro transcribed (IVT) RNA encoding a CAR. In certain embodiments, the composition of the present invention comprises IVT RNA encoding multiple antigens. In certain embodiments, the composition of the present invention comprises IVT RNA encoding an adjuvant. In certain embodiments, the composition of the present invention comprises IVT RNA encoding one or more antigens and one or more adjuvants.

[0230] Nucleoside-modified RNA In certain embodiments, the composition comprises nucleoside-modified RNA. In certain embodiments, the composition comprises nucleoside-modified mRNA. Compared to unmodified mRNA, nucleoside-modified mRNA has certain advantages, including, for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, which is incorporated herein by reference in its entirety.

[0231] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiological pathways, is translated very efficiently and almost immediately after delivery, and functions as a template for continuous in vivo protein production that lasts for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The small amount of mRNA required to exert a physiological effect makes the mRNA applicable to human therapy. In certain embodiments, immune cells expressing mRNA molecules encoding a CAR are directed to target cells expressing an antigen specifically bound by the CAR.

[0232] In certain applications, expressing a protein by delivering encoding mRNA offers numerous advantages compared to methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to the cell, thus avoiding all the side effects associated with plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA-based and viral-based vectors, mRNA does not carry the risk of 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 encoding mRNA. In certain embodiments, using mRNA instead of protein also offers many advantages. While circulating proteins often have short half-lives, requiring frequent administration of proteins, mRNA provides a template for continuous protein production over several days. There are problems with protein purification, and proteins may contain aggregates and other impurities that can cause harmful effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0233] In certain embodiments, nucleoside-modified RNA contains pseudouridine, a naturally occurring modified nucleoside. In certain embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0234] The presence of modified nucleosides, including pseudouridine, in RNA has been shown to suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed pseudouridine-containing RNA encoding proteins can be translated more efficiently than RNA without modified nucleosides or RNA containing modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it has been shown that the presence of pseudouridine improves RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and weakens both PKR activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established, which is crucial for obtaining pseudouridine-containing RNA that has excellent translational ability and does not exhibit innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). When pseudouridine-containing RNA encoding erythropoietin, purified by HPLC, was administered to mice and macaques, a significant increase in serum EPO levels occurred (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0235] The present invention encompasses RNA molecules, oligoribonucleotide molecules, and polyribonucleotide molecules containing pseudouridine or modified nucleosides. In certain embodiments, the composition comprises an isolated nucleic acid encoding an antigen or antigen-binding molecule, wherein the nucleic acid comprises pseudouridine or modified nucleosides. In certain embodiments, the composition comprises a vector comprising an isolated nucleic acid encoding an antigen, an antigen-binding molecule, an adjuvant, or a combination thereof, wherein the nucleic acid comprises pseudouridine or modified nucleosides.

[0236] In certain embodiments, the nucleoside-modified RNA of the present invention is IVT RNA. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In some embodiments, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In some embodiments, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0237] In a particular form, the modified nucleoside is m 1 a Mori 3 It is Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudolidine). In some embodiments, the modified nucleoside is m 1 It is Ψ(1-methylpseudolidine). In some embodiments, the modified nucleoside is Ψm(2'-O-methylpseudolidine). In some embodiments, the modified nucleoside is m 5 D(5-methyldihydrouridine) is used. In some embodiments, the modified nucleoside is m 3 The modified nucleoside is Ψ(3-methylpseudridine). In some embodiments, the modified nucleoside is an unmodified pseudouridine moiety. In some embodiments, the modified nucleoside is any monophosphate, diphosphate, or triphosphate of the above pseudouridines. In some embodiments, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0238] In some embodiments, the modified nucleoside in the nucleoside-modified RNA of the present invention is uridine (U). In some embodiments, the modified nucleoside is cytidine (C). In some embodiments, the modified nucleoside is adenosine (A). In other embodiments, the modified nucleoside is guanosine (G).

[0239] In some embodiments, the modified nucleoside of the present invention is m 5 It is C(5-methylcytidine). In some embodiments, the modified nucleoside is m 5U(5-methyluridine) is used. In some embodiments, the modified nucleoside is m 6 A(N 6 (-methyladenosine). In some embodiments, the modified nucleoside is s 2 It is U(2-thiouridine). In some embodiments, the modified nucleoside is Ψ(pseudolidine). In some embodiments, the modified nucleoside is Um(2'-O-methyluridine).

[0240] In other embodiments, the modified nucleoside is m 1 A(1-methyladenosine); m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N) 6 -methyladenosine);i 6 A(N 6 -Isopentenyladenosine);ms 2 i6A(2-methylthio-N) 6 Isopentenyladenosine; io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A(2-methylthio-N) 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A(N 6 -Glycinylcarbamoyladenosine); t 6 A(N 6 -Threonylcarbamoyladenosine);ms 2 t 6 A(2-methylthio-N) 6 -Threonylcarbamoyladenosine);m 6 t 6 A(N 6 -methyl-N 6 -Threonylcarbamoyladenosine);hn 6 A(N 6 -Hydroxynorvalylcarbamoyladenosine); ms 2 hn 6 A(2-methylthio-N) 6-Hydroxynorvalylcarbamoyladenosine);Ar(p)(2'-O-ribosyladenosine (phosphorus));I(inosine);m 1 I(1-methylinosine); m 1 Im(1,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine); ac 4 C(N 4 -acetylcytidine);f 5 C(5-formylcytidine); m 5 Cm(5,2'-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lysidine); m 1 G(1-methylguanosine); m 2 G(N 2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine); m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine);Gr(p)(2'-O-ribosylguanosine (phospho));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(low-modified hydroxywybutosine);imG(wyosine);mimG(methylwyosine);Q(queosine);oQ(epoxyqueosine);galQ(galactosylqueosine);manQ(mannosylqueosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Alkaeosin); D (Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine); m5 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-oxyacetate methyl ester); chm 5 U(5-(carboxyhydroxymethyl)uridine)); mchm 5 U(5-(carboxyhydroxymethyl)uridinemethyl 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 4C(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-demethylyosin); imG2(isoyosin); or ac 6 A(N 6 -Acetyladenosine)

[0241] In some embodiments, the nucleoside-modified RNA of the present invention includes a combination of two or more of the above modifications. In some embodiments, the nucleoside-modified RNA includes a combination of three or more of the above modifications. In some embodiments, the nucleoside-modified RNA includes a combination of more than three of the above modifications.

[0242] In some embodiments, 0.1% to 100% of the residues in the nucleoside-modified material of the present invention are modified (for example, by the presence of pseudouridine or the presence of a modified nucleoside base). In some embodiments, 0.1% of the residues are modified. In some embodiments, the fraction of modified residues is 0.2%. In some embodiments, the fraction is 0.3%. In some embodiments, the fraction is 0.4%. In some embodiments, the fraction is 0.5%. In some embodiments, the fraction is 0.6%. In some embodiments, the fraction is 0.8%. In some embodiments, the fraction is 1%. In some embodiments, the fraction is 1.5%. In some embodiments, the fraction is 2%. In some embodiments, the fraction is 2.5%. In some embodiments, the fraction is 3%. In some embodiments, the fraction is 4%. In some embodiments, the fraction is 5%. In some embodiments, the fraction is 6%. In some embodiments, the fraction is 8%. In some embodiments, the percentage is 10%. In some embodiments, the percentage is 12%. In some embodiments, the percentage is 14%. In some embodiments, the percentage is 16%. In some embodiments, the percentage is 18%. In some embodiments, the percentage is 20%. In some embodiments, the percentage is 25%. In some embodiments, the percentage is 30%. In some embodiments, the percentage is 35%. In some embodiments, the percentage is 40%. In some embodiments, the percentage is 45%. In some embodiments, the percentage is 50%. In some embodiments, the percentage is 60%. In some embodiments, the percentage is 70%. In some embodiments, the percentage is 80%. In some embodiments, the percentage is 90%. In some embodiments, the percentage is 100%.

[0243] In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 3%. In some embodiments, the percentage is less than 1%. In some embodiments, the percentage is less than 2%. In some embodiments, the percentage is less than 4%. In some embodiments, the percentage is less than 6%. In some embodiments, the percentage is less than 8%. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 12%. In some embodiments, the percentage is less than 15%. In some embodiments, the percentage is less than 20%. In some embodiments, the percentage is less than 30%. In some embodiments, the percentage is less than 40%. In some embodiments, the percentage is less than 50%. In some embodiments, the percentage is less than 60%. In some embodiments, the percentage is less than 70%.

[0244] In some embodiments, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In some embodiments, the fraction of a given nucleotide that is modified is 0.2%. In some embodiments, the fraction is 0.3%. In some embodiments, the fraction is 0.4%. In some embodiments, the fraction is 0.5%. In some embodiments, the fraction is 0.6%. In some embodiments, the fraction is 0.8%. In some embodiments, the fraction is 1%. In some embodiments, the fraction is 1.5%. In some embodiments, the fraction is 2%. In some embodiments, the fraction is 2.5%. In some embodiments, the fraction is 3%. In some embodiments, the fraction is 4%. In some embodiments, the fraction is 5%. In some embodiments, the fraction is 6%. In some embodiments, the fraction is 8%. In some embodiments, the fraction is 10%. In some embodiments, the percentage is 12%. In some embodiments, the percentage is 14%. In some embodiments, the percentage is 16%. In some embodiments, the percentage is 18%. In some embodiments, the percentage is 20%. In some embodiments, the percentage is 25%. In some embodiments, the percentage is 30%. In some embodiments, the percentage is 35%. In some embodiments, the percentage is 40%. In some embodiments, the percentage is 45%. In some embodiments, the percentage is 50%. In some embodiments, the percentage is 60%. In some embodiments, the percentage is 70%. In some embodiments, the percentage is 80%. In some embodiments, the percentage is 90%. In some embodiments, the percentage is 100%.

[0245] In some embodiments, the fraction of a given nucleotide that is modified is less than 8%. In some embodiments, the fraction is less than 10%. In some embodiments, the fraction is less than 5%. In some embodiments, the fraction is less than 3%. In some embodiments, the fraction is less than 1%. In some embodiments, the fraction is less than 2%. In some embodiments, the fraction is less than 4%. In some embodiments, the fraction is less than 6%. In some embodiments, the fraction is less than 12%. In some embodiments, the fraction is less than 15%. In some embodiments, the fraction is less than 20%. In some embodiments, the fraction is less than 30%. In some embodiments, the fraction is less than 40%. In some embodiments, the fraction is less than 50%. In some embodiments, the fraction is less than 60%. In some embodiments, the fraction is less than 70%.

[0246] In some embodiments, the nucleoside-modified RNA of the present invention is translated more efficiently in cells than unmodified RNA molecules having the same sequence. In some embodiments, the nucleoside-modified RNA exhibits enhanced ability to be translated by target cells. In some embodiments, translation is enhanced by a factor of 2 compared to its unmodified counterpart. In some embodiments, translation is enhanced by a factor of 3. In some embodiments, translation is enhanced by a factor of 5. In some embodiments, translation is enhanced by a factor of 7. In some embodiments, translation is enhanced by a factor of 10. In some embodiments, translation is enhanced by a factor of 15. In some embodiments, translation is enhanced by a factor of 20. In some embodiments, translation is enhanced by a factor of 50. In some embodiments, translation is enhanced by a factor of 100. In some embodiments, translation is enhanced by a factor of 200. In some embodiments, translation is enhanced by a factor of 500. In some embodiments, translation is enhanced by a factor of 1000. In some embodiments, the translation is augmented by a factor of 2000. In some embodiments, the augmentation is between 10 and 1000. In some embodiments, the augmentation is between 10 and 100. In some embodiments, the augmentation is between 10 and 200. In some embodiments, the augmentation is between 10 and 300. In some embodiments, the augmentation is between 10 and 500. In some embodiments, the augmentation is between 20 and 1000. In some embodiments, the augmentation is between 30 and 1000. In some embodiments, the augmentation is between 50 and 1000. In some embodiments, the augmentation is between 100 and 1000. In some embodiments, the augmentation is between 200 and 1000. In some embodiments, the translation is augmented by any other significant amount or range of amounts.

[0247] In some embodiments, the nucleoside-modified RNA of the present invention encoding an antigen induces a significantly greater adaptive immune response than an unmodified in vitro synthetic RNA molecule having the same sequence. In some embodiments, the modified RNA molecule exhibits a twice as large an adaptive immune response as its unmodified counterpart. In some embodiments, the adaptive immune response increases by a factor of three. In other embodiments, the adaptive immune response increases by a factor of five. In some embodiments, the adaptive immune response increases by a factor of seven. In some embodiments, the adaptive immune response increases by a factor of ten. In some embodiments, the adaptive immune response increases by a factor of fifteen. In other embodiments, the adaptive immune response increases by a factor of twenty. In some embodiments, the adaptive immune response increases by a factor of fifty. In some embodiments, the adaptive immune response increases by a factor of 100. In some embodiments, the adaptive immune response increases by a factor of 200. In some embodiments, the adaptive immune response increases by a factor of 500. In some embodiments, the adaptive immune response increases by a factor of 1000. In some embodiments, the adaptive immune response increases by a factor of 2000. In other embodiments, the adaptive immune response increases by a different factor.

