Compositions and methods for t cell targeting of extrahcpatic delivery of therapeutic agents

Abstract

The present disclosure relates, in part, to immune cell-targeted lipid nanoparticle (LNP) compositions and methods of their use for the in vivo delivery of nucleic acid molecules and / or therapeutic agents to target cells. In certain embodiments, the target cells are located in the spleen of a subject. In certain embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR). In certain embodiments, the target cells are T cells. In certain embodiments, the present disclosure relates to the use of the LNP described herein for the treatment, prevention, and / or amelioration of diseases and / or disorders, including but not limited to cancer.

Description

[0001] Statement regarding federally funded research or development This invention was completed with government funding from the TR002776 project granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0002] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 581876, filed September 11, 2023, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Background Technology

[0003] Chimeric antigen receptor (CAR) T-cell therapy has reshaped the landscape of cancer immunotherapy. Six FDA-approved CAR T-cell immunotherapies are currently used to treat relapsed or refractory acute lymphoblastic leukemia (ALL), B-cell lymphoma, and multiple myeloma, with applications in other cancers under investigation. Currently, the production of these potent autologous cell therapies relies on complex ex vivo cell engineering processes.

[0004] In short, the collected patient T cells are isolated, virally modified to express a transmembrane CAR construct, and then re-infused 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 virally induced, it is permanent and potent. After clearing cancerous cells, these CAR T cells can also attack healthy B cells, leading to adverse reactions such as cytokine release syndrome, long-term B cell regeneration disorders, and pancytopenia, exposing patients to the risk of serious infections.

[0005] Therefore, there is a need in the art for new methods to engineer CAR T cells that provide potent cancer cell killing while mitigating the severity of these off-target effects. This disclosure addresses and satisfies this unmet need. Summary of the Invention

[0006] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising: (a) An ionizable lipid compound having the structure of formula (I) or a salt thereof: Formula (I), Among them, A1, A2, L1, L2, L3, L4, L5, L6, R1, R2, R 3a R 3b R 4a R 4b R 5a R5b 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 As defined elsewhere in this document; and Compounds having the structure of formula (I) or their salts comprise about 25 mol% to about 45 mol% of LNP; (b) 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), wherein DOPE comprises approximately 10 mol% to approximately 20 mol% of LNP. (c) Cholesterol lipids, of which cholesterol lipids account for approximately 40 mol% to approximately 50 mol% of LNP; (d) Polyethylene glycol (PEG) conjugated lipids and / or their modified derivatives, wherein the PEG conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to about 5.0 mol% of the LNP; and (e) A cell-targeting domain that is specific to binding to molecules on the surface of target cells, wherein the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0007] In some embodiments, the ionizable lipid of formula (I) is 1,1'-((2-(2-(4-(2-((2-(2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azanediyl)bis(tetradecane-2-ol). (C14-4).

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

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

[0010] In another aspect, this disclosure provides a method for delivering at least one selected from nucleic acid molecules and therapeutic agents to target cells, the method comprising administering to a subject a therapeutically effective amount of at least one LNP of this disclosure and / or a pharmaceutical composition of this disclosure. Attached Figure Description

[0011] The accompanying drawings illustrate various embodiments of this application by way of example rather than limitation.

[0012] Figure 1A : Schematic diagrams of conventional CAR T-cell engineering methods and exemplary CAR T-cell engineering methods of this disclosure. In conventional LNP-based T-cell transfection methods, LNPs bind to patient T cells in vitro to generate CAR T cells. In Ab-LNP-based T-cell transfection of this disclosure, Ab-LNPs are administered to the patient to generate in vivo CAR T-cell therapy, wherein the antibody promotes T-cell targeting. Figure 1B : Non-restrictive schematic diagram of Ab-LNP formation. An ethanol phase containing LNP components and water containing mRNA cargo are combined via microfluidic mixing to generate mal-LNPs with a mal-PEG surface. These bind to cleaved and reduced antibody fragments, which conjugate with the mal-LNPs to form Ab-LNPs. Figure 1C The structures of clinically standard MC3 lipids and C14-4 ionizable lipids used to generate certain exemplary LNPs of this disclosure, and representative IVIS images of their respective in vivo performance, illustrate extrahepatic delivery using C14-4.

[0013] Figures 2A-2E Exemplary Ab-LNP showed increased size and efficacy compared to mal-LNP. Figure 2A-2CBy keeping the excipient ratio constant while varying the mal-PEG:PEG ratio, a mal-LNP library was prepared, allowing different amounts of anti-CD5 human antibody to bind to the mal-LNP surface. Figure 2A Then, the library was measured using DLS to observe the results compared to the 1:5 mal-LNP (shown). Figure 2B The dimensional changes of representative DLS curves before and after the concatenation of ) and Ab-LNP were analyzed, and the mean diameter (mean peak intensity) was summarized (n=3, error bars = standard deviation). Statistical analysis included Bonferroni-corrected one-way ANOVA, compared with B10, p<0.001 Figure 2C ). Figure 2D-2E In Jurkat cells (CD5) + In this study, mal-LNP and Ab-LNP libraries were screened at a dose of 50 ng / 60,000 cells to measure 24-hour luciferase-encoded mRNA delivery and cell viability (n=3 biological replicates, error bars=standard deviation). Luminescence measurements were normalized to B10 delivery, and viability was normalized to untreated cells. Statistical analysis included Bonferroni-corrected one-way ANOVA, compared to B10, p<0.001.

[0014] Figures 3A-3C Dose response and kinetics of Jurkat transfection using an exemplary 1:5 LNP. Figures 3A-3B Jurkat cells were treated with mRNA encoding luciferase for 24 hours using 1:5 mal-LNP and Ab-LNP within the mRNA dose range to express luciferase. Figure 3A ) and survival rate ( Figure 3B The efficacy and biocompatibility of Ab-LNP were confirmed (n=3 biological replicates, error bars = standard deviation). Results of luminescence measurements were normalized to B10 delivery, and viability was normalized to untreated cells. Results for each treatment group within each dose were compared in a Bonferroni-corrected one-way ANOVA between two measurements, compared to B10. p<0.05. Figure 3C Jurkat cells were treated with 1:5 LNP at 50 ng / 60,000 cells for 0–24 hours to express luciferase (n = 3 biological replicates, error bars = standard deviation). Statistical analysis included Bonferroni-corrected two-way ANOVA, compared with the same dose of B10. p<0.05.

[0015] Figures 4A-4CCompared to standard LNP formulations, Ab-LNPs exhibit biodistribution that favors spleen delivery. Figure 4A Representative DLS curves of the exemplary LNP treatment group, showing the size change after antibody conjugation. Figure 4B Representative IVIS images of organs collected from mice 6 hours after intravenous injection of certain exemplary LNPs containing 0.6 mg / kg mRNA encoding luciferase. Figure 4C The luminescence measurements of the liver, spleen, and lymph nodes (LNs) on IVIS images were normalized to the background (n=4 biological replicates, error bars = standard deviation). The normalized luminescence ratios of the spleen and liver for each mouse were also summarized. Statistical analyses included Dunnett-corrected one-way ANOVA, compared to the MC3 treatment group. p<0.05, p<0.001.

[0016] Figures 5A-5H Compared with untargeted LNPs, Ab-LNPs showed improved T cell delivery in vivo. Figures 5A-5D : such as representative experimental schemes ( Figure 5A As shown in the figure, 6 hours after intravenous injection of 0.6 mg / kg LNP containing mRNA encoding GFP, B cells (CD19) + ), macrophages (CD11b + ) and T cells (CD3) + ) in blood ( Figure 5B ),spleen( Figure 5C ) and lymph nodes ( Figure 5D Transfection rates were measured using flow cytometry (n=4 biological replicates, error bars = standard deviation). Statistical analysis included Dunnett-corrected two-way ANOVA for significant comparisons. p<0.05, p<0.0001. Figure 5E-5H : such as representative experimental schemes ( Figure 5E As shown in the figure, after intravenous injection of 0.6 mg / kg LNP containing mRNA encoding GFP, T cells at different time points ( Figure 5F ), macrophages ( Figure 5G ) and B cells ( Figure 5H Transfection rates in blood and spleen were measured using flow cytometry (n=4 biological replicates, error bars = standard deviation). Unless otherwise stated, statistical analyses included Tukey-corrected two-way ANOVA, compared with B10 at the same time point. p<0.05, p<0.001.

[0017] Figures 6A-6C Exemplary dose increases of CD3-LNP showed signs of T cell population reduction and toxicity; 24 hours after intravenous injection of different doses of LNPs encapsulating GFP mRNA, the transfection rate of T cells in the blood ( Figure 6A ), normalized T cell count ( Figure 6B ) and measurement of serum ALT and AST levels ( Figure 6C (n=3). T cell population was measured as a percentage of single cell populations, with T cell counts, ALT, and AST normalized to the PBS-treated group. Statistical analysis included Dunnett-corrected two-way ANOVA, compared to PBS. p<0.05, p<0.001.

[0018] Figures 7A-7B Ab-LNP generates functional CAR T cells in vivo. Figure 7A Transfection rate and mean fluorescence intensity (CAR staining) of T cells in blood at different time points after low-dose (0.5 mg / kg) and high-dose (2 mg / kg) CAR mRNA delivery (n=4). Statistical analysis included Dunnett-corrected two-way ANOVA, compared with PBS at the same time point. p<0.05, p<0.0001. Figure 7B In these identical treatment groups, B cell depletion, an indicator of 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. Other LNP groups containing mRNA encoding luciferase were included as negative controls for CAR function (n=4). Statistical analyses included a Tukey-corrected two-way ANOVA, comparing CAR activity to PBS at the same time point. p<0.05, p<0.0001.

[0019] Figures 8A-8C Cytokine levels in mouse serum after CAR mRNA treatment. Serum IL-6 levels at different time points after CAR mRNA delivery at low (0.5 mg / kg) and high (2 mg / kg) doses. Figure 8A ), GM-CSF ( Figure 8B ) and TNF-α Figure 8C The concentration of ) was normalized using PBS-treated mice (n=4). Statistical analysis included Dunnett-corrected two-way ANOVA. p<0.05, p<0.0001. Detailed Implementation

[0020] Reference will now be made to certain embodiments of the disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

[0021] In this document, values ​​expressed in range format should be interpreted flexibly, including not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges contained within the range, as if each value and subrange were explicitly listed. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(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%) within the indicated range. Unless otherwise stated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0022] In this document, unless the context clearly specifies otherwise, the terms “a,” “an,” or “the” are used to include one or more. Unless otherwise stated, the term “or” is used to refer to a non-exclusive “or.” The expressions “at least one of A and B” or “at least one of A or B” have the same meaning as “A, B, or A and B.” Furthermore, it should be understood that the wording or terminology used herein (unless otherwise defined) is descriptive and not restrictive. The use of any section headings is for the purpose of aiding reading the document and should not be construed as restrictive; information relating to a section heading may appear within or outside that particular section. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference.

[0023] In the methods described herein, actions may be performed in any order unless the timing or sequence of operations is explicitly stated. Furthermore, specific actions may be performed simultaneously unless the explicit language of the claims states that they may be performed separately. For example, the claimed action of doing X and the claimed action of doing Y may be performed simultaneously in a single operation, and the resulting process will fall within the literal scope of the claimed method.

[0024] describe As noted elsewhere in this article, there is a need in the art for novel approaches to engineer CAR T cells that deliver potent cancer killing while mitigating the severity of these off-target effects. Compared to viral transduction, mRNA-based cell engineering offers several advantages. From a production perspective, mRNA avoids common concerns associated with viral vectors, such as limited payload, insertional mutations, and in vivo immunogenicity. Furthermore, mRNA induces transient CAR expression, which mitigates the risks associated with long-term CAR T cell activity. Specifically, this transient CAR expression allows for time-controlled CAR T cell therapy to prevent the long-term presence of CAR T cells in the absence of cancer cells. Overall, this potential has led to the evaluation of mRNA CAR T cell therapies for a variety of cancers, including melanoma, Hodgkin's lymphoma, and ALL, demonstrating their ability to alleviate short-term disease burden as effectively as virally engineered CAR T cells. However, the rapid degradation and poor transmembrane penetration of mRNA necessitate optimized delivery methods for generating these mRNA CAR T cells.

[0025] One promising mRNA delivery strategy is the use of nanoparticles, as they can mitigate cytotoxicity, stabilize mRNA cargo, and enhance intracellular delivery. Specifically, lipid nanoparticles (LNPs) have been used as a platform for the potent delivery of mRNA to various cell types because their ionizable lipids are charged in acidic environments to facilitate endosome escape. Furthermore, their clinical applications have received multiple FDA approvals, including Alnylam's Onpattro siRNA LNP therapeutic vaccine and Moderna and Pfizer BioNTech's COVID-19 mRNA LNP vaccine, making them a potentially ideal platform for delivering mRNA to other immune cells such as T cells. Previous work has demonstrated potent T cell transfection using such LNPs and, through excipient screening, established an optimized LNP platform for the in vitro generation of mRNA-based CAR T cells.

[0026] However, despite the therapeutic efficacy of in vitro CAR T-cell engineering, its associated costs and complexities may hinder its widespread clinical application. Therefore, it is necessary to investigate alternative production strategies, such as in vivo engineering of CAR T cells. Delivering CAR mRNA to T cells in vivo, thereby avoiding the processes of leukocyte removal and in vitro T-cell expansion, would simplify production and eliminate the need for patient-specific manufacturing. Figure 1A However, many obstacles complicate the transfection of T cells in vivo, including the sustained motility of circulating T cells, which may shorten the duration of LNP-T cell interactions, and the clearance of LNPs by the liver, which may limit their bioavailability.

[0027] To overcome this first hurdle and improve LNP-T cell binding, other nanoparticle platforms utilize antibody conjugation to alter biodistribution, enhance specificity to minimize off-target effects, and improve delivery to target cell populations both in vitro and in vivo. In T cell applications, many of these benefits have been demonstrated by targeting various receptors, including CD3, CD8, CD4, CD7, CD5, Nrp1, and... 7) Polymerized NPs or LNPs. However, no study has yet directly compared the efficacy of these different antibodies against different T-cell markers to identify the best candidates for targeting in vivo mRNA delivery.

[0028] Furthermore, most antibody-based targeting strategies do not utilize nanoparticle platforms also designed for delivery to extrahepatic tissues. Therefore, the task of targeting nanoparticle platforms is not only to reach the desired cell population but also to overcome hepatic accumulation. As described in this paper, an antibody-conjugated targeting strategy is applied to the LNP platform, which enables extrahepatic delivery to aid in in vivo T-cell targeting.

[0029] Therefore, in one aspect, this disclosure relates to the design, development, and evaluation of antibody-coated LNP platforms (Ab-LNP) for T cell targeting, and demonstrates their potential in in vivo CAR T cell engineering. To generate Ab-LNP, a previously established B10 LNP platform containing C14-4 ionizable lipids was modified to include maleimide-functionalized PEG (mal-PEG), as previous work has validated this antibody-nanoparticle conjugation strategy. Figure 1B To make this method applicable to B10 LNPs, the optimal excipient molar ratio of mal-PEG was determined through in vitro screening in Jurkat cells, which exhibited enhanced T cell transfection with minimal toxicity. These Ab-LNPs were then explored for in vivo T cell transfection.

[0030] This study only examined pan-T cell markers and did not include T cell subsets (such as CD4) that might be detrimental to immunotherapies such as CAR T cell therapy. + or CD8 + T cells). Specifically, Ab-LNPs targeting pan-T cell markers CD3, CD5, and CD7 were formulated, and their biodistribution was compared with B10 LNPs and clinically relevant DLin-MC3-DMA LNPs. Although many untargeted LNPs have been reported to be primarily transported to the liver, suggesting their tropism is opposite to the desired immune cell delivery, all C14-4 LNPs showed a bias towards spleen delivery, while Ab-LNPs showed low levels of liver delivery, indicating their ability to bypass this organ ( Figure 1CWhen specifically observing immune cell populations, untargeted LNPs could not demonstrate T cell transfection in vivo; only CD3-LNPs and higher doses of CD7-LNPs demonstrated significant T cell delivery.

[0031] The in vivo delivery of non-restricted, exemplary therapeutic cargo (i.e., mRNA encoding CD19CAR) by these two Ab-LNPs was then explored. Both platforms generated large numbers of circulating CAR T cells, with the CD3-LNPs generating CAR T cells persisting for up to 60 hours post-administration. Furthermore, these Ab-LNPs led to significant depletion of circulating B cells, demonstrating their efficacy in eliminating the B cell population and thus demonstrating their therapeutic potential for B-cell carcinoma. Although many cytokines are associated with the cytokine release syndrome observed in patients receiving CAR T-cell therapy, this article explored three relevant cytokines: IL-6, TFN-α, and GM-CSF. The transient, dose-dependent elevation of these cytokine levels in serum supports the potential of Ab-LNPs to alleviate cytokine release and allow for repeated administration.

[0032] In summary, we evaluated T cell transfection using three Ab-LNP platforms targeting pan-T cell markers, revealing 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 validating Ab-LNP as a platform for in vivo CAR T cell production.

[0033] Therefore, in one aspect, the present invention provides an exohepatic Ab-LNP platform for in vivo CAR T-cell engineering. Exemplary Ab-LNPs were screened in Jurkat cells using an anti-CD5 antibody to determine optimized antibody densities by incorporating different proportions of mal-PEG with PEG into the LNP formulation. These Ab-LNPs were then generated using antibodies against CD3, CD5, or CD7 pan-T-cell markers, demonstrating the platform's versatility while allowing comparisons of these T-cell targets. In vivo screening of these LNPs using different reporter cargoes showed that C14-4 LNP achieved a higher proportion of spleen delivery than the MC3 clinical standard compared to liver delivery, and that the Ab-LNPs exhibited T-cell transfection capability. Specifically, CD3-LNP and CD7-LNP achieved significant transfection in circulating T cells and were specific relative to other immune cell types. These two Ab-LNP platforms were then formulated with CAR-encoded mRNA and achieved significant CAR positivity in vivo, accompanied by potent B-cell exhaustion. Therefore, the development of Ab-LNPs, including CD3- and CD7-LNPs, has proven to be a means of generating functional CAR-T cells in vivo. Thus, Ab-LNPs show promise as a platform for CAR T-cell engineering and other T-cell engineering applications.

[0034] definition The term “about” as used herein may allow for the degree of variability of a value or range, such as within 10%, 5%, or 1% of the limit of the value or range, and may include the exact value or range.

[0035] The term "adjuvant" as used in this article is defined as any molecule that enhances an antigen-specific adaptive immune response.

[0036] As used herein, the term "alkenyl" refers to straight-chain, branched, and cycloalkyl groups as defined herein, except that there is 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, etc.

[0037] As used herein, the term "alkoxy" refers to an oxygen atom bonded 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, pentoxy, hexoxy, and so on. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and so on. Examples of cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, and so on. An alkoxy group may include 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 include double or triple bonds, and may also include heteroatoms. For example, allyloxy or methoxyethoxy are also alkoxy groups as understood herein, as are methylenedioxy groups where two adjacent atoms of the structure are substituted with them.

[0038] As used herein, the term "alkyl" refers to straight-chain and branched alkyl and cycloalkyl groups having 1 to 40 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, or, in some embodiments, 1 to 8 carbon atoms. Examples of straight-chain alkyl groups include those having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. As used herein, the term "alkyl" includes n-alkyl, isoalkyl, and trans-isoalkyl groups, as well as other branched forms of alkyl. Representative substituted alkyl groups may be substituted once or more with any of the groups listed herein, such as amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen.

[0039] As used herein, the term "alkynyl" refers to straight-chain and branched alkyl groups, where at least one triple bond exists between two carbon atoms. Therefore, an alkynyl group has 2 to 40 carbon atoms, 2 to approximately 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, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3), etc.

[0040] As used herein, the term "amine" refers to primary, secondary, and tertiary amines having, for example, the formula N(group)3, wherein each group may be independently H or non-H, such as alkyl, aryl, etc. Amines include, but are not limited to, R-NH2, such as alkylamines, aromatic amines, and alkylaromatic amines; R2NH, wherein each R is independently selected, such as dialkylamines, diarylamines, arylalkylamines, heterocyclic amines, etc.; and R3N, wherein each R is independently selected, such as trialkylamines, dialkylaromatic amines, alkyldiaromatic amines, triaromatic amines, etc. The term "amine" also includes ammonium ions as used herein.

[0041] The term "amino group" used in this article refers to -NH2, -NHR, -NR2, and -NR3. + Substituents of the form, wherein each R is independently chosen, and each has a protonated form (but -NR3). + Except that it cannot be protonated. Therefore, any compound substituted with an amino group can be considered an amine. The term "amino" as used herein can refer to a primary, secondary, tertiary, or quaternary amino group. The "alkylamino" group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0042] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphonic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and palmitoylphosphatidylglycerol (POPG), as well as other anionic modifying groups that bind to neutral lipids.

[0043] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, or they can be immunoreactive portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies used in this invention can exist in various 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, New York; 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).

[0044] The term "antibody fragment" refers to a portion of a complete antibody, specifically the antigenic determination variable region of the complete 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.

[0045] The term "antibody heavy chain" as used in this article refers to the larger of the two polypeptide chains present in the native conformation of all antibody molecules.

[0046] The term "antibody light chain" as used in this article refers to the smaller of the two polypeptide chains present in the native conformation of all antibody molecules. The κ and λ light chains refer to the two main isotypes of antibody light chains.

[0047] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage. The term should also be understood to refer to an antibody produced by synthesizing a DNA molecule encoding an antibody, wherein the DNA molecule expresses an antibody protein or the amino acid sequence of a specified antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and known in the art. The term should also be understood to refer to an antibody produced by synthesizing an RNA molecule encoding an antibody. This RNA molecule expresses an antibody protein or the amino acid sequence of a specified antibody, wherein the RNA is obtained by transcribing DNA (synthesized or cloned) or using other techniques available and known in the art.

