Compositions and methods for inhibiting signal regulatory protein alpha (SIRP alpha) gene expression

Abstract

Provided herein in various embodiments are methods and compositions comprising a Signal Regulating Protein Alpha (SIRP Alpha) therapeutic agent. In certain embodiments, the present disclosure provides methods and compositions for modulating the expression of SIRP [alpha]. In certain embodiments, the methods and compositions are useful for the treatment of SIRP [alpha] mediated diseases or disorders.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 369,926, filed on July 29, 2022. The entire teachings of the above application are incorporated herein by reference.

[0003] Incorporation of XML Materials by Reference

[0004] This application incorporates by reference the sequence listing contained in the following concurrently filed Extensible Markup Language (XML) file:

[0005] a) File name: 00502374001.xml; created on June 27, 2023, size: 338132 bytes. Background Art

[0006] 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 (SIRP) receptor family and immune regulation. Members of the SIRP family have highly conserved extracellular regions but different transmembrane regions with opposite (i.e. inhibitory vs. activating) signaling potentials.

[0007] The interaction of SIRPα with CD47 is an important myeloid cell immune checkpoint. The interaction of SIRPα with CD47 provides a downregulatory signal that inhibits host cell phagocytosis. On the other hand, SIRPβ triggers cell activation signals by binding to the transmembrane adaptor protein DNA-X activating protein (DAP12). Therefore, the SIRP family belongs to a class of proteins known as paired receptors.

[0008] SIRPα and SIRPβ are both expressed on myeloid cells, while 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, although SIRPγ has a 10-fold weaker affinity for CD47 than SIRPα. Binding of CD47 to SIRPγ has been shown to mediate adhesion between T cells and antigen-presenting cells (APCs) and endothelial cells, leading to T cell activation, proliferation, and transendothelial migration.

[0009] Therefore, there is a need for compositions and methods that modulate SIRPα / CD47 interaction without altering SIRPβ or SIRPγ function to treat many SIRPα-associated diseases (eg, cancer immunotherapy). Summary of the invention

[0010] On the one hand, the present invention provides a method for reducing the expression of signal regulatory 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 regulatory protein alpha (SIRPα). In some embodiments, the nucleic acid is a double-stranded ribonucleic acid (dsRNA), an antisense oligomer (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), a circular RNA, a peptide nucleic acid (PNA), a locked nucleic acid (LNA) or a combination thereof.

[0011] In other aspects, the present 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.

[0012] In another aspect, the present disclosure provides a composition comprising: a nanocarrier selected from the group consisting of lipids, polymers, and lipid-polymer hybrids, and a nucleic acid that reduces the 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.

[0013] In some embodiments, the present disclosure provides a method of treating cancer. In some embodiments, the method is used to treat a disease or condition mediated by SIRPα.

[0014] Additionally, the present disclosure provides methods of making and using the methods and compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The foregoing will become apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead being placed upon illustrating the embodiments.

[0016] Figure 1 A sequence alignment between SEQ ID NO: 1 (NCBI Reference No. NP_001035111.1), SEQ ID NO: 2 (NCBI Reference No. NP_006056.2), and SEQ ID NO: 3 (NCBI Reference No. NP_061026.2) is shown. SIRP family proteins share a highly conserved N-terminal extracellular domain. The N-terminal signal sequence is boxed and the transmembrane region is underlined.

[0017] Figure 2A sequence alignment between SEQ ID NO: 4 (NCBI Reference NM_001040022.1), SEQ ID NO: 5 (NCBI Reference NM_006065.5) and SEQ ID NO: 6 (NCBI Reference NM_018556.4) is shown. SIRPα siRNA target sequences are underlined and variants with a minor allele frequency (MAF) > 0.05 are boxed.

[0018] Figure 3 Shown are CD45 gene-silenced macrophages among mouse peritoneal immune cells.

[0019] Figure 4 Dose- and time-dependent SIRPα gene silencing in murine peritoneal macrophages in vivo was demonstrated.

[0020] Figure 5A It was shown that siSIRPα-LNP reduced the expression level of SIRPα on human primary macrophages. Figure 5B Shows Figure 5A quantification.

[0021] Fig. 6A A shift in macrophage phenotype toward M1 is shown. Figure 6B It was shown that after silencing SIRPα expression in primary human macrophages by siSIRPα-LNP, the macrophage phenotype shifted from M2 phenotype. Figure 6C showed that SIRPα silencing increased the expression of the class I antigen-presenting molecule HLA-A2 in primary human macrophages.

[0022] Fig. 7A Shown, using one-way ANOVA with Tukey's multiple comparison test, that siRNA sequences were able to silence SIRPα expression on primary human macrophages in vitro. Figure 7B Shown, by two-way ANOVA using Sidak's multiple comparison test, that various lipids were able to form siSIRPα-LNPs in vitro and silence SIRPα expression on primary human macrophages.

[0023] Fig. 8A Maintaining viability of primary human macrophages with various siRNA sequences and lipids is shown. Figure 8B Maintaining viability of primary human macrophages with FL-A, MC3, and Lipid 5 lipids is shown.

[0024] Fig. 9A Several siRNAs were shown to enhance antigen presentation (HLA-DR expression). Fig. 9BIt was shown that various siRNAs can promote M1 polarization (CD86 expression). sihSIRPα18 is more effective than other siRNAs. RiM = RNAiMAX, a commercial transfection reagent.

[0025] Fig. 10A It was shown that various lipids were able to form siSIRPα-LNPs that promoted the expression of antigen presenting proteins (HLA-DR). Fig. 10B It was shown that various lipids were able to form siSIRPα-LNPs that promoted the expression of M1 phenotype proteins (CD86).

[0026] Fig.11A A schematic diagram of the experimental setup is shown. Fig. 11B Quantification of SIRPα silencing in macrophage populations as determined by flow cytometry is shown. Because the anti-SIRPα used for blocking (as an experimental treatment) inhibited the binding of the SIRPα staining antibody used in flow cytometry, SIRPα expression levels could not be determined under anti-SIRPα conditions (labeled NA in the bar graph).

[0027] Fig. 12A Shown is a shift in macrophage phenotype toward an M1 phenotype (CD86) following silencing of SIRPα expression in primary human macrophages by siSIRPα-LNP, but not following anti-SIRPα treatment. Data were collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells that had been pretreated with anti-HER2 or IgG isotype control antibodies. SKOV-3 pretreatment is indicated on the x-axis and macrophage pretreatment is indicated in the legend for each bar graph. Fig. 12B The levels of CD86 expression in the CD45+ macrophage population measured by flow cytometry (mean fluorescence intensity, MFI) are shown. siSIRPα-LNP+aHER2 synergistically promotes phagocytosis of ovarian cancer by primary human macrophages. Blocking SIRPα by pre-treatment of macrophages with anti-SIRPα did not improve phagocytosis compared with pre-treatment with isotype control antibody (labeled as IgG cont.) or siSIRPα. Fig. 12C CFSE fluorescence intensity within the CD45+ macrophage population quantified by flow cytometry is shown. Silencing of SIRPα with siSIRPα, but not blocking with anti-SIRPα, increases expression of the class I antigen-presenting molecule HLA-A2 in primary human macrophages. Data were collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells that had been pretreated with anti-HER2 or IgG isotype control antibodies.

[0028] Fig.13 A schematic diagram of the experimental setup is shown.

[0029] Figures 14A-14BIt was shown that blocking SIRPα with anti-SIRPα promoted reciprocal phagocytosis (macrophages engulfing other macrophages) compared to silencing SIRPα with siSIRPα. Fig.14A Flow cytometry data quantifying the percentage of macrophages labeled with a violet cell tracker compared to their initial seeding density (40%) are shown. Purple macrophages treated with siRNA-LNPs are not engulfed by other (green, i.e., untreated) macrophages {there is little depletion of the purple macrophage population compared to the initial seeding density}. Purple macrophages treated with anti-SIRPα or its isotype (IgG) control are engulfed by other macrophages (green, i.e., untreated) {in co-cultures pretreated with anti-SIRPα or IgG control, purple macrophages are heavily depleted). Fig. 14B It is shown that macrophages treated with antibodies (anti-SIRPα or its IgG control) (purple) are more actively engulfed by other macrophages (ie, mutually engulfed) than macrophages treated with siRNA-LNPs (green).

[0030] Fig.15A SKOV-3 human ovarian carcinoma was shown to express CD47. Fig. 15B Shows Fig.15A quantification. Fig. 15C Shown is the expression of HER2 in SKOV-3 human ovarian cancer. Fig.15D Shows Fig. 15C quantification.

[0031] Fig.16 A schematic diagram of the cancer cell phagocytosis assay is shown.

[0032] Fig.17A It was shown that siSIRPα-LNP reduced the expression level of SIRPα on human primary macrophages. Fig. 17B Shows Fig.17A quantification.

[0033] Fig.18A It was shown that siSIRPα-LNP+aHER2 synergistically promoted the phagocytosis of ovarian cancer by primary human macrophages. Fig.18B The percentage of macrophages positive for CFSE cancer cell tracer (indicative of phagocytosis) was quantified.

[0034] Figures 19A-19C Figure 3 shows that silencing of SIRPα expression in primary human macrophages by siSIRPα-LNPs results in a shift in macrophage phenotype from an M2 phenotype to an M1 phenotype. Data were collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells that had been pretreated with anti-HER2 or IgG isotype control antibodies. Fig.19A CD206 is shown. Fig.19B CD163 is shown. Fig.19C CD86 is shown.

[0035] Fig. 20 SIRPα silencing is shown to increase the expression of the class II antigen presenting molecule HLA-DR in primary human macrophages. Data were collected from co-cultures of primary human macrophages with SKOV-3 ovarian cancer cells that had been pretreated with anti-HER2 or IgG isotype control antibodies.

[0036] Fig.21A Macrophage cross-presentation experiments are shown. Fig.21B Shown are flow cytometry data demonstrating SIRPα gene silencing in a macrophage population. Fig. 21C Flow cytometry data showing the expression of M1 phenotype markers are shown.

[0037] Fig.22A showed that SIRPα gene silencing increased macrophage phagocytosis of B16 melanoma cells. Fig. 22B Cross-presentation of the model B16 melanoma antigen (OVA) on MHC class I molecules is shown.

[0038] Figures 23A-23C showed that SIRPα gene silencing increased cross-presentation of cancer antigens by macrophages. Fig.23A The percentage of cross-presenting macrophages is shown. Fig. 23B The calculated number of macrophages positive for both CSFE and MHC1-OVA (double positive) is shown. Fig.23C Shows Fig. 23B The data in were divided by the number of CSFE-positive macrophages and multiplied by 100.

[0039] Figures 24A-24B It was shown that various siRNA sequence-LNP formulation combinations were able to silence SIRPα expression on THP-1-derived macrophages in vitro. Fig.24A 50 nM siRNA is shown. Fig. 24B 5 nM siRNA is shown. DETAILED DESCRIPTION

[0040] A description of exemplary embodiments follows.

[0041] Several aspects of the present disclosure are described below with reference to examples that are only used for illustrative purposes. It should be appreciated that many specific details, relationships, and methods are stated to provide a complete understanding of the present disclosure. However, those of ordinary skill in the relevant art will readily recognize that the present disclosure can be implemented without one or more specific details, and can also be implemented with other methods, schemes, reagents, cell lines, and animals. The present disclosure is not limited by the order of the actions or events shown, because some actions can occur in different orders and / or occur simultaneously with other actions or events. In addition, not all actions, steps, or events shown need to be implemented according to the method of the present disclosure. Many techniques and procedures described or quoted herein are fully understood and generally adopted by those skilled in the art using conventional methodology.

[0042] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, for clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing significant differences from those commonly understood in the art. It should also be understood that terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with their meanings in the context of the relevant technology and / or as otherwise defined herein.

[0043] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] As used herein, the indefinite articles "a", "an", and "the" are to be understood as including plural references unless the context clearly indicates otherwise.

[0045] In this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of, for example, a stated integer or step or group of integers but not the exclusion of any other integer or step. When used herein, the term "comprises" may be replaced by the terms "containing" or "including".

