Compositions and methods for inhibiting the expression of the signal regulatory protein alpha (SIRPα) gene

Abstract

In various embodiments, provided herein are methods and compositions comprising signal-regulatory protein alpha (SIRPα) therapeutics. In certain embodiments, the present disclosure provides methods and compositions for modulating SIRPα expression. In certain embodiments, the methods and compositions are useful for treating SIRPα-mediated diseases or conditions.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 369,926, filed July 29, 2022. The entire teachings of the above application(s) are incorporated herein by reference.

[0002] Incorporating XML material by reference This application incorporates by reference the sequence listing contained in the following extensible Markup Language (XML) file being submitted concurrently with this application: a) File name: 00502374001.xml, Creation date: June 27, 2023, Size: 338,132 bytes. [Background technology]

[0003] Signal regulatory protein alpha (SIRPA or SIRPα), signal regulatory protein beta (SIRPB or SIRPβ), and signal regulatory protein gamma (catus or SIRPγ) are all members of the signal regulatory protein (SIRP) family of receptors, which are involved in immune regulation. Members of the SIRP family have highly conserved extracellular domains but distinct transmembrane regions with opposing (i.e., inhibitory versus activating) signal transduction capabilities.

[0004] The interaction of SIRPα with CD47 is an important immune checkpoint in myeloid cells. SIRPα interaction with CD47 provides a downregulatory signal that inhibits phagocytosis of host cells. SIRPβ, on the other hand, triggers cell activation signals through its association with the transmembrane adaptor protein DNA-X activation protein (DAP12). Thus, the SIRP family is classified into a group of proteins called paired receptors.

[0005] Both SIRPα and SIRPβ are expressed in cells of the myeloid lineage, whereas SIRPγ is expressed on T cells, natural killer (NK) cells, and natural killer T (NKT) cells. SIRPβ has no known natural ligand. Both SIRPα and SIRPγ bind to CD47, but SIRPγ binds to CD47 with 10-fold weaker affinity than SIRPα. Binding of CD47 to SIRPγ has been shown to mediate adhesion of T cells to antigen-presenting cells (APCs) and endothelial cells, leading to T cell activation, proliferation, and transendothelial migration. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, compositions and methods for modulating SIRPα / CD47 interactions without altering the function of SIRPβ or SIRPγ are needed to treat many SIRPα-associated diseases (e.g., cancer immunotherapy). [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a method for reducing the expression of signal-regulating protein alpha (SIRPα) in a cell, comprising contacting the cell with a lipid nanoparticle composition comprising a nucleic acid that reduces the expression of signal-regulating protein alpha (SIRPα). In some embodiments, the nucleic acid is double-stranded ribonucleic acid (dsRNA), antisense oligomer (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), circular RNA, peptide-nucleic acid (PNA), locked nucleic acid (LNA), or a combination thereof.

[0008] In yet another aspect, the disclosure provides a composition comprising a nucleic acid that reduces expression of signal-regulatory protein alpha (SIRPα), wherein the nucleic acid comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 18-227.

[0009] In another aspect, the present disclosure provides a composition comprising a nanocarrier selected from the group consisting of a lipid, a polymer, and a lipid-polymer hybrid; and a nucleic acid that reduces expression of signal-regulatory protein alpha (SIRPα), wherein the concentration of SIRPα in cells contacted with the composition is reduced compared to the concentration of SIRPα in otherwise identical cells.

[0010] In some embodiments, the disclosure provides methods of treating cancer, hi some embodiments, the methods are for treating a SIRPα-mediated disease or condition.

[0011] Additionally, the present disclosure provides methods of making and using the methods and compositions disclosed herein.

[0012] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings, in which like reference characters designate like parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows a sequence alignment between SEQ ID NO: 1 (NCBI Ref. No. NP_001035111.1), SEQ ID NO: 2 (NCBI Ref. No. NP_006056.2), and SEQ ID NO: 3 (NCBI Ref. No. NP_061026.2). SIRP family proteins share a highly conserved N-terminal extracellular domain. The N-terminal signal sequence is boxed, and the transmembrane region is underlined.

[0014] [Figure 2]Figure 2 shows a sequence alignment between SEQ ID NO: 4 (NCBI Ref. No. NM_001040022.1), SEQ ID NO: 5 (NCBI Ref. No. NM_006065.5), and SEQ ID NO: 6 (NCBI Ref. No. NM_018556.4). SIRPα siRNA target sequences are underlined, and variants with a minor allele frequency (MAF) > 0.05 are boxed.

[0015] [Figure 3] FIG. 3 shows macrophage-specific CD45 gene silencing in mouse peritoneal immune cells.

[0016] [Figure 4] FIG. 4 shows dose- and time-dependent SIRPα gene silencing in mouse peritoneal macrophages in vivo.

[0017] [Figure 5] Figure 5A shows that siSIRPα-LNP reduced SIRPα expression levels on human primary macrophages, and Figure 5B shows the quantification of Figure 5A.

[0018] [Figure 6] Figure 6A shows a shift in macrophage phenotype to M1. Figure 6B shows a shift in macrophage phenotype away from the M2 phenotype after siSIRPα-LNP silencing of SIRPα expression in primary human macrophages. Figure 6C shows that SIRPα silencing increases the expression of the class I antigen-presenting molecule HLA-A2 in primary human macrophages.

[0019] [Figure 7]Figure 7A shows that siRNA sequences can silence SIRPα expression on primary human macrophages in vitro using one-way ANOVA and Tukey's multiple comparison test. Figure 7B shows that various lipids can form siSIRPα-LNPs and silence SIRPα expression on primary human macrophages in vitro using two-way ANOVA and Sidak's multiple comparison test.

[0020] [Figure 8] Figure 8A shows that the viability of primary human macrophages is maintained with various siRNA sequences and lipids, and Figure 8B shows that the viability of primary human macrophages is maintained with FL-A, MC3, and lipid 5 lipids.

[0021] [Figure 9] Figure 9A shows that multiple siRNAs can promote antigen presentation (HLA-DR expression). Figure 9B shows that multiple siRNAs can promote M1 polarization (CD86 expression). sihSIRPα18 is more potent than other siRNAs. RiM = RNAiMAX, a commercially available transfection reagent.

[0022] [Figure 10] Figure 10A shows that multiple lipids can form siSIRPα-LNPs that promote the expression of antigen-presenting proteins (HLA-DR), and Figure 10B shows that multiple lipids can form siSIRPα-LNPs that promote the expression of M1 phenotype proteins (CD86).

[0023] [Figure 11] Figure 11A shows a schematic diagram of the experimental setup. Figure 11B shows quantification of SIRPα silencing within macrophage populations as assessed by flow cytometry. Because the anti-SIRPα used for blocking (as an experimental treatment) inhibited binding of the SIRPα staining antibody used in flow cytometry, SIRPα expression levels could not be determined in the anti-SIRPα condition (marked NA in the bar graph).

[0024] [Figure 12] Figure 12A shows a shift in macrophage phenotype to an M1 phenotype (CD86) following siSIRPα-LNP silencing of SIRPα expression in primary human macrophages, but not following anti-SIRPα treatment. Data collected from cocultures of primary human macrophages with SKOV-3 ovarian cancer cells pretreated with either an anti-HER2 antibody or an IgG isotype control antibody. SKOV-3 pretreatment is indicated on the x-axis, and macrophage pretreatment is indicated in the legend of each bar graph. Figure 12B shows CD86 expression levels (median fluorescence intensity, MFI) within the CD45+ macrophage population as measured by flow cytometry. siSIRPα-LNP + aHER2 synergistically promotes ovarian cancer phagocytosis by primary human macrophages. Blocking SIRPα by pretreating macrophages with anti-SIRPα does not improve phagocytosis compared to pretreatment with an isotype control antibody (denoted IgG cont.) or siSIRPα. Figure 12C shows CFSE fluorescence intensity within the CD45+ macrophage population, as quantified by flow cytometry. SIRPα silencing with siSIRPα, but not blocking with anti-SIRPα, increases expression of the class I antigen-presenting molecule HLA-A2 in primary human macrophages. Data collected from cocultures of primary human macrophages with SKOV-3 ovarian cancer cells pretreated with either anti-HER2 antibody or an IgG isotype control antibody.

[0025] [Figure 13] A schematic diagram of the experimental setup is shown.

[0026] [Figure 14]Figures 14A-14B show that blocking SIRPα with anti-SIRPα promotes mutual phagocytosis (macrophages engulfing other macrophages) compared to silencing SIRPα with siSIRPα. Figure 14A shows flow cytometry data quantifying the percentage of macrophages labeled with a violet cell tracker compared to the initial seeding density (40%). Violet macrophages treated with siRNA-LNPs are not engulfed by other (green, i.e., untreated) macrophages {negatively, there is little depletion of the violet macrophage population compared to the initial seeding density}. Violet macrophages treated with anti-SIRPα or its isotype (IgG) control are engulfed by other macrophages (green, i.e., untreated) {negatively, there is substantial depletion of violet macrophages in co-cultures pretreated with anti-SIRPα or IgG control}. Figure 14B shows that macrophages (purple) treated with antibodies (anti-SIRPα or its IgG control) are more actively phagocytosed by other macrophages (green) than are macrophages treated with siRNA-LNPs (i.e., mutual phagocytosis).

[0027] [Figure 15] Figure 15A shows that SKOV-3 human ovarian cancer expresses CD47. Figure 15B shows quantification of Figure 15A. Figure 15C shows HER2 expression in SKOV-3 human ovarian cancer. Figure 15D shows quantification of Figure 15C.

[0028] [Figure 16] FIG. 16 shows a schematic diagram of the cancer cell phagocytosis assay.

[0029] [Figure 17] Figure 17A shows that siSIRPα-LNP reduced SIRPα expression levels on human primary macrophages. Figure 17B shows the quantification of Figure 17A.

[0030] [Figure 18]Figure 18A shows that siSIRPα-LNP+aHER2 synergistically promotes ovarian cancer phagocytosis by primary human macrophages. Figure 18B quantifies the percentage of macrophages positive for CFSE cancer cell tracker (indicating phagocytosis).

[0031] [Figure 19] Figures 19A-19C show a shift in macrophage phenotype toward an M1 and away from an M2 phenotype following siSIRPα-LNP silencing of SIRPα expression in primary human macrophages. Data collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells pretreated with either anti-HER2 antibody or an IgG isotype control antibody. Figure 19A shows CD206. Figure 19B shows CD163. Figure 19C shows CD86.

[0032] [Figure 20] Figure 20 shows that SIRPα silencing increases the expression of the class II antigen-presenting molecule HLA-DR in primary human macrophages. Data collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells pretreated with either an anti-HER2 antibody or an IgG isotype control antibody.

[0033] [Figure 21] Figure 21A shows a schematic diagram of a macrophage cross-presentation experiment. Figure 21B shows flow cytometry data demonstrating SIRPα gene silencing within macrophage populations. Figure 21C shows flow cytometry data demonstrating M1 phenotype marker expression.

[0034] [Figure 22] Figure 22A shows that SIRPα gene silencing increases macrophage phagocytosis of B16 melanoma cells. Figure 22B shows cross-presentation of a model B16 melanoma antigen (OVA) on MHC class I molecules.

[0035] [Figure 23] Figures 23A-23C show that SIRPα gene silencing increases cross-presentation of cancer antigens by macrophages. Figure 23A shows the percent cross-presenting macrophages. Figure 23B shows the calculated number of macrophages that were positive for both CSFE and MHC1-OVA (double positives), and Figure 23C shows the data from Figure 23B divided by the number of macrophages that were positive for CSFE, multiplied by 100.

[0036] [Figure 24] Figures 24A-24B show that various siRNA sequence-LNP formulation combinations can silence SIRPα expression on THP-1-derived macrophages in vitro. Figure 24A shows 50 nM siRNA. Figure 24B shows 5 nM siRNA. DETAILED DESCRIPTION OF THE INVENTION

[0037] A description of an exemplary embodiment follows.

[0038] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present disclosure. However, one of ordinary skill in the relevant art will readily recognize that the present disclosure can be practiced without one or more of the specific details, or can be practiced using other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the illustrated order of acts or events, as some acts may occur in a different order and / or simultaneously with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to implement a methodology in accordance with the present disclosure. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those of ordinary skill in the art using conventional methodology.

[0039] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Furthermore, it is understood that terms, e.g., those defined in commonly used dictionaries, should be construed as having a meaning consistent with the meaning in the context of the relevant art and / or as otherwise defined herein.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0041] As used herein, the indefinite articles "a," "an," and "the" are to be understood to include plural references unless the context clearly dictates otherwise.

[0042] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise," as well as variations such as "comprises" and "comprising," are understood to mean, for example, the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including."

[0043] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments of the present disclosure, any of the terms "comprising," "containing," "including," and "having" can, in some embodiments, be replaced with the terms "consisting of" or "essentially consisting of" to change the scope of the disclosure.

[0044] As used herein, the conjunction "and / or" between multiple recited elements is understood to encompass both individual and combined alternatives. For example, when two elements are joined by "and / or," the first alternative refers to the applicability of the first element without the second element. The second alternative refers to the applicability of the second element without the first element. The third alternative refers to the applicability of the first element and the second element together. Any one of these alternatives is understood to fulfill the meaning, and therefore the requirement, of the term "and / or" as used herein. The simultaneous applicability of more than one alternative is also understood to be within the meaning, and therefore the requirement, of the term "and / or."

[0045] When lists are presented, unless otherwise stated, each individual element of the list, and every combination of the list, should be understood to be a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0046] Receptor basal activity Some cell surface receptors have constitutive (i.e., intrinsic or basal) activity that activates intracellular signaling in the absence of a ligand. This basal activity is defined as the probability of a receptor existing in an active state in the absence of a ligand. Although antibody therapeutics have been shown to be useful for sterically blocking receptor-ligand interactions, they are not believed to be able to inhibit the basal activity of a receptor.

[0047] Nucleic acid therapeutics, such as siRNA therapeutics, are a promising approach because they can reduce the number of receptors on the cell surface and constitutive signaling.

[0048] Signal Regulatory Protein Alpha (SIRPα) Signal regulatory protein alpha or SIRPα (also called CD172a or SHPS-1) is a transmembrane protein expressed in myeloid cells.

[0049] The SIRPα-CD47 immune checkpoint has been targeted by numerous blocking agents (e.g., anti-CD47 antibodies, anti-SIRPα antibodies, and SIRPα fusion proteins), some of which are being evaluated in clinical trials (e.g., Hu5F9-G4, TTI-621, and ALX148).