[0248] In some embodiments, “inducing a significantly greater number of adaptive immune responses” refers to a detectable increase in the adaptive immune response. In some embodiments, the term refers to a multiplicative increase in the adaptive immune response (e.g., one of the multiplicative increases listed above). In some embodiments, the term refers to an increase such that nucleoside-modified RNA can be administered at lower doses or frequencies than unmodified RNA molecules of the same species, while still inducing an effective adaptive immune response. In some embodiments, the increase is such that nucleoside-modified RNA can be administered using a single dose to induce an effective adaptive immune response.

[0249] In some embodiments, the nucleoside-modified RNA of the present invention exhibits significantly lower innate immunogenicity than unmodified in vitro synthetic RNA molecules having the same sequence. In some embodiments, the modified RNA molecule exhibits an innate immune response that is half that of its unmodified counterpart. In some embodiments, the innate immunogenicity is reduced to one-third. In some embodiments, the innate immunogenicity is reduced to one-fifth. In some embodiments, the innate immunogenicity is reduced to one-seventh. In some embodiments, the innate immunogenicity is reduced to one-tenth. In some embodiments, the innate immunogenicity is reduced to one-fifteenth. In some embodiments, the innate immunogenicity is reduced to one-twentieth. In some embodiments, the innate immunogenicity is reduced to one-fiftieth. In some embodiments, the innate immunogenicity is reduced to one-hundredth. In some embodiments, the innate immunogenicity is reduced to one-twenty-fif In some embodiments, innate immunogenicity is reduced to 1 / 1000th. In some embodiments, innate immunogenicity is reduced to 1 / 2000th. In some embodiments, innate immunogenicity is reduced by a different multiple.

[0250] In some embodiments, “significantly lower innate immunogenicity” means a detectable reduction in innate immunogenicity. In some embodiments, the term means a multiplier reduction in innate immunogenicity (e.g., one of the multiplier reductions listed above). In some embodiments, the term means a reduction such that an effective amount of nucleoside-modified RNA can be administered without eliciting a detectable innate immune response. In some embodiments, the term means a reduction such that nucleoside-modified RNA can be repeatedly administered without inducing an innate immune response sufficient to detectably reduce the production of recombinant protein. In some embodiments, the reduction is such that nucleoside-modified RNA can be repeatedly administered without inducing an innate immune response sufficient to eliminate the detectable production of recombinant protein.

[0251] Polypeptide therapeutic substances In other relevant aspects, therapeutic substances include isolated peptides that modulate a target. For example, in certain embodiments, the peptides of the present invention directly inhibit or activate a target by binding to it, thereby modulating the target's normal functional activity. In certain embodiments, the peptides of the present invention modulate a target by competing with endogenous proteins. In certain embodiments, the peptides of the present invention modulate the activity of a target by acting as a transdominant-negative variant.

[0252] Variants of the polypeptide therapeutic substance may include: (i) having one or more amino acid residues substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), wherein such substituted amino acid residues may or may not be encoded by the genetic code; (ii) having one or more modified amino acid residues, e.g., residues modified by the attachment of a substituted group; (iii) the polypeptide being an alternative splice variant of the polypeptide of the present invention; (iv) a polypeptide fragment; and / or (v) the polypeptide fused with another polypeptide, such as a leader sequence or secretion sequence, or a sequence employed for purification (e.g., a His tag) or a sequence employed for detection (e.g., an Sv5 epitope tag). Fragments may include polypeptides prepared via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of the art based on the teachings herein.

[0253] CAR agonist In certain embodiments, the mRNA molecule of the present invention encodes a chimeric antigen receptor (CAR). In certain embodiments, the CAR includes an antigen-binding domain. In certain embodiments, the antigen-binding domain is a targeting domain, which directs T cells expressing the CAR to a predetermined target cell or tissue. For example, in certain embodiments, the targeting domain includes an antibody, antibody fragment, or peptide that specifically binds to one expressed on a pathogenic organism or tumor cell, thereby directing T cells expressing the CAR to a cell or tissue expressing the antigen.

[0254] In certain embodiments, the present invention relates to an immune cell targeting LNP comprising an active agent, wherein the active agent comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In certain embodiments, the active agent comprises an mRNA molecule encoding a CAR. In certain embodiments, the active agent comprises a modified nucleoside mRNA molecule encoding a CAR.

[0255] In various embodiments, a CAR can be a "first-generation," "second-generation," "third-generation," "fourth-generation," or "fifth-generation" CAR (e.g., Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. See 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009).

[0256] The “first-generation” CARs for use in the present invention comprise an antigen-binding domain fused to a transmembrane domain, such as a single-chain variable fragment (scFv), the transmembrane domain being fused to the cytoplasmic / intracellular domain of a T cell receptor chain. “First-generation” CARs typically possess an intracellular domain from the CD3ζ chain, which is the primary signaling factor from the endogenous T cell receptor (TCR). “First-generation” CARs can provide de novo antigen recognition through their CD3ζ chain signaling domain in a single fusion molecule, independently of HLA-mediated antigen presentation, and can induce activation of both CD4+ and CD8+ T cells.

[0257] The “second-generation” CAR for use in this invention comprises an antigen-binding domain, e.g., a single-chain variable fragment (scFv), fused with an intracellular signaling domain capable of activating T cells and a costimulatory domain designed to enhance the potency and persistence of T cells (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Thus, CAR design can combine two physiologically mediated functions, antigen recognition and signal transduction, by two separate complexes: the TCR heterodimer and the CD3 complex. The “second-generation” CAR contains intracellular domains from various costimulatory molecules, e.g., CD28, 4-1BB, ICOS, OX40, and similar molecules, in the cytoplasmic tail of the CAR to provide additional signaling to the cell.

[0258] "Second-generation" CARs provide both co-stimulation, for example, via the CD28 domain or 4-1BB domain, and activation, for example, via the CD3ζ signaling domain. Preclinical studies have shown that "second-generation" CARs can enhance the antitumor activity of T cells. For example, robust efficacy of "second-generation" CAR-modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0259] "Third-generation" CARs offer multiple co-stimuli, for example, by including both the CD28 domain and the 4-1BB domain, and activation, for example, by including the CD3ζ activation domain.

[0260] "Fourth-generation" CARs provide co-stimulation, for example, by the CD28 domain or the 4-1BB domain, and activation, for example, by the CD3ζ signaling domain in addition to constitutive or inducible chemokine components.

[0261] "Fifth-generation" CARs provide co-stimulation, for example, by the CD28 domain or 4-1BB domain, and activation, for example, by the CD3ζ signaling domain, constitutive or inducible chemokine components, and the intracellular domain of cytokine receptors, such as IL-2Rβ.

[0262] In various embodiments, CARs can be included in multivalent CAR systems, such as dual-CAR or "tandem-CAR" systems. Multivalent CAR systems include systems or cells containing multiple CARs, and systems or cells containing bivalent / bispecific CARs that target more than one antigen.

[0263] In the embodiments disclosed herein, a CAR generally comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, as described above. In certain non-limiting embodiments, the antigen-binding domain is an scFv specific to binding to a surface antigen of a target cell (e.g., a pathogen or tumor cell).

[0264] combination In certain embodiments, the composition of the present invention comprises a combination of active substances described herein. In certain embodiments, the composition comprising a combination of active substances described herein has an additive effect, where the overall effect of the combination is approximately equal to the sum of the effects of each individual active substance. In other embodiments, the composition comprising a combination of active substances described herein has a synergistic effect, where the overall effect of the combination is greater than the sum of the effects of each individual active substance.

[0265] A composition containing a combination of active substances includes the individual active substances in any preferred ratio. For example, in a particular embodiment, the composition contains two individual active substances 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 included.

[0266] Cell targeting domain In various aspects of the present invention, the LNP of the present invention is conjugated to a targeting domain that is specific to binding to a receptor on a target cell.

[0267] In certain embodiments, the target cells are stem cells. Exemplary stem cells that can be targeted by the compositions of the present invention include, but are not limited to, hematopoietic stem cells and stem cells related to hematopoietic stem cells (e.g., myeloid stem cells and lymphoid stem cells).

[0268] In certain configurations, the target cells are peripheral blood mononuclear cells (PBMCs).

[0269] In some cases, the target cells are immune cells. Exemplary immune cells that can be targeted by the compositions of the present invention include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells, dendritic cells, macrophages, monocytes, neutrophils, eosinophils, and basophils. In certain embodiments, the immune cells are T cells. In some embodiments, the T cells that can be targeted using the compositions of the present invention may be CD4+ or CD8+, and may include helper T cells (CD4+), cytotoxic T cells (also called cytotoxic T lymphocytes, CTLs; CD8- T cells), as well as memory T cells, including central memory T cells (TCMs), stem memory T cells (TSCMs), stem cell-like memory T cells (or stem-like memory T cells), and effector memory T cells, e.g., T EM Cells and T EMRA This may include, but is not limited to, (CD45RA+) cells, effector T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, Tfh (follicular helper) cells, regulatory T cells, natural killer T cells, mucosa-associated invariant T cells (MAIT), and γδ T cells. Major T cell subtypes include T N (Naive), T SCM (Stem cell memory), T CM (Central Memory), T TM (Transitional memory), T EM(Effects memory), and T TE This includes terminal effectors (TCR transgenic T cells), T cells redirected for universal cytokine-mediated killing (TRUCK), tumor-infiltrating T cells (TILs), CAR-T cells, or any T cells that can be used to treat a disease or disorder.

[0270] In certain embodiments, the T cells of the present invention are immunostimulatory cells, i.e., cells that mediate an immune response. Exemplary immunostimulatory T cells include, but are not limited to, helper T cells (CD4+), cytotoxic T cells (also known as cytotoxic T lymphocytes, CTLs; CD8+ T cells), as well as memory T cells, including central memory T cells (TCMs), stem memory T cells (TSCMs), stem cell-like memory T cells (or stem-like memory T cells), and effector memory T cells, such as TEM cells and TEMRA (CD45RA+) cells, effector T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, Tfh (follicular helper) cells, natural killer T cells, mucosa-associated invariant T cells (MAITs), and γδ T cells.

[0271] In a particular embodiment, the T cell targeting domain is CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, C It binds to D154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, or CCR7.

[0272] In a particular embodiment, the present invention relates to a composition comprising a combination of delivery vehicles conjugated to an immune cell targeting domain for targeting multiple types of immune cells. In a particular embodiment, the combination comprises two or more immune cell targeting delivery vehicles that target two or more immune cell antigens. In a particular embodiment, the two or more immune cell antigens are CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD1 The following are selected from 54, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7. In certain embodiments, the combination comprises two or more T cell targeting delivery vehicles that target the surface antigens of CD4+ T cells and CD8+ T cells. In certain embodiments, the combination comprises two or more T cell targeting delivery vehicles that target CD4 and CD8.

[0273] In certain embodiments, the targeting domain is conjugated to the LNP of the present invention. Exemplary methods of conjugation may include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic ("van der Waals") interactions. In certain embodiments, the conjugation is reversible, such that the delivery vehicle can be detached from the targeting domain upon exposure to certain conditions or chemicals. In some embodiments, the conjugation is irreversible, such that the delivery vehicle does not dissociate from the targeting domain under normal conditions.

[0274] In some embodiments, the conjugation includes a covalent bond between the activated polymer-conjugate lipid and the targeting domain. The term "activated polymer-conjugate lipid" refers to a molecule comprising a lipid moiety and a polymer moiety that has been activated via the addition of a functional group to the polymer-conjugate lipid by a first coupling group. In certain embodiments, the activated polymer-conjugate lipid includes a first coupling group capable of reacting with a second coupling group. In certain embodiments, the activated polymer-conjugate lipid is an activated pegylated lipid. In certain embodiments, the first coupling group is bonded to the lipid moiety of the pegylated lipid. In some embodiments, the first coupling group is bonded to the polyethylene glycol moiety of the pegylated lipid. In certain embodiments, the second functional group is covalently attached to the targeting domain.

[0275] The first and second coupling groups can be any functional groups known to those skilled in the art for forming a covalent bond together, for example, under mild reaction or physiological conditions. In some embodiments, the first or second coupling group is selected from the group consisting of maleimide, N-hydroxysuccinimide (NHS) esters, carbodiimide, hydrazide, pentafluorophenyl (PFP) ester, phosphine, hydroxymethylphosphine, psoralen, imide ester, pyridyl disulfide, isocyanate, vinyl sulfone, α-haloacetyl, aryl azide, acyl azide, alkyl azide, diaziline, benzophenone, epoxide, carbonate, anhydride, sulfonyl chloride, cyclooctin, aldehyde, and sulfhydryl groups. In some embodiments, the first or second coupling group is selected from the group consisting of free amines (-NH2), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group reactive to a sulfhydryl group, such as maleimide, pyridyl disulfide, or haloacetyl. In a particular embodiment, the first coupling group is maleimide.

