MIRNA TO CONTROL THE EXPRESSION OF IN VIVO ADMINISTRATION CONSTRUCTS

Abstract

Compositions and methods for making and using engineered phagocytic cells that express a chimeric antigen receptor having an enhanced phagocytic activity for stable and durable expression are described and can be suitably used for immunotherapy in cancer or infection.

Description

MIRNA TO CONTROL THE EXPRESSION OF IN VIVO ADMINISTRATION CONSTRUCTS CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 489,307, filed March 9, 2023, U.S. Provisional Application No. 63 / 582,220, filed September 12, 2023, and U.S. Provisional Application No. 63 / 593,159, filed October 25, 2023, each of which is incorporated herein by reference in its entirety. FUNDAMENTALS OF THE INVENTION

[0002] Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. Although myeloid cells have many functions, including phagocytosis and their ability to activate T cells, harnessing these functions for therapeutic uses has remained elusive. New avenues are therefore being sought to utilize other cell types for the development of enhanced therapeutics, including, but not limited to, T-cell malignancies.

[0003] Manipulated myeloid cells can also be short-lived in vivo, phenotypically diverse, sensitive, plastic, and often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes has been difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with the transfection of myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells, such as monocytes / macrophages, comprise many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer to these cells an inefficient process. This is especially true for activated macrophages that undergo a dramatic change in their physiology after exposure to Petition 870250099881, dated 10 / 31 / 2025, page 9 / 364 2 / 178 immune or inflammatory stimuli. Viral transduction into these cells was hampered because macrophages are late-stage cells that generally do not divide; therefore, some of the vectors that rely on integration into a replicative genome have found limited success. Furthermore, macrophages are quite responsive to “danger signals,” and therefore several of the original viral vectors that were used for gene transfer induced potent antiviral responses in these cells, making these vectors unsuitable for gene delivery. SUMMARY

[0004] The present disclosure relates to immunotherapy using myeloid cells, including phagocytic cells of the immune system, particularly macrophages. It is an object of the invention disclosed herein to take advantage of one or more of these functions of myeloid cells for therapeutic uses. For example, it is an object of the invention disclosed herein to express a recombinant polypeptide in myeloid cells when systemically distributed in vivo. In one aspect, with the aid of recombinant technology, a manipulated nucleic acid (e.g., mRNA) encoding the recombinant polypeptide is administered in vivo; the manipulated nucleic acid comprises sequences incorporated into the 5' and / or 3' untranslated regions that allow inhibition of the recombinant polypeptide expression in non-myeloid cells. For example, it is an object of the invention disclosed herein to minimize off-target effects in the manipulation of myeloid cells in vivo.For example, it is an objective of the invention disclosed in this document to exploit the phagocytic activity of myeloid cells, including manipulated myeloid cells, for therapeutic uses. For example, it is an objective of the invention disclosed in this document to exploit the ability of myeloid cells, including manipulated myeloid cells, to promote T cell activation. For example, it is an objective of the invention disclosed in this document to exploit the ability of myeloid cells, including manipulated myeloid cells, to promote T cell activation. Petition 870250099881, dated 10 / 31 / 2025, page 10 / 364 3 / 178 to promote the secretion of tumoricidal molecules. For example, it is an objective of the invention disclosed in this document to exploit the ability of myeloid cells, including manipulated myeloid cells, to promote the recruitment and trafficking of immune cells and molecules. For example, it is an objective of the invention disclosed in this document to generate activated myeloid cells in vivo. The present disclosure provides innovative methods and compositions that can successfully process a myeloid cell to express an exogenous nucleic acid in vivo, for example, an exogenous recombinant nucleic acid comprising a sequence encoding a polypeptide that has a therapeutic effect. For example, innovative methods and compositions are provided in this document by which recombinant nucleic acids administered to an organism can preferentially or specifically express themselves in myeloid cells in vivo.

[0005] This disclosure involves producing and using engineered myeloid cells (e.g., CD14+ cells, such as macrophages or other phagocytic cells) that can attack and kill (ATAK) diseased cells directly and / or indirectly, such as cancer cells and infected cells. Engineered myeloid cells, such as monocytes and macrophages and other phagocytic cells, can be prepared by incorporating nucleic acid sequences (e.g., mRNA, plasmids, viral constructs) encoding a gene of interest, such as a chimeric fusion protein (CFP) that has an extracellular binding domain specific for disease-associated antigens (e.g., cancer antigens), into cells using, for example, nucleic acid technology, synthetic nucleic acids, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection.It has been found that myeloid cells can be manipulated to have a wide and diverse range of activities. For example, it has been found that myeloid cells can be manipulated to express a chimeric fusion protein (CFP) containing an antigen-binding domain. Petition 870250099881, dated 10 / 31 / 2025, page 11 / 364 4 / 178 a wide and diverse range of activities. For example, myeloid cells have been found to be manipulated to have enhanced phagocytic activity so that, after CFP binding to an antigen on a target cell, the cell exhibits increased phagocytosis of the target cell. Myeloid cells have also been found to be manipulated to promote T cell activation so that, after CFP binding to an antigen on a target cell, the cell promotes the activation of T cells, such as T cells in the tumor microenvironment. Manipulated myeloid cells can be manipulated to promote the secretion of tumor molecules so that, after CFP binding to an antigen on a target cell, the cell promotes the secretion of tumor molecules from nearby cells.Manipulated myeloid cells can be manipulated to promote the recruitment and trafficking of immune cells and molecules so that, after CFP binding to an antigen on a target cell, the cell promotes the recruitment and trafficking of immune cells and molecules to the target cell or a tumor microenvironment.

[0006] This disclosure is based on the important finding that myeloid cells can be manipulated to express a gene of interest and can be targeted to a site of inflammation, infection, or tumor. Manipulated myeloid cells, therefore, can overcome at least some of the limitations of CAR-T cells, including being readily recruited to solid tumors; with a manipulated survival duration, thus reducing the risk of prolonged persistence resulting in aplasia and immunodeficiency; myeloid cells cannot be contaminated with T cells; myeloid cells can avoid fratricide, for example, because they do not express the same antigens as malignant T cells; and myeloid cells have a multitude of antitumor functions that can be deployed. In some respects, manipulated myeloid-derived cells may be safer immunotherapy tools for targeting and destroying diseased cells.However, cell manipulation. Petition 870250099881, dated 10 / 31 / 2025, page 12 / 364 5 / 178 myeloid cells to enhance gene expression for non-viral DNA therapy remains a significant challenge.

[0007] Furthermore, myeloid cells, such as macrophages, have been found ubiquitously in the tumor environment (TME) and are notably the most abundant cells in some tumor types. As part of their role in the immune system, myeloid cells, such as macrophages, are naturally involved in the elimination of diseased cells. The present invention relates to harnessing the function of myeloid cells specifically for targeting, killing, and direct and / or indirect elimination of diseased cells, as well as delivery payloads such as antigens and cytokines.

[0008] Manipulated myeloid cells can also be short-lived in vivo, phenotypically diverse, sensitive, plastic, and often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes has been difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with the transfection of myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells, such as monocytes / macrophages, comprise many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer to these cells an inefficient process. This is especially true for activated macrophages that undergo a dramatic change in their physiology after exposure to immune or inflammatory stimuli.Viral transduction into these cells was hampered because macrophages are late-stage cells that generally do not divide; therefore, some of the vectors that rely on integration into a replicative genome have met with limited success. Furthermore, macrophages are quite responsive to “danger signals,” and thus several of the original viral vectors used for gene transfer induced potent antiviral responses in these cells, making these... Petition 870250099881, dated 10 / 31 / 2025, page 13 / 364 6 / 178 vectors unsuitable for gene distribution.

[0009] This disclosure provides innovative methods and compositions that can successfully electroporate, transfect, or transduce a myeloid cell, or induce a genetic modification in a myeloid cell, with the aim of enhancing a functional aspect of a myeloid cell, additionally, without compromising the cell's differentiation capacity, maturation potential, and / or plasticity. A composition comprising a nucleic acid comprising (i) a DNA sequence encoding an mRNA or (ii) the mRNA sequence, wherein the mRNA sequence comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the 5' UTR is at least 45 nucleotides long and a sequence encoding a target gene or protein is located between them. In some embodiments, the 5' UTR sequence and / or the 3' UTR sequence may comprise a non-native sequence, that is, a sequence that is not present in an unmodified transcript.In some embodiments, the nucleic acid sequence is a recombinant nucleic acid sequence. In some embodiments, the nucleic acid or nucleic acid sequence is manipulated. In some embodiments, the nucleic acid or nucleic acid sequence is synthetic. In some embodiments, the nucleic acid or nucleic acid sequence is transcribed in vitro. In some embodiments, the nucleic acid or nucleic acid sequence is isolated or purified.

[0010] In some embodiments, the nucleic acid, for example, a manipulated nucleic acid, an in vitro transcribed mRNA (IVT), a synthetic or modified nucleic acid, as described in this document, is not conjugated or associated with a lipid nanoparticle (LNP). In some embodiments, the manipulated nucleic acid is systematically delivered in vivo to alter myeloid cells in vivo, the manipulated nucleic acid being encapsulated in a nanoparticle. In some embodiments, the nanoparticle comprises non-lipid polymeric compounds. Petition 870250099881, dated 10 / 31 / 2025, page 14 / 364 7 / 178

[0011] In some embodiments, the manipulated nucleic acid is formulated into a lipid nanoparticle for systemic delivery to alter myeloid cells in vivo.

[0012] In some embodiments, the nucleic acid, for example, a manipulated nucleic acid, an mRNA transcribed in vitro, a synthetic or modified nucleic acid, as described in this document, is electroporated into a cell. In some embodiments, the nucleic acid, for example, an IVT mRNA, comprises a 3' UTR and a 5' UTR. In some embodiments, the 3' UTR sequence is followed by a poly A sequence. In some embodiments, the poly A sequence is at least 100 nucleotides long. In some embodiments, the poly A sequence is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides long. In some embodiments, the poly-A sequence is greater than 200 nucleotides in length. In some embodiments, within the 5' UTR, a translation initiation site is at least 15 nucleotides downstream of the 5' end of the mRNA.In some embodiments, a translation initiation site is at least 20 nucleotides downstream of the 5' end of the mRNA. In some embodiments, the translation initiation site is at least 25 nucleotides downstream of the ribosome binding site. In some embodiments, the translation initiation site is at least 30 nucleotides downstream of the ribosome binding site. In some embodiments, the 5' end of the nucleic acid comprises a methyl guanylate cap. In some embodiments, the nucleic acid comprises a single translation initiation site. In some embodiments, the mRNA coding sequence is 100–10,000 nucleotides in length. In some embodiments, the nucleic acid sequence comprises a 5' UTR sequence selected from the sequences described in this document. In some embodiments, the nucleic acid sequence comprises a 3' UTR sequence selected from the sequences described in this document. A [description of a specific sequence] is provided in this document. Petition 870250099881, dated 10 / 31 / 2025, page 15 / 364 8 / 178 composition comprising a cell comprising the composition described in the section above, wherein in some embodiments the cell is a myeloid cell, a CD14+ cell, a CD16- cell or a CD14+ / CD16- cell or the cell is a T cell. A pharmaceutical composition comprising the composition described above is also provided herein; and a pharmaceutically acceptable excipient.

[0013] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a myeloid cell is provided in this document, the method comprising bringing a heterogeneous population of cells comprising a myeloid cell and a non-myeloid cell into contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences; wherein the exogenous polypeptide is expressed in the myeloid cell of the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the non-myeloid cell of the heterogeneous cell population. “Bringing into contact” includes contacting the heterogeneous population on the outer surface of a cell in the heterogeneous cell population.

[0014] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a myeloid cell is provided in this document, the method comprising placing a heterogeneous population of cells comprising a myeloid cell and a non-myeloid cell in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the exogenous polypeptide is expressed in the myeloid cell of the heterogeneous cell population, and wherein the Petition 870250099881, dated 10 / 31 / 2025, page 16 / 364 9 / 178 exogenous polypeptide is not substantially expressed in the non-myeloid heterogeneous cell population.

[0015] In one aspect, a method of expressing an exogenous polypeptide of a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences; and wherein the recombinant nucleic acid lacks a sequence encoding a viral protein or portion thereof, and wherein the exogenous polypeptide is expressed in the cell that is the first cell type of the heterogeneous population of cells, and wherein the exogenous polypeptide is not substantially expressed in the cell that is the second cell type of the heterogeneous population of cells.

[0016] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the composition lacks a sequence encoding a viral protein or portion thereof, and wherein the exogenous polypeptide is expressed in the cell that is the first cell type of the heterogeneous cell population, and wherein the exogenous polypeptide is not Petition 870250099881, dated 10 / 31 / 2025, page 17 / 364 10 / 178 substantially expressed in the cell that is the second cell type in the heterogeneous cell population.

[0017] In one aspect, a method for expressing a chimeric fusion protein from a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the chimeric fusion protein, and (b) one or more miRNA target sequences; and wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous population of cells, and wherein the chimeric fusion protein is not substantially expressed in the cell that is the second cell type of the heterogeneous population of cells.

[0018] In one aspect, a method for expressing a chimeric fusion protein of a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous population of cells, and wherein the chimeric fusion protein is not substantially expressed in the cell that is the second cell type of the heterogeneous population of cells.

[0019] In some forms, the non-myeloid cell of the population Petition 870250099881, dated 10 / 31 / 2025, page 18 / 364 11 / 178 heterogeneous cells express a microRNA (miRNA) comprising a sequence that is reverse complementary to at least one or more miRNA target sequences, or a fragment thereof.

[0020] In some embodiments, the non-myeloid cell of the heterogeneous cell population comprises any of the following: a hepatocyte, a lymphoid cell (e.g., T cell, B cell), an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell, and a skeletal cell. A myeloid cell may be, for example, a macrophage, a monocyte, a dendritic cell, a monocyte-derived cell, or a CD14+ cell.

[0021] In some forms, the myeloid cell from the heterogeneous cell population does not substantially express a miRNA that is reverse complementary to at least one or more target miRNA sequences.

[0022] In some embodiments, the recombinant nucleic acid is mRNA.

[0023] In some embodiments, the exogenous polypeptide is a recombinant polypeptide that activates a myeloid cell when expressed in the myeloid cell ex vivo.

[0024] In some embodiments, activating the myeloid cell involves activating or increasing the chemotaxis of the myeloid cell compared to a cell that does not express the recombinant polypeptide.

[0025] In some modalities, activating the myeloid cell involves activating the phagocytosis of the myeloid cell.

[0026] In some embodiments, the exogenous polypeptide comprises a recombinant receptor protein, a recombinant cytokine, chemokine, growth factor, or a portion thereof.

[0027] In some embodiments, the composition comprising the recombinant nucleic acid further comprises a carrier of Petition 870250099881, dated 10 / 31 / 2025, page 19 / 364 12 / 178 nucleic acid distribution.

[0028] In some embodiments, the nucleic acid delivery vehicle comprises a lipid molecule.

[0029] In some embodiments, the nucleic acid delivery vehicle comprises a cationic lipid. In some embodiments, the nucleic acid delivery vehicle comprises a lipid nanoparticle. In some embodiments, the nucleic acid delivery vehicle is a polymer.

[0030] In one aspect, a composition comprising one or more recombinant polynucleic acids engineered to express a sequence encoding an exogenous polypeptide in a myeloid cell is provided in this document, the recombinant nucleic acid comprising the sequence encoding the exogenous polypeptide, and one or more miRNA target sequences, wherein one or more miRNA target sequences comprise a sequence that is reverse complementary to at least one miRNA sequence or fragment thereof that is expressed in a non-myeloid cell and is absent in a myeloid cell.

[0031] In some embodiments, one or more recombinant polynucleic acids comprise (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide.

[0032] In some embodiments, one or more recombinant polynucleic acids comprise a vector comprising the sequence encoding the human polypeptide and one or more miRNA target sequences.

[0033] In some embodiments, one or more recombinant polynucleic acids are one mRNA molecule.

[0034] In some embodiments, mRNA comprises a 5' UTR and a 3' UTR.

[0035] In some modes, one or more target sequences of Petition 870250099881, dated 10 / 31 / 2025, p. 20 / 364 13 / 178 miRNAs are incorporated into the UTR 5' and UTR 3'.

[0036] In some embodiments, each of the one or more miRNA target sequences comprises a target sequence for a miRNA expressed in a non-myeloid cell and in which the expression of the miRNA is absent in a myeloid cell.

[0037] In some embodiments, the miRNA target sequence comprises a target sequence for a miRNA that is expressed in a hepatocyte, a lymphoid cell (e.g., T cell, B cell), an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell, or a skeletal cell.

[0038] In some embodiments, the miRNA target sequence comprises a target sequence for a miRNA that is expressed in an epithelial cell and a hepatocyte. In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 4. In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 5.

[0039] In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 6. In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 7. In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 8. In some embodiments, the miRNA target sequence comprises the sequence established in SEQ ID NO: 9. In some embodiments, the 3' mRNA UTR comprises one or more sequences selected from SEQ ID NOs: 5, 7, and 9. In some embodiments, the 5' mRNA UTR comprises one or more sequences selected from SEQ ID NOs: 4, 6, and 8.

[0040] In some embodiments, the composition additionally comprises a nucleic acid delivery vehicle.

[0041] In some embodiments, the acid delivery vehicle Petition 870250099881, dated 10 / 31 / 2025, p. 21 / 364 14 / 178 nucleic acid comprises a lipid molecule.

[0042] In some embodiments, the nucleic acid delivery vehicle comprises a cationic lipid.

[0043] In some embodiments, the nucleic acid delivery vehicle comprises a lipid nanoparticle.

[0044] In some embodiments, the nucleic acid delivery vehicle is a polymer.

[0045] In some embodiments, the exogenous polypeptide is a therapeutic polypeptide.

[0046] In some embodiments, the exogenous polypeptide comprises a receptor protein for a cytokine, chemokine, growth factor, or a portion thereof.

[0047] In some embodiments, the exogenous polypeptide comprises a chimeric antigen receptor.

[0048] In one aspect, a method of expressing an exogenous polypeptide in a subject's myeloid cell is provided in this document, the method comprising administering to the subject the composition as defined in any of the embodiments above; wherein, after administration, the exogenous polypeptide is expressed in the subject's myeloid cell and substantially not expressed in a subject's non-myeloid cell.