[0050] However, while these treatments work well in combination with antibodies that sterically block CD47-SIRPα and opsonize target cells, neither CD47-SIRPα blockade nor target opsonization is sufficient to efficiently transport target cells into macrophages. Furthermore, the use of multiple blocking agents has been shown to result in heterotrimeric interactions, described as the "scorpion effect" (Kurlander, RJ "Blockade of Fc receptor-mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens," The Journal of Immunology 131.1 (1983): 140-147) (incorporated herein by reference in its entirety). Furthermore, anti-SIRPα antibodies cause macrophage opsonization, leading to mutual phagocytosis, in which macrophages are engulfed by other macrophages.

[0051] Furthermore, the gene encoding human SIRPα is reported to be highly polymorphic, as is the extracellular domain of SIRPα. Therefore, effective therapeutic targeting of SIRPα across diverse patient populations requires pan-allelic SIRPα inhibition while maintaining the specificity of SIRPα for other SIRP family molecules.

[0052] Therefore, there is a need to develop therapeutic agents that can be designed to cover polymorphic variability while avoiding regions of high allele frequency.

[0053] One such approach is the use of nucleic acid therapeutics, such as siRNA, directed against SIRPα. Although siRNA directed against SIRPα has been shown to promote macrophage polarization from an anti-inflammatory M2 to a pro-inflammatory M1 phenotype, currently available siRNA directed against SIRPα has not been optimized for specificity and potency, and to date, siRNA directed against SIRPα has not been formulated as lipid nanoparticles for in vivo or clinical use.

[0054] Therefore, there is a strong demand for nucleic acid therapeutic compositions and methods designed to target the transmembrane region, cytoplasmic region, or even the untranslated region of mRNA. Furthermore, by combining nucleic acid therapeutics with an appropriate delivery vehicle, such as lipid nanoparticles, cell type-specific delivery of nucleic acid therapeutics can be achieved, thereby reducing off-target effects.

[0055] Compositions of the present disclosure In some embodiments, the present disclosure provides a composition that is a pharmaceutically acceptable composition.

[0056] In one aspect, the disclosure provides a composition comprising a nucleic acid that reduces expression of signal-regulatory protein alpha (SIRPα), wherein the nucleic acid comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 18-227. In some embodiments, the polynucleotide is at least 80% identical to at least one of SEQ ID NOs: 140-227.

[0057] As used herein, the term "sequence identity" refers to the degree to which two sequences have the same residues at the same positions when aligned to achieve the maximum level of identity, expressed as a percentage. In aligning and comparing sequences, typically, one sequence is designated as a reference sequence, to which a test sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as the percentage of positions over the entire length of the reference sequence where the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the maximum level of identity. As an example, two sequences are considered to have 70% sequence identity if, when aligned to achieve the maximum level of identity, the test sequence has the same nucleotide residues at 70% of the same positions over the entire length of the reference sequence.

[0058] Alignment of sequences for comparison to achieve the maximum level of identity can be readily performed by one of skill in the art using an appropriate alignment method or algorithm, in some cases the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). In some embodiments, codon-optimized sequences for efficient expression in different cells, tissues, and / or organisms reflect the codon usage patterns in such cells, tissues, and / or organisms, including conservative (or non-conservative) amino acid substitutions that do not adversely affect normal activity.

[0059] Thus, in some embodiments, the polynucleotide is at least about 70% identical to at least one of SEQ ID NOs: 18-227, e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NOs: 18-227. In certain embodiments, the polynucleotide is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 18-227. In some embodiments, the polynucleotide is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NOs: 18-227. In certain embodiments, the polynucleotide has about 70 to 100%, e.g., about 75 to 100%, 75 to 99%, 80 to 100%, 80 to 98%, 85 to 100%, 85 to 97%, 90 to 100%, 90 to 96%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% sequence identity to at least one of SEQ ID NOs: 18 to 227.

[0060] In still further embodiments, the polynucleotide is at least about 70% identical to at least one of SEQ ID NOs: 140-227, e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NOs: 140-227. In certain embodiments, the polynucleotide is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 140-227. In some embodiments, the polynucleotide is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NOs: 140-227. In certain embodiments, the polynucleotide has about 70 to 100%, e.g., about 75 to 100%, 75 to 99%, 80 to 100%, 80 to 98%, 85 to 100%, 85 to 97%, 90 to 100%, 90 to 96%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% sequence identity to at least one of SEQ ID NOs: 140 to 227.

[0061] In some embodiments, in the compositions of the disclosure, contacting a cell with a nucleic acid reduces the concentration of SIRPα compared to the concentration of SIRPα in an otherwise identical cell.

[0062] As used herein, the terms "reduce" or "reducing" refer to modulation that lowers SIRPα levels (e.g., levels prior to or in the absence of modulation by an agent). In some embodiments, an agent (e.g., a composition) reduces the level of SIRPα by at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In certain embodiments, the agent (e.g., composition) reduces SIRPα levels by at least about 5%, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In certain embodiments, the agent (e.g., composition) reduces SIRPα levels by at least about 5%, e.g., by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%.

[0063] In some embodiments, SIRPα concentrations are measured using a cell-based functional assay.

[0064] The present disclosure also provides a composition comprising a nanocarrier selected from the group consisting of a lipid, a polymer, and a lipid-polymer hybrid and a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic, wherein the concentration of SIRPα in a cell contacted with the composition is reduced compared to the concentration of SIRPα in an otherwise identical cell. In some embodiments, the SIRPα therapeutic is a double-stranded ribonucleic acid (dsRNA), an antisense oligomer (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a circular RNA, a peptide-nucleic acid (PNA), a locked nucleic acid (LNA), or a combination thereof.

[0065] In certain embodiments of the present disclosure, the SIRPα therapeutic in the composition comprises a polynucleotide having at least about 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, wherein the contiguous nucleotide sequence is at least about 15 nucleotides in length. In yet further embodiments, the SIRPα therapeutic in the composition comprises a polynucleotide having at least about 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, wherein the contiguous nucleotide sequence is about 15-30 nucleotides in length. In yet further embodiments, the SIRPα therapeutic in the composition comprises a polynucleotide having at least about 80% identity to at least one of SEQ ID NOs:18-139.

[0066] In some embodiments, the composition further comprises a 2'-deoxythymidine-3'-phosphate 3' overhang or a 2'-deoxythymidine-5'-phosphate-phosphorothioate 3' overhang. In still further embodiments, the polynucleotide sequence further comprises at least one modified pyrimidine. The modified pyrimidine can be 2'-O-methylcytidine-3'-phosphate or 2'-O-methyluridine-3'-phosphate. In some embodiments, the SIRPα therapeutic agent is an siRNA duplex. In some embodiments, the siRNA duplex comprises a polynucleotide sequence hybridized to a complementary sequence.

[0067] In some embodiments, the concentration of the SIRPα therapeutic agent is at least about 25 nM, 2.5 nM, 250 pM, or 25 pM. In some embodiments, the composition is formulated as a pharmaceutical composition. In yet further embodiments, the pharmaceutical composition is an advanced therapy medicinal product. Examples of advanced therapy medicinal products include, but are not limited to, somatic cell therapy medicinal products, tissue engineering products, gene therapy medicinal products, tumor vaccines, or combinations thereof.

[0068] Methods of the present disclosure In some embodiments, the present disclosure provides methods of treatment and methods of enhancing the efficacy of treatment comprising administration of the compositions described herein.

[0069] As used herein, "therapy," "treat," "treating," or "treatment" means inhibiting or alleviating a condition in a subject in need thereof. For example, therapy or treatment refers to either (i) preventing symptoms associated with a disease or disorder, (ii) postponing the onset of symptoms associated with a disease or disorder, and / or (iii) reducing the severity of such symptoms that occur or are expected to occur with the disease or disorder. The term includes ameliorating or managing existing symptoms, preventing further symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, the term indicates that a beneficial result is produced in at least some subjects (e.g., humans) treated. Many therapies or treatments are effective in some, but not all, of the subjects who receive the treatment or treatment.

[0070] As used herein, the term "effective amount" refers to an amount of a composition that, when administered alone or in combination to a cell, tissue, or subject, is effective to achieve the desired therapy or treatment under the conditions of administration. For example, an effective amount is an amount sufficient to generate an immune response that results in the effectiveness of the therapy or treatment. The effectiveness of the therapy or treatment (e.g., induction of a humoral immune response and / or a cellular immune response) can be determined by a suitable method known in the art.

[0071] In some embodiments, the present disclosure provides uses of compositions or medicaments for treating signal-regulatory protein alpha (SIRPα)-mediated diseases or conditions. In some embodiments, the present disclosure provides methods for reducing signal-regulatory protein alpha (SIRPα) expression in a cell, the method comprising contacting the cell with a lipid nanoparticle composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic.

[0072] As used herein, a "subject" or "patient" includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), and livestock (e.g., birds, pigs, cattle (dairy cows, bulls, steers, or heifers), sheep, goats, horses, etc.), as well as non-domestic animals. In some embodiments, the subject is a mammal (e.g., a non-human mammal). In some embodiments, the subject is a human. In still further embodiments, the subject of the present disclosure may be a cell, cell culture, tissue, organ, or organ system. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an infant, child, adolescent, or adult.

[0073] As used herein, "administering" or "administration" refers to taking steps to deliver a composition to a subject. Administration can occur, for example, once, multiple times, and / or over one or more extended periods of time. Administration includes both direct administration, including self-administration, and indirect administration, which involves prescribing or instructing a subject to ingest a composition. For example, as used herein, a person who instructs a subject (e.g., a patient) to self-administer a composition (e.g., a drug) or to have another administer the composition, and / or a person (e.g., a physician) who provides a prescription for the composition to the subject, is administering the composition to the subject.

[0074] In some aspects, the present disclosure provides a method of treating a signal-regulatory protein alpha (SIRPα)-mediated disease or condition, comprising administering to a subject in need thereof a lipid nanoparticle (LNP) composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic.

[0075] In yet a further aspect, the present disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a lipid nanoparticle (LNP) composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic. In some embodiments of the present disclosure, the method relates to cancer treatment. Cancer treatments provided by the present disclosure include carcinoma, sarcoma, melanoma, lymphoma, and / or leukemia. In some embodiments, the method of the present disclosure can be replaced, advanced, or followed by another treatment regimen. In some embodiments, the other treatment regimen can include, but is not limited to, chemotherapy, radiation therapy, surgery, hormone therapy, biological response modifier therapy, immunotherapy, and / or bone marrow transplantation.

[0076] In some embodiments, the methods of the present disclosure provide for the treatment of SIRPα-mediated diseases or conditions. Examples of SIRPα-mediated diseases and / or conditions include, but are not limited to, acute myeloid leukemia, adenosquamous lung carcinoma, atypical meningioma, B-cell acute lymphoblastic leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, bladder transitional cell carcinoma, brain glioblastoma, breast cancer, breast ductal adenocarcinoma, Burkitt's lymphoma, cecal adenocarcinoma, cervical squamous cell carcinoma, chronic lymphocytic leukemia, clear cell renal carcinoma, colon adenocarcinoma, cutaneous melanoma, endometrioid adenocarcinoma, esophageal adenocarcinoma, esophageal squamous cell carcinoma, gastric adenocarcinoma, gastric cancer, glioma, head and neck squamous cell carcinoma, These include hepatobiliary and pancreatic neoplasms, hepatoblastoma, hepatocellular carcinoma, HER2-positive breast cancer, renal neoplasms, large cell lung cancer, lobular breast carcinoma, pancreatic adenocarcinoma, lung cancer, lymphoid neoplasms, melanoma, multiple myeloma, nasopharyngeal squamous cell carcinoma, non-small cell lung carcinoma, oral squamous cell carcinoma, ovarian endometrial adenocarcinoma with squamous differentiation, ovarian serous adenocarcinoma, pancreatic apical cell carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumor, renal papillary carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, skin cancer, small cell lung carcinoma, squamous cell lung carcinoma, thyroid cancer, thyroid neoplasm, and uterine carcinosarcoma.

[0077] In some embodiments, the present disclosure provides a composition that is a pharmaceutically acceptable composition.

[0078] As used herein, the term "pharmaceutically acceptable" refers to a species that is, within the scope of sound medical judgment, suitable for use without undue toxicity, irritation, allergic response, etc., and for which a reasonable benefit / risk ratio is warranted. For example, a substance is pharmaceutically acceptable if it can be contacted with cells, tissues, or organs of animals or humans without undue toxicity, irritation, allergic response, immunogenicity, or other untoward reaction, and if it is suitable for use in dosage forms in accordance with a dosing schedule and in amounts to be used, for which a reasonable benefit / risk ratio is warranted.

[0079] The desired dose can be conveniently administered as a single dose, e.g., the agent is administered once daily, or as multiple doses administered at appropriate intervals, e.g., the agent is administered 2, 3, 4, 5, 6, or more times daily. The daily dose can be divided into several, e.g., 2, 3, 4, 5, 6, or more, administrations, particularly when relatively large amounts are administered, or where deemed appropriate. Typically, the composition is administered about 1 to about 6 times (e.g., 1, 2, 3, 4, 5, or 6 times) per day, or alternatively, as an infusion (e.g., continuous infusion). In some embodiments, the interval can be daily, weekly, biweekly, monthly, quarterly, every 2 or 3 months, yearly, or twice yearly, or any combination thereof.

[0080] It is well within the ability of one of ordinary skill in the art to determine the dosage and route of administration for a particular drug, patient, and disease or condition. Preferably, the dosage causes no or minimal adverse side effects.

[0081] A lower or higher dose than the above dose may be required. The specific dosage and treatment regimen for any particular subject depends on various factors, such as the activity of the specific drug employed, age, body weight, general health, sex, diet, administration time, excretion rate, drug combination, severity and course of the disease, condition or symptom, the subject's predisposition to the disease, condition or symptom, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of drug in the composition also depends on the specific drug in the composition.

[0082] In some embodiments, the concentration of one or more active agents provided in the composition is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09% , 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% w / w, w / v or v / v and / or greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.01% w / w, w / v or v / v.

[0083] In some embodiments, the concentration of one or more active agents provided in the composition is in the range of about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.05% to about 25%, about 0.1% to about 20%, about 0.15% to about 15%, or about 1% to about 10% w / w, w / v, or v / v. In some embodiments, the concentration of one or more active agents provided in the composition is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.05% to about 2.5%, or about 0.1% to about 1% w / w, w / v, or v / v.