[0276] In certain embodiments, the second coupling group is a sulfhydryl group. The sulfhydryl group can be introduced on the targeting domain using any method known to those skilled in the art. In certain embodiments, the sulfhydryl group is located on a free cysteine ​​residue. In certain embodiments, the sulfhydryl group is exposed by reduction of a disulfide on the targeting domain, for example, through a reaction with 2-mercaptoethylamine. In certain embodiments, the sulfhydryl group is introduced by a chemical reaction, such as a reaction between a free amine and 2-iminotilane or N-succinimidyl S-acetylthioacetate (SATA).

[0277] In some embodiments, polymer-conjugated lipids and targeting domains are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves reactions between 1,3-dipole functional groups, such as azides, nitrile oxides, nitrones, isocyanides, and links, and alkene or alkyne dipoles. Exemplary dipoles include, but are not limited to, any strained cycloalkenes and cycloalkynes known to those skilled in the art, including cyclooctin, dibenzocyclooctin, monofluorinated cyclooctin, difluorinated cyclooctin, and biarylazacyclooctinone.

[0278] In certain embodiments, the targeting domain is conjugated to the LNP using maleimide conjugation.

[0279] Targeting Domains In certain embodiments, the composition includes a targeting domain that directs a delivery vehicle toward target immune cells. The targeting domain may include nucleic acids, peptides, antibodies, small molecules, organic molecules, inorganic molecules, glycans, sugars, hormones, and the like, which target particles to sites where the therapeutic substance is particularly needed. In certain embodiments, the particles involve multivalent targeting, where the particles include multiple targeting mechanisms as described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target related to a site requiring the active substance contained within the delivery vehicle. For example, the targeting domain may be selected to recognize a ligand that acts as a cell surface marker on target cells related to a particular disease condition. Such a target may be a protein, protein fragment, antigen, or other biomolecule related to the site being targeted. In some embodiments, the targeting domain is an affinity ligand that specifically binds to the target. In certain embodiments, the target (e.g., an antigen) binds to a site requiring treatment with the active substance. In some embodiments, the targeting domain may be copolymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to a composition containing a delivery vehicle, for example, through a chemical reaction between the targeting domain and the composition containing the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. The targeting domain of the present invention includes, but is not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0280] In various embodiments, the targeting domain binds to cell surface molecules of the target cell. For example, in various embodiments, the targeting domain binds to cell surface molecules of endothelial cells, stem cells, or immune cells.

[0281] peptide In certain embodiments, the targeting domain of the present invention comprises a peptide. In certain embodiments, the targeting domain, which is a peptide, specifically binds to a target of interest.

[0282] The peptides of the present invention can 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 resin, and purified by preparative high-performance liquid chromatography. Automated synthesis can be achieved, for example, by using the ABI 431 A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0283] The peptide can alternatively be produced by recombinant means or by cleavage from a longer polypeptide chain. The composition of the peptide can be determined by amino acid analysis or sequencing.

[0284] Variants of the peptide according to the present invention may include: (i) one or more amino acid residues being substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), wherein such substituted amino acid residues may or may not be encoded by the genetic code; (ii) having one or more modified amino acid residues, e.g., residues modified by the attachment of a substituted group; (iii) the peptide being an alternative splice variant of the peptide of the present invention; (iv) a peptide fragment; and / or (v) the peptide being fused with another peptide, such as a reader sequence or secretion sequence, or a sequence employed for purification (e.g., a His tag) or a sequence employed for detection (e.g., an Sv5 epitope tag). Fragments may include peptides prepared by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of the art based on the teachings herein.

[0285] As is well known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conserved amino acid substitutions with the sequence of the second peptide. A variant is defined as one which contains a peptide sequence different from the original sequence, preferably with less than 40% of residues per target segment differing from the original sequence, more preferably with less than 25% of residues per target segment differing from the original sequence, more preferably with less than 10% of residues per target segment differing from the original protein sequence, and most preferably with only a few residues per target segment differing from the original protein sequence, while at the same time being sufficiently homologous to the original sequence to maintain the functionality of the original sequence. The present invention contains amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar to or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined by 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)].

[0286] The peptides of the present invention can be post-translation modified. For example, post-translation modifications that fall 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 investigated by adding canine microsomal membrane or Xenopus egg extract (U.S. Patent No. 6,103,489) to a standard translation reaction.

[0287] The peptides of the present invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0288] nucleic acid In certain embodiments, the targeting domain of the present invention comprises an isolated nucleic acid, such as DNA oligonucleotides and RNA oligonucleotides. In certain embodiments, the targeting domain, which is a nucleic acid, specifically binds to a target. For example, in certain embodiments, the nucleic acid comprises a nucleotide sequence that specifically binds to a target.

[0289] The nucleotide sequence of the targeting domain, which is a nucleic acid, may alternatively include sequence variations to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid functions like the original and specifically binds to the target in question.

[0290] In the sense used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein if the nucleotide sequence has a degree of identity of at least 60%, preferably at least 70%, preferably at least 85%, and more preferably at least 95% with respect to the nucleotide sequences described herein. Other possible modifications include the insertion of one or more nucleotides into a sequence, the addition of one or more nucleotides to any of the ends of a sequence, or the deletion of one or more nucleotides at any end or in the middle of a sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods widely known to those skilled in the art. Identity between two amino acid sequences is preferably determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0291] antibody In certain embodiments, the targeting domain of the present invention comprises an antibody or antibody fragment. In certain embodiments, the targeting domain, which is an antibody, specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, their Fab fragments and single-chain Fv(scFv) fragments, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0292] The antibodies may be intact monoclonal or polyclonal antibodies, immunologically active fragments (e.g., Fab fragments or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778), or chimeric antibodies, such as those containing the binding specificity of a mouse antibody but with the remainder being of human origin. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeric antibodies, can be prepared using methods known to those skilled in the art.

[0293] Such antibodies can be produced in a variety of ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell cultures, and recombinant expression in transgenic animals. The selection of the production method depends on several factors, including the desired antibody structure, the importance of carbohydrate moiety on the antibody, the ease of culture and purification, and cost. Many different antibody structures, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies containing components from different species, can be produced using standard expression techniques. Small-sized antibody fragments, such as Fab and Fv fragments, which lack effector function and have limited pharmokinetic activity, can be produced in bacterial expression systems. Single-chain Fv fragments exhibit low immunogenicity.

[0294] antigen The present invention provides compositions that induce an immune response in a target. In certain embodiments, the composition comprises immune cell-targeting LNPs, which include nucleic acid molecules encoding antigen-specific chimeric antigen receptors (CARs).

[0295] In certain embodiments, the antigen comprises polypeptides or peptides associated with pathogens or tumor cells, and as a result, in vivo-modified immune cells expressing CARs are then targeted to the antigen, inducing an immune response against the antigen and therefore against the pathogen or tumor cells.

[0296] In certain embodiments, antigens recognized by CARs encoded by nucleic acid molecules include proteins, peptides, fragments thereof, variants thereof, or combinations thereof from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals.

[0297] In certain embodiments, the antigen includes a tumor-specific antigen or a tumor-associated antigen, and as a result, immune cells expressing the CAR are directed toward tumor cells expressing the antigen.

[0298] Viral antigens In certain embodiments, the antigen comprises a viral antigen, a fragment thereof, or a variant thereof. In certain embodiments, the viral antigen comprises the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae. It is a virus from one of the following families: Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae.In certain aspects, viral antigens include papillomaviruses, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (major and minor pox), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hair cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), and rabies virus. It is derived from the following viruses: Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex virus type 1 (oral herpes), herpes simplex virus type 2 (genital herpes), varicella (varicella-zoster, also known as chickenpox), cytomegalovirus (CMV), such as human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, chikungunya virus, lassa virus, arenavirus, severe acute respiratory syndrome (SARS) virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or oncogenic viruses.

[0299] Parasite antigens In certain embodiments, the antigen includes a parasitic antigen or a fragment or variant thereof. In certain embodiments, the parasite is a protozoan, helminth, or ectoparasite. In certain embodiments, the helminth (i.e., worm) is a flatworm (e.g., trematodes and tapeworms), acanthocephalus, or roundworm (e.g., pinworm). In certain embodiments, the ectoparasite is a louse, flea, tick, or mites.

[0300] In certain aspects, parasites are any parasite that causes the following diseases: Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, raccoon ascariasis, Chagas disease, liver fluke infection, Cochliomyia, cryptosporidiosis, diphyllobothrium infection, dung worm infection, echinococcosis, elephantiasis, pinworm infection, phylariasis, hypertrophic fluke infection, filariasis, giardiasis, gnathostomiasis, membranoid tapeworm infection, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, Yokogawa fluke infection, fly larva infection, onchocerciasis, lice infection, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinellosis, and whipworm infection.

[0301] In certain aspects, parasites include Acanthamoeba, Anisakis, Ascaris lumbricoides, horseflies, Balantidium coli, bed bugs, Cestoda (tapeworms), scrub typhus mites, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworms, Leishmania, Linguatula serrata, liver flukes, Loa loa, Paragonimus (lung flukes), pinworms, and Plasmodium falciparum (malaria parasite). These include falciparum, schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0302] bacterial antigen In certain embodiments, the antigen includes a bacterial antigen, or a fragment or variant thereof. In certain embodiments, the bacteria include the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, and Fibrobacter. It is from one of the following phyla: Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0303] In certain embodiments, bacteria are either Gram-positive or Gram-negative. In certain embodiments, bacteria are either aerobic or anaerobic. In certain embodiments, bacteria are either autotrophic or heterotrophic. In certain embodiments, bacteria are either mesophilic, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or serophilic.

[0304] In certain embodiments, bacteria include anthrax bacteria, antibiotic-resistant bacteria, pathogenic bacteria, foodborne pathogens, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Bacillus tetanus. In certain embodiments, bacteria include mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0305] fungal antigen In certain embodiments, the antigen includes a fungal antigen, or a fragment or variant thereof. In certain embodiments, the fungus includes species of the genus Aspergillus, Blastomyces dermatitidis, Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, species of Fusarium, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, and Sporothrix schenkyi. It belongs to the genus Exserohilum (schenckii) or Cladosporium.

[0306] Tumor antigen In certain embodiments, the antigen includes tumor antigens, such as tumor-associated antigens or tumor-specific antigens. In the context of the present invention, “tumor antigen,” “hyperproliferative disorder antigen,” or “antigen associated with hyperproliferative disorder” refers to an antigen common to a given hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkins lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0307] Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T-cell-mediated immune response. In certain embodiments, the tumor antigens of the present invention comprise one or more antigenic cancer epitopes immunogenically recognized by tumor-infiltrating lymphocytes (TILs) derived from mammalian cancerous tumors. The selection of antigens will depend on the specific type of cancer to be treated or prevented by the compositions of the present invention.

[0308] Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigens (CEAs), β-human chorionic gonadotropins, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, survivorbin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0309] In certain embodiments, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules such as the oncogene HER-2 / Neu / ErbB-2. Another group of target antigens are oncoemetic antigens such as carcinoembryonic antigens (CEAs). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy using monoclonal antibodies, but the success has been limited.

[0310] The types of tumor antigens referred to in this invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and do not appear on other cells in the body. TAA-associated antigens are not specific to tumor cells and instead are expressed on normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are usually present at extremely low levels on normal cells but are expressed at much higher levels on tumor cells.

[0311] Non-limiting examples of TSA antigens or TAA antigens include: differentiation antigens such as MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, as well as tumor-specific multisystem antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, and HER-2 / neu; intrinsic tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA This includes 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophyllin C associated protein, TAAL6, TAG72, TLP, and TPS.

[0312] Adjuvant In certain embodiments, the composition comprises an adjuvant. In certain embodiments, the composition comprises a nucleic acid molecule encoding the adjuvant. In certain embodiments, the nucleic acid molecule encoding the adjuvant is IVT RNA. In certain embodiments, the nucleic acid molecule encoding the adjuvant is nucleoside-modified mRNA.

[0313] Exemplary adjuvants include, but are not limited to, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-inducing chemokines (CTACK), epithelial thymic expression chemokines (TECK), mucosa-associated epithelial chemokines (MEC), IL-12, IL-15, MHC, CD80, CD86, and optionally IL-15 with a deleted signal sequence and containing a signal peptide from IgE. Other genes that could be useful adjuvants include those encoding the following: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, variant forms of IL-18, CD40, C D40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4 , DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon-responsive genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and their functional fragments.