[0046] As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the terms "comprising," "containing," "including," and "having," whenever used herein in the context of aspects or embodiments of the present disclosure, may be replaced with the terms "consisting of" or "consisting essentially of" in some embodiments to alter the scope of the present disclosure.

[0047] As used herein, the connection term "and / or" between multiple listed elements is understood to cover both individual and combined options. For example, where two elements are combined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within this meaning and therefore satisfies the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within this meaning and therefore satisfies the requirements of the term "and / or".

[0048] When a list is given, it is understood that each individual element of the list and each combination of the list is a separate embodiment unless otherwise specified. For example, a list of embodiments expressed as "A, B, or C" should be interpreted to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0049] Receptor basal activity

[0050] Some cell surface receptors have constitutive (i.e., intrinsic or basal) activity that activates intracellular signaling in the absence of a ligand. The 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 spatially blocking receptor-ligand interactions, antibody therapeutics are not considered to be able to inhibit receptor basal activity.

[0051] Nucleic acid therapeutics, such as siRNA therapeutics, represent a promising approach because such therapies can reduce the number of receptors on the cell surface as well as constitutive signaling.

[0052] Signal regulatory protein α (SIRPα)

[0053] Signal regulatory protein alpha or SIRPα (also known as CD172a or SHPS-1) is a transmembrane protein expressed on myeloid cells.

[0054] The SIRPα-CD47 immune checkpoint is the target of many blocking agents (e.g., anti-CD47 and anti-SIRPα antibodies and SIRPα fusion proteins), some of which are being evaluated in clinical trials (e.g., Hu5F9-G4, TTI-621, and ALX148).

[0055] However, although these therapies block CD47-SIRPα spatially and work well in combination with antibodies that regulate target cells, blocking CD47-SIRPα and regulating target cells are not sufficient to effectively engulf target cell macrophages. In addition, the use of a variety of blockers has been shown to cause heterotrimeric interactions, which have been 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) (the entire contents of which are incorporated herein by reference). In addition, anti-SIRPα antibodies cause opsonization of macrophages, resulting in mutual phagocytosis, in which macrophages are engulfed by other macrophages.

[0056] Furthermore, the gene encoding human SIRPα has been reported to be highly polymorphic. This is also true for the extracellular domain of SIRPα. Therefore, effective therapeutic targeting of SIRPα across different patient populations requires pan-allelic SIRPα inhibition while maintaining the specificity of SIRPα relative to other SIRP family molecules.

[0057] Therefore, there is a need to develop therapeutics that can be designed to cover polymorphic variability while avoiding regions with high allele frequencies.

[0058] One such approach is the use of nucleic acid therapeutics, such as siRNA against SIRPα. siRNA against SIRPα has been shown to promote polarization of macrophages from an anti-inflammatory M2 to a pro-inflammatory M1 phenotype. However, currently available siRNA against SIRPα has not been optimized for specificity and potency, and to date no siRNA against SIRPα has been formulated in lipid nanoparticles for in vivo or clinical use.

[0059] Therefore, there is a strong need for nucleic acid therapeutic compositions and methods designed to target the transmembrane region, cytoplasmic region or even the untranslated region of mRNA. In addition, by combining nucleic acid therapeutics with suitable delivery carriers such as lipid nanoparticles, cell type-specific delivery of nucleic acid therapeutics can be achieved, reducing off-target effects.

[0060] Compositions of the present disclosure

[0061] In some embodiments, the present disclosure provides compositions that are pharmaceutically acceptable compositions.

[0062] 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 has at least 80% identity to at least one of SEQ ID NOs: 140-227.

[0063] As used herein, the term "sequence identity" refers to the extent to which two sequences have identical residues at the same position when the sequences are aligned to achieve a maximum level of identity expressed as a percentage. For sequence alignment and comparison, one sequence is typically 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 a percentage of a position over the entire length of the reference sequence, wherein the reference sequence and the test sequence share the same nucleotides or amino acids when aligning the reference sequence and the test sequence to achieve the maximum level of identity. For example, when a test sequence has identical nucleotide residues at 70% of the same position over the entire length of the reference sequence when aligned to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0064] Those of ordinary skill in the art can easily use appropriate alignment methods or algorithms to compare sequences for comparison to obtain the maximum level of identity. In some cases, alignment can include introducing a gap to provide the maximum level of identity. Examples include Smith & Waterman, Adv. Appl. Math. 2:482 (1981) local homology algorithm, Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) homology alignment algorithm, Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988) similarity search method, computerized implementation of these algorithms (Wisconsin genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wis GAP, BESTFIT, FASTA and TFASTA), and visual inspection (generally referring to 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 containing conservative (or non-conservative) amino acid substitutions that do not adversely affect normal activity.

[0065] Thus, in some embodiments, the polynucleotide has at least about 70% identity to at least one of SEQ ID NOs: 18-227, such as 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 has 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 70-100% sequence identity to at least one of SEQ ID NOs: 18-227, e.g., about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100%.

[0066] In still further embodiments, the polynucleotide has at least about 70% identity to at least one of SEQ ID NOs: 140-227, such as 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 has 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 70-100% sequence identity to at least one of SEQ ID NOs: 140-227, e.g., about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100%.

[0067] In some embodiments, the compositions of the present disclosure, wherein contacting a cell with the nucleic acid reduces the concentration of SIRPα compared to the concentration of SIRPα in an otherwise identical cell.

[0068] As used herein, the term "reducing" refers to a regulation that reduces the concentration of SIRPα (e.g., the level before or in the absence of an agent regulation). In some embodiments, the agent (e.g., a composition) reduces the concentration of SIRPα by at least about 5%, for example, 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., a composition) reduces the concentration of SIRPα by at least about 5%, for example, 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 the concentration 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%.

[0069] In some embodiments, the concentration of SIRPα is measured using a cell-based functional assay.

[0070] The present disclosure also provides a composition comprising: a nanocarrier selected from the group consisting of lipids, polymers, and lipid-polymer hybrids, and a nucleic acid signal regulatory protein alpha (SIRPα) therapeutic agent; wherein the concentration of SIRPα in cells contacted with the composition is reduced compared to the concentration of SIRPα in other otherwise identical cells. In some embodiments, 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.

[0071] In certain embodiments of the present disclosure, the composition wherein the SIRPα therapeutic 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, wherein the contiguous nucleotide sequence is at least about 15 nucleotides in length. In still other embodiments, the composition wherein the SIRPα therapeutic agent 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 still other embodiments, the composition wherein the SIRPα therapeutic agent comprises a polynucleotide having at least about 80% identity to at least one of SEQ ID NOs: 18-139.

[0072] In some embodiments, the composition further comprises a 2'-deoxythymidine-3'-phosphate 3' overhang or a 2'-deoxythymidine-5'-phosphate-thiophosphate 3' overhang. In yet other 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 a siRNA duplex. In some embodiments, the siRNA duplex comprises a polynucleotide sequence that hybridizes to a complementary sequence.

[0073] 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 further embodiments, the pharmaceutical composition is an advanced therapeutic drug product. Examples of advanced therapeutic drug products include, but are not limited to, somatic cell therapy drug products, tissue engineering products, gene therapy drug products, tumor vaccines, or combinations thereof.

[0074] Methods of the present disclosure

[0075] In some embodiments, the present disclosure provides methods of treatment and methods of enhancing the efficacy of treatment comprising administering the compositions described herein.

[0076] As used herein, "therapy", "treat / treating / treatment" means to inhibit or alleviate the condition of a subject in need thereof. For example, therapy or treatment refers to any of the following: (i) preventing symptoms associated with a disease or condition; (ii) delaying the development of symptoms associated with a disease or condition; and / or (iii) reducing or reducing the severity of such symptoms that are expected to develop with the disease or condition. The term includes improving or controlling existing symptoms, preventing additional symptoms, and improving or preventing the underlying causes of these symptoms. Therefore, the term indicates that a beneficial result is conferred on at least some of the subjects (e.g., humans) being treated. Many therapies or treatments are effective for some but not all subjects undergoing the therapy or treatment.

[0077] As used herein, the term "effective amount" refers to the amount of a composition that is effective to achieve the desired therapy or treatment under the conditions of administration when administered alone or in combination to a cell, tissue, or subject. For example, an effective amount is an amount sufficient to produce an immune response to bring about a therapy or treatment effect. The effectiveness of a therapy or treatment (e.g., eliciting a humoral and / or cellular immune response) can be determined by suitable methods known in the art.

[0078] In some embodiments, the present disclosure provides a composition or agent for treating a disease or condition mediated by signal regulatory protein alpha (SIRPα). In some embodiments, the present disclosure provides a method for reducing the expression of signal regulatory protein alpha (SIRPα) in a cell, comprising contacting the cell with a lipid nanoparticle composition comprising a nucleic acid signal regulatory protein alpha (SIRPα) therapeutic agent.

[0079] As used herein, "subject" or "patient" includes humans, livestock, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), and livestock (e.g., chickens, pigs, cattle (e.g., cows, bulls, steers, or heifers), sheep, goats, horses, etc.), as well as non-livestock. In some embodiments, the subject is a mammal (e.g., a non-human mammal). In some embodiments, the subject is a human. In further embodiments, the subject of the present disclosure can be a cell, cell culture, tissue, organ, or organ system. In some embodiments, the subject is a male. In some embodiments, the subject is a female. In various embodiments, the subject is an infant, a teenager, a young adult, or an adult.

[0080] As used herein, "administering" or "administration" refers to taking steps to deliver a composition to a subject. Administration is performed, for example, once, multiple times, and / or over one or more extended periods of time. Administration includes direct administration (including self-administration) and indirect administration (including the act of prescribing or instructing a subject to take a composition). For example, as used herein, a subject (e.g., a patient) is instructed to self-administer a composition (e.g., a drug) or another person administers the composition and / or provides a prescription for the composition to the subject (e.g., a physician) administers the composition to the subject.

[0081] In some aspects, the present disclosure provides a method of treating a disease or condition mediated by signal regulatory protein alpha (SIRPα), comprising administering a lipid nanoparticle (LNP) composition comprising a nucleic acid signal regulatory protein alpha (SIRPα) therapeutic agent to a subject in need thereof.

[0082] In more aspects, the present disclosure provides a method for treating cancer, comprising administering a lipid nanoparticle (LNP) composition comprising a nucleic acid signal regulatory protein alpha (SIRPα) therapeutic agent to a subject in need thereof. In some embodiments of the present disclosure, the method relates to the treatment of cancer. The cancer treatments provided by the present disclosure include carcinoma, sarcoma, melanoma, lymphoma and / or leukemia. In some embodiments, the methods of the present disclosure may replace, continue or follow another treatment regimen. In some embodiments, another treatment regimen may include, but is not limited to, chemotherapy, radiotherapy, surgery, hormone therapy, biological response modifier therapy, immunotherapy and / or bone marrow transplantation.

[0083] In certain aspects, the methods disclosed herein provide 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 cancer, atypical meningioma, B-cell acute lymphoblastic leukemia, basal cell carcinoma, bile duct cancer, bladder cancer, bladder transitional cell carcinoma, glioblastoma, breast cancer, mammary duct adenocarcinoma, Burkitts lymphoma, cecal adenocarcinoma, cervical squamous cell carcinoma, chronic lymphoid leukemia, clear cell renal carcinoma, colon adenocarcinoma, skin melanoma, endometrioid adenocarcinoma, esophageal adenocarcinoma, esophageal squamous cell carcinoma, gastric adenocarcinoma, gastric cancer, glioma, head and neck squamous cell carcinoma. , hepatobiliary tumors, hepatoblastoma, hepatocellular carcinoma, HER2-positive breast cancer, kidney tumors, large cell lung cancer, lobular breast cancer, lung adenocarcinoma, lung cancer, lymphoid tumors, melanoma, multiple myeloma, nasopharyngeal squamous cell carcinoma, non-small cell lung cancer, oral squamous cell carcinoma, ovarian endometrial adenocarcinoma with squamous differentiation, ovarian serous adenocarcinoma, pancreatic acinar cell carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumors, papillary renal cell carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, skin cancer, small cell lung cancer, squamous cell lung cancer, thyroid cancer, thyroid tumors, and uterine carcinosarcoma.