[0084] In certain embodiments, the compositions can be administered parenterally or non-parenterally, including by any method, such as aerosol inhalation, injection, infusion, ingestion, transfusion, implantation, or transplantation. For example, the compositions described herein can be administered to a patient intraarterially, intradermally, subcutaneously, intratumorally, intramedullary, intranodal, intramuscularly, intravenously (i.v.), intranasally, intrathoracically, or intraperitoneally. In one aspect, the compositions of the present disclosure are administered intravenously. In one aspect, the compositions of the present disclosure are administered to a subject by intramuscular or subcutaneous injection. The compositions can be injected, for example, directly into a tumor, lymph node, tissue, organ, or site of infection.

[0085] In some embodiments, the compositions described herein are used in combination with other known drugs and therapeutic agents. As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject during the course of the subject's treatment, for example, two or more treatments are delivered after the subject is diagnosed with a disease and before the disease is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the different treatments (e.g., additional therapeutic agents) can be administered simultaneously or sequentially.

[0086] In some embodiments, the disclosure provides a method of making a lipid nanoparticle formulation to obtain a signal-regulating protein alpha (SIRPα) therapeutic, wherein the SIRPα therapeutic comprises a polynucleotide having at least about 80% identity to the consecutive sequence SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17; diluting the polynucleotide with a citrate buffer to form an aqueous phase; solubilizing a mixture of an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) to form an ethanol phase; mixing the aqueous phase and the ethanol phase to form a precipitate; and separating the precipitate to obtain the lipid nanoparticle formulation.

[0087] In some embodiments, the present disclosure provides compositions and methods for altering homeostatic control mechanisms to modulate phagocytic activity.

[0088] In some embodiments, the present disclosure provides compositions and methods for macrophage activation. In some embodiments, macrophage activation is characterized by: i) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin 1-beta (IL-1b), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); ii) decreased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, TGFβ, and / or IL-10; iii) decreased expression and / or secretion of at least one site selected from the group consisting of IL-1b, TNF-α, IL-12, IL-18, GM-CSF, CCL3, CCL4, and IL-23. iv) an increase in the ratio of IL-1b, IL-6, and / or TNF-α expression to IL-10 expression; v) an increase in CD8+ cytotoxic T cell activation; vi) an increase in the recruitment of CD8+ cytotoxic T cell activation; vii) an increase in CD4+ helper T cell activity; viii) an increase in the recruitment of CD4+ helper T cell activity; ix) an increase in NK cell activity; x) an increase in NK cell recruitment; xi) an increase in neutrophil activity; xii) an increase in macrophage and / or dendritic cell activity; and / or xiii) an increase in the inflammatory phenotype of myeloid cells as assessed by a spindle-shaped morphology, flattened appearance, and / or an increase in the number of dendrites as assessed microscopically.

[0089] Lipid nanoparticles (LNPs) In some embodiments of the present disclosure, the composition is a lipid nanoparticle composition. The lipid nanoparticle composition comprises an ionizable lipid, a phospholipid, a sterol, a polymer-conjugated lipid, or any combination thereof. In embodiments of the present disclosure, the amount of ionizable lipid relative to the total mass of the composition is about 20 mol% to about 80 mol%, about 35 mol% to about 70 mol%, or about 40 mol% to about 65 mol%. The amount of cholesterol relative to the total mass of the composition is about 10 mol% to about 60 mol%, about 20 mol% to about 55 mol%, or about 25 mol% to about 50 mol%.

[0090] In some embodiments of the present disclosure, the amount of phospholipid relative to the total weight of the composition is about 3 mol% to about 55 mol%, for example, about 0.25 mol% to about 12 mol%, about 0.5 mol% to about 6 mol%, or about 1 mol% to about 3 mol%.

[0091] Sterols are disclosed in U.S. Patent Application Publication No. 2023 / 0210993A1. Examples of sterols include cholesterol, phytosterols (such as sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether.

[0092] Polymer-conjugated lipids are disclosed in International Publication No. WO2023 / 114943A2. The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. Examples of polymer-conjugated lipids include PEGylated lipids. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0093] Ionizable lipids In some embodiments of the present disclosure, the ionizable lipid is a lipid having at least one biodegradable group. The ionizable lipid may be a lipid having at least one ionizable amino group and at least one biodegradable group. Examples of such biodegradable groups include groups represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

[0094] In some embodiments, the lipid composition includes that of U.S. Patent Application Publication No. 2022 / 0096381, the contents of which are incorporated herein by reference. For example, a lipid represented by formula (1) or a salt thereof can be used as the ionizable lipid. [ka]

[0095] In the formula, X is -NR 1 - or -O-, R 1 is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 - represents a group represented by R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or the following formula: [ka] R represents a group represented by 22 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, and R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L 2 -R 32 - represents a group represented by R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O- or the following formula [ka] R represents a group represented by 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, and R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R11 and R 12 each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted; R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and R 8 The groups in any one or more pairs of the groups may be linked together to form a 4- to 7-membered ring which may contain an O atom, and the substituents on the alkyl group having 1 to 18 carbon atoms which may be substituted include a hydroxyl group, a carboxyl group, -NR 45 R 46 an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or —O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 -OR 44 is a group represented by R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 or -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 -OR 44 is a group represented by R 41 , R 42 , R 43 , R 44, R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; a, b, c, and d each independently represent an integer of 0 to 3, a+b is 1 or more, and c+d is 1 or more.

[0096] In some embodiments, R1 represents a hydrocarbon group having 6 to 24 carbon atoms. R2 and R3 represent a hydrocarbon group having 3 to 24 carbon atoms, and the hydrocarbon group is an alkyl group, an alkenyl group, an alkynyl group, an alkyl group, or an alkenyl group. R 1 and a hydrocarbon group having 6 to 24 carbon atoms represented by the formula: 2 and R 3 The hydrocarbon group having 3 to 24 carbon atoms represented by the formula (I) is an alkyl group, an alkenyl group, an alkynyl group, an alkyl group, or an alkenyl group. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched, or may be linear or cyclic.

[0097] The alkyl group having 6 to 24 carbon atoms may be an alkyl group having 6 to 20 carbon atoms, and the alkyl group having 3 to 24 carbon atoms may be an alkyl group having 6 to 20 carbon atoms. Specific examples include a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (e.g., a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (e.g., a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a nonadecyl group, and an icosyl group.

[0098] The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be linear or branched, or chain or cyclic. The alkenyl group having 6 to 24 carbon atoms may be an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms may be an alkenyl group having 6 to 20 carbon atoms. Specific examples include a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group (e.g., a (Z)-hexadeca-9-enyl group), a hexadecadienyl group, a heptadecenyl group (e.g., a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (e.g., a (Z)-heptadeca-8-enyl group), and a hexadecadienyl group (e.g., a (Z)-hexadeca-9-enyl group). Examples include an (8Z,11Z)-heptadeca-8,11-dienyl group, an octadecenyl group (for example, a (Z)-octadec-9-enyl group), an octadecadienyl group (for example, a (9Z,12Z)-octadeca-9,12-dienyl group), a nonadecenyl group, an icosenyl group (for example, a (Z)-ico-11-enyl group), and an icosadienyl group (for example, a (11Z,14Z)-icosa-11,14-dienyl group).

[0099] An alkynyl group having 6 to 24 carbon atoms may be an alkynyl group having 6 to 20 carbon atoms, and an alkynyl group having 3 to 24 carbon atoms may be an alkynyl group having 6 to 20 carbon atoms. Specific examples include hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl groups. All of the above alkenyl groups may have one double bond or two double bonds. All of the above alkynyl groups may have one triple bond or two triple bonds.

[0100] R 21 and R 31The hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) may be an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms may be linear or branched, or may be linear or cyclic. The alkyl group having 10 to 24 carbon atoms may be an alkyl group having 12 to 24 carbon atoms. Specific examples thereof include a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (e.g., a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (e.g., a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, and a 1-hexylnonyl group. Examples of alkenyl groups include 2-hexyloctyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylnonyl, 2-heptylundecyl, 3-heptyldecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, or may be linear or cyclic.Specific examples thereof include a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (for example, a (Z)-tridec-8-enyl group), a tetradecenyl group (for example, a tetradec-9-enyl group), a pentadecenyl group (for example, a (Z)-pentadecen-8-enyl group), a hexadecenyl group (for example, a (Z)-hexadec-9-enyl group), a heptadecenyl group (for example, a (Z)-hexadec-9-enyl group), a hexadecenyl group (for example, a (Z)-hexadec-9-enyl group), a hexadecenyl group (for example, a (Z)-hexadec-9-enyl group), a hexadecenyl group (for example, a (Z)-hexadec-9-enyl group), a hexadecenyl group (for example, a (Z)-hexadec-8-enyl group), a hex ... Examples of the alkynyl group include an oxadecadienyl group, a heptadecenyl group (e.g., a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (e.g., a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (e.g., a (Z)-octadeca-9-enyl group), and an octadecadienyl group (e.g., a (9Z,12Z)-octadeca-9,12-dienyl group). The alkynyl group having 10 to 24 carbon atoms may be linear or branched, or may be chain or cyclic. Specific examples include a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. All of the above alkenyl groups may have one double bond or two double bonds. All of the above alkynyl groups can have one triple bond or two triple bonds.

[0101] R 22 and R 32The divalent hydrocarbon linking group having 1 to 18 carbon atoms represented by the formula (I) may be an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms may be linear or branched, or chain or cyclic. The number of carbon atoms in the alkylene group may be 1 to 12, 1 to 10, or 2 to 10. Specific examples include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, an undecamethylene group, and a dodecamethylene group. The alkenylene group having 2 to 18 carbon atoms may be linear or branched, or chain or cyclic. The number of carbon atoms in the alkenylene group may be 1 to 12 or 2 to 10.

[0102] -O(CO)O-, -O(CO)- and (CO)O- are L 1 and -O(CO)- and -(CO)O- are in the range of L 1 -O(CO)O-, -O(CO)- and -(CO)O- can be in the range of L 2 and -O(CO)- and -(CO)O- are in the range of L 2 may be substituted, R 4 , R 6 , R 9 , R 10 , R 11 and R 12 The alkyl group having 1 to 18 carbon atoms represented by the formula (I) may be linear or branched, or chain or cyclic. The number of carbon atoms in the alkyl group may be 1 to 12. Specific examples include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl groups. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, or -O(CO)OR. 41 , -O(CO)-R42 , -(CO)OR 43 -OR 44 a group represented by -O(CO)-R 42 Or -(CO)OR 43 A group represented by the formula:

[0103] may be substituted, R 5 , R 7 , and R 8 The alkyl group having 1 to 18 carbon atoms represented by the formula (I) may be linear or branched, or chain or cyclic. The number of carbon atoms in the alkyl group may be 1 to 12, or 1 to 8. Specific examples include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl groups. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, or -O(CO)OR. 41 , -O(CO)-R 42 , -(CO)OR 43 -OR 44 or a group represented by -O(CO)-R 42 , -(CO)OR 43 -OR 44 A group represented by the formula:

[0104] Examples of 4- to 7-membered rings that may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. Examples of 4- to 7-membered rings include a 6-membered ring, a piperidine ring, and a morpholine ring.

[0105] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12When the optionally substituted alkyl group having 1 to 18 carbon atoms has a substituted or unsubstituted aryl group as a substituent, the number of carbon atoms in the aryl group may be 6 to 22, 6 to 18, or 6 to 10. Specific examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group. Substituents on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 or -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 -OR 44 Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group.

[0106] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 When the optionally substituted alkyl group having 1 to 18 carbon atoms has a substituted or unsubstituted heteroaryl group as a substituent, the number of carbon atoms in the heteroaryl group is 1 to 12 or 1 to 6. Specific examples of the heteroaryl group include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, and an oxazolyl group. Substituents on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 or -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 OR 44Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, and a pyridonyl group.

[0107] R 41 , R 42 , R 43 , R 44 , R 45 and R 46Examples of hydrocarbon groups having 1 to 18 carbon atoms represented by the formula (I) include alkyl groups having 1 to 18 carbon atoms, alkenyl groups having 2 to 18 carbon atoms, or alkynyl groups having 2 to 18 carbon atoms, as well as alkyl groups having 1 to 18 carbon atoms or alkenyl groups having 2 to 18 carbon atoms. The alkyl groups having 1 to 18 carbon atoms may be linear or branched, or may be linear or cyclic. The number of carbon atoms in the alkyl groups is 3 to 18 or 5 to 18. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (e.g., 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl groups. Alkenyl groups having 2 to 18 carbon atoms may be linear or branched, or may be linear or cyclic. The number of carbon atoms in an alkenyl group is 3 to 18 or 5 to 18. Specific examples thereof include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (for example, a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (for example, a (Z)-trideca-8-enyl group), a tetradecenyl group (for example, a tetradec-9-enyl group), a pentadecenyl group (for example, a (Z)-pentadeca-8-enyl group), Examples of alkynyl groups include alkynyl groups (e.g., (Z)-hexadecenyl groups), hexadecadienyl groups, heptadecenyl groups (e.g., (Z)-heptadeca-8-enyl groups), heptadecadienyl groups (e.g., (8Z,11Z)-heptadeca-8,11-dienyl groups), octadecenyl groups (e.g., (Z)-octadeca-9-enyl groups), and octadecadienyl groups (e.g., (9Z,12Z)-octadeca-9,12-dienyl groups). Alkynyl groups having 2 to 18 carbon atoms may be linear or branched, or open-chain or cyclic.The number of carbon atoms in the alkynyl group is 3 to 18 or 5 to 18. Specific examples thereof include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group.

[0108] X is -NR 1 -, R 1 is a hydrocarbon group having 6 to 24 carbon atoms or R 21 -L 1 -R 22 In this case, R 2 and R 3 one of which represents a hydrogen atom and the other represents a hydrocarbon group having 6 to 24 carbon atoms or R 31 -L 2 -R 32 - can represent a group represented by the formula:

[0109] When X represents -O-, R 2 and R 3 are each independently a hydrocarbon group having 6 to 24 carbon atoms or R 31 -L 2 -R 32 R can represent a group represented by the formula -. 4 , R 6 , R 9 , R 10 , R 11 , and R 12 can each represent a hydrogen atom. 5 represents a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, -O(CO)-R 42 Or -(CO)OR 43 R may be an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 18 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group. 5 is an alkyl group, R 5 is R 4 , R 6 , R10 and R 12 can be linked to form a ring which may contain an O atom. In particular, R 5 is an alkyl group having 1 to 18 carbon atoms, -O(CO)-R 42 Or -(CO)OR 43 an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 12 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, and an alkyl group having 1 to 18 carbon atoms or -O(CO)-R 42 OR-(CO)OR 43 It may be an alkyl group having 1 to 18 carbon atoms which may be substituted with

[0110] R 7 and R 8 are each independently a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, or —O(CO)—R 42 Or -(CO)OR 43 Alternatively, R may represent an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group. 7 and R 8 can be linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0111] R 5 is R 7 or R 8 It is not connected to R 7 or R 8 does not form a ring.