[0049] In one aspect, a method is provided in this document for producing a composition to selectively express an exogenous polypeptide in a myeloid cell, the method comprising: (i) synthesizing a polynucleotide sequence that is a reverse complement of a miRNA that is expressed in a non-myeloid cell, wherein the expression of the miRNA is absent in a non-myeloid cell; (ii) synthesizing a manipulated polynucleic acid comprising a sequence encoding the exogenous polypeptide and a miRNA target sequence comprising the polynucleotide sequence that is the reverse complement. Petition 870250099881, dated 10 / 31 / 2025, page 22 / 364 15 / 178 of the miRNA in (i), wherein the sequence encoding the exogenous polypeptide and the target miRNA sequence are operationally linked.

[0050] This document provides a method for treating a subject with a disease or condition comprising administering the pharmaceutical composition described herein to a subject in need. This document also provides a method for expressing a nucleic acid-encoded protein in a cell, the method comprising (a) incorporating into the cell ex vivo a nucleic acid comprising (i) DNA encoding an mRNA or (ii) the mRNA, and (b) expressing a protein encoded by a sequence of the mRNA; wherein the mRNA comprises (i) a 5' UTR sequence; a sequence encoding a protein or polypeptide and (ii) a 3' UTR sequence, wherein the expression of the protein or polypeptide is detectable for at least 24 hours, at least 48 hours, or at least 72 hours after (a).

[0051] This document provides a method for expressing a nucleic acid-encoded protein in a cell, the method comprising (a) incorporating into the cell ex vivo a nucleic acid comprising (i) DNA encoding an mRNA or (ii) the mRNA and (b) expressing a protein encoded by a sequence of the mRNA; wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the 5' UTR is at least 45 nucleotides long.

[0052] In some embodiments, the 3' UTR is followed by a poly A tail and the poly A tail is enzymatically added to the 3' end of the mRNA. In some embodiments, the 3' UTR is followed by a poly A tail and the poly A tail is an encoded poly A tail. Incorporation by Reference

[0053] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually referenced. Petition 870250099881, dated 10 / 31 / 2025, p. 23 / 364 16 / 178 indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE FIGURES

[0054] The novel features of the invention are presented in particular in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the invention are used and the appended figures.

[0055] FIG. 1 shows data indicating that the 3' UTR of β-globin with four miR122-5p binding sites suppresses mRNA expression in hepatocytes (Huh-7 cells), but not in primary monocytes.

[0056] FIG. 2 shows data indicating that the 3' UTR of β-globin with four miR192-5p binding sites suppresses mRNA expression in hepatocytes, but not in primary monocytes.

[0057] FIG. 3 represents data indicating the 3' UTR of β-globin with four miR192-3p binding sites. No significant change was observed with and without the miRNA.

[0058] FIG. 4 shows data indicating that the 3' UTR of β-globin with four miR27b-5p binding sites prolongs mRNA expression specifically in primary monocytes.

[0059] FIG. 5 shows data indicating that the UTR 3' of β-globin with four miR27b-3p binding sites suppresses mRNA expression in primary hepatocytes and monocytes (without cell specificity).

[0060] FIG. 6 shows data indicating the 3' UTR of β-globin with four miR10a-5p binding sites. No significant effect with or without miRNA was observed.

[0061] FIG. 7 shows the 3' UTR of β-globin with four miR10a-3p binding sites. No significant effect with or without miRNA was observed.

[0062] FIG. 8 shows data indicating that GC33-CD89 lambda 10 and lambda 4 demonstrate prolonged expression in primary monocytes. Petition 870250099881, dated 10 / 31 / 2025, p. 24 / 364 17 / 178

[0063] FIG. 9A shows AI-based mRNA sequence enhancement and folding to provide greater mRNA stability.

[0064] FIG. 9B (left) shows data illustrating an exemplary screening of various codon- and sequence-optimized mRNA structures to prepare long-term stable mRNA constructs with different UTRs; several combinations for constructs were generated using AI algorithms.

[0065] FIG. 9B (right) shows that microRNA sites stabilize mRNA in myeloid cells, which is confirmed in several cell types. Chimeric receptor expression continued beyond 7 days after mRNA introduction (e.g., transfection) when miRNA was administered.

[0066] FIG. 10 shows data indicating results obtained from unprecedentedly high longevity of primary monocytes, improving mRNA coding sequence along with the incorporation of microRNA sites, as shown in the figures.

[0067] FIGS. 11A and 11B demonstrate the experimental setup and constructs used to understand whether UTRs with miRNA sequences influence the half-life of a generic mRNA. For this construct, luciferase (F. Luc) was used as a surrogate marker for mRNA comprising the engineered UTRs. Myeloid cells were cultured ex vivo, as indicated. The cells are from a human donor.

[0068] FIG. 12 demonstrates the mode of differentiation of myeloid cell types into iDCs and macrophages from immature cells isolated in culture while expressing mRNA-encoded luciferase with specialized UTRs.

[0069] FIG. 13 shows the expression of markers for distinct cell types of myeloid cells that express F. Luc mRNA.

[0070] FIG. 14 shows data indicating prolonged expression of f. Luc expressing mRNA, endowed with the modified UTR. Petition 870250099881, dated 10 / 31 / 2025, page 25 / 364 18 / 178 DETAILED DESCRIPTION

[0071] All terms are intended to be understood as they would be understood by one skilled in the art. Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by one skilled in the art to which the disclosure pertains.

[0072] The section headings used in this document are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0073] Although several features of this disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although this disclosure may be described in this document in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment.

[0074] Myeloid cells have recently been shown to provide a strong platform for therapeutic intervention in several diseases, including cancer, because these cells can chemotactically migrate to a locus of infection, inflammation, and tumor; they can phagocytose and kill bacteria and parasites; they can eliminate dead or apoptotic cells; and they can attack tumor cells or cells harboring a mutation or an aberrant phenotype. A myeloid cell is generally short-lived within the body. A myeloid cell can be manipulated ex vivo to enhance one or more of the functions related to phagocytosis or chemotaxis and introduced into a subject with a disease, so that the manipulated myeloid cell targets and attacks diseased cells in the subject, such as tumor cells or infected cells. However, myeloid cells are complex and can rapidly undergo a change of state in which their effectiveness as a phagocytic cell may be diminished.In this regard, manipulating or manipulating a myeloid cell may represent... Petition 870250099881, dated 10 / 31 / 2025, page 26 / 364 19 / 178 several challenges. For example, introducing plasmid DNA into a myeloid cell can lead to the transformation of a monocyte cell into an M2 phenotype, where the cells may lose chemotactic movement and surface expression of chemoattractant receptors. A monocyte or macrophage with an altered phenotype after the introduction of DNA comprising a gene of interest may become less effective in its innate functions, such as phagocytosis and chemotaxis.

[0075] Methods and compositions for manipulating a myeloid cell are provided in this document. In some embodiments, the manipulation is ex vivo. In some embodiments, an exogenous polynucleotide sequence can be introduced in vivo that is designed to be specifically uptaken and expressed by a myeloid cell in vivo. In some embodiments, the design of the exogenous polynucleotide sequence, for example, a recombinant polynucleotide sequence, is such that it allows the expression of the polypeptide encoded by the exogenous polynucleic acid in a myeloid cell and prevents the expression of the polypeptide encoded by the exogenous polynucleic acid in a non-myeloid cell. Importantly, compositions and methods are provided in this document for expressing a polynucleotide sequence in a myeloid cell. The method and compositions described in this document allow the preferential and specific expression of a polypeptide encoded by the recombinant polynucleic acid.

[0076] The myeloid cell expressing the exogenous polynucleotide sequence must retain its chemotactic, phagocytic, and cytotoxic function in order to perform a therapeutically effective function in a living body.

[0077] Polynucleotide compositions are provided in this document for enhanced expression of a protein or polypeptide encoded in a myeloid cell. In some embodiments, the aim is for a polynucleic acid to be preferentially expressed in a myeloid cell residing in a heterogeneous population of cells. Petition 870250099881, dated 10 / 31 / 2025, page 27 / 364 20 / 178 comprising myeloid cells and non-myeloid cells. In some embodiments, a myeloid cell is a monocyte, a monocyte-derived macrophage, a macrophage, a CD14+ cell, a CD14+ and CD16+ cell, or a CD14+ and CD16- cell. In some embodiments, the non-myeloid cell type comprises an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a T cell, a B cell, an NK cell, a muscle cell, an ocular cell, a nerve cell, a brain cell, a skeletal cell, or a hepatocyte.

[0078] In some forms, the polynucleotide is messenger RNA.

[0079] This disclosure is directed to a non-viral method and compositions for expressing exogenous genetic material, such as a polynucleotide, in a myeloid cell to ensure robust and prolonged expression in a myeloid cell, and to ensure that the myeloid cell expressing the exogenous genetic material is fully functional and capable of actively migrating to the site of infection or inflammation or tumor, that it responds to chemotactic signals in vivo so that it is localized, for example, to a tumor, and that it is actively phagocytic.

[0080] Prolonged expression of a polynucleic acid in a myeloid cell has been found to be highly challenging. First, there is a technical difficulty associated with the transfection of monocytes and macrophages. As professional phagocytes, monocytes and macrophages are endowed with many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer to these cells an inefficient process. Monocytes and macrophages undergo a dramatic change in their physiology after exposure to immune or inflammatory stimuli. Viral transduction from these cells has been hampered because macrophages are late-stage cells that generally do not divide; therefore, some of the vectors that rely on integration into a replicative genome have found limited success. Furthermore, macrophages are Petition 870250099881, dated 10 / 31 / 2025, page 28 / 364 21 / 178 quite responsive to “danger signals” and therefore several of the original viral vectors that were used for gene transfer induced potent antiviral responses in these cells, making these vectors unsuitable for gene delivery. For multiple reasons, as is known to those versed in the art, alternatives to viral gene delivery are always sought for safety reasons. However, most of the original transfection techniques had only limited success with macrophages. This is due to the need for fairly high gene copy numbers required to efficiently transfect the cells and the relatively high degree of toxicity associated with the process by which the host cell membrane is made permeable to DNA.The various transfection methods used to introduce foreign DNA into mammalian cells include: DEAE-dextran, calcium phosphate coprecipitation, cationic lipid carriers, and physical disruption of host cell membranes by electroporation or nucleofection. All of these approaches have been used with varying degrees of success in macrophages.

[0081] Methods and compositions are provided in this document for producing engineered myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages) designed to specifically express a gene of interest (GOI), such as a protective gene in a disease. A gene of interest may be a gene or a polynuclear acid carrying a sequence encoding the corrected wild-type protein sequence that can replace a non-functional mutant sequence in a subject not expressing a functional protein. A gene of interest may be an immunoprotective gene, a chemokine, a cytokine, a secreted protein, a cytoplasmic protein, a membrane protein, or a nuclear protein. In some embodiments, a gene of interest is a chimeric fusion protein (CFP), alternatively termed a chimeric antigen receptor (CAR). Petition 870250099881, dated 10 / 31 / 2025, page 29 / 364 22 / 178

[0082] In one embodiment, a myeloid cell can be manipulated to express a CAR (chimeric antigen receptor), also called a chimeric fusion protein (CFP), which can bind to a target protein or biomolecule or a target cell. These manipulated myeloid cells can therefore attack and kill target cells directly (e.g., by phagocytosis) and / or indirectly (e.g., by T cell activation). In some embodiments, the target cell is a cancerous cell.

[0083] Although a polynucleotide encoding a CFP (or CAR) may be used throughout the descriptive report as an exemplary embodiment, the composition and methods disclosed in this document are applicable to expressing a polynucleotide encoding any gene of interest, for example, a synthetic polynucleotide.

[0084] Although cancer is an exemplary embodiment described in detail in this disclosure, the methods and technologies described herein are contemplated to be useful in targeting an infected or otherwise diseased cell within the body. In some embodiments, the methods and compositions can be used to introduce any polynucleotide sequence encoding a protein to enhance macrophage function, such as a transcription enhancer, or a cytokine or chemokine. The method can be used to target the myeloid cell to a pathogen and engulf the pathogen while simultaneously presenting suitable antigens to T cells to induce the lymphocyte cascade and protective immunity. In some embodiments, an exemplary pathogen might be Bacillus anthracis, Clostridium botulinum, Fracisella tularensis, Variola major, Salmonella sp., Mycobacterium tuberculosis, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica or protozoans, for example, Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma gondi, Naegleria fowleri, Balamuthia mandrillaris or fungi.

[0085] In some modalities, the methods and compositions Petition 870250099881, dated 10 / 31 / 2025, page 30 / 364 23 / 178 disclosed in this document may be useful in generating engineered myeloid cells to express a gene to compensate for a deficient gene or to replace a mutant gene product with a correct wild-type protein, such as in monogenic disorders.

[0086] Compositions and methods for treating diseases or conditions, such as cancer, are provided in this document. The compositions and methods provided in this document utilize human myeloid cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages, to target diseased cells, such as cancer cells. The compositions and methods provided in this document can be used to eliminate diseased cells, such as cancer cells and / or diseased tissue, by a variety of mechanisms, including T cell activation and recruitment, effector immune cell activation (e.g., CD8 T cell and NK cell activation), antigen cross-presentation, enhanced inflammatory responses, reduction of regulatory T cells, and phagocytosis. For example, myeloid cells can be used to sustain immune responses against cancer cells.

[0087] In one aspect, a method for expressing a polynucleic acid in a myeloid cell, such as a human CD14+ myeloid cell, is provided in this document. In some embodiments, a manipulated polynucleic acid is provided in this document. In some embodiments, the manipulated polynucleic acid is suitable for efficient expression in a polypeptide encoded by the manipulated polynucleic acid in a myeloid cell. In some embodiments, the polynucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the mRNA encodes a protein that is expressed in the myeloid cell. In some embodiments, the mRNA-encoded protein is a cytoplasmic protein. In some embodiments, the mRNA-encoded protein is a nuclear protein. In some embodiments, the mRNA-encoded protein is a protein of Petition 870250099881, dated 10 / 31 / 2025, page 31 / 364 24 / 178 membrane. In some embodiments, the protein encoded by mRNA is a secreted protein.

[0088] In some embodiments, an mRNA comprising a sequence encoding a gene of interest is expressed in a myeloid cell, wherein the mRNA is modified for efficient expression in a myeloid cell. In some embodiments, the mRNA is modified at the termini for enhanced and / or prolonged expression in a myeloid cell. In some embodiments, the termini include a 5' termini and / or a 3' termini modification.

[0089] In some embodiments, the modification may be in the mRNA chemistry, which is in the mRNA nucleotides. In some embodiments, the modification is introduced into the mRNA at the time of mRNA formation. In some embodiments, the modification is introduced into the mRNA after the mRNA is formed.

[0090] In one embodiment, the poly A tail of the mRNA is designed to improve the expression efficiency of a polypeptide encoded by a sequence within the mRNA in a myeloid cell.

[0091] In some embodiments, the 5' UTR is specifically designed or chosen to improve the expression efficiency of a polypeptide encoded by a sequence within mRNA in a myeloid cell.

[0092] In some embodiments, the 3' UTR is specifically designed or chosen to improve the expression efficiency of a polypeptide encoded by a sequence within mRNA in a myeloid cell.

[0093] Also provided herein is a pharmaceutical composition comprising a composition described herein, such as a manipulated nucleic acid described herein, a vector described herein, a polypeptide described herein or a cell described herein; and a pharmaceutically prepared excipient. Petition 870250099881, dated 10 / 31 / 2025, p. 32 / 364 25 / 178 acceptable. The term “nucleic acid” can be used to designate a nucleic acid that is synthesized, recombinant, manipulated, or transcribed in vitro. In some embodiments, a nucleic acid does not occur naturally.

[0094] This document provides a composition comprising a nucleic acid comprising: (i) DNA encoding an mRNA or (ii) the mRNA, wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the 5' UTR is at least 45 nucleotides long and a sequence encoding a protein or polypeptide, wherein the protein or polypeptide encoded by the nucleic acid when expressed in the myeloid cell is detectable in the cell for at least up to 72 hours.This document provides a composition comprising a nucleic acid comprising: an mRNA wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein (i) and (ii) flank (iii) a sequence encoding a protein or polypeptide; wherein the 5' UTR sequence is selected from sequences provided in this document; and the 3' UTR sequence is selected from sequences provided in this document; wherein the protein or polypeptide encoded by the nucleic acid when expressed in the myeloid cell is detectable in the cell for at least up to 72 hours.This document provides a composition comprising a nucleic acid comprising: (i) DNA encoding an mRNA or (ii) the mRNA, wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the 5' UTR is at least 45 nucleotides long and a sequence encoding a protein or polypeptide, and wherein the mRNA comprises an enzymatically added polyA tail; wherein the protein or polypeptide encoded by the nucleic acid, when expressed in the myeloid cell, is detectable in the cell for at least up to 72 hours. In some embodiments, the composition is not conjugated or associated with a lipid nanoparticle (LNP). In some embodiments, the nucleic acid comprises one or more modified nucleotide bases, wherein one. Petition 870250099881, dated 10 / 31 / 2025, page 33 / 364 26 / 178 of the total number of uridine bases is modified to a pseudouridine, a 1-methyl-pseudouridine, or a 5-methoxyuridine. In some embodiments, less than 50% of the total number of uridine bases are modified to a pseudouridine, a 1-methyl-pseudouridine, or a 5-methoxyuridine. In some embodiments, the protein or polypeptide encoded by the nucleic acid, when expressed in the myeloid cell, is detectable in the cell for at least 72 hours. In some embodiments, the 5' UTR is at least 20 nucleotides long. In some embodiments, the 5' UTR is at least 30 nucleotides long. In some embodiments, the 5' UTR is at least 60 nucleotides long. In some embodiments, the 5' UTR is at least 100 nucleotides long.

[0095] In one aspect, methods and compositions are disclosed in this document for in vivo expression, the method comprises the expression of a recombinant polypeptide encoded by a recombinant nucleic acid preferentially or specifically in myeloid cells, avoiding off-target expression in one or more non-myeloid cell types.

[0096] In one embodiment, compositions and methods are provided in this document for manipulating the 3' UTR of recombinant nucleic acid so that the recombinant nucleic acid is degraded in certain non-myeloid cells, or is not translated to generate the recombinant polypeptide in certain non-myeloid cells. In some embodiments, the certain non-myeloid cells are abundantly present in the vicinity of myeloid cells or may be specific cell types in which the recombinant polypeptide is particularly undesirable or harmful. Alternatively, in some embodiments, compositions and methods are provided in this document for manipulating the 5' UTR of recombinant nucleic acid so that the recombinant nucleic acid is degraded in certain non-myeloid cells, or is not translated to generate the recombinant polypeptide in certain non-myeloid cells. Petition 870250099881, dated 10 / 31 / 2025, page 34 / 364 27 / 178 translated to generate the recombinant polypeptide in certain non-myeloid cells. In some embodiments, the non-myeloid cell where the expression of the recombinant polypeptide is undesired is an epithelial cell, or a cell of epithelial lineage.