[0084] In some embodiments, the present disclosure provides compositions that are pharmaceutically acceptable compositions.

[0085] As used herein, the term "pharmaceutically acceptable" refers to a substance that is suitable for use within the scope of sound medical judgment without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. For example, a substance is pharmaceutically acceptable when it is suitable for use in contact with animal or human cells, tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity or other adverse reactions, and its amount is used in a dosage form according to a dosing regimen, and is commensurate with a reasonable benefit / risk ratio.

[0086] The desired dose can be conveniently administered in a single dose, for example, so that the medicament is administered once a day, or with a multiple dose administered at appropriate intervals, for example, so that the medicament is administered 2, 3, 4, 5, 6 or more times a day. The daily dose can be divided into several portions, for example 2, 3, 4, 5, 6 or more, particularly when administered in a relatively large amount, when it is considered appropriate. Typically, the composition is administered about 1 to about 6 times (for example, 1, 2, 3, 4, 5 or 6 times) a day, or is administered as an infusion (for example, continuous infusion). In some embodiments, the interval can be every day, every week, every two weeks, every month, every quarter, every two or three months, once a year or twice a year, or any combination thereof.

[0087] Determining the dosage and route of administration for a particular agent, patient, and disease or condition is well within the capabilities of one skilled in the art. Preferably, the dosage causes no or minimal adverse side effects.

[0088] Doses lower or higher than the above doses may be required. The specific dosage and treatment regimen for any particular subject will depend on a variety of factors, such as the activity of the specific agent used, age, body weight, general health, sex, diet, time of administration, excretion rate, drug combination, severity and course of the disease, disorder or symptom, the subject's treatment of the disease, disorder or symptom, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of the agent in the composition will also depend on the specific agent in the composition.

[0089] In some embodiments, the concentration of one or more active agents provided in the composition is less than 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.

[0090] 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.

[0091] In certain embodiments, the administration of the composition can be carried out in any manner, such as by parenteral or non-parenteral administration, including by aerosol inhalation, injection, infusion, ingestion, blood transfusion, implantation or transplantation. For example, the composition described herein can be administered to the patient via artery, intradermal, subcutaneous, intratumor, intramedullary, intranodal, intramuscular, by intravenous (iv) injection, intranasal, intrathecal or intraperitoneal. In one aspect, the composition of the present disclosure is administered intravenously. In one aspect, the composition of the present disclosure is administered to the subject by intramuscular or subcutaneous injection. The composition can be, for example, directly injected into a tumor, lymph node, tissue, organ or infection site.

[0092] In some embodiments, the compositions described herein are used in combination with other known agents and therapies. As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the course of treatment of the subject, such as after the subject has been diagnosed with a disease and before the disease has been cured or eliminated or treatment has stopped for other reasons. In some embodiments, different treatments (e.g., additional therapeutic agents) may be administered simultaneously or sequentially.

[0093] In some embodiments, the present disclosure provides a method for preparing a lipid nanoparticle formulation, comprising: obtaining a signal regulatory protein alpha (SIRPα) therapeutic agent, wherein the SIRPα therapeutic agent 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 in a citrate buffer to form an aqueous phase; dissolving 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; mixing the aqueous phase with the ethanol phase, wherein a precipitate is formed; and isolating the precipitate to obtain a lipid nanoparticle formulation.

[0094] In some embodiments, the present disclosure provides compositions and methods for altering homeostatic control mechanisms to modulate phagocytic activity.

[0095] In some embodiments, the present disclosure provides compositions and methods for macrophage activation. In some embodiments, macrophage activation: increasing the inflammatory phenotype of myeloid cells by: i) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHCI, interleukin 1-β (IL-1b), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF and / or tumor necrosis factor α (TNF-α); ii) decreased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, TGFb and / or IL-10; iii) a protein selected from IL-1b, TNF-a, IL-12, IL-18, GM-CSF, CCL3, CCL4, and IL-1. -23; iv) increased secretion of at least one cytokine or chemokine of the group consisting of IL-1b, IL-6, and / or TNF-a and the expression of IL-10; v) increased CD8+ cytotoxic T cell activation; vi) increased recruitment of CD8+ cytotoxic T cell activation; vii) increased CD4+ helper T cell activity; viii) increased recruitment of CD4+ helper T cell activity; ix) increased NK cell activity; x) increased NK cell recruitment; xi) increased neutrophil activity; xii) increased macrophage and / or dendritic cell activity; and / or xiii) increased spindle morphology, spindle appearance, smoothness of appearance and / or number of dendrites.

[0096] Lipid Nanoparticles (LNP)

[0097] 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-coupled lipid, or any combination thereof. In embodiments of the present disclosure, the amount of the ionizable lipid is about 20 mol% to about 80 mol%, about 35 mol% to about 70 mol%, or about 40 mol% to about 65 mol% relative to the total mass of the composition. Relative to the total mass of the composition, the amount of cholesterol is about 10 mol% to about 60 mol%, about 20 mol% to about 55 mol%, or about 25 mol% to about 50 mol%.

[0098] In some embodiments of the present disclosure, the amount of phospholipids is about 3 mol% to about 55 mol% relative to the total mass of the composition. The amount of phospholipids is, 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% relative to the total mass of the composition.

[0099] Sterols are disclosed in US2023 / 0210993 A1. Examples of sterols include cholesterol, phytosterols (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholesterone, cholestenone, coprolol, cholesteryl-2'hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether.

[0100] Polymer-conjugated lipids are disclosed in WO 2023 / 114943 A2. The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0101] Ionizable lipids

[0102] In some embodiments of the present disclosure, the ionizable lipid is a lipid having at least one biodegradable group. The ionizable lipid can be a lipid having at least one ionizable amino group and at least one biodegradable group. Examples of the above-mentioned biodegradable groups include groups represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

[0103] In some embodiments, the lipid composition includes those of US2022 / 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 an ionizable lipid:

[0104]

[0105] In this formula, X represents -NR 1 -or-O-;R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -represented by a group, wherein R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or is represented by the following formula represents a group, and R 22 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms; R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32-represented by a group, wherein R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or is represented by the following formula The group represented by R 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms; R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , 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 one or more pairs may be linked to each other 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 are hydroxyl, carboxyl, -NR 45 R 46 represents an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or -O(CO)OR 41 、-O(CO)OR 42 , -(CO)OR 43 OR 44 A group represented by 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 alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, -NR 45 R 46 The amino group represented by -O(CO)O–R41 、-O(CO)–R 42 、-(CO)O–R 43 or-O–R 44 A group represented by 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.

[0106] In some embodiments, R 1 represents a hydrocarbon group having 6 to 24 carbon atoms. 2 and R 3 represents a hydrocarbon group having 3 to 24 carbon atoms, wherein the hydrocarbon group is an alkyl group, an alkenyl group, an alkynyl group, an alkyl group or an alkenyl group. 1 A hydrocarbon group having 6 to 24 carbon atoms represented by R 2 and R 3 The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be straight chain or branched chain, or may be chain or cyclic.

[0107] 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. Specifically, examples thereof include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (e.g., 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (e.g., 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, nonadecyl, eicosyl, and the like.

[0108] The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be straight chain or branched, or may be 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. Specifically, examples thereof include hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl (e.g., (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (e.g., (Z)-heptadec-8-enyl), heptadecenyl (e.g., (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (e.g., (Z)-octadec-9-enyl), octadecadienyl (e.g., (9Z,12Z)-octadecadienyl (9,12-dienyl), nonadecenyl, eicosenyl (e.g., (Z)-eicosadienyl), eicosadienyl (e.g., (11Z,14Z)-eicosadienyl) and the like.

[0109] The alkynyl group with 6 to 24 carbon atoms can be an alkynyl group with 6 to 20 carbon atoms, and the alkynyl group with 3 to 24 carbon atoms can be an alkynyl group with 6 to 20 carbon atoms. Specifically, examples thereof include hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, octadecynyl, etc. All of the above alkenyl groups can have one double bond or two double bonds. All of the above alkynyl groups can have one triple bond or two triple bonds.

[0110] By R 21 and R 31The hydrocarbon group having 1 to 24 carbon atoms represented by the invention 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 straight chain or branched chain, or may be chain or cyclic. The alkyl group having 10 to 24 carbon atoms may be an alkyl group having 12 to 24 carbon atoms. Specifically, examples thereof include decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (e.g., 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (e.g., 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, 2-butyloctyl, 1-pentylhexyl, 2-pentylheptyl, 3-pentyloctyl, 1-hexylheptyl, 1-hexylnonyl, 2 -hexyloctyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylnonyl, 2-heptylundecyl, 3-heptyldodecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, 2-(4,4-dimethylpent-2-yl)-5,7,7-trimethyloctyl, etc. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, or may be chain or cyclic. Specifically, examples thereof include decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (e.g., (Z)-tridec-8-enyl), tetradecenyl (e.g., tetradec-9-enyl), pentadecenyl (e.g., (Z)-pentadeca-8-enyl), hexadecenyl (e.g., (Z)-hexadec-9-enyl), hexadecadienyl, heptadecaenyl (e.g., (Z)-heptadec-8-enyl), heptadecadienyl (e.g., (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (e.g., (Z)-octadec-9-enyl), octadecadienyl (e.g., (9Z,12Z)-octadec-9,12-dienyl), etc. The alkynyl group having 10 to 24 carbon atoms may be linear or branched, or may be chain-like or cyclic. Specifically, examples thereof include decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecenyl, octadecynyl, etc. 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.

[0111] By R 22 and R 32The divalent hydrocarbon linking group represented by 1 to 18 carbon atoms can be an alkylene group with 1 to 18 carbon atoms or an alkenylene group with 2 to 18 carbon atoms. The alkylene group with 1 to 18 carbon atoms can be straight or branched, or can be chain-like or cyclic. The number of carbon atoms in the alkylene group can be 1 to 12, 1 to 10, or 2 to 10. Specifically, examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, elevenmethylene, dodecamethylene, etc. The alkenylene group with 2 to 18 carbon atoms can be straight or branched, or can be chain-like or cyclic. The number of carbon atoms in the alkenylene group can be 1 to 12, or 2 to 10.

[0112] -O(CO)O-, -O(CO)-, and -(CO)O- can be 1 In the range of L 1 -O(CO)O-, -O(CO)-, and -(CO)O- can be in the range of L 2 In the range of L 2 can be replaced and by R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 The alkyl group having 1 to 18 carbon atoms represented by alkyl may be straight or branched, or may be chain or cyclic. The number of carbon atoms in the alkyl group may be 1 to 12. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. In the case where the alkyl group has a substituent, the substituent is a hydroxyl group, a carboxyl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by -O(CO)-R 42 or-(CO)OR 43 Represents the group.

[0113] can be replaced and by R 5 , R 7 and R 8The alkyl group having 1 to 18 carbon atoms represented by alkyl may be straight or branched, or may be chain or cyclic. The number of carbon atoms in the alkyl group may be 1 to 12, or may be 1 to 8. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. In the case where the alkyl group has a substituent, the substituent is a hydroxyl group, a carboxyl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by -O(CO)-R 42 、-(CO)OR 43 OR 44 Represents the group.

[0114] Examples of the 4- to 7-membered ring which may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring and an azepane ring. The 4- to 7-membered ring may be a 6-membered ring, a piperidine ring or a morpholine ring.

[0115] In R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 In the case where an alkyl group having 1 to 18 carbon atoms which may be substituted 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. Specifically, examples of the aryl group include phenyl, naphthyl, anthracenyl, phenanthryl, and the like. The substituent on the aryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, a -NR 45 R 46 Amino group, or -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 , or -OR 44 The group represented by is a hydroxyl group or a carboxyl group. Specifically, examples of the substituted aryl group include hydroxyphenyl, carboxyphenyl and the like.

[0116] In R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R11 , and R 12 In the case where an alkyl group having 1 to 18 carbon atoms which may be substituted 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. Specifically, examples of the heteroaryl group include pyridyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, oxazolyl, and the like. The substituent on the heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, a -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、(CO)OR 43 OR 44 Specifically, examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxylpyridyl group, a pyridonyl group, and the like.