[0112] a+b is 1 or 2, or 1. c+d is 1 or 2, or 1.

[0113] The compound represented by formula (1) is a compound represented by formula (21). [ka]

[0114] In the formula, R 2 and R 3 each independently represents a hydrocarbon group containing one or more unsaturated bonds and having 3 to 24 carbon atoms, or R 2 and R 3 are each independently R 31 -L 2 -R 32 -, or R 2 and R 3 One of them is R 31 -L 2 -R 32 -, and the other represents a hydrocarbon group having 3 to 24 carbon atoms; R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or the following formula [ka] R represents a group represented by 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, and R 5 is -O(CO)-R 42 OR-(CO)OR 43 represents an alkyl group having 1 to 18 carbon atoms, which may be substituted with 42 and R 43 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms.

[0115] In equation (21), R 2 and R 3 One of them is R 31 -L 2 -R 32 and the other is a hydrocarbon group having 3 to 24 carbon atoms. In formula (21), L2 can represent -O(CO)- or -(CO)O-.

[0116] In some embodiments, an ionizable lipid suitable for use in the present disclosure is an ionizable lipid of formula (2) or a pharmaceutically acceptable salt thereof: [ka]

[0117] During the ceremony, R 51 and R 52 are each independently R 35 is a hydrocarbon group having 1 to 21 carbon atoms optionally substituted with R 35 is a hydroxyl group, -G20-CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 and G 20 is —(CO)O— or —O(CO)—, R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms; R 58 and R 59 are each independently hydrogen or optionally R 36 is a cyclic hydrocarbon group having 3 to 6 carbon atoms substituted with R 60 is a hydrocarbon group having 1 to 18 carbon atoms, R 36 is -N(R 61 )(R 62 ), or -G 20 -R 65 and R 61 and R 62 are each independently hydrogen or a cyclic hydrocarbon group having 3 to 6 carbon atoms, R 65 is a hydrocarbon group having 1 to 18 carbon atoms, -L 40 -CH(R 66 )(R 67 ), or -G 20-R 66 and L 40 is a divalent hydrocarbon group containing 1 to 6 carbon atoms, R 66 and R 67 are each independently a hydrocarbon group containing 1 to 10 carbon atoms or an alkoxy group containing 1 to 10 carbon atoms, L 10 is a divalent hydrocarbon group containing 1 to 18 carbon atoms, G 30 is -S(CO)N(R 64 )- and R 64 -L 30 -G 20 -CH(R 55 )(R 56 ) and a' is 0 or 1; G 10 is G 20 , -O(CO)O-, or -N(R 63 )C(O)—, c' is 0 or 1, R 63 is a hydrocarbon group containing 1 to 18 carbon atoms, L 20 is a divalent hydrocarbon group containing 1 to 6 carbon atoms, b' is 0 or 1, R 53 , R 54 , and R 57 are each independently hydrogen or R 36 is a hydrocarbon group containing 1 to 18 carbon atoms optionally substituted with L 30 is a single bond or a hydrocarbon group containing 1 to 18 carbon atoms.

[0118] The compound represented by formula (2) is represented by formula (2a) [ka] It can be expressed as:

[0119] During the ceremony, R 51 and R 52 are each independently R 35 is a hydrocarbon group having 1 to 21 carbon atoms optionally substituted with R 35 is a hydroxyl group or -G 20 -CH(R 55 )(R 56 ) and G 20 is —(CO)O— or —O(CO)—, R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms; L 10 is a divalent hydrocarbon group containing 1 to 18 carbon atoms, G 10 is —(CO)O— or —O(CO)—, R 53 , R 54 , and R 57 are each independently hydrogen or a hydrocarbon group containing 1 to 18 carbon atoms.

[0120] The compound represented by formula (2) is represented by formula (2b) [ka] It can be expressed as:

[0121] During the ceremony, R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms, L 10 is a divalent hydrocarbon group containing 1 to 18 carbon atoms, G 10 is -O(CO)O-, L 20 is a divalent hydrocarbon group containing 1 to 6 carbon atoms, R 53 , R 54 , and R 57 are each independently hydrogen or R36 is a hydrocarbon group containing 1 to 18 carbon atoms optionally substituted with R 36 is -O(CO)-R 65 and R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) and L 40 is a divalent hydrocarbon group containing 1 to 6 carbon atoms, R 66 and R 67 are each independently an alkoxy group containing 1 to 10 carbon atoms.

[0122] The compound represented by formula (2) is represented by formula (2c)

[0123] [ka] It can be expressed as:

[0124] During the ceremony, R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms, L 10 is a divalent hydrocarbon group containing 1 to 18 carbon atoms, G 10 is -N(R 63 )C(O)—, R 63 is a hydrocarbon group containing 1 to 18 carbon atoms, R 53 , R 54 , and R 57 are each independently hydrogen or R 36 is a hydrocarbon group containing 1 to 18 carbon atoms optionally substituted with R 36 is -(CO)OR 65 and R 65 -L 40-CH(R 66 )(R 67 ) and L 40 is a divalent hydrocarbon group containing 1 to 6 carbon atoms, R 66 and R 67 are each independently a hydrocarbon group containing 1 to 10 carbon atoms.

[0125] The compound represented by formula (2) is represented by formula (2d) [ka] It can be expressed as:

[0126] During the ceremony, R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms, L 10 is a divalent hydrocarbon group containing 1 to 18 carbon atoms, G 30 is -S(CO)NR 64 - and R 64 -L 30 -G 20 -CH(R 55 )(R 56 ) and L 30 is a single bond or a hydrocarbon group containing 1 to 18 carbon atoms, G 20 is —(CO)O—, R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms; G 10 is —(CO)O—, R 53 , R 54 and R 57 are each independently hydrogen or a hydrocarbon group containing 1 to 18 carbon atoms.

[0127] R 51 or R52The hydrocarbon group having 1 to 21 carbon atoms is preferably an alkyl group having 1 to 21 carbon atoms, an alkenyl group having 2 to 21 carbon atoms, or an alkynyl group having 2 to 21 carbon atoms, and more preferably an alkyl group having 1 to 21 carbon atoms or an alkenyl group having 2 to 21 carbon atoms. The alkyl group having 1 to 21 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21. Examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. Alkenyl groups having 2 to 18 carbon atoms may be linear or branched, open-chain or cyclic. The number of carbon atoms is preferably 3 to 21, more preferably 5 to 18. Examples include an allyl group, a prenyl group, a pentanyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-trideca-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadeca-8-enyl group), ), hexadecenyl group (preferably (Z)-hexadecan-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably (Z)-heptadecan-8-enyl group), heptadecadienyl group (preferably (8Z,11Z)-heptadecan-8,11-dienyl group), octadecenyl group (preferably (Z)-octadecan-9-enyl group), and octadecadienyl group (preferably (9Z,12Z)-octadecan-9,12-dienyl group). The alkynyl group having 2 to 21 carbon atoms may be linear or branched, and may be linear or cyclic.The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21. Examples include a propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, nonynyl group, decynyl group, undecynyl group, dodecynyl group, tetradecynyl group, pentadecynyl group, hexadecynyl group, heptadecynyl group, and octadecynyl group. Examples of hydrocarbon groups having 1 to 18 carbon atoms include those having 1 to 18 carbon atoms specifically listed among hydrocarbon groups having 1 to 21 carbon atoms.

[0128] The cyclic hydrocarbon group is preferably a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, a cycloalkynyl group having 3 to 10 carbon atoms, or an aryl group having 6 to 10 carbon atoms.

[0129] The hydrocarbon group having 1 to 6 carbon atoms is preferably an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms can be linear or branched, and can be chain-like or cyclic. Specific examples thereof include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, and hexyl. The alkenyl group having 2 to 6 carbon atoms can be linear or branched, and can be chain-like or cyclic. Specific examples thereof include allyl, prenyl, pentenyl, and hexenyl. The alkynyl group having 2 to 6 carbon atoms can be linear or branched, and can be chain-like or cyclic. Specific examples thereof include propargyl, butynyl, pentynyl, and hexynyl.

[0130] R 66 and R 67The hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and is preferably an alkyl group having 1 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group. The alkenyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), and decenyl. The alkynyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl.

[0131] The compounds represented by formula (1), (2), (2a), (2c) or (2d) can form salts.

[0132] Examples of salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0133] Examples of salts of acidic groups include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, ammonium salts, and nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N′-dibenzylethylenediamine.

[0134] Among the above salts, for example, pharmacologically acceptable salts can be used. In some embodiments, the lipid represented by formula (1) and its production method are described in International Publication No. WO2019 / 235635A1 and International Publication No. WO2021 / 095876A1 (these are incorporated herein by reference in their entirety).

[0135] In some embodiments of the present disclosure, the lipid or salt represented by formula (1) is the lipid, FL-A:

[0136] [ka]

[0137] In some embodiments of the present disclosure, the ionizable lipid may be the following lipid:

[0138] MC3, ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate) International Publication No. WO2010 / 054405A1:

[0139] [ka]

[0140] L-319, (bis[(Z)-non-2-enyl]9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) International Publication Nos. WO2011 / 153493A2, WO2013 / 086354A1, and WO2013 / 086322A1:

[0141] [ka]

[0142] ALC-0315, (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) International Publication No. WO2017 / 075331A1:

[0143] [ka]

[0144] SM-102, (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) International Publication No. WO2017 / 099823A1:

[0145] [ka]

[0146] Lipid 5, (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) International Publication No. WO2017 / 099823A1:

[0147] [ka]

[0148] Lipid 29, (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727:

[0149] [ka]

[0150] ATX-100, (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) International Publication No. WO2019 / 191780A1:

[0151] [ka]

[0152] Lipid A9, (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) International Publication No. WO2017 / 004143A1:

[0153] [ka]

[0154] Lp01, ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl](9Z,12Z)-octadeca-9,12-dienoate) International Publication No. WO2015 / 09534A2, International Publication No. WO2020 / 219876A1:

[0155] [ka]

[0156] TCL053, ([2-[4-(dimethylamino)butanoyloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl](Z)-tetradec-9-enoate) International Publication No. WO2020 / 032184A1:

[0157] [ka]

[0158] cKK-E12:

[0159] [ka]

[0160] C12-200:

[0161] [ka]

[0162] 306Oi10:

[0163] [ka]

[0164] 93-O17S:

[0165] [ka]

[0166] YSK05:

[0167] [ka]

[0168] Exemplary ionizable lipids of formula (II) include: MC3, ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoic acid) International Publication No. WO2010 / 054405A1:

[0169] [ka]

[0170] L-319, (bis[(Z)-non-2-enyl]9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) International Publication Nos. WO2011 / 153493A2, WO2013 / 086354A1, and WO2013 / 086322A1:

[0171] [ka]

[0172] ALC-0315, (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) International Publication No. WO2017 / 075331A1:

[0173] [ka]

[0174] SM-102, (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) International Publication No. WO2017 / 099823A1:

[0175] [ka]

[0176] Lipid 5, (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) International Publication No. WO2017 / 099823A1:

[0177] [ka]

[0178] Lipid 29, (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727:

[0179] [ka]

[0180] ATX-100, (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) International Publication No. WO2019 / 191780A1:

[0181] [ka]

[0182] Lipid A9, (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) International Publication No. WO2017 / 004143A1:

[0183] [ka]

[0184] Lp01, ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl](9Z,12Z)-octadeca-9,12-dienoate) International Publication No. WO2015 / 09534A2, International Publication No. WO2020 / 219876A1:

[0185] [ka]

[0186] TCL053, ([2-[4-(dimethylamino)butanoyloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl](Z)-tetradec-9-enoate) International Publication No. WO2020 / 032184A1:

[0187] [ka]

[0188] TCL065, ([2-[5-(dimethylamino)pentanoyloxymethyl]-3-(3-pentyloctanoyloxy)-2-(3-pentyloctanoyloxymethyl)propyl]-3-pentyloctanoate) International Publication No. WO2020 / 032184A1:

[0189] [ka]

[0190] Lipid 9, ([(6Z,16Z)-12-[6-(dimethylamino)hexanoyloxy]docosa-6,16-dien-11-yl)(Z)-undec-5-enoate) International Publication No. WO2021 / 188389A2:

[0191] [ka]

[0192] Lipid 19, ([(6Z,16Z)-12-[6-(dimethylamino)hexanoyloxy]docosa-6,16-dien-11-yl)(9Z,12Z)-octadeca-9,12-dienoate) International Publication No. WO2021 / 188389A2:

[0193] [ka]

[0194] GCL1, ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate) International Publication No. WO2020 / 219941A1:

[0195] [ka]

[0196] CL4H6, ([7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl](Z)-octadec-9-enoate):

[0197] [ka]

[0198] Example method mRNA isolation mRNA was isolated using the Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen #61012) according to the manufacturer's instructions. Briefly, cells were harvested and lysed in 100 μl of lysis / binding buffer (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% lithium dodecyl sulfate (LiDS), 5 mM dithiothreitol (DTT)). 20 μl of magnetic bead suspension was added to a PCR plate, and the lysed cells were added to the beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 100 μl of wash buffer A (10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mM EDTA, 0.1% LiDS) and then twice with 100 μl of wash buffer B (10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mM EDTA). Next, 20 μl of elution buffer (10 mM Tris-HCl, pH 7.5) was added, and the mRNA was eluted at 80°C. The beads were captured on a magnetic stand, and 20 μl of the supernatant was transferred to another 96-well plate.

[0199] cDNA synthesis cDNA was synthesized using the ABI High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814) according to the manufacturer's instructions. Briefly, 10 μl of master mix containing 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of 10× random primers, 1 μl of reverse transcriptase, and 4.2 μl of water per reaction was added to 10 μl of mRNA isolated using the protocol described above. The plate was sealed, mixed, and incubated in a thermal cycler for 10 minutes at room temperature, followed by 2 hours at 37°C and 5 minutes at 85°C.

[0200] Real-time PCR Two microliters of cDNA was added to a master mix containing 0.5 μl of ACTB TaqMan probe (Applied Biosystems #Hs99999903_m1) or 0.5 μl of SIRPA TaqMan probe (Applied Biosystems #Hs00388955_m1) and 5 μl of TaqMan Fast Advanced Master Mix (Applied Biosystems #4444556) per well in a 384-well plate. Real-time PCR was performed in a Light Cycler 480 (Roche). Each duplex was tested in two to three independent transfections, and each transfection was assayed in duplicate unless otherwise noted.