[0048] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response may involve antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant DNA or RNA, or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA containing a nucleotide sequence or a portion thereof encoding a protein that elicits an adaptive immune response therefore encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, arranged in various combinations to elicit the desired immune response. Moreover, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is apparent that antigens can be synthesized or can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0049] As used herein, the term "aryl" refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in its ring. Therefore, aryl includes, but is not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrene, triphenylenyl, pyrene, napthacenyl, chrysenyl, biphenylene, anthracene, and naphthyl. In some embodiments, the aryl group contains about 6 to about 14 carbons in the cyclic portion of the group. The aryl group can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be monosubstituted or substituted more than once, such as, but not limited to, phenyl groups substituted at any one or more positions at the 2-, 3-, 4-, 5-, or 6-positions of the benzene ring, or naphthyl groups substituted at any one or more positions at the 2- to 8-positions.

[0050] As used herein, the term "cationic lipid" refers to any of many lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., about 7.0). Cationic lipids comprising alkyl chains having multiple unsaturated sites (e.g., at least two or three unsaturated sites) have been found to be particularly useful for forming lipid particles with increased membrane fluidity. Many cationic lipids and related analogues that are also useful in this disclosure are described in U.S. Patent 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 PCT Publication WO 96 / 10390, the entire contents of which are incorporated herein by reference for all purposes. Non-limiting examples of cationic lipids are described herein. In some cases, cationic lipids include protonable tertiary amine (e.g., titratable pH) head groups, C 18 The lipids consist of alkyl chains, head groups, ether bonds between alkyl chains, and 0 to 3 double bonds. Examples of such lipids include, for instance, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0051] As used herein, the term "cycloalkyl" refers to a cycloalkyl group, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group may have 3 to about 8-12 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, such as, but not limited to, norbornel, adamantyl, bornel, camphenyl, isocamphenyl, and carenyl, and fused rings, such as, but not limited to, decalinyl, etc. Cycloalkyl groups also include rings substituted with straight-chain or branched alkyl groups as defined herein. Representative substituted cycloalkyl groups may be mono- or poly-substituted, such as, but not limited to, 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl groups or mono-, di-, or tri-substituted norbornyl or cycloheptyl groups, which may be substituted, for example, amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination refers to cyclic alkenyl groups.

[0052] "Disease" is a state of health in animals in which they are unable to maintain homeostasis, and their health continues to deteriorate if the disease is not treated. In contrast, "disorder" is a state of health in which animals are able to maintain homeostasis, but their health is not as good as it would be without disorder. Disorders do not necessarily lead to further decline in an animal's health if left untreated.

[0053] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient quantity of an agent to provide the desired biological outcome. This outcome may be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired biological systemic change. The appropriate therapeutic amount in any individual case can be determined by a person skilled in the art using routine laboratory procedures.

[0054] Specifically, in the case of mRNA, the “effective amount” or “therapeutic effective amount” of the therapeutic nucleic acid associated with the mRNA is an amount sufficient to produce the desired effect, for example, an amount of protein in which the mRNA is directed to express a protein that causes the desired biological effect in an 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 cell types in vivo, and the therapeutic effective amount of mRNA is an amount that produces an amount of the protein encoding the 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 cell types of healthy individuals. For example, in some embodiments, the expressed protein is a protein normally expressed in cell types in vivo, and the therapeutic effective amount of mRNA is an amount that produces a similar level of expression in individuals with abnormal expression of the protein (i.e., protein-deficient individuals) as observed in healthy individuals. Suitable analyses for measuring the expression of mRNA or protein include, but are not limited to, dot blot hybridization, Northern hybridization, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic analysis known to those skilled in the art.

[0055] As used herein, the term "encode" refers to a product (e.g., protein and RNA) specified by a given nucleotide sequence in a nucleic acid (i.e., DNA and / or RNA) during transcription or translation of DNA or RNA, respectively. In some embodiments, the term "encode" refers to an RNA sequence specified by transcription of a DNA sequence. In some embodiments, the term "encode" refers to an amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In some embodiments, the term "encode" refers to an amino acid sequence specified by transcription of DNA into mRNA and subsequent translation of mRNA encoded by the DNA sequence. In some embodiments, the encoded product may include a direct transcription or translation product. In some embodiments, the encoded product may include post-translational modifications understood or reasonably expected by those skilled in the art.

[0056] An "expression vector" is a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other expression elements may be provided by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as visceral, plasmid (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0057] The term "fully encapsulated" means that the active agent or therapeutic agent in the lipid particles is not significantly degraded after exposure to serum or by nucleases or proteases that would significantly degrade free DNA, RNA, or proteins. In a fully encapsulated system, in a treatment that would typically degrade 100% of the free active agent or therapeutic agent, preferably less than about 25% of the active agent or therapeutic agent in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particles is degraded. In the context of nucleic acid therapeutics, full encapsulation can be determined by OLIGREEN® assay. OLIGREEN® is a highly sensitive fluorescent nucleic acid staining agent for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen, Carlsbad, California). "Fully encapsulated" also means that the lipid particles are serum stable, i.e., they do not rapidly degrade into their component parts upon administration in vivo.

[0058] Unless otherwise stated, the terms “halo,” “halogen,” or “halide” as used herein refer to a fluorine, chlorine, bromine, or iodine atom, either on its own or as part of another substituent.

[0059] As used herein, the term "haloalkyl" includes monohaloalkyl, polyhaloalkyl in which all halogen atoms may be the same or different, and perhaloalkyl in which all hydrogen atoms are replaced by halogen atoms (such as fluorine). Examples of haloalkyl groups include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and so on.

[0060] As used herein, the term "helper lipid" refers to a lipid that enhances the effectiveness of delivering lipid particles (such as cationic lipid particles) to a target site, preferably to cells. Helper lipids can be neutral, positively charged, or negatively charged. In some embodiments, helper lipids are neutral or negatively charged. Non-limiting examples of helper lipids include 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) in healthy animals.

[0061] As used herein, the term "heteroaryl" refers to an aromatic ring compound containing five or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S; for example, a heteroaryl ring can have five to about 8-12 ring members. A heteroaryl is a type of heterocyclic group having an aromatic electronic structure. A heteroaryl, called a C2-heteroaryl, can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms, and so on. Similarly, a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so on. The sum of the number of carbon atoms and the number of heteroatoms equals the total number of ring atoms. Heteroaryl groups include, but are not limited to, the following groups: pyrrole, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, thiophene, benzothiophene, benzofuranyl, indolyl, azaindolyl, indolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridyl, isoxazolopyridyl, thianaphthyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Heteroaryl groups may be unsubstituted or substituted with groups as discussed herein. Representative substituted heteroaryl groups may be substituted once or multiple times with groups such as those listed herein.

[0062] Other examples of aryl and heteroaryl groups include, but are not limited to, phenyl, biphenyl, indene, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazole, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracene (1-anthrayl, 2-anthrayl, 3-anthrayl), thiophene (2-thiophene, 3-thiophene), furanyl (2-furanyl, 3-furanyl), indole, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthyl, isonindanyl, diphenylmethyl, acridineyl, thiazolyl, pyrrole (2-pyrrole), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), and triazolyl (1,2,3-triazolyl- 1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl, 1,2,4-triazol-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), quinolinyl (2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 5-quinolinyl, 6-quinolinyl, 7-quinolinyl, 8-quinolinyl), isoquinolinyl (1-isoquinolinyl) 3-Isoquinolinyl, 4-Isoquinolinyl, 5-Isoquinolinyl, 6-Isoquinolinyl, 7-Isoquinolinyl, 8-Isoquinolinyl), 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) 2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophene (2-benzo[b]thiophene, 3-benzo[b]thiophene, 4-benzo[b]thiophene, 5-benzo[b]thiophene, 6-benzo[b]thiophene, 7-benzo[b]thiophene), 2,3-dihydro-benzo[b]thiophene, (2-(2,3-dihydro-benzo[b]thiophene), 3-(2,3-dihydro-benzo[b]thiophene), 4-(2,3-dihydro-benzo[b]thiophene), 5-(2,3-dihydro-benzo[b]thiophene), 6-(2,3-dihydro-benzo[b]thiophene), 7 ...3-Dihydro-benzo[b]thiophene), indole (1-indole, 2-indole, 3-indole, 4-indole, 5-indole, 6-indole, 7-indole), indazole (1-indole, 3-indole, 4-indole, 5-indole, 6-indole, 7-indole), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl) azole group, 6-benzimidazolyl group, 7-benzimidazolyl group, 8-benzimidazolyl group), benzoxazolyl group (1-benzoxazolyl group, 2-benzoxazolyl group), benzothiazolyl group (1-benzothiazolyl group, 2-benzothiazolyl group, 4-benzothiazolyl group, 5-benzothiazolyl group, 6-benzothiazolyl group, 7-benzothiazolyl group), carbazole group (1-carbazole group, 2-carbazole group, 3-carbazole group, 4-carbazole group), 5H- Dibenzo[b,f]azapyridine (5H-dibenzo[b,f]azapyr-1-yl, 5H-dibenzo[b,f]azapyr-2-yl, 5H-dibenzo[b,f]azapyr-3-yl, 5H-dibenzo[b,f]azapyr-4-yl, 5H-dibenzo[b,f]azapyr-5-yl), 10,11-dihydro-5H-dibenzo[b,f]azapyridine (10,11) -dihydro-5H-dibenzo[b,f]aza-1-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-2-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-3-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-4-yl, 10,11-dihydro-5H-dibenzo[b,f]aza-5-yl, etc.

[0063] As used herein, the term "heterocycloalkyl" refers to an aliphatic, partially unsaturated, or fully saturated 3- to 14-membered ring system, including monocyclic, bicyclic, and tricyclic systems of 3 to 8 atoms, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl group may include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be substituted. Representative heterocycloalkyl groups include, but are not limited to, the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolylyl, imidazolinyl, imidazoalkyl, piperidinyl, piperazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazoalkyl, isothiazolyl, and tetrahydrofuranyl.

[0064] As used herein, the term "heterocyclyl" refers to an aromatic or non-aromatic ring compound containing three or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. Thus, a heterocyclyl group can be a cycloheteroalkyl or heteroaryl group, or, if polycyclic, any combination thereof. In some embodiments, a heterocyclyl group comprises 3 to about 20 ring members, while other such groups have 3 to about 15 ring members. A heterocyclyl group referred to as a C2-heterocyclyl group can be a 5-ring having two carbon atoms and three heteroatoms, a 6-ring having two carbon atoms and four heteroatoms, and so on. Similarly, a C4-heterocyclyl group can be a 5-ring having one heteroatom, a 6-ring having two heteroatoms, and so on. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclyl ring may also include one or more double bonds. A heteroaryl ring is one embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused-ring species, including those comprising fused-ring aromatic and non-aromatic groups. For example, dioxolane and phenyldioxolane systems (methylenedioxanyl ring systems) are heterocyclic groups within the meaning of this document. This phrase also includes polycyclic systems containing heteroatoms, such as, but not limited to, quinuclidyl groups. Heterocyclic groups may be unsubstituted or may be substituted as discussed herein. Heterocyclic groups include, but are not limited to, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophene, benzothiophene, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, inzolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazolepyridinyl, isoxazolpyridinyl, thianaphthyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Representative substituted heterocyclic groups may be monosubstituted or substituted multiple times, for example, but not limited to, piperidinyl or quinolinyl, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted by groups as listed herein.

[0065] As used herein, "homologous" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when a position is occupied by the same base or amino acid monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine. The 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 compared, multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, comparisons are made when two sequences are aligned to obtain the maximum homology.

[0066] 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 equal to or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably neutral at or above 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 all lipids to be present in a charged or neutral form. Typically, ionizable lipids have a pK of a protonable group. a It ranges from about 4 to about 7.

[0067] An "immunogen" is any substance introduced into the body to produce 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.

[0068] As used in this article, "immune cell" refers to any cell involved in the organization of the immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (such as dendritic cells and macrophages), monocytes, neutrophils, eosinophils, basophils, etc.

[0069] "Isolated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," while the same nucleic acids or peptides partially or completely isolated from their native coexisting substances are "isolated." Isolated nucleic acids or proteins can exist in a largely purified form or in non-natural environments, such as host cells.

[0070] The term “lipid” refers to a group of organic compounds, including but not limited to fatty acid esters, characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, which include fats, oils and waxes; (2) “complex lipids”, which include phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0071] As used herein, the term "conjugated lipid" refers to a lipid conjugated to one or more polymer groups that inhibits the aggregation of lipid particles. Such lipid conjugations include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugations), PEG-lipid conjugations such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramides (e.g., U.S. Patent 5,885,613, the entire contents of which are incorporated herein by reference for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be directly conjugated to lipids or linked to lipids via a linker. Any linker suitable for coupling PEG to lipids can be used, including, for example, ester-free linkers and ester-containing linkers. In a preferred embodiment, an ester-free linker is used.

[0072] As used herein, “lipid encapsulated” can refer to lipid particles that provide an active agent or therapeutic agent (such as a nucleic acid (e.g., a protein cargo)) with full encapsulation, partial encapsulation, or both. 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).

[0073] The term "lipid nanoparticle" refers to a particle having a size of at least one nanometer scale (e.g., 1-1000 nm) that comprises one or more lipids and / or additives.

[0074] As used herein, the term "lipid particle" refers to a lipid formulation that can be used to deliver an active agent or therapeutic agent (such as a nucleic acid (e.g., mRNA)) to a target of interest. In the lipid particles of this disclosure (which are typically formed from cationic lipids, non-cationic lipids, and conjugated lipids to prevent particle aggregation), the active agent or therapeutic agent can be encapsulated within the lipid, thereby protecting the formulation from enzymatic degradation.

[0075] In the context of this invention, the following abbreviations for common nucleosides (nucleobases bound to ribose or deoxyribose via N-glycosidic bonds) are used: "A" for adenosine, "C" for cytidine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0076] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences in degenerate form that encode the same amino acid sequence. The phrase "nucleotide sequence that encodes a protein or an RNA" may also contain introns, provided that the nucleotide sequence encoding the protein may contain introns in some form.

[0077] As used herein, the term "modulating" refers to a detectable increase or decrease in a subject's response level compared to the response level of a subject without treatment or the compound, and / or compared to the response level of other subjects who are otherwise identical but untreated. This term includes interfering with and / or influencing natural signals or responses to thereby mediate a beneficial therapeutic response in the subject (preferably a human).

[0078] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences in degenerate form that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns. Furthermore, nucleotide sequences may contain modified nucleosides capable of being translated by cellular translation mechanisms. For example, an mRNA in which all uridines are replaced by pseudouridine, 1-methylpseudouridine, or another modified nucleoside.

[0079] The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterions at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalins, cholesterol, cerebrosides, and diacylglycerols.

[0080] The term "non-cationic lipid" refers to any amphiphilic lipid as well as any other neutral or anionic lipid.

[0081] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting 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 operably linked to the second nucleotide sequence. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA or RNA sequences are contiguous and, where two protein-coding regions need to be linked, are located within the same reading frame.

[0082] The terms “patient,” “subject,” or “individual,” etc., are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, applicable to the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0083] The term "polymer conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer conjugated lipid is a PEGylated lipid. 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-carboxyl-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, and so on.

[0084] The term "polynucleotide" as used herein is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, the terms nucleic acid and polynucleotide are used interchangeably herein. Those skilled in the art will generally know that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR™, as well as synthetic methods.

[0085] In some cases, the polynucleotides or nucleic acids of the present invention are “nucleoside-modified nucleic acids,” which refer to nucleic acids containing at least one modified nucleoside. “Modified nucleoside” refers to a nucleoside that has been modified. For example, more than one hundred different nucleoside modifications have been identified in RNA (Rozenski et al., 1999, The RNA Modification Database: updated 1999. Nucl Acids Res 27:196-197).

[0086] In some implementations, "pseudouridine" refers to m 1 acp 3 (1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In some embodiments, this term refers to m 1 (1-Methylpseudouridine). In some embodiments, this term refers to... m(2'-O-methylpseudouridine). In some embodiments, this term refers to m 5 D(5-methyldihydrouridine). In some embodiments, this term refers to m 3 (3-Methylpseudouridine). In some embodiments, this term refers to the unmodified pseudouridine moiety. In some embodiments, this term refers to any of the above-described pseudouridine monophosphate, diphosphate, or triphosphate. In some embodiments, this term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0087] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably 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 limitation on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes 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 (which are also commonly referred to in the art, for example, peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art, for example, proteins, of which there are many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and so on. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0088] As used in this article, the term "promoter" is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism required to initiate polynucleotide sequence-specific transcription. For example, a promoter recognized by bacteriophage RNA polymerase and used to produce mRNA via in vitro transcription.

[0089] As used herein, the term "specifically binds" refers to antibodies that recognize a specific antigen but substantially do not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen of one species may also bind to antigens of one or more other species. However, this cross-species reaction itself does not change the antibody's specific classification. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reaction itself does not change the antibody's specific classification. In some cases, the terms "specific binding" or "specifically binding" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that this interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than the usual protein. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0090] The term “substituted” as used herein in conjunction with the definition of a molecule or organic group means a state in which one or more hydrogen atoms are replaced by one or more non-hydrogen atoms. The terms “functional group” or “substituent” as used herein refer to a group that can be substituted into or be substituted into 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, alkoxy, aryloxy, arylalkoxy, oxygen (carbonyl), and carboxyl groups including carboxylic acids, carboxylates, and carboxyl esters; sulfur atoms in groups such as thiols, alkyl and arylsulfides, sulfoxides, sulfones, sulfonyl groups, and sulfonamides; nitrogen atoms in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and heteroatoms in various other groups. Non-limiting examples of substituents that can bind to substituted carbon (or other) atoms include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O (oxo), S (thiocarbonyl), 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, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0-2 N(R)C(O)R、(CH2) 0-2 N(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, where R can be a hydrogen or carbon-based moiety; for example, R can be hydrogen, (C1-C 100 ) hydrocarbon group, alkyl group, acyl group, cycloalkyl group, aryl group, aralkyl group, heterocyclic group, heteroaryl group or heteroaryl group; or, wherein the two R groups bonded to the nitrogen atom or adjacent nitrogen atom may together with one or more nitrogen atoms form a heterocyclic group.

[0091] The term "therapeutic" as used in this article refers to treatment and / or prevention. It refers to achieving a therapeutic effect by suppressing, reducing, alleviating, or eradicating at least one sign or symptom of a disease or disorder.

[0092] The term "therapeutically effective amount" refers to the amount of a test compound that will elicit a biological or medical response in the tissue, system, or subject sought by a researcher, veterinarian, physician, or other clinician. The term "therapeuticly effective amount" includes, when administered, an amount sufficient to prevent the development of one or more signs or symptoms of the disorder or disease being treated, or to alleviate them to some extent. Therapeuticly effective amounts will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0093] As used in this article, “treat” a disease means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0094] As used in this article, the terms "transfected," "transformed," or "transduced" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary test cells and their progeny.

[0095] The phrase “under transcriptional control” or “operatively linked” used in this article refers to the promoter being in the correct position and orientation relative to the polynucleotide to control the transcriptional initiation of RNA polymerase and the expression of the polynucleotide.

[0096] A "vector" is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, 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, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0097] Scope: In this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format description is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of scope should be considered as having specifically disclosed all possible sub-scopes and the various numerical values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the various numbers within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0098] Lipids and lipid nanoparticles (LNPs) In one aspect, the present invention provides a lipid nanoparticle (LNP) composition. In some embodiments, the LNP is an immune cell-targeted LNP.

[0099] In some embodiments, LNP comprises an ionizable lipid compound having the structure of formula (I) or a salt thereof. Formula (I), in: A1 and A2 are independently selected from CH, N, and P; L1 and L6 are each independently selected from CR 19 and N; Each occurrence of L2 and L5 is independently selected from -CH2- and -CHR. 19 -、-O-、-NH- and -NR 19 -; L3 and L4 are each independently selected from -CH2- and -CHR. 19 -、-O-、-NH- and -NR 19 -; 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 R13a R 13b R 14a R 14b R 15a R 15b R 16a R 16b R 17 R 18 and R 19 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C3-C) 12 cycloalkyl), optionally substituted C2-C 12 Heterocyclic alkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional replacement of C5-C) 12 Cycloalkenyl), optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloalkynyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C6-C) 12 Cyclo-alkynyl), optionally substituted C6-C 10 Aryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C6-C) 10 aryl), optionally substituted C2-C 12 heteroaryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 (heteroaryl), C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid ester, ester, -Y(R) 20 ) z` (R 21 ) z`` -Ester, -Y(R) 20 ) z` (R 21 ) z`` -NO2, -CN and sulfoxy groups, Or selected from 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 The two geminal substituents can combine with the C atoms they are bonded to to form C=O; Each occurrence of Y is independently selected from C, N, O, S, and P; R 20 and R 21 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters, esters, -NO2, -CN, and sulfinyl groups. Or R 20 and R 21 It can combine with the Y atoms it is attached to 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 The compounds having the structure of formula (I) or their salts account for about 25 mol% to about 35 mol% of the LNP.

[0100] In some embodiments, the LNP contains at least one accessory lipid.

[0101] In some implementations, LNP includes cholesterol lipids.

[0102] In some embodiments, LNPs include polyethylene glycol (PEG) conjugated lipids and / or their modified derivatives.

[0103] In some embodiments, the LNP includes a cell-targeting domain specific for binding to molecules on the surface of target cells. In other embodiments, the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0104] In some embodiments, the auxiliary lipid is dioleoylphosphatidylethanolamine (DOPE). In some embodiments, DOPE accounts for about 10 mol% to about 20 mol% of the LNP. In some embodiments, DOPE accounts for less than 10 mol% to about 20 mol% of the LNP. In some embodiments, DOPE accounts for more than 10 mol% to about 20 mol% of the LNP.

[0105] In some embodiments, cholesterol accounts for about 40 mol% to about 50 mol% of LNP. In some embodiments, cholesterol accounts for less than 40 mol% to about 50 mol% of LNP. In some embodiments, cholesterol accounts for more than 40 mol% to about 50 mol% of LNP.