[0117] As R 41 , R 42 , R 43 , R 44 , R 45 , and R 46The alkyl group represented by the invention is a hydrocarbon group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkynyl group having 2 to 18 carbon atoms, and an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be straight or branched, or may be chain-like or cyclic. The number of carbon atoms in the alkyl group is 3 to 18, or 5 to 18. Specifically, examples thereof 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, octadecyl, etc. The alkenyl group having 2 to 18 carbon atoms may be straight or branched, or may be chain-like or cyclic. The number of carbon atoms in the alkenyl group is 3 to 18, or 5 to 18. Specifically, examples thereof include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (e.g., (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (e.g., (Z)-tridec-8-enyl), tetradecenyl (e.g., tetradec-9-enyl), pentadecenyl (e.g., (Z)-pentadecan ... Alkynyl groups include, for example, cyclopentyl ...

[0118] The X indicates -NR 1 -In the case of R 1 It may represent a hydrocarbon group having 6 to 24 carbon atoms or a 21 -L 1 -R 22 - represents a group. In this case, R 2 and R 3 One of them represents a hydrogen atom, and the other represents a hydrocarbon group having 6 to 24 carbon atoms or a 31 -L 2 -R 32-represented group.

[0119] In the case where X represents -O-, R 2 and R 3 may each independently represent a hydrocarbon group having 6 to 24 carbon atoms or a 31 -L 2 -R 32 -represented by a group. 4 , R 6 , R 9 , R 10 , R 11 , and R 12 may each represent a hydrogen atom. 5 It may be a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or -O(CO)-R 42 or-(CO)OR 43 An alkyl group having 1 to 18 carbon atoms which may be substituted, 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 In the case of an alkyl group, R 5 Can be used with R 4 , R 6 , R 10 , and R 12 are connected to form a ring which may contain an O atom. 5 is an alkyl group having 1 to 18 carbon atoms, which may be -O(CO)-R 42 or-(CO)OR 43 substituted alkyl groups having 1 to 18 carbon atoms, alkyl groups having 1 to 12 carbon atoms which may be substituted with aryl groups, or alkyl groups having 1 to 8 carbon atoms which may be substituted with hydroxyl groups, and alkyl groups having 1 to 18 carbon atoms or alkyl groups having -O(CO)-R 42 or-(CO)OR 43 A substituted alkyl group having 1 to 18 carbon atoms.

[0120] R 7 and R 8 may each independently represent a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, which may be -O(CO)-R 42 or-(CO)OR 43 substituted alkyl group having 1 to 18 carbon atoms, alkyl group having 1 to 8 carbon atoms which may be substituted by an aryl group, or alkyl group having 1 to 8 carbon atoms which may be substituted by a hydroxyl group. 7 and R 8 They may be linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0121] R 5Not with R 7 or R 8 connected and not with R 7 or R 8 Form a ring.

[0122] a+b can be 1 or 2, or 1. c+d can be 1 or 2, or 1.

[0123] The compound represented by formula (1) is a compound represented by formula (21).

[0124]

[0125] In this 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 Each independently represents R 31 -L 2 -R 32 - represented by a group, or R 2 and R 3 One of the representations is represented by R 31 -L 2 -R 32 - represents a group, the other represents a hydrocarbon group having 3 to 24 carbon atoms, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or the following formula The group shown, R 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R 5 Indicates that -O(CO)-R 42 or-(CO)OR 43 Substituted alkyl having 1 to 18 carbon atoms, wherein R 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.

[0126] In formula (21), R 2 and R 3 One of them can be R 31 -L 2 -R 32 -, and the other is a hydrocarbon group having 3 to 24 carbon atoms. In formula (21), L2 may represent -O(CO)- or -(CO)O-.

[0127] In some embodiments, the ionizable lipid suitable for use in the present disclosure is an ionizable lipid of formula (2) or a pharmaceutically acceptable salt thereof:

[0128]

[0129] in

[0130] R 51 and R 52 Each independently is optionally replaced by R 35 a substituted hydrocarbon group having 1 to 21 carbon atoms;

[0131] R 35 is hydroxyl, –G20–CH(R 55 )(R 56 ),–N(R 58 )(R 59 ) or –G 20 –R 60 ;

[0132] G 20 is –(CO)O– or –O(CO)–;

[0133] R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms;

[0134] R 58 and R 59 are each independently hydrogen or a cyclic hydrocarbon group having 3 to 6 carbon atoms, optionally replaced by R 36 replace;

[0135] R 60 is a hydrocarbon group having 1 to 18 carbon atoms;

[0136] R 36 Yes – N(R 61 )(R 62 ) or –G 20 –R 65 ;

[0137] R 61 and R 62 are each independently hydrogen or a cyclic hydrocarbon group having 3 to 6 carbon atoms;

[0138] R 65 is a hydrocarbon group having 1 to 18 carbon atoms, –L 40 –CH(R 66 )(R 67 ) or –G 20 –R 66 ;

[0139] L40 is a divalent hydrocarbon radical containing 1 to 6 carbon atoms;

[0140] R 66 and R 67 Each is independently a hydrocarbon group containing 1 to 10 carbon atoms or an alkoxy group containing 1 to 10 carbon atoms;

[0141] L 10 is a divalent hydrocarbon radical containing 1 to 18 carbon atoms;

[0142] G 30 Yes – S(CO)N(R 64 )–;

[0143] R 64 Yes – L 30 –G 20 –CH(R 55 )(R 56 );

[0144] a' is 0 or 1;

[0145] G 10 It's G 20 , –O(CO)O– or –N(R 63 )C(O)–;

[0146] c' is 0 or 1;

[0147] R 63 is a hydrocarbon group containing 1 to 18 carbon atoms;

[0148] L 20 is a divalent hydrocarbon radical containing 1 to 6 carbon atoms;

[0149] b' is 0 or 1;

[0150] R 53 , R 54 and R 57 are each independently hydrogen or optionally replaced by R 36 Substituted hydrocarbon groups containing 1 to 18 carbon atoms; and

[0151] L 30 It is a single bond or a hydrocarbon group containing 1 to 18 carbon atoms.

[0152] The compound represented by formula (2) can be represented by formula (2a)

[0153]

[0154] in

[0155] R 51 and R 52 Each independently is optionally replaced by R35 a substituted hydrocarbon group having 1 to 21 carbon atoms;

[0156] R 35 is hydroxyl or –G 20 –CH(R 55 )(R 56 );

[0157] G 20 is –(CO)O– or –O(CO)–;

[0158] R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms;

[0159] L 10 is a divalent hydrocarbon radical containing 1 to 18 carbon atoms;

[0160] G 10 is –(CO)O– or –O(CO)–; and

[0161] R 53 , R 54 and R 57 Each is independently hydrogen or a hydrocarbon group containing 1 to 18 carbon atoms.

[0162] The compound represented by formula (2) can be represented by formula (2b)

[0163]

[0164] in

[0165] R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms;

[0166] L 10 is a divalent hydrocarbon radical containing 1 to 18 carbon atoms;

[0167] G 10 is –O(CO)O–;

[0168] L 20 is a divalent hydrocarbon radical containing 1 to 6 carbon atoms;

[0169] R 53 , R 54 and R 57 are each independently hydrogen or optionally replaced by R 36 Substituted hydrocarbon groups containing 1 to 18 carbon atoms;

[0170] R 36 Yes–O(CO)–R 65 ;

[0171] R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 –CH(R 66 )(R 67 );

[0172] L 40 is a divalent hydrocarbon radical containing 1 to 6 carbon atoms; and

[0173] R 66 and R 67 Each is independently an alkoxy group having 1 to 10 carbon atoms.

[0174] The compound represented by formula (2) can be represented by formula (2c)

[0175]

[0176] in

[0177] R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms;

[0178] L 10 is a divalent hydrocarbon radical containing 1 to 18 carbon atoms;

[0179] G 10 Yes – N(R 63 )C(O)–;

[0180] R 63 is a hydrocarbon group containing 1 to 18 carbon atoms;

[0181] R 53 , R 54 and R 57 are each independently hydrogen or optionally replaced by R 36 Substituted hydrocarbon groups containing 1 to 18 carbon atoms;

[0182] R 36 Yes–(CO)OR 65 ;

[0183] R 65 Yes – L 40 –CH(R 66 )(R 67 );

[0184] L 40 is a divalent hydrocarbon radical containing 1 to 6 carbon atoms; and

[0185] R 66 and R 67Each is independently a hydrocarbon group containing 1 to 10 carbon atoms.

[0186] The compound represented by formula (2) can be represented by formula (2d)

[0187]

[0188] in

[0189] R 51 and R 52 are each independently a hydrocarbon group having 1 to 21 carbon atoms;

[0190] L 10 is a divalent hydrocarbon radical containing 1 to 18 carbon atoms;

[0191] G 30 Yes – S(CO)NR 64 –

[0192] R 64 Yes – L 30 –G 20 –CH(R 55 )(R 56 )

[0193] L 30 is a single bond or a hydrocarbon containing 1 to 18 carbon atoms;

[0194] G 20 is –(CO)O–;

[0195] R 55 and R 56 are each independently hydrogen or a hydrocarbon group having 1 to 18 carbon atoms;

[0196] G 10 is –(CO)O–; and

[0197] R 53 , R 54 and R 57 Each is independently hydrogen or a hydrocarbon group containing 1 to 18 carbon atoms.

[0198] R 51 or R 52The 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, 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 straight or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, more preferably 5 to 21 carbon atoms. 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, heptadecanyl and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be straight or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, more preferably 5 to 18. Examples include allyl, prenyl, pentanyl group), hexenyl, heptenyl, octenyl, nonenyl (preferably, (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably, (Z)-tridec-8-enyl), tetradecenyl (preferably, tetradec-9-enyl), pentadecenyl (preferably, (Z)-pentadeca-8-enyl), hexadecenyl (preferably, (Z)-hexadec-9-enyl), hexadecadienyl, heptadecaenyl (preferably, (Z)-heptadec-8-enyl), heptadecadienyi (preferably, (8Z,11Z)-heptadec-8,11-dienyi), octadecenyl (preferably, (Z)-octadec-9-enyl), octadecadienyl (preferably, (9Z,12Z)-octadecadienyl) and the like. Alkynyl with 2 to 21 carbon atoms can be straight or branched, and can be chain or cyclic. Carbon number is preferably 3 to 21, more preferably 5 to 21 carbon atoms. Examples include propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecinyl, octadecynyl, etc. Examples of hydrocarbon radicals with 1 to 18 carbon atoms include those example groups specifically listed in hydrocarbon radicals with 1 to 21 carbon atoms.

[0199] As the cycloalkyl group, 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 is preferred.

[0200] For the hydrocarbon group with 1 to 6 carbon atoms, it is preferred to have an alkyl group with 1 to 6 carbon atoms, an alkenyl group with 2 to 6 carbon atoms or an alkynyl group with 2 to 6 carbon atoms, and more preferably an alkyl group with 1 to 6 carbon atoms or an alkenyl group with 2 to 6 carbon atoms. The alkyl group with 1 to 6 carbon atoms can be straight or branched, and can be chain or cyclic. Its specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl and hexyl. The alkenyl group with 2 to 6 carbon atoms can be straight or branched, and can be chain or cyclic. Its specific examples include allyl, isoprenyl, pentenyl and hexenyl. The alkynyl group with 2 to 6 carbon atoms can be straight or branched, and can be chain or cyclic. Its specific examples include propargyl, butynyl, pentynyl and hexynyl.

[0201] R 66 and R 67 The hydrocarbon radical having 1 to 10 carbon atoms is preferably an alkyl radical having 1 to 10 carbon atoms, an alkenyl radical having 2 to 10 carbon atoms, or an alkynyl radical having 2 to 10 carbon atoms, preferably an alkyl radical having 1 to 10 carbon atoms or an alkenyl radical having 2 to 10 carbon atoms. The alkyl radical having 1 to 10 carbon atoms may be straight or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10 carbon atoms. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl and decyl. The alkenyl radical having 2 to 10 carbon atoms may be straight 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, isopentadienyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl) and decenyl. Alkynyl groups with 2 to 10 carbon atoms may be straight or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10 carbon atoms. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.