[0201] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with the same concentration of siRNA for luciferase or mock-transfected cells. IC50 values were calculated using Graphpad Prism software.

[0202] Lipid Chemical Structure In some embodiments, the lipid chemical is 2-butyloctyl 3-ethyl-12-hexyl-6-(2-(octanoyloxy)ethyl)-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate, as described in FL-A or International Publication No. WO 2019 / 235635, which is incorporated by reference in its entirety:

[0203] [ka]

[0204] In some embodiments, the lipid chemical is MC3 or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, as described in International Publication No. WO 2010 / 054405, which is incorporated by reference in its entirety:

[0205] [ka]

[0206] In some embodiments, the lipid chemical is lipid 5 or heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, as described in International Publication No. WO 2017 / 099823, which is incorporated by reference in its entirety:

[0207] [ka]

[0208] In some embodiments, the lipid chemical is distearoylphosphatidylcholine (DSPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine:

[0209] [ka]

[0210] In some embodiments, the lipid chemical is cholesterol:

[0211] [ka]

[0212] In some embodiments, the lipid chemical is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000):

[0213] [ka]

[0214] Lipid nanoparticle (LNP) formulations siRNA was diluted with 10 mM citrate buffer (pH 3.0) (aqueous phase), and an appropriate amount of lipid was co-dissolved in 200-proof ethanol (ethanol phase). Nanoparticles formulated using a microfluidic device were synthesized with a v / v ratio of 3:1 between the aqueous and ethanol phases. The LNPs were then dialyzed overnight at 4°C or room temperature against PBS in a 20 kDa MWCO cassette.

[0215] Particle size measurement LNP particle size and PDI (polydispersity index) were obtained using a Zetasizer (Malvern). For size measurements, LNPs were diluted 1 / 200 v / v in PBS, and z-average values were reported. For zeta potential measurements, LNPs were diluted 1 / 200 v / v in 0.1x PBS.

[0216] Quantification of siRNA concentration and encapsulation The siRNA concentration in the dialyzed particles was determined using a modified Quant-iT RiboGreen RNA assay (Thermo Fisher). Nanoparticle dilutions of approximately 1 ng / pL siRNA were made in TE buffer (pH 8.5), and siRNA standards ranging from 2 ng / pL to 0.125 ng / pL were generated. 50 pL of each solution was added to separate wells in a 96-well black polystyrene plate. 50 pL of TE buffer was added to each well. The plate was incubated at 37°C for 15 minutes with shaking at 350 rpm. After incubation, diluted RiboGreen reagent was added (100 pL per well), and the plate was incubated for 3 minutes as before. RiboGreen fluorescence was measured using a Tecan plate reader according to the accompanying protocol, and the siRNA standards were used to determine nanoparticle siRNA concentration. Note that two standards were generated: one with and one without Triton-X. The particles in TE buffer were used to determine the unencapsulated siRNA concentration and TE-TX, and the encapsulation efficiency was determined by the following formula:

[0217]

number

[0218] Sequence alignment The amino acid and mRNA sequences were aligned using Clustal Omega.

[0219] [Example 1] siRNA targeting SIRPα In certain embodiments of the present disclosure, siRNA duplexes were designed to target human or mouse transcripts annotated in the NCBI Gene Database (Tables 1 and 2). In further embodiments, the present disclosure contemplates the design of siRNA duplexes that target transcripts of domestic pets, e.g., domestic cats or domestic dogs, annotated in the NCBI Gene Database (Table 3).

[0220] Table 1: Human SIRPα mRNA target sequence [Table 1]

[0221] Table 2: Mouse SIRPα mRNA target sequence [Table 2]

[0222] Table 3: Cat and dog SIRPα mRNA target sequences [Table 3]

[0223] To mitigate the risk of off-target gene silencing (e.g., silencing of SIRPβ and / or SIRPγ), the cDNA sequences of human SIRPα, SIRPβ, and SIRPγ were aligned using Clustal W2 and sequence identity was calculated (Figure 2). Additionally, single nucleotide polymorphism (SNP) analysis of human SIRPα was performed based on data available in EnsEMBL (enssembl.org).

[0224] As used herein, the term "sequence identity" refers to the degree to which two sequences have the same residues at the same positions when aligned to achieve the maximum level of identity, expressed as a percentage. In aligning and comparing sequences, typically, one sequence is designated as a reference sequence, to which a test sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as the percentage of positions over the entire length of the reference sequence at which the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the maximum level of identity. As an example, two sequences are considered to have 70% sequence identity if, when aligned to achieve the maximum level of identity, the test sequence has the same nucleotide residues at 70% of the same positions over the entire length of the reference sequence.

[0225] Alignment of sequences for comparison to achieve the maximum level of identity can be readily performed by one of skill in the art using an appropriate alignment method or algorithm, in some cases the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). In some embodiments, codon-optimized sequences for efficient expression in different cells, tissues, and / or organisms reflect the codon usage patterns in such cells, tissues, and / or organisms, including conservative (or non-conservative) amino acid substitutions that do not adversely affect normal activity.

[0226] Eighteen variants with a global mutation allele frequency (MAF) greater than 0.05 were extracted (Table 4) and mapped onto the SIRPα sequence (Figure 2). Variants with a minor allele frequency (MAF) greater than 0.05 are boxed.

[0227] Human SIRPα siRNA target sequences were identified (Table 5) and mapped onto the SIRPα sequence (Figure 2, underlined). Mouse SIRPα siRNA target sequences were also identified (Table 6). siRNAs with greater than 75% sequence identity were predicted to be cross-reactive. In some embodiments, siRNA sequence identity with human SIRPβ and SIRPγ is less than 75% to minimize off-target silencing. In some embodiments, the siRNA target sequence is located within SEQ ID NO: 4. In some embodiments, the siRNA target sequence is between 361 and 1875 within SEQ ID NO: 4. In some embodiments, the siRNA target sequence is between 1438 and 1875 within SEQ ID NO: 4. In some embodiments, the siRNA target sequence is a region within SEQ ID NO: 4 that is unique to SIRPα. In some embodiments, the siRNA target sequence is within the perimembrane domain, transmembrane domain, and / or cytoplasmic domain of SIRPα. In some embodiments, siRNA sequence identity with feline and canine SIRPα is greater than 75% for species cross-reactivity.

[0228] Table 4: Human SIRPα variants [Table 4]

[0229] In some embodiments, the present disclosure provides modifications of siRNA sequences. In some embodiments, the modified siRNA sequence comprises at least one nucleotide overhang covalently attached to the 3'-end of the sense sequence, the antisense sequence, or both the sense and antisense sequences. In some embodiments, the modified siRNA sequence comprises two nucleotide overhangs covalently attached to the 3'-end of the siRNA sense sequence, the antisense sequence, or both the sense and antisense sequences. In some embodiments, the nucleotide overhang comprises at least one artificial nucleotide. In some embodiments, the nucleotide overhang comprises at least two artificial nucleotides. In some embodiments, the overhang is U, T, UU, TT, dT, dTdT, sdT, dTsdT, sdTsdT, or sdTdT. Non-limiting examples of modified siRNA sequences are found in Tables 7 and 8.

[0230] In yet another embodiment, the present disclosure provides the design and use of siRNA sequences that comprise one or more modified nucleotides.In some embodiments, the modified nucleotide is in siRNA sense sequence.In some embodiments, the modified nucleotide is in siRNA antisense sequence.In still another embodiment, the modified nucleotide is present in both siRNA sense sequence and antisense sequence.

[0231] In some embodiments, the modified nucleotide is chemically modified. Chemical modifications can include phosphate backbone modifications (e.g., phosphorothioate or boranophosphate linkages), ribose ring modifications, such as 2'-O-methyl and / or 2'-fluoro and / or 4'-thio modifications, and locked or unlocked nucleic acids. Other modifications include pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-aminouridine, 5-methyluridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, 5-methylcytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine. pseudoisocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, 5-methylpseudoisocytidine, N6-methyladenosine, 7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0232] In some embodiments, the modified nucleotide is 2'-O-methyladenosine (mA), 2'-O-methylcytidine (mC), 2'-O-methylguanidine (mG), or 2'-O-methyluridine (mU). Non-limiting examples of modified siRNA sequences are found in Tables 7 and 8.

[0233] In another aspect, the disclosure provides designs and uses of siRNA duplexes. In some embodiments, the siRNA duplex comprises SEQ ID NO: 18 and SEQ ID NO: 19. In some embodiments, the siRNA duplex comprises SEQ ID NO: 20 and SEQ ID NO: 21. In some embodiments, the siRNA duplex comprises SEQ ID NO: 22 and SEQ ID NO: 23. In some embodiments, the siRNA duplex comprises SEQ ID NO: 24 and SEQ ID NO: 25. In some embodiments, the siRNA duplex comprises SEQ ID NO: 26 and SEQ ID NO: 27. In some embodiments, the siRNA duplex comprises SEQ ID NO: 28 and SEQ ID NO: 29. In some embodiments, the siRNA duplex comprises SEQ ID NO: 30 and SEQ ID NO: 31. In some embodiments, the siRNA duplex comprises SEQ ID NO: 32 and SEQ ID NO: 33. In some embodiments, the siRNA duplex comprises SEQ ID NO: 34 and SEQ ID NO: 35. In some embodiments, the siRNA duplex comprises SEQ ID NO: 36 and SEQ ID NO: 37. In some embodiments, the siRNA duplex comprises SEQ ID NO: 38 and SEQ ID NO: 39. In some embodiments, the siRNA duplex comprises SEQ ID NO: 40 and SEQ ID NO: 41. In some embodiments, the siRNA duplex comprises SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the siRNA duplex comprises SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the siRNA duplex comprises SEQ ID NO:46 and SEQ ID NO:47. In some embodiments, the siRNA duplex comprises SEQ ID NO:48 and SEQ ID NO:49. In some embodiments, the siRNA duplex comprises SEQ ID NO:50 and SEQ ID NO:51. In some embodiments, the siRNA duplex comprises SEQ ID NO:52 and SEQ ID NO:53. In some embodiments, the siRNA duplex comprises SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the siRNA duplex comprises SEQ ID NO:56 and SEQ ID NO:57. In some embodiments, the siRNA duplex comprises SEQ ID NO:58 and SEQ ID NO:59. In some embodiments, the siRNA duplex comprises SEQ ID NO:60 and SEQ ID NO:61. In some embodiments, the siRNA duplex comprises SEQ ID NO:62 and SEQ ID NO:63. In some embodiments, the siRNA duplex comprises SEQ ID NO:64 and SEQ ID NO:65. In some embodiments, the siRNA duplex comprises SEQ ID NO:66 and SEQ ID NO:67.In some embodiments, the siRNA duplex comprises SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, the siRNA duplex comprises SEQ ID NO:70 and SEQ ID NO:71. In some embodiments, the siRNA duplex comprises SEQ ID NO:72 and SEQ ID NO:73. In some embodiments, the siRNA duplex comprises SEQ ID NO:74 and SEQ ID NO:75. In some embodiments, the siRNA duplex comprises SEQ ID NO:76 and SEQ ID NO:77. In some embodiments, the siRNA duplex comprises SEQ ID NO:78 and SEQ ID NO:79. In some embodiments, the siRNA duplex comprises SEQ ID NO:80 and SEQ ID NO:81. In some embodiments, the siRNA duplex comprises SEQ ID NO:82 and SEQ ID NO:83. In some embodiments, the siRNA duplex comprises SEQ ID NO:84 and SEQ ID NO:85. In some embodiments, the siRNA duplex comprises SEQ ID NO:86 and SEQ ID NO:87. In some embodiments, the siRNA duplex comprises SEQ ID NO:88 and SEQ ID NO:89. In some embodiments, the siRNA duplex comprises SEQ ID NO:90 and SEQ ID NO:91. In some embodiments, the siRNA duplex comprises SEQ ID NO:92 and SEQ ID NO:93. In some embodiments, the siRNA duplex comprises SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the siRNA duplex comprises SEQ ID NO:96 and SEQ ID NO:97. In some embodiments, the siRNA duplex comprises SEQ ID NO:98 and SEQ ID NO:99. In some embodiments, the siRNA duplex comprises SEQ ID NO:100 and SEQ ID NO:101. In some embodiments, the siRNA duplex comprises SEQ ID NO:102 and SEQ ID NO:103. In some embodiments, the siRNA duplex comprises SEQ ID NO:104 and SEQ ID NO:105. In some embodiments, the siRNA duplex comprises SEQ ID NO:106 and SEQ ID NO:107. In some embodiments, the siRNA duplex comprises SEQ ID NO:108 and SEQ ID NO:109. In some embodiments, the siRNA duplex comprises SEQ ID NO:110 and SEQ ID NO:111. In some embodiments, the siRNA duplex comprises SEQ ID NO:112 and SEQ ID NO:113. In some embodiments, the siRNA duplex comprises SEQ ID NO:114 and SEQ ID NO:115. In some embodiments, the siRNA duplex comprises SEQ ID NO:116 and SEQ ID NO:117.In some embodiments, the siRNA duplex comprises SEQ ID NO: 118 and SEQ ID NO: 119. In some embodiments, the siRNA duplex comprises SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, the siRNA duplex comprises SEQ ID NO: 122 and SEQ ID NO: 123. In some embodiments, the siRNA duplex comprises SEQ ID NO: 124 and SEQ ID NO: 125. In some embodiments, the siRNA duplex comprises SEQ ID NO: 126 and SEQ ID NO: 127. In some embodiments, the siRNA duplex comprises SEQ ID NO: 128 and SEQ ID NO: 129. In some embodiments, the siRNA duplex comprises SEQ ID NO: 130 and SEQ ID NO: 131. In some embodiments, the siRNA duplex comprises SEQ ID NO: 132 and SEQ ID NO: 133. In some embodiments, the siRNA duplex comprises SEQ ID NO: 134 and SEQ ID NO: 135. In some embodiments, the siRNA duplex comprises SEQ ID NO: 136 and SEQ ID NO: 137. In some embodiments, the siRNA duplex comprises SEQ ID NO: 138 and SEQ ID NO: 139. In some embodiments, the siRNA duplex comprises SEQ ID NO: 140 and SEQ ID NO: 141. In some embodiments, the siRNA duplex comprises SEQ ID NO: 142 and SEQ ID NO: 143. In some embodiments, the siRNA duplex comprises SEQ ID NO: 144 and SEQ ID NO: 145. In some embodiments, the siRNA duplex comprises SEQ ID NO: 146 and SEQ ID NO: 147. In some embodiments, the siRNA duplex comprises SEQ ID NO: 148 and SEQ ID NO: 149. In some embodiments, the siRNA duplex comprises SEQ ID NO: 150 and SEQ ID NO: 151. In some embodiments, the siRNA duplex comprises SEQ ID NO: 152 and SEQ ID NO: 153. In some embodiments, the siRNA duplex comprises SEQ ID NO: 154 and SEQ ID NO: 155. In some embodiments, the siRNA duplex comprises SEQ ID NO: 156 and SEQ ID NO: 157. In some embodiments, the siRNA duplex comprises SEQ ID NO: 158 and SEQ ID NO: 159. In some embodiments, the siRNA duplex comprises SEQ ID NO: 160 and SEQ ID NO: 161. In some embodiments, the siRNA duplex comprises SEQ ID NO: 162 and SEQ ID NO: 163. In some embodiments, the siRNA duplex comprises SEQ ID NO: 164 and SEQ ID NO: 165. In some embodiments, the siRNA duplex comprises SEQ ID NO:166 and SEQ ID NO:167.In some embodiments, the siRNA duplex comprises SEQ ID NO: 168 and SEQ ID NO: 169. In some embodiments, the siRNA duplex comprises SEQ ID NO: 170 and SEQ ID NO: 171. In some embodiments, the siRNA duplex comprises SEQ ID NO: 172 and SEQ ID NO: 173. In some embodiments, the siRNA duplex comprises SEQ ID NO: 174 and SEQ ID NO: 175. In some embodiments, the siRNA duplex comprises SEQ ID NO: 176 and SEQ ID NO: 177. In some embodiments, the siRNA duplex comprises SEQ ID NO: 178 and SEQ ID NO: 179. In some embodiments, the siRNA duplex comprises SEQ ID NO: 180 and SEQ ID NO: 181. In some embodiments, the siRNA duplex comprises SEQ ID NO: 182 and SEQ ID NO: 183. In some embodiments, the siRNA duplex comprises SEQ ID NO: 184 and SEQ ID NO: 185. In some embodiments, the siRNA duplex comprises SEQ ID NO: 186 and SEQ ID NO: 187. In some embodiments, the siRNA duplex comprises SEQ ID NO: 188 and SEQ ID NO: 189. In some embodiments, the siRNA duplex comprises SEQ ID NO: 190 and SEQ ID NO: 191. In some embodiments, the siRNA duplex comprises SEQ ID NO: 192 and SEQ ID NO: 193. In some embodiments, the siRNA duplex comprises SEQ ID NO: 194 and SEQ ID NO: 195. In some embodiments, the siRNA duplex comprises SEQ ID NO: 196 and SEQ ID NO: 197. In some embodiments, the siRNA duplex comprises SEQ ID NO: 198 and SEQ ID NO: 199. In some embodiments, the siRNA duplex comprises SEQ ID NO: 200 and SEQ ID NO: 201. In some embodiments, the siRNA duplex comprises SEQ ID NO: 202 and SEQ ID NO: 203. In some embodiments, the siRNA duplex comprises SEQ ID NO: 204 and SEQ ID NO: 205. In some embodiments, the siRNA duplex comprises SEQ ID NO: 206 and SEQ ID NO: 207. In some embodiments, the siRNA duplex comprises SEQ ID NO: 208 and SEQ ID NO: 209. In some embodiments, the siRNA duplex comprises SEQ ID NO: 210 and SEQ ID NO: 211. In some embodiments, the siRNA duplex comprises SEQ ID NO: 212 and SEQ ID NO: 213. In some embodiments, the siRNA duplex comprises SEQ ID NO: 214 and SEQ ID NO: 215. In some embodiments, the siRNA duplex comprises SEQ ID NO:216 and SEQ ID NO:217.In some embodiments, the siRNA duplex comprises SEQ ID NO: 218 and SEQ ID NO: 219. In some embodiments, the siRNA duplex comprises SEQ ID NO: 220 and SEQ ID NO: 221. In some embodiments, the siRNA duplex comprises SEQ ID NO: 222 and SEQ ID NO: 223. In some embodiments, the siRNA duplex comprises SEQ ID NO: 224 and SEQ ID NO: 225. In some embodiments, the siRNA duplex comprises SEQ ID NO: 226 and SEQ ID NO: 227.