[0314] Pharmaceutical composition Formulations of the pharmaceutical compositions described herein may be prepared by any method known or to be developed in the art of pharmacology. Generally, such preparation methods include the steps of associating the active ingredient with a carrier or one or more other auxiliary components, and then, if necessary or desirable, forming or packaging the product into desired single-dose or multi-dose units.

[0315] The descriptions of pharmaceutical compositions provided herein focus primarily on those suitable for ethical administration to humans, but 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 a veterinary pharmacologist of ordinary knowledge can design and carry out such modifications, if any, by ordinary experimentation. The subjects to whom the pharmaceutical compositions of the present invention are intended to be administered include, but are not limited to, humans and other primates, non-human primates, and mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0316] Pharmaceutical compositions useful in the methods of the present invention may be prepared, packaged, or marketed as formulations suitable for ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intraventricular, intradermal, intramuscular, or other routes of administration. Other formulations intended include projected nanoparticles, liposome preparations, resealed erythrocytes containing active ingredients, and immunogenicity-based formulations.

[0317] The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk as single unit doses or as multiple single unit doses. As used herein, “unit dose” means a specific amount of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, or a convenient fraction of such a dose, for example, half or one-third of such a dose.

[0318] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of the present invention will vary depending on the attributes, size, and state of the object being treated, and further depending on the route through which the composition is administered. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0319] The pharmaceutical composition of the present invention may further contain, in addition to the active ingredient, one or more additional pharmaceutically active substances.

[0320] Controlled-release or sustained-release formulations of the pharmaceutical composition of the present invention can be produced using prior art.

[0321] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical penetration of the target tissue and administration of the pharmaceutical composition through the penetration into the tissue. Thus, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of the composition through a surgical incision, application of the composition through a non-surgical wound penetrating the tissue, and similar methods. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreous, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0322] Formulations of pharmaceutical compositions suitable for parenteral administration include an active ingredient combined with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in forms suitable for bolus or continuous administration. Formulations for injection may be prepared, packaged, or sold in unit dosage forms, for example, ampoules or multi-dose containers containing preservatives. 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 include one or more additional components, including, but not limited to, suspension agents, stabilizing agents, or dispersing agents. In certain embodiments of parenteral administration formulations, 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.

[0323] The pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile aqueous or oily suspensions or solutions for injection. These suspensions or solutions may be formulated according to known techniques and may contain additional components, in addition to the active ingredient, such as dispersing agents, wetting agents, or suspension agents described herein. Such sterile injection formulations may be prepared using non-toxic, parenterally-administrable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solutions, and fixative oils such as synthetic monoglycerides or synthetic diglycerides. Other useful, parentally-administrable formulations include those containing the active ingredient in microcrystalline form in liposome preparations or as components of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion-exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0324] The pharmaceutical compositions of the present invention may be prepared, packaged, or marketed in formulations suitable for pulmonary administration via the buccal oral cavity. Such formulations may comprise dry particles containing the active ingredient, the dry particles having a diameter in the range of about 0.5 to about 7 nanometers, and preferably 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 to which a flow of propellant can be directed to disperse the powder, or for administration using a self-propelled solvent / powder dispensing vessel such as a device comprising the active ingredient dissolved or suspended in a low-boiling point propellant in a sealed container. Preferably, such powder comprises particles, where 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. More preferably, 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. The dried powder composition preferably contains a solid fine powder diluent such as sugar and is conveniently provided in unit dose form.

[0325] Low-boiling-point propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may constitute 50-99.9% (w / w) of the composition, and the active ingredient may constitute 0.1-20% (w / w) of the composition. The propellant may further include additional components such as liquid nonionic or solid anionic surfactants, or solid diluents (preferably having a particle size of the same order as the particles containing the active ingredient).

[0326] Formulations of pharmaceutical compositions suitable for parenteral administration include an active ingredient combined with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in forms suitable for bolus or continuous administration. Formulations for injection may be prepared, packaged, or sold in unit dosage forms, for example, ampoules or multi-dose containers containing preservatives. 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 include one or more additional components, including, but not limited to, suspension agents, stabilizing agents, or dispersing agents. In certain embodiments of parenteral administration formulations, 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.

[0327] The pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile aqueous or oily suspensions or solutions for injection. These suspensions or solutions may be formulated according to known techniques and may contain additional components, in addition to the active ingredient, such as dispersing agents, wetting agents, or suspension agents as described herein. Such sterile injection formulations may be prepared using non-toxic, parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solutions, and fixative oils such as synthetic monoglycerides or synthetic diglycerides. Other useful parenterally administered formulations include those containing the active ingredient in microcrystalline form in liposome preparations or as components of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0328] As used herein, “additional components” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binding agents; lubricants; sweeteners; flavoring agents; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspension agents; dispersants or wetting agents; emulsifiers, lubricants; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymer or hydrophobic materials. Other “additional components” 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.

[0329] Treatment method In one aspect, the disclosure of the present invention provides a method for delivering at least one selected from the group consisting of nucleic acid molecules and therapeutic substances to target cells. In a particular embodiment, the method includes administering a therapeutically effective amount of at least one LNP of the disclosure of the present invention to the target.

[0330] In certain embodiments, the LNP comprises at least one ionizable lipid.

[0331] In certain embodiments, the LNP comprises at least one helper lipid.

[0332] In certain embodiments, LNPs include cholesterol lipids.

[0333] In certain embodiments, the LNP comprises polyethylene glycol (PEG) conjugated lipids and / or modified derivatives thereof.

[0334] In certain embodiments, the LNP includes a cell-targeting domain that is specific for binding to surface molecules of target cells. In certain embodiments, the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0335] The present invention provides a method for delivering an active substance to targeted immune cells. In some embodiments, the active substance is a diagnostic active substance for detecting at least one marker associated with a disease or disorder. In some embodiments, the active substance is a therapeutic substance for treating or preventing a disease or disorder. Accordingly, in some embodiments, the present invention provides a method for diagnosing, treating, or preventing a disease or disorder, comprising the step of administering an effective amount of a composition comprising one or more diagnostic or therapeutic substances, one or more adjuvants, or a combination thereof.

[0336] In some embodiments, the method provides for delivering a composition for gene editing or genetic manipulation to targeted immune cells in order to treat or prevent a disease or disorder. Exemplary diseases or disorders include, but are not limited to, pathogenic diseases and disorders, as well as cancer.

[0337] In some embodiments, the method provides immunity to an infectious disease or a disease or disorder associated with an infectious agent in a target. Thus, the present invention provides a method for treating or preventing infectious diseases or disorders associated with infectious agents. For example, the method may be used to treat or prevent viral, bacterial, fungal, or parasitic infections, depending on the type of antigen in the administered composition. Exemplary antigens and associated infectious diseases, diseases, and tumors are described elsewhere herein.

[0338] The present invention also relates in part to a method for treating cancer and related diseases or disorders in a subject where such treatment is needed, comprising the administration of a composition comprising at least one immune cell-targeting LNP comprising a nucleic acid molecule encoding a CAR specific for binding to a tumor antigen for the treatment of cancer or related diseases or disorders. Exemplary cancers that can be treated using the compositions and methods of the present invention include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendiceal cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain and spinal cord tumors, brainstem glioma, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, rhabdoid tumors, embryonic tumors of the central nervous system, central nervous system lymphomas, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant glioma Oma, cervical cancer, pediatric visual tract tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenic leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumor, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, mycosis, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational cancer cancer), gestational trophoblastoma, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual tract glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal (renal cell) carcinoma, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histhiosibutoma and osteosarcoma of bone, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, oral cancer (mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myelogenic leukemia, myeloid leukemia, myeloma, myeloproliferative disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity Cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovaries, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumors, low-grade ovarian tumors, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis / ureteral cancer, airway cancers involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland This includes, but is not limited to, fluid gland cancer, sarcoma, Sézary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pinealoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, renal pelvis-ureteral transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0339] In certain embodiments, the composition is administered to a target subject having an infection, disease, or cancer. In certain embodiments, the composition is administered to a subject at risk of developing an infection, disease, or cancer. For example, the composition may be administered to a subject at risk of coming into contact with a virus, bacteria, fungus, parasite, or similar organism.

[0340] In certain embodiments, the method includes administering an immune cell targeting LNP composition comprising one or more nucleic acid molecules for the treatment or prevention of a disease or disorder. In certain embodiments, one or more nucleic acid molecules encode a therapeutic substance for the treatment of a disease or disorder. In certain embodiments, one or more nucleic acid molecules encode an active agent (e.g., an mRNA molecule encoding a chimeric antigen receptor) for targeting T cells to antigens expressed by pathogens or cancer cells.

[0341] In certain embodiments, the compositions of the present invention may be administered in combination with additional therapeutic substances, adjuvants, or combinations thereof. For example, in certain embodiments, the method includes administering an LNP composition comprising nucleic acid molecules encoding one or more active agents for targeting immune cells to a target pathogen or tumor cell, and a second LNP comprising nucleic acid molecules encoding one or more adjuvants. In certain embodiments, the method includes administering a single LNP composition comprising nucleic acid molecules encoding one or more active agents for targeting immune cells to a target pathogen or tumor cell, and nucleic acid molecules encoding one or more adjuvants.

[0342] In a particular embodiment, the method includes administering to a subject a plurality of nucleoside-modified nucleic acid molecules that encode a plurality of active agents, adjuvants, or combinations thereof for targeting immune cells to a target pathogen or tumor cell.

[0343] In certain embodiments, the method of the present invention enables the sustained expression of an active agent or adjuvant for targeting immune cells to a target pathogen or tumor cell, as described herein, for at least several days after administration. However, the method also provides transient expression in certain embodiments, because in certain embodiments, the nucleic acid is not integrated into the target genome.

[0344] In certain embodiments, the method comprises the step of administering nucleoside-modified RNA, which provides stable expression of an active agent or adjuvant for targeting immune cells to a target pathogen or tumor cell, as described herein.

[0345] The administration of the composition of the present invention in a treatment method can be achieved in a number of different ways using methods known in the art. In certain embodiments, the method of the present invention includes systemic administration of the subject, including, for example, enteral or parenteral administration. In certain embodiments, the method includes intradermal delivery of the composition. In some embodiments, the method includes intravenous delivery of the composition. In some embodiments, the method includes intramuscular delivery of the composition. In certain embodiments, the method includes subcutaneous delivery of the composition. In certain embodiments, the method includes inhalation of the composition. In certain embodiments, the method includes intranasal delivery of the composition.

[0346] It will be understood that the compositions of the present invention may be administered to a subject either alone or in combination with another active substance.

[0347] Therefore, the therapeutic and preventive methods of the present invention encompass the use of pharmaceutical compositions, which encode active agents, adjuvants, or combinations thereof, for targeting immune cells to target pathogens or tumor cells, as described herein, in order to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention may be administered to deliver doses ranging from ng / kg / day to 100 mg / kg / day. In certain embodiments, the present invention envisions the administration of doses resulting in concentrations of the compounds of the present invention of 10 nM to 10 μM in mammals.

[0348] Typically, the dosage that can be administered to a mammal, preferably a human, in the method of the present invention ranges from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, but the exact dosage administered will vary depending on any number of factors, including but not limited to the type of mammal and the type of disease condition being treated, the age of the mammal, and the route of administration. Preferably, the dosage of the compound varies from about 0.1 μg to about 10 mg per kilogram of body weight of the mammal. More preferably, the dosage varies from about 1 μg to about 1 mg per kilogram of body weight of the mammal.

[0349] The composition may be administered to mammals several times a day, or it may be administered at a less frequent frequency, for example, once a day, once a week, once every two weeks, once a month, or even less frequently, for example, once every few months, or even once a year or less. The frequency of administration will be readily apparent to those skilled in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0350] In certain embodiments, administration of the immunogenic composition or vaccine of the present invention may be carried out by a single dose or boosted by multiple doses.

[0351] In certain embodiments, the present invention includes a method comprising the step of administering one or more compositions encoding one or more active agents or adjuvants for targeting immune cells to a target pathogen or tumor cell, as described herein. In certain embodiments, the method has an additive effect, where the overall effect of administering the combination is approximately equal to the sum of the effects of administering each active agent or adjuvant for targeting immune cells to a target pathogen or tumor cell. In other embodiments, the method has a synergistic effect, where the overall effect of administering the combination is greater than the sum of the effects of administering each active agent or adjuvant for targeting immune cells to a target pathogen or tumor cell. [Examples]

[0352] Various aspects of this application can be better understood by referring to the following examples provided for illustrative purposes. The scope of this application is not limited to the examples given herein.

[0353] material and method Ionizable lipids and mRNA synthesis To prepare C14-4 ionizable lipids, n Addition chemistry was performed according to methods known to those skilled in the art. Briefly, a polyamine core (Enamine Inc., Monmouth Junction, NJ) was combined with an excess of epoxide-terminated C14 alkyl chains (epoxytetradecane, Sigma Aldrich, St. Louis, MO) with gentle stirring at 80°C for 48 hours. The crude product was then dried using Rotovap R-300 (Buchi, New Castle, DE) and resuspended in ethanol for use in LNP formulation.