[0202] The compound represented by formula (1), (2), (2a), (2c) or (2d) may form a salt.

[0203] Examples of the salt in the basic group include: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed 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 formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0204] Examples of the salt in the acidic group include salts formed with alkali metals such as sodium and potassium; salts formed with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts formed with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine (1-ephenamine) and N,N'-dibenzylethylenediamine.

[0205] Among the above salts, for example, pharmacologically acceptable salts can be used. In some embodiments, the lipid represented by formula (1) and a method for preparing the same are described in WO 2019 / 235635 A1 and WO 2021 / 095876 A1 (which are incorporated herein by reference in their entirety).

[0206] In some embodiments of the present disclosure, the lipid or salt represented by formula (1) is lipid FL-A:

[0207]

[0208] In some embodiments of the present disclosure, the ionizable lipids may be the following lipids.

[0209] MC3; (4-(dimethylamino)butyric acid [(6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl] ester) WO 2010 / 054405A1:

[0210]

[0211] L-319; (9-[4-(dimethylamino)butyryloxy]heptadecanedioic acid bis[(Z)-non-2-enyl] ester) WO 2011 / 153493 A2, WO 2013 / 086354 A1, WO 2013 / 086322 A1:

[0212]

[0213] ALC-0315; (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) WO 2017 / 075331A1:

[0214]

[0215] SM-102; (8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester) WO 2017 / 099823A1:

[0216]

[0217] Lipid 5; (8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester) WO 2017 / 099823A1:

[0218]

[0219] Lipid 29; (8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobutene-1-yl]amino]propyl]amino]octanoic acid undecane-3-yl ester) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727:

[0220]

[0221] ATX-100; (4-[3-(dimethylamino)propylsulfonylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butyric acid pentadecane-8-yl ester) WO 2019 / 191780 A1:

[0222]

[0223] Lipid A9; (10-[3-(dimethylamino)propyl-nonanoylamino]azelaic acid bis(2-butyloctyl) ester) WO2017 / 004143A1:

[0224]

[0225] Lp01; ((9Z,12Z)-octadec-9,12-dienoic acid [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctyloxybutyryloxy)propyl] ester) WO 2015 / 09534 A2, WO 2020 / 219876 A1:

[0226]

[0227] TCL053; ((Z)-tetradec-9-enoic acid [2-[4-(dimethylamino)butyryloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl] ester) WO 2020 / 032184 A1:

[0228]

[0229] cKK-E12:

[0230]

[0231] C12-200:

[0232]

[0233] 306Oi10:

[0234]

[0235] 93-O17S:

[0236]

[0237] YSK05:

[0238]

[0239] Examples of ionizable lipids of formula (II) include:

[0240] MC3; (4-(dimethylamino)butyric acid [(6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl] ester) WO 2010 / 054405A1:

[0241]

[0242] L-319; (9-[4-(Dimethylamino)butyryloxy]heptadecanedioic acid bis[(Z)-non-2-enyl] ester)

[0243] WO 2011 / 153493A2, WO 2013 / 086354A1, WO 2013 / 086322A1:

[0244]

[0245] ALC-0315; (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) WO 2017 / 075331A1:

[0246]

[0247] SM-102; (8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester) WO 2017 / 099823A1:

[0248]

[0249] Lipid 5; (8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester) WO 2017 / 099823A1:

[0250]

[0251] Lipid 29; (8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobutene-1-yl]amino]propyl]amino]octanoic acid undecane-3-yl ester) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727:

[0252]

[0253] ATX-100; (4-[3-(dimethylamino)propylsulfonylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butyric acid pentadecane-8-yl ester) WO 2019 / 191780 A1:

[0254]

[0255] Lipid A9; (10-[3-(dimethylamino)propyl-nonanoylamino]azelaic acid bis(2-butyloctyl) ester) WO2017 / 004143A1:

[0256]

[0257] Lp01; ((9Z,12Z)-octadec-9,12-dienoic acid [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctyloxybutyryloxy)propyl] ester) WO 2015 / 09534 A2, WO 2020 / 219876 A1:

[0258]

[0259] TCL053; ((Z)-tetradec-9-enoic acid [2-[4-(dimethylamino)butyryloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl] ester) WO 2020 / 032184 A1:

[0260]

[0261] TCL065; (3-pentyloctanoic acid [2-[5-(dimethylamino)pentanoyloxymethyl]-3-(3-pentyloctanoyloxy)-2-(3-pentanoyloxymethyl)propyl] ester) WO 2020 / 032184 A1:

[0262]

[0263] Lipid 9; ((Z)-undec-5-enoic acid [(6Z,16Z)-12-[6-(dimethylamino)hexanoyloxy]docosa-6,16-dien-11-yl] ester) WO 2021 / 188389 A2:

[0264]

[0265] Lipid 19; ((9Z,12Z)-octadecadienoic acid [(6Z,16Z)-12-[6-(dimethylamino)hexanoyloxy]docosa-6,16-dien-11-yl] ester) WO 2021 / 188389 A2:

[0266]

[0267] GCL1; (5-(dimethylamino)pentanoic acid [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] ester) WO 2020 / 219941 A1:

[0268]

[0269] CL4H6; ((Z)-octadec-9-enoic acid [7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] ester):

[0270]

[0271] Example

[0272] method

[0273] mRNA isolation

[0274] Using Dynabeads TM mRNADIRECT TMPurification kit (Invitrogen #61012) is separated mRNA according to manufacturer's instructions. In brief, harvest cells and lyse in 100 μ l 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 μ l magnetic bead suspension is added to PCR plate, and then the lysed cells are added to the magnetic beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads are washed 2 times with 100 μ l washing buffer A (10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mM EDTA, 0.1% LiDS), and then washed 2 times with 100 μ l washing 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 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.

[0275] cDNA synthesis

[0276] cDNA was synthesized using ABI high capacity cDNA reverse transcription kit (Applied Biosystems #4368814) according to the manufacturer's instructions. In brief, the master mix of 10 μl of 10X buffer, 0.8 μl of 25X dNTP, 2 μl of 10X random primers, 1 μl of reverse transcriptase and 4.2 μl of water per reaction was added to the 10 μl mRNA solution separated using the above protocol. The plate was sealed, mixed, and incubated at room temperature for 10 minutes on a thermal cycler, then incubated at 37°C for 2 hours and at 85°C for 5 minutes.

[0277] Real-time PCR

[0278] 2 μl 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). Unless otherwise stated, each duplex was tested in two or three independent transfections, and each transfection was assayed in duplicate.

[0279] 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 against luciferase or mock-transfected cells. IC50s were calculated using GraphpadPrism software.

[0280] Lipid chemical structure

[0281] In some embodiments, the lipid chemical is FL-A or 3-ethyl-12-hexyl-6-(2-(octanoyloxy)ethyl)-10-oxo-9,11-dioxa-3,6-diazaheneicostan-21-oic acid 2-butyl octyl ester described in WO2019 / 235635 (incorporated herein by reference in its entirety):

[0282]

[0283] In some embodiments, the lipid chemical is MC3 or (4-(dimethylamino)butyric acid [(6Z,9Z,28Z,31Z)-heptatriacontria-6,9,28,31-tetraen-19-yl] ester, which is described in WO2010 / 054405 (incorporated herein by reference in its entirety):

[0284]

[0285] In some embodiments, the lipid chemical is Lipid 5 or octadecyl-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, which is described in WO2017 / 099823 (incorporated herein by reference in its entirety):

[0286]

[0287] In some embodiments, the lipid chemical is distearoylphosphatidylcholine (DSPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine:

[0288]

[0289] In some embodiments, the lipid chemical is cholesterol:

[0290]

[0291] In some embodiments, the lipid chemical is 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000 (DMG-PEG2000):

[0292]

[0293] Lipid Nanoparticle (LNP) Formulation

[0294] siRNA was diluted in 10 mM citrate buffer, pH 3.0, (aqueous phase), while an appropriate amount of lipid was co-dissolved in 200 standard strength ethanol (ethanol phase). Nanoparticles prepared by microfluidic devices were synthesized in a 3:1 v / v ratio of aqueous phase to ethanol phase. LNPs were then dialyzed against PBS in a 20 kDa MWCO box at 4 ° C or room temperature for overnight.

[0295] Particle size measurement

[0296] LNP particle size and PDI (polydispersity index) were obtained using a Zetasizer (Malvern). For size measurements, LNPs were diluted in PBS at a ratio of 1 / 200 v / v and z average values ​​were reported. For zeta potential measurements, LNPs were diluted in 0.1X PBS at a ratio of 1 / 200 v / v.

[0297] Quantification of siRNA concentration and encapsulation

[0298] The siRNA concentration in the dialyzed particles was determined by a modified Quant-iT RiboGreen RNA assay. Nanoparticle dilutions of approximately 1 ng pL-l siRNA were prepared in TE buffer (pH 8.5), and siRNA standards ranging from 2 ng pL-l to 0.125 ng pL-l were prepared. 50 pL of each solution was added to a separate well in a 96-well black polystyrene plate. 50 μL of TE buffer was added to each well. The plate was incubated at 37 ° C for 15 minutes while shaking at 350 rpm. After incubation, diluted RiboGreen reagent (100 pL per well) was added, and the plate was incubated for 3 minutes as described above. RiboGreen fluorescence was measured using a Tecan plate reader according to the provided protocol, and the nanoparticle siRNA concentration was determined using siRNA standards. It should be noted that two separate standards were developed: with and 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 equation:

[0299]

[0300] Sequence alignment

[0301] Amino acid and mRNA sequences were aligned using Cluster ω.

[0302] Example 1: siRNA targeting SIRPα

[0303] In certain embodiments of the present disclosure, siRNA duplexes are 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 targeting household pets, such as domestic cat or dog transcripts annotated in the NCBI gene database (Table 3).

[0304] Table 1: Human SIRPα mRNA target sequences

[0305] SEQ ID NO: NCBI reference number SEQ ID NO.4 NM_001040022.1 SEQ ID NO.7 NM_001040023.2 SEQ ID NO.8 NM_001330728.1 SEQ ID NO.9 NM_080792.3

[0306] Table 2: Mouse SIRPα mRNA target sequences

[0307] SEQ ID NO: NCBI reference number SEQ ID NO.10 NM_001177647.2 SEQ ID NO.11 NM_001291019.1 SEQ ID NO.12 NM_001291020.1 SEQ ID NO.13 NM_001291021.1 SEQ ID NO.14 NM_001291022.1 SEQ ID NO.15 NM_001355158.1

[0308] Table 3: Cat and dog SIRPα mRNA target sequences

[0309] SEQ ID NO: NCBI reference number Species SEQ ID NO.16 XM_045053803.1 Domestic cat (Felis catus) SEQ ID NO.17 XM_038433331.1 Domestic dog (Canis lupus familiaris)

[0310] To mitigate the risk of off-target gene silencing (e.g., silencing of SIRPβ and / or SIRPγ), human SIRPα, SIRPβ, and SIRPγ cDNA sequences were aligned using ClustalW2 and sequence identity was calculated ( Figure 2 In addition, single nucleotide polymorphism (SNP) analysis of human SIRPα was performed based on data provided by EnsEMBL (EnsEMBL.org).

[0311] As used herein, the term "sequence identity" refers to the extent to which two sequences have identical residues at the same position when the sequences are aligned to achieve a maximum level of identity expressed as a percentage. For sequence alignment and comparison, one sequence is typically 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 a percentage of a position over the entire length of the reference sequence, wherein the reference sequence and the test sequence share the same nucleotides or amino acids when aligning the reference sequence and the test sequence to achieve the maximum level of identity. For example, when a test sequence has identical nucleotide residues at 70% of the same position over the entire length of the reference sequence when aligned to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0312] Those of ordinary skill in the art can easily use appropriate alignment methods or algorithms to compare sequences for comparison to obtain the maximum level of identity. In some cases, alignment can include introducing a gap to provide the maximum level of identity. Examples include Smith & Waterman, Adv. Appl. Math. 2:482 (1981) local homology algorithm, Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) homology alignment algorithm, Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988) similarity search method, computerized implementation of these algorithms (Wisconsin genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wis GAP, BESTFIT, FASTA and TFASTA), and visual inspection (generally referring to 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 containing conservative (or non-conservative) amino acid substitutions that do not adversely affect normal activity.