[0097] In one embodiment, the nucleic acid is an mRNA comprising a poly-A sequence that is enzymatically added. In some embodiments, the nucleic acid is an mRNA comprising a poly-A sequence that is encoded by the plasmid comprising the template for generating the mRNA by in vitro transcription (IVT). The template for IVT is a linearized plasmid. Typically, the length of the poly-A is controlled when encoded by the plasmid and is less controlled when enzymatically added. An mRNA product comprising an enzymatically added poly-A tail can be tailored at best to contain a narrow range of A residue numbers. The mRNA transcribed in vitro is subsequently purified beforehand.

[0098] In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid and at least one of: a non-cationic lipid, a neutral lipid, cholesterol, and a conjugated lipid. In some embodiments, the LNP is less than 250 nm in diameter. In some embodiments, the LNP is less than 250 nm in diameter. In some embodiments, the LNP is less than 150 nm in diameter. In some embodiments, the LNP is about 100 nm in diameter. In some embodiments, the nucleic acid is encapsulated in a liposome.

[0099] In some embodiments, the nucleic acid comprises a poly-A sequence downstream of the UTR 3' sequence. In some embodiments, the poly-A sequence is at least 50 nucleotides long. In some embodiments, the poly-A sequence is at least 60, 70, 80, or 90 nucleotides long. Petition 870250099881, dated 10 / 31 / 2025, p. 35 / 364 28 / 178 nucleotides in length. In some embodiments, the poly A sequence is at least 100 nucleotides long. In some embodiments, the poly A sequence is at least 110 nucleotides long. In some embodiments, the poly A sequence is at least 120 nucleotides long. In some embodiments, the poly A sequence is at least 130 nucleotides long. In some embodiments, the poly A sequence is at least 140 nucleotides long. In some embodiments, the poly A sequence is at least 150, 160, 170, 180, 190, or 200 nucleotides long. In some embodiments, within the 5' UTR, a translation initiation site is at least 15 nucleotides downstream from the 5' end. In some embodiments, the translation initiation site is at least 20 nucleotides downstream from the 5' end. In some embodiments, the translation initiation site is at least 25 nucleotides downstream from the 5' end.In some embodiments, the translation initiation site is at least 30 nucleotides downstream from the 5' end. In some embodiments, the nucleic acid comprises a single translation initiation site.

[0100] In some embodiments, the nucleic acid comprises a 5' methyl guanylate cap. In some embodiments, the nucleic acid comprises a Cap 0 structure. In some embodiments, the Cap 0 structure is introduced into the mRNA cotranscriptionally using an antireverse cap analog (ARCA).

[0101] In some embodiments, the nucleic acid is 20-20,000 nucleotides long. In some embodiments, the nucleic acid is 20-22 nucleotides long. In some embodiments, the nucleic acid is 20-50 nucleotides long. In some embodiments, the nucleic acid is 20-100 nucleotides long. In some embodiments, the nucleic acid is a small interfering RNA. In some embodiments, the nucleic acid is an inhibitory RNA. In some embodiments, the nucleic acid is a tRNA. In some embodiments, the nucleic acid is Petition 870250099881, dated 10 / 31 / 2025, p. 36 / 364 29 / 178 single-stranded. In some forms, the nucleic acid is double-stranded.

[0102] In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is 50-20,000 nucleotides long.

[0103] In some embodiments, the nucleic acid is a manipulated nucleic acid. In some embodiments, the nucleic acid is recombinant nucleic acid. In some embodiments, the nucleic acid is mRNA transcribed in vitro. In some embodiments, the nucleic acid is a synthesized nucleic acid.

[0104] In some forms, the nucleic acid is isolated.

[0105] In some forms, the nucleic acid is purified.

[0106] In some embodiments, the nucleic acid comprises at least 1 modified nucleotide.

[0107] In some embodiments, the nucleic acid comprises at least 10% modified nucleotides.

[0108] In some embodiments, the nucleic acid comprises at least 20% modified nucleotides.

[0109] In some embodiments, the nucleic acid comprises at least 30%, 40%, or 50% modified nucleotides. In some embodiments, less than 70% of the uridine residues in the nucleic acid are modified.

[0110] In some embodiments, less than 50% of the uridine residues in the nucleic acid are modified.

[0111] In some embodiments, the modified nucleotide is a pseudouridine, 1-methyl-pseudouridine or a 5-methoxyuridine that replaces a uridine.

[0112] In some embodiments, nucleic acid comprises about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about Petition 870250099881, dated 10 / 31 / 2025, p. 37 / 364 30 / 178 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40% or more of the uridine residues of the nucleic acid that are modified to a pseudouridine, 1-methyl-pseudouridine, or a 5-methoxyuridine.

[0113] This document provides a composition comprising a cell comprising the composition according to any of the embodiments described above.

[0114] In some forms, the cell is a myeloid cell, a CD14+ cell, a CD16- cell, or a CD14+ / CD16- cell.

[0115] In some forms, the cell is a T cell.

[0116] In some modalities, protein expression in the cell is detectable for at least 72 hours.

[0117] This document provides a pharmaceutical composition comprising the composition according to any of the embodiments described above; and a pharmaceutically acceptable excipient.

[0118] This document provides a method for treating a subject with a disease or condition comprising administering the pharmaceutical composition described above to a subject in need.

[0119] This document provides a method for expressing a nucleic acid-encoded protein in a CD14+ cell, the method comprising: (a) incorporating into the CD14+ cell a nucleic acid comprising (i) DNA encoding an mRNA or (ii) mRNA wherein the mRNA comprises a sequence encoding the protein, and (b) expressing the protein encoded by a sequence of the mRNA; wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the expression of the protein is detectable in the CD14+ cell for at least 72 hours after (a).

[0120] In some modalities, protein expression is detectable according to an immunoassay up to at least 72 hours after (a). Petition 870250099881, dated 10 / 31 / 2025, page 38 / 364 31 / 178

[0121] In some modalities, protein expression is detectable in at least 20% of cells after at least 72 hours after (a).

[0122] This document provides a method for expressing a nucleic acid-encoded protein in a CD14+ cell, the method comprising incorporating into the CD14+ cell a nucleic acid comprising (i) DNA encoding an mRNA or (ii) the mRNA and expressing a protein encoded by a sequence of the mRNA; wherein the mRNA comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence flanking a protein-encoding sequence; wherein the 5' UTR is at least 45 nucleotides long.

[0123] In some embodiments, the nucleic acid comprises a 5' UTR sequence selected from the sequences provided in this document. In some embodiments, the nucleic acid sequence comprises a 3' UTR sequence selected from the sequences provided in this document.

[0124] In some embodiments, the nucleic acid comprises a poly A tail, wherein the poly A tail is enzymatically added to the 3' end of the mRNA or is an encoded poly A tail.

[0125] In some embodiments, the nucleic acid sequence comprises a 5' UTR sequence, and the 3' UTR can be tested and paired into a construct in order to maximize the expression level and stability of the mRNA over time. This document provides a method for expressing a nucleic acid in a myeloid cell such that the nucleic acid is detectable in the cell for at least 72 hours after the introduction of the nucleic acid into the myeloid cell. This document provides a method for expressing an mRNA encoding a protein in a myeloid cell such that the protein is detectable in the cell for at least 72 hours after the introduction of the mRNA into the myeloid cell. In some embodiments, the 5' UTR has at least 50, at least 60, at least 70, Petition 870250099881, dated 10 / 31 / 2025, page 39 / 364 32 / 178 at least 80 or at least 100 nucleotides in length. In some embodiments, the nucleic acid comprises a poly-A tail of 50200 adenylate residues (SEQ ID NO: 27) and in which the poly-A tail is enzymatically added to the mRNA.

[0126] In some embodiments, mRNA is targeted for specific expression in myeloid cells. In some embodiments, mRNA encodes a sequence that is specifically expressed in a myeloid cell.

[0127] In some embodiments, mRNA is introduced into the myeloid cell as a naked nucleic acid.

[0128] In some embodiments, mRNA is introduced into the myeloid cell via a delivery vehicle, for example, an LNP. In some embodiments, the lipid nanoparticle comprises a cationic lipid and at least one of: a non-cationic lipid, a neutral lipid, cholesterol, and a conjugated lipid.

[0129] In some embodiments, incorporation involves transfection. In some embodiments, incorporation involves electroporation.

[0130] In some embodiments, incorporation involves culturing the cell in the presence of nucleic acid, where the nucleic acid is present at a concentration of no more than about 500 micrograms / mL.

[0131] In some embodiments, incorporation comprises culturing the cell in the presence of nucleic acid, wherein the nucleic acid is present at a concentration of no more than about 250 micrograms / mL. In some embodiments, incorporation comprises culturing the cell in the presence of nucleic acid, wherein the nucleic acid is present at a concentration of no more than about 100 micrograms / mL. In some embodiments, incorporation comprises culturing the cell in the presence of nucleic acid, wherein the nucleic acid is Petition 870250099881, dated 10 / 31 / 2025, page 40 / 364 33 / 178 present at a concentration of at most about 50 micrograms / mL.

[0132] In some embodiments, incorporation involves culturing the cell in the presence of nucleic acid, wherein the nucleic acid is present at a concentration of about 1 microgram / mL to about 500 micrograms / mL.

[0133] In some embodiments, incorporation involves culturing the cell in the presence of nucleic acid, wherein the nucleic acid is present at a concentration of about 10 micrograms / mL to about 100 micrograms / mL.

[0134] In some embodiments, incorporation involves culturing the cell in the presence of nucleic acid, wherein the nucleic acid is present at a concentration of about 10 micrograms / mL to about 50 micrograms / mL. In some embodiments, incorporation involves incorporation in vivo. In some embodiments, the nucleic acid comprises a sequence encoding a membrane protein. In some embodiments, the nucleic acid comprises a sequence encoding a cytosolic protein. In some embodiments, the nucleic acid comprises a sequence encoding a recombinant protein. In some embodiments, the nucleic acid is mRNA, wherein the mRNA is 50–20,000 nucleotides in length.

[0135] In some embodiments, the nucleic acid comprises at least 1 modified nucleotide. In some embodiments, the nucleic acid comprises at least 10% modified nucleotides. In some embodiments, the nucleic acid comprises at least 20% modified nucleotides. In some embodiments, the nucleic acid comprises at least 30%, 40%, or 50% modified nucleotides. In some embodiments, less than 70% of the uridine residues in the nucleic acid are modified. In some embodiments, less than 50% of the uridine residues in the nucleic acid are modified. In some embodiments, the modified nucleotide is a pseudouridine, 1-methyl-pseudouridine, or a Petition 870250099881, dated 10 / 31 / 2025, page 41 / 364 34 / 178 5-methoxyuridine replacing uridine.

[0136] In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid and at least one of: a non-cationic lipid, a neutral lipid, cholesterol, and a conjugated lipid. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, and a conjugated lipid. In some embodiments, the conjugated lipid is a PEGylated lipid. In some embodiments, the PEG fraction is PEG-2000.

[0137] In some embodiments, after incorporation of the nucleic acid into an ex vivo CD14+ cell population, at least 80% of the cells in the CD14+ cell population express the nucleic acid-encoded protein within 24 hours. In some embodiments, after incorporation of the nucleic acid into an ex vivo CD14+ cell population, at least 70% of the population express the nucleic acid-encoded protein after 48 hours. In some embodiments, the protein is detectable in CD14+ cells up to 72 hours after nucleic acid incorporation. In some embodiments, the protein is detectable in CD14+ cells up to 96 hours after nucleic acid incorporation. In some embodiments, the protein is detectable in CD14+ cells up to 5 days after nucleic acid incorporation.

[0138] Reference in the descriptive report to “some embodiments”, “an embodiment”, “an embodiment” or “other embodiments” means that a feature, structure or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of this disclosure. As used in this descriptive report and claims, the words “comprising” (and any form of “comprising”, such as “comprising” and “comprises”), “having” (and any form of having, such as “having” and “has”), “including” (and any form of including, such as Petition 870250099881, dated 10 / 31 / 2025, p. 42 / 364 35 / 178 “include” and “includes”) or “containing” (and any form of containing, such as “contains” and “contains”) are inclusive or open-ended and do not exclude additional and unmentioned method elements or steps. It is contemplated that any embodiment discussed in this descriptive report may be implemented in relation to any disclosure method or composition and vice versa. Furthermore, disclosure compositions may be used to achieve disclosure methods.

[0139] The term “about” or “approximately,” as used in this document when referring to a measurable value, such as a parameter, quantity, time duration, and the like, may encompass variations of + / -30% or less, + / -20% or less, + / -10% or less, + / -5% or less, or + / -1% or less of and from the specified value, to the extent that such variations are appropriate to be carried out in this disclosure. It should be understood that the value to which the modifier “about” or “approximately” refers is also specifically disclosed.

[0140] An “antigen-presenting cell” or “APC,” as used in this document, includes professional antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). An APC may express major histocompatibility complex (MHC) molecules and may display MHC-complexed antigens on its surface that can be recognized by T cells and trigger T cell activation and an immune response. Professional antigen-presenting cells, notably dendritic cells, play a key role in stimulating unexposed T cells. Antigen-presenting cells Petition 870250099881, dated 10 / 31 / 2025, page 43 / 364 36 / 178 Non-professional cells, such as fibroblasts, can also contribute to this process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens onto MHC class I molecules. The antigens that give rise to proteins that are recognized in association with MHC class I molecules are generally proteins that are produced within cells, and these antigens are processed and associate with MHC class I molecules.

[0141] A “biological sample” can refer to any tissue, cell, fluid or other material derived from an organism.

[0142] The term “epitope” can refer to any protein determinant, such as a sequence or structure or amino acid residues, capable of binding to an antibody or antibody-binding fragment, a T cell receptor, and / or an antibody-like molecule. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge characteristics. A “T cell epitope” may refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0143] A manipulated cell, such as a manipulated myeloid cell, may refer to a cell that has at least one exogenous nucleic acid sequence in the cell, even if transiently expressed. The expression of an exogenous nucleic acid can be accomplished by various methods described elsewhere and encompasses methods known in the art. This disclosure relates to the preparation and use of manipulated cells, for example, manipulated myeloid cells, such as manipulated phagocytic cells. This disclosure relates, inter alia, to a manipulated cell comprising an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP). Petition 870250099881, dated 10 / 31 / 2025, page 44 / 364 37 / 178

[0144] The term “immune response” includes, but is not limited to, T cell-mediated, NK cell-mediated, and / or B cell-mediated immune responses. These responses can be influenced by the modulation of T cell co-stimulation and NK cell co-stimulation. Exemplary immune responses include T cell responses, for example, cytokine production and cellular cytotoxicity. In addition, immune responses include immune responses that are indirectly affected by NK cell activation, B cell activation, and / or T cell activation, for example, antibody production (humoral responses) and activation of cytokine-responsive cells, for example, macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens from an intruding organism. Unlike the innate immune system, the adaptive immune system is highly specific to a pathogen.Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted by B cells into body fluids bind to pathogen-derived antigens, leading to the elimination of the pathogen through a variety of mechanisms, for example, complement-mediated lysis.

[0145] In cell-mediated immune reactions, T cells capable of destroying other cells are activated. For example, if proteins associated with a disease are present in a cell, they can be proteolytically fragmented into peptides within the cell. Specific cellular proteins can then bind to the antigen or to a peptide formed in this way and transport them to the cell surface, where they can be presented to molecular defense mechanisms, such as T cells. Cytotoxic T cells can recognize these antigens and kill cells harboring these antigens.

[0146] A “ligand” can refer to a molecule that is capable of becoming Petition 870250099881, dated 10 / 31 / 2025, page 45 / 364 38 / 178 to link or form a complex with another molecule, such as a receptor. A ligand may include, but is not limited to, a protein, a glycoprotein, a carbohydrate, a lipoprotein, a hormone, a fatty acid, a phospholipid, or any component that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor may have promiscuous ligand binding, in which case it may bind to multiple ligands that share at least one similarity in structural configuration, charge distribution, or any other physicochemical characteristic. A ligand may be a biomolecule. A ligand may be an abiotic material. For example, a ligand may be a negatively charged particle that is a ligand for the MARCO scavenger receptor. For example, a ligand may be TiO2, which is a ligand for the SRA1 scavenger receptor.

[0147] The term “major histocompatibility complex (MHC)”, “MHC molecule”, or “MHC protein” can refer to a protein capable of binding to an antigenic peptide and presenting the antigenic peptide to T lymphocytes. Such antigenic peptides may represent T cell epitopes. The human MHC is also called the HLA complex. Thus, the terms “human leukocyte antigen (HLA)”, “HLA molecule”, or “HLA protein” are used interchangeably with the terms “major histocompatibility complex (MHC)”, “MHC molecule”, and “MHC protein”. HLA proteins can be classified as HLA class I or HLA class II. The structures of the proteins of the two HLA classes are very similar; however, they have very different functions. HLA class I proteins are present on the surface of almost all cells in the body, including most tumor cells.HLA class I proteins are loaded with antigens that generally originate from endogenous proteins or pathogens present within cells and are then presented to unexposed or cytotoxic T lymphocytes (CTLs). HLA class II proteins are present on cells. Petition 870250099881, dated 10 / 31 / 2025, page 46 / 364 39 / 178 antigen-presenting cells (APCs), including, but not limited to, dendritic cells, B cells, and macrophages. They primarily present peptides that are processed from external antigen sources, for example, outside the cells, to helper T cells.

[0148] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can absorb entities by phagocytosis into phagosomes—although B cells exhibit more general endocytosis into endosomes—which fuse with lysosomes whose acidic enzymes cleave the absorbed protein into many different peptides. Autophagy is another source of HLA class II peptides. The most studied HLA subclass II genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0149] The presentation of peptides by HLA class II molecules to CD4+ helper T cells can lead to immune responses to foreign antigens. Once activated, CD4+ T cells can promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells can also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. HLA class II molecules are typically heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than the peptide-binding grooves of class I.

[0150] HLA alleles are normally expressed in a codominant manner. For example, each person carries 2 alleles of each of the 3 class I genes (HLA-A, HLA-B, and HLA-C) and therefore can express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPA1 and DPB1, which encode α and β chains), HLA-DQ genes (DQA1 and DQB1, for α and β chains), one HLA-DRa gene (DRA1), and one or more HLA-OPβ genes (DRB1 and DRB3, -4 or -5). HLA-DRB1, for example, has almost 400 known alleles. This means that a Petition 870250099881, dated 10 / 31 / 2025, p. 47 / 364 40 / 178 A heterozygous individual can inherit six or eight functional HLA class II alleles: three or more from each parent. Thus, HLA genes are highly polymorphic; there are many different alleles in different individuals within a population. The genes that encode HLA proteins have many possible variations, allowing each person's immune system to react to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which receives a particular number. In some modalities, the HLA class I alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01 (non-classical). In some forms, the HLA class II alleles are HLADRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLADRB*07:01.