[0234] Table 5: Human SIRPα 19-mer mRNA target sequence [Table 5] JPEG2025526439000054.jpg231170JPEG2025526439000055.jpg231170

[0235] n / a means that either sequence has an insertion or deletion (no or 0% identity).

[0236] Table 6: Mouse SIRPα 19-mer mRNA target sequence [Table 6] JPEG2025526439000057.jpg231170JPEG2025526439000058.jpg231170

[0237] n / a means that sequence identity cannot be calculated due to insertions or deletions in either sequence

[0238] Table 7: Human SIRPα siRNA duplexes and sequences [Table 7] JPEG2025526439000060.jpg220170JPEG2025526439000061.jpg220170

[0239] Table 8: Mouse SIRPα siRNA duplexes and sequences [Table 8] JPEG2025526439000063.jpg231170JPEG2025526439000064.jpg231170

[0240] Table 9: Comparison of mouse SIRPα 19-mer mRNA target sequences [Table 9]

[0241] [Example 2] Dual response screening of human SIRPα siRNA duplexes and variants siRNA-modified variants derived from the siRNA duplex with high efficiency and specificity for human SIRPα RNA knockdown were designed according to the sequence and chemical modifications (described in Example 1), and the resulting duplex variants were further tested in THP-1-derived macrophages.

[0242] THP-1 monocytes were cultured under standard culture conditions and transferred to 96-well plates at a density of 10,000–15,000 cells per well. THP-1 monocytes were differentiated into macrophages by incubation with 100 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 hours followed by a 24-hour recovery period. SIRPα siRNA duplexes were transfected into THP-1 macrophages using Viromer Blue (0.5 μl / well). A total of 30 siRNA duplexes, including variants from the original modified siRNA duplex, were transfected into THP-1 macrophage cells for further validation. SIRPα siRNA was transfected at final concentrations of 5 nM and 25 nM, respectively. Anti-fLuc siRNA duplexes were used as a negative control. After 48 hours of incubation, total mRNA was extracted and purified, and cDNA was synthesized by reverse transcription. SIRPα expression levels were quantified by real-time PCR. The information and sequences of these siRNA duplexes are included in Tables 10 and 11.

[0243] Table 10: Relative fold change in SIRPα mRNA levels [Table 10]

[0244] Table 11: Relative fold change in SIRPα mRNA levels [Table 11]

[0245] [Example 3] Dose response of selected human SIRPα siRNA duplexes and variants Human SIRPα siRNA duplexes and variants that resulted in significant reductions in SIRPα mRNA levels in the dual-dose screen were selected and further tested for dose response. In addition to these duplexes, 10 other siRNA duplexes were also tested. THP-1 monocytes were cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000–15,000 cells per well. THP-1 monocytes were differentiated into macrophages by incubation with 100 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 hours followed by a 24-hour recovery period. THP-1 macrophages were transfected with SIRPα siRNA duplexes using Viromer Blue (0.5 μl / well). The doses of each SIRPα siRNA duplex were 25 nM, 2.5 nM, 250 pM, and 25 pM. After 48 hours of incubation, treated cells were harvested and residual SIRPα mRNA levels in each condition were measured by RT-qPCR. IC of each SIRPα siRNA duplex 50 The values were determined and the respective dose responses are shown in Tables 12 and 13.

[0246] Table 12: IC of SIRPα siRNA duplexes 50 value [Table 12]

[0247] Table 13: IC of SIRPα siRNA duplexes 50 value [Table 13]

[0248] [Example 4] Dual response screening of mouse SIRPα siRNA duplexes and variants Modified siRNA variants derived from siRNA duplexes with high efficiency and specificity for mouse SIRPα RNA knockdown were designed according to the sequence and chemical modifications (described in Example 1). The resulting duplex variants were further tested in the J774 mouse macrophage cell line. J774 cells were cultured using standard culture conditions and plated into 96-well plates at a density of 10,000–15,000 cells per well. SIRPα siRNA duplexes were introduced into J774 macrophages using Viromer Blue (0.5 μl / well). A total of 36 siRNA duplexes, including variants from the original modified siRNA duplex, were transfected into J774 cells for further validation. SIRPα siRNA was transfected at final concentrations of 5 nM and 25 nM, respectively. Anti-fLuc siRNA duplexes were used as a negative control. After 48 hours of incubation, total mRNA was extracted and purified, and cDNA was synthesized by reverse transcription. SIRPα expression levels were quantified by real-time PCR. The information and sequences of these siRNA duplexes are included in Tables 14 and 15.

[0249] Table 14: Relative fold change in SIRPα mRNA levels [Table 14]

[0250] Table 15: Relative fold change in SIRPα mRNA levels [Table 15]

[0251] [Example 5] Dose response of selected mouse SIRPα siRNA duplexes and variants Mouse SIRPα siRNA duplexes and variants that resulted in significant reductions in SIRPα mRNA levels in the dual-dose screen were selected and further tested for dose response. J774 mouse macrophage cell line was cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000-15,000 cells per well. J774 cells were transfected with SIRPα siRNA duplexes using Viromer Blue (0.5 μl / well). Doses of each SIRPα siRNA duplex included 25 nM, 2.5 nM, 250 pM, and 25 pM. After 48 hours of incubation, treated cells were harvested, and residual SIRPα mRNA levels in each condition were measured by RT-qPCR. The IC of each SIRPα siRNA duplex was 50 Values were determined and the respective dose responses are shown in Table 16.

[0252] Table 16: IC of SIRPα siRNA duplexes 50 value [Table 16]

[0253] [Example 6] Formulation of siRNA in lipid nanoparticles (LNPs) LNPs were synthesized with a molar ratio of 50:10:38.5:1.5 of ionizable lipid:1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol:1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and a weight ratio of 10:1 of ionizable lipid:siRNA.

[0254] Particle size and PDI values were obtained with a Zeta Sizer (Malvern), and siRNA concentration and encapsulation efficiency were assessed with a modified Ribo-Green assay (Thermo Fisher).

[0255] Table 17: Composition of LNP-formulated SIRPα siRNA [Table 17]

[0256] Table 18: Physicochemical properties of LNPs [Table 18]

[0257] [Example 7] LNPs enable myeloid cell-specific RNA delivery in vivo To investigate which immune cell types LNPs can deliver siRNA to, we prepared LNPs containing siRNA against CD45 (siCD45). CD45, a cell surface tyrosine phosphatase, was chosen because it is ubiquitously expressed on all immune cell types and can therefore be used to test gene silencing in different immune cell subsets. Mice were intraperitoneally injected with siCD45-LNPs or PBS.

[0258] siCD45 siRNA sequence Sense: mCmUGGmCmUGAAmUmUmUmCAGAGmCA (SEQ ID NO: 228)-dTdT, Antisense: UGCUCUGAAAUUmCAGCmCAG (SEQ ID NO: 229)-dTdT

[0259] Three days after administration, peritoneal lavage was performed to collect immune cells from the peritoneal cavity. These immune cells were stained for markers of different immune cell subsets and analyzed by flow cytometry.

[0260] Surprisingly, macrophages and their precursors (Mono / Macs) showed statistically significant CD45 gene silencing at 0.01 mg / kg, whereas lymphocytes, e.g., B cells and T cells, did not show significant gene silencing (Figure 3).

[0261] This macrophage-tropic property of LNPs should be useful to further suppress off-target gene silencing of SIRPγ in T cells.

[0262] [Example 8] siSIRPα-LNP exhibits gene silencing in mice To optimize the dosage and schedule of siSIRPα-LNP in vivo, mice were intraperitoneally injected with siSIRPα-LNP, siLuc-LNP, or PBS. 3 or 7 days after administration, gene silencing in peritoneal immune cells was analyzed by flow cytometry. siSIRPα-LNP demonstrated dose-dependent SIRPα gene silencing, with an effective dose 50 (ED50) of 0.00025 mg / kg (Figure 4).

[0263] [Example 9] siSIRPα-LNP promotes the M1 phenotype in primary human macrophages To investigate whether SIRPα gene silencing on macrophages can reprogram the phenotype of tumor-associated macrophages from M2 to M1, the expression levels of macrophage polarization markers and antigen-presenting molecules were assessed.

[0264] Human primary monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using a pan-monocyte isolation kit (Miltenyi Biotec). These monocytes were differentiated into macrophages with M-CSF (20 ng / ml for 4 days, followed by 40 ng / ml for 4 days) and then transfected with FL-A LNP-formulated siSIRPα or siLuc (50 nM siRNA dose) on day 8. These cells were harvested 3 days after transfection and analyzed by flow cytometry. 20 ng / mL M-CSF was maintained in the culture medium after transfection.

[0265] SIRPα siRNA demonstrated approximately 90% SIRPα gene silencing in human primary macrophages (Figures 5A and 5B). Furthermore, SIRPα silencing reduced M2 marker expression (CD163) and increased M1 marker expression (CD86). These results strongly suggest that SIRPα siRNA can reprogram the tumor microenvironment (Figures 6A-6C). Furthermore, the upregulation of antigen-presenting molecules (MHC-I) suggests that SIRPα siRNA can enhance antigen presentation on macrophages and other myeloid cells (Figures 6A-6C).

[0266] [Example 10] The siSIRPα sequence and LNP formulation are generalizable (1) To investigate whether other siRNA sequences and LNP formulations could silence SIRPα expression and reprogram the phenotype of tumor-associated macrophages from M2 to M1, we evaluated the expression levels of macrophage polarization markers and antigen-presenting molecules by various siRNA sequences and LNP formulations.

[0267] Human primary monocytes were isolated from peripheral blood mononuclear cells using a Pan Monocyte Isolation Kit, Human (Miltenyi Biotec). These monocytes were differentiated into macrophages with M-CSF (20 ng / ml for 4 days, followed by 40 ng / ml for 4 days) and then transfected with LNP-formulated siSIRPα or siLuc (50 nM siRNA dose) on day 8. Lipofectamine® RNAiMax (RIM)-formulated siSIRPα and siLuc were also tested. These cells were harvested 3 days post-transfection and analyzed by flow cytometry. 20 ng / mL M-CSF was maintained in the culture medium after transfection.

[0268] The experimental protocol is further described below in Example 12.