[0313] Eighteen variants with a global mutant allele frequency (MAF) greater than 0.05 were extracted (Table 4) and mapped on the SIRPα sequence ( Figure 2 ). Variants with minor allele frequency (MAF) > 0.05 are boxed.

[0314] Human SIRPα siRNA target sequences were identified (Table 5) and mapped on the SIRPα sequence ( Figure 2; underlined). Mouse SIRPα siRNA target sequences were also identified (Table 6). siRNAs with more than 75% sequence identity are predicted to be cross-reactive. In some embodiments, the sequence identity of the siRNA to human SIRPβ and SIRPγ is less than 75% to minimize off-target silencing. In some embodiments, the siRNA target sequence is within SEQ ID NO:4. In some embodiments, the siRNA target sequence is between 361-1875 in SEQ ID NO:4. In some embodiments, the siRNA target sequence is between 1438-1875 in SEQ ID NO:4. In some embodiments, the siRNA target sequence is a region unique to SIRPα in SEQ ID NO:4. In some embodiments, the siRNA target sequence is within the peri-membrane, transmembrane and / or cytoplasmic domains of SIRPα. In some embodiments, for species cross-reactivity, the siRNA sequence identity to cat and dog SIRPα is greater than 75%.

[0315] Table 4: Human SIRPα variants

[0316] Variant ID Global minor allele frequency (MAF) Genome coordinates (Chr: bp) Allele rs1057114 0.4321 20:1915243 G / A / C rs114499682 0.4093 20:1915414 T / C rs115287948 0.4093 20:1915413 G / A / C rs139878822 0.3894 20:1915406-1915408 CGA / - rs138283486 0.3756 20:1915304 C / A / G rs146163282 0.3756 20:1915306 T / A / C / G rs149634649 0.3756 20:1915305 C / A / T rs17853847 0.3369 20:1915196 T / C / G rs1555770973 0.3229 20:1915318-1915319 AC / G rs17855612 0.2634 20:1915189 C / A / T rs1135192 0.2354 20:1915148 G / A rs143735290 0.2312 20:1915150 T / C rs146490405 0.231 20:1915151 G / A / T rs17855610 0.2157 20:1915174 C / G / T rs17855611 0.2153 20:1915180 G / A / C rs17855609 0.2111 20:1915167 A / G / T rs17853846 0.2109 20:1915169 A / G / T rs17855614 0.09585 20:1915319 C / A

[0317] In some embodiments, the disclosure provides the modification of siRNA sequence.In some embodiments, the modified siRNA sequence comprises at least one nucleotide protruding end covalently attached to the 3' end of sense sequence, antisense sequence or both sense and antisense sequence.In some embodiments, the modified siRNA sequence comprises a dinucleotide protruding end covalently attached to the 3' end of siRNA sense sequence, antisense sequence or both sense and antisense sequence.In some embodiments, the nucleotide protruding end comprises at least one artificial nucleotide.In some embodiments, the nucleotide protruding end comprises at least two artificial nucleotides.In some embodiments, the protruding end is U, T, UU, TT, dT, dTdT, sdT, dTsdT, sdTsdT or sdTdT.Non-limiting examples of modified siRNA sequences can be found in Table 7 and Table 8.

[0318] In further embodiments, the disclosure provides the design and use of siRNA sequences comprising one or more modified nucleotides. In some embodiments, the modified nucleotides are in the siRNA sense sequence. In some embodiments, the modified nucleotides are in the siRNA antisense sequence. In further embodiments, the modified nucleotides are siRNA sense and antisense sequences.

[0319] In some embodiments, the modified nucleotide is chemically modified. Chemical modifications can include modifications of the phosphate backbone (e.g., phosphorothioate bonds or boranoate bonds), 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 can include pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-aminouridine, 5-methyluridine, 2-thiouridine pseudouridine, 4-thiouridine pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, 5-methylcytidine, N4-methylcytidine, 2-thiouridine, 5-azacytidine, 5-hydroxycytidine, 5 -aminocytidine, 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.

[0320] In some embodiments, the modified nucleotide is 2'-O-methyladenosine (mA); 2'-O-methylcytidine (mC), 2'-O-methylguanosine (mG) or 2'-O-methyluridine (mU). Non-limiting examples of modified siRNA sequences can be found in Tables 7 and 8.

[0321] On the other hand, the present disclosure provides the design and use 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.

[0322] Table 5: Human SIRPα 19-mer mRNA target sequences

[0323]

[0324]

[0325]

[0326]

[0327] n / a indicates that the sequence has an insertion or deletion (no identity or 0%).

[0328] Table 6: Mouse SIRPα 19-mer mRNA target sequences

[0329]

[0330]

[0331] n / a means that sequence identity could not be calculated due to insertions or deletions in either sequence.

[0332] Table 7: Human SIRPα siRNA duplexes and sequences

[0333]

[0334]

[0335] Table 8: Mouse SIRPα siRNA duplexes and sequences

[0336]

[0337]

[0338] Table 9: Comparison of mouse SIRPα 19-mer mRNA target sequences

[0339]

[0340] Example 2: Dual response screening of human SIRPα siRNA duplexes and variants

[0341] siRNA modified variants derived from siRNA duplexes were designed based on sequence and chemical modifications (as described in Example 1) with high efficiency and specificity for human SIRPα RNA knockdown, and the resulting duplex variants were further tested in a THP-1 derived macrophage cell line.

[0342] THP-1 monocytes were cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000-15,000 cells / well. THP-1 monocytes were differentiated into macrophages by incubation with 100ng / mL phorbol 12-myristic acid 13-acetate (PMA) for 24 hours and a subsequent recovery period of 24 hours. THP-1 macrophages were transfected with SIRPα siRNA duplexes using Viromer Blue (0.5μl / well). A total of 30 siRNA duplexes, including variants from the original modified siRNA duplexes, were transfected into THP-1 macrophages and further verified. SIRPα siRNA was transfected at final concentrations of 5nM and 25nM, respectively. Anti-fLuc siRNA duplexes were used as negative controls. After incubation for 48 hours, 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.

[0343] Table 10: Relative fold change of SIRPα mRNA levels

[0344] siRNA duplex ID 25nM 5nM sihSIRPα_1 0.09 0.15 sihSIRPα_2 0.08 0.14 sihSIRPα_3 0.02 0.09 sihSIRPα_4 0.03 0.09 sihSIRPα_5 0.16 0.48 sihSIRPα_6 0.11 0.18 sihSIRPα_7 0.05 0.16 sihSIRPα_8 0.08 0.09 sihSIRPα_9 0.11 0.28 sihSIRPα_10 0.21 0.94 sihSIRPα_11 0.05 0.13 sihSIRPα_12 0.05 0.14 sihSIRPα_13 0.05 0.15 sihSIRPα_14 0.43 0.75 sihSIRPα_15 0.10 0.34 sihSIRPα_16 0.04 0.13 sihSIRPα_17 0.05 0.17 sihSIRPα_18 0.01 0.08 sihSIRPα_19 0.03 0.15 siLuc 1.00 1.00

[0345] Table 11: Relative fold change of SIRPα mRNA levels

[0346] siRNA duplex ID 25nM 5nM sihSIRPα_1-PM 0.07 0.60 sihSIRPα_2-PM 0.17 0.60 sihSIRPα_3-PM 0.02 0.56 sihSIRPα_4-PM 0.05 0.60 sihSIRPα_7-PM 0.13 0.74 sihSIRPα_8-PM 0.06 0.77 sihSIRPα_12-PM 0.07 0.68 sihSIRPα_13-PM 0.07 0.66 sihSIRPα_17-PM 0.21 0.71 sihSIRPα_18-PM 0.01 0.72 sihSIRPα_19-PM 0.05 0.75 siLuc 1.00 1.00

[0347] Example 3: Dose response of selected human SIRPα siRNA duplexes and variants

[0348] Human SIRPα siRNA duplexes and variants that resulted in a significant reduction in SIRPα mRNA levels in a double-dose screen were selected and further tested for dose response. In addition to these duplexes, 10 siRNA duplexes were tested. THP-1 monocytes were cultured using standard culture conditions and transferred to a 96-well plate at a density of 10,000-15,000 cells / well. THP-1 monocytes were differentiated into macrophages by incubation with 100ng / mL phorbol 12-myristic acid 13-acetate (PMA) for 24 hours and a subsequent recovery period of 24 hours. THP-1 macrophages were transfected with SIRPα siRNA duplexes using Viromer Blue (0.5μl / well). The doses of each SIRPα siRNA duplex included 25nM, 2.5nM, 250pM, and 25pM. After incubation for 48 hours, the treated cells were harvested and the remaining SIRPα mRNA levels were measured under each condition by RT-qPCR. Determination of the IC of each SIRPα siRNA duplex 50 The values ​​for each dose response are shown in Tables 12 and 13.

[0349] Table 12: IC of SIRPα siRNA duplexes 50 value

[0350]

[0351] Table 13: IC of SIRPα siRNA duplexes 50 value

[0352]

[0353] Example 4: Dual response screening of mouse SIRPα siRNA duplexes and variants

[0354] siRNA modified variants derived from siRNA duplexes were designed according to sequence and chemical modification (as described in Example 1), which have high efficiency and specificity for mouse SIRPαRNA knockout, and the resulting duplex variants were further tested in the J774 mouse macrophage cell line. J774 cells were cultured using standard culture conditions and transferred to a 96-well plate at a density of 10,000-15,000 cells / well. J774 macrophages were transfected with SIRPαsiRNA duplexes using Viromer Blue (0.5 μl / well). A total of 36 siRNA duplexes, including variants from the original modified siRNA duplexes, were transfected into J774 cells and further verified. SIRPαsiRNA was transfected at final concentrations of 5nM and 25nM, respectively. Anti-fLuc siRNA duplexes were used as negative controls. After incubation for 48 hours, 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.

[0355] Table 14: Relative fold change of SIRPα mRNA levels

[0356] siRNA duplex ID 25nM 5nM simSIRPα_1 0.53 0.77 simSIRPα_2 0.84 0.85 simSIRPα_3 0.21 0.33 simSIRPα_4 0.36 0.68 simSIRPα_5 0.18 0.39 simSIRPα_6 0.30 0.60 simSIRPα_7 0.15 0.50 simSIRPα_8 0.21 0.48 simSIRPα_9 1.40 0.81 simSIRPα_10 0.19 0.43 simSIRPα_11 0.18 0.67 simSIRPα_12 0.13 0.49 simSIRPα_13 0.09 0.55 simSIRPα_14 0.23 0.70 simSIRPα_15 0.13 0.44 simSIRPα_16 0.26 0.63 siLuc 1.00 1.00

[0357] Table 15: Relative fold change of SIRPα mRNA levels

[0358] siRNA duplex ID 25nM 2.5nM simSIRPα_3-PM 0.03 0.06 simSIRPα_5-PM 0.05 0.21 simSIRPα_10-PM 0.15 0.19 simSIRPα_11-PM 0.15 0.09 simSIRPα_13-PM 0.24 0.13 simSIRPα_17-PM 0.80 1.01 simSIRPα_18-PM 0.09 0.31 simSIRPα_19-PM 0.05 0.12 simSIRPα_20-PM 0.10 0.25 simSIRPα_21-PM 0.06 0.07 simSIRPα_22-PM 0.08 0.13 simSIRPα_23-PM 0.04 0.21 simSIRPα_24-PM 0.55 0.78 simSIRPα_25-PM 0.04 0.10 simSIRPα_26-PM 0.37 0.74 simSIRPα_27-PM 0.53 0.83 simSIRPα_28-PM 0.23 0.45 simSIRPα_29-PM 0.09 0.12 simSIRPα_30-PM 0.07 0.21 simSIRPα_31-PM 0.03 0.05 siLuc 1.00 1.00

[0359] Example 5: Dose response of selected mouse SIRPα siRNA duplexes and variants

[0360] Mouse SIRPα siRNA duplexes and variants that resulted in a significant reduction in SIRPα mRNA levels in a double-dose screen were selected and further tested for dose response. The J774 mouse macrophage cell line was cultured using standard culture conditions and transferred to a 96-well plate at a density of 10,000-15,000 cells / well. J774 cells were transfected with SIRPα siRNA duplexes using Viromer Blue (0.5 μl / well). The doses of each SIRPα siRNA duplex included 25nM, 2.5nM, 250pM, and 25pM. After 48 hours of incubation, the treated cells were harvested and the remaining SIRPα mRNA levels were measured under each condition by RT-qPCR. The IC of each SIRPα siRNA duplex was determined. 50 The values ​​for each dose response are shown in Table 16.