[0151] A “myeloid cell” can broadly refer to cells of the myeloid lineage of the hematopoietic cell system and may exclude, for example, the lymphocytic lineage. Myeloid cells comprise, for example, cells of the granulocyte lineage and monocyte lineages. Myeloid cells are differentiated from common progenitors derived from hematopoietic stem cells in the bone marrow. Commitment to myeloid cell lineages can be governed by the activation of distinct transcription factors, and consequently, myeloid cells can be characterized as cells with a level of plasticity, which can be described as the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited into local tissues through various chemokine receptors on their surface. Myeloid cells respond to various cytokines and chemokines.

[0152] A myeloid cell, for example, can be a cell that originates in the bone marrow from a hematopoietic stem cell under the influence of one or more cytokines and chemokines, such as G-CSF, GMCSF, Flt3L, CCL2, VEGF, and S100A8 / 9. In some embodiments, the cell Petition 870250099881, dated 10 / 31 / 2025, p. 48 / 364 41 / 178 Myeloid is a precursor cell. In some embodiments, the myeloid cell may be a cell with characteristics of a common myeloid progenitor, or a granulocyte progenitor, a myeloblast cell, or a monocyte dendritic cell progenitor, or a combination thereof. A myeloid may include a granulocyte or a monocyte or a precursor cell thereof. A myeloid may include an immature granulocyte, an immature monocyte, an immature macrophage, an immature neutrophil, and an immature dendritic cell. A myeloid may include a monocyte or a pre-monocytic cell or a monocyte precursor. In some cases, a myeloid cell, as used in this document, may refer to a monocyte with an MO phenotype, an M1 phenotype, or an M2 phenotype. A myeloid cell may include a dendritic cell (DC), a mature DC, a monocyte-derived DC, a plasmacytoid DC, a pre-dendritic cell, or a DC precursor.A myeloid cell may include a neutrophil, which may be a mature neutrophil, a neutrophil precursor, or a polymorphonuclear cell (PMN). A myeloid cell may include a macrophage, a monocyte-derived macrophage, a tissue macrophage, a macrophage of an MO phenotype, an M1, or an M2. A myeloid cell may include a tumor-infiltrating monocyte (TIM). A myeloid cell may include a tumor-associated monocyte (TAM). A myeloid cell may include a myeloid-derived suppressor cell (MDSC). A myeloid cell may include a tissue-resident macrophage. A myeloid cell may include a tumor-associated DC (TADC). Consequently, a myeloid cell can express one or more cell surface markers, for example, CD11b, CD14, CD15, CD16, CD38, CCR5, CXCR1, CXCR2, CXCR4, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR4, CCR7, CX3CR1, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, a protein from the SIGLEC family, and a protein from the CLEC family.In some cases, a myeloid cell can be characterized by high or low expression of one or more cell surface markers, for example, CD11b, CD14, CD15, CD16. Petition 870250099881, dated 10 / 31 / 2025, p. 49 / 364 42 / 178 CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or a combination thereof.

[0153] “Phagocytosis” is used interchangeably with “engulfment” and can refer to a process by which a cell engulfs a particle, such as a cancerous cell or an infected cell. This process can give rise to an internal compartment (phagosome) containing the particle. This process can be used to ingest and / or remove a particle, such as a cancerous cell or an infected cell, from the body. A phagocytic receptor may be involved in the phagocytosis process. The phagocytosis process can be closely coupled to an immune response and antigen presentation. The processing of exogenous antigens follows their uptake into professional antigen-presenting cells by some type of endocytic event. Phagocytosis can also facilitate antigen presentation. For example, antigens from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0154] A “polypeptide” may refer to a molecule containing amino acids linked together by a peptide bond, such as a glycoprotein, a lipoprotein, a cellular protein, or a membrane protein. A polypeptide may comprise one or more subunits of a protein. A polypeptide may be encoded by a nucleic acid. In some embodiments, the polypeptide may comprise more than one peptide sequence in a single amino acid chain, which may be separated by a spacer, a linker, or a peptide cleavage sequence. A polypeptide may be a fused polypeptide. A polypeptide may comprise one or more domains, modules, or fractions.

[0155] A “receptor” can refer to a chemical structure composed of a polypeptide that transduces a signal, such as a polypeptide that transduces an extracellular signal into a cell. A receptor Petition 870250099881, dated 10 / 31 / 2025, page 50 / 364 43 / 178 can serve to transmit information within a cell, a cell formation, or an organism. A receptor comprises at least one receptor unit and may contain two or more receptor units, where each receptor unit comprises a protein molecule, for example, a glycoprotein molecule. A receptor may contain a structure that binds to a ligand and may form a complex with the ligand. Signaling information may be transmitted by a conformational change of the receptor after binding to the ligand on the surface of a cell.

[0156] The term “antibody” refers to a class of proteins that are generally known as immunoglobulins, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, IgA (including IgAI and IgA2), IgD, IgE, IgM and IgY. The term “antibody” includes, but is not limited to, complete antibodies, single-chain antibodies, single-domain antibodies (sdAb) and antigen-binding fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd (consisting of Vh and Ch1), single-chain variable fragment (scFv), single-chain antibodies, disulfide-linked variable fragment (dsFv) and fragments comprising a Vl and / or Vh domain. Antibodies may be of any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, may comprise variable regions alone or in combination with one or more of a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain.Also included are any combinations of variable regions and hinge region, CH1, CH2, and CH3 domains. Antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that, for example, bind specifically to an HLA-associated polypeptide or an HLA-peptide complex.

[0157] A nucleic acid, as described in this document, may contain a nucleotide sequence that does not occur naturally. A engineered nucleic acid may be synthesized in the laboratory. A nucleic acid Petition 870250099881, dated 10 / 31 / 2025, page 51 / 364 44 / 178 can be prepared using recombinant DNA technology, for example, enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. A nucleic acid can be DNA, RNA, analogs thereof, or a combination thereof. Recombinant DNA can be transcribed ex vivo or in vitro to generate messenger RNA (mRNA). An mRNA, as described in this document, can be isolated, purified, and used to electroporate or transfect a cell. An mRNA, as described in this document, can be an mRNA transcribed in vitro. A nucleic acid, such as an mRNA, described in this document can be synthetic. A nucleic acid, such as an mRNA, described in this document can be encoded by a vector. A nucleic acid, such as an mRNA, described in this document can encode a protein or polypeptide.

[0158] The process of introducing or incorporating a nucleic acid into a cell can be through electroporation, transformation, transfection, or transduction. Transformation is the process of uptake of foreign nucleic acid by a bacterial cell. This process is adapted for plasmid DNA propagation, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that absorb extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the introduction of small molecules, such as DNA, RNA, or antibodies, into eukaryotic cells. Transfection can also refer to the introduction of bacteriophages into bacterial cells."Transduction" is primarily used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to natural infections of humans or animals with wild-type viruses.

[0159] The term “vector” can refer to a nucleic acid molecule capable of autonomous replication in a host cell and that Petition 870250099881, dated 10 / 31 / 2025, page 52 / 364 Patent 45 / 178 allows the cloning of nucleic acid molecules. As is known to those skilled in the art, a vector includes, but is not limited to, a plasmid, cosmid, phagemid, viral vectors, phage vectors, yeast vectors, mammalian vectors, and the like. For example, a vector for exogenous gene transformation may be a plasmid. In certain embodiments, a vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for replication and / or maintenance of the nucleic acid sequence in a host cell. In some embodiments, a vector or plasmid provided in this document is an expression vector. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences to which they are operationally linked. In some embodiments, an expression vector or plasmid is in the form of circular double-stranded DNA molecules.A vector or plasmid may or may not be integrated into the genome of a host cell. In some embodiments, nucleic acid sequences from a plasmid are not integrated into a host cell genome or chromosome after introduction. For example, the plasmid may comprise elements for transient expression or stable expression of nucleic acid sequences, such as genes or open reading frames harbored by the plasmid, in a host cell. In some embodiments, a vector is a transient expression vector. In some embodiments, a vector is a stably expressed vector that replicates autonomously in a host cell. In some embodiments, nucleic acid sequences from a plasmid are integrated into a host cell genome or chromosome after introduction into the host cell.The expression vectors that can be used in the methods disclosed in this document include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors. A vector can be a DNA or RNA vector. In some embodiments, a vector is provided in this document. Petition 870250099881, dated 10 / 31 / 2025, page 53 / 364 46 / 178 document is an RNA vector that is capable of integrating into the genome of a host cell after introduction into the host cell (e.g., by reverse transcription), for example, a retroviral vector or a lentiviral vector. Other forms of expression vectors known to those skilled in the art that serve equivalent functions may also be used, for example, self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are attached.

[0160] The terms “spacer” or “linker,” as used in reference to a fusion protein / polypeptide, refer to a peptide sequence that links two other peptide sequences of the fusion protein / polypeptide. In some embodiments, a linker or spacer has no specific biological activity beyond linking or preserving some minimum distance or other spatial relationship between proteins or RNA sequences. In some embodiments, the constituent amino acids of a spacer may be selected to influence some property of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Linkers suitable for use in an embodiment of the present disclosure are well known to those skilled in the art and include, but are not limited to, linear or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.In some embodiments, a linker is used to separate two or more polypeptides, for example, two antigenic peptides by a sufficient distance to ensure that each antigenic peptide folds appropriately. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity to develop an ordered secondary structure. The amino acids in the flexible linker protein / peptide region may include Gly, Asn, and Ser, or any permutation of amino acid sequences containing Gly, Asn, and Ser. Petition 870250099881, dated 10 / 31 / 2025, page 54 / 364 47 / 178 Ser. Other nearly neutral amino acids, such as Thr and Ala, can also be used in the linker sequence.

[0161] The terms “treat”, “treated”, “treating”, “treatment” and similar terms are intended to refer to reducing, preventing or improving a disorder and / or symptoms associated with it (e.g., a neoplasm or tumor or infectious agent or an autoimmune disease). “Treating” may refer to administering therapy to a subject after the onset, or suspected onset, of a disease (e.g., cancer or infection by an infectious agent or an autoimmune disease). “Treating” includes the concepts of “relieve”, which may refer to decreasing the frequency of occurrence or recurrence, or the severity, of any symptoms or other disease-related effects and / or side effects associated with the therapy.The term “treating” also encompasses the concept of “managing,” which refers to reducing the severity of a disease or disorder in a patient, for example, prolonging life or extending the survivability of a patient with the disease, or delaying its recurrence, for example, extending the remission period in a patient who has suffered from the disease. It is appreciated that, while not preventing, the treatment of a disorder or condition does not require that the disorder, condition, or symptoms associated with it be completely eliminated. The term “preventing,” “preventing,” “prevention,” and their grammatical equivalents, as used in this document, may refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who has not developed such symptoms at the time administration of an agent or compound begins.In certain embodiments, treating a subject or patient, as described in this document, comprises administering a therapeutic composition, such as a drug, a metabolite, a preventative component, a nucleic acid, a peptide, or a protein that encodes or forms a drug, a metabolite, or a preventative component. In some embodiments, treating comprises administering a... Petition 870250099881, dated 10 / 31 / 2025, page 55 / 364 48 / 178 cell or a population of cells to a subject in need. In some embodiments, treatment comprises administering to the subject one or more manipulated cells described in this document, for example, one or more manipulated myeloid cells, such as phagocytic cells. Treatment comprises treating a disease or condition or syndrome, which may be a pathological disease, condition or syndrome, or a latent disease, condition or syndrome. In some cases, treatment, as used in this document, may comprise administering a therapeutic vaccine. In some embodiments, the manipulated phagocytic cell is administered to a patient or subject. In some embodiments, a cell administered to a human subject results in reduced immunogenicity. For example, a manipulated phagocytic cell may lead to no or reduced graft-versus-host disease (GVHD) or fratricidal effect.In some embodiments, a manipulated cell administered to a human subject is immunocompatible with the subject (that is, with a corresponding HLA subtype that is naturally expressed in the subject). The subject-specific HLA alleles or HLA genotype of a subject can be determined by any method known in the art.In exemplary embodiments, the methods include determining polymorphic gene types, which may involve generating an alignment of reads extracted from a sequencing dataset to a gene reference set comprising allele variants of the polymorphic gene; determining a first posterior likelihood or a posterior likelihood derived score for each allele variant in the alignment; identifying the allele variant with a maximum first posterior likelihood or posterior likelihood derived score as a first allele variant; identifying one or more overlapping reads that aligned with the first allele variant and one or more other allele variants; and determining a second posterior likelihood or posterior likelihood derived score for one or more other variants. Petition 870250099881, dated 10 / 31 / 2025, p. 56 / 364 49 / 178 allele using a weighting factor, identify a second allele variant by selecting the allele variant with a second maximum posterior probability or posterior probability derived score, the first and second allele variants defining the gene type for the polymorphic gene, and provide an output of the first and second allele variants.

[0162] A “fragment” may refer to a portion of a protein or nucleic acid. In some embodiments, a fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of a reference protein or nucleic acid.

[0163] The terms “isolated,” “purified,” “biologically pure,” and their grammatical equivalents refer to material that is free to varying degrees from the components that normally accompany it as found in its native state. “Isolated” denotes a degree of separation from the original source or environment. “Purity” denotes a degree of separation that is greater than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials so that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analytical chemical techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" may denote that a nucleic acid or protein essentially produces a band on an electrophoretic gel. For a protein that can undergo modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be purified separately.

[0164] The terms “neoplasia” or “cancer” refer to any Petition 870250099881, dated 10 / 31 / 2025, page 57 / 364 50 / 178 A disease that is caused by or results from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is a non-limiting example of a neoplasm or cancer. The terms “cancer” or “tumor” or “hyperproliferative disorder” refer to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancerous cells are frequently in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancerous cell, such as a leukemia cell.

[0165] The term “vaccine” should be understood to mean a composition for generating immunity for the prophylaxis and / or treatment of diseases (e.g., neoplasm / tumor / infectious agents / autoimmune diseases). Consequently, vaccines as used in this document are medicaments comprising manipulated nucleic acids, or cells comprising and expressing a nucleic acid, and intended for use in humans or animals to generate a specific protective and defensive substance by vaccination. A “vaccine composition” may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of this disclosure relate to the use of technology in the preparation of a phagocytic cell-based vaccine.

[0166] The term “pharmaceutically acceptable” refers to a regulatory agency approved or approvable by the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, including humans. A “pharmaceutically acceptable excipient, carrier, or diluent” refers to an excipient, carrier, or diluent that can be administered to a subject along with an agent and that does not destroy the pharmacological activity. Petition 870250099881, dated 10 / 31 / 2025, page 58 / 364 51 / 178 of this and is non-toxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0167] MicroRNAs (miRNAs) can be described as a class of small non-coding RNAs that play important roles in the regulation of gene expression. Most miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs and, finally, mature miRNAs. miRNAs have an average length of 22 nucleotides. Most miRNAs are transcribed from DNA sequences into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and mature miRNAs. In most cases, miRNAs interact with the 3' UTR of target mRNAs to suppress expression.

[0168] In most cases, miRNAs interact with the 3' untranslated region (UTR 3') of target mRNAs to induce mRNA degradation and translation repression. Although target gene mRNA levels may not be altered by miRNA overexpression, reduced protein levels are expected. However, miRNA interaction with other regions, including the 5' UTR, coding sequence, and gene promoters, has also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as the subcellular location of miRNAs, the abundance of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. The miRNA sequence (typically 22 base pairs) is partially complementary to the binding sites on the 3' UTR of genes, often with full complementarity to 7 or 8 nucleotides in the "seed region" (residues 2-9) of the miRNA.Genetic suppression in mammals is believed to occur primarily through the inhibition of translation. However, miRNAs in mammals also cause mRNA decline. The miRNA target genes identified to date have been primarily involved in vital biological processes, such as development. Petition 870250099881, dated 10 / 31 / 2025, page 59 / 364 52 / 178 apoptosis, neural patterning, and viral infection. miRNAs are transcribed by RNA polymerase II as part of delimited and polyadenylated primary transcripts (pri-miRNAs) that may be protein-coding or non-coding. The primary transcript is cleaved by the enzyme Drosha ribonuclease III to produce a stem-loop precursor of approximately 70 nt (pre-miRNA), which is further cleaved by cytoplasmic ribonuclease Dicer to generate the mature miRNA and antisense miRNA star products (miRNA*). The mature miRNA is incorporated into an RNA-induced silencer complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in inhibition or destabilization of the target mRNA translation.

[0169] Nucleic acid molecules useful in dissemination methods include, but are not limited to, any nucleic acid molecule with activity or encoding a polypeptide. Polynucleotides with substantial identity to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridizing” refers to when nucleic acid molecules pair up to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various stringency conditions. (See, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). For example, the strict salt concentration may typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate.Low-rigor hybridization can be achieved in the absence of an organic solvent, for example, formamide, while high-rigor hybridization can be achieved in the presence of at least about 35% formamide, or at least about 50% formamide. Conditions of... Petition 870250099881, dated 10 / 31 / 2025, page 60 / 364 53 / 178 Strict temperatures may typically include temperatures of at least about 30 °C, at least about 37 °C, or at least about 42 °C. Additional variable parameters, such as hybridization time, detergent concentration, for example, sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of rigor are achieved by combining these various conditions as needed. In one exemplary embodiment, hybridization may occur at 30 °C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization may occur at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In another illustrative embodiment, hybridization can occur at 42 °C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA.Useful variations in these conditions will be readily apparent to those skilled in the art. For most applications, the washing steps following hybridization can also vary in rigor. The rigor of washing conditions can be defined by the salt concentration and temperature. As above, the rigor of washing can be increased by decreasing the salt concentration or increasing the temperature. For example, the rigorous salt concentration for the washing steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Rigorous temperature conditions for the washing steps can include a temperature of at least about 25 °C, at least about 42 °C, or at least about 68 °C. In exemplary embodiments, the washing steps can occur at 25 °C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.In other exemplary embodiments, the washing steps may occur at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the... Petition 870250099881, dated 10 / 31 / 2025, page 61 / 364 54 / 178 washing steps can occur at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations in these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0170] “Substantially identical” refers to a polypeptide or nucleic acid molecule exhibiting at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described in this document) or nucleic acid sequence (e.g., any of the nucleic acid sequences described in this document). Such a sequence may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., Genetics Computer Group sequence analysis software package, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis., programs 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX).This software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an illustrative approach to determining the degree of identity, a BLAST program can be used, with a... Petition 870250099881, dated 10 / 31 / 2025, p. 62 / 364 55 / 178 probability score between e-3 and em° indicating a closely related sequence. A “reference” is a standard of comparison. It will be understood that the numbering of specific positions or residues in the respective sequences depends on the particular protein or polypeptide and the numbering scheme used. The numbering may be different, for example, in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect the numbering. One skilled in the art will be able to identify the respective residue in any homologous protein and in the respective coding nucleic acid by methods well known in the art, for example, by sequence alignment with a reference sequence and determination of homologous residues.