[0106] In some embodiments, the conjugated lipids include PEG-conjugated lipids. In some embodiments, the PEG-conjugated lipids comprise a mixture of lipids conjugated to PEG and lipids conjugated to maleimide-substituted PEG. In some embodiments, the PEG-conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to about 5.0 mol% of the LNP. In some embodiments, the PEG-conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to less than 5.0 mol% of the LNP. In some embodiments, the PEG-conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to less than 5.0 mol% of the LNP.

[0107] In some implementations, R1, R2, R 17 R 18 and R 19 Each is independently selected from 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) 15CH3, 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 CH3.

[0108] In some implementations, 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 and R 16b Each is independently selected from H and OCH2CH3.

[0109] In some implementations, m is 0. In some implementations, m is 1. In some implementations, m is 2. In some implementations, m is 3. In some implementations, m is 4. In some implementations, m is 5. In some implementations, n is 0. In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3. In some implementations, n is 4. In some implementations, n is 5. In some implementations, o is 0. In some implementations, o is 1. In some implementations, o is 2. In some implementations, o is 3. In some implementations, o is 4. In some implementations, o is 5. In some implementations, p is 0. In some implementations, p is 1. In some implementations, p is 2. In some implementations, p is 3. In some implementations, p is 4. In some implementations, p is 5. In some implementations, q is 0. In some implementations, q is 1. In some implementations, q is 2. In some implementations, q is 3. In some implementations, q is 4. In some implementations, q is 5. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 5. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, u is 4. In some embodiments, u is 5. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, v is 4. In some embodiments, v is 5. In some implementations, w is 0. In some implementations, w is 1. In some implementations, w is 2. In some implementations, w is 3. In some implementations, w is 4. In some implementations, w is 5. In some implementations, x is 0. In some implementations, x is 1. In some implementations, x is 2. In some implementations, x is 3. In some implementations, x is 4. In some implementations, x is 5.

[0110] In some embodiments, the compound of formula (I) is: Equation (II).

[0111] In some embodiments, the compound of formula (I) is: Formula (III).

[0112] In some embodiments, the compound of formula (I) is: Formula (IV).

[0113] In some embodiments, the compound of formula (I) is: Formula (V).

[0114] In some embodiments, the compound of formula (I) is: Formula (VI).

[0115] In some embodiments, the compound of formula (I) is: Equation (VII).

[0116] In some 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 selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; a 1 a 2 a 3 a 4 and a 5 Each can be 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 can be 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 can be independently 0, 1, 2, 3, 4 or 5.

[0117] In some embodiments, the compound of formula (I) is: Formula (VIII).

[0118] In some embodiments, the compound of formula (I) is: Formula (IX).

[0119] In some embodiments, the compound of formula (I) is: Formula (X).

[0120] In some embodiments, the compound of formula (I) is: Formula (XI).

[0121] In some embodiments, the compound of formula (I) is: Formula (XII).

[0122] In some embodiments, the compound of formula (I) is: Formula (XIII).

[0123] In some embodiments, the compound of formula (I) is: Formula (XIV).

[0124] In some embodiments, the compound of formula (I) is: Formula (XV).

[0125] In some embodiments, the compound of formula (I) is: Formula (XVI).

[0126] In some embodiments, in compounds of formulas (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), and (XVI), the following definitions apply independently: R1, R2, R3, R4, and R5 are each independently selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; and a 1 a 2 a 3 a 4 and a 5 Each can be 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.

[0127] In some embodiments, R1, R2, R3, R4 and R5 are each independently selected from H, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, isobutyl and sec-butyl.

[0128] In some implementations, a 1 It is 0. In some implementations, a 1 It is 1. In some implementations, a 1 It is 2. In some implementations, a 1 It is 3. In some implementations, a 1 It is 4. In some implementations, a 1 It is 5. In some implementations, a 1 It is 6. In some implementations, a 1 It is 7. In some implementations, a 1 It is 8. In some implementations, a 1 It is 9. In some implementations, a 1 It is 10. In some implementations, a 1 It is 11. In some implementations, a 1 It is 12. In some implementations, a 1 It is 13. In some implementations, a 1 It is 14. In some implementations, a 1 It is 15. In some implementations, a 1 It is 16. In some implementations, a 1 It is 17. In some implementations, a 1 It is 18. In some implementations, a 1 It is 19. In some implementations, a 1 It is 20. In some implementations, a 1 It is 21. In some implementations, a 1 It is 22. In some implementations, a 1 It is 23. In some implementations, a 1It is 24. In some implementations, a 1 It is 25. In some implementations, a 2 It is 0. In some implementations, a 2 It is 1. In some implementations, a 2 It is 2. In some implementations, a 2 It is 3. In some implementations, a 2 It is 4. In some implementations, a 2 It is 5. In some implementations, a 2 It is 6. In some implementations, a 2 It is 7. In some implementations, a 2 It is 8. In some implementations, a 2 It is 9. In some implementations, a 2 It is 10. In some implementations, a 2 It is 11. In some implementations, a 2 It is 12. In some implementations, a 2 It is 13. In some implementations, a 2 It is 14. In some implementations, a 2 It is 15. In some implementations, a 2 It is 16. In some implementations, a 2 It is 17. In some implementations, a 2 It is 18. In some implementations, a 2 It is 19. In some implementations, a 2 It is 20. In some implementations, a 2 It is 21. In some implementations, a 2 It is 22. In some implementations, a 2 It is 23. In some implementations, a 2 It is 24. In some implementations, a 2 It is 25. In some implementations, a 3 It is 0. In some implementations, a 3 It is 1. In some implementations, a 3 It is 2. In some implementations, a 3 It is 3. In some implementations, a 3 It is 4. In some implementations, a 3 It is 5. In some implementations, a 3 It is 6. In some implementations, a 3 It is 7. In some implementations, a 3 It is 8. In some implementations, a 3 It is 9. In some implementations, a3 It is 10. In some implementations, a 3 It is 11. In some implementations, a 3 It is 12. In some implementations, a 3 It is 13. In some implementations, a 3 It is 14. In some implementations, a 3 It is 15. In some implementations, a 3 It is 16. In some implementations, a 3 It is 17. In some implementations, a 3 It is 18. In some implementations, a 3 It is 19. In some implementations, a 3 It is 20. In some implementations, a 3 It is 21. In some implementations, a 3 It is 22. In some implementations, a 3 It is 23. In some implementations, a 3 It is 24. In some implementations, a 3 It is 25. In some implementations, a 4 It is 0. In some implementations, a 4 It is 1. In some implementations, a 4 It is 2. In some implementations, a 4 It is 3. In some implementations, a 4 It is 4. In some implementations, a 4 It is 5. In some implementations, a 4 It is 6. In some implementations, a 4 It is 7. In some implementations, a 4 It is 8. In some implementations, a 4 It is 9. In some implementations, a 4 It is 10. In some implementations, a 4 It is 11. In some implementations, a 4 It is 12. In some implementations, a 4 It is 13. In some implementations, a 4 It is 14. In some implementations, a 4 It is 15. In some implementations, a 4 It is 16. In some implementations, a 4 It is 17. In some implementations, a 4 It is 18. In some implementations, a 4 It is 19. In some implementations, a 4 It is 20. In some implementations, a 4It is 21. In some implementations, a 4 It is 22. In some implementations, a 4 It is 23. In some implementations, a 4 It is 24. In some implementations, a 4 It is 25. In some implementations, a 5 It is 0. In some implementations, a 5 It is 1. In some implementations, a 5 It is 2. In some implementations, a 5 It is 3. In some implementations, a 5 It is 4. In some implementations, a 5 It is 5. In some implementations, a 5 It is 6. In some implementations, a 5 It is 7. In some implementations, a 5 It is 8. In some implementations, a 5 It is 9. In some implementations, a 5 It is 10. In some implementations, a 5 It is 11. In some implementations, a 5 It is 12. In some implementations, a 5 It is 13. In some implementations, a 5 It is 14. In some implementations, a 5 It is 15. In some implementations, a 5 It is 16. In some implementations, a 5 It is 17. In some implementations, a 5 It is 18. In some implementations, a 5 It is 19. In some implementations, a 5 It is 20. In some implementations, a 5 It is 21. In some implementations, a 5 It is 22. In some implementations, a 5 It is 23. In some implementations, a 5 It is 24. In some implementations, a 5 It is 25.

[0129] In some implementations, b 1 It is 0. In some implementations, b 1 It is 1. In some implementations, b 1 It is 2. In some implementations, b 1 It is 3. In some implementations, b 1 It is 4. In some implementations, b 1 It is 5. In some implementations, b2 It is 0. In some implementations, b 2 It is 1. In some implementations, b 2 It is 2. In some implementations, b 2 It is 3. In some implementations, b 2 It is 4. In some implementations, b 2 It is 5. In some implementations, b 3 It is 0. In some implementations, b 3 It is 1. In some implementations, b 3 It is 2. In some implementations, b 3 It is 3. In some implementations, b 3 It is 4. In some implementations, b 3 It is 5. In some implementations, b 4 It is 0. In some implementations, b 4 It is 1. In some implementations, b 4 It is 2. In some implementations, b 4 It is 3. In some implementations, b 4 It is 4. In some implementations, b 4 It is 5. In some implementations, b 5 It is 0. In some implementations, b 5 It is 1. In some implementations, b 5 It is 2. In some implementations, b 5 It is 3. In some implementations, b 5 It is 4. In some implementations, b 5 It is 5.

[0130] In some implementations, c 1 It is 0. In some implementations, c 1 It is 1. In some implementations, c 1 It is 2. In some implementations, c 1 It is 3. In some implementations, c 1 It is 4. In some implementations, c 1 It is 5. In some implementations, c 2 It is 0. In some implementations, c 2 It is 1. In some implementations, c 2 It is 2. In some implementations, c 2 It is 3. In some implementations, c 2 It is 4. In some implementations, c 2 It is 5.

[0131] In some implementations, d 1It is 0. In some implementations, d 1 It is 1. In some implementations, d 1 It is 2. In some implementations, d 1 It is 3. In some implementations, d 1 It is 4. In some implementations, d 1 It is 5. In some implementations, d 2 It is 0. In some implementations, d 2 It is 1. In some implementations, d 2 It is 2. In some implementations, d 2 It is 3. In some implementations, d 2 It is 4. In some implementations, d 2 It is 5. In some implementations, d 3 It is 0. In some implementations, d 3 It is 1. In some implementations, d 3 It is 2. In some implementations, d 3 It is 3. In some implementations, d 3 It is 4. In some implementations, d 3 It is 5. In some implementations, d 4 It is 0. In some implementations, d 4 It is 1. In some implementations, d 4 It is 2. In some implementations, d 4 It is 3. In some implementations, d 4 It is 4. In some implementations, d 4 It is 5.

[0132] In some embodiments, the ionizable lipid of formula (I) comprises 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azinediyl)bis(tetradecane-2-ol): (C14-4).

[0133] In some implementations, the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.5.

[0134] In some embodiments, the PEG-conjugated lipids include C14-PEG: .

[0135] In some embodiments, the modified derivatives of the PEG-conjugated lipids include 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[maleimide (polyethylene glycol)-2000] (ammonium salt) (mPEG): .

[0136] In some embodiments, the total PEG comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio ranging from more than about 1:1 to about 1:10 (mPEG:PEG).

[0137] In some embodiments, the total PEG comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio selected from 1:3, 1:5, 1:7 and 1:10 (mPEG:PEG).

[0138] In some embodiments, maleimide PEG is covalently conjugated to a cell-targeting domain that is specific for binding to molecules on the surface of target cells. In some embodiments, the covalent conjugation includes a covalent bond between the α-carbon of the maleimide carbonyl group and the thiol moiety of the cell-targeting domain. In some embodiments, the covalent conjugation occurs via a [1,4]-conjugation addition (i.e., Michael addition) between the maleimide of the maleimide PEG and the thiol of the cysteine ​​residue of the cell-targeting domain.

[0139] In some implementations, LNPs are delivered to the spleen in a larger proportion than those to the liver.

[0140] In some implementations, the target cells are selected from stem cells, peripheral blood mononuclear cells, and immune cells.

[0141] In some embodiments, the LNP also includes at least one selected from nucleic acid molecules and therapeutic agents.

[0142] In some embodiments, the LNP also includes at least one reagent selected from mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

[0143] In some implementations, LNPs include nucleic acid molecules.

[0144] In some implementations, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0145] In some implementations, the nucleic acid molecule is selected from cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecules and target nucleic acids, or any combination thereof.

[0146] In some implementations, nucleic acid molecules encode chimeric antigen receptors (CARs).

[0147] In some implementations, CARs are specific for binding to surface antigens of pathogenic or tumor cells.

[0148] In some implementations, the cell-targeting domain that binds to surface molecules specific to target cells is an immune cell-targeting domain that specifically binds to T cells.

[0149] In some embodiments, the surface molecules of the target cells are selected from 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, and CD154. 4. At least one of 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.

[0150] As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH value drops below the pK of the lipid's ionizable group, but gradually becomes more neutral at higher pH values. Below the pK, the lipid is capable of binding to negatively charged nucleic acids. In some embodiments, cationic lipids include zwitterionic lipids that are positively charged when the pH value decreases.

[0151] In some embodiments, cationic lipids include any one of a variety of lipid species that carry a net positive charge at a selected pH (such as physiological pH). Such lipids include, but are not limited to: N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearate-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-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA), disteaamide-glycylcarboxylic acid spermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-(dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristoxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In addition, various commercially available cationic lipid formulations can be used in this invention. These include, for example: LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphatidylethanolamine (DOPE), available from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleoyloxy)propyl)-N-(2-(spermine carboxamido)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA) and (DOPE), available from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic liposome containing distearate-glycyl carboxyspermine (DOGS) in ethanol, available from Promega, Madison, Wisconsin). The following lipids are cationic lipids and carry a positive charge at pH levels below physiological levels: DODAP, DODMA, DMDMA, 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLenDMA).

[0152] In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids available for use in this invention include those described in WO2012 / 016184, the entire contents of which are incorporated herein by reference. Representative amino lipids include, but are not limited to: 1,2-dilinoleoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoxy-3-morpholinylpropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinolethio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1 2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleoamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA) and 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0153] In some embodiments, the lipids are polyethylene glycol-modified lipids, including but not limited to DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxyl-NHS, DOPE-PEG-carboxylic acid, and DSPE-PEG-carboxylic acid.

[0154] The term "neutral lipid" refers to any of a variety of lipids that exist as uncharged or neutral zwitterions at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0155] Exemplary neutral lipids include, for example: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), and dioleoylphosphatidylethanolamine (DPPE). Myristoyl phosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), distearyl-phosphatidylethanolamine (DSPE)-maleimide-PEG, distearyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), stearoyloleoylphosphatidylcholine (SOPC), and 1,2-ditransoleoyl-sn-glycerol-3-phosphatidylethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC).

[0156] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0157] A steroid is a compound that contains the following carbon skeleton: .

[0158] In some embodiments, the steroid or steroid analogue is cholesterol. In some embodiments, the molar ratio of cationic lipids is...

[0159] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphonic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and palmitoylphosphatidylglycerol (POPG), as well as other anionic modifying groups that bind to neutral lipids.

[0160] The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that comprises both a lipid moiety and a polyethylene glycol moiety. 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-carboxyl-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, and so on.

[0161] In some embodiments, the LNP comprises additional stabilizing lipids, which are polyethylene glycol-lipids (PEGylated lipids). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In some embodiments, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)] 2000 [Carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises polyethylene glycol-modified diacylglycerol (PEG-DAG), such as 1-(monomethoxy polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-S-DAG, such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyethylene glycol-modified ceramide (PEG-cer), or PEG-dialkoxypropylcarbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0162] In some embodiments, additional lipids are present in the LNP in an amount of about 1 mol% to about 10 mol%. In some embodiments, additional lipids are present in the LNP in an amount of about 1 mol% to about 5 mol%. In some embodiments, additional lipids are present in the LNP in an amount of about 1 mol% or about 2.5 mol%.

[0163] The term "lipid nanoparticles" refers to particles having at least one nanoscale size (e.g., 1-1000 nm) and containing one or more lipids, such as lipids of formula (I)-(XVI).

[0164] In various embodiments, the average diameter of the lipid nanoparticles is about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0165] In various embodiments, the lipids or LNPs of the present invention are substantially non-toxic.

[0166] In various embodiments, the lipids or LNPs described herein are formulated for stability targeting immune cells in vivo.

[0167] In some embodiments, the LNP formulated for in vivo immune cell targeting stability comprises C14-4 at a concentration ranging from about 10 mol% to about 45 mol%. In some embodiments, C14-4 is present at a molar ratio of about 40%.

[0168] In some embodiments, the stable LNP formulated for targeting immune cells in vivo comprises phospholipids in concentrations ranging from about 10 mol% to about 45 mol%. In some embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), and the DOPE is present in a molar ratio of about 25 or a molar percentage of about 25%.

[0169] In some embodiments, the stable LNP formulated for targeting immune cells in vivo comprises cholesterol lipids in concentrations ranging from about 5 mol% to about 50 mol%. In some embodiments, cholesterol is present at a molar ratio of about 30 or a molar percentage of about 30%.

[0170] In some embodiments, the stable LNP formulated for targeting immune cells in vivo comprises a total PEG concentration ranging from about 0.5 mol% to about 12.5 mol%. In some embodiments, the total PEG is present at a molar ratio of about 2.5 or a molar percentage of about 2.5%.

[0171] In some embodiments, the stable LNP formulated for targeting immune cells in vivo comprises ionizable lipid C14-4, DOPE, cholesterol, and total PEG, wherein the C14-4:DOPE:cholesterol:total PEG is present in a molar ratio of about 40:25:30:2.5 or a molar percentage of about 40%:25%:30%:2.5%.

[0172] In some embodiments, the total PEG comprises maleimide PEG (mPEG) and PEG in a molar ratio of about 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 therebetween. In some embodiments, the LNP comprises a total PEG in a molar ratio of about 2.5, wherein the total PEG comprises mPEG and PEG in a molar ratio of 1:3. In some embodiments, the LNP comprises a total PEG in a molar ratio of about 2.5, and the total PEG comprises PEG and mPEG in a molar ratio of 1:5. In some embodiments, the LNP comprises a total PEG in a molar ratio of about 2.5, and the total PEG comprises PEG and mPEG in a molar ratio of 1:7. In some embodiments, the LNP comprises a total PEG in a molar ratio of about 2.5, and the total PEG comprises PEG and mPEG in a molar ratio of 1:10.

[0173] Small molecule therapeutic agents In various embodiments, the reagent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent 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 some embodiments, the small molecule therapeutic agent comprises organic molecules, inorganic molecules, biomolecules, synthetic molecules, etc.

[0174] Combinatorial libraries of molecularly diverse chemical compounds for treating various diseases and conditions are well known in the art, as are methods for preparing such libraries. These methods may utilize 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 sequences, deconvolution strategies, labeling techniques, and generating unbiased molecular libraries for lead discovery that are compared with biased structures used for lead development. In some embodiments of the invention, combinatorial techniques are used to synthesize and / or identify therapeutic agents.

[0175] In general methods of small library synthesis, an activated core molecule condenses with multiple structural units to produce a covalently linked core-structural unit assemblies, forming a combined library. The shape and rigidity of the core determine the orientation of the structural units in shape space. Libraries can be biased by targeting characterized biological structures (“focused libraries”) by altering the core, bonds, or structural units, or by using a flexible core for synthesis with less structural bias. In some embodiments of the invention, therapeutic agents are synthesized via small library synthesis.

[0176] The small molecules and small molecule compounds described herein may exist in the form of salts, even if salts are not described, and it should be understood that the invention covers all salts and solvates of the therapeutic agents described herein, as well as non-salt and non-solvent forms of the therapeutic agents, as fully understood by those skilled in the art. In some embodiments, the salts of the therapeutic agents of the present invention are pharmaceutically acceptable salts.

[0177] In the event that any therapeutic agent described herein may exist in tautomeric forms, each tautomeric form is intended to be included in the invention, even if only one or some tautomeric forms may be explicitly described. For example, when the 2-hydroxypyridinyl moiety is described, the corresponding 2-pyridone tautomeric form is also intended to be included.

[0178] This invention also includes any or all stereochemical forms of the therapeutic agents, including any enantiomers or diastereomers. The description of structures or names herein is intended to cover all possible stereoisomers of the described therapeutic agents. All forms of the therapeutic agents are also covered by this invention, such as crystalline or amorphous forms of the therapeutic agents. It is also intended to include compositions comprising the therapeutic agents of the invention, such as compositions of substantially pure therapeutic agents including their specific stereochemical forms, or compositions comprising mixtures of the therapeutic agents of the invention in any proportion, including two or more stereochemical forms, such as racemic or non-racemic mixtures.

[0179] This invention also includes any or all active analogs or derivatives, such as prodrugs, of any therapeutic agent described herein. In some embodiments, the therapeutic agent is a prodrug. In some embodiments, the small molecule described herein is a derivatized candidate. Thus, in certain circumstances, analogs of the small molecule described herein with modulating potency, selectivity, and solubility are included, providing useful leads for drug discovery and drug development. Therefore, in certain circumstances, new analogs are designed considering drug delivery, metabolism, novelty, and safety issues during the optimization process.

[0180] In some cases, the small molecule therapeutic agents described herein are derivatives or analogs of known therapeutic agents, as is well known in the fields of combinatorial chemistry and medicinal chemistry. Analogs or derivatives can be prepared by adding and / or substituting functional groups at various positions. Therefore, the small molecules described herein can be converted into derivatives / analogs using well-known chemical synthetic processes. For example, all hydrogen atoms or substituents can be selectively modified to produce new analogs. Furthermore, linking atoms or groups can be modified to have longer or shorter linking groups with a carbon backbone or heteroatoms. Additionally, cyclic groups can be changed to have different numbers of cyclic atoms and / or contain heteroatoms. Furthermore, aromatic rings can be converted to alicyclic rings and vice versa. For example, the ring can be 5-7 atoms and can be a carbon ring or a heterocyclic ring.