[0269] All SIRPα siRNA sequences formulated with FL-A and all LNPs formulated with sihSIRPα_18-PM (si18) demonstrated approximately 50% SIRPα gene silencing in human primary macrophages while maintaining nearly 100% cell viability (Figures 7A and 7B, 8A and 8B). Furthermore, among all siRNAs tested, si18 demonstrated the most significant upregulation of M1 markers (CD86) and MHC-II antigen-presenting molecules (HLA-DR), suggesting that si18 has higher immunogenicity than other siRNAs (Figures 9A and 9B). Regarding ionizable lipids, FL-A induced the most significant upregulation of CD86 and HLA-DR, indicating that FL-A is the most suitable for macrophage delivery for cancer applications (Figures 10A and 10B). Based on these results, sihSIRPα_18-PM formulated in FL-A was selected for more detailed evaluation (hereafter simply referred to as siSIRPα-LNP).

[0270] Table 19: List of siRNA-LNPs tested in human primary macrophages [Table 19]

[0271] [Example 11] The siSIRPα sequence and LNP formulation are generalizable (2) To investigate whether other siRNA sequences and LNP formulations can silence SIRPα expression, we evaluated the SIRPα expression levels of various siRNA sequences and LNP formulations using THP-1-derived macrophages. The following ionizable lipids were tested, and the LNP formulations are listed in Table 20.

[0272] FL-A (2-butyloctyl 3-ethyl-12-hexyl-6-(2-(octanoyloxy)ethyl)-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate) described in WO 2019 / 235635A1:

[0273] [ka]

[0274] GCL1, described in International Publication No. WO 2020 / 219941 A1 ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate):

[0275] [ka]

[0276] Lipid A9, (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate), as described in International Publication No. WO 2017 / 004143 A1:

[0277] [ka]

[0278] ALC-0315, described in International Publication No. WO 2017 / 075331 A1 (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate):

[0279] [ka]

[0280] Lipid 5, described in International Publication No. WO 2017 / 099823 A1 (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate):

[0281] [ka]

[0282] Table 20: List of siRNA-LNPs tested in THP-1 derived macrophages [Table 20]

[0283] The experimental protocol was as described in Example 3. The doses of each SIRPα siRNA duplex included 50 nM and 5 nM.

[0284] All SIRPα siRNA sequences and LNP formulations demonstrated greater than 50% SIRPα gene silencing in THP-1-derived macrophages at 50 nM (Figures 24A and 24B). Based on these results, it was concluded that the siRNA sequences selected in the screen were potent enough to demonstrate gene silencing when formulated with various ionizable lipids.

[0285] [Example 11] Comparison of siRNA and antibodies against SIRPα Ovarian cancer phagocytosis To compare siSIRPα-driven SIPRa gene silencing with antibody blocking of SIRPα activity, primary human macrophages were pretreated with siSIRPα-LNP (or siLuc-LNP as a negative control) or anti-SIRPα (or an isotype (IgG) control antibody) before coculture with human ovarian cancer cells (SKOV-3). These pretreated macrophages were also labeled with violet BMQC cell tracker fluorescent dye (ThermoFisher Scientific #C10094, Invitrogen) before initiating coculture. SKOV-3 ovarian cancer cells were stained with green fluorescent CFSE cell tracker (BioLegend). In some conditions, SKOV-3 cells were pretreated with anti-HER2 antibody (as in Example 12). Macrophages and SKOV-3 ovarian cancer cells were seeded at a 1:2 ratio and cocultured for 2.5 hours (Figures 11A and 11B). Flow cytometry was used to quantify SIRPα silencing, expression levels of macrophage polarization markers and antigen-presenting molecules, and phagocytosis.

[0286] siSIRPα treatment resulted in over 80% SIRPα gene silencing in primary human macrophages (Figures 11A and 11B). SIRPα gene silencing with siSIRPα also increased M1 marker expression compared to SIRPα blocking with a blocking antibody (Figures 12A-12C). The intensity of CFSE fluorescence within the macrophage population indicates phagocytosis by macrophages. The increase in CFSE fluorescence within the CD45+ macrophage population after siSIRPα treatment indicates that siSIRPα treatment increased SKOV-3 ovarian cancer phagocytosis by macrophages compared to blocking SIRPα with a blocking antibody (Figures 12A-12C). Furthermore, the expression level of class 1 antigen-presenting molecule (HLA-A2) within the CD45+ macrophage population was increased after siSIRPα treatment compared to treatment with a SIRPα blocking antibody (Figures 12A-12C).

[0287] Mutual phagocytosis Mutual phagocytosis is the engulfment of macrophages by other macrophages. To compare mutual phagocytosis after treatment with either siSIRPα or an anti-SIRPα blocking antibody, primary human macrophages were treated with siSIRPα-LNP, siLuc-LNP (as a negative control), anti-SIRPα, or an IgG control antibody (as a negative control). Treated macrophages were labeled with violet BMQC cell tracker fluorescent dye (Invitrogen by ThermoFisher Scientific #C10094) and co-cultured with untreated macrophages. Untreated macrophages were labeled with green fluorescent CFSE cell tracker (BioLegend). Violet (i.e., pre-treated) and green (i.e., untreated) macrophages were plated at a 1:1.5 ratio and incubated as co-cultures for 2.5 hours (Figure 13). Next, the remaining violet- and green-labeled macrophages were quantified using flow cytometry to assess their mutual phagocytosis.

[0288] Macrophages treated with either anti-SIRPα blocking antibody or IgG control antibody were substantially depleted after 2.5 hours of coculture, whereas macrophages treated with siSIRPα-LNP or siLuc-LNP were not (Figures 14A and 14B). This indicates that treatment with anti-SIRPα blocking antibody renders macrophages more susceptible to mutual phagocytosis than treatment with siSIRPα. The mutual phagocytic activity of violet (i.e., pre-treated) macrophages was quantified as the percentage of violet macrophages positive for the CFSE cell tracker. Macrophages pre-treated with anti-SIRPα blocking antibody exhibited greater phagocytosis of other (i.e., green) macrophages than macrophages pre-treated with siSIRPα (Figures 14A and 14B).

[0289] [Example 13] siSIRPα-LNP plus aHER2 antibody enhances phagocytosis of ovarian cancer cells by primary human macrophages To determine whether SIRPα gene silencing on macrophages synergizes with antibody therapeutics, primary human macrophages transfected with siRNA-LNPs (siSIRPα or siLUC as a negative control) were co-cultured with human ovarian cancer cells (SKOV-3 cells) pre-incubated with antibody therapeutics (aHER2).

[0290] Here, we observed that SKOV-3 human ovarian cancer cells express high levels of the SIRPα ligand CD47 (Figures 15A and 15B), suggesting that CD47-SIRPα interaction may contribute to macrophage inhibition and the ability of SKOV-3 ovarian cancer cells to evade host immune defenses. Engagement of CD47 with macrophage-expressed SIRPα triggers an inhibitory cascade, i.e., a "don't eat me" off-signal, that prevents macrophage phagocytosis of CD47-bearing target (SKOV-3) cells. Conversely, antibodies capable of engaging Fc receptors on macrophages trigger a stimulatory cascade, i.e., an "eat me" on-signal, that leads to macrophage activation and phagocytosis (Figure 16). SKOV-3 ovarian cancer cells express HER2 (Figures 15C and 15D), suggesting that anti-HER2 antibodies opsonize SKOV-3 cells, engage Fc receptors on macrophages, and provide a positive signal for phagocytosis (Figure 16).

[0291] Primary human monocytes were isolated from peripheral blood mononuclear cells and differentiated into macrophages as described in Example 12. Primary human macrophages were transfected with siRNA-LNP as described in Example 12 and used for coculture (described below) 3 days after transfection. SKOV-3 ovarian cancer cells were labeled with the fluorescent cell tracker CFSE (BioLegend) and preincubated with an anti-HER2 antibody or an IgG isotype control antibody prior to coculture. After coculture of SKOV-3 cancer cells with transfected macrophages for 2 hours, flow cytometry was used to quantify the level of SIRPα silencing (Figures 17A and 17B), the percentage of macrophages positive for the CFSE cancer cell tracker (indicating phagocytosis) (Figures 18A and 18B), the expression of M1 vs. M2 macrophage phenotype markers (Figures 19A-19C), and the expression of antigen-presenting molecules (Figure 20).

[0292] siSIRPα-LNP resulted in >90% SIRPα gene silencing in primary human macrophages (Figures 17A and 17B). SIRPα silencing in combination with anti-HER2 cancer treatment synergistically promoted macrophage phagocytosis of SKOV-3 ovarian cancer cells (Figures 18A and 18B). The level of phagocytosis observed after siSIRPα + aHER2 treatment (approximately 70%) exceeded that observed after siSIRPα silencing or anti-HER2 treatment alone (Figures 18A and 18B). Furthermore, SIRPα silencing reduced M2 marker expression (CD206, CD163 (Figures 19A and 19B)) and increased M1 marker expression (CD86) (Figure 19C). These results strongly suggest that SIRPα siRNA can reprogram the tumor microenvironment. The upregulation of class II antigen-presenting molecules (HLA-DR) (FIG. 20) suggests that SIRPα siRNA may enhance antigen presentation by primary human macrophages.

[0293] [Example 14] siSIRPα-LNP promotes cross-presentation of phagocytosed antigens in vitro Cross-presentation is the ability of certain professional antigen-presenting cells (mainly macrophages and dendritic cells) to internalize, process, and present extracellular antigens on MHC class I molecules. Because antigen cross-presentation has the potential to initiate antigen-specific CD8 T cell responses, stimulating macrophages to cross-present tumor antigens has attracted considerable interest.

[0294] To determine whether SIRPα gene silencing promotes tumor antigen cross-presentation by macrophages, mouse peritoneal cells treated with intraperitoneal injection of siSIRPα-LNP (or siLUC-LNP as a negative control) were harvested 3 days after siRNA treatment and placed in coculture with B16 mouse melanoma cells (Figures 21A-21C). B16 melanoma cells expressed ovalbumin (OVA), used as a model cancer antigen. To track B16 melanoma cells and quantify phagocytosis, B16 cells were stained with CSFE cell tracker before being placed in coculture. After overnight coculture of labeled B16 melanoma cells with mouse ip cells, flow cytometry was used to identify macrophages, evaluate SIRPα silencing within the macrophage population, quantify B16 melanoma phagocytosis and cancer antigen presentation on MHC class I, and assess phenotype.

[0295] Intraperitoneal injection of siSIRPα-LNP resulted in significant silencing of SIRPα expression on mouse peritoneal macrophages and increased CD86 expression, indicating a shift to an M1 phenotype (Figures 21A-C). Macrophage phagocytosis of B16 antigen was quantified as the percentage of macrophages positive for the B16 cell tracker CFSE. siSIRPα-LNP treatment significantly increased the percentage of macrophages exhibiting B16 phagocytosis (Figures 22A and 22B). Importantly, macrophage presentation of a model antigen (OVA) on MHC class I was also significantly increased by siSIRPα-LNP treatment (Figures 22A and 22B). Cross-presentation requires internalization of extracellular antigens followed by presentation on MHC class I. Therefore, to quantify cross-presentation more directly, we simultaneously assessed B16 antigen uptake (as indicated by CFSE) and MHC class I OVA presentation on macrophages using flow cytometry (Figures 23A-C). The percentage of cross-presenting macrophages was quantified as the fraction of CFSE macrophages that were also MHC class I-OVA positive.

[0296] % cross-presentation = (MHC-OVA+CFSE+) / (CFSE+) × 100

[0297] The percentage of cross-presenting macrophages was significantly increased after intraperitoneal treatment with siSIRPα-LNP (Figures 23A-23C). Collectively, these results strongly suggest that SIRPα siRNA promotes macrophage activation (M1 phenotype) and cross-presentation of cancer antigens by macrophages, ultimately promoting potent anti-cancer immune responses in cancer patients.

[0298] References

[0299] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. References Barclay, A. Neil, and Deborah Hatherley. "The counterbalance theory for evolution and function of paired receptors." Immunity 29.5 (2008): 675-678. Dietrich, Jes, et al. "Cutting edge: signal-regulatory protein β1 is a DAP12-associated activating receptor expressed in myeloid cells." The Journal of Immunology 164.1 (2000): 9-12. Kurlander, R. J. "Blockade of Fc receptor-mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens." The Journal of Immunology 131.1 (1983): 140-147. Madeira, Faebio, et al. "The EMBL-EBI search and sequence analysis tools APIs in 2019." Nucleic acids research 47.W1 (2019): W636-W641. Muntjewerff, Elke M., Luca D. Meesters, and Geert Van den Bogaart. "Antigen cross-presentation by macrophages." Frontiers in Immunology 11 (2020): 1276. Piccio, Laura, et al. "Adhesion of human T cells to antigen-presenting cells through SIRPβ2-CD47 interaction costimulates T-cell proliferation." Blood 105.6 (2005): 2421-2427. Stefanidakis, Michael, et al. "Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro." Blood, The Journal of the American Society of Hematology 112.4 (2008): 1280-1289. US 2018 / 0312600 Voets, Erik, et al. "Functional characterization of the selective pan-allele anti-SIRPα antibody ADU-1805 that blocks the SIRPα-CD47 innate immune checkpoint." Journal for immunotherapy of cancer 7.1 (2019): 1-15. WO 2010 / 054405 A1 WO 2017 / 178653 A2 WO 2019 / 235635 A1 Ye, Xiaojing, et al. "Signal regulatory protein α associated with the progression of oral leukoplakia and oral squamous cell carcinoma regulates phenotype switch of macrophages." Oncotarget 7.49 (2016): 81305.

[0300] While exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

1. A method for reducing the expression of signal-regulatory protein alpha (SIRPα) in a cell, the method comprising contacting the cell with a nanoparticle composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic agent.

2. 2. The method of claim 1, wherein the SIRPα therapeutic agent is a double-stranded ribonucleic acid (dsRNA), an antisense oligomer (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a circular RNA, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a combination thereof.

3. 3. The method of claim 1 or 2, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is at least about 15 nucleotides in length.

4. 4. The method of claim 3, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is about 15-30 nucleotides in length.

5. 5. The method of claim 4, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 18-139.

6. The method of claim 5, wherein the polynucleotide sequence further comprises a 2'-deoxythymidine-3'-phosphate 3' overhang or a 2'-deoxythymidine-5'-phosphate-phosphorothioate 3' overhang.

7. The method of claim 6, wherein the polynucleotide sequence further comprises at least one modified pyrimidine.

8. 8. The method of claim 7, wherein at least one modified pyrimidine is 2'-O-methyl cytidine-3'-phosphate or 2'-O-methyl uridine-3'-phosphate.