[0354] mRNA encoding firefly luciferase (luc), mRNA encoding green fluorescent protein (GFP), and mRNA encoding anti-mouse CD19(1D3) specific chimeric antigen receptor (CAR) were all generated from linearized, in vitro transcribed (IVT) template plasmids carrying a T7 promoter, 5'UTR and 3'UTR elements, and a 101-nucleotide poly(A) tail. Cloning and endotoxin-free plasmid preparation services were provided by GenScript (Piscataway, NJ). mRNA was synthesized using the MEGAScript T7 kit (Invitrogen AMB13345), incorporating m1Ψ-5'-triphosphate (TriLink N-1081) as a substitute for UTP in the IVT reaction. Capping of IVT mRNA was co-transcribed using CleanCap (TriLink, San Diego, CA), a trinucleotide cap1 analog. mRNA was purified by cellulose purification according to standard procedures known to those skilled in the art. All mRNA samples were analyzed by agarose gel electrophoresis and then stored at -20°C.

[0355] Antibody treatment The antibodies used in the studies described herein included anti-human CD5 (mouse anti-human, UCHT2, ThermoFisher, Waltham, MA, USA), anti-mouse CD3 (hamster anti-mouse, CD3ε, BioXCell, Lebanon, NH, USA), anti-mouse CD5 (rat anti-mouse, 53-7.3, Biolegend, San Diego, CA, USA), and anti-mouse CD7 (mouse anti-mouse, 2AE46, Proteintech, Rosemont, IL, USA). Anti-human CD5 antibodies were cleaved using IdeZ, anti-mouse CD5 antibodies using pepsin, and anti-mouse CD7 antibodies using ficin. Mouse CD3 antibodies were provided in F(ab)2 format and therefore only reduction was performed.

[0356] IdeZ protease (New England Biolabs, Ipswich, MA, USA) was used according to the manufacturer's instructions, with 1 μL of IdeZ added per 15 μg of antibody during a 90-minute reaction at 37°C. Antibodies cleaved with pepsin (Pierce Fab2 Micro Preparation Kit, ThermoFisher) and ficin (Pierce Mouse IgG1 Fab an Fab2 Micro Preparation Kit, ThermoFisher) were microfabricated using the microfabrication kits and according to the manufacturer's instructions.

[0357] Next, antibody fragments containing anti-mouse CD3 F(ab)2 were reduced with dithiothreitol (DTT) via incubation at 25°C for 30 minutes. The DTT was then removed by centrifugation using a 10 kDa filter (Millipore Sigma, St. Louis, MO, USA), and the antibody product was resuspended in 100 μL of PBS.

[0358] Formulation and Characterization of LNPs LNPs were synthesized according to methods known to those skilled in the art, using a microfluidic device for combining an aqueous phase containing mRNA with an ethanol phase containing lipid and cholesterol components. The aqueous phase contained mRNA in 10 mM citrate buffer. The ethanol phase contained ionizable lipid C14-4, 1,2-dioleoyl-sn glycero-3-johoe-tanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma, St. Louis, MO), and lipid-anchored polyethylene glycol (PEG) (Avanti Polar Lipids), as well as lipid-anchored PEG-maleimide (mal-PEG, Avanti Polar Lipids), in a molar ratio of ionizable lipid 35:DOPE 16:cholesterol 46.5:total PEG 2.5. The aqueous and ethanol phases were then mixed in a microfluidic device in a 3:1 ratio using a 33DS syringe pump (Harvard Apparatus, Holliston, MA). After synthesis, LNPs were dialyzed against PBS for 2 hours before sterilization using a 0.22 μm filter and subsequent antibody conjugation.

[0359] Next, the LNPs were analyzed in triplicates using dynamic light scattering (DLS) performed with a Zetasizer Nano (Malver Instruments, Malvern, UK) to determine their diameter (z-mean) and polydispersion index (PDI). The mRNA concentration of the LNPs was measured via A260 absorbance using an Infinite M Plex plate reader (Tecan, Morrisville, NC).

[0360] Ab-LNP conjugation Each antibody treated with mal-LNPs, combined with cleaved and reduced antibody fragments in a 1:1 maleimide-to-antibody fragment molar ratio, was estimated to yield up to four antibody fragments. After 1 hour at room temperature, the mal-LNPs and antibodies were left overnight at 4°C to complete the reaction. To isolate Ab-LNPs from unreacted antibody fragments containing the Fc region, Ab-LNPs were passed through a Sephadex G-75 bead (Millipore Sigma) column and collected in fractions of approximately 200 μL. Any fraction containing mRNA, measured via A260 / A280 reading on an Infinite M Plex plate reader (Tecan), was pooled as the final Ab-LNP product.

[0361] cell culture Immortalized human T cell line Jurkat cells (ATCC number TIB-152) were cultured in RPMI-1640 containing L-glutamine (ThermoFisher) supplemented with 10% bovine serum and 1% penicillin-streptomycin.

[0362] In vitro luciferase assay and toxicity assay Jurkat cells were plated in 96-well plates at a rate of 60,000 cells per 60 μL before treatment with LNP. After 24 hours, or at various time points as otherwise indicated, cells were centrifuged at 300 g for 7 minutes and resuspended in 50 μL of 1× lysis buffer (Promega, Madison, WI) and 100 μL of luciferase assay substrate (Promega) to determine luciferase mRNA delivery. The luminescence signal was then measured using an Infinite M Plex plate reader (Tecan) and normalized to either the untreated or control group as described. To quantify cytotoxicity after 24 hours, each well was treated with 60 μL of CellTiter-Glo (Promega). After a 10-minute incubation, luminescence corresponding to ATP production was quantified using an Infinite M Plex plate reader (Tecan), and the signal from each group was normalized to the untreated cells.

[0363] In vivo in vivo distribution and CARdel All treatments were administered via tail vein injection in volumes of <200 μL.

[0364] For mice treated with luciferase mRNA: Ten minutes before euthanasia, mice were intraperitoneally injected with 150 mg / kg of D-luciferin potassium salt (Biotium, Fremont, CA) to visualize the luminescence signal. Subsequently, luciferase imaging of collected organs was performed using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). Image analysis was completed using LivingImage software (PerkinElmer).

[0365] For mice treated with GFP mRNA or CAR mRNA: Blood samples were collected from the posterior orbit into blood collection tubes, centrifuged (8 minutes, 750 × g) to remove serum, and then repeatedly replenished with 1 × erythrocyte lysis buffer (Invitrogen). At the time of collection, the spleen and lymph nodes were homogenized, and for spleen samples, any red blood cells were lysed using erythrocyte lysis buffer. After this treatment, all cells from the blood, spleen, and lymph nodes were resuspended as single-cell suspensions in 0.6% PBSA for staining and further analysis.

[0366] Both GFP mRNA-treated and CAR mRNA-treated samples were stained for flow cytometry using the following markers: CD3 (T cells), CD19 (B cells), and CD11b (monocytes / macrophages). Fluorescence staining for these markers included AF700-CD3 (ThermoFisher), APC-CD3 (ThermoFisher), eFluor450-CD19 (ThermoFisher), PE-eF610-CD11b (ThermoFisher), and PE-CD11b (ThermoFisher). CAR mRNA-treated samples were also stained with biotinylated mouse CD19 protein (Sino Biological, Wayne, PA, USA), followed by staining with either streptavidin-AF488 (ThermoFisher) or streptavidin-FITC (Avantor, Radnor, PA, USA). All dyes were diluted according to the manufacturer's recommendations and used to stain cell samples on ice for 20-30 minutes, followed by two washes with PBSA. The cells were then subjected to a BD LSR II flow cytometer (BD Biosciences, Macquarie Park, NSW, Australia). Standard gating was performed to remove doublets, and cell populations were identified as those positive for their markers and negative for staining the remaining cell populations. For example, T cells were identified as CD3 + CD19 - CD11b - They were identified as follows: When observing the depletion of T cells and B cells, these predetermined cell populations were quantified as a fraction of their cell type within a single cell population.

[0367] Toxicity assays and cytokine assays First, to isolate the serum, whole blood was centrifuged at 750 × g for 8 minutes, and the supernatant was collected. This serum was then diluted according to the manufacturer's instructions for the following assays. These assays quantified the concentrations of AST, ALT, IL-6, TNF-α, and GM-CSF. AST and ALT were quantified using a colorimetric assay kit (Cayman Chemicals, Ann Arbor, Michigan, USA) performed according to the manufacturer's recommendations. IL-6, TNF-α, and GM-CSF were quantified using a colorimetric Qantikine ELISA kit (bio-techne, R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

[0368] Example 1: In vitro design and screening of Ab-LNP libraries for T cell targeting. Conventional LNP formulations contain four components: (i) ionizable lipids to provide pH-dependent charge changes that promote endosomal escape and potent intracellular delivery; (ii) cholesterol for stability and membrane fusion; (iii) phospholipids for structural support and endosomal escape; and (iv) lipid-anchored polyethylene glycol (PEG) to prevent aggregation and promote stability. However, in order to produce the antibody-coated LNPs (Ab-LNPs) of the present invention for targeting applications, this conventional formulation has been modified to include maleimide-functionalized lipid-anchored PEG (mal-PEG) as a small portion of the total PEG content. This is because the addition of maleimide to the surface of the LNP (mal-LNP) allows for antibody binding to the surface via thiol-maleimide interactions.

[0369] Therefore, to produce antibody fragments that can bind to the surface of mal-LNP, the entire antibody was cleaved and reduced (Figure 1B). Specifically, the Fab and Fc regions of the CD3, CD5, and CD7 antibodies were separated using IdeZ, pepsin, and ficin, and the resulting fragments were then reduced using dithiothreitol (DTT) to expose the free thiols on the Fab fragments. These antibody fragments then bound to the surface of mal-LNP to form Ab-LNP, and any unconjugated antibody fragments (e.g., both the Fc region and unconjugated Fab) were removed using size exclusion chromatography.

[0370] First, to determine the optimal amount of mal-PEG to incorporate, four exemplary mal-LNP formulations containing luciferase-encoding mRNA were prepared by keeping the molar ratio of excipients constant and changing only the mal-PEG to PEG ratio. This allowed for the conjugation of various amounts of antibody onto the mal-LNP surface (Figures 2A-2C). Next, the mal-LNPs were conjugated with antibodies against human CD5, and their sizes were measured using dynamic light scattering (DLS) before and after conjugation (Figures 2A-2E). DLS measurements revealed that while all Ab-LNPs increased in size compared to LNPs without mal-PEG (B10), mal-LNPs did not significantly increase in diameter compared to B10. Therefore, the size increase observed for Ab-LNPs was attributable to antibody conjugation.

[0371] Next, using their size increase as evidence of conjugation success, Ab-LNPs were screened for mRNA delivery and toxicity in Jurkat cells, a CD5+ human T cell line. This screening used luciferase-encoding mRNA as the model cargo because it requires intracellular delivery and translation to produce a luciferase protein capable of interacting with luciferin reagents, thus allowing the luminescence signal to function as a measure of functional mRNA delivery. When this mRNA delivery was quantified normalized against the baseline B10 treatment group, Ab-LNPs achieved a 15-fold increase in luciferase mRNA delivery compared to B10 without significant toxicity (Figures 2D-2E). All Ab-LNPs resulted in a significant enhancement of transfection regardless of the mal-PEG to PEG ratio, illustrating the impact of antibody targeting even at lower antibody densities. However, when observing relative delivery across mPEG:PEG ratios, 1:3 and 1:5 LNP formulations yielded the highest normalized luminescence, while 1:7 and 1:10 LNP formulations tended to show decreased delivery. This reflects the tendency that a higher antibody-to-LNP ratio leads to better cell uptake, and this improvement plateaued at the 1:5 mPEG:PEG ratio. Therefore, the 1:5 LNP formulation achieved the highest delivery (i.e., the same as the 1:3 LNP formulation) while using less antibody. Consequently, the 1:5 LNP formulation was used in subsequent studies.

[0372] To further characterize the performance of these Ab-LNPs, both dose-response and transfection kinetics were assessed. Across a range of doses, Ab-LNPs maintained a significant increase in luminescence up to high doses that also resulted in a significant decrease in cell viability compared to both non-targeting B10 and non-targeting mal-LNP formulations (Figures 3A-3B). Increased toxicity of LNPs was observed only in the Ab-LNP group; no significant toxicity was observed in either the B10 or mal-LNP groups. Improved mRNA delivery was observed as early as 4 hours at a dose of 50 ng mRNA, indicating that Ab-LNPs can rapidly enhance delivery (Figure 3C).

[0373] In summary, these results demonstrate an improvement in Ab-LNP performance compared to non-targeting LNP platforms, justifying further exploration of this Ab-LNP platform.