[0171] A polypeptide (e.g., protein or peptide) or nucleic acid (e.g., transcript, e.g., mRNA, miRNA, siRNA, etc.) may be represented as “substantially expressed” in a cell or tissue, which may refer to the level of expression of the polypeptide or nucleic acid where the level of expression is substantial, e.g., readily detectable and / or at least within a comparable range relative to expression in another reference cell or tissue. The expression “substantially” as in “substantially expressed” may mean in one aspect that the expression is detectable or the element is expressed in a detectable manner.In some cases, substantially expressed is relative to one where it is not substantially expressed, while the former has higher expression than the latter, where the latter refers to an element where it is not substantially expressed and may not be detectable by common experimental procedures known in the art or relevant to the context. In some cases, the expression may be at least 10 times greater when referred to as “substantially expressed” compared to a reference expression level that may be within a detectable (but low) range and which is not substantially expressed. Petition 870250099881, dated 10 / 31 / 2025, p. 63 / 364 56 / 178

[0172] The term “subject” or “patient” may refer to an organism, such as an animal (e.g., a human) that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but is not limited to, a human or non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0173] The term “therapeutic effect” may refer to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasm, tumor, or infection by an infectious agent or an autoimmune disease) or its associated pathology. “Therapeutically effective amount,” as used in this document, may refer to an amount of an agent that is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survival capacity of the patient with such disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond what would be expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian skilled in the art may readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required. Untranslated regions (UTRs) 5' and 3', poly-A tail, 5' and 3' CAP of an mRNA

[0174] In one aspect, a manipulated polynucleotide construct for non-viral gene distribution and expression is disclosed in this document, including factors that could potentially affect the stability and translation efficiency of mRNA, for example, features related to a 5' untranslated region (5' UTR), a 3' UTR, and polyadenylation (poly A tail). The untranslated regions at the 5' and 3' ends flanking the coding sequence are critical for the expression of the encoded polypeptide when introduced into a cell. Of these, the 5' UTR Petition 870250099881, dated 10 / 31 / 2025, page 64 / 364 57 / 178 is responsible for the efficient assembly of the ribosome at the translation initiation codon. In some embodiments, the manipulated polynucleotide comprises a manipulated untranslated region (UTR).

[0175] Naturally occurring mRNAs contain a 5' cap structure that helps stabilize the RNA and is fundamental to eukaryotic gene expression (Shuman, S. et al. Mol. Microbiol. 1995, 17, 405-410). These mRNAs also contain a 3' poly A tail. Both modifications stabilize the protein-encoding mRNA. The 5' end modification may comprise a guanosine- or purine-based cap structure that increases the expression of RNA-encoded products (e.g., protein), decreases immunogenicity, and / or improves RNA stability. The 3' end modifications may comprise cis-diol modification at the 3' and 2' positions of the RNA terminal ribose, for example, by inserting a ring atom between these positions or by replacing one or both hydroxyl groups with other substituents, to improve RNA stability (e.g., by slowing the degradation rate).The 5'-cap structure found at the 5' end of eukaryotic messenger RNAs (mRNAs) and many viral RNAs consists of an N7-methylguanosine nucleoside linked to the 5'-terminal nucleoside of the pre-mRNA via a 5'-5' triphosphate linkage. This cap structure fulfills many functions that ultimately lead to mRNA translation. RNA capping is also important for other processes such as RNA splicing and export from the nucleus, and for preventing mRNA recognition by the cellular innate immunity machinery (Daffis, S. et al Nature 2010, 468, 4526; Zist, R. et al Nat. Immunol. 2011, 12, 137-43).

[0176] The 3' UTR can determine the half-life of an mRNA in a cell. mRNA degradation is regulated through protein complexes that bind to regions within the 3' UTR and the poly-A tail. For example, regulatory sequences in the 3' UTR containing adenylate-uridylate-rich elements (AU-rich elements; AREs) can destabilize Petition 870250099881, dated 10 / 31 / 2025, page 65 / 364 58 / 178 mRNAs. The most important regulatory elements involved are the poly-A tail and other cis elements, but multiple and diverse regulatory mechanisms have been described (Oktaba et al., 2015; Yue et al., 2018). miRNA and miRNA target sequences for negative regulation of expression in non-myeloid cell types.

[0177] This disclosure provides systems (including methods and compositions) for regulating mRNA expression in a cell that is not the desired “target cell” for mRNA expression. In some embodiments, this can be achieved by the careful inclusion of miRNA target regions in the mRNA whose expression is intended to be regulated. Canonical miRNA binding involves base pairing of the miRNA seed region (nucleotides 2-8) with complementary target sites. To take advantage of the effect of one or more cell-type-specific miRNAs, it would be necessary to have identified the miRNA target sequences of the miRNA seed region specific to the miRNA. Furthermore, it is necessary to have determined the miRNAs that are specific to the cell type other than the desired cell type, in order to use the miRNAs to regulate mRNA expression.In some embodiments, the mRNA is a recombinant mRNA comprising one or more miRNA target sites, such that a cell expressing a miRNA that interacts with the miRNA target site can cause the mRNA to decay or prevent translation of the mRNA. In one embodiment, the present disclosure is about expressing a nucleic acid sequence encoding a polypeptide from a recombinant nucleic acid in a specific target cell, in a situation, for example, where the recombinant nucleic acid is introduced systemically into an organism while simultaneously preventing the expression of the encoded polypeptide in a cell that is not the specific target cell (non-target cell or not the “desired” cell type). In another embodiment, an accidental uptake of a recombinant mRNA comprising a sequence encoding the polypeptide by a cell in which the recombinant mRNA is not. Petition 870250099881, dated 10 / 31 / 2025, page 66 / 364 59 / 178 desired expression can be subjected to a suppressive uptake effect by adopting a measure in the recombinant mRNA design to include miRNA target sites in the recombinant mRNA that would allow naturally occurring miRNA in the undesired cell type to degrade the recombinant mRNA or to prevent translation of the protein that is encoded by a sequence within the recombinant mRNA. In this approach, a negative regulation or negative control for recombinant mRNA expression is achieved by including miRNA target sites for one or more miRNAs that are prevalent in the undesired cell types but are absent in the cell type where the recombinant mRNA is desired to be expressed.

[0178] In one embodiment, the desired cell type to express the sequence is a myeloid cell, for example, a macrophage or monocyte. In some embodiments, the myeloid cell is a dendritic cell. In some embodiments, the myeloid cell is a CD14+ cell. In one embodiment, the undesired cell is an epithelial cell. In some embodiments, the undesired cell is a hepatocyte. In some embodiments, the desired cell is a myeloid cell, wherein a myeloid cell for the purposes of the present description is a monocyte-derived macrophage, a monocyte, a dendritic cell, and all the cells described above combined, in which the recombinant nucleic acid is desired to be expressed. On many occasions, an exogenous nucleic acid is absorbed into an abundant cell type and produces an off-target effect. This measure allows for the minimization of off-target effects of the administered recombinant nucleic acid.Epithelial cells, hepatocytes, and smooth muscle cells are abundant in living tissues. Therefore, systemically administered recombinant nucleic acids may produce off-target effects in these cells when the goal is for the recombinant nucleic acids to be expressed in a specific cell type other than these cells. Petition 870250099881, dated 10 / 31 / 2025, page 67 / 364 60 / 178

[0179] Candidate miRNAs for which target miRNA sequences are incorporated into mRNA constructs for selective mRNA expression in myeloid cells, and relative destruction or suppression of expression in non-myeloid cells must have at least 10 times higher expression in non-myeloid cells compared to myeloid cells. More consistently, the expression of candidate miRNAs is negligible or undetectable in myeloid cells. For the purposes of this description, a relative expression level of a candidate miRNA is at least 10 times lower in a myeloid cell compared to a non-myeloid cell.For example, the candidate miRNA is at least 10, 10, 1, 10, 2, 10, 3, 10, 4, 10, 5, 10, 6, 10, 7, 10, 8, 10, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 times more abundant in a non-myeloid cell, for example, a hepatocyte, than in a myeloid cell, such as a monocyte-derived myeloid cell.

[0180] In some embodiments, the prevalent miRNA in an undesired cell type is miR-192b. In some embodiments, the prevalent miRNA in an undesired cell type is miR-27b. In some embodiments, the prevalent miRNA in an undesired cell type is miR10a. In some embodiments, the recombinant mRNA comprises a miRNA target site for a miRNA that is highly prevalent in hepatocytes and epithelial cells and absent in myeloid cells. For example, in some embodiments, the miRNA is hsa-miR-192b (abbreviated as miR192b). For example, in one embodiment, the miRNA is hsa-miR-27b (abbreviated as miR-27b). For example, in one embodiment, the miRNA is hsa-miR-10a (abbreviated as miR-10a).

[0181] According to studies in the literature, miR-192 is highly expressed in hepatocytes and epithelial cells, and absent in myeloid cells. Cellular ontology information for miR-192 indicates significantly high expression of the microRNA in endodermal cells, cells Petition 870250099881, dated 10 / 31 / 2025, p. 68 / 364 61 / 178 epithelial and hepatocytes from human donor samples. Similarly, miR-27b is highly expressed in hepatocytes, endodermal cells, and endoepithelial cells. CD14+ myeloid cells, for example, monocytes, macrophages, and dendritic cells, show very low to almost negligible expression in human donors compared to non-myeloid cells, such as epithelial cells or hepatocytes. For example, in one study, dendritic cells derived from several donors show a lack of expression of miR-27b and miR-192b. For example, macrophages derived from monocytes from donor 1 and donor 2 show a miR-192b expression level of 179 and 266 arbitrary units, respectively; CD14+ monocytes from donor 1 show a miR-192b expression level of 1,355 arbitrary units; CD14+ monocytes from donor 2 show a miR-192b expression level of 1,644 arbitrary units.Compared to non-myeloid cells: miR-192b expression in donor 1 CD8+ T cells was 6,099 arbitrary units; in donor 2 CD8+ T cells it was 6,547 arbitrary units; in donor 3 CD8+ T cells it was 13,812 arbitrary units; in donor 1 CD4+ T cells it was 6,894 arbitrary units; in donor 2 CD4+ T cells it was 6,850 arbitrary units; in donor 1 CD19+ B cells it was 6,894 arbitrary units; in donor 2 CD19+ B cells it was 6,460 arbitrary units; in donor 2 NK cells it was 11,304 arbitrary units; in donor 3 NK cells it was 11,536 arbitrary units; The count in hepatocytes from donor 1 was 278,282 arbitrary units; in hepatocytes from donor 2 it was 238,786 arbitrary units; and in intestinal epithelial cells from donor 1 it was 303,408.miR-192b, therefore, appears to be a good candidate for suppressing the expression of recombinant polynuclear acid-encoded protein in hepatocytes and epithelial cells, while its expression would remain unaffected in myeloid cells. This is because miR-mediated downregulation of recombinant nucleic acid expression occurs naturally in cells that are not the desired cells. Petition 870250099881, dated 10 / 31 / 2025, page 69 / 364 Selective cellular metabolism of recombinant nucleic acid can be achieved in 62 / 178 cases.

[0182] The expression of miR-27b in CD14+ monocytes from donor 1 is 5,154 arbitrary units, in donor 2 is 5,119 arbitrary units, and in donor 3 is 4,492 arbitrary units; macrophages derived from monocytes from donor 1 are 2,143; from donor 2 is 718; and from donor 3 is 1,721 arbitrary units. In comparison, expression in hepatocytes was 91,758; 99,780 and 94,187 in the three donors, respectively. Therefore, miR-27b appears to be a good candidate for suppressing the expression of recombinant polynuclear acid-encoded protein in hepatocytes and epithelial cells, while its expression would remain unchanged in myeloid cells.

[0183] miR-10a expression appears to be significantly higher in many types of epithelial cells, hepatic sinusoidal endothelial cells, and muscle cells, while it is expressed at very low to almost negligible levels in dendritic cells, monocyte-derived macrophages, and CD14+ monocytes. For example, miR-10a expression in CD14+ monocytes is 1,258 and 939 arbitrary sample units from donor 1 and donor 2, respectively; monocyte-derived macrophages from donor 1 show an expression level of 2,127 arbitrary units, from donor 2 was 316 arbitrary units, and from donor 3 was 1,303 arbitrary units. In comparison, expression in renal epithelial cells was 242,330; in renal proximal tubular epithelial cells was 222,685; The number of renal glomerular endothelial cells was 125,314; and the number of hepatic sinusoidal epithelial cells was 140.929 arbitrary units from donor 1; and similarly, donor 2 cells showed high expression of miR-10a in epithelial, hepatic, and smooth muscle cells. For example, in renal proximal tubular epithelial cells, the expression was 207,925 and in renal mesangial cells it was 180,568 arbitrary units from donor 2 samples. In colonic smooth muscle cells, donor 1 expressed 171,804 arbitrary units and donor 2 expressed . Petition 870250099881, dated 10 / 31 / 2025, page 70 / 364 63 / 178 209,172 arbitrary units. These indicate that miR-10a appears to be a good candidate for suppressing the expression of recombinant polynucleic acid-encoded protein in non-myeloid cells, such as hepatocytes and epithelial and smooth muscle cells, while its expression would remain unaffected in myeloid cells.

[0184] In some embodiments, the exemplary human microRNA miR-192 (NR_029578) 110 bp RNA GCCGAGACCG AGTGCACAGG GCTCTGACCT ATGAATTGAC AGCCAGTGCT CTCGTCTCCC CTCTGGCTGC CAATTCCATA GGTCACAGGT ATGTTCGCCT CAATGCCAGC (SEQ ID NO: 1).

[0185] In some embodiments, the exemplary human microRNA miR-27b (NR_029665) 97 bp is: ACCTCTCTAA CAAGGTGCAG AGCTTAGCTG ATTGGTGAAC AGTGATTGGT TTCCGCTTTG TTCACAGTGG CTAAGTTCTG CACCTGAAGA GAAGGTG (SEQ ID NO: 2). In some embodiments, the exemplary human microRNA miR-10a (NR_029608) 110 bp RNA GATCTGTCTG TCTTCTGTAT ATACCCTGTA GATCCGAATT TGTGTAAGGA ATTTTGTGGT CACAAATTCG TATCTAGGGG AATATGTAGT TGACATAAAC ACTCCGCTCT (SEQ ID NO: 3).

[0186] In one embodiment, the recombinant mRNA may be designed to comprise one or more miRNA targets (e.g., targets for one or more different miRNAs). In one embodiment, the recombinant mRNA may be designed to comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNA targets for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different miRNAs, respectively. In some embodiments, the recombinant mRNA may be designed to comprise at least miRNA targets for at least 2 miRNAs. In some embodiments, the recombinant mRNA may be designed to comprise at least miRNA targets for at least 3 miRNAs.

[0187] In some embodiments, recombinant mRNA comprises Petition 870250099881, dated 10 / 31 / 2025, page 71 / 364 64 / 178 a miRNA target site in a 5' UTR site of the recombinant mRNA, designed in this document as miRNA-5p. In some embodiments, the recombinant mRNA comprises a miRNA target site in a 3' UTR site of the recombinant mRNA, designed in this document as miRNA-3p. In some embodiments, the recombinant mRNA comprises a miRNA target site in a 5' UTR and a miRNA target site in a 3' UTR site of the recombinant mRNA. In some embodiments, the recombinant mRNA comprises one or more miRNA target sites in a 5' UTR and one or more miRNA target sites in a 3' UTR site of the recombinant mRNA.

[0188] In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-192b and a second target site in the 3' UTR for miR-27b and / or miR-10a. In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-192b (e.g., miR-192b-5p), a second target site in the 3' UTR for miR-27b (e.g., miR-27b-3p), and a third target site in the 3' UTR for miR-10a (e.g., miR-10a-3p). In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-27b and a second target site in the 3' UTR for miR-192b and / or miR-10a. In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-27b (e.g., miR27b-5p), a second target site in the 3' UTR for miR-192b (e.g., miR192b-3p), and a third target in the 3' UTR for miR-10a (e.g., miR10a-3p).In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-10a (e.g., miR10a-5p), a second target site in the 3' UTR for miR-192b (e.g., miR192b-3p), and a third target in the 3' UTR for miR-27a (e.g., miR27b-3p).

[0189] In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-27b and a second target site in the 3' UTR for miR-192b and / or miR-10a. In some embodiments, the Petition 870250099881, dated 10 / 31 / 2025, p. 72 / 364 65 / 178 recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-27b (e.g., miR-27b-5p), a second target site in the 3' UTR for miR-192b (e.g., miR-192b-3p), and a third target in the 3' UTR site for miR-1 Oa (e.g., miR-10a-3p). In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-1 Oa (e.g., miR-10a-5p), a second target site in the 3' UTR for miR-192b (e.g., miR-192b-3p), and a third target in the 3' UTR site for miR-27a (e.g., miR-27b-3p).

[0190] In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-192b (e.g., miR-192-5p) and a second target site in the 5' UTR for miR-27b (e.g., miR-27b-5p) and a third target site in the 3' UTR for miR-10a (e.g., miR-10a-3p).

[0191] In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-27b (e.g., miR27b-5p) and a second target site in the 5' UTR for miR-10a (e.g., miR10a-5p) and a third target site in the 3' UTR for miR-192b (e.g., miR192b-3p).

[0192] In some embodiments, the recombinant mRNA comprises a first miRNA target site in the 5' UTR for miR-192b (e.g., miR-192b-5p) and a second target site in the 5' UTR for miR-10a (e.g., miR-10a-5p) and a third target site in the 3' UTR for miR-27b (e.g., miR-27b-3p).

[0193] In some embodiments, there are two, three or more miRNA target sequences in the 5' UTR of recombinant mRNA.

[0194] In some embodiments, there are two, three or more miRNA target sequences in the 3' UTR of recombinant mRNA.