[0361] Table 16: IC of SIRPα siRNA duplexes 50 value

[0362]

[0363] Example 6: Formulation of siRNA using lipid nanoparticles (LNPs)

[0364] LNPs were synthesized with a molar ratio of ionizable lipid: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC): cholesterol: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) of 50:10:38.5:1.5, wherein the weight ratio of ionizable lipid: siRNA was 10:1.

[0365] Particle size and PDI values ​​were obtained by Zeta Sizer (Malvern), and siRNA concentration and encapsulation efficiency were assessed by a modified Ribo-Green assay (Thermo Fisher).

[0366] Table 17: Composition of LNP formulated SIRPα siRNA

[0367]

[0368] Table 18: Physicochemical properties of LNP

[0369] Particle size (nm) PDI Encapsulation efficiency (%) LNP1 125.3 0.05 94 LNP2 137.1 0.10 92 LNP3 136.4 0.05 93 LNP4 92.6 0.05 97 LNP5 107.2 0.22 95 LNP6 116.7 0.07 93 LNP7 95.1 1.2 92 LNP8 100.1 0.8 90

[0370] Example 7: LNPs enable in vivo delivery of myeloid cell-specific RNA

[0371] To examine the immune cell types to which LNPs could deliver siRNA, siRNA-containing LNPs targeting CD45 (siCD45) were prepared. 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 injected intraperitoneally with siCD45-LNPs or PBS.

[0372] siCD45 siRNA sequence sense: mCmUGGmCmUGAAmUmUmUmCAGAGmCA (SEQ ID NO: 228)-dTdT, antisense: UGCUCUGAAAUUmCAGCmCAG (SEQ ID NO: 229)-dTdT

[0373] Three days after administration, peritoneal lavage was performed to harvest immune cells in the peritoneal cavity, and these immune cells were stained for markers of different immune cell subsets and analyzed by flow cytometry.

[0374] Notably, macrophages and their precursors (Mono / Macs) showed statistically significant CD45 gene silencing at 0.01 mg / kg, whereas lymphocytes such as B cells and T cells did not show significant gene silencing ( Figure 3 ).

[0375] This macrophage-tropic property of LNPs should be exploited to further inhibit off-target gene silencing of SIRPγ in T cells.

[0376] Example 8: siSIRPα-LNP shows gene silencing in mice

[0377] To optimize the dosage and dosing schedule of siSIRPα-LNP in vivo, mice were injected intraperitoneally with siSIRPα-LNP, siLuc-LNP, or PBS. Gene silencing in peritoneal immune cells was analyzed by flow cytometry 3 or 7 days after administration. siSIRPα-LNP showed dose-dependent SIRPα gene silencing, with an effective dose 50 (ED50) of 0.00025 mg / kg ( Figure 4 ).

[0378] Example 9: siSIRPα-LNP promotes M1 phenotype in primary human macrophages

[0379] To investigate whether SIRPα gene silencing on macrophages could reprogram the tumor-associated macrophage phenotype from M2 to M1, the expression levels of macrophage polarization markers and antigen-presenting molecules were evaluated.

[0380] Primary human monocytes were isolated from peripheral blood mononuclear cells by a human 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 siSIRPα or siLuc (50 nM siRNA dose) formulated with FL-ALNP on day 8. These cells were harvested on day 3 after transfection and analyzed by flow cytometry. After transfection, 20 ng / mL M-CSF was maintained in the culture medium.

[0381] SIRPα siRNA showed approximately 90% silencing of SIRPα gene in primary human macrophages ( Figure 5A , 5B). In addition, SIRPα silencing reduced M2 marker expression (CD163) and increased M1 marker (CD86). These results strongly suggest that SIRPα siRNA can reprogram the tumor microenvironment ( Figures 6A-6C In addition, upregulation of antigen presenting molecules (MHC-I) suggests that SIRPα siRNA can enhance antigen presentation on macrophages and other myeloid cells ( Figures 6A-6C ).

[0382] Example 10: siSIRPα sequences and LNP formulations are generalizable (1)

[0383] To investigate whether other siRNA sequences and LNP formulations could silence SIRPα expression and reprogram the tumor-associated macrophage phenotype from M2 to M1, the expression levels of macrophage polarization markers and antigen-presenting molecules with various siRNA sequences and LNP formulations were evaluated.

[0384] Human primary monocytes were isolated from peripheral blood mononuclear cells by human 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 LNP-formulated siSIRPα or siLuc (50 nM siRNA dose) on day 8. siSIRPα and siLuc formulated with RNAiMax (RIM). The cells were harvested on day 3 post-transfection and analyzed by flow cytometry. After transfection, 20 ng / mL M-CSF was maintained in the culture medium.

[0385] The experimental protocol is further described in Example 12 below.

[0386] All SIRPα siRNA sequences formulated with FL-A and all LNPs formulated with sihSIRPα_18-PM (si18) showed approximately 50% SIRPα gene silencing on human primary macrophages while maintaining almost 100% cell viability ( Fig. 7A , 7B ; Fig. 8A , 8B ). In addition, among all the siRNAs tested, si18 showed the most significant upregulation of M1 markers (CD86) and MHC-II antigen presenting molecules (HLA-DR). It appears that si18 has higher immunogenicity than other siRNAs ( Fig. 9A , 9B Regarding ionizable lipids, FL-A induced the most significant upregulation of CD86 and HLA-DR, indicating that FL-A is most suitable for macrophage delivery for cancer applications ( Fig. 10A , 10B Based on these results, sihSIRPα_18-PM formulated with FL-A was selected for more detailed evaluation (hereinafter referred to as siSIRPα-LNP).

[0387] Table 19: List of siRNA-LNPs tested in human primary macrophages

[0388]

[0389] Example 11: siSIRPα sequences and LNP formulations are generalizable (2)

[0390] To investigate whether other siRNA sequences and LNP formulations could silence SIRPα expression, THP-1 derived macrophages were used to evaluate SIRPα expression levels with various siRNA sequences and LNP formulations. The following ionizable lipids were tested and the LNP formulations are listed in Table 20.

[0391] FL-A (2-butyloctyl 3-ethyl-12-hexyl-6-(2-(octanoyloxy)ethyl)-10-oxo-9,11-dioxa-3,6-diazaheneicosane-21-acid salt) as described in WO 2019 / 235635 A1:

[0392]

[0393] GCL1; (5-(dimethylamino)pentanoic acid [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] ester), described in WO 2020 / 219941 A1:

[0394]

[0395] Lipid A9; (10-[3-(dimethylamino)propyl-nonanoylamino]azelaic acid bis(2-butyloctyl) ester), described in WO 2017 / 004143A1:

[0396]

[0397] ALC-0315; (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate), described in WO 2017 / 075331 A1:

[0398]

[0399] Lipid 5; (8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester), described in WO 2017 / 099823A1:

[0400]

[0401] Table 20: List of siRNA-LNPs tested in THP-1 derived macrophages

[0402]

[0403] The experimental protocol was as described in Example 3. The doses of each SIRPα siRNA duplex included 50 nM and 5 nM.

[0404] All SIRPα siRNA sequences and LNP formulations showed more than 50% SIRPα gene silencing on THP-1 derived macrophages at 50 nM ( Fig.24A , 24B ). Based on these results, it can be concluded that the siRNA sequences selected in the screen are potent enough to exhibit gene silencing when formulated with various ionizable lipids.

[0405] Example 11: Comparison of siRNA and anti-SIRPα antibody

[0406] Ovarian cancer phagocytosis

[0407] To compare siSIRPα gene silencing driven by siSIRPα with antibody blockade of SIRPα activity, primary human macrophages were pretreated with siSIRPα-LNPs (or siLuc-LNPs as a negative control) or anti-SIRPα (or isotype (IgG) control antibody) prior to co-culture with human ovarian cancer cells (SKOV-3). These pre-treated macrophages were also labeled with purple BMQC cell tracking fluorescent dye (Invitrogen, ThermoFisher Scientific #C10094) prior to co-culture. SKOV-3 ovarian cancer cells were stained with green fluorescent CFSE cell tracer (BioLegend). Under some conditions, SKOV-3 cells were pretreated with anti-HER2 antibodies (as in Example 12). Macrophages and SKOV-3 ovarian cancer cells were seeded at a ratio of 1:2 and incubated as co-culture for 2.5 hours ( Fig.11A , 11B ). Flow cytometry was used to quantify SIRPα silencing, expression levels of macrophage polarization markers and antigen-presenting molecules, and phagocytosis.

[0408] Treatment with siSIRPα resulted in >80% silencing of SIRPα gene in primary human macrophages ( Fig.11A , 11B ). SIRPα gene silencing with siSIRPα also increased M1 marker expression compared to blocking SIRPα with a blocking antibody ( Figures 12A-12C ). The intensity of CFSE fluorescence in the macrophage population indicates the phagocytosis of macrophages. Compared with blocking SIRPα with blocking antibodies, the increased CFSE fluorescence in the CD45+ macrophage population after siSIRPα treatment indicates that siSIRPα treatment increases the phagocytosis of SKOV-3 ovarian cancer by macrophages ( Figures 12A-12C In addition, the expression level of class 1 antigen presenting molecules (HLA-A2) in the CD45+ macrophage population was increased after siSIRPα treatment compared with treatment with SIRPα blocking antibody ( Figures 12A-12C ).

[0409] Mutual phagocytosis

[0410] Mutual phagocytosis is the engulfment of macrophages by other macrophages. To compare mutual phagocytosis after treatment with siSIRPα or anti-SIRPα blocking antibody, primary human macrophages were treated with siSIRPα-LNP, siLuc-LNP (as negative control), anti-SIRPα or IgG control antibody (as negative control). Treated macrophages were labeled with purple BMQC cell tracking fluorescent dye (Invitrogen, ThermoFisher Scientific #C10094) and placed in co-culture with untreated macrophages. Untreated macrophages were labeled with green fluorescent CFSE cell tracer (BioLegend). Purple (i.e., pretreated) macrophages and green (i.e., untreated) macrophages were seeded at a ratio of 1:1.5 and incubated as co-culture for 2.5 hours ( Fig.13 ). The remaining purple- and green-labeled macrophages were then quantified by flow cytometry and mutual phagocytosis was assessed.

[0411] Macrophages treated with anti-SIRPα blocking antibody or IgG control antibody were substantially depleted after 2.5 h of co-culture, whereas macrophages treated with siSIRPα-LNP or siLuc-LNP were not depleted ( Fig.14A , 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 purple (i.e., pretreated) macrophages was quantified as the percentage of purple macrophages that were positive for the CFSE cell tracer. Macrophages pretreated with anti-SIRPα blocking antibody showed more phagocytosis of other (i.e., green) macrophages than macrophages pretreated with siSIRPα ( Fig.14A , 14B ).

[0412] Example 13: siSIRPα-LNP+aHER2 antibody promotes phagocytosis of ovarian cancer cells by primary human macrophages

[0413] To determine whether SIRPα gene silencing on macrophages synergizes with antibody therapeutics, primary human macrophages that had been transfected with siRNA-LNPs (siSIRPα or siLUC as negative controls) were co-cultured with human ovarian cancer cells (SKOV-3 cells) that had been pre-incubated with antibody therapeutics (aHER2).