9. 9. The method of claim 8, wherein the SIRPα therapeutic agent is an siRNA duplex.

10. The method of claim 9 , wherein the siRNA duplex comprises a polynucleotide sequence hybridized to a complementary sequence.

11. The siRNA duplexes are selected from the group consisting of SEQ ID NO:140 and SEQ ID NO:141, SEQ ID NO:142 and SEQ ID NO:143, SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:147, SEQ ID NO:148 and SEQ ID NO:149, SEQ ID NO:150 and SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:153, SEQ ID NO:154 and SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157, SEQ ID NO:158 and SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:162 and SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:167, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:170 and SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173, SEQ ID NO:174 and SEQ ID NO:175, SEQ ID NO:176 and SEQ ID NO:177, SEQ ID NO:178 and SEQ ID NO:179, SEQ ID NO:180 and SEQ ID NO:181, SEQ ID NO:182 and SEQ ID NO:183, SEQ ID NO:

11. The method of claim 10, comprising SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:186 and SEQ ID NO:187, SEQ ID NO:188 and SEQ ID NO:189, SEQ ID NO:190 and SEQ ID NO:191, SEQ ID NO:192 and SEQ ID NO:193, SEQ ID NO:194 and SEQ ID NO:195, SEQ ID NO:196 and SEQ ID NO:197, SEQ ID NO:198 and SEQ ID NO:199, SEQ ID NO:200 and SEQ ID NO:201, SEQ ID NO:202 and SEQ ID NO:203, SEQ ID NO:204 and SEQ ID NO:205, SEQ ID NO:206 and SEQ ID NO:207, SEQ ID NO:208 and SEQ ID NO:209, SEQ ID NO:210 and SEQ ID NO:211, SEQ ID NO:212 and SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, SEQ ID NO:216 and SEQ ID NO:217, SEQ ID NO:218 and SEQ ID NO:219, SEQ ID NO:220 and SEQ ID NO:221, SEQ ID NO:222 and SEQ ID NO:223, SEQ ID NO:224 and SEQ ID NO:225, or SEQ ID NO:226 and SEQ ID NO:

227.

12. 12. The method of claim 11, wherein the siRNA duplex comprises SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:206 and SEQ ID NO:207, or SEQ ID NO:228 and SEQ ID NO:

229.

13. 13. The method of claim 12, wherein the lipid nanoparticles comprise an ionizable lipid, a phospholipid, a sterol, a polymer-conjugated lipid, or any combination thereof.

14. 14. The method of claim 13, wherein the ionizable lipid is selected from the group consisting of FL-A, MC3, lipid 5, lipid A9, ALC-0315, and GCL1.

15. 15. The method of claim 14, wherein the concentration of the SIRPα therapeutic agent is at least about 25 nM, 2.5 nM, 250 pM, or 25 pM.

16. A composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic, wherein the SIRPα therapeutic comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 18-227.

17. The composition of claim 16, wherein the polynucleotide is at least 80% identical to at least one of SEQ ID NOs: 140-227.

18. 18. The composition of claim 17, wherein contacting a cell with the SIRPα therapeutic agent reduces the concentration of SIRPα compared to the concentration of SIRPα in otherwise identical cells.

19. The composition of claim 18, wherein the concentration of SIRPα is measured using a cell-based functional assay.

20. 1. A composition comprising a nanocarrier selected from the group consisting of a lipid, a polymer, and a lipid-polymer hybrid, wherein the nanocarrier encapsulates a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic agent, and wherein the concentration of SIRPα in cells contacted with the composition is reduced compared to the concentration of SIRPα in otherwise identical cells.

21. 21. The composition of claim 20, wherein the SIRPα therapeutic agent is a double-stranded ribonucleic acid (dsRNA), an antisense oligomer (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a circular RNA, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a combination thereof.

22. 22. The composition of claim 21, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is at least about 15 nucleotides in length.

23. 23. The composition of claim 22, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is about 15-30 nucleotides in length.

24. 24. The composition of claim 23, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 95% identity to at least one of SEQ ID NOs: 18-139.

25. 25. The composition of claim 24, wherein the polynucleotide sequence further comprises a 2'-deoxythymidine-3'-phosphate 3' overhang or a 2'-deoxythymidine-5'-phosphate-phosphorothioate 3' overhang.

26. 26. The composition of claim 25, wherein the polynucleotide sequence further comprises at least one modified pyrimidine.

27. 27. The composition of claim 26, wherein at least one modified pyrimidine is 2'-O-methylcytidine-3'-phosphate or 2'-O-methyluridine-3'-phosphate.

28. 28. The composition of claim 27, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 140-227.

29. 29. The composition of claim 28, wherein the SIRPα therapeutic agent is an siRNA duplex.

30. 30. The composition of claim 29, wherein the siRNA duplex comprises a polynucleotide sequence hybridized to a complementary sequence.

31. The siRNA duplexes are selected from the group consisting of SEQ ID NO:140 and SEQ ID NO:141, SEQ ID NO:142 and SEQ ID NO:143, SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:147, SEQ ID NO:148 and SEQ ID NO:149, SEQ ID NO:150 and SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:153, SEQ ID NO:154 and SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157, SEQ ID NO:158 and SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:162 and SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:167, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:170 and SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173, SEQ ID NO:174 and SEQ ID NO:175, SEQ ID NO:176 and SEQ ID NO:177, SEQ ID NO:178 and SEQ ID NO:179, SEQ ID NO:180 and SEQ ID NO:181, SEQ ID NO:182 and SEQ ID NO:183, SEQ ID NO:

31. The composition of claim 30, comprising SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:186 and SEQ ID NO:187, SEQ ID NO:188 and SEQ ID NO:189, SEQ ID NO:190 and SEQ ID NO:191, SEQ ID NO:192 and SEQ ID NO:193, SEQ ID NO:194 and SEQ ID NO:195, SEQ ID NO:196 and SEQ ID NO:197, SEQ ID NO:198 and SEQ ID NO:199, SEQ ID NO:200 and SEQ ID NO:201, SEQ ID NO:202 and SEQ ID NO:203, SEQ ID NO:204 and SEQ ID NO:205, SEQ ID NO:206 and SEQ ID NO:207, SEQ ID NO:208 and SEQ ID NO:209, SEQ ID NO:210 and SEQ ID NO:211, SEQ ID NO:212 and SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, SEQ ID NO:216 and SEQ ID NO:217, SEQ ID NO:218 and SEQ ID NO:219, SEQ ID NO:220 and SEQ ID NO:221, SEQ ID NO:222 and SEQ ID NO:223, SEQ ID NO:224 and SEQ ID NO:225, or SEQ ID NO:226 and SEQ ID NO:

227.

32. 32. The composition of claim 31 , wherein the siRNA duplex comprises SEQ ID NO: 144 and SEQ ID NO: 145, SEQ ID NO: 160 and SEQ ID NO: 161, SEQ ID NO: 168 and SEQ ID NO: 169, SEQ ID NO: 184 and SEQ ID NO: 185, SEQ ID NO: 206 and SEQ ID NO: 207, or SEQ ID NO: 228 and SEQ ID NO:

229.

33. 33. The composition of claim 32, wherein the composition is a lipid nanoparticle composition.

34. 34. The composition of claim 33, wherein the lipid nanoparticles comprise an ionizable lipid, a phospholipid, a sterol, a polymer-conjugated lipid, or a combination thereof.

35. The composition of claim 34, wherein the lipid nanoparticles comprise at least one selected from the group consisting of FL-A, MC3, lipid 5, lipid A9, ALC-0315, GCL1, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000.

36. 26. The composition of claim 25, wherein the concentration of the SIRPα therapeutic agent is at least about 25 nM, 2.5 nM, 250 pM, or 25 pM.

37. 37. The composition of claim 36, wherein the composition is formulated as a pharmaceutical composition.

38. 38. The composition of claim 37, wherein the advanced therapy medicinal product is a somatic cell therapy medicinal product, a tissue engineering product, a gene therapy medicinal product, a tumor vaccine, or a combination thereof.

39. A method for treating a signal-regulatory protein alpha (SIRPα)-mediated disease or condition, comprising administering to a subject in need thereof a lipid nanoparticle (LNP) composition comprising a nucleic acid signal-regulatory protein alpha (SIRPα) therapeutic agent.

40. A method of treating cancer, comprising administering to a subject in need thereof a lipid nanoparticle (LNP) composition comprising a nucleic acid signal-regulating protein alpha (SIRPα) therapeutic agent.

41. 41. The method of claim 40, wherein the SIRPα therapeutic agent is a double-stranded ribonucleic acid (dsRNA), an antisense oligomer (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a circular RNA, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a combination thereof.

42. 42. The method of claim 41, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is at least 15 nucleotides in length.

43. 43. The method of claim 42, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to a contiguous sequence within SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or a combination thereof, and wherein the contiguous nucleotide sequence is about 15-30 nucleotides in length.

44. 44. The method of claim 43, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity to at least one of SEQ ID NOs: 18-139.

45. 45. The method of claim 44, wherein the polynucleotide sequence further comprises a 2'-deoxythymidine-3'-phosphate 3' overhang or a 2'-deoxythymidine-5'-phosphate-phosphorothioate 3' overhang.

46. 46. The method of claim 45, wherein the polynucleotide sequence further comprises at least one modified pyrimidine.

47. 47. The method of claim 46, wherein at least one modified pyrimidine is 2'-O-methyl cytidine-3'-phosphate or 2'-O-methyl uridine-3'-phosphate.

48. 48. The method of claim 47, wherein the SIRPα therapeutic agent is an siRNA duplex.

49. 49. The method of claim 48, wherein the siRNA duplex comprises a polynucleotide sequence hybridized to a complementary sequence.

50. siRNA duplexes containing SEQ ID NO:140 and SEQ ID NO:141, SEQ ID NO:142 and SEQ ID NO:143, SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:147, SEQ ID NO:148 and SEQ ID NO:149, SEQ ID NO:150 and SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:153, SEQ ID NO:154 and SEQ ID NO:155, SEQ ID NO:156 and SEQ ID NO:157, SEQ ID NO:158 and SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:162 and SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:167, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:170 and SEQ ID NO:171, SEQ ID NO:172 and SEQ ID NO:173, SEQ ID NO:174 and SEQ ID NO:175, SEQ ID NO:176 and SEQ ID NO:177, SEQ ID NO:178 and SEQ ID NO:179, SEQ ID NO:180 and SEQ ID NO:181, SEQ ID NO:182 and SEQ ID NO:183, 50. The method of claim 49, comprising SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:186 and SEQ ID NO:187, SEQ ID NO:188 and SEQ ID NO:189, SEQ ID NO:190 and SEQ ID NO:191, SEQ ID NO:192 and SEQ ID NO:193, SEQ ID NO:194 and SEQ ID NO:195, SEQ ID NO:196 and SEQ ID NO:197, SEQ ID NO:198 and SEQ ID NO:199, SEQ ID NO:200 and SEQ ID NO:201, SEQ ID NO:202 and SEQ ID NO:203, SEQ ID NO:204 and SEQ ID NO:205, SEQ ID NO:206 and SEQ ID NO:207, SEQ ID NO:208 and SEQ ID NO:209, SEQ ID NO:210 and SEQ ID NO:211, SEQ ID NO:212 and SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, SEQ ID NO:216 and SEQ ID NO:217, SEQ ID NO:218 and SEQ ID NO:219, SEQ ID NO:220 and SEQ ID NO:221, SEQ ID NO:222 and SEQ ID NO:223, SEQ ID NO:224 and SEQ ID NO:225, or SEQ ID NO:226 and SEQ ID NO:

227.

51. 51. The method of claim 50, wherein the siRNA duplex comprises SEQ ID NO:144 and SEQ ID NO:145, SEQ ID NO:160 and SEQ ID NO:161, SEQ ID NO:168 and SEQ ID NO:169, SEQ ID NO:184 and SEQ ID NO:185, SEQ ID NO:206 and SEQ ID NO:207, or SEQ ID NO:228 and SEQ ID NO:

229.

52. 52. The method of claim 51, wherein the lipid nanoparticle (LNP) comprises an ionizable lipid, a phospholipid, a sterol, a polymer-conjugated lipid, or any combination thereof.

53. 53. The method of claim 52, wherein the ionizable lipid is selected from the group consisting of FL-A, MC3, lipid 5, lipid A9, ALC-0315, and GCL1.

54. 54. The method of claim 53, wherein the concentration of the SIRPα therapeutic agent is at least about 25 nM, 2.5 nM, 250 pM, or 25 pM.

55. The SIRPα-mediated disease or condition is selected from the group consisting of acute myeloid leukemia, adenosquamous lung carcinoma, atypical meningioma, B-cell acute lymphoblastic leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, transitional cell carcinoma of the bladder, glioblastoma of the brain, breast cancer, ductal adenocarcinoma, Burkitt's lymphoma, cecal adenocarcinoma, cervical squamous cell carcinoma, chronic lymphocytic leukemia, clear cell renal carcinoma, colon adenocarcinoma, cutaneous melanoma, endometrioid adenocarcinoma of the endometrioid adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastric adenocarcinoma, gastric cancer, glioma, head and neck squamous cell carcinoma, hepatobiliary neoplasms, hepatoblastoma, hepatocellular carcinoma, HCC, and glioma.

55. The method of claim 54, wherein the cancer is selected from the group consisting of ER2-positive breast cancer, renal neoplasm, large cell lung cancer, lobular breast cancer, pancreatic adenocarcinoma, lung cancer, lymphoid neoplasm, melanoma, multiple myeloma, nasopharyngeal squamous cell carcinoma, non-small cell lung carcinoma, oral squamous cell carcinoma, ovarian endometrial adenocarcinoma with squamous differentiation, ovarian serous adenocarcinoma, pancreatic apical cell carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumor, renal papillary carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, skin cancer, small cell lung carcinoma, squamous cell lung carcinoma, thyroid cancer, thyroid neoplasm, and uterine carcinosarcoma.

56. 1. A method of producing a lipid nanoparticle formulation, comprising: a) obtaining a signal regulatory protein alpha (SIRPα) therapeutic agent, the SIRPα therapeutic comprises a polynucleotide having at least 80% identity to the consecutive sequence SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17; b) diluting the polynucleotide with a citrate buffer to form an aqueous phase; c) solubilizing a mixture of ionizable lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) to form an ethanol phase; d) mixing the aqueous phase and the ethanol phase to form a precipitate; e) separating the precipitate to obtain the lipid nanoparticle formulation; A method comprising:

57. 25. The use according to any one of claims 1 to 24 for treating a signal regulatory protein alpha (SIRPα) mediated disease or condition.

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