[0195] In one aspect, a method is provided for the expression of an exogenous polypeptide, in which a recombinant nucleic acid is brought into contact with a heterogeneous population of cells comprising Petition 870250099881, dated 10 / 31 / 2025, page 73 / 364 66 / 178 a myeloid cell and a non-myeloid cell, wherein the expression of the exogenous polypeptide encoded by the recombinant nucleic acid occurs in the myeloid cell and does not occur in the non-myeloid cells of the heterogeneous cell population. The method comprises placing the heterogeneous cell population in contact with a composition comprising recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences described above; wherein the exogenous polypeptide is expressed in the myeloid cell of the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the non-myeloid cells of the heterogeneous cell population. In some embodiments, the exogenous polypeptide is expressed at a level that is at least 10 times higher in the myeloid cell than in the non-myeloid cells of the heterogeneous cell population.In some embodiments, the exogenous polypeptide not being substantially expressed in non-myeloid cells may refer to the exogenous polypeptide being expressed at least 10 times less in non-myeloid cells compared to myeloid cells from the heterogeneous cell population. In some embodiments, the exogenous polypeptide is expressed at least 10 to 100 times higher in myeloid cells than in non-myeloid cells from the heterogeneous cell population. In some embodiments, the expression of the exogenous polypeptide is undetectable in non-myeloid cells from the heterogeneous cell population. In some embodiments, the recombinant nucleic acid is mRNA.

[0196] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a myeloid cell is provided in this document, the method comprising placing a heterogeneous population of cells comprising a myeloid cell and a non-myeloid cell in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid Petition 870250099881, dated 10 / 31 / 2025, page 74 / 364 67 / 178 comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the exogenous polypeptide is expressed in the myeloid cell from the heterogeneous cell population, and wherein the exogenous polypeptide encoded by the recombinant nucleic acid sequence is not substantially expressed in the non-myeloid cells from the heterogeneous cell population. In some embodiments, the polypeptide required for the expression of the exogenous polypeptide is a polypeptide that is a regulator, for example, an enhancer, a transcription inducer or a transcription repressor, an inducer for an inducible promoter of the exogenous polypeptide that is expressed in the myeloid cell; miRNA-mediated suppression of the expression of such polypeptide would result in the lack of expression of the exogenous polypeptide.In some embodiments, the inducible promoter is operationally linked to the sequence encoding the exogenous polypeptide. For example, an inducible promoter is a tetracycline-inducible promoter. In some embodiments, the recombinant nucleic acid is DNA or RNA. In some embodiments, the RNA is polycistronic RNA. In some embodiments, the DNA is plasmid DNA.

[0197] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences; and wherein the recombinant nucleic acid lacks a sequence encoding a viral protein or portion thereof, and wherein Petition 870250099881, dated 10 / 31 / 2025, page 75 / 364 68 / 178 The exogenous polypeptide is expressed in the cell that is the first cell type in the heterogeneous cell population, and the exogenous polypeptide is not substantially expressed in the cell that is the second cell type in the heterogeneous cell population. In some embodiments, the first cell type is a myeloid cell type and the second cell type is a non-myeloid cell type. In some embodiments, the non-myeloid cell in the heterogeneous cell population comprises any of a hepatocyte, a lymphoid cell (e.g., T cell, B cell), an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell, and a skeletal cell. A myeloid cell may be, for example, a macrophage, a monocyte, a dendritic cell, a monocyte-derived cell, or a CD14+ cell.

[0198] In one aspect, a method for expressing an exogenous polypeptide of a recombinant nucleic acid in a first cell type is provided in this document, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide;wherein the composition lacks a sequence encoding a viral protein or portion thereof, and wherein the exogenous polypeptide is expressed in the cell that is the first cell type in the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the cell that is the second cell type in the heterogeneous cell population.

[0199] In one aspect, a method is provided in this document of Petition 870250099881, dated 10 / 31 / 2025, page 76 / 364 69 / 178 Expression of a chimeric fusion protein from a recombinant nucleic acid in a first cell type, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the chimeric fusion protein, and (b) one or more miRNA target sequences; and wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous cell population, and wherein the chimeric fusion protein is not substantially expressed in the cell that is the second cell type of the heterogeneous cell population. In some embodiments, the first cell type is a myeloid cell type. In some embodiments, the second cell type is a non-myeloid cell type.In some forms, the non-myeloid cell type includes an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell, a skeletal cell, or a hepatocyte.

[0200] In one aspect, a method is provided in this document for expressing a chimeric fusion protein of a recombinant nucleic acid in a first cell type, the method comprising placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant nucleic acid comprises (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences, and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous population of cells, Petition 870250099881, dated 10 / 31 / 2025, page 77 / 364 70 / 178 and in which the chimeric fusion protein is not substantially expressed in the cell that is the second cell type in the heterogeneous cell population. In one aspect, the first cell type is a myeloid cell type. In some embodiments, the second cell type is a non-myeloid cell type.

[0201] In some embodiments, one or more target regulatory factors may be co-expressed. Exemplary miRNA target regulatory factors may include siRNA, shRNA, and / or miRNA. In some embodiments, siRNA, shRNA, and / or miRNA target RNA. In some embodiments, the mixture comprises an RNA mimic, which is a sequence that is analogous to another RNA sequence. In some embodiments, the mixture comprises an agent or small molecule fraction that regulates target miRNA levels. In some embodiments, these components will be administered together. In some embodiments, these components may be administered separately.

[0202] In some embodiments, the expression of the polypeptide encoded by the recombinant nucleic acid, as designed above, occurs in a myeloid cell; whereas, it is not substantially expressed in the non-myeloid heterogeneous cell population. In some embodiments, the expression of a recombinant nucleic acid polypeptide, as designed above, is not substantially expressed when the expression is at least 10 times lower in a second cell compared to a first cell where it is substantially expressed. In some embodiments, the expression of a recombinant nucleic acid polypeptide with the miRNA target sequences is at least 10 times lower in the undesired cells expressing the miRNA, compared to a desired cell in which the miRNA is absent.In some embodiments, the expression of a recombinant nucleic acid polypeptide with miRNA target sequences is at least 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. Petition 870250099881, dated 10 / 31 / 2025, page 78 / 364 71 / 178 75, 80, 85, 90, 95, 100 or more times lower in undesired cells expressing the miRNA, compared to a desired cell in which the miRNA is absent. In some embodiments, the expression of a recombinant nucleic acid polypeptide with the miRNA target sequences is undetectable in the undesired cell types.

[0203] In some embodiments, the potentiation of the expression of a polypeptide encoded by a polynucleotide can be achieved using non-native or synthetic 5' and 3' UTRs. In some embodiments, recombinant polynucleotides are manipulated with non-native or synthetic 5' and 3' UTRs that greatly potentiate the expression of a protein-coding sequence comprised in a nucleic acid when introduced into a myeloid cell. In some embodiments, the 5' UTR can be encoded within a plasmid in which the DNA sequence encoding an exogenous protein-coding sequence is incorporated for distribution and expression. Similarly, in some embodiments, the 3' UTR can also be encoded within a plasmid in which the DNA sequence encoding an exogenous protein-coding sequence is incorporated for distribution and expression. In some embodiments, the nucleic acid is an IVT RNA.In some embodiments, the 5' and / or 3' UTR can be added to mRNA comprising the sequence encoding an exogenous protein in vitro via an enzymatic reaction. A poly-A tail can be enzymatically added in vitro to an RNA or can be an encoded poly-A tail, such as by an IVT template. In some embodiments, the enzymatically added poly-A tails can be tailored to have a desired length.

[0204] A suitable 5' cap structure is important in the synthesis of functional messenger RNA. In some embodiments, the 5' cap comprises a guanosine triphosphate arranged as GpppG at the 5' terminus of the nucleic acid.

[0205] In some embodiments, mRNA comprises a cap 7Petition 870250099881, of 10 / 31 / 2025, p. 79 / 364 72 / 178 methylguanosine 5', m7-GpppG. A 7-methylguanosine 5' cap can increase mRNA translation efficiency and prevent 5'-3' mRNA exonuclease degradation. In some embodiments, the mRNA comprises an “anti-reverse” cap analog (ARCA, m7,3'-O GpppG).

[0206] In some embodiments, the guanosine cap is a CapO structure.

[0207] In some embodiments, the guanosine cap is a Cap 1 structure.

[0208] In addition to its essential role in the cap-dependent initiation of protein synthesis, the mRNA cap can also function as a protective group for 5' to 3' exonuclease cleavage and a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation, and nuclear export. It acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5' to 3' loop of mRNA during translation. Three enzymatic activities are required to generate the Cap 0 structure, namely RNA triphosphatase (TPase), RNA guanyltransferase (GTase), and guanine-N7 methyltransferase (guanine-N7 MTase). Each of these enzymatic activities performs an essential step in converting the 5' triphosphate of the nascent RNA to the Cap 0 structure. RNA TPase removes the γ-phosphate from the 5' triphosphate to generate RNA 5' diphosphate. GTase transfers a GMP group from GTP to the 5' diphosphate via a covalent lysine-GMP intermediate.Guanine-N7 MTase then adds a methyl group to the N7 amine of the guanine cap to form the cap 0 structure. For the cap 1 structure, m7G-specific 2I methyltransferase (2I MTase) methylates the +1 ribonucleotide at the 2'0 position of the ribose to generate the cap 1 structure. The nuclear RNA capping enzyme interacts with the polymerase subunit of the RNA polymerase II complex on the phosphorylated Ser5 repeats of the C-terminal heptad repeats. RNA guanine-N7 methyltransferase also interacts with the phosphorylated heptad repeats of RNA polymerase II. In some embodiments, the cap is a G-quadruplex cap. In some... Petition 870250099881, dated 10 / 31 / 2025, page 80 / 364 In 73 / 178 embodiments, the 5' cap is added to the 5' end of mRNA by cotranscriptional capping, in which a cap analog is introduced into the transcription reaction along with the four standard nucleotide triphosphates at an optimized cap analog to GTP ratio of 4:1. This allows the transcript to begin with the cap structure in a large proportion of the synthesized RNA molecules. This approach produces a mixture of transcripts, of which 80% have caps, and the remainder have 5' triphosphate ends. In some embodiments, the cap analogs used in cotranscriptional RNA capping are the standard 7-methylguanosine (m7G) cap analog.

[0209] In some embodiments, the cap analogs used in cotranscriptional RNA capping are the anti-reverse cap analog (ARCA), also known as the 7-meGpppG 3' 0-me cap analog. ARCA is methylated at the 3' position of m7G, preventing RNA elongation by forming a phosphodiester bond at this position. Thus, transcripts synthesized using ARCA contain 5'-m7G cap structures in the correct orientation, with the 7-methylated G as the terminal residue. In contrast, the m7G cap analog can be incorporated in either the correct or reverse orientation.

[0210] In one embodiment, the 5' UTR comprises about 50 nucleotides. In some embodiments, the 5' UTR comprises fewer than 50 nucleotides. In one embodiment, the 5' UTR may comprise more than 50 nucleotides. In one embodiment, the 5' UTR comprises at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0211] In some embodiments, the 5' UTR comprises at least 45 nucleotides in length. In one embodiment, the 5' UTR comprises a translation initiation site that is at least 15 nucleotides downstream from the 5' end. In some embodiments, the 5' UTR comprises at least 45 nucleotides in length. In one embodiment, the 5' UTR comprises a translation initiation site that is by Petition 870250099881, dated 10 / 31 / 2025, page 81 / 364 74 / 178 minus 20 nucleotides downstream of the 5' end. In some embodiments, the 5' UTR comprises at least 45 nucleotides in length. In one embodiment, the 5' UTR comprises a translation initiation site that is at least 25 nucleotides downstream of the 5' end. In some embodiments, the 5' UTR comprises at least 45 nucleotides in length. In one embodiment, the 5' UTR comprises a translation initiation site that is at least 30 nucleotides downstream of the 5' end.

[0212] In some embodiments, the 5' UTR comprises at least 45 nucleotides in length. In one embodiment, the 5' UTR comprises a single translation initiation site.

[0213] In some embodiments, the addition of a poly-A tail at the end of transcription can be done enzymatically. For example, the poly-A tail can be made using E. coli poly(A) polymerase. Alternatively, for example, the poly-A tail can be made using Saccharomyces (yeast) poly(A) polymerase. The length of the introduced poly-A tail can be optimized by titrating the reaction.

[0214] In some embodiments, the nucleic acid comprises about 100-250 polyadenosyl (poly(A)) residues.

[0215] In some embodiments, the length of poly A is between 50-200 adenosine nucleotide residues (SEQ ID NO: 27).

[0216] In some embodiments, the length of poly A is at least about 120 adenosine nucleotide residues.

[0217] In some embodiments, the length of poly A is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or about 250 adenosine residues.

[0218] In some embodiments, the 5' and 3' UTRs for stabilizing nucleic acid within the cell and ensuring enhanced expression of the exogenous protein comprise any of the sequences described in this document. In some embodiments, the cell is a myeloid cell. Petition 870250099881, dated 10 / 31 / 2025, page 82 / 364 75 / 178 In some modalities, the cell is a myeloid precursor cell. In some modalities, the cell is a CD14+ cell. In some modalities, the cell is a CD14+ / CD16- cell.

[0219] In some embodiments, an mRNA with 50 nucleotides 20,000 nucleotides can be expressed in myeloid cells using the methods described in this document. In some embodiments, the mRNA comprises a coding region of about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000 or about 20,000 nucleotides.

[0220] In some embodiments, replacing the 5' UTR and / or the 3' UTR improves the expression of the encoded protein or polypeptide by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more compared with standard UTRs.

[0221] In some embodiments, replacing the 5' UTR and / or the 3' UTR improves the duration of expression of the encoded protein or polypeptide in the myeloid cell by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more compared to Standard UTRs.

[0222] In some modalities, protein expression is detectable according to an immunoassay after at least 72 hours after Petition 870250099881, dated 10 / 31 / 2025, page 83 / 364 76 / 178 (a). In some embodiments, protein expression is detectable in at least 10% to at least 50% of cells according to an immunoassay after at least 72 hours after (a). In some embodiments, protein expression is detectable in at least 40% of cells according to an immunoassay after at least 72 hours after (a). In some embodiments, protein expression is detectable in at least 30% of cells according to an immunoassay after at least 72 hours after (a). In some embodiments, protein expression is detectable in at least 20% of cells after at least 72 hours after (a). In some embodiments, protein expression is detectable according to an immunoassay after at least 48 hours after (a). In some embodiments, protein expression is detectable in at least 10% to at least 90% of cells according to an immunoassay after at least 48 hours after (a).In some embodiments, protein expression is detectable in at least 80% of cells, at least 70% of cells, at least 60% of cells, at least 50% of cells, at least 40% of cells, at least 30% of cells, or at least 20% of cells according to an immunoassay after at least 48 hours after (a).

[0223] In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after the incorporation of the nucleic acid into the cell. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, up to and / or at least 96 hours after the incorporation of the nucleic acid into the cell. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, up to and / or at least 3 days after incorporation. Petition 870250099881, dated 10 / 31 / 2025, page 84 / 364 77 / 178 of the nucleic acid in the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 4 days after the incorporation of the nucleic acid into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 5 days after the incorporation of the nucleic acid into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 6 days after the incorporation of the nucleic acid into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 7 days after the incorporation of the nucleic acid into the cell.In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 8 days after the nucleic acid is incorporated into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 9 days after the nucleic acid is incorporated into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, up to and / or at least 10 days or more after the nucleic acid is incorporated into the cell. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, in at least 10% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by a Petition 870250099881, dated 10 / 31 / 2025, page 85 / 364 78 / 178 immunoassay, in at least 20% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, in at least 30% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, in at least 40% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, in at least 50% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, Petition 870250099881, dated 10 / 31 / 2025, page 86 / 364 79 / 178 or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, in at least 60% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, in at least 70% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a nucleic acid-encoded protein is detectable, such as by an immunoassay, in at least 80% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or 10 or more days after the incorporation of the nucleic acid into the cell population. In some embodiments, the expression of a protein encoded by the nucleic acid is detectable, such as by an immunoassay, in at least 90% of the cells in a cell population up to and / or at least 24, 25, 26, 27, 28, 29, 30, Petition 870250099881, dated 10 / 31 / 2025, page 87 / 364 80 / 178 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 hours, or up to and / or at least 3, 4, 5, 6, 7, 8, 9 or more days after the incorporation of the nucleic acid into the cell population.

[0224] In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell comprises a cell infected with a pathogen. In some embodiments, the target cell is a cancerous cell. In some embodiments, the target cell is a cancerous cell that is a lymphocyte. In some embodiments, the target cell is a cancerous cell that is an ovarian cancerous cell. In some embodiments, the target cell is a cancerous cell that is a breast cancer cell. In some embodiments, the target cell is a cancerous cell that is a pancreatic cell. In some embodiments, the target cell is a cancerous cell that is a glioblastoma cell.

[0225] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is an unmodified mRNA. In some embodiments, the nucleic acid is a modified mRNA. In some embodiments, the nucleic acid is a circRNA. In some embodiments, the nucleic acid is a tRNA. In some embodiments, the nucleic acid is a microRNA. In some embodiments, the nucleic acid is a manipulated nucleic acid. In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, the nucleic acid is a nucleic acid transcribed in vitro.

[0226] Also provided in this document is a vector comprising a nucleic acid sequence encoding a CFP described in this document. In some embodiments, the vector is a viral vector. Petition 870250099881, dated 10 / 31 / 2025, p. 88 / 364 81 / 178 In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operationally linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is polycistronic. In some embodiments, each of at least one nucleic acid sequence is operationally linked to a separate promoter. In some embodiments, the vector further comprises one or more internal ribosome entry sites (IRESs). In some embodiments, the vector further comprises a 5' UTR and / or a 3' UTR flanking at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0227] Also provided in this document is a nucleic acid-encoded polypeptide of a composition described in this document.

[0228] Also provided in this document is a cell comprising a composition described in this document, a vector described in this document, or a polypeptide described in this document. In some embodiments, the cell is a phagocytic cell. In some embodiments, the cell is a stem cell-derived cell, a myeloid cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglia, or an astrocyte. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is an M1 cell. In some embodiments, the cell is an M2 cell. In some embodiments, the cell is an M1 macrophage cell. In some embodiments, the cell is an M2 macrophage cell. In some embodiments, the cell is an M1 myeloid cell. In some embodiments, the cell is an M2 myeloid cell. mRNA nucleotide modifications Petition 870250099881, dated 10 / 31 / 2025, page 89 / 364 82 / 178

[0229] In some aspects, the oligonucleotide described in this document comprises at least one chemical modification. A chemical modification may be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof. In some cases, a modification is a chemical modification. In one embodiment, the modification may be a phosphonate modification. In one aspect, the phosphonate modification is a phosphorothioate (PS Rp isomer). In one aspect, the phosphonate modification is a phosphorothioate (PS Sp isomer).