[0181] As used herein, the terms "analog," "analogue," or "derivative" refer to a compound or molecule produced from a parent compound or molecule through one or more chemical reactions. Therefore, an analog may have a structure similar to, or be based on, the small molecule therapeutic agents described herein, but differ in certain components or structural composition, and its metabolic effects may be similar or opposite. Analogs or derivatives of any small molecule inhibitor according to the present invention can be used to treat diseases or disorders.

[0182] In some embodiments, the small molecule therapeutic agents described herein can be independently derivatized or analogues can be prepared by modifying hydrogen groups independently of each other with other substituents. That is, each atom on each molecule can be modified independently relative to other atoms on the same molecule. Any conventional modifications used to generate derivatives / analogs can be used. For example, atoms and substituents can independently comprise hydrogen, alkyl, aliphatic, straight-chain aliphatic, aliphatic with chain heteroatoms, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic with one or more heteroatoms, aromatic, heteroaromatic, polyaromatic, polyamino acid, peptide, polypeptide, combinations thereof, halogen, halogenated aliphatic, etc. Furthermore, any cyclic group on the compound can be derivatized to increase and / or decrease ring size and to change the main chain atom to a carbon atom or a heteroatom.

[0183] Nucleic acid therapy In some embodiments, the compositions of the present invention comprise in vitro transcribed (IVT) RNA molecules. For example, in some embodiments, the compositions of the present invention comprise IVT RNA molecules encoding reagents. In some embodiments, the IVT RNA molecules of the compositions are nucleoside-modified mRNA molecules. In some embodiments, the reagents are used to target immune cells to pathogens or tumor cells of interest. In some embodiments, the IVT RNA molecules encode chimeric antigen receptors (CARs).

[0184] In some embodiments, the CAR is specific for binding to one or more antigens. In some embodiments, the antigen includes at least one viral antigen, bacterial antigen, fungal antigen, parasitic antigen, influenza antigen, tumor-associated antigen, tumor-specific antigen, or any combination thereof.

[0185] However, the present invention is not limited to any particular reagent or combination of reagents. In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises a nucleic acid molecule encoding an adjuvant. In some embodiments, the composition comprises nucleoside-modified RNA encoding an adjuvant.

[0186] In some embodiments, the composition comprises at least one RNA molecule encoding a combination of at least two reagents. In some embodiments, the composition comprises a combination of two or more RNA molecules encoding a combination of two or more reagents.

[0187] In some 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 against viruses, bacteria, fungi, parasites, cancer, etc., in a subject. In some embodiments, the method includes administering to the subject a composition comprising one or more LNP molecules formulated for targeting immune cells in vivo, the LNP molecules comprising one or more RNAs encoding at least one antigen, adjuvant, or a combination thereof.

[0188] In some embodiments, the present invention provides a method for gene editing of immune cells of a subject. For example, the method may be used to deliver one or more components of a gene editing system (e.g., components of a CRISPR system) to the immune cells of a subject. In some embodiments, the method includes administering to a subject a composition comprising one or more ionizable LNP molecules formulated for targeted T cell delivery, and comprising one or more nucleoside-modified RNA molecules for gene editing.

[0189] In some embodiments, the method includes administering the composition to a subject. In some embodiments, the method includes administering multiple doses to a subject. In some embodiments, the method includes administering a single dose of the composition, wherein the single dose is effective in delivering the targeted therapeutic agent.

[0190] In other relevant aspects, the therapeutic agent is an isolated nucleic acid. In some embodiments, the isolated nucleic acid molecule is either a DNA molecule or an RNA molecule. In some embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA, or miRNA molecule. In some embodiments, the isolated nucleic acid molecule encodes a therapeutic peptide, such as a thrombotic regulatory protein, an endothelial protein C receptor (EPCR), an antithrombotic protein including plasminogen activator and its mutants, or an antioxidant protein including catalase, superoxide dismutase (SOD), and iron chelating proteins. In some embodiments, the therapeutic agent is an siRNA, miRNA, shRNA, or antisense molecule that inhibits target nucleic acids, including nucleic acids encoding proteins involved in the exacerbation of the pathological process.

[0191] In some embodiments, the nucleic acid contains a promoter / regulatory sequence that enables the nucleic acid to direct its expression. Therefore, the present invention includes expression vectors and methods for introducing exogenous nucleic acids into cells and simultaneously expressing them within the cells, such as those described by Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York) and Ausubel et al. (1997, Molecular Cloning: A Laboratory Manual, John Wiley & Sons, New York), as well as those described elsewhere herein.

[0192] In some implementations, siRNA is used to reduce the level of target proteins. RNA interference (RNAi) is a phenomenon that introduces double-stranded RNA (dsRNA) into various organisms and cell types, leading to the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short interfering RNAs, or siRNAs, by a ribonuclease called Dicer. The siRNA then assembles with protein components to form an RNA-induced silencing complex (RISC), which unfolds in the process. The activated RISC then binds to the complementary transcript via base-pairing interactions between the antisense strand of the siRNA and the mRNA. The bound mRNA is cleaved, and the sequence-specific degradation of the mRNA results in gene silencing. For example, see 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, TIG14(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) described a chemical modification of siRNA that facilitates intravenous systemic delivery. Optimizing siRNA requires consideration of overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' dangling nucleotides. For example, see Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods for reducing PTPN22 levels using RNAi technology.

[0193] In one aspect, the present invention includes a vector comprising siRNA or an antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide. The incorporation of the desired polynucleotide into the vector and the choice of the vector are well known in the art, for example, as described in Sambrook et al. (2012) and Ausubel et al. (1997) and elsewhere herein.

[0194] In some embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) therapeutic agent. shRNA molecules are well known in the art and target the mRNA of a target, thereby reducing the expression of the target. In some embodiments, the encoded shRNA is expressed by a cell and then processed into siRNA. For example, in some cases, the cell possesses a natural enzyme (such as dicer) that cleaves the shRNA to form siRNA.

[0195] To assess the expression of siRNA, shRNA, or antisense polynucleotides, the expression vector introduced into cells may also contain a selective marker gene or a reporter gene, or both, to facilitate the identification of expressing cells from a cell population seeking transfection or infection using the delivery vector of the present invention. In other embodiments, the selective marker may be carried on a separate DNA fragment or contained within the delivery vector. Both the selective marker and the reporter gene may be flanked by suitable regulatory sequences to enable their expression in host cells. Useful selective markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0196] Therefore, in one aspect, the delivery vector may comprise a vector containing a nucleotide sequence or construct to be delivered. The choice of vector will depend on the host cell into which it will be subsequently introduced. In a particular embodiment, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a particular embodiment, the expression vector is selected from viral vectors, bacterial vectors, and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be used in the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are commercially available.

[0197] For example, the vector that introduces the nucleic acid sequence can be a plasmid, which, when introduced into a cell, may or may not integrate into the host cell's genome. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the present invention or gene constructs of the present invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0198] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In one particular embodiment, the vector is a vector that can be used to transform animal cells.

[0199] In some implementations, the recombinant expression vector may also contain nucleic acid molecules encoding peptides or peptide-like molecules.

[0200] Promoters can be promoters naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a polynucleotide sequence, located downstream or upstream of that sequence. Optionally, certain advantages can be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a polynucleotide sequence in their natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters and enhancers isolated from any other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers that are not "naturally present," i.e., those containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to synthesizing nucleic acid sequences for the production of promoters and enhancers, recombinant cloning and / or nucleic acid amplification technologies (including PCR™) can be used in conjunction with the compositions disclosed herein to produce sequences (US Patent 4,683,202, US Patent 5,928,906). Furthermore, control sequences that guide the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts can also be considered.

[0201] Of course, it is important to use promoters and / or enhancers that effectively guide the expression of DNA fragments in the selected cell type, organelle, and organism for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression; see, for example, Sambrook et al. (2012). The promoters employed can be constitutive, tissue-specific, inducible, and / or, under appropriate conditions, advantageous for guiding high-level expression of the introduced DNA fragment, such as in the large-scale production of recombinant proteins and / or peptides. Promoters can be heterologous or endogenous.

[0202] Recombinant expression vectors can also contain selective marker genes, which aids in host cell selection. Suitable selective marker genes are protein-encoding genes, such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as the Fc portion of immunoglobulins, preferably IgG. Selective markers can be introduced from the nucleic acid of interest into separate vectors.

[0203] After the siRNA polynucleotide is generated, those skilled in the art will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, siRNA polynucleotides can be further designed to resist degradation by modifying them to include thiophosphates or other linkages, methylphosphonates, sulfones, sulfates, ketones, dithiophosphates, phosphoramidates, phosphates, etc. (see, for example, 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 (1989)).

[0204] Any polynucleotide can be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, adding flanking sequences at the 5' and / or 3' ends; using thiophosphates or 2'O-methyl groups in the backbone instead of phosphodiester linkages; and / or including non-traditional bases such as inosine, queuosine, and wybutosine, as well as acetylation, methylation, thiolation, and other modifications of adenine, cytidine, guanine, thymine, and uridine.

[0205] In some embodiments of the invention, the antisense nucleic acid sequence expressed by the plasmid vector is used as a therapeutic agent to inhibit the expression of the target protein. The antisense expression vector is used to transfect mammalian cells or mammals themselves, thereby resulting in a reduction in the endogenous expression of the target protein.

[0206] Antisense molecules and their use in suppressing 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 specific mRNA molecule, as the term is defined elsewhere herein (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule, thereby suppressing gene translation.

[0207] The use of antisense methods to suppress gene translation is known in the art, as described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). As taught in Inoue, 1993, U.S. Publication No. 5,190,931, such antisense molecules can be delivered to cells via gene expression using DNA encoding antisense molecules.

[0208] Optionally, the antisense molecules of the present invention can be synthesized and then provided to cells. Preferably, antisense oligomers of about 10 to about 30 nucleotides, more preferably about 15 nucleotides, are preferred because they are readily synthesized and introduced into target cells. The synthetic antisense molecules contemplated in this invention include oligonucleotide derivatives known in the art that exhibit improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0209] In some embodiments of the invention, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes that can be used to inhibit the expression of target molecules can be designed by incorporating the target sequence into a basic ribozyme structure, such as one that is complementary to the mRNA sequence encoding the target molecule. Ribozymes targeting target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or they can be expressed by the DNA genes encoding them.

[0210] In some embodiments, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, wherein a guide RNA (gRNA) targeting a gene encoding a target molecule and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the target gene. In some embodiments, the therapeutic agent comprises gRNA or a nucleic acid molecule encoding gRNA. In some embodiments, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0211] In some embodiments, the formulation comprises miRNA or a miRNA mimic. In some embodiments, the formulation comprises a nucleic acid molecule encoding a miRNA or a miRNA mimic.

[0212] miRNAs are small non-coding RNA molecules that can induce post-transcriptional silencing of specific genes in a cell by inhibiting translation or degrading target mRNAs. miRNAs can be perfectly complementary to their target nucleic acids or have non-complementary regions, resulting in a "bulge" in the non-complementary region. miRNAs can suppress gene expression by inhibiting translation, for example, when the miRNA is not perfectly complementary to the target nucleic acid, or by inducing degradation of the target RNA; degradation is believed to occur only when the miRNA binds to its target in a perfectly complementary manner. This disclosure may also include double-stranded precursors of miRNAs. The length of miRNAs or pri-miRNAs can be 18-100 nucleotides or 18-80 nucleotides. The length of mature miRNAs can be 19-30 nucleotides or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. Pre-miRNAs typically have a length of about 70-100 nucleotides and exhibit a hairpin conformation. miRNAs are generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNAs into functional miRNAs. The hairpin or mature microRNA, or pri-miRNA reagents of this disclosure can be synthesized in vivo via cell-based systems or in vitro via chemical synthesis.

[0213] In various embodiments, the reagent comprises an oligonucleotide containing a nucleotide sequence of a disease-associated miRNA. In some embodiments, the oligonucleotide comprises a nucleotide sequence of a disease-associated miRNA in mature or hairpin-form premicroRNA. In other embodiments, combinations of oligonucleotides comprising sequences of one or more disease-associated miRNAs, any premiRNA, any fragment, or any combination thereof are contemplated.

[0214] miRNAs can be synthesized to include modifications that impart desired characteristics. For example, such modifications can improve stability, hybridization thermodynamics with target nucleic acids, targeting specific tissues or cell types, or cell permeability, for example, through mechanisms that are dependent on or independent of endocytosis.

[0215] Modifications can also improve sequence specificity, thereby reducing off-target effects. Methods for synthesis and chemical modification are described in more detail below. If desired, miRNA molecules can be modified to stabilize the miRNA against degradation, extend its half-life, or otherwise enhance its efficacy. Desired modifications are described, for example, in U.S. Patent Publications 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, the entire contents of each of which are incorporated herein by reference. To enhance nuclease resistance and / or binding affinity to targets, the single-stranded oligonucleotide reagents of this disclosure may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphate thioester linkages. Including locked nucleic acids (LNAs), vinyl nucleic acids (ENAs), such as 2'-4'-vinyl-bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to targets. Adding pyranose to the oligonucleotide backbone can also reduce endonuclease cleavage. Oligonucleotides can be further modified by including a 3' cationic group or by reversing the 3'-terminal nucleoside linker to the 3'-3' end. Alternatively, the 3' end can be blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' exonuclease cleavage. While not theoretically constrained, the 3' may inhibit exonuclease cleavage by sterically blocking the binding of exonucleases to the 3' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5' exonucleases.

[0216] In some embodiments, the miRNA comprises a 2'-modified oligonucleotide containing oligodeoxynucleotide internucleotides, some or all of which are modified with phosphate thioesters for nuclease resistance. The presence of methylphosphonate modification increases the affinity of the oligonucleotide for its target RNA, thereby reducing IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It should be understood that the methods and reagents of this disclosure can be used in conjunction with any techniques that may be developed to enhance the stability or efficacy of repressive nucleic acid molecules.

[0217] miRNA molecules include nucleotide oligomers containing a modified backbone or non-natural internucleotide links. Oligomers with modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides without a phosphorus atom in their internucleotide backbone are also considered nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, phosphoramidates, thiophosphatates, thioalkylphosphonates, thioalkylphosphate triester polymers, and boron phosphates. Various salts, mixed salts, and free acid forms are also included.

[0218] The miRNAs described herein, whether in mature or hairpin form, can be provided as naked oligonucleotides. In some cases, formulations that facilitate the delivery of miRNAs or other nucleotide oligomers to cells may be required (see, for example, U.S. Patent Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, the entire contents of each of which are incorporated herein by reference).

[0219] In some instances, the miRNA composition is at least partially crystalline, homogeneously crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another instance, the miRNA composition is in an aqueous phase, such as in a solution comprising water. The aqueous phase or crystalline composition may be incorporated into a delivery carrier, such as liposomes (particularly for the aqueous phase) or particles (e.g., microparticles suitable for the crystalline composition). Typically, the miRNA composition is formulated in a manner compatible with the intended method of administration. The miRNA composition may be formulated in combination with another reagent, such as another therapeutic agent or a reagent for stabilizing oligonucleotides, such as a protein complexed with an oligonucleotide reagent. Other reagents include chelating agents, such as EDTA (e.g., for removing divalent cations, such as Mg), salts, and RNase inhibitors (e.g., broad-spectrum specific RNase inhibitors). In some embodiments, the miRNA composition includes another miRNA, such as a second miRNA composition (e.g., a microRNA different from the first). Other formulations may include at least three, five, ten, twenty, fifty, or one hundred or more different oligonucleotide species.

[0220] In some embodiments, the composition comprises an oligonucleotide composition that mimics miRNA activity. In some embodiments, the composition comprises an oligonucleotide having nucleobase identity with the nucleobase sequence of the miRNA and is therefore designed to mimic miRNA activity. In some embodiments, the oligonucleotide composition mimicking miRNA activity comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin or a processed miRNA double-strand.

[0221] In some embodiments, the oligonucleotide shares identity with the nucleobase sequence of an endogenous miRNA or miRNA precursor. The selected oligonucleotide used in the compositions of the present invention can be of one of several lengths. Such an oligonucleotide can be 7 to 100 linked nucleosides. For example, an oligonucleotide sharing nucleobase identity with a miRNA can be 7 to 30 linked nucleosides. An oligonucleotide sharing identity with a miRNA precursor is at most 100 linked nucleosides. In some embodiments, the oligonucleotide comprises 7 to 30 linked nucleosides. In some embodiments, the oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In some embodiments, the oligonucleotide comprises 19 to 23 linked nucleosides. In some embodiments, the oligonucleotide is 40 to 50, 60, 70, 80, 90, or 100 linked nucleosides.

[0222] In some embodiments, the oligonucleotide has a sequence that is identical to the miRNA or its precursor. The nucleotide sequence of the mature miRNA described herein and its corresponding stem-loop sequence are sequences found in miRBase, an online searchable database of miRNA sequences and annotations. Entry in the miRBase sequence database represents a predicted hairpin portion of the miRNA transcript (stem-loop), containing information about the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (premiRNAs) and may in some cases include flanking sequences of the presumed primary transcript and the presumed primary transcript. The miRNA nucleotide sequence described herein includes any version of the miRNA, including sequences described in miRBase sequence database version 10.0 and sequences described in any earlier releases of the miRBase sequence database. The release of the sequence database may lead to the renaming of certain miRNAs. The release of the sequence database may lead to variations in the mature miRNA sequence. The compositions of the present invention comprise oligomeric compounds containing oligonucleotides that are identical to any nucleotide sequence version of the miRNA described herein.

[0223] In some embodiments, the oligonucleotide has a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to that of the miRNA in 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 nucleotides. Therefore, in some embodiments, the oligonucleotide's nucleotide sequence may have one or more nucleotides that are different from those of the miRNA.

[0224] In some embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof. For example, the composition may comprise a viral vector, plasmid, granule, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in desired mammalian cells or tissues.

[0225] vaccine In some embodiments, the present invention provides immunogenic compositions for inducing or activating an immune response in a subject. For example, in some embodiments, the immunogenic composition is a vaccine. As used herein, an “immunogenic composition” may comprise an LNP that includes 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 specific embodiments, the composition comprises or encodes all or part of any peptide antigen or antigen-binding molecule, or an immunogenic 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.

[0226] The composition of the vaccines of the present invention may vary in their nucleic acid components. In non-limiting examples, the nucleic acids encoding antigens or antigen-binding molecules may also be formulated together with adjuvants. It will be understood, of course, that the various compositions described herein may further comprise additional components. The vaccines of the present invention and their various components may be prepared and / or administered by any of the methods disclosed herein or any methods that a person skilled in the art would become aware of in view of this disclosure.

[0227] 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 have or are at risk of developing or susceptible to disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the obvious clinical manifestation of the disease, thereby preventing the disease or disorder or optionally delaying its progression. In the context of the medical field, the term "prevention" encompasses any activity that reduces the burden of death or morbidity caused by disease. Prevention may be carried out at primary, secondary, and tertiary levels of prevention. Primary prevention avoids the development of disease, while secondary and tertiary prevention encompass activities aimed at preventing disease progression and the onset of symptoms, as well as reducing the negative impacts of a diagnosed disease by restoring function and reducing disease-related complications.

[0228] Nucleic acid In some embodiments, the present invention includes an ionizable LNP molecule formulated for targeted delivery to T cells in vivo, comprising or encapsulating one or more nucleic acid molecules. In some embodiments, the nucleic acid molecule is an mRNA molecule. In some embodiments, the mRNA molecule encodes a CAR. In some embodiments, the nucleoside-modified mRNA molecule encodes a CAR. In some embodiments, the present invention includes a nucleoside-modified mRNA molecule encoding an adjuvant.

[0229] As described herein, the nucleotide sequence encoding a CAR can optionally include sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that all nucleotides encode a polypeptide according to the invention. Therefore, the scope of the invention includes nucleotide sequences substantially homologous to the nucleotide sequences described herein and encoding antigens or antigen-binding molecules or adjuvants of interest.

[0230] Furthermore, the scope of this invention includes nucleotide sequences that encode amino acid sequences substantially homologous to the amino acid sequences described herein and retain the immunogenic function of the original amino acid sequences.

[0231] As used herein, an amino acid sequence is considered "substantially homologous" to any of the amino acid sequences described herein when it has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with any of the amino acid sequences described herein. The identity between two amino acid sequences is preferably determined using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0232] In some embodiments, the present invention relates to a construct comprising a nucleotide sequence encoding a CAR. In some embodiments, the construct comprises multiple nucleotide sequences encoding multiple antigens. For example, in some 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 some embodiments, the present invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In some embodiments, the construct comprises a first nucleotide sequence encoding a CAR and a second nucleotide sequence encoding an adjuvant.

[0233] In some embodiments, the composition comprises a plurality of constructs, each construct encoding one or more antigens. In some 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 some embodiments, the composition comprises: a first construct containing a nucleotide sequence encoding a CAR; and a second construct containing a nucleotide sequence encoding an adjuvant.

[0234] In another specific embodiment, the construct is operatively bound to a translation control element. The construct may incorporate a operatively bound regulatory sequence for expressing the nucleotide sequence of the present invention, thereby forming an expression cassette.

[0235] carrier The nucleic acid sequence encapsulated in the immune cell-targeting LNP molecule of the present invention can be obtained using recombination methods known in the art, such as screening a library from cells expressing the gene, deriving the gene from a known vector containing the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the nucleic acid molecule of interest can be synthesized.

[0236] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0237] In some embodiments, the compositions of the present invention comprise in vitro transcribed (IVT) RNA encoding a CAR. In some embodiments, the compositions of the present invention comprise IVT RNA encoding multiple antigens. In some embodiments, the compositions of the present invention comprise IVT RNA encoding an adjuvant. In some embodiments, the compositions of the present invention comprise IVT RNA encoding one or more antigens and one or more adjuvants.

[0238] Nucleoside-modified RNA In some embodiments, the composition comprises nucleoside-modified RNA. In some embodiments, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA has particular advantages over unmodified mRNA, including, for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA for use in the present invention is further described in U.S. Patent No. 8,278,036 (which is incorporated herein by reference in its entirety).