[0374] Example 2: In vivo distribution of a specific exemplary Ab-LNP that targets T cells While enhancing mRNA delivery with minimal cellular toxicity in vitro is significant for improving current ex vivo T cell engineering practices, this study attempted to explore the potential of these Ab-LNP platforms for in vivo T cell engineering. For this in vivo study, antibodies against mouse CD3 (CD3-LNP), mouse CD5 (CD5-LNP), and mouse CD7 (CD7-LNP) were incorporated into Ab-LNPs as representative pan-T cell markers. These Ab-LNPs were compared to mal-LNPs with a mal-PEG:PEG ratio of 1:5 (mal-B10), B10 LNPs, and DLin-MC3-DMA(MC3)LNPs, the clinical standard used in FDA-approved therapies. Each of these LNP groups was formulated by encapsulating luciferase-encoding mRNA and characterized via DLS to determine their size (Figure 4A). As expected, this revealed that MC3, B10, and mal-B10 were similar in size, and their size was increased compared to the Ab-LNP group. Next, each of the LNP groups was administered intravenously to mice at a dose of 0.6 mg / kg.

[0375] Six hours later, in vivo distribution was assessed using an in vivo imaging system (IVIS) to capture luminescence signals indicating functional mRNA delivery to major organs (Figure 4B). These images were then used to quantify the luminescence signals from each organ, revealing differences in in vivo distribution among LNP platforms (Figure 4C). mRNA delivery by the standard MC3 LNP resulted primarily in liver delivery, with minimal delivery in both the spleen and lymph nodes (LNs). The remaining LNPs, all containing C14-4 ionizable lipids, primarily resulted in splenic delivery, and all treatment groups achieved enhanced mRNA delivery compared to the MC3 group. Therefore, this ionizable lipid itself may be advantageous for LNPs targeting immune cells.

[0376] Furthermore, among these C14-4 LNPs, the B10 LNP group and the mal-B10 LNP group resulted in higher liver delivery compared to MC3, while Ab-LNP did not result in increased liver transfection, suggesting that the presence of antibodies on the LNP surface may help bypass some of the liver delivery. Luminescence at LNs across all groups was significantly lower than signals from the liver and spleen, although there was a significant increase in delivery from the mal-B10, CD3-LNP, and CD7-LNP groups.

[0377] Since the majority of delivery across all treatment groups occurred in the liver and spleen, the normalized luminescence signals from these organs were then summarized by comparing the spleen versus liver signals. In this comparison, higher values ​​indicate a bias towards splenic delivery compared to hepatic transfection, which may be beneficial for reaching immune cells. In this comparison, MC3 yielded the lowest value, as it achieved no splenic delivery at all, and B10 also failed to significantly improve splenic delivery compared to hepatic delivery. However, the mal-LNP and Ab-LNP groups achieved significantly higher ratios, indicating increased potential for them to reach immune cells. These results suggest that C14-4 LNP enhanced in vivo mRNA delivery compared to MC3, and that Ab-LNP, as the only platform that increased splenic delivery without increasing hepatic delivery, shows great potential.

[0378] To elucidate how this organ-level in vivo distribution impacts delivery to specific immune cells, we next evaluated LNPs for their immune cell transfection in blood, spleen, and LN. Here, LNPs were formulated using GFP-encoding mRNA to enable cell-level analysis using flow cytometry. Six hours after intravenous administration of 0.6 mg / kg of mRNA, GFP expression in cells from blood, spleen, and LN was measured in B cell populations, T cell populations, and macrophage populations (Figures 5A-5D). The majority of immune cell transfection occurred in the blood for all treatment groups, and transfection rates varied across immune cell types. B10 LNPs demonstrated minimal transfection across all immune cell types, while mal-B10 LNPs tended to increase macrophage transfection, but without significant specificity.

[0379] While all three exemplary Ab-LNPs exhibited their highest transfection rates in T cells, only CD3-LNP achieved a significant transfection rate in T cells compared to B cells and macrophages, reaching an average of 6.5% GFP positivity. The same T-cell specificity was not observed in the spleen, where the majority of transfection occurred in macrophages. Here, B10 was the only LNP evaluated that did not show increased delivery to macrophages, but no treatment group exceeded a transfection rate of more than 1%, indicating that delivery to any immune cell was minimal overall. Furthermore, no significant immune cell transfection was observed in LNs, which supported findings from the in vivo distribution of luciferase. Therefore, these results support the use of Ab-LNPs to achieve delivery to immune cells, and CD3-LNP emerged as the most promising platform for transfecting circulating T cells.

[0380] Since CD3-LNP achieved potent and specific transfection of circulating T cells at 6 hours, we then explored its in vivo distribution at the cellular level at later time points. GFP expression of T cells, macrophages, and B cells in blood, spleen, and LN was assessed over 48 hours after intravenous administration of either the B10 group or the CD3-LNP group (Figure 5E-5H). Across these organs, mRNA delivery was observed primarily in blood and spleen for both LNP groups, and transfection of immune cells in LN was minimal, even at later time points. However, the LNP groups achieved transfection in different immune cell populations. In T cells, B10 LNP treatment resulted in minimal transfection in both blood and spleen, while CD3-LNP achieved significantly potent transfection. In blood, GFP expression in mice treated with CD3-LNP decreased over time (a predictable consequence of transient mRNA expression), increasing post-activation T cell motility and the potential for CD3 interaction-related T cell depletion. In the spleen, GFP expression in T cells significantly increased at 24 hours, reaching an average of 11% positivity. This represents a remarkable improvement compared to the 4% delivery observed with previously described CD3-targeting MC3 LNPs, likely due to the observed bias of C14-4 towards the spleen compared to MC3 in terms of in vivo distribution. Overall, these T cell populations treated with CD3-LNP showed the highest GFP positivity rate compared to other immune cell types examined, supporting the T cell specificity of CD3-LNPs.

[0381] Observation of macrophage and B cell populations revealed several additional trends. In macrophages, increased GFP expression in the blood was observed in both the B10 and CD3-LNP groups, with B10 achieving significantly higher GFP expression than CD3-LNP at 48 hours. However, in the spleen, both LNP groups demonstrated modest transfection in macrophages, with GFP expression decreasing over time and no significant difference between treatment groups. In B cells, modest transfection was observed from both LNP groups, but CD3-LNP showed increased delivery in both blood and spleen compared to B10. At 24 hours, B cell transfection in the spleen significantly increased, reflecting the increase observed for T cell transfection at this point, although the majority of transfection was still T cell-specific. These results suggest that CD3-LNP maintained T cell specificity over time, resulting in the greatest transfection of circulating T cells in the blood at an earlier stage, while B10 mostly led to macrophage transfection, with delivery to T cells being negligible.

[0382] Example 3: Ab-LNP demonstrates dose-dependent mRNA delivery to T cells in vivo with minimal toxicity. The in vivo distribution of Ab-LNPs across organs and immune cell types, when combined, encouraged further exploration of these targeting platforms to observe the dose impact on transfection and toxicity. In these experiments, IgG antibodies were conjugated to LNPs to create non-specific Ab-LNP control groups along with B10 LNPs, and blood was collected 24 hours after intravenous injection to assess T cell transfection (Figure 6A). Since neither the non-targeting control (i.e., B10 and IgG-LNP) resulted in significant GFP expression, it was suggested that specific antibody targeting may be necessary to achieve T cell transfection, even at high doses of mRNA. Ab-LNPs showed increased GFP expression, but CD5-LNPs did not achieve significant T cell transfection, even at the highest dose of 2.4 mg / kg. However, both CD3-LNPs and CD7-LNPs induced significant GFP expression at the highest doses, and CD3-LNPs demonstrated potent delivery across all doses. This comparison confirmed that CD3-LNP and CD7-LNP are promising platforms for T cell targeting.

[0383] However, when the T cell population was quantified in these treatment groups, CD3-LNP-treated mice had significantly fewer circulating T cells than untreated mice (Figure 6B). This phenomenon of T cell depletion following CD3 antibody exposure, even in the absence of the Fc region, has been widely reported as a transient effect induced by CD3-T cell interaction, but this phenomenon has not been characterized in many CD3-targeting NP studies. A moderate decrease in T cell count was observed at one dose of CD5-LNP, suggesting that this depletion of circulating T cells may also be induced by other antibodies or T cell interactions, but no such decrease was observed at any dose of CD7-LNP or with any non-targeting LNP.

[0384] To further investigate the biocompatibility of these LNPs beyond immune cell interactions, we then assessed serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for signs of toxicity. Variations in ALT and AST levels were minimal across doses and treatment groups, with only the highest dose of CD3-LNP inducing significant changes (Figure 6C). Therefore, all intermediate doses of LNP treatment did not result in signs of hepatotoxicity, and Ab-LNPs generally did not cause higher toxicity than non-targeting LNPs, allowing for the use of increased doses in subsequent experiments.

[0385] Example 4: Ab-LNP generates functional CAR T cells in vivo. Since CD3-LNP and high doses of CD7-LNP achieved potent delivery to circulating T cells, these Ab-LNPs were then used to deliver non-limiting therapeutic cargo (i.e., mRNA encoding chimeric antigen receptors (CARs)). The CAR constructs used in these experiments targeted mouse CD19, meaning that delivery of functional CARs would result in the elimination of circulating B cells. Because controlling the concentration of circulating CAR T cells may be beneficial in mitigating cytokine release and subsequent side effects, B10, CD3-LNP, or CD7-LNP were used here at either low (0.5 mg / kg) or high (2.0 mg / kg) doses to deliver CAR mRNA and evaluate the ability of these LNPs to transfect T cells in a dose-dependent manner.

[0386] Following intravenous administration, circulating T cells were evaluated for CAR expression over 60 hours to observe both relative delivery across the LNP platform and the duration of CAR expression (Figure 7A). Measurement of CAR expression was accompanied by a comparison of mean fluorescence intensity (MFI) of CAR staining within the T cell population. At the earliest 12-hour time point, both doses of Ab-LNP led to significant CAR expression, with CD3-LNP reaching 15% and 17% positivity in circulating T cells at their lower and higher doses, respectively, and CD7-LNP reaching 5% and 6% positivity at their higher doses. The B10 treatment group reached 2% CAR positivity at its higher dose, but this was not a significant transfection rate compared to the background from PBS-treated mice. Similarly, only the doses of CD3-LNP and the higher doses of CD7-LNP achieved increased MFI values ​​in circulating T cells, indicating more potent CAR expression in these groups. At 36 hours, CAR positivity and MFI values ​​were lower, and only high-dose CD3-LNP maintained a significant percentage of CAR T cells (7%). By 60 hours, no group maintained significant CAR expression, successfully demonstrating mRNA transientity. However, MFI measurements showed that high-dose CD3-LNP continued to increase even at 60 hours, indicating that a small, persistent CAR-positive population in this treatment group may maintain potent expression.

[0387] In summary, while B10 LNP failed to generate a significant CAR T cell population, Ab-LNP demonstrated robust transfection. These findings highlight the importance of a targeted LNP approach for achieving in vivo T cell transfection using therapeutic mRNA cargo.

[0388] Next, B cell elimination was measured to assess whether the observed CAR positivity indicated the production of functional CAR T cells. Although the B cells observed in this experiment were not cancerous, circulating B cell depletion indicates CAR function, as CARs target and can eliminate the CD19+ cell population. Here, to account for any changes in the circulating B cell population due to the presence of LNPs, B10 LNPs containing luciferase mRNA (Luc) were also administered to mice at both low and high doses, and B cell depletion was calculated compared to PBS-treated mice (Figure 7B). At the earliest 12-hour time point, both doses of the Ab-LNP group demonstrated a significant reduction in circulating B cells, with only the high dose of B10 resulting in B cell depletion. CD3-LNPs, at their low and high doses, were able to achieve reductions of 49% and 56% in circulating B cells, respectively, while CD7-LNPs resulted in reductions of 35% and 52%. At high doses, B10 LNP achieved a 46% reduction in circulating T cells, but the population recovered by the next 36-hour mark. In contrast, at 36 hours, Ab-LNP resulted in more persistent B cell depletion; CD3-LNP reduced the B cell population by 58% and 90% for their low and high mRNA doses, respectively, while CD7-LNP maintained a more modest reduction of 30% and 47%. By 60 hours, the circulating B cell population recovered with low-dose CD7-LNP, but was maintained with only a 32% reduction at high doses. However, CD3-LNP continued to demonstrate a significant reduction in B cells, with reductions of 45% and 56% at low and high doses, respectively. Throughout the dose and time points, no B cell depletion was observed in the Luc mRNA LNP group, indicating that these reductions were CAR-mRNA dependent. In summary, these data indicate that the CAR expression observed in T cells corresponded to functional CAR T cell activity in vivo. Both CD3-LNP and CD7-LNP achieved significant B cell depletion over 36 hours, with CD3-LNP demonstrating long-term depletion at 60 hours.