[0414] Here, it was observed that SKOV-3 human ovarian cancer cells expressed high levels of the SIRPα ligand CD47 ( Fig.15A , 15B), suggesting that the CD47-SIRPα interaction may contribute to macrophage inhibition, and the ability of SKOV-3 ovarian cancer to evade host immune defenses. Engagement of CD47 to SIRPα expressed by macrophages triggers an inhibitory cascade that prevents macrophage phagocytosis of target (SKOV-3) cells bearing CD47, i.e., a “don’t eat me” OFF signal. Conversely, antibodies capable of engaging Fc receptors on macrophages trigger a stimulatory cascade that leads to macrophage activation and phagocytosis, i.e., an “eat me” ON signal ( Fig.16 ). SKOV-3 ovarian cancer cells express HER2 ( Fig. 15C , 15D ) showed that anti-HER2 antibodies could opsonize SKOV-3 cells, engage Fc receptors on macrophages, and provide a positive signal for phagocytosis ( Fig.16 ).

[0415] 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-LNPs as described in Example 12 and used for co-culture 3 days after transfection (as described below). SKOV-3 ovarian cancer cells were labeled with the fluorescent cell tracer CFSE (BioLegend) and pre-incubated with anti-HER2 or IgG isotype control antibodies before co-culture. Flow cytometry was used to quantify the level of SIRPα silencing after SKOV-3 cancer cells were co-cultured with transfected macrophages for 2 hours ( Fig.17A , 17B ), the percentage of CFSE cancer cell tracer-positive macrophages (surface phagocytosis) ( Fig.18A , 18B ), expression of M1 relative to M2 macrophage phenotype markers ( Figures 19A-19C ) and expression of antigen presenting molecules ( Fig. 20 ).

[0416] siSIRPα-LNP resulted in more than 90% SIRPα gene silencing in primary human macrophages ( Fig.17A , 17B ). Combination of SIRPα silencing and anti-HER2 cancer therapy synergistically promotes macrophage phagocytosis of SKOV-3 ovarian cancer cells ( Fig.18A , 18B The level of phagocytosis observed after siSIRPα+aHER2 treatment (approximately 70%) exceeded that observed after siSIRPα silencing or anti-HER2 treatment alone ( Fig.18A , 18B In addition, SIRPα silencing reduced the expression of M2 markers (CD206, CD163 ( Fig.19A , 19B)) and increased expression of M1 markers (CD86)( Fig.19C These results strongly suggest that SIRPα siRNA can reprogram the tumor microenvironment. Upregulation of class II antigen presenting molecules (HLA-DR) ( Fig. 20 ) suggested that SIRPα siRNA could enhance antigen presentation in primary human macrophages.

[0417] Example 14: siSIRPα-LNP promotes phagocytic antigen cross-presentation in vitro

[0418] Cross-presentation is the ability of certain professional antigen-presenting cells (primarily macrophages and dendritic cells) to internalize, process, and present extracellular antigens on MHC class I molecules. Because antigen cross-presentation has the ability to elicit antigen-specific CD8 T cell responses, it is important to stimulate macrophages to cross-present tumor antigens.

[0419] To determine whether SIRPα gene silencing promotes cross-presentation of tumor antigens by macrophages, peritoneal cells from mice treated with intraperitoneal injection of siSIRPα-LNPs (or siLUC-LNPs as negative control) were harvested 3 days after siRNA treatment and placed in co-culture with B16 mouse melanoma cells ( Figures 21A-21C ). B16 melanoma cells express ovalbumin (OVA), which is used as a model cancer antigen. To track B16 melanoma cells and quantify phagocytosis, B16 cells were stained with CSFE cell tracer before being placed in co-culture. After overnight co-culture of labeled B16 melanoma cells with murine IP cells, flow cytometry was used to identify macrophages and macrophages within macrophage populations: SIRPα silencing was assessed, phagocytosis of B16 melanoma was quantified, and cancer antigen presentation on MHC class I was assessed, and phenotype was assessed.

[0420] Intraperitoneal injection of siSIRPα-LNPs resulted in a significant silencing of SIRPα expression on murine peritoneal macrophages and increased their CD86 expression, indicating a shift toward an M1 phenotype ( Fig.21A -C). Macrophage phagocytosis of B16 antigen was quantified as the percentage of macrophages positive for the B16 cell tracer CFSE. siSIRPα-LNP treatment significantly increased the percentage of macrophages exhibiting B16 phagocytosis ( Fig.22A , 22B Importantly, macrophage presentation of a model antigen (OVA) on MHC class I was also significantly increased by siSIRPα-LNP treatment ( Fig.22A , 22BCross-presentation requires internalization of extracellular antigen and its subsequent presentation on MHC class I. Therefore, to more directly quantify cross-presentation, flow cytometry was used to simultaneously assess B16 antigen uptake (indicated by CFSE) and OVA presentation on MHC class I on macrophages ( Fig.23A -C). The percentage of cross-presenting macrophages was quantified as the fraction of CFSE+ macrophages that were also MHC class I-OVA positive.

[0421] % cross presentation = (MHC-OVA + CFSE +) / (CFSE +) × 100

[0422] The percentage of cross-presenting macrophages increased significantly after intraperitoneal siSIRPα-LNP treatment ( Figures 23A-23C ). Taken together, these results strongly suggest that SIRPα siRNA can promote macrophage activation (M1 phenotype) and cancer antigen cross-presentation by macrophages, ultimately promoting a strong anticancer immune response in cancer patients.

[0423] References:

[0424] Barclay, A. Neil, and Deborah Hatherley. "The counterbalance theory forevolution and function of paired receptors." Immunity 29.5 (2008): 675-678.

[0425] Dietrich,Jes,et al."Cutting edge:signal-regulatory proteinβ1is aDAP12-associated activating receptor expressed in myeloid cells."The Journal of Immunology 164.1(2000):9-12.

[0426] Kurlander, RJ "Blockade of Fc receptor-mediated binding to U-937cells by murine monoclonal antibodies directed against a variety of surface antigens." The Journal of Immunology 131.1 (1983): 140-147.

[0427] Madeira,Fábio,et al."The EMBL-EBI search and sequence analysis toolsAPIs in 2019."Nucleic acids research 47.W1(2019):W636-W641.

[0428] Muntjewerff,Elke M.,Luca D.Meesters,and Geert Van den Bogaart."Antigen cross-presentation by macrophages."Frontiers in Immunology 11(2020):1276.

[0429] Piccio,Laura,et al."Adhesion of human T cells to antigen-presentingcells through SIRPβ2-CD47interaction costimulates T-cell proliferation."Blood105.6(2005):2421-2427.

[0430] Stefanidakis,Michael,et al."Endothelial CD47 interaction with SIRPγis required for human T-cell transendothelial migration under shear flowconditions in vitro."Blood,The Journal of the American Society of Hematology112.4(2008):1280-1289.

[0431] US2018 / 0312600

[0432] Voets, Erik, et al. "Functional characterization of the selective pan-allele anti-SIRPαantibody ADU-1805 that blocks the SIRPα–CD47 innate immune checkpoint." Journal for immunotherapy of cancer 7.1 (2019): 1-15.

[0433] WO 2010 / 054405 A1

[0434] WO 2017 / 178653 A2

[0435] WO 2019 / 235635 A1

[0436] Ye, Xiaojing, et al. "Signal regulatory proteinα associated with the progression of oral leukoplakia and oral squamous cell carcinoma regulatesphenotype switch of macrophages." Oncotarget 7.49(2016):81305.

[0437] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0438] 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 as encompassed by the following 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. The method of claim 1, wherein the SIRPα therapeutic agent is double-stranded ribonucleic acid (dsRNA), antisense oligomer (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), circular RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA) or a combination thereof.

3. The method of claim 1 or 2, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity with a consecutive 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 consecutive nucleotide sequence is at least about 15 nucleotides in length.

4. The method of claim 3, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity with a consecutive 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 consecutive nucleotide sequence is approximately 15-30 nucleotides in length. 5 . The method of claim 4 , wherein the SIRPα therapeutic 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. The method of claim 7, wherein the at least one modified pyrimidine is 2'-O-methylcytidine-3'-phosphate or 2'-O-methyluridine-3'-phosphate.

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

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

11. The method of claim 10, wherein the siRNA duplex comprises 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 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: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: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: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: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; 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. 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. The method of claim 12, wherein the lipid nanoparticles comprise ionizable lipids, phospholipids, sterols, polymer-conjugated lipids, or any combination thereof.

14. The method of claim 13, wherein the ionizable lipid is selected from the group consisting of FL-A; MC3; Lipid 5; LipidA9; ALC-0315; and GCL1.

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

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

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

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

22. A composition according to claim 21, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity with a consecutive 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 consecutive nucleotide sequence is at least 15 nucleotides in length.

23. A composition according to claim 22, wherein the SIRPα therapeutic agent comprises a polynucleotide having at least 80% identity with a consecutive 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 consecutive nucleotide sequence is approximately 15-30 nucleotides in length.

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

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

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

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

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

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

31. The composition of claim 30, wherein the siRNA duplex comprises 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 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: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: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: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:199 and SEQ ID NO:190; 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; 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. 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. The composition of claim 32, wherein the composition is a lipid nanoparticle composition.

34. The composition of claim 33, wherein the lipid nanoparticles comprise ionizable lipids, phospholipids, sterols, polymer-conjugated lipids, or a combination thereof.

35. The composition of claim 34, wherein the lipid nanoparticles comprise at least one substance 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. 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. The composition of claim 36, wherein the composition is formulated as a pharmaceutical composition.

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

39. A method for treating a disease or condition mediated by signal regulatory protein alpha (SIRPα), 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 signal regulatory protein alpha (SIRPα) therapeutic agent.

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

42. The method of claim 41, wherein the SIRPα therapeutic 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, wherein the contiguous nucleotide sequence is at least 15 nucleotides in length.

43. The method of claim 42, wherein the SIRPα therapeutic 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, wherein the contiguous nucleotide sequence is approximately 15-30 nucleotides in length.

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

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

47. The method of claim 46, wherein the at least one modified pyrimidine is 2'-O-methylcytidine-3'-phosphate or 2'-O-methyluridine-3'-phosphate.

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

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

50. The method of claim 49, wherein the siRNA duplex comprises 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 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: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: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: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: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; 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. 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. 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. The method of claim 52, wherein the ionizable lipid is selected from FL-A; MC3; Lipid 5; LipidA9; ALC-0315; and GCL1.

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 method of claim 54, wherein the SIRPα-mediated disease or condition is selected from the group consisting of acute myeloid leukemia, adenosquamous lung cancer, atypical meningioma, B-cell acute lymphoblastic leukemia, basal cell carcinoma, bile duct cancer, bladder cancer, bladder transitional cell carcinoma, glioblastoma, breast cancer, mammary duct adenocarcinoma, Burkitts lymphoma, cecal adenocarcinoma, cervical squamous cell carcinoma, chronic lymphatic leukemia, clear cell renal carcinoma, colon adenocarcinoma, skin melanoma, endometrioid adenocarcinoma, esophageal adenocarcinoma, esophageal squamous cell carcinoma, gastric adenocarcinoma, gastric cancer, glioma tumors, head and neck squamous cell carcinoma, hepatobiliary tumors, hepatoblastoma, hepatocellular carcinoma, HER2-positive breast cancer, kidney tumors, large cell lung cancer, lobular breast cancer, lung adenocarcinoma, lung cancer, lymphoid tumors, melanoma, multiple myeloma, nasopharyngeal squamous cell carcinoma, non-small cell lung cancer, oral squamous cell carcinoma, ovarian endometrial adenocarcinoma with squamous differentiation, ovarian serous adenocarcinoma, pancreatic acinar cell carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumors, papillary renal cell carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, skin cancer, small cell lung cancer, squamous cell lung cancer, thyroid cancer, thyroid tumors, and uterine carcinosarcoma.

56. A method for preparing a lipid nanoparticle formulation, comprising: a) obtaining a signal regulatory protein alpha (SIRPα) therapeutic agent, wherein 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 citrate buffer to form an aqueous phase; c) dissolving 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 with the ethanol phase, wherein a precipitate is formed; e) separating the precipitate to obtain a lipid nanoparticle preparation.

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

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