[0239] In some embodiments, the nucleoside-modified mRNA does not activate any pathophysiological pathways, is highly effective and translates almost immediately after delivery, and serves as a template for continuous protein production in vivo for several days (Karikó et al., 2008, Mol Ther 16:1833-1840; Karikó et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert its physiological effects is very small, making it suitable for human treatment. In some embodiments, immune cells containing mRNA molecules encoding CAR are directed to cells of interest expressing antigens that are specifically bound by CAR.

[0240] In some cases, expressing proteins by delivering mRNA offers several advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the desired protein-coding sequence is the only substance delivered to the cell, thus avoiding all the side effects associated with the plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA-based and virus-based vectors, mRNA does not carry the risk of being incorporated into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of mRNA. In some implementations, using mRNA instead of protein also has several advantages. Proteins often have short half-lives during circulation, thus protein therapy requires frequent dosing, while mRNA provides a template for continuous protein production over several days. Protein purification is problematic; they may contain aggregates and other impurities, leading to adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0241] In some embodiments, the nucleoside-modified RNA includes the naturally occurring modified nucleoside pseudouridine. In some embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Karikó et al., 2008, MolTher 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Karikó et al., 2011, Nucleic Acids Research 39:e142; Karikó et al., 2012, Mol Ther 20:948-953; Karikó et al., 2005, Immunity 23:165-175).

[0242] It has been demonstrated that the presence of modified nucleosides in RNA, including pseudouridine, inhibits its innate immunogenicity (Karikó et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed RNA encoding proteins containing pseudouridine can be translated more efficiently than RNA without modified nucleosides or containing other modified nucleosides (Karikó et al., 2008, Mol Ther16:1833-1840). Subsequently, it has been shown that the presence of pseudouridine increases RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and attenuates PKR activation and translational repression (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 RNAs with excellent translational potential and no innate immunogenicity (Karikó et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified pseudouridine-containing RNAs encoding erythropoietin to mice and macaques resulted in a significant increase in serum EPO levels (Karikó et al., 2012, Mol Ther 20:948-953), thus demonstrating that pseudouridine-containing mRNAs are suitable for in vivo protein therapy.

[0243] This invention includes RNA, oligonucleotides, and polynucleotide molecules comprising pseudouridine or modified nucleosides. In some embodiments, the composition comprises isolated nucleic acids encoding antigens or antigen-binding molecules, wherein the nucleic acids comprise pseudouridine or modified nucleosides. In some embodiments, the composition comprises a carrier comprising isolated nucleic acids encoding antigens, antigen-binding molecules, adjuvants, or combinations thereof, wherein the nucleic acids comprise pseudouridine or modified nucleosides.

[0244] In some embodiments, the nucleoside-modified RNA of the present invention is IVT RNA. For example, in some 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.

[0245] In some embodiments, the modified nucleoside is m 1 acp 3 Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In some embodiments, the modified nucleoside is m 1Ψ(1-methylpseudouridine). In some embodiments, the modifying nucleoside is Ψm(2'-O-methylpseudouridine). In some embodiments, the modifying nucleoside is m. 5 D (5-methyldihydrouridine). In some embodiments, the modified nucleoside is m 3 Ψ (3-methylpseuuridine). In some embodiments, the modified nucleoside is the unmodified pseudouridine moiety. In some embodiments, the modified nucleoside is any of the above-described pseudouridine monophosphate, diphosphate, or triphosphate. In some embodiments, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0246] 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 another embodiment, the modified nucleoside is guanosine (G).

[0247] In some embodiments, the modified nucleoside of the present invention is m 5 C (5-methylcytidine). In some embodiments, the modified nucleoside is m 5 U (5-methyluridine). In some embodiments, the modified nucleoside is m 6 A (N 6 -methyladenosine). In some embodiments, the modified nucleoside is S-methyladenosine. 2 U (2-thiouridine). In some embodiments, the modifying nucleoside is Ψ (pseudouridine). In some embodiments, the modifying nucleoside is Um (2'-O-methyluridine).

[0248] In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A (2-methylthio-N) 6 -methyladenosine); i 6 A (N 6 -Isopentenyl adenosine); ms 2 i6A (2-methylthio-N) 6 Isopentenyl adenosine; 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 -glycylcarbamoyladenosine); t6 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 -hydroxyn-valinecarbamoyladenosine); ms 2 hn 6 A (2-methylthio-N) 6 -hydroxyvaline carbamoyl adenosine); Ar(p)(2'-O-ribosyl adenosine (phosphate)); I (inosine); m 1 I (1-methylinosine); m 1 Im (1,2'-O-dimethylinosine); m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C (2-thiocytidine); ac 4 C (N 4 -Acetylcytidine); f 5 C (5-formylcytidine); m 5 Cm (5,2'-O-dimethylcytidine); ac 4 Cm (N 4 -acetyl-2'-O-methylcytidine); k 2 C (Laishidin); m 1 G(1-methylguanosine); m 2 G (N2-methylguanosine); m 7 G (7-methylguanosine); Gm (2'-O-methylguanosine); m 2 2G (N 2 N 2 -dimethylguanosine); m 2 Gm (N 2 ,2'-O-dimethylguanosine); m 2 2Gm (N 2 N 2 ,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxy-wybutosine); OHyW (hydroxy-wybutosine); OHyW (Insufficiently modified hydroxywyosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosylqueuosine); manQ (mannosylqueuosine); preQ0 (7-cyano-7-deazonoguanosine); preQ1 (7-aminomethyl-7-deazonoguanosine); G + (archaeosine); D (dihydrouridine); m 5 Um (5,2'-O-dimethyluridine); s 4 U (4-thiouridine); m 5 s 2 U (5-methyl-2-thiouridine); s 2 Um (2-thio-2'-O-methyluridine); acp 3 U (3-(3-amino-3-carboxypropyl)uridine); ho 5 U (5-hydroxyuridine); mo 5 U (5-methoxyuridine); cmo 5 U (uridine 5-oxyacetic acid); mcmo 5 U (uridine 5-oxyacetic acid methyl ester); chm 5 U (5-(carboxyhydroxymethyl)uridine)); mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U (5-methoxycarbonylmethyluridine); mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U (5-methoxycarbonylmethyl-2-thiouridine); nm 5 s 2 U (5-aminomethyl-2-thiouridine); mnm 5 U (5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U (5-methylaminomethyl-2-selenoside); 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 5s 2 U (5-Carboxymethylaminomethyl-2-thiouridine); m 6 2A (N 6 N 6 -dimethyladenosine); Im (2'-O-methylinosine); m 4 C (N 4 -methylcytidine); m 4 Cm (N 4 ,2'-O-dimethylcytidine); hm 5 C (5-hydroxymethylcytidine); m 3 U (3-methyluridine); cm 5 U (5-carboxymethyluridine); m 6 Am (N 6 ,2'-O-dimethyladenosine); m 6 2Am (N 6 N 6 (,O-2'-trimethyladenosine); m 2,7 G (N 2 ,7-Dimethylguanosine); m 2,2,7 G (N 2 N 2 ,7-Trimethylguanosine); m 3 Um (3,2'-O-dimethyluridine); m 5 D (5-methyldihydrouridine); f 5 Cm(5-formyl-2'-O-methylcytidine); m 1 Gm (1,2'-O-dimethylguanosine); m 1 Am (1,2'-O-dimethyladenosine); τm 5 U(5-taurinomethyluridine); τm 5 s 2 U (5-Tauratemethyl-2-thiouridine); imG-14 (4-Demethylwyoside); imG2 (Isowyoside); or ac 6 A (N 6 - Acetyladenosine.

[0249] In some embodiments, the nucleoside-modified RNA of the present invention comprises a combination of two or more of the above-described modifications. In some embodiments, the nucleoside-modified RNA comprises a combination of three or more of the above-described modifications. In some embodiments, the nucleoside-modified RNA comprises a combination of more than three of the above-described modifications.

[0250] In some embodiments, 0.1% to 100% of the residues in the nucleoside modification of the present invention are modified (e.g., due to the presence of pseudouridine or modified nucleoside bases). 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 fraction is 10%. In some embodiments, the fraction is 12%. In some embodiments, the fraction is 14%. In some embodiments, the fraction is 16%. In some embodiments, the fraction is 18%. In some embodiments, the fraction is 20%. In some embodiments, the fraction is 25%. In some embodiments, the fraction is 30%. In some embodiments, the fraction is 35%. In some embodiments, the fraction is 40%. In some embodiments, the fraction is 45%. In some embodiments, the fraction is 50%. In some embodiments, the fraction is 60%. In some embodiments, the fraction is 70%. In some embodiments, the fraction is 80%. In some embodiments, the fraction is 90%. In some embodiments, the fraction is 100%.

[0251] In some implementations, the score is less than 5%. In some implementations, the score is less than 3%. In some implementations, the score is less than 1%. In some implementations, the score is less than 2%. In some implementations, the score is less than 4%. In some implementations, the score is less than 6%. In some implementations, the score is less than 8%. In some implementations, the score is less than 10%. In some implementations, the score is less than 12%. In some implementations, the score is less than 15%. In some implementations, the score is less than 20%. In some implementations, the score is less than 30%. In some implementations, the score is less than 40%. In some implementations, the score is less than 50%. In some implementations, the score is less than 60%. In some implementations, the score is less than 70%.

[0252] In some embodiments, 0.1% of residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In some embodiments, the fraction of the modified given nucleotide 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 fraction is 12%. In some embodiments, the fraction is 14%. In some embodiments, the fraction is 16%. In some embodiments, the fraction is 18%. In some embodiments, the fraction is 20%. In some embodiments, the fraction is 25%. In some embodiments, the fraction is 30%. In some implementations, the fraction is 35%. In some implementations, the fraction is 40%. In some implementations, the fraction is 45%. In some implementations, the fraction is 50%. In some implementations, the fraction is 60%. In some implementations, the fraction is 70%. In some implementations, the fraction is 80%. In some implementations, the fraction is 90%. In some implementations, the fraction is 100%.

[0253] In some embodiments, the fraction of the given modified nucleotide 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%.

[0254] In some embodiments, the nucleoside-modified RNA of the present invention exhibits higher translation efficiency in cells than unmodified RNA molecules of the same sequence. In some embodiments, the nucleoside-modified RNA demonstrates enhanced translation ability by target cells. In some embodiments, translation is enhanced by 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by 3-fold. In some embodiments, translation is enhanced by 5-fold. In some embodiments, translation is enhanced by 7-fold. In some embodiments, translation is enhanced by 10-fold. In some embodiments, translation is enhanced by 15-fold. In some embodiments, translation is enhanced by 20-fold. In some embodiments, translation is enhanced by 50-fold. In some embodiments, translation is enhanced by 100-fold. In some embodiments, translation is enhanced by 200-fold. In some embodiments, translation is enhanced by 500-fold. In some embodiments, translation is enhanced by 1000-fold. In some embodiments, translation is enhanced by 2000-fold. In some embodiments, the fold increase is 10-1000-fold. In some embodiments, the fold increase is 10-200-fold. In some embodiments, the fold increase is 10-300-fold. In some embodiments, the multiplier is 10-500 times. In some embodiments, the multiplier is 20-1000 times. In some embodiments, the multiplier is 30-1000 times. In some embodiments, the multiplier is 50-1000 times. In some embodiments, the multiplier is 100-1000 times. In some embodiments, the multiplier is 200-1000 times. In some embodiments, the translation enhances any other significant amount or range of amounts.

[0255] In some embodiments, the nucleoside-modified antigen-encoding RNA of the present invention induces a significantly greater adaptive immune response compared to unmodified in vitro synthesized RNA molecules of the same sequence. In some embodiments, the modified RNA molecules exhibit an adaptive immune response twice as large as their unmodified counterparts. In some embodiments, the adaptive immune response is increased by 3-fold. In another embodiment, the adaptive immune response is increased by 5-fold. In some embodiments, the adaptive immune response is increased by 7-fold. In some embodiments, the adaptive immune response is increased by 10-fold. In some embodiments, the adaptive immune response is increased by 15-fold. In another embodiment, the adaptive immune response is increased by 20-fold. In some embodiments, the adaptive immune response is increased by 50-fold. In some embodiments, the adaptive immune response is increased by 100-fold. In some embodiments, the adaptive immune response is increased by 2000-fold. In some embodiments, the adaptive immune response increases by other folds.

[0256] In some embodiments, "inducing a significantly greater adaptive immune response" refers to a detectable increase in the adaptive immune response. In some embodiments, the term refers to a fold increase in the adaptive immune response (e.g., a fold increase of 1 as listed above). In some embodiments, the term refers to an increase such that nucleoside-modified RNA can be administered at a lower dose or frequency than the same type of unmodified RNA molecule while still inducing an effective adaptive immune response. In some embodiments, the increase enables nucleoside-modified RNA to induce an effective adaptive immune response with a single dose.

[0257] In some embodiments, the nucleoside-modified RNA of the present invention exhibits significantly less innate immunogenicity compared to unmodified in vitro synthesized RNA molecules of the same sequence. In some embodiments, the modified RNA molecules exhibit twice the innate immune response of their unmodified counterparts. In some embodiments, innate immunogenicity is reduced by 3-fold. In some embodiments, innate immunogenicity is reduced by 5-fold. In some embodiments, innate immunogenicity is reduced by 7-fold. In some embodiments, innate immunogenicity is reduced by 10-fold. In some embodiments, innate immunogenicity is reduced by 15-fold. In some embodiments, innate immunogenicity is reduced by 20-fold. In some embodiments, innate immunogenicity is reduced by 50-fold. In some embodiments, innate immunogenicity is reduced by 100-fold. In some embodiments, innate immunogenicity is reduced by 200-fold. In some embodiments, innate immunogenicity is reduced by 500-fold. In some embodiments, innate immunogenicity is reduced by 1000-fold. In some embodiments, innate immunogenicity is reduced by 2000-fold. In some embodiments, innate immunogenicity is reduced by other folds.

[0258] In some embodiments, "exhibiting significantly less innate immunogenicity" means a detectable reduction in innate immunogenicity. In some embodiments, the term refers to a fold reduction in innate immunogenicity (e.g., a fold reduction of 1 as listed above). In some embodiments, the term refers to a reduction such that an effective amount of nucleoside-modified RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that repeated administration of nucleoside-modified RNA can be administered without triggering an innate immune response sufficient to detectably reduce the production of recombinant protein. In some embodiments, the reduction is such that repeated administration of nucleoside-modified RNA can be administered without triggering an innate immune response sufficient to eliminate detectable production of recombinant protein.

[0259] peptide therapy In other related aspects, therapeutic agents include peptides that modulate the separation of a target. For example, in some embodiments, the peptides of the present invention directly inhibit or activate a target by binding to it, thereby modulating the normal functional activity of the target. In some embodiments, the peptides of the present invention modulate the target by competing with endogenous proteins. In some embodiments, the peptides of the present invention modulate the activity of the target by acting as a trans-dominant-negative mutant.

[0260] Variants of the peptide therapeutic agent may be (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code; (ii) variants in which one or more modified amino acid residues are present, such as residues modified by linking substituents; (iii) variants in which the peptide is an optional splice variant of the peptide of the present invention; (iv) fragments of the peptide; and / or (v) variants in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence for purification (e.g., His-tag) or for detection (e.g., Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translational modified or chemically modified. Based on the teachings herein, these variants are considered to fall within the scope of those skilled in the art.

[0261] CAR agents In some embodiments, the mRNA molecule of the present invention encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is a targeting domain, wherein the targeting domain directs CAR-expressing T cells to specific cells or tissues of interest. For example, in some embodiments, the targeting domain includes an antibody, antibody fragment, or peptide that specifically binds to an antigen expressed on a pathogenic object or tumor cell, thereby directing CAR-expressing T cells to cells or tissues expressing the antigen.

[0262] In some embodiments, the present invention relates to an immune cell-targeting LNP containing an agent comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the agent comprises an mRNA molecule encoding a CAR. In some embodiments, the agent comprises a modified nucleoside mRNA molecule encoding a CAR.

[0263] In various implementations, the CAR can be a “first-generation,” “second-generation,” “third-generation,” “fourth-generation,” or “fifth-generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensenet 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. 173:2143-2150 (2004); Sadelainet al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009)).

[0264] The "first-generation" CAR used in this invention includes an antigen-binding domain, for example, a single-stranded variable fragment (scFv) fused to a transmembrane domain of a cytoplasmic / intracellular domain of the same T cell receptor chain. The "first-generation" CAR generally has an intracellular domain of the CD3ζ-chain, which is the primary signaling medium for endogenous T cell receptor (TCR) signaling. The "first-generation" CAR provides de novo antigen recognition and induces activation of CD4+ and CD8+ T cells through its CD3ζ-chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0265] The "second-generation" CAR used in this invention comprises an antigen-binding domain, for example, a single-stranded variable fragment (scFv) fused with an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to enhance T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Therefore, CAR design can combine antigen recognition with signal transduction, both physiologically performed by two separate complexes: the TCR heterodimer and the CD3 complex. The "second-generation" CAR contains intracellular domains derived from various co-stimulatory molecules, such as CD28, 4-1BB, ICOS, OX40, etc., from the cytoplasmic tail of the CAR, to provide additional signals to the cell.

[0266] "Second-generation" CARs provide co-stimulation (e.g., via the CD28 or 4-1BB domains) and activation (e.g., via the CD3ζ signaling domain). Preclinical studies have shown that "second-generation" CARs can enhance the antitumor activity of T cells. For example, "second-generation" CAR-modified T cells have demonstrated robust efficacy in CD19 molecular clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0267] The “third-generation” CAR provides multiple co-stimulation (e.g., by including CD28 and 4-1BB domains) and activation (e.g., by including the CD3ζ activation domain).

[0268] The “fourth generation” CAR provides co-stimulation (e.g., via the CD28 or 4-1BB domains) and activation (e.g., via the CD3ζ signaling domain in addition to constitutive or inducible chemokine components).

[0269] The "fifth-generation" CAR provides co-stimulation (e.g., via the CD28 or 4-1BB domain) and activation (e.g., via the CD3ζ signaling domain, constitutive or inducible chemokine components, and intracellular domains of cytokine receptors (e.g., IL-2Rβ).

[0270] In various implementations, the CAR may be included in a multivalent CAR system, such as a dual CAR or tandem CAR system. Multivalent CAR systems include systems or cells containing multiple CARs, as well as systems or cells containing bivalent / bispecific CARs targeting more than one antigen.

[0271] In the embodiments disclosed herein, as described above, a CAR typically comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain. In a specific, non-limiting embodiment, the antigen-binding domain is an scFv specific to binding to a surface antigen of a target cell of interest (e.g., a pathogen or tumor cell).

[0272] combination In some embodiments, the compositions of the present invention comprise a combination of agents described herein. In some embodiments, the compositions comprising a combination of agents described herein have an additive effect, wherein the total effect of the combination is approximately equal to the sum of the effects of the individual agents. In other embodiments, the compositions comprising a combination of agents described herein have a synergistic effect, wherein the total effect of the combination is greater than the sum of the effects of the individual agents.

[0273] A composition comprising a combination of agents, including individual agents in any suitable proportion. For example, in some embodiments, the composition comprises two individual agents in a 1:1 ratio. However, the combination is not limited to any specific proportion. Rather, any proportion that has proven effective is included.

[0274] Cell-targeting domain In various embodiments of the present invention, the LNP of the present invention is conjugated with a targeting domain that is specific to the receptor binding to the target cell.

[0275] In some 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 associated with hematopoietic stem cells (e.g., myeloid stem cells and lymphoid stem cells).

[0276] In some implementations, the target cells are peripheral blood mononuclear cells (PBMCs).

[0277] In a single cell, the target cell is an immune cell. Exemplary immune cells that can be targeted by the compositions according to the 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 some embodiments, the immune cell is a T cell. In some embodiments, the T cells that can be targeted by the compositions of the invention may be CD4+ or CD8+, and may include, but are not limited to: T helper cells (CD4+); cytotoxic T cells (also known as cytotoxic T lymphocytes, CTL; CD8- T cells); and memory T cells, including central memory T cells (TCM), stem cell memory T cells (TSCM), stem cell-like memory T cells (or stem cell-like memory T cells), and effector memory T cells, such as T cells. EM Cells and T EMRA(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 inertial 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 (Effective memory) and T TE (End-stage effect), TCR transgenic T cells, T cells redirected for universal cytokine-mediated killing (TRUCK), tumor-infiltrating T cells (TIL), CAR-T cells, or any T cell that can be used to treat a disease or disorder.

[0278] In some embodiments, the T cells of the present invention are immunostimulatory cells, i.e., cells that mediate immune responses. Examples of immunostimulatory T cells include, but are not limited to: T helper cells (CD4+); cytotoxic T cells (also known as cytotoxic T lymphocytes, CTL; CD8+ T cells); and memory T cells, including central memory T cells (TCM), stem cell memory T cells (TSCM), stem cell-like memory T cells (or stem cell-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 (follicle helper) cells, natural killer T cells, mucosa-associated inertial T cells (MAIT), and γδ T cells.

[0279] In some implementations, the T cell targeting domain binds to 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, and CD153. CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, L AG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6 or CCR7.

[0280] In some embodiments, the present invention relates to a composition comprising a combination of delivery agents conjugated to an immune cell targeting domain for targeting a variety of immune cells. In some embodiments, the composition comprises two or more immune cell targeting delivery agents targeting two or more immune cell antigens. In some embodiments, the two or more immune cell antigens are selected from 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, CD15 3. CD154, 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 some embodiments, the combination comprises two or more T cell-targeting delivery agents that target the surface antigens of CD4+ T cells and CD8+ T cells. In some embodiments, the combination comprises two or more T cell-targeting delivery agents that target CD4 and CD8.

[0281] In some embodiments, the target 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 some embodiments, the conjugation is reversible, such that the delivery medium can dissociate from the target domain upon exposure to certain conditions or chemicals. In some embodiments, the conjugation is irreversible, such that the delivery medium does not dissociate from the target domain under normal conditions.