[0230] In some respects, the phosphate group of a chemically modified nucleotide may be modified by replacing one or more of the oxygens with a different substituent. In some respects, the chemically modified nucleotide may include the replacement of an unmodified phosphate moiety with a modified phosphate, as described in this document. In some respects, modification of the phosphate backbone may include changes that result in an uncharged ligand or a charged ligand with an asymmetric charge distribution. Examples of modified phosphate groups may include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranephosphates, boranephosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates, and phosphotriesters.In some respects, one of the non-bridged phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (where R can be, for example, hydrogen, alkyl, or aryl), C (for example, an alkyl group, an aryl group, and the like), H, NR2 (where R can be, for example, hydrogen, alkyl, or aryl), or (where R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group can be achiral. However, replacing one of the non-bridged oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral. A phosphorus atom in a phosphate group modified in this way is a stereogenic center. Petition 870250099881, dated 10 / 31 / 2025, page 90 / 364 83 / 178 stereogenic phosphorus atoms may possess either the “R” (Rp in this document) or “S” (Sp in this document) configuration. In some cases, the oligonucleotide comprises stereopure nucleotides comprising either the S conformation of phosphorothioate or the R conformation of phosphorothioate. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90% or more. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 95%. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 96%. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 97%. In some aspects, the chiral phosphate product is present in a diastereomeric excess of 98%. In some respects, the chiral phosphate product is present in a diastereomeric excess of 99%.In some respects, both non-bridged oxygens of phosphorodithioates can be replaced by sulfur. The phosphorus center in phosphorodithioates can be achiral, which prevents the formation of oligoribonucleotide diastereomers. In some respects, modifications to one or both non-bridged oxygens can also include the substitution of the non-bridged oxygens by a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl). In some respects, the phosphate linker can also be modified by the substitution of a bridged oxygen (i.e., the oxygen that links the phosphate to the nucleoside) by nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates). The substitution can occur at one or both linking oxygens.

[0231] In one embodiment, the modification may be a ribose modification. In one aspect, the ribose modification is a 2'O-methyl (2'-OME) modification. In one aspect, the ribose modification is a 2'-O-methoxyethyl (2'-O-MOE) modification. In one aspect, the ribose modification is 2'-deoxy-2'-fluoro (2'-F). In one aspect, the ribose modification is fluoro 2' Petition 870250099881, dated 10 / 31 / 2025, page 91 / 364 84 / 178 arabino (2'-Ara-F). In one aspect, the ribose modification is 2'-O-benzyl. In one aspect, the ribose modification is 2'-O-methyl-4-pyridine (2'-OCH2Py(4)). In one aspect, the ribose modification is a blocked nucleic acid (LNA). In one aspect, the ribose modification may be a base modification. In one aspect, the base modification is pseudouridine (Ψ). In one aspect, the ribose modification is 2'-thiouridine (s2U). In one aspect, the ribose modification is N6'-methyladenosine (m6C). In one aspect, the ribose modification is 5'-methylcytidine (m5C). In one aspect, the ribose modification is 5'-fluoro-2'-dioxyuridine. In one aspect, the ribose modification is N-ethylpiperidine (adenine modified by 7'-EAA triazole). In one aspect, the ribose modification is adenine modified by 6'-N-ethylpiperidine triazole. In one aspect, the ribose modification is 6-phenylpyrrolocytosine. In one aspect, the ribose modification is 2'-4'-difluorotolyl ribonucleoside (rF).In one aspect, the ribose modification is 5'-nitroindole.

[0232] In some respects, the chemical modification described in this document comprises modification of the nucleotide base (e.g., the nucleobase). Exemplary nucleobases may include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases may be modified or substituted in the nucleic acids described in this document. The nucleotide nucleobase may be independently selected from a purine, a pyrimidine, a purine, or a pyrimidine analog. In some respects, the nucleobase may be natural or synthetic derivatives of a base.

[0233] In some respects, the chemical modification described in this document comprises the modification of a uracil. In some respects, the oligonucleotide described in this document comprises at least one chemically modified uracil. Exemplary chemically modified uracil may include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4 Petition 870250099881, dated 10 / 31 / 2025, page 92 / 364 85 / 178 thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g. 5-iodo-uridine or 5-bromo-uridine), 3-methyluridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, acid methyl ester Uridine 5-oxyacetic acid, 5-carboxymethyl-uridine, 1-carboxymethylpseudouridine, 5-carboxy-hydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 5methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propyni lurid i na, 1 -propynyl pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio1-methyl-pseudouridine,3-methyl-pseudouridina, 2-thio-1-methyl-pseudouridina, 1methyl-1 -deaza-pseudouridina, 2-thio-1 -methyl-1 -deaza-pseudouridina, dihydroundina, di-hidropseudoundina, 5,6-dihydrouridina, 5-methyl-dihydrouridina, 2-thio-dihydrouridina, 2-thio-dihydrouridina, 2-methoxyuridina, 2-methoxy-4-thio-uridina, 4-methoxy-pseudouridina, 4-methoxy-2-thiopseudouridina, N1-methyl-pseudouridina, 3-(3-amino-3-carboxypropyl)uridina, 1-methyl-3-(3-amino-3-carboxypropy pseudouridina, 5(isopentenylaminomethyl)uridina, 5-(isopentenylaminomethia])-2-thio-uridina, atio-uridina, 2'-O-metil-uridina, 5,2'-O-dimethyl-uridina, 2'-0-metilpseudouridina, 2-thio-2'-O-metil-uridina, 5-methoxycarbonylmetil-2'-O-metiluridina, 5-carbamoylmetil-2'-O-metil-uridina, 5-carboxymetilaminometil-2'-O-metil-uridina, 3,2'-O-metil-uridina, 5-(isopentenylaminometil)-2'-O-metiluridina, l-thio-uridina, desoxitimida, 2'-F-ara-uridina, 2'-F-uridina, 2'-OH-arauridina, 5-(2-carbomethoxyvinyl)uridina, 5-[3-( lE-propenylamino)uridina, pyrazolo[3,4-d]pyrimidines, xanthine and hypoxanthine.,

[0234] In some respects, the chemical modification described in this document involves the modification of a cytosine. In some respects, Petition 870250099881, dated 10 / 31 / 2025, page 93 / 364 86 / 178 The oligonucleotide described in this document comprises at least one chemically modified cytosine.Examples of chemically modified cytosines may include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methylcytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularin, 5-azazebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocitidine, 4methoxy-1-methyl-pseudoisocitidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-Phara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0235] In some respects, the chemical modification described in this document comprises the modification of an adenine. In some respects, the oligonucleotide described in this document comprises at least one chemically modified adenine.Exemplary chemically modified adenine may include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, 2-methyladenine, N6-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxy-isopentenyl)adenosine, 2-methylthio-N6(cis-hydroxy-isopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6, N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl. Petition 870250099881, dated 10 / 31 / 2025, page 94 / 364 87 / 178 adenosine, N6-acetyladenine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, α-thioadenosine, 2'-O-methyladenosine, N6, 2'-O-dimethyladenosine, N6-Methyl-2'-deoxyadenosine, N6, N6, 2'-O-trimethyladenosine, 1,2'O-dimethyladenosine, 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-ara-adenosine, 2'-Fadenosine, 2'-OH-ara-adenosine and N6-(19-amino-pentaoxanonadecyl)adenosine.

[0236] In some respects, the chemical modification described in this document comprises modifying a guanine. In some respects, the oligonucleotide described in this document comprises at least one chemically modified guanine. Examples of chemically modified guanine may include inosine, 1-methyl-inosine, viosine, methylviosine, 4-demethyl-viosine, isoviosine, vibutosine, peroxyvibutosine, hydroxyvibutosine, unmodified hydroxyvibutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-azaguanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-azaguanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2, N2-dimethylguanosine, N2, 7-dimethyl-guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxoguanosine, 7-methyl-8-oxo-guanosine, 1-methio-guanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methylguanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methylguanosine, 1-methyl-2'-O-methyl-guanosine, N2, 7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, I, 2'-O-dimethyl-inosine, 6-O-phenyl-2'-deoxy-inosine, 2'-Oribosylguanosine, 1-thio-guanosine, 6-O-methyguanosine, O6-Methyl-2'deoxyguanosine, 2'-F-ara-guanosine and 2'-F-guanosine.,

[0237] In some forms, the modification is at a terminal nucleotide.

[0238] In some modalities, the modification is in a Petition 870250099881, dated 10 / 31 / 2025, page 95 / 364 88 / 178 internal nucleotide.

[0239] In some forms, the mRNA is not modified.

[0240] In some embodiments, mRNA comprises 1, 2, or 3 or more modified nucleotides.

[0241] In some cases, a modification may be permanent. In other cases, a modification may be transient. In some cases, multiple modifications are made to the nucleic acid. Nucleic acid modification may alter the physicochemical properties of a nucleotide, such as its conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.

[0242] In some embodiments, a recombinant nucleic acid of the dissemination encodes a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, one or more intracellular signaling domains comprise a phagocytosis signaling domain or phagocytosis enhancer domain, for example, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRy, FcRa, or FcRs. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from OO3ζ. In some embodiments, one or more intracellular signaling domains additionally comprise a pro-inflammatory signaling domain.In some modalities, the pro-inflammatory signaling domain includes a PI3-kinase (PI3K) recruitment domain.

[0243] In some embodiments, the CFP comprises: (a) an extracellular domain comprising: (i) an scFv that specifically binds to CD5 (anti-CD5 binding domain), or an scFv that binds to HER2, or TROP2, or GPC3; and (ii) a CD8-derived hinge domain; a domain of Petition 870250099881, dated 10 / 31 / 2025, page 96 / 364 89 / 178 hinge derived from CD28 or at least a portion of an extracellular domain of CD68; (b) a transmembrane domain of CD8, a transmembrane domain of CD28, a transmembrane domain of CD2, or a transmembrane domain of CD68; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRa, FcRy, or FcRe, and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain, or (B) derived from CD40.In some embodiments, the CFP comprises, as an alternative (c) to the above: an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from an intracellular domain of the phagocytic receptor and (ii) a second intracellular signaling domain derived from an intracellular domain of the phagocytic receptor of the sequestering receptor comprising: (A) comprising a PI3K recruitment domain, or (B) derived from CD40. Table 1A lists an illustrative set of various domains and combinations thereof that have been used to design the CFPs described in this document. In some embodiments, the CFP comprises an intracellular signaling domain derived from an intracellular signaling domain of an innate immune receptor.

[0244] In some embodiments, the polynucleic acid is an mRNA. In some embodiments, the polynucleic acid is a circRNA. In some embodiments, the polynucleic acid is a viral vector. In some embodiments, the polynucleic acid is delivered via a viral vector.

[0245] In some embodiments, the myeloid cell is a CD14+ cell, a CD14+ / CD16- cell, a CD14+ / CD16+ cell, a CD14- / CD16+ cell, a CD14- / CD16- cell, a dendritic cell, an MO macrophage, an M2 macrophage, an M1 macrophage, or a myeloid cell in Petition 870250099881, dated 10 / 31 / 2025, page 97 / 364 90 / 178 mosaic / macrophage / dendritic cell.

[0246] In one aspect, a method of treating cancer in a human subject in need is provided in this document comprising administering a pharmaceutical composition to the human subject, the pharmaceutical composition comprising: (a) a myeloid cell comprising a polynucleic acid sequence, wherein the polynucleic acid sequence comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operationally linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier; wherein the myeloid cell expresses the CFP.

[0247] In some embodiments, after CFP binds to CD5 expressed by a target cancer cell, the killing or phagocytic activity of the myeloid cell subject is increased by more than 20% compared to a myeloid cell that does not express CFP. In some embodiments, tumor growth is inhibited in the human subject.

[0248] In some forms, the cancer is a CD5+ cancer. In some forms, the cancer is leukemia, T-cell lymphoma, or B-cell lymphoma.

[0249] In some embodiments, the anti-CD5 binding domain is a CD5-binding protein comprising an antigen-binding fragment of an antibody, an scFv domain, a Fab fragment, or an sdAb domain. In some embodiments, the anti-CD5 binding domain is a protein or fragment thereof that binds to CD5, such as a CD5 ligand (e.g., a natural CD5 ligand).

[0250] In some embodiments, the CFP additionally comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, wherein one or more intracellular signaling domains comprise a signaling domain of Petition 870250099881, dated 10 / 31 / 2025, page 98 / 364 91 / 178 phagocytosis and in which a wild-type protein comprising the intracellular domain does not comprise the extracellular domain.

[0251] In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than MegflO, MerTk, FcRo, and Bail. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRy, FcRa, or FcRs.

[0252] In some embodiments, one or more intracellular signaling domains additionally comprise pro-inflammatory signaling domains. In some embodiments, the pro-inflammatory signaling domain comprises a PI3-kinase (PI3K) recruitment domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the extracellular domain comprises a CD8-derived hinge domain, a CD28-derived hinge domain, or at least a portion of a CD68 extracellular domain.

[0253] In some embodiments, the CFP comprises: (a) an extracellular domain comprising: (i) an scFv that specifically binds to CD5, and (ii) a hinge domain derived from CD8, a hinge domain derived from CD28, or at least a portion of an extracellular domain of CD68; (b) a transmembrane domain of CD8, a transmembrane domain of CD28, a transmembrane domain of CD2, or a transmembrane domain of CD68; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRy or FcRs, and (ii) a second intracellular signaling domain that: (A) comprises a PI3K recruitment domain, or (B) is derived from CD40. In some embodiments, the nucleic acid is mRNA or circRNA. In some forms, the myeloid cell is a CD14+ cell, a cell Petition 870250099881, dated 10 / 31 / 2025, page 99 / 364 92 / 178 CD14+ / CD16-, a CD14+ / CD16+ cell, a CD14- / CD16+ cell, a CD14- / CD16- cell, a dendritic cell, an MO macrophage, an M2 macrophage, an M1 macrophage, or a mosaic myeloid cell / macrophage / dendritic cell.

[0254] In some embodiments, the method further comprises administering an additional therapeutic agent selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits a GTPase, an agent that promotes the disassembly of F-actin, an agent that promotes the recruitment of PI3K to the PFP, an agent that promotes PI3K activity, an agent that promotes the production of 3,4,5-trisphosphate phosphatidylinositol, an agent that promotes the activity of ARHGAP12, an agent that promotes the activity of ARHGAP25, an agent that promotes the activity of SH3BP1, an agent that promotes the sequestration of lymphocytes in primary and / or secondary lymphoid organs, an agent that increases the concentration of unexposed T cells and central memory T cells in secondary lymphoid organs, and any combination thereof.

[0255] In some embodiments, the myeloid cell additionally comprises: (a) an endogenous peptide or protein that dimerizes with CFP, (b) a non-endogenous peptide or protein that dimerizes with CFP; and / or (c) a second recombinant nucleic acid sequence, wherein the second recombinant nucleic acid sequence comprises a sequence encoding a peptide or protein that interacts with CFP; wherein the dimerization or interaction enhances phagocytosis by the myeloid cell expressing CFP compared with a myeloid cell not expressing CFP.

[0256] In some forms, the myeloid cell exhibits (i) increased effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-trisphosphate production, Petition 870250099881, dated 10 / 31 / 2025, page 100 / 364 93 / 178 phagocytosis with the target cell expressing the antigen, resistance to CD47-mediated inhibition of phagocytosis, resistance to LILRB1-mediated inhibition of phagocytosis, or any combination thereof; and / or (ii) an increase in the expression of IL-1, IL-3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, a TNF family of cytokines, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, LCR, LCR, GMCSF, IL-17, IP-10, RANTES, an interferon, MHC class I protein, MHC class II protein, CD40, CD48, CD58, CD80, CD86, CD112, CD155, a death receptor of the TRAIL / TNF family, TGFβ, B7-DC, B7-H2, LIGHT, HVEM, TL1A, 41BBL, OX40L, GITRL, CD30L, TIM1, TIM4, SLAM, PDL1, an MMP (by For example, MMP2, MMP7 and MMP9) or any combination thereof.

[0257] In some embodiments, the intracellular signaling domain is derived from a phagocytic or binding receptor or in which the intracellular signaling domain comprises a phagocytosis activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from MegflO, MerTk, FcR-alpha or Bail. In some embodiments, the intracellular signaling domain is derived from a protein, such as a receptor (e.g., a phagocytic receptor), selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, sequestering receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fea I receptor, CR1, CD35, CD3ζ, a complement receptor, CR3, CR4, Tim-1, Tim-4, and CD169.In some modalities, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some modalities, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain. Petition 870250099881, dated 10 / 31 / 2025, page 101 / 364 94 / 178

[0258] In some embodiments, the intracellular signaling domain is derived from a receptor containing an ITAM domain.

[0259] In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from MegflO, MerTk, FcRa or Bail.

[0260] In some embodiments, the intracellular signaling domain is derived from a receptor, such as a phagocytic receptor, selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, sequestering receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fea I receptor, CR1, CD35, CD3C, CR3, CR4, Tim-1, Tim-4, and CD169. In some modalities, the intracellular signaling domain includes a pro-inflammatory signaling domain.

[0261] In some embodiments, the intracellular signaling domain is derived from a receptor, such as a phagocytic receptor, selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, sequestering receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fea I receptor, CR1, CD35, CD3C, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor other than a phagocytic receptor selected from MegflO, MerTk, FcRa, or Bail.In some modalities, the intracellular signaling domain comprises an intracellular signaling domain. Petition 870250099881, dated 10 / 31 / 2025, page 102 / 364 95 / 178 derived from a phagocytic receptor selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, sequestering receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha I receptor, CR1, CD35, Oϋ3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular signaling domain comprises a scavenger receptor-derived intracellular signaling domain. In some embodiments, the intracellular domain comprises a CD47 inhibition domain. In some embodiments, the intracellular domain comprises a Rac inhibition domain, a Cdc42 inhibition domain, or a GTPase inhibition domain.In some embodiments, the Rac inhibition domain, the Cdc42 inhibition domain, or the GTPase inhibition domain inhibits Rac, Cdc42, or GTPase in a phagocytic cup of a cell expressing PFP. In some embodiments, the intracellular domain comprises an F-actin disassembly activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibition domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibition domain. In some embodiments, the intracellular domain comprises at least one ITAM domain. In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ITAM domains.In some embodiments, the intracellular domain comprises at least one ITAM domain selected from an ITAM domain of CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc receptor chain epsilon 1, Fc receptor chain epsilon 2, Fc receptor chain gamma 1, Fc receptor chain. Petition 870250099881, dated 10 / 31 / 2025, page 103 / 364 96 / 178 gamma 2a, Fc gamma 2b 1 receptor chain, Fc gamma 2b2 receptor chain, Fc gamma 3a receptor chain, Fc gamma 3b receptor chain, Fc beta 1 receptor chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof with at least one, but not more than 20 modifications. In some embodiments, at least one ITAM domain comprises a phosphorylation site of an Src family kinase. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprises a Factin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0262] In some embodiments, the intracellular domain does not comprise a domain derived from a CD3 zeta intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from a MerTK intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from a TLR4 intracellular domain. In some embodiments, the intracellular domain comprises a CD47 inhibition domain. In some embodiments, the intracellular signaling domain comprises an integrin-activating domain, such as the PSGL-1 intracellular region. In some embodiments, the intracellular signaling domain comprises a Rap1 GTPase-activating domain, such as that of EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from paxillin. In some embodiments, the intracellular signaling domain activates focal adhesion kinase.In some modalities, the intracellular signaling domain is derived from a single phagocytic receptor. In some modalities, the intracellular signaling domain is derived from a single sequestering receptor. In some modalities, the intracellular domain comprises a phagocytosis-enhancing domain. Petition 870250099881, dated 10 / 31 / 2025, page 104 / 364 97 / 178

[0263] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAKI, MDA-5, an IFN receptor, STING, a member of the NLRP family, NLRP1-14, NOD1, NOD2, Pirin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tankl-binding kinase (TBK), a caspase domain, a procaspase binding domain, or any combination thereof.