[0389] In addition to confirming functional CAR T cell production in vivo, serum was collected to observe the impact of LNP treatment and dose on cytokine production. IL-6, TNF-α, and GM-CSF were assessed as representative cytokines produced during adverse events such as cytokine release syndrome (Figures 8A-8C). At 12 hours, CD3-LNP resulted in the highest levels of all three cytokines, which was expected as CD3-LNP was the most potent platform for CAR T cell production. By 36 hours, serum levels of IL-6 had decreased, with only the high dose of CD3-LNP showing an increase in concentration compared to PBS. However, serum levels of GM-CSF and TNF-α remained elevated, with GM-CSF significantly increased in the high-dose groups of CD3-LNP, CD7-LNP, and B10 LNP, and TNF-α significantly increased in both doses of CD3-LNP and the high dose of B10 LNP. By 60 hours, all cytokine levels had returned to normal, except in the high-dose CD3-LNP group, which maintained elevated TNF-α serum concentrations. Additionally, since Luc mRNA LNP did not induce any increase in cytokine levels at any time point, it is suggested that the base LNP formulation alone is not inflammatory. Throughout this data, the higher dose of LNP resulted in increased serum cytokine levels, supporting the potential of these LNP platforms for mitigating cytokine release in CAR T cell production.

[0390] Here, lower doses of CD3-LNP resulted in lower serum cytokine levels while still producing potent CAR T cells, and CD7-LNP, while still achieving high levels of B cell depletion, did not lead to significant increases in IL-6 and TNF-α. Furthermore, the transient nature of these increased cytokine levels supports the exploration of repeated dosing to maintain CAR positivity without reaching extremely high cytokine concentrations in serum. Taken together, these results demonstrate the ability of these Ab-LNP platforms to achieve potent CAR T cell production in vivo with the potential for regulating cytokine production.

[0391] Listed aspects The following exemplary embodiments are provided, but their numbering should not be interpreted as indicating a level of importance. Embodiment 1 provides the following: Lipid nanoparticle (LNP) composition including the following: (a) Equation (I): An ionizable lipid compound having the structure TIFF2026531625000024.tif41147 or a salt thereof, During the ceremony, A1 and A2 are each independently selected from the group consisting of CH, N, and P; L1 and L6 are each independent of CR 19 Selected from the group consisting of and N; The occurrences of L2 and L5 are independently -CH2- and -CHR 19 -, -O-, -NH-, and -NR 19 -Selected from the group consisting of; L3 and L4 are independently -CH2- and -CHR 19 -, -O-, -NH-, and -NR 19 -Selected from the group consisting of; R1, R2, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16a , R 16b , R 17 , R 18 , and R 19 Each occurrence is independently of H, halogen, or possibly substituted C1-C 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(C3~C may be replaced) 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(C2~C may be substituted) 12 Heterocycloalkyl), C2-C (may be substituted) 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, -Y(R 20 ) z` (R 21 ) z`` -(C5~C may be substituted) 12 Cycloalkenyl) may be substituted C2-C 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, -Y(R 20 ) z` (R 21 ) z``-(C6~C may be substituted) 12 Cycloalkynyl) may be substituted C6-C 10 Ariel, -Y(R 20 ) z` (R 21 ) z`` -(C6~C may be substituted) 10 (aryl), may be substituted C2-C 12 Heteroaryl, -Y(R 20 ) z` (R 21 ) z`` -(C2~C may be substituted) 12 Heteroaryl), C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z` (R 21 ) z`` -ester, -Y(R 20 ) z` (R 21 ) z`` Selected from the group consisting of -NO2, -CN, and sulfoxy, Or, R 3a and R 3b , R 4a and R 4b , R 5a and R 5b , R 6a and R 6b , R 7a and R7b , R 8a and R 8b , R 9a and R 9b , R 10a and R 10b , R 11a and R 11b , R 12a and R 12b , R 13a and R 13b , R 14a and R 14b , or R 15a and R 15b Two more selected geminal substituents can combine with the carbon atom to which they are bonded to form a C=O group; Each occurrence of Y is independently selected from the group consisting of C, N, O, S, and P; R 20 and R 21 Each occurrence is independently of H, halogen, or possibly substituted C1-C 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, C6-C (may be substituted) 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Selected from the group consisting of alkoxy, carboxyl, carboxylate, ester, -NO2, -CN, and sulfoxy, Or, R 20 and R 21 These can combine with the Y atom to which they are bonded to form C=O; Each occurrence of z` and z`` is independently 0, 1, or 2; Each occurrence of m, n, o, p, q, r, s, t, u, v, w, and x is independently 0, 1, 2, 3, 4, or 5; and A compound having the structure of formula (I) or a salt thereof constitutes about 25 mol% to about 45 mol% of the LNP. The ionizable lipid compound or a salt thereof; (b) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) which constitutes about 10 mol% to about 20 mol% of the LNP; (c) Cholesterol lipids that constitute approximately 40 mol% to approximately 50 mol% of the LNP; (d) polyethylene glycol (PEG) conjugate lipids and / or modified derivatives thereof constituting about 0.5 mol% to about 5.0 mol% of the LNP; and (e) A cell targeting domain that is specific to binding to surface molecules of a target cell, and is covalently conjugated to at least one component of the LNP. Embodiment 2 provides the following: The ionizable lipid compounds of formula (I) are as follows: Selected from the group consisting of TIFF2026531625000025.tif48139TIFF2026531625000026.tif198128TIFF2026531625000027.tif46128, During the ceremony, R1, R2, R3, R4, R5, R6, and R7 are each independently H, halogen, or C1-C which may be substituted. 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, may be substituted C6~C 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; b 1 , b 2 , b 3 , b 4 , and b 5Each of these is independently 0, 1, 2, 3, 4, or 5; c 1 and c 2 Each is independently 0, 1, 2, 3, 4, or 5; and d 1 d 2 d 3 , and d 4 Each of these is independently 0, 1, 2, 3, 4, or 5. LNP in embodiment 1. Embodiment 3 provides the following: The ionizable lipid compounds of formula (I) are as follows: Selected from the group consisting of TIFF2026531625000028.tif50144TIFF2026531625000029.tif193149TIFF2026531625000030.tif189128, During the ceremony, R1, R2, R3, R4, and R5 are each independently H, halogen, or C1-C which may be substituted. 28 Alkyl, possibly substituted C3-C 12 Cycloalkyl, possibly substituted C2-C 12 Heterocycloalkyl, possibly substituted C2-C 28 Alkenyl, C5-C may be substituted. 12 Cycloalkenyl, C2-C may be substituted. 28 Alkinyl, may be substituted C6-C 12 Cycloalkynyl, C6-C (may be substituted) 10 Aryl, possibly substituted C2-C 12 Heteroaryl, C1~C 28 Alkoxycarbonyl, linear C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1~C 28 Aminoalkyl, C2~C 28 Aminoalkenyl, C2~C 28 Aminoalkynyl, C6~C 10 Aminoaryl, aminoacetate, acyl, OH, C1-C28 Hydroxyalkyl, C2-C 28 Hydroxyalkenyl, C2-C 28 Hydroxyalkynyl, C6~C 10 Hydroxyaryl, C1-C 28 Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; and a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. LNP in embodiment 1. Embodiment 4 provides the following: The ionizable lipid of formula (I) is 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azandiyl)bis(tetradecane-2-ol): An LNP of any one of embodiments 1 to 3, including TIFF2026531625000031.tif47128. Embodiment 5 provides the following: An LNP according to any one of embodiments 1 to 4, wherein the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.5. Embodiment 6 provides the following: The aforementioned PEG conjugate lipid is C14-PEG: An LNP of any one of embodiments 1 to 5, including TIFF2026531625000032.tif23140. Embodiment 7 provides the following: The modified derivative of the PEG conjugate lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000(ammonium salt)(mPEG): An LNP of any one of embodiments 1 to 6, including TIFF2026531625000033.tif19145. Embodiment 8 provides the following: The LNP according to embodiment 7, wherein the total PEG conjugate lipids contain a mixture of mPEG and PEG in a ratio of approximately 1:1 to approximately 1:10 (mPEG:PEG). Embodiment 9 provides the following: An LNP according to embodiment 7 or 8, wherein the total PEG conjugate lipids contain a mixture of maleimide PEG (mPEG) and PEG in a ratio selected from the group consisting of 1:3, 1:5, 1:7, and 1:10 (mPEG:PEG). Embodiment 10 provides the following: The LNP is one of any one of embodiments 1 to 9, wherein the target cell is selected from the group consisting of stem cells, peripheral blood mononuclear cells, and immune cells. Embodiment 11 provides the following: One of the LNPs in any of embodiments 1 to 10, which is delivered to the spleen in a larger proportion than to the liver. Embodiment 12 provides the following: An LNP according to any one of embodiments 1 to 11, further comprising at least one selected from the group consisting of nucleic acid molecules and therapeutic substances. Embodiment 13 provides the following: An LNP of any one of embodiments 1 to 12, further comprising at least one active substance selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies. Embodiment 14 provides the following: An LNP according to embodiment 13, comprising a nucleic acid molecule. Embodiment 15 provides the following: The LNP according to embodiment 14, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule. Embodiment 16 provides the following: LNP according to embodiment 14 or 15, wherein the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagonist, antisense molecule, and targeted nucleic acid, or any combination thereof. Embodiment 17 provides the following: The nucleic acid molecule is an LNP in any one of embodiments 14 to 16, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR). Embodiment 18 provides the following: The LNP according to embodiment 17, wherein the CAR is specific to binding to surface antigens of pathogenic cells or tumor cells. Embodiment 19 provides the following: An LNP in any one of embodiments 1 to 18, wherein the cell targeting domain, which is specific to the binding surface molecule of the target cell, is an immune cell targeting domain that is specific to binding to T cells. Embodiment 20 provides the following: An LNP in any one of embodiments 7 to 19, wherein the cell-targeting domain, which is specific to the binding surface molecule of the target cell, is covalently conjugated to an mPEG. Embodiment 21 provides the following: The surface molecules of the target cell are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD2 An LNP of any one of embodiments 1 to 20, which is at least one selected from the group consisting of 54, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7. Embodiment 22 provides the following: A pharmaceutical composition comprising at least one LNP of any one of embodiments 1 to 21 and a pharmaceutically acceptable carrier. Embodiment 23 provides the following: A pharmaceutical composition according to embodiment 22, further comprising an adjuvant. Embodiment 24 provides the following: A pharmaceutical composition according to embodiment 22 or 23, which is a vaccine. Embodiment 25 provides the following: A method for delivering at least one selected from the group consisting of nucleic acid molecules and therapeutic substances to target cells, comprising the step of administering to the subject a therapeutically effective amount of at least one LNP of any one of embodiments 1 to 21 and / or any one of the pharmaceutical compositions of embodiments 22 to 24. Embodiment 26 provides the following: The method according to embodiment 25, wherein the therapeutic substance is at least one selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies. Embodiment 27 provides the following: The method according to embodiment 25, wherein the nucleic acid molecule is at least one selected from the group consisting of DNA molecules and RNA molecules. Embodiment 28 provides the following: The method according to embodiment 25, wherein the nucleic acid molecule is at least one selected from the group consisting of cDNA, mRNA, miRNA, siRNA, antagonist, antisense molecule, and targeted nucleic acid. Embodiment 29 provides the following: The method according to embodiment 25, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR). Embodiment 30 provides the following: The method according to embodiment 29, wherein the CAR is specific to binding to surface antigens of pathogenic cells or tumor cells. Embodiment 31 provides the following: The method according to any one of embodiments 25 to 30, wherein the target cells are selected from the group consisting of stem cells, peripheral blood mononuclear cells, and immune cells. Embodiment 32 provides the following: The method according to embodiment 30, wherein the CAR includes a cell-targeting domain specific to binding to T cells. Embodiment 33 provides the following: The aforementioned cell targeting domains are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, A method according to embodiment 32, which is specific to binding to at least one selected from the group consisting of CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7. Embodiment 34 provides the following: The method according to any one of embodiments 25 to 33, wherein the LNP or its composition further comprises an adjuvant. Embodiment 35 provides the following: A method according to any one of embodiments 25 to 34, wherein the nucleic acid molecule and / or therapeutic substance is at least partially encapsulated within the LNP. Embodiment 36 provides the following: Any one of embodiments 25 to 35 for treating, preventing, and / or improving at least one selected from the group consisting of viral infections, bacterial infections, fungal infections, parasitic infections, cancer, or cancer-related diseases or disorders.

[0392] The terms and expressions used herein are for illustrative purposes only, not restrictive ones, and the use of such terms and expressions is not intended to exclude any of the exhibited and described features or any equivalents thereof, and it is recognized that various modifications are possible within the scope of the embodiments of this application. Therefore, although this application describes specific embodiments and optional features, it should be understood that modifications and changes to the compositions, methods, and concepts disclosed herein can be made by those skilled in the art, and that such modifications and changes are considered to be within the scope of the embodiments of this application.