[0282] In some embodiments, the conjugation includes a covalent bond between an activated polymer-conjugated lipid and a targeting domain. The term "activated polymer-conjugated lipid" refers to a molecule comprising a lipid moiety and a polymer moiety, the polymer moiety being activated by functionalization of the polymer-conjugated lipid with a first coupling group. In some embodiments, the activated polymer-conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In some embodiments, the activated polymer-conjugated lipid is an activated polyethylene glycol-modified lipid. In some embodiments, the first coupling group is bound to the lipid moiety of the polyethylene glycol-modified lipid. In some embodiments, the first coupling group is bound to the polyethylene glycol moiety of the polyethylene glycol-modified lipid. In some embodiments, the second functional group is covalently linked to the targeting domain.

[0283] The first and second coupling groups can be any functional groups known to those skilled in the art, for example, that form a covalent bond together under mild reaction conditions or physiological conditions. In some embodiments, the first or second coupling group is selected from maleimide, N-hydroxysuccinimide (NHS) ester, carbodiimide, acylhydrazine, pentafluorophenyl (PFP) ester, phosphine, hydroxymethylphosphine, psoralen, imide ester, pyridyl disulfide, isocyanate, vinyl sulfone, α-haloacetyl, aryl azide, acyl azide, alkyl azide, diazirines, benzophenone, epoxide, carbonate, acid anhydride, sulfonyl chloride, cyclooctyne, aldehyde, and mercapto. In some embodiments, the first or second coupling group is selected from free amine (–NH2), free mercapto (–SH), free hydroxide group (–OH), carboxyl group, acylhydrazine, and alkoxyamine. In some embodiments, the first coupling group is a functional group reactive to a thiol group, such as maleimide, pyridyl disulfide, or haloacetyl. In some embodiments, the first coupling group is maleimide.

[0284] In some embodiments, the second coupling group is a thiol group. The thiol group can be mounted on the target domain using any method known to those skilled in the art. In some embodiments, the thiol group is present on a free cysteine ​​residue. In some embodiments, the thiol group is manifested by reduction of a disulfide on the target domain, such as by reaction with 2-mercaptoethylamine. In some embodiments, the thiol group is mounted by a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0285] In some embodiments, the polymer-conjugated lipids and targeted domains are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves reactions between functional groups having 1,3-dipolar atoms (such as azides, nitrile oxides, nitrones, isocyanates, etc.) and alkene or alkyne dipolarophiles. Exemplary dipolarophiles include any strained cyclic olefins and cyclic olefins known to those skilled in the art, including but not limited to cyclooctene, dibenzocyclooctene, monofluorinated cyclooctene, difluorinated cyclooctene, and biarylazacyclooctynone.

[0286] In some implementations, maleimide conjugation is used to conjugate the target domain with the LNP.

[0287] Targeted structural domain In some embodiments, the composition includes a targeting domain that directs the delivery agent to target immune cells. The targeting domain may include nucleic acids, peptides, antibodies, small molecules, organic molecules, inorganic molecules, glycans, sugars, hormones, etc., which target the particles to sites specifically requiring a therapeutic agent. In some embodiments, the particles include multivalent targets, wherein the particles incorporate the multiple targeting mechanisms described herein. In some embodiments, the targeting domain of the delivery agent specifically binds to a target associated with a site for which an agent needs to be included in the delivery agent. For example, the targeting domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a specific disease state. Such a target may be a protein, a protein fragment, an antigen, or other biomolecule associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand that specifically binds to a target. In some embodiments, the target (e.g., an antigen) is associated with a site requiring treatment with an agent. In some embodiments, the targeting domain may copolymerize with the composition containing the delivery agent. In some embodiments, the targeting domain may be covalently linked to the composition containing the delivery agent, such as through a chemical reaction between the targeting domain and the composition containing the delivery agent. In some embodiments, the targeting domain is an additive in the delivery medium. The targeting domains of the present invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0288] In various embodiments, the targeting domain binds to cell surface molecules of the cell of interest. For example, in various embodiments, the targeting domain binds to cell surface molecules of endothelial cells, stem cells, or immune cells.

[0289] peptides In some embodiments, the targeting domain of the present invention includes a peptide. In some embodiments, the peptide targeting domain specifically binds to a target of interest.

[0290] The peptides of this invention can be prepared using chemical methods. For example, the peptides can be synthesized using solid-phase technology (Roberge JY et al. (1995) Science 269: 202-204), cleaved from a resin, and purified by preparative high-performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0291] Peptides can also be prepared through recombinant methods or by cleaving longer polypeptides. The composition of peptides can be confirmed by amino acid analysis or sequencing.

[0292] Variants of the peptide according to the invention may be (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code; (ii) variants in which one or more modified amino acid residues are present, such as residues modified by linking substituents; (iii) variants in which the peptide is an optional splice variant of the peptide of the invention; (iv) fragments of the peptide; and / or (v) variants in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence for purification (e.g., His-tag) or for detection (e.g., Sv5 epitope tag). Fragments comprise peptides generated by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translational modified or chemically modified. Based on the teachings herein, these variants are considered to fall within the scope of those skilled in the art.

[0293] As is 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 a second peptide. A variant is defined as a peptide sequence that differs from the original sequence, preferably less than 40% of residues per segment of interest compared to the original sequence, more preferably less than 25% of residues per segment of interest compared to the original sequence, even more preferably less than 10% of residues per segment of interest compared to the original sequence, and most preferably only a few residues per segment of interest compared to the original protein sequence, while being sufficiently homologous to the original sequence to preserve its functionality. This invention includes 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 known to those skilled in the art. 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)] is preferably used to determine the identity between two amino acid sequences.

[0294] The peptides of this invention can be post-translational modified. For example, post-translational modifications falling within the scope of this invention include signal peptide cleavage, glycosylation, acetylation, isoprenelation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding canine microsomal membranes or Xenopus egg extracts to a standard translation reaction (US Patent No. 6,103,489).

[0295] The peptides of the present invention may include non-natural amino acids formed by post-translational modification or by introducing non-natural amino acids during translation.

[0296] Nucleic acid In some embodiments, the targeting domain of the present invention comprises isolated nucleic acids, including, for example, DNA oligonucleotides and RNA oligonucleotides. In some embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in some embodiments, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0297] The nucleotide sequence of a nucleic acid targeting domain can optionally include sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid functions as the original nucleic acid and specifically binds to the target of interest.

[0298] For the purposes of this specification, a nucleotide sequence is considered "substantially homologous" to any of the nucleotide sequences described herein when it has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with any of the nucleotide sequences described herein. Other examples of possible modifications include inserting one or more nucleotides into the sequence, adding one or more nucleotides at any end of the sequence, or deleting one or more nucleotides at any end of the sequence or within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods known to those skilled in the art. 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)] is preferably used to determine the identity between two amino acid sequences.

[0299] Antibody In some embodiments, the targeting domain of the present invention includes an antibody or antibody fragment. In some embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, their Fab and single-chain Fv (scFv) fragments, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0300] Antibodies can be complete monoclonal or polyclonal antibodies, as well as immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778), or chimeric antibodies, such as antibodies containing mouse antibody-binding specificity, but with the remainder derived from human. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art.

[0301] These 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 choice of manufacturing method depends on several factors, including the desired antibody structure, the importance of the carbohydrate moiety on the antibody, ease of culture and purification, and cost. A variety of different antibody structures can be generated using standard expression techniques, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies containing components from different species. Small-sized antibody fragments, such as Fab and Fv fragments, may be generated in bacterial expression systems; these fragments lack effector function and have limited pharmacokinetic activity. Single-chain Fv fragments exhibit low immunogenicity.

[0302] antigen This invention provides compositions for inducing an immune response in a subject. In some embodiments, the composition comprises an immune cell-targeting LNP containing a nucleic acid molecule encoding a chimeric antigen receptor (CAR) that is specific to the antigen.

[0303] In some implementations, the antigen includes a polypeptide or peptide associated with pathogens or tumor cells, such that in vivo modified immune cells expressing CAR then target the antigen, inducing an immune response against the antigen, and thus inducing an immune response against pathogens or tumor cells.

[0304] In some implementations, the antigens recognized by the CAR encoded by the nucleic acid molecule include proteins, peptides, fragments thereof, or variants thereof, or combinations thereof, from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals.

[0305] In some implementations, the antigen includes a tumor-specific antigen or a tumor-associated antigen, such that immune cells expressing CAR are directed to tumor cells expressing the antigen.

[0306] Viral antigens In some embodiments, the antigen includes a viral antigen, a fragment thereof, or a variant thereof. In some embodiments, the viral antigen originates from a virus belonging to one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In some implementations, the viral antigens are derived from papillomaviruses (e.g., human papillomavirus (HPV)), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (major and minor smallpox), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, norovirus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, etc. 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), herpes zoster virus (varicella-zoster virus, 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 type 2 (SARS-CoV-2), or oncogenic viruses.

[0307] parasite antigen In some embodiments, the antigen includes a parasitic antigen or a fragment or variant thereof. In some embodiments, the parasite is a protozoan, worm, or ectoparasite. In some embodiments, the worm is a flatworm (e.g., flukes and tapeworms), an acanthocephalan, or a roundworm (e.g., pinworm). In some embodiments, the ectoparasite is a lice, flea, tick, or mite.

[0308] In some embodiments, the parasite is any parasite that causes the following diseases: Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, bulgocytosis, baculocystitis, baculocystitis, chagas disease, clonorchiasis, trypanosomes, cryptosporidiosis, sparganosis, draconis disease, echinococcosis, elephantiasis, pinworm infection, liver fluke infection, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenolesia, isosporidiosis, Oncomelania flavomarginata fever, leishmaniasis, Lyme disease, malaria, metaclonal infection, myiasis, onchocerciasis, lice infestation, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, and whipworm infection.

[0309] In some embodiments, the parasites are Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidiumcoli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, and Paragonimus. Lung flukes, pinworms, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0310] bacterial antigens In some embodiments, the antigen includes a bacterial antigen or a fragment or variant thereof. In some embodiments, the bacteria belong to any of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, and Elusimicrob. ia), Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericulates, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0311] In some embodiments, the bacteria are Gram-positive or Gram-negative bacteria. In some embodiments, the bacteria are aerobic or anaerobic bacteria. In some embodiments, the bacteria are autotrophic or heterotrophic bacteria. In some embodiments, the bacteria are mesophiles, neutrophils, extremophiles, acidophiles, alkaliphiles, thermophiles, psychrophiles, halophiles, or osmophiles.

[0312] In some embodiments, the bacteria are anthrax bacteria, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacteria, Staphylococcus bacteria, Streptococcus bacteria, or tetanus bacteria. In some embodiments, the bacteria are mycobacteria, Clostridium tetani, Yersiniapestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0313] Fungal antigens In some embodiments, the antigen includes a fungal antigen or a fragment or variant thereof. In some embodiments, the fungus is Aspergillus, Blastomyces dermatitidis, Candidayeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0314] Tumor antigens In some embodiments, the antigen includes tumor antigens, which include, for example, tumor-associated antigens or tumor-specific antigens. In the context of this invention, "tumor antigen," "hyperplastic disorder antigen," or "antigen associated with hyperplastic disorder" refers to an antigen commonly found in a specific hyperplastic disorder. In some aspects, the hyperplastic disorder antigens of this invention are derived from cancer, including but not limited to primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0315] Tumor antigens are proteins produced by tumor cells that trigger immune responses, particularly T-cell-mediated immune responses. In some embodiments, the tumor antigens of the present invention comprise one or more antigenic cancer epitopes that are immunogenically recognized by tumor-infiltrating lymphocytes (TILs) derived from mammalian cancer tumors. The choice of antigen will depend on the specific type of cancer to be treated or prevented by the compositions of the present invention.

[0316] Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muta-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostate-specific protein (prostein), PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0317] In some implementations, tumor antigens comprise one or more antigenic oncotopes associated with malignancy. Malignant tumors express a variety of proteins that can act 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 are transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute a truly tumor-specific immunoglobulin antigen, which is unique to that specific tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphomas. Some of these antigens (idiotypic CEA, HER-2, CD19, CD20) have been used as targets for monoclonal antibody passive immunotherapy, but with limited success.

[0318] The tumor antigens mentioned in this invention can also be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and do not occur on other cells in the body. TAA-associated antigens are not specific to tumor cells; on the contrary, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. The expression of this antigen on a tumor can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0319] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as 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 major 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-linkin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 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 / cyclic protein C-related protein, TAAL6, TAG72, TLP, and TPS.

[0320] adjuvant In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises a nucleic acid molecule encoding the adjuvant. In some embodiments, the nucleic acid molecule encoding the adjuvant is IVT RNA. In some embodiments, the nucleic acid molecule encoding the adjuvant is nucleoside-modified mRNA.

[0321] 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-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with a deleted signal sequence and optionally containing a signal peptide derived from IgE. Other genes that can serve as 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, p150.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutants of IL-18, CD40, CD40L, and angiogenesis. Factors, 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, cysteine ​​ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig and their functional fragments.

[0322] Pharmaceutical Composition The formulations of the pharmaceutical compositions described herein can be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve the step of associating the active ingredient with a carrier or one or more other auxiliary components, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose units.

[0323] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to a wide variety of animals. It is well understood that modifications to make a pharmaceutical composition suitable for administration to humans can be made to suit a wide variety of animals, and that such modifications can be designed and performed by a reasonably skilled veterinary pharmacist through routine (if any) experimentation. This is in consideration of the fact that the pharmaceutical compositions of the present invention can be administered to, but is not limited to, humans and other primates and mammals, including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0324] Pharmaceutical compositions that can be used in the methods of this invention can be prepared, packaged, or sold in formulations suitable for ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, sublingual, intravenous, intraventricular, intradermal, intramuscular, or other routes of administration. Other formulations considered include projected nanoparticles, liposome preparations, released erythrocytes containing the active ingredient, and immunogenic formulations.

[0325] The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk as a single unit dose or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose (dosage) of the active ingredient to be applied to the subject or a convenient fraction of such dose, for example, half or one-third of such dose.

[0326] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any other components in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the treated subject and, more particularly, on the route of administration of the composition. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0327] In addition to the active ingredient, the pharmaceutical compositions of the present invention may also contain one or more other pharmaceutical active agents.

[0328] Controlled-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques.

[0329] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by making a physical opening (breaching) into the tissue of the target and administering the pharmaceutical composition through an opening in the tissue. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, administration through a surgical incision, administration through a non-surgical wound that penetrates the tissue. Specifically, parenteral administration is considered to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0330] Formulations of pharmaceutical compositions suitable for parenteral administration comprise an active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives. Parenteral administration formulations include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, ointments, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form to be reconstituted with a suitable mediator (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally.

[0331] Pharmaceutical compositions can be prepared, packaged, or sold as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known techniques and may contain additional components such as dispersants, wetting agents, or suspending agents as described herein, in addition to the active ingredient. Such sterile injectable formulations can be prepared using non-toxic, parenteral-acceptable diluents or solvents, such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other useful parenteral-application formulations include those containing an active ingredient in microcrystalline form, as a liposome preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may contain pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, microsoluble polymers, or microsoluble salts.

[0332] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in formulations suitable for oral administration to the lungs. Such formulations may comprise dry particles containing the active ingredient and having a diameter ranging from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers. These compositions are conveniently available in dry powder form for application using devices including dry powder reservoirs—to which the propellant flow can be directed to disperse the powder—or using self-propelled solvent / powder dispensing containers, such as those containing the active ingredient dissolved or suspended in a low-boiling-point propellant in a sealed container. Preferably, the powder comprises particles, wherein at least 98% by weight of the particles have a diameter greater than 0.5 nanometers, and at least 95% by weight of the particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer, and at least 90% by weight of the particles have a diameter less than 6 nanometers. The dry powder composition preferably comprises a solid fine powder diluent such as sugar and is conveniently provided in unit doses.

[0333] Low-boiling-point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant comprises 50 to 99.9% (w / w) of the composition, and the active ingredient comprises 0.1 to 20% (w / w) of the composition. The propellant may further contain additional components, such as liquid nonionic or solid anionic surfactants or solid diluents (preferably having a particle size on the same order of magnitude as the particles containing the active ingredient).

[0334] Formulations of pharmaceutical compositions suitable for parenteral administration comprise an active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives. Parenteral administration formulations include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, ointments, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form to be reconstituted with a suitable mediator (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally.

[0335] Pharmaceutical compositions can be prepared, packaged, or sold as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known techniques and may contain additional components such as dispersants, wetting agents, or suspending agents as described herein, in addition to the active ingredient. Such sterile injectable formulations can be prepared using non-toxic, parenteral-acceptable diluents or solvents, such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other useful parenteral-application formulations include those containing an active ingredient in microcrystalline form, as a liposome preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may contain pharmaceutically acceptable polymers or hydrophobic materials such as emulsions, ion exchange resins, microsoluble polymers, or microsoluble salts.

[0336] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous mediators and solvents; oily mediators and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulsifiers; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0337] Treatment In one aspect, this disclosure provides a method for delivering at least one selected from nucleic acid molecules and therapeutic agents to target cells. In some embodiments, the method includes administering a therapeutically effective amount of at least one LNP of this disclosure to a subject.

[0338] In some embodiments, the LNP comprises at least one ionizable lipid.

[0339] In some embodiments, the LNP contains at least one accessory lipid.

[0340] In some implementations, LNP contains cholesterol lipids.

[0341] In some embodiments, the LNP comprises polyethylene glycol (PEG) conjugated lipids and / or their modified derivatives.

[0342] In some embodiments, the LNP includes a cell-targeting domain specific for binding to molecules on the surface of target cells. In some embodiments, the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0343] This invention provides a method for delivering a reagent to immune cells of a target subject. In some embodiments, the reagent is a diagnostic reagent for detecting at least one biomarker associated with a disease or disorder. In some embodiments, the reagent is a therapeutic agent for treating or preventing a disease or disorder. Therefore, in some embodiments, this invention provides a method for diagnosing, treating, or preventing a disease or disorder, the method comprising administering an effective amount of a composition comprising one or more diagnostic or therapeutic agents, one or more adjuvants, or a combination thereof.

[0344] In some embodiments, the method provides delivery of a composition for gene editing or genetic manipulation to target immune cells of a subject to treat or prevent a disease or disorder. Exemplary diseases or disorders include, but are not limited to, pathogenic diseases and disorders, and cancer.

[0345] In some embodiments, the method provides a target subject with immunity against an infection or disease or disorder associated with an infectious agent. Therefore, the present invention provides a method for treating or preventing an infection or disease or disorder associated with an infectious agent. For example, depending on the type of antigen in the applied composition, the method can be used to treat or prevent viral, bacterial, fungal, or parasitic infections. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0346] This invention also relates in part to methods for treating associated cancers and diseases or disorders in persons in need of treatment, the methods comprising administering a composition comprising at least one immune cell-targeting LNP, the immune cell-targeting LNP comprising a nucleic acid molecule encoding a CAR that is specific for binding to a tumor antigen, to treat associated cancers or diseases or disorders. Exemplary cancers that can be treated with the compositions and methods of this invention include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendiceal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brainstem gliomas, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, atypical teratoid / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system, lymphomas of the central nervous system, cerebellar astrocytoma, astrocytoma / malignant glioma of the brain, cervical cancer, and childhood vision disorders. Sensory pathway tumors, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelogenous disorders, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumors, extracranial cancer, extragonadal germ cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungooides, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational cancer. Cancer), gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal (renal cell) carcinoma, Langerhans cell cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medullary oral carcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic occult primary squamous neck cancer, oral cancerCancer), multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myeloid leukemia, pleurodesis, myeloma, myeloproliferative disorders, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's 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, ovary, ovary Cancer, ovarian epithelial cancer, ovarian germ cell tumor, low-potency ovarian tumor, pancreatic cancer, papilloma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal cell tumor, pineal blastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor, plasma cell tumor / multiple myeloma, pleural pulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) carcinoma, renal pelvis and ureter cancer, 15 Respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, Seserie 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, squamous neck cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineal blastoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, endometrial sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Woldanstrom macroglobulinemia and nephroblastoma.

[0347] In some embodiments, the composition is applied to a target subject suffering from an infection, disease, or cancer. In other embodiments, the composition is applied to a subject at risk of developing an infection, disease, or cancer. For example, the composition may be applied to a subject at risk of contact with viruses, bacteria, fungi, parasites, etc.

[0348] In some embodiments, the method includes administering an immune cell-targeting LNP composition comprising one or more nucleic acid molecules for treating or preventing a disease or disorder. In some embodiments, the one or more nucleic acid molecules encode a therapeutic agent for treating a disease or disorder. In some embodiments, the one or more nucleic acid molecules encode an agent for targeting T cells to an antigen expressed by a pathogen or cancer cell (e.g., an mRNA molecule encoding a chimeric antigen receptor).

[0349] In some embodiments, the compositions of the present invention may be administered in combination with other therapeutic agents, adjuvants, or combinations thereof. For example, in some embodiments, the method includes administering an LNP composition comprising a nucleic acid molecule encoding one or more agents for targeting immune cells to pathogens or tumor cells of interest and a second LNP comprising a nucleic acid molecule encoding one or more adjuvants. In some embodiments, the method includes administering a single LNP composition comprising a nucleic acid molecule encoding one or more agents for targeting immune cells to pathogens or tumor cells of interest and a nucleic acid molecule encoding one or more adjuvants.

[0350] In some embodiments, the method includes administering to a subject a variety of nucleoside-modified nucleic acid molecules that encode multiple agents, adjuvants, or combinations thereof for targeting immune cells to pathogens or tumor cells of interest.

[0351] In some embodiments, the method of the present invention allows the agents or adjuvants described herein for targeting immune cells to pathogens or tumor cells of interest to be expressed continuously for at least several days after administration. However, in some embodiments, the method also provides transient expression, for example, in some embodiments where the nucleic acid is not integrated into the target genome.

[0352] In some embodiments, the method includes administering nucleoside-modified RNA that provides stable expression of the agent or adjuvant described herein for targeting immune cells to pathogens or tumor cells of interest.