[0264] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a connexin protein (Cx). For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43, Cx46, Cx37, Cx40, Cx33, Cx50, Cx59, Cx62, 0x32, 0x26, 0x31, 0x30.3, 0x31.1, 0x30, 0x25, 0x45, 0x47, 0x31.3, 0x36, 0x31.9, 0x39, 0x40.1, or 0x23. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from 0x43.

[0265] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a SIGLEC protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Siglec-1 (Sialoadesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-13, Siglec-14, Siglec-15, Siglec-16, or Siglec-17.

[0266] In some forms, the intracellular domain comprises Petition 870250099881, dated 10 / 31 / 2025, page 105 / 364 98 / 178 a signaling domain, such as an intracellular signaling domain, derived from a C-type lectin protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain, derived from a mannose receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain, derived from an asialoglycoprotein receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from macrophage galactose-like lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K), DAP12 myeloid-associated lectin (MDL)-1 (CLEC5A), a DC-associated C-type lectin 1 subfamily protein (Dectin1), dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid-type C lectin receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, a DC immunoreceptor subfamily protein (DCIR),DCIR / CLEC4A, Dectin 2 / CLEC6A, Blood DC antigen 2 (BDCA2) (CLEC4C), Minculo (macrophage-inducible C-type lectin) (CLEC4E), a NOD-like receptor protein, NOD-like receptor MHC Class II transactivator (CIITA), IPAF, BIRC1, a RIG-I receptor protein (RLR), RIG-I, MDA5, LGP2, NAIP5 / Birc1e, an NLRP protein, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, an NLR protein, NOD1 or NOD2, or any combination thereof.

[0267] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a cell adhesion molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from IgCAMs, a cadherin, an integrin, a lectin-type C-domain protein (CTLD), and / or a proteoglycan molecule. For example, the intracellular domain may comprise a signaling domain, such as a signaling domain Petition 870250099881, dated 10 / 31 / 2025, page 106 / 364 99 / 178 intracellular, derived from an E-cadherin, a P-cadherin, an N-cadherin, an R-cadherin, a B-cadherin, a T-cadherin, or an M-cadherin. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a selectin, such as an E-selectin, an L-selectin, or a P-selectin.

[0268] In some embodiments, the CFP does not comprise a complete intracellular signaling domain. In some embodiments, the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length.

[0269] In some embodiments, the nucleic acid encodes an extracellular FcRa chain domain, a transmembrane FcRa chain domain, and / or an intracellular FcRa chain domain. In some embodiments, the nucleic acid encodes an extracellular FcRp chain domain, a transmembrane αοΡβ chain domain, and / or an intracellular FcRp chain domain. In some embodiments, the FcRa chain or the FcRp chain forms a complex with FcRy when expressed in a cell. In some embodiments, the FcRa chain or the FcRp chain forms a complex with endogenous FcRy when expressed in a cell. In some embodiments, the FcRa chain or the FcRp chain does not incorporate into a cell membrane of a cell that does not express FcRy. In some embodiments, the FcRa chain does not comprise an intracellular FcRa chain signaling domain. In some embodiments, the FcRp chain does not comprise an intracellular FcRp chain signaling domain.In some embodiments, the nucleic acid encodes an extracellular TREM domain, a transmembrane TREM domain, and / or an intracellular TREM domain. In some embodiments, TREM is TREM1, TREM2, or TREM3. Petition 870250099881, dated 10 / 31 / 2025, page 107 / 364 100 / 178

[0270] In some embodiments, after CFP binding to target cell antigen, the killing activity of a CFP-expressing cell is increased by at least more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000% compared to a cell that does not express CFP.

[0271] A target cell is, for example, a cancerous cell. In some embodiments, the manipulated myeloid cell, after engulfing a cancerous cell, can present a cancerous antigen on its cell surface to activate a T cell. An “antigen” is a molecule capable of stimulating an immune response. Antigens recognized by T cells, whether helper T lymphocytes (T helper cells (TH)) or cytotoxic T lymphocytes (CTLs), are not recognized as intact proteins, but rather as small peptides that associate with MHC proteins (such as MHC class I or class II proteins) on the cell surface. During the course of a naturally occurring immune response, antigens that are recognized in association with MHC class II molecules on antigen-presenting cells (APCs) are acquired from outside the cell, internalized, and processed into small peptides that associate with MHC class II molecules.

[0272] In some embodiments, after CFP binding to target cell antigen, the killing activity of a CFP-expressing cell is increased by at least more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to a cell that does not express CFP. In some modalities, CFP is incorporated. Petition 870250099881, dated 10 / 31 / 2025, p. 108 / 364 101 / 178 functionally in a cell membrane when CFP is expressed in the cell. In some embodiments, after CFP binds to target cell antigen, the killing activity of a CFP-expressing cell is increased by at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, or 100-fold compared to a cell that does not express CFP.

[0273] In some modes, the target cell that expresses the antigen is a cancerous cell.

[0274] In some embodiments, a cell expressing CFP exhibits an increase in phagocytosis of a target cell expressing the antigen compared to a cell not expressing CFP. In some embodiments, a cell expressing CFP exhibits at least a 1.1-fold increase in phagocytosis of a target cell expressing the antigen compared to a cell not expressing CFP. In some embodiments, a cell expressing CFP exhibits at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increase in phagocytosis of a target cell expressing the antigen compared to a cell not expressing CFP. In some embodiments, a cell expressing CFP exhibits an increase in the production of a cytokine compared to a cell not expressing CFP.In some modalities, the cytokine is selected from the group consisting of IL-1, IL-3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, an interferon, and combinations thereof. In some modalities, a cell expressing CFP exhibits increased effector activity compared to a cell not expressing CFP. In some... Petition 870250099881, dated 10 / 31 / 2025, p. 109 / 364 In some modalities, a cell expressing CFP exhibits increased cross-presentation compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased expression of an MHC class II protein compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased expression of CD80 compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased expression of CD86 compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased expression of MHC class I protein compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased expression of TRAIL / TNF family death receptors compared to a cell not expressing CFP.In some modalities, a cell expressing CFP exhibits increased B7-H2 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased LIGHT expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased HVEM expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased CD40 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased TL1A expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased 41BBL expression compared to a cell not expressing CFP.In some modes, a cell expressing CFP exhibits increased OX40L expression compared to a cell that does not express CFP. In some modes, a cell that... Petition 870250099881, dated 10 / 31 / 2025, page 110 / 364 Cells expressing CFP exhibit increased expression of GITRL death receptors compared to cells that do not express CFP. In some modalities, a cell expressing CFP exhibits increased expression of CD30L compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased expression of TIM4 compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased expression of TIM1 ligand compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased expression of SLAM compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased expression of CD48 compared to a cell that does not express CFP.In some modalities, a cell expressing CFP exhibits increased CD58 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased CD155 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased CD112 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased PDL1 expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased B7-DC expression compared to a cell not expressing CFP. In some modalities, a cell expressing CFP exhibits increased respiratory disruption compared to a cell not expressing CFP.In some modalities, a cell expressing CFP exhibits increased ROS production compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased iNOS production compared to a cell that does not. Petition 870250099881, dated 10 / 31 / 2025, page 111 / 364 104 / 178 expresses CFP. In some modalities, a cell expressing CFP exhibits increased iNOS production compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased extracellular vesicle production compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased trogocytosis with a target cell expressing the antigen compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased resistance to CD47-mediated inhibition of phagocytosis compared to a cell that does not express CFP. In some modalities, a cell expressing CFP exhibits increased resistance to LILRB1-mediated inhibition of phagocytosis compared to a cell that does not express CFP.In some forms, a cell expressing CFP exhibits an increase in the production of phosphatidylinositol 3,4,5-trisphosphate.

[0275] In some embodiments, the extracellular domain of a CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRyl, FcRyIIA, FcRyIIB, FcRyllC, FcRyIIIA, FcRylllB, FcRn, TRIM21, FcRL5-binding domain. In some embodiments, the extracellular domain of a CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of a CFP comprises an FcRa, FcRp, FcRc, or FcRy extracellular domain. In some embodiments, the extracellular domain comprises an FcRa (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an FcRp extracellular domain. In some embodiments, the extracellular domain comprises an FCER1A extracellular domain. In some embodiments, the extracellular domain comprises an extracellular domain FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B. In some embodiments, the extracellular domain comprises an integrin domain or Petition 870250099881, dated 10 / 31 / 2025, page 112 / 364 105 / 178 an integrin receptor domain. In some embodiments, the extracellular domain comprises one or more α1, α2, β1, β3, α4, β5, β6, β7, β8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7 or β8 integrin domains.

[0276] In some embodiments, the CFP additionally comprises an extracellular domain operationally linked to the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the extracellular domain additionally comprises an extracellular domain of a receptor, a hinge, a spacer, and / or a linker. In some embodiments, the extracellular domain comprises an extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor as the receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises an extracellular domain of a sequestering receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an extracellular domain of an immunoglobulin or an immunoglobulin hinge region.In some embodiments, the extracellular domain comprises a phagocytic engulfment domain. In some embodiments, the extracellular domain comprises a structure capable of multimeric assembly. In some embodiments, the extracellular domain comprises a scaffold for multimerization. In some embodiments, the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the extracellular antigen-binding domain binds specifically to the antigen of a target cell. In some embodiments, the extracellular antigen-binding domain comprises an antibody domain. In some... Petition 870250099881, dated 10 / 31 / 2025, page 113 / 364 In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, wherein the antibody domain comprises a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a Vh domain, a Vl domain, a VNAR domain, a Vhh domain, a bispecific antibody, a diabody, or a functional fragment, or a combination thereof. In some embodiments, the extracellular antigen-binding domain comprises a linker, an extracellular domain of a receptor, or an adapter. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain that is specific for a single antigen.In some embodiments, the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, wherein each of the at least two extracellular antigen-binding domains is specific for a different antigen.

[0277] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogenic antigen, or an autoimmune antigen. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen is a T-lymphocyte antigen. In some embodiments, the antigen is an extracellular antigen. In some embodiments, the antigen is an intracellular antigen.In some embodiments, the antigen is selected from the group consisting of a Thymidine Kinase (TK1) antigen, Hypoxanthine-Guanine Phosphoribosyltransferase (HPRT), Tyrosine Kinase-like Receptor 1 Orphan (ROR1), Mucin-1, Mucin-16 (MUC16), MUC1, Epidermal Growth Factor Receptor VIII (EGFRvIII), Mesothelin, Human Epidermal Growth Factor Receptor 2 (HER2), EBNA-1, LEMD1, Phosphatidylserine, Carcinoembryonic Antigen (CEA), B-Cell Maturation Antigen (BCMA), Glycan 3 (GPC3), Follicular Stimulating Hormone Receptor, Fibroblast Activating Protein (FAP), Hepatocellular Carcinoma. Petition 870250099881, dated 10 / 31 / 2025, page 114 / 364 107 / 178 Producer of Erythropoietin A2 (EphA2), EphB2, a Natural Killer Group 2D (NKG2D) ligand, Disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, an integrin receptor, PRSS21, VEGFR2, ΡϋΟΡΡβ, SSEA-4, EGFR, NCAM, prostate, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is an antigen of a protein selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD56. In some embodiments, the antigen is an ovarian cancer antigen or a T-cell lymphoma antigen. In some embodiments, the antigen is an integrin receptor antigen.In some embodiments, the antigen is an antigen of an integrin receptor or integrin selected from the group consisting of α1, α2, α1β, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, and β8. In some embodiments, the antigen is an antigen of an integrin receptor ligand. In some embodiments, the antigen is an antigen of fibronectin, vitronectin, collagen, or laminin. In some embodiments, the antigen-binding domain may bind to two or more different antigens.

[0278] In some embodiments, the antigen-binding domain comprises a self-antigen or fragment thereof, such as Dsg1 or Dsg3. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain or an antibody domain wherein the antibody domain binds to a self-antigen, such as Dsg1 or Dsg3.

[0279] In some embodiments, the transmembrane domain and the extracellular antigen-binding domain are operationally linked via a ligand. In some embodiments, the transmembrane domain Petition 870250099881, dated 10 / 31 / 2025, page 115 / 364 108 / 178 and the extracellular antigen-binding domain are operationally linked via a ligand, such as a hinge region of CD8a, IgG1, or IgG4.

[0280] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0281] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises an FcR transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRy transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRa transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRp transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRs transmembrane domain.

[0282] Methods for myeloid-specific expression that can be used regardless of whether the nucleic acid is distributed as mRNA or DNA are contemplated in this document. Consequently, in one aspect, CFP is designed so that it takes advantage of the presence of an endogenous myeloid protein for its own expression and functionality. In one aspect, myeloid-specific expression of CFP is achieved through a design in which CFP is expressed on the membrane only when it dimerizes (or multimerizes) with a monocyte-specific protein. In some embodiments, the myeloid-specific functionality of CFP is achieved through a design in which CFP is expressed on the membrane only when it dimerizes (or multimerizes) with a monocyte-specific protein. In some embodiments, the transmembrane domain Petition 870250099881, dated 10 / 31 / 2025, p. 116 / 364 109 / 178 comprises a multimerization domain, such that the transmembrane domain multimerizes specifically with a cell in which the expression of the chimeric receptor is desired, for example – a myeloid cell. For example, the transmembrane domain multimerizes with an endogenous transmembrane protein of a myeloid cell, and the multimerization facilitates the expression and / or stabilization of the chimeric receptor. Alpha FcR receptors oligomerize with gamma FcR receptors for membrane expression in myeloid cells and / or function. Several Fc receptors (FcRs) are endogenously expressed in monocytes and myeloid cells. Exemplary FcR domain sequences used in the construction of chimeric fusion protein receptors disclosed in this document are provided below:

[0283] An exemplary human CD16 TM sequence embedded in a CFP (includes an extracellular hinge domain) GLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSV (SEQ ID NO: 28); wherein the extracellular hinge domain is GLAVSTISSFFPPGYQV (SEQ ID NO: 29) and the TM domain is SFCLVMVLLFAVDTGLYFSV (SEQ ID NO: 30). An exemplary human CD16 cytoplasmic domain comprises the sequence KTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 31). An exemplary human CD89 TM sequence embedded in a CFP (includes an extracellular hinge domain) comprises a sequence IHQDYTTQNLIRMAVAGLVLVALLAILV (SEQ ID NO: 32) wherein the extracellular hinge domain is IHQDYTTQN (SEQ ID NO: 33) and the TM domain is LIRMAVAGLVLVALLAILV (SEQ ID NO: 34). An exemplary human CD89 cytoplasmic domain comprises a sequence ENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 35).

[0284] In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of the selected Fc gamma R1 protein (CD64). In Petition 870250099881, dated 10 / 31 / 2025, page 117 / 364 110 / 178 In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of the selected Fc gamma RIIIA (CD16) protein. In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of the selected FcQRIIA (CD32a) protein. In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of the selected Fc alpha R1 (CD89) protein. Any of the domains in the CFP described herein may be of a suitable mammalian origin, depending on or independently of the origin of any other adjacent domains. Non-human mammalian transmembrane domains are contemplated within the scope of this disclosure. In some embodiments, the CFP comprises an intracellular domain of the FcyR1 (CD64), FcyRIIIA (CD16), or FcyRIIA (CD32a) protein from which the TMD is derived.In some forms, the TMD has a sequence that is identical to the TMD sequence of the FcyR1 (CD64), FcyRIIIA (CD16), or FcyRIIA (CD32a) protein.

[0285] In some embodiments, the transmembrane domain comprises a transmembrane domain of a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5. In some embodiments, the transmembrane domain oligomerizes with a transmembrane domain of an endogenous receptor when CFP is expressed in a cell. In some embodiments, the transmembrane domain oligomerizes with a transmembrane domain of an exogenous receptor when CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with a transmembrane domain of an endogenous receptor when CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with a transmembrane domain of an exogenous receptor when CFP is expressed in a cell. In some embodiments, the transmembrane domain is derived from a protein that is different from the protein from which the intracellular signaling domain is derived. Petition 870250099881, dated 10 / 31 / 2025, page 118 / 364 111 / 178 In some embodiments, the transmembrane domain is derived from a protein that is different from the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises a transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain are derived from the same protein. In some embodiments, the transmembrane domain is derived from the same protein as the intracellular signaling domain. In some embodiments, the nucleic acid encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0286] In some embodiments, the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids long. In some embodiments, the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids long.

[0287] In some embodiments, the nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition additionally comprises a pro-inflammatory nucleotide or a nucleotide in the nucleic acid, for example, ATP, ADP, UTP, UDP and / or UDP-glucose.

[0288] In some embodiments, the composition additionally comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine, cytokine. In some embodiments, the chemokine is selected from the group consisting of IL-1, IL-3, IL-5, IL-6, IL-8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES and interferon. In some embodiments, the cytokine is selected from the group consisting of IL1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, LCR, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon. Petition 870250099881, dated 10 / 31 / 2025, p. 119 / 364 112 / 178

[0289] In some embodiments, myeloid cells are specifically targeted for delivery. Myeloid cells can be targeted using specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymeric structures to affect myeloid cell function. In some embodiments, the targeting structures are multilayered, for example, of one or more layers of PLGA and one or more layers of PVA. In some embodiments, the targeting structures are assembled in an order for layered activity.In some embodiments, the targeted polymeric structures are organized into specifically shaped components, such as labile structures that can adhere to a myeloid cell surface and distribute one or more components, such as growth factors and cytokines, in order to maintain the myeloid cell in a microenvironment that provides a specific