Claims

1. Lipid nanoparticle (LNP) composition including the following: (a) Equation (I): An ionizable lipid compound having the structure or a salt thereof, During the ceremony, A 1 and A 2 Each is independently selected from the group consisting of CH, N, and P; L 1 and L 6 Each of them operates independently, CR 19 Selected from the group consisting of and N; L 2 and each occurrence of L 5 is independently selected from -CH 2 -, -CHR 19 -, -O-, -NH-, and -NR 19 -; each occurrence is selected from the group consisting of; L 3 and L 4 Each of them operates independently, -CH 2 -, -CHR 19 -, -O-, -NH-, and -NR 19 - Selected from the group consisting of; R 1 , R 2 , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16a , R 16b , R 17 , R 18 , and R 19 Each occurrence is independently H, halogen, or possibly substituted C. 1 ~C 28 Alkyl, possibly substituted C 3 ~C 12 Cycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(C may be replaced) 3 ~C 12 Cycloalkyl, may be substituted C 2 ~C 12 Heterocycloalkyl, -Y(R 20 ) z` (R 21 ) z`` -(C may be replaced) 2 ~C 12 (heterocycloalkyl), optionally substituted C 2 ~C 28 Alkenyl, C may be substituted 5 ~C 12 Cycloalkenyl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C 5 to C 12 cycloalkenyl), optionally substituted C 2 to C 28 alkynyl, optionally substituted C 6 to C 12 cycloalkynyl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C 6 to C 12 cycloalkynyl), optionally substituted C 6 to C 10 aryl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C 6 to C 10 aryl), optionally substituted C 2 to C 12 heteroaryl, -Y(R 20 ) z` (R 21 ) z`` -(optionally substituted C 2 to C 12 heteroaryl), C 1 to C 28 alkoxycarbonyl, linear C 1 to C 28 alkoxycarbonyl, branched C 1 to C 28 alkoxycarbonyl, C(=O)NH 2 , NH 2 , C 1 to C 28 aminoalkyl, C 2 to C 28 aminoalkenyl, C 2 to C 28 aminoalkynyl, C 6 to C 10 aminoaryl, aminoacetate, acyl, OH, C 1 to C 28 hydroxyalkyl, C 2 to C 28 Hydroxyalkenyl, C 2 ~C 28 Hydroxyalkynyl, C 6 ~C 10 Hydroxyaryl, C 1 ~C 28 Alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z` (R 21 ) z`` -ester, -Y(R 20 ) z` (R 21 ) z`` , -NO 2 Selected from the group consisting of , -CN, and sulfoxy, Or, R 3a and R 3b , R 4a and R 4b , R 5a and R 5b , R 6a and R 6b , R 7a and R 7b , R 8a and R 8b , R 9a and R 9b , R 10a and R 10b , R 11a and R 11b , R 12a and R 12b , R 13a and R 13b , R 14a and R 14b , or R 15a and R 15b Two more selected geminal substituents can, together with the carbon atom to which they are bonded, form a C=O group; Each occurrence of Y is independently selected from the group consisting of C, N, O, S, and P; R 20 and R 21 Each occurrence is independently H, halogen, or possibly substituted C. 1 ~C 28 Alkyl, possibly substituted C 3 ~C 12 Cycloalkyl, may be substituted C 2 ~C 12 Heterocycloalkyl, optionally substituted C 2 ~C 28 Alkenyl, C may be substituted 5 ~C 12 Cycloalkenyl, may be substituted C 2 ~C 28 Alkinyl, C may be substituted. 6 ~C 12 Cycloalkynyl, possibly substituted C 6 ~C 10 Aryl, C may be substituted. 2 ~C 12 Heteroaryl, C 1 ~C 28 Alkoxycarbonyl, linear C 1 ~C 28 Alkoxycarbonyl, branched C 1 ~C 28 Alkoxycarbonyl, C(=O)NH 2 NH 2 , C 1 ~C 28 Aminoalkyl, C 2 ~C 28 Aminoalkenyl, C 2 ~C 28 Aminoalkynyl, C 6 ~C 10 Aminoaryl, aminoacetate, acyl, OH, C 1 ~C 28 Hydroxyalkyl, C 2 ~C 28 Hydroxyalkenyl, C 2 ~C 28 Hydroxyalkynyl, C 6 ~C 10 Hydroxyaryl, C 1 ~C 28 Alkoxy, carboxyl, carboxylate, ester, -NO 2 Selected from the group consisting of , -CN, and sulfoxy, Or, R 20 and R 21 They can combine with the Y atom to which they are bonded to form C=O; Each occurrence of z` and z'' is independently 0, 1, or 2; Each occurrence of m, n, o, p, q, r, s, t, u, v, w, and x is independently 0, 1, 2, 3, 4, or 5; and A compound having the structure of formula (I) or a salt thereof constitutes about 25 mol% to about 45 mol% of the LNP. The ionizable lipid compound or a salt thereof; (b) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) constituting about 10 mol% to about 20 mol% of the LNP; (c) Cholesterol lipids that constitute approximately 40 mol% to approximately 50 mol% of the LNP; (d) polyethylene glycol (PEG) conjugate lipids and / or modified derivatives thereof constituting about 0.5 mol% to about 5.0 mol% of the LNP; and (e) A cell targeting domain that is specific to binding to surface molecules of a target cell, and is covalently conjugated to at least one component of the LNP.

2. The ionizable lipid compounds of formula (I) are as follows: Selected from the group consisting of, During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 These are H, halogen, and possibly substituted C, each independently. 1 ~C 28 Alkyl, possibly substituted C 3 ~C 12 Cycloalkyl, may be substituted C 2 ~C 12 Heterocycloalkyl, optionally substituted C 2 ~C 28 Alkenyl, C may be substituted 5 ~C 12 Cycloalkenyl, may be substituted C 2 ~C 28 Alkinyl, C may be substituted. 6 ~C 12 Cycloalkynyl, may be substituted C 6 ~C 10 Aryl, C may be substituted. 2 ~C 12 Heteroaryl, C 1 ~C 28 Alkoxycarbonyl, linear C 1 ~C 28 Alkoxycarbonyl, branched C 1 ~C 28 Alkoxycarbonyl, C(=O)NH 2 NH 2 , C 1 ~C 28 Aminoalkyl, C 2 ~C 28 Aminoalkenyl, C 2 ~C 28 Aminoalkynyl, C 6 ~C 10 Aminoaryl, aminoacetate, acyl, OH, C 1 ~C 28 Hydroxyalkyl, C 2 ~C 28 Hydroxyalkenyl, C 2 ~C 28 Hydroxyalkynyl, C 6 ~C 10 Hydroxyaryl, C 1 ~C 28 Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; b 1 , b 2 , b 3 , b 4 , and b 5 Each of these is independently 0, 1, 2, 3, 4, or 5; c 1 and c 2 Each of them is independently 0, 1, 2, 3, 4, or 5; and d 1 d 2 d 3 , and d 4 Each of these is independently 0, 1, 2, 3, 4, or 5. The LNP according to claim 1.

3. The ionizable lipid compounds of formula (I) are as follows: Selected from the group consisting of, During the ceremony, R 1 , R 2 , R 3 , R 4 , and R 5 These are H, halogen, and possibly substituted C, each independently. 1 ~C 28 Alkyl, possibly substituted C 3 ~C 12 Cycloalkyl, may be substituted C 2 ~C 12 Heterocycloalkyl, optionally substituted C 2 ~C 28 Alkenyl, C may be substituted 5 ~C 12 Cycloalkenyl, may be substituted C 2 ~C 28 Alkinyl, C may be substituted. 6 ~C 12 Cycloalkynyl, possibly substituted C 6 ~C 10 Aryl, C may be substituted. 2 ~C 12 Heteroaryl, C 1 ~C 28 Alkoxycarbonyl, linear C 1 ~C 28 Alkoxycarbonyl, branched C 1 ~C 28 Alkoxycarbonyl, C(=O)NH 2 NH 2 , C 1 ~C 28 Aminoalkyl, C 2 ~C 28 Aminoalkenyl, C 2 ~C 28 Aminoalkynyl, C 6 ~C 10 Aminoaryl, aminoacetate, acyl, OH, C 1 ~C 28 Hydroxyalkyl, C 2 ~C 28 Hydroxyalkenyl, C 2 ~C 28 Hydroxyalkynyl, C 6 ~C 10 Hydroxyaryl, C 1 ~C 28 Selected from the group consisting of alkoxys, carboxyls, carboxylates, and esters; and a 1 a 2 a 3 a 4 , and a 5 Each of these is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. The LNP according to claim 1.

4. The ionizable lipid of formula (I) is 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azandiyl)bis(tetradecane-2-ol): An LNP according to any one of claims 1 to 3, including the LNP described in any one of claims 1 to 3.

5. The LNP according to any one of claims 1 to 4, wherein the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.

5.

6. The aforementioned PEG conjugate lipid is C14-PEG: An LNP according to any one of claims 1 to 5, including the LNP described in any one of claims 1 to 5.

7. The modified derivative of the PEG conjugate lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000(ammonium salt)(mPEG): An LNP according to any one of claims 1 to 6, including the LNP described in any one of claims 1 to 6.

8. The LNP according to claim 7, wherein the total PEG conjugate lipids comprise a mixture of mPEG and PEG in a ratio of about 1:1 to about 1:10 (mPEG:PEG).

9. The LNP according to claim 7 or 8, wherein the total PEG conjugate lipid comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio selected from the group consisting of 1:3, 1:5, 1:7, and 1:10 (mPEG:PEG).

10. The LNP according to any one of claims 1 to 9, wherein the target cells are selected from the group consisting of stem cells, peripheral blood mononuclear cells, and immune cells.

11. The LNP according to any one of claims 1 to 10, which is delivered to the spleen in a larger proportion than to the liver.

12. The LNP according to any one of claims 1 to 11, further comprising at least one selected from the group consisting of nucleic acid molecules and therapeutic substances.

13. The LNP according to any one of claims 1 to 12, further comprising at least one active agent selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

14. The LNP according to claim 13, comprising a nucleic acid molecule.

15. The LNP according to claim 14, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

16. The LNP according to claim 14 or 15, wherein the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagonistol, antisense molecules, and targeted nucleic acids, or any combination thereof.

17. The LNP according to any one of claims 14 to 16, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

18. The LNP according to claim 17, wherein the CAR is specific to binding to surface antigens of pathogenic cells or tumor cells.

19. The LNP according to any one of claims 1 to 18, wherein the cell targeting domain, which is specific to the binding surface molecule of the target cell, is an immune cell targeting domain that is specific to binding to T cells.

20. The LNP according to any one of claims 7 to 19, wherein the cell targeting domain, which is specific to the binding surface molecule of the target cell, is covalently conjugated to the mPEG.

21. The surface molecules of the target cell are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD25 The LNP according to any one of claims 1 to 20, which is at least one selected from the group consisting of 4, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7.

22. A pharmaceutical composition comprising at least one LNP according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition according to claim 22, further comprising an adjuvant.

24. A pharmaceutical composition according to claim 22 or 23, which is a vaccine.

25. A method for delivering at least one selected from the group consisting of nucleic acid molecules and therapeutic substances to target cells, comprising the step of administering to the subject a therapeutically effective amount of at least one LNP according to any one of claims 1 to 21 and / or a pharmaceutical composition according to any one of claims 22 to 24.

26. The method according to claim 25, wherein the therapeutic substance is at least one selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

27. The method according to claim 25, wherein the nucleic acid molecule is at least one selected from the group consisting of DNA molecules and RNA molecules.

28. The method according to claim 25, wherein the nucleic acid molecule is at least one selected from the group consisting of cDNA, mRNA, miRNA, siRNA, antagonist, antisense molecule, and targeted nucleic acid.

29. The method according to claim 25, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

30. The method according to claim 29, wherein the CAR is specific to binding to surface antigens of pathogenic cells or tumor cells.

31. The method according to any one of claims 25 to 30, wherein the target cells are selected from the group consisting of stem cells, peripheral blood mononuclear cells, and immune cells.

32. The method according to claim 30, wherein the CAR includes a cell-targeting domain specific to binding to T cells.

33. The aforementioned cell targeting domains are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, C The method according to claim 32, which is specific to binding to at least one selected from the group consisting of D254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7.

34. The method according to any one of claims 25 to 33, wherein the LNP or its composition further comprises an adjuvant.

35. The method according to any one of claims 25 to 34, wherein the nucleic acid molecule and / or therapeutic substance is at least partially encapsulated within the LNP.

36. The method according to any one of claims 25 to 35 for treating, preventing and / or improving at least one selected from the group consisting of viral infections, bacterial infections, fungal infections, parasitic infections, cancer, or cancer-related diseases or disorders.

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