[0353] The administration of the compositions of the present invention in therapeutic methods can be achieved in many different ways using methods known in the art. In some embodiments, the methods of the present invention include systemic administration to the subject, including, for example, enteric or parenteral administration. In some embodiments, the methods include intradermal delivery of the composition. In some embodiments, the methods include intravenous delivery of the composition. In some embodiments, the methods include intramuscular delivery of the composition. In some embodiments, the methods include subcutaneous delivery of the composition. In some embodiments, the methods include inhalation of the composition. In some embodiments, the methods include intranasal delivery of the composition.

[0354] It should be understood that the compositions of the present invention can be applied to the target alone or in combination with other agents.

[0355] Therefore, the treatment and prevention methods of the present invention encompass the practice of the methods using pharmaceutical compositions coded herein for targeting immune cells to pathogens or tumor cells of interest, adjuvants, or combinations thereof. Pharmaceutical compositions used to practice the present invention may be administered at doses from ng / kg / day to 100 mg / kg / day. In some embodiments, the present invention contemplates administration in mammals at doses resulting in concentrations of the compounds of the present invention from 10 nM to 10 μM.

[0356] Generally, the compound can be applied to mammals in the method of the present invention, preferably in a range of 0.01 μg to 50 mg per kilogram of mammalian body weight. The precise dosage will vary depending on any number of factors, including but not limited to the type of mammal and the type of disease state being treated, the age of the mammal, and the route of administration. Preferably, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. More preferably, the dosage will vary from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0357] The composition can be administered to mammals frequently, several times a day, or less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. The frequency of dosage will be apparent to those skilled in the art and will depend on a variety of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0358] In some embodiments, the administration of the immunogenic composition or vaccine of the present invention may be performed by a single administration or by multiple administrations.

[0359] In some embodiments, the invention includes a method comprising administering one or more compositions encoding one or more agents or adjuvants described herein for targeting immune cells to pathogens or tumor cells of interest. In some embodiments, the method has an additive effect, wherein the total effect of administering the combination is approximately equal to the sum of the effects of administering the individual agents or adjuvants for targeting immune cells to pathogens or tumor cells of interest. In other embodiments, the method has a synergistic effect, wherein the total effect of administering the combination is greater than the sum of the effects of administering the individual agents or adjuvants for targeting immune cells to pathogens or tumor cells of interest.

[0360] Example Various embodiments of this application can be better understood by referring to the following examples, which are provided by way of illustration. The scope of this application is not limited to the embodiments given herein.

[0361] Materials and Methods Ionizable lipid and mRNA synthesis Using methods known to those skilled in the art, S n 2. Addition chemistry preparation of C14-4 ionizable lipids. Briefly, a polyamine core (Enamine Inc., Monmouth Junction, NJ) was gently stirred with an excess of an epoxide-terminated C14 alkyl chain (epoxytetradecane, Sigma Aldrich, St. Louis, MO) 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 the formulation of LNPs.

[0362] mRNAs encoding firefly luciferase (luc), green fluorescent protein (GFP), and anti-mouse CD19 (1D3) specific chimeric antigen receptor (CAR) were generated from linearized in vitro transcription (IVT) template plasmids carrying a T7 promoter, 5' and 3' UTR elements, and a 101-nucleotide long poly(A) tail. GenScript (Piscataway, NJ) provided cloning and endotoxin-free plasmid preparation services. mRNAs were synthesized using the MEGAScript T7 kit (Invitrogen AMB13345), with m1Ψ-5'-triphosphate (TriLink N-1081) added as a UTP substitute to the IVT reaction. IVT mRNAs were co-transcribed and capped using the trinucleotide cap1 analog CleanCap (TriLink, San Diego, CA). The mRNAs were purified by cellulose purification according to standard procedures known to those skilled in the art. All mRNAs were analyzed by agarose gel electrophoresis and then stored at -20°C.

[0363] Antibody treatment The antibodies used in this study 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). The anti-human CD5 antibody was cleaved using IdeZ, the anti-mouse CD5 antibody was cleaved using pepsin, and the anti-mouse CD7 antibody was cleaved using figase. The mouse CD3 antibody was provided in F(ab)2 form and was therefore reduced only.

[0364] Use IdeZ protease (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions, adding 1 μL per 15 μg antibody. L IdeZ, reacted at 37°C for 90 minutes. Antibodies cleaved with pepsin (Pierce Fab2 Micropreparation Kit, ThermoFisher) and figase (Pierce Mouse IgG1 Fab and Fab2 Micropreparation Kit, ThermoFisher) were processed using a microfabrication kit and following the manufacturer’s instructions.

[0365] The antibody fragments, including anti-mouse CD3 F(ab)2, were then reduced using dithiothreitol (DTT) by incubation at 25°C for 30 minutes. 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.

[0366] LNP formulations and characteristics LNPs were synthesized according to methods known to those skilled in the art, using a microfluidic device to combine an aqueous phase containing mRNA with ethanol containing lipid and cholesterol components. The aqueous phase consisted of mRNA in 10 mM citrate buffer. The ethanol phase contained ionizable lipid C14-4, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma, St. Louis, MO), lipid-anchored polyethylene glycol (PEG) (Avanti Polar lipid), and lipid-anchored PEG-maleimide (mal-PEG, Avanti Polar Lipids) in a molar ratio of 35 ionizable lipids: 16 DOPE: 46.5 cholesterol: 2.5 total PEG. The aqueous and ethanol phases were then mixed in a microfluidic device at a 3:1 ratio using a 33DS syringe pump (Harvard Apparatus, Holliston, MA). After synthesis, LNP was dialyzed with PBS for 2 hours, then sterilized with a 0.22µm filter, followed by antibody conjugation.

[0367] The LNPs were then analyzed three times using dynamic light scattering (DLS) on a Zetasizer Nano (Malver Instruments, Malvern, UK) to determine their diameter (z-mean) and polydispersity index (PDI). The mRNA concentration of the LNPs was measured using the A260 absorbance on an Infinite M Plex microplate reader (Tecan, Morrisville, NC).

[0368] Ab-LNP fusion Mal-LNP binds to cleaved and reduced antibody fragments at a molar ratio of 1:1 with maleimide, estimating a maximum of four antibody fragments per treatment. After incubation at room temperature for 1 hour, mal-LNP and antibody are incubated overnight at 4°C to complete the reaction. To isolate Ab-LNP from unreacted antibody fragments (including the Fc region), Ab-LNP is passed through a Sephadex G-75 bead column (Millipore Sigma) at approximately 200 μL. The L fractions were collected. Then, any fractions containing mRNA, measured by A260 / A280 readings on an Infinite M Plex microplate reader (Tecan), were combined to form the final Ab-LNP product.

[0369] Cell culture Jurkat cells (ATCC ID: TIB-152) are an immortalized human T cell line cultured in RPMI-1640 medium (ThermoFisher) containing L-glutamine, supplemented with 10% bovine serum and 1% penicillin-streptomycin.

[0370] In vitro luciferase and toxicity test Jurkat cells were seeded at 60,000 cells per 60 µL in 96-well plates, with three replicates per treatment, and then treated with LNP. To measure luciferase mRNA delivery at 24 hours (or other specified time points), cells were centrifuged at 300 g for 7 min and resuspended in 50 µL of 1X lysis buffer (Promega, Madison, WI) and 100 µL of luciferase assay substrate (Promega). Chemiluminescence signals were measured using an Infinite M Plex microplate reader (Tecan) and then normalized to the untreated or control groups as described above. To quantify cytotoxicity at 24 hours, 60 µL of CellTiter-Glo reagent (Promega) was added to each well. After 10 min of incubation, the chemiluminescence corresponding to ATP production was quantified using an Infinite M Plex microplate reader (Tecan), and the signals were normalized to the untreated cells.

[0371] In vivo biodistribution and CAR delivery All treatments were administered via tail vein injection in volumes less than 200 µL.

[0372] For mice receiving luciferase mRNA: 10 minutes prior to euthanasia, mice were intraperitoneally injected with 150 mg / kg of D-luciferin potassium salt (Biotium, Fremont, CA) to detect luminescence. Organs were then imaged using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). Image analysis was performed using LivingImage software (PerkinElmer).

[0373] For mice receiving GFP or CAR mRNA: Blood samples were collected via retroorbital sampling into blood collection tubes, centrifuged (8 min, 750 xg) to remove serum, and then repeatedly added with 1x erythrocyte lysis buffer (Invitrogen). Spleens and lymph nodes were collected and homogenized; for spleen samples, erythrocytes were lysed using erythrocyte lysis buffer. After the above treatment, cells from blood, spleen, and lymph nodes were resuspended in 0.6% PBSA to prepare single-cell suspensions for staining and further analysis.

[0374] Both GFP and CAR mRNA-treated samples were stained by flow cytometry using the following markers: CD3 (T cells), CD19 (B cells), and CD11b (monocytes / macrophages). The fluorescent dyes used 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 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 washing twice in PBSA. Cells were analyzed using a BD LSR II flow cytometer (BD Biosciences, Macquarie Park, NSW, Australia). Standard gating was performed to exclude paired cells, and cell populations were identified based on their marker positivity and the negative staining of other cell populations. For example, T cells were identified as CD3+. + CD19 - CD11b - When observing T cell and B cell exhaustion, the number of these specific cell populations is quantified as the proportion of that cell type in a single cell population.

[0375] Toxicity and cytokine detection To first separate the serum, whole blood was centrifuged at 750 xg for 8 minutes, and the supernatant was collected. The 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) according to the manufacturer's recommendations. IL-6, TNF-α, and GM-CSF were quantified using a colorimetric Qantikine ELISA kit (bio-techne, R&Dsystems, Minneapolis, MN, USA) according to the manufacturer's instructions.

[0376] Example 1: In vitro design and screening of T cell-targeting Ab-LNP libraries Traditional LNP formulations comprise four components: (i) ionizable lipids to provide pH-dependent charge changes, facilitating endosome escape and potent intracellular delivery; (ii) cholesterol for stability and membrane fusion; (iii) phospholipids for structural support and endosome escape; and (iv) lipid-anchored polyethylene glycol (PEG) to prevent aggregation and promote stability. However, to produce the antibody-coated LNPs (Ab-LNPs) of this disclosure for targeted applications, these conventional formulations are modified to include maleimide-functionalized lipid-anchored PEG (mal-PEG) as part of the total PEG content, because the addition of maleimide to the LNP (mal-LNP) surface allows the antibody to bind to the surface via thiol-maleimide interactions.

[0377] Therefore, in order to generate antibody fragments capable of binding to the surface of mal-LNP, the entire antibody is cleaved and reduced ( Figure 1B Specifically, IdeZ, pepsin, and figase were used to separate the Fab and Fc regions of CD3, CD5, and CD7 antibodies. The resulting fragments were then reduced with dithiothreitol (DTT) to reveal free thiol groups on the Fab fragments. These antibody fragments were then bound to the surface of mal-LNPs to form Ab-LNPs, and any unconjugated antibody fragments (e.g., Fc regions and unconjugated Fab regions) were removed using size exclusion chromatography.

[0378] To first determine the optimal amount of mal-PEG to be incorporated, four exemplary mal-LNP formulations encapsulating luciferase-encoding mRNA were produced by keeping the excipient molar ratio constant and only varying the mal-PEG to PEG ratio, allowing different amounts of antibody to conjugate to the mal-LNP surface. Figure 2A-2C Then, mal-LNP was conjugated with anti-human CD5 antibody, and its size was measured using dynamic light scattering (DLS) before and after conjugation. Figures 2A-2E When compared to LNPs without mal-PEG (B10), DLS measurements showed that the size of all Ab-LNPs increased relative to B10, while the diameter of LNPs with mal-PEG did not increase significantly. Therefore, the observed increase in Ab-LNP size is attributed to antibody conjugation.

[0379] Use their size increase as evidence of successful conjugation, then screen Ab-LNPs on CD5. +mRNA delivery and toxicity in the human T cell line Jurkat. In this screening, luciferase-encoded mRNA was used as a model cargo because it requires intracellular delivery and translation to produce a luciferase protein capable of interacting with a luciferin reagent, thus allowing the luminescent signal to serve as a measure of functional mRNA delivery. When this mRNA delivery was quantified as normalized to the standard B10 treatment group, Ab-LNP was able to achieve up to a 15-fold increase in luciferase mRNA delivery compared to B10, without significant toxicity. Figure 2D-2E Regardless of the mal-PEG:PEG ratio, all Ab-LNPs significantly enhanced transfection, indicating that antibody targeting has an effect even at lower antibody densities. However, when observing relative delivery with respect to the mal-PEG:PEG ratio, the 1:3 and 1:5 LNP formulations produced the highest normalized luminescence, while the 1:7 and 1:10 LNP formulations tended to decrease delivery. This reflects the tendency for more antibody per LNP, leading to better cellular uptake, an improvement that plateaued at a 1:5 mPEG:PEG ratio. Therefore, the 1:5 LNP formulation achieved the highest delivery rate (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.

[0380] To further characterize the performance of these Ab-LNPs, dose-response and transfection kinetics were evaluated. Within a certain dose range, the luminescence of Ab-LNPs remained significantly increased compared to both non-targeted B10 and mal-LNP formulations, until high doses were reached, which also led to a significant decrease in cell viability. Figures 3A-3B Increased toxicity of LNP was observed only in the Ab-LNP group; no significant toxicity was observed in the B10 or mal-LNP groups. Improved mRNA delivery was also observed as early as 4 hours at a 50 ng mRNA dose, indicating that Ab-LNP can rapidly enhance delivery. Figure 3C ).

[0381] In summary, these results confirm the improved performance of Ab-LNP compared to the untargeted LNP platform, which warrants further exploration of this Ab-LNP platform.

[0382] Example 2: Biodistribution of certain exemplary Ab-LNPs targeting T cells While enhanced mRNA delivery exhibits minimal cytotoxicity in vitro and has the potential to improve current ex vivo T cell engineering practices, this study sought to explore the potential of these Ab-LNP platforms in in vivo T cell engineering. In this in vivo study, Ab-LNPs were combined with antibodies against mouse CD3 (CD3-LNP), CD5 (CD5-LNP), and CD7 (CD7-LNP) as representative pan-T cell markers. These Ab-LNPs were compared with mal-LNP (mal-B10), B10 LNP, and DLin-MC3-DMA (MC3) LNP at a 1:5 mal-PEG:PEG ratio, with DLin-MC4-DMA being a clinically recognized standard of care approved by the FDA. Each of these LNP groups was formulated to encapsulate luciferase-encoded mRNA and characterized by DLS to determine its size. Figure 4A This indicates that MC3, B10, and mal-B10 are similar in size, as expected, while the size increases in the Ab-LNP group. Each LNP group was then administered intravenously to mice at a dose of 0.6 mg / kg.

[0383] Six hours later, biodistribution was assessed using an in vivo imaging system (IVIS) to capture luminescent signals indicating the delivery of functional mRNA to major organs. Figure 4B These images were then used to quantify the luminescence signal from each organ, revealing differences in biodistribution on the LNP platform. Figure 4C The standard MC3 LNP mRNA delivery primarily resulted in liver delivery, with minimal delivery to the spleen and lymph nodes (LNs). The remaining LNPs contained C14-4 ionizable lipids, primarily leading to spleen delivery, and each treatment group achieved enhanced mRNA delivery compared to the MC3 group. Therefore, these ionizable lipids themselves may be beneficial for LNPs targeting immune cells.

[0384] Furthermore, among these C14-4 LNPs, the B10 and mal-B10 LNP groups resulted in higher liver delivery compared to MC3, while Ab-LNP did not lead to increased liver transfection, which may indicate that the presence of antibodies on the LNP surface helps bypass part of the liver for delivery. The luminescence of all LN groups was significantly lower than that of the liver and spleen, but delivery was significantly increased in the mal-B10, CD3-LNP, and CD7-LNP groups.

[0385] Since the majority of delivery occurred in the liver and spleen across all treatment groups, the normalized luminescent signaling of these organs was summarized by comparing spleen and liver signals. In this comparison, higher values ​​indicated a bias towards spleen delivery relative to liver transfection, which may be beneficial for reaching immune cells. MC3 produced the lowest values ​​in this comparison because it failed to achieve any spleen delivery, and B10 also failed to significantly improve spleen delivery relative to the liver. However, the ratios in the mal-LNP and Ab-LNP groups were significantly higher, suggesting they have greater potential for reaching immune cells. These results indicate that C14-4 LNP enhances in vivo mRNA delivery compared to MC3, and Ab-LNP shows great promise as the only platform that increases spleen delivery without increasing liver delivery.

[0386] To elucidate how this organ-level biodistribution influences specific immune cell delivery, the transfection of LNPs into immune cells in blood, spleen, and lung nuclei (LN) was evaluated. Here, LNPs were formulated using GFP-encoded mRNA for cellular-level analysis using flow cytometry. Six hours after intravenous administration of 0.6 mg / kg mRNA, GFP expression was measured in blood, spleen, and LN cells in B cell, T cell, and macrophage populations. Figures 5A-5D Most of the immune cell transfection in all treatment groups occurred in the blood, and the transfection rates varied among different immune cell types. B10 LNP showed the lowest transfection rate among all immune cell types, and mal-B10 LNP tended to increase the transfection rate in macrophages, but this was not significantly specific.

[0387] Although all three exemplary Ab-LNPs showed the highest transfection rates in T cells, only CD3-LNP achieved a significant transfection rate in T cells compared to B cells and macrophages, averaging 6.5% GFP positivity. No similar T cell specificity was observed in the spleen; most transfection occurred in macrophages. Here, B10 was the only LNP that did not show increased delivery to macrophages, although the transfection rate in all treatment groups did not exceed 1%, indicating minimal overall delivery to any immune cell type. Furthermore, no significant immune cell transfection was observed in LNs, supporting the finding of luciferase biodistribution. Therefore, these results support the use of Ab-LNPs for immune cell delivery, with CD3-LNP representing the most promising platform for transfecting circulating T cells.

[0388] With CD3-LNP achieving potent and specific transfection of circulating T cells within 6 hours, cellular biodistribution was explored at a later time point. Forty-eight hours after intravenous administration of B10 or CD3-LNP, GFP expression in T cells, macrophages, and B cells in blood, spleen, and lymphocytes was assessed. Figure 5E-5H In these organs, mRNA delivery was primarily observed in the blood and spleen in both LNP groups, with minimal transfection of immune cells in the LNs even at longer time points. However, transfection was achieved in different immune cell populations in the LNP groups. In T cells, B10 LNP treatment resulted in low transfection rates in both the blood and spleen, while CD3-LNP achieved significantly potent transfection. In the blood, GFP expression in mice treated with CD3-LNP decreased over time (the expected outcome of transient mRNA expression), T cell motility increased after activation, and the likelihood of T cell exhaustion associated with CD3 interaction increased. In the spleen, GFP expression in T cells increased significantly after 24 hours, with a mean positivity rate of 11%. This is a significant improvement compared to the previously reported 4% delivery rate of CD3-targeting MC3 LNPs, likely due to the spleen bias observed in the biodistribution of C14-4 compared to MC3. In summary, these T cell populations treated with CD3-LNP exhibited the highest GFP positivity rate compared to other immune cell types examined, supporting the T cell specificity of CD3-LNP.

[0389] Observation of macrophage and B cell populations revealed some additional trends. In macrophages, increased GFP expression in the blood was observed in both the B10 and CD3-LNP groups, with B10 showing significantly higher GFP expression than CD3-LNP at 48 hours. However, in the spleen, both LNP groups showed moderate transfection in macrophages, with GFP expression decreasing over time, and no difference between treatment groups. In B cells, both LNP groups showed moderate transfection, but CD3-LNP showed increased delivery in both the blood and spleen compared to B10. At 24 hours, B cell transfection in the spleen was significantly increased, reflecting the increased T cell transfection observed at this time point, although most transfection remained T cell-specific. These results suggest that CD3-LNP maintains T cell specificity over time, with maximum transfection of circulating T cells in the blood occurring at earlier time points, while B10 primarily leads to macrophage transfection, with negligible T cell delivery.

[0390] Example 3: Ab-LNP delivers dose-dependent mRNA to T cells in vivo with minimal toxicity. In summary, the biodistribution of Ab-LNPs in organs and immune cell types encourages further exploration of these targeting platforms to observe the effects of dosage on transfection and toxicity. In these experiments, IgG antibodies conjugated to LNPs, along with B10 LNPs, formed a nonspecific Ab-LNP control group. Blood was collected 24 hours after intravenous injection to assess T cell transfection. Figure 6ANeither the untargeted controls (i.e., B10 and IgG-LNP) produced significant GFP expression, suggesting that even at high doses of mRNA, specific antibody targeting may be required to achieve T cell transfection. Ab-LNP showed increased GFP expression, although CD5-LNP did not achieve significant T cell transfection even at the highest dose of 2.4 mg / kg. However, both CD3-LNP and CD7-LNP induced significant GFP expression at the highest dose, with CD3-LNP demonstrating potent delivery at all doses. This comparison supports CD3-LNP and CD7-LNP as promising platforms for T cell targeting.

[0391] However, when the T cell populations in these treatment groups were quantified, the circulating T cells in mice treated with CD3-LNP were significantly fewer than in untreated mice. Figure 6B Even in the absence of the Fc region, T cell exhaustion following exposure to CD3 antibodies has been widely reported as a transient effect induced by CD3-T cell interactions, although it has not been characterized in many studies of CD3-targeted LNPs. A modest decrease in T cell counts was observed in a single dose of CD5-LNP, suggesting that such exhaustion of circulating T cells could also be caused by other antibodies or T cell interactions, but this was not observed in any CD7-LNP dose or any non-targeted LNP.

[0392] To further investigate the biocompatibility of these LNPs beyond immune cell interactions, toxic indicators of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assessed. ALT and AST levels showed minimal variation across different dose and treatment groups, with only the highest dose of CD3-LNP inducing significant changes. Figure 6C Therefore, no signs of hepatotoxicity were observed in any of the moderate-dose LNP treatments, confirming that Ab-LNPs generally do not produce greater toxicity than untargeted LNPs and allowing for the use of higher doses in subsequent experiments.

[0393] Example 4: Ab-LNP generates functional CAR T cells in vivo Because CD3-LNP and high-dose CD7-LNP achieve potent delivery to circulating T cells, these Ab-LNPs have subsequently been used to deliver unrestricted therapeutic cargo (i.e., mRNA encoding a chimeric antigen receptor (CAR)). The CAR constructs used in these experiments target mouse CD19, meaning that functional CAR delivery leads to the elimination of circulating B cells. Here, CAR mRNA was delivered using B10, CD3-LNP, or CD7-LNP at low (0.5 mg / kg) or high (2.0 mg / kg) doses to evaluate the ability of these LNPs to transfect T cells in a dose-dependent manner, as controlling the concentration of CAR T cells in circulation may be beneficial in mitigating cytokine release and its subsequent side effects.

[0394] Following intravenous administration, CAR expression in circulating T cells was assessed within 60 hours to observe the relative delivery rates and duration of CAR expression across different LNP platforms. Figure 7A CAR expression was measured along with a comparison of mean fluorescence intensity (MFI) of CAR staining in T cell populations. At the earliest time point of 12 hours, both doses of Ab-LNP resulted in significant CAR expression, with CD3-LNP positivity rates of 15% and 17% in circulating T cells at low and high doses, respectively, and CD7-LNP positivity rates of 5% and 6%, respectively. The B10 treatment group achieved a 2% CAR positivity rate at the high dose, which was not a significant transfection rate compared to the background of PBS-treated mice. Similarly, only the CD3-LNP dose and the high dose of CD7-LNP achieved increased MFI values ​​in circulating T cells, indicating more potent CAR expression in these groups. At 36 hours, both CAR positivity rates and MFI values ​​were low, with only the high-dose CD3-LNP maintaining a significant percentage of CAR T cells (7%). By 60 hours, no group maintained significant CAR expression, successfully demonstrating the transient nature of mRNA. However, in MFI measurements, high-dose CD3-LNP showed an increase even after 60 hours, suggesting that a small number of persistent CAR-positive clusters in this treatment group may maintain strong expression.

[0395] In summary, these findings demonstrate the importance of targeted LNP methods for achieving in vivo therapeutic mRNA cargo T cell transfection, as B10 LNPs fail to generate a significant CAR T cell population, while Ab-LNPs exhibit potent transfection.

[0396] Next, to assess whether the observed CAR positivity indicated the production of functional CAR T cells, B cell elimination was measured. Although the B cells observed in this experiment were not cancer cells, the depletion of circulating B cells indicated CAR function, as it was able to target and eliminate CD19. +Cell population. Here, B10 LNPs containing luciferase mRNA (Luc) were also administered to mice at both low and high doses to account for any changes in the circulating B cell population due to the presence of LNPs, and B cell exhaustion was calculated compared to PBS-treated mice. Figure 7B At the earliest time point of 12 hours, both doses of the Ab-LNP group showed a significant reduction in circulating B cells, with only the high-dose B10 leading to B cell exhaustion. At its low and high doses, CD3-LNP achieved a 49% and 56% reduction in circulating B cells, respectively, while CD7-LNP resulted in a 35% and 52% reduction. At the high dose, B10 LNP achieved a 46% reduction in circulating T cells, but recovered at the next time point of 36 hours. In contrast, at 36 hours, Ab-LNP caused more sustained B cell exhaustion, with CD3-LNP reducing the B cell population by 58% and 90% at low and high doses of mRNA, respectively, while CD7-LNP maintained a more moderate reduction of 30% and 47%. By 60 hours, the circulating B cell population had recovered with the low-dose CD7-LNP, while the high-dose maintained only a 32% reduction. However, CD3-LNP continued to show a significant reduction in B cells, with low and high doses reducing them by 45% and 56%, respectively. No B cell exhaustion was observed in the Luc mRNA LNP group at all doses and time points, indicating that these reductions were CAR-mRNA dependent. Taken together, these data suggest that the CAR expression observed in T cells corresponds to functional CAR T cell activity in vivo. Both CD3-LNP and CD7-LNP achieved significant B cell exhaustion within 36 hours, with CD3-LNP showing prolonged exhaustion at 60 hours.

[0397] In addition to confirming the production of functional CAR T cells in vivo, serum was collected to observe the effects of LNP treatment and dosage 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-8CAt 12 hours, CD3-LNP produced the highest levels of all three cytokines, as expected, given its status as the most potent platform for CAR T cell production. By 36 hours, serum IL-6 levels had decreased, with only high-dose CD3-LNP showing an increase compared to PBS. However, serum levels of GM-CSF and TNF-α remained elevated, with significant increases in GM-CSF in the high-dose CD3-LNP, CD7-LNP, and B10 LNP groups, and significant increases in TNF-α in both doses of CD3-LNP and the high-dose B10 LNP group. By 60 hours, all cytokine levels returned to normal, except for the high-dose CD3-LNP group, which maintained elevated serum TNF-α concentrations. Furthermore, Luc mRNA LNP did not lead to any increase in cytokine levels at any time point, indicating that the basal LNP formulation itself is not inflammatory. Throughout the data, higher doses of LNP resulted in elevated serum cytokine levels, supporting the potential of these LNP platforms for CAR T cell production to mitigate cytokine release.

[0398] Here, lower doses of CD3-LNP resulted in lower serum cytokine levels while still producing potent CAR-T cells, while CD7-LNP achieved high levels of B cell depletion without a significant increase in IL-6 or TNF-α. Furthermore, the transient nature of these elevated cytokine levels supports exploring repeated dosing to maintain CAR positivity without reaching extremely high serum cytokine concentrations. In conclusion, these results confirm the ability of these Ab-LNP platforms to achieve potent CAR T cell production in vivo and their potential for modulated cytokine production.

[0399] List of implementation methods The following exemplary implementations are provided, and their numbers should not be interpreted as indicating a level of importance: Embodiment 1 provides a lipid nanoparticle (LNP) composition comprising: (a) An ionizable lipid compound having the structure of formula (I) or a salt thereof: Formula (I) in: A1 and A2 are each independently selected from CH, N, and P; L1 and L6 are each independently selected from CR 19 and N; Each occurrence of L2 and L5 is independently selected from -CH2- and -CHR. 19 -、-O-、-NH- and -NR 19 -; L3 and L4 are each independently selected from -CH2- and -CHR. 19-、-O-、-NH- and -NR 19 -; 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 and R 19 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C3-C) 12 cycloalkyl), optionally substituted C2-C 12 Heterocyclic alkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional replacement of C5-C) 12 Cycloalkenyl), optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloalkynyl, -Y(R) 20 )z` (R 21 ) z`` -(Optional substitution of C6-C) 12 Cyclo-alkynyl), optionally substituted C6-C 10 Aryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C6-C) 10 aryl), optionally substituted C2-C 12 heteroaryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 (heteroaryl), C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid ester, ester, -Y(R) 20 ) z` (R 21 ) z`` -Ester, -Y(R) 20 ) z` (R 21 ) z`` -NO2, -CN and sulfinyl groups, Or selected from 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 R11a and R 11b R 12a and R 12b R 13a and R 13b R 14a and R 14b or R 15a and R 15b The two geminal substituents can combine with the C atoms they are bonded to to form C=O; Each occurrence of Y is independently selected from C, N, O, S, and P; R 20 and R 21 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters, esters, -NO2, -CN, and sulfinyl groups. Or R 20 and R 21 It can combine with the Y atoms they are bonded to 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 The compounds having the structure of formula (I) or their salts comprise approximately 25 mol% to approximately 45 mol% of the LNP. (b) 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), wherein DOPE comprises approximately 10 mol% to approximately 20 mol% of LNP. (c) Cholesterol lipids, of which cholesterol lipids account for approximately 40 mol% to approximately 50 mol% of LNP; (d) Polyethylene glycol (PEG) conjugated lipids and / or their modified derivatives, wherein the PEG conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to about 5.0 mol% of the LNP; and (e) A cell-targeting domain that is specific to binding to molecules on the surface of target cells, wherein the cell-targeting domain is covalently conjugated to at least one component of the LNP.

[0400] Embodiment 2 provides the LNP described in Embodiment 1, wherein the ionizable lipid compound of formula (I) is selected from: Equation (II), Equation (III), Formula (IV), Equation (V), Formula (VI), Equation (VII), in: R1, R2, R3, R4, R5, R6, and R7 are each independently selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, straight-chain C1-C28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; a 1 a 2 a 3 a 4 and a 5 Each can be 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 can be 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 can be independently 0, 1, 2, 3, 4 or 5.

[0401] Embodiment 3 provides the LNP described in Embodiment 1, wherein the ionizable lipid compound of formula (I) is selected from: Equation (VIII), Equation (IX), Equation (X), Formula (XI), Formula (XII), Formula (XIII), Formula (XIV), Equation (XV), and Formula (XVI), in: R1, R2, R3, R4, R5, R6, and R7 are each independently selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; and a 1 a 2 a 3 a 4 and a 5 Each can be 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.

[0402] Embodiment 4 provides an LNP according to any one of Embodiments 1-3, wherein the ionizable lipid of formula (I) comprises 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azanediyl)bis(tetradecane-2-ol): (C14-4).

[0403] Embodiment 5 provides an LNP as described in any one of Embodiments 1-4, wherein the molar ratio of (a):(b):(c):(d) in the LNP is approximately 35:16:46.5:2.5.

[0404] Embodiment 6 provides an LNP as described in any one of Embodiments 1-5, wherein the PEG-conjugated lipid comprises C14-PEG: .

[0405] Embodiment 7 provides an LNP according to any one of Embodiments 1-6, wherein the modified derivative of the PEG-conjugated lipid comprises 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[maleimide (polyethylene glycol)-2000] (ammonium salt) (mPEG): .

[0406] Embodiment 8 provides the LNP described in Embodiment 7, wherein the total PEG-conjugated lipid comprises a mixture of mPEG and PEG in a ratio ranging from about 1:1 to about 1:10 (mPEG:PEG).

[0407] Embodiment 9 provides the LNP described in Embodiment 7 or 8, wherein the total PEG-conjugated lipid comprises a mixture of maleimide PEG (mPEG) and PEG in a ratio selected from 1:3, 1:5, 1:7 and 1:10 (mPEG:PEG).

[0408] Embodiment 10 provides an LNP as described in any one of Embodiments 1-9, wherein the target cells are selected from stem cells, peripheral blood mononuclear cells, and immune cells.

[0409] Embodiment 11 provides an LNP as described in any one of Embodiments 1-10, wherein the proportion of LNP delivered to the spleen is greater than that to the liver.

[0410] Embodiment 12 provides an LNP as described in any one of Embodiments 1-11, wherein the LNP further comprises at least one selected from nucleic acid molecules and therapeutic agents.

[0411] Embodiment 13 provides an LNP as described in any one of Embodiments 1-12, wherein the LNP further comprises at least one reagent selected from mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

[0412] Embodiment 14 provides the LNP described in Embodiment 13, wherein the LNP comprises a nucleic acid molecule.

[0413] Embodiment 15 provides the LNP described in Embodiment 14, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0414] Implementation 16 provides the LNP described in Implementation 14 or 15, wherein the nucleic acid molecule is selected from cDNA, mRNA, miRNA, siRNA, modified RNA, antagonism, antisense molecules and target nucleic acids or any combination thereof.

[0415] Embodiment 17 provides an LNP as described in any one of Embodiments 14-16, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

[0416] Embodiment 18 provides the LNP described in Embodiment 17, wherein the CAR specifically binds to the surface antigen of pathogenic cells or tumor cells.

[0417] Embodiment 19 provides an LNP as described in any one of Embodiments 1-18, wherein the cell-targeting domain that binds to surface molecules specific to target cells is an immune cell-targeting domain that specifically binds to T cells.

[0418] Embodiment 20 provides an LNP as described in any one of Embodiments 7-19, wherein the cell-targeting domain of the target cell-specific binding surface molecule is covalently conjugated with mPEG.

[0419] Embodiment 21 provides the LNP described in any one of Embodiments 1-20, wherein the surface molecules of the target cells are selected from 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, At least one of CD153, CD154, 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.

[0420] Embodiment 22 provides a pharmaceutical composition comprising at least one LNP according to any one of Embodiments 1-21 and a pharmaceutically acceptable carrier.

[0421] Embodiment 23 provides the pharmaceutical composition of Embodiment 22, wherein the composition further comprises an adjuvant.

[0422] Embodiment 24 provides the pharmaceutical composition described in Embodiment 22 or 23, wherein the pharmaceutical composition is a vaccine.

[0423] Embodiment 25 provides a method for delivering at least one selected from nucleic acid molecules and therapeutic agents to target cells, the method comprising administering to the subject a therapeutically effective amount of at least one LNP according to any one of Embodiments 1-21 and / or a pharmaceutical composition according to any one of Embodiments 22-24.

[0424] Implementation 26 provides the method of implementation 25, wherein the therapeutic agent is at least one selected from mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins, and antibodies.

[0425] Embodiment 27 provides the method of Embodiment 25, wherein the nucleic acid molecule is at least one selected from DNA molecules and RNA molecules.

[0426] Implementation 28 provides the method of implementation 25, wherein the nucleic acid molecule is at least one selected from cDNA, mRNA, miRNA, siRNA, antagonism, antisense molecules and target nucleic acids.

[0427] Implementation 29 provides the method of implementation 25, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

[0428] Implementation 30 provides the method of implementation 29, wherein the CAR specifically binds to the surface antigen of pathogenic cells or tumor cells.

[0429] Embodiment 31 provides the method of any one of Embodiments 25-30, wherein the target cells are selected from stem cells, peripheral blood mononuclear cells, and immune cells.

[0430] Implementation 32 provides the method of implementation 30, wherein the CAR includes a cell-targeting domain that specifically binds to T cells.

[0431] Implementation 33 provides the method described in Implementation 32, wherein the cell-targeting domain specifically binds to a subset of 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, C At least one of D153, CD154, 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.

[0432] Embodiment 34 provides the method of any one of Embodiments 25-33, wherein the LNP or a composition thereof further comprises an adjuvant.

[0433] Embodiment 35 provides the method of any one of Embodiments 25-34, wherein the nucleic acid molecule and / or therapeutic agent are at least partially encapsulated within the LNP.

[0434] Embodiment 36 provides a method according to any one of Embodiments 25-35, wherein the method treats, prevents and / or improves at least one selected from viral infection, bacterial infection, fungal infection, parasitic infection, cancer or cancer-related disease or disorder.

[0435] The terminology and expressions used herein are for descriptive purposes only and not for limitation. Their use is not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications may be possible within the scope of embodiments of this application. Therefore, it should be understood that although this application describes specific embodiments and optional features, modifications and variations can be made to the compositions, methods, and concepts disclosed herein by those skilled in the art, and such modifications and variations are considered to be within the scope of embodiments of this application.

Claims

1. A lipid nanoparticle (LNP) composition comprising: (a) An ionizable lipid compound having the structure of formula (I) or a salt thereof: Formula (I) in: A1 and A2 are each independently selected from CH, N, and P; L1 and L6 are each independently selected from CR 19 and N; Each occurrence of L2 and L5 is independently selected from -CH2- and -CHR. 19 -、-O-、-NH- and -NR 19 -; L3 and L4 are each independently selected from -CH2- and -CHR. 19 -、-O-、-NH- and -NR 19 -; 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 and R 19 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C3-C) 12 cycloalkyl), optionally substituted C2-C 12 Heterocyclic alkyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional replacement of C5-C) 12 Cycloalkenyl), optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloalkynyl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C6-C) 12 Cyclo-alkynyl), optionally substituted C6-C 10 Aryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C6-C) 10 aryl), optionally substituted C2-C 12 heteroaryl, -Y(R) 20 ) z` (R 21 ) z`` -(Optional substitution of C2-C) 12 (heteroaryl), C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid ester, ester, -Y(R) 20 ) z` (R 21 ) z`` -Ester, -Y(R) 20 ) z` (R 21 ) z`` -NO2, -CN and sulfinyl groups, Or selected from 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 The two geminal substituents can combine with the C atoms they are bonded to to form C=O; Each occurrence of Y is independently selected from C, N, O, S, and P; R 20 and R 21 Each occurrence of is independently selected from H, halogen, or optionally substituted C1-C. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters, esters, -NO2, -CN, and sulfinyl groups. Or R 20 and R 21 It can combine with the Y atoms they are bonded to 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 Compounds having the structure of formula (I) or their salts comprise about 25 mol% to about 45 mol% of LNP; (b) 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), wherein DOPE comprises approximately 10 mol% to approximately 20 mol% of LNP. (c) Cholesterol lipids, of which cholesterol lipids account for approximately 40 mol% to approximately 50 mol% of LNP; (d) Polyethylene glycol (PEG) conjugated lipids and / or their modified derivatives, wherein the PEG conjugated lipids and / or their modified derivatives comprise about 0.5 mol% to about 5.0 mol% of the LNP; and (e) A cell-targeting domain that is specific to binding to molecules on the surface of target cells, wherein the cell-targeting domain is covalently conjugated to at least one component of the LNP.

2. The LNP according to claim 1, wherein the ionizable lipid compound of formula (I) is selected from: Equation (II), Equation (III), Formula (IV), Equation (V), Formula (VI), Equation (VII), in: R1, R2, R3, R4, R5, R6, and R7 are each independently selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; a 1 a 2 a 3 a 4 and a 5 Each can be 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 can be 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 can be independently 0, 1, 2, 3, 4 or 5.

3. The LNP according to claim 1, wherein the ionizable lipid compound of formula (I) is selected from: Equation (VIII), Equation (IX), Equation (X), Formula (XI), Formula (XII), Formula (XIII), Formula (XIV), Formula (XV), Formula (XVI), in: R1, R2, R3, R4, R5, R6, and R7 are each independently selected from H, halogens, or optionally substituted C1-C atoms. 28 Alkyl, optionally substituted C3-C 12 cycloalkyl, optionally substituted C2-C 12 Heterocyclic alkyl groups, optionally substituted C2-C 28 alkenyl, optionally substituted C5-C 12 Cycloalkenyl, optionally substituted C2-C 28 Alkyne group, optionally substituted C6-C 12 Cycloynyl, optionally substituted C6-C 10 Aryl, optionally substituted C2-C 12 heteroaryl, C1-C 28 Alkoxycarbonyl, straight-chain C1-C 28 Alkoxycarbonyl, branched C1-C 28 Alkoxycarbonyl, C(=O)NH2, NH2, C1-C 28 aminoalkyl, C2-C 28 amino-alkenyl, C2-C 28 amino-alkynyl, C6-C 10 Aminoaryl, aminoacetic acid ester, acyl, OH, C1-C 28 Hydroxyalkyl, C2-C 28 Hydroxyolefin, C2-C 28 Hydroxyalkynyl group, C6-C 10 Hydroxyaryl, C1-C 28 Alkoxy, carboxyl, carboxylic acid esters and esters; and a 1 a 2 a 3 a 4 and a 5 Each can be 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.

4. The LNP according to any one of claims 1-3, wherein the ionizable lipid of formula (I) comprises 1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1-yl)ethoxy)ethyl)azanediyl)bis(tetradecane-2-ol): (C14-4)。 5. The LNP according to any one of claims 1-4, wherein the molar ratio of (a):(b):(c):(d) in the LNP is about 35:16:46.5:2.

5.

6. The LNP according to any one of claims 1-5, wherein the PEG-conjugated lipid comprises C14-PEG: 。 7. The LNP according to any one of claims 1-6, wherein the modified derivative of the PEG-conjugated lipid comprises 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[maleimide (polyethylene glycol)-2000] (ammonium salt) (mPEG): 。 8. The LNP of claim 7, wherein the total PEG-conjugated lipid comprises a mixture of mPEG and PEG in a ratio ranging from about 1:1 to about 1:10 (mPEG:PEG).

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

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

11. The LNP according to any one of claims 1-10, wherein the proportion of LNP delivered to the spleen is greater than that to the liver.

12. The LNP according to any one of claims 1-11, wherein the LNP further comprises at least one selected from nucleic acid molecules and therapeutic agents.

13. The LNP according to any one of claims 1-12, wherein the LNP further comprises at least one reagent selected from mRNA, siRNA, microRNA, CRISPR-Cas9, small molecules, proteins and antibodies.

14. The LNP of claim 13, wherein the LNP comprises 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 cDNA, mRNA, miRNA, siRNA, modified RNA, anta-coma, antisense molecules and targeting nucleic acids or any combination thereof.

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

18. The LNP of claim 17, wherein the CAR specifically binds to the surface antigen of pathogenic cells or tumor cells.

19. The LNP according to any one of claims 1-18, wherein the cell-targeting domain that binds to surface molecules specific to target cells is an immune cell-targeting domain that specifically binds to T cells.

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

21. The LNP according to any one of claims 1-20, wherein the target cell surface molecules are selected from 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, CD16, CD17, CD18, CD19, CD103, CD119, CD126, CD150 ... At least one of the following: CD153, CD154, 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.

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

23. The pharmaceutical composition of claim 22, wherein the composition further comprises an adjuvant.

24. The pharmaceutical composition according to claim 22 or 23, wherein the pharmaceutical composition is a vaccine.

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

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

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

28. The method of claim 25, wherein the nucleic acid molecule is selected from at least one of cDNA, mRNA, miRNA, siRNA, anta-coma, antisense molecules, and target nucleic acids.

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

30. The method of claim 29, wherein the CAR specifically binds to the surface antigen of pathogenic cells or tumor cells.

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

32. The method of claim 30, wherein the CAR comprises a cell-targeting domain that specifically binds to T cells.

33. The method of claim 32, wherein the cell-targeting domain specifically binds to a subset of 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, CD1 53, at least one of CD154, 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.

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

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

36. The method according to any one of claims 25-35, wherein the method treats, prevents and / or improves at least one selected from viral infection, bacterial infection, fungal infection, parasitic infection, cancer or cancer-related disease or disorder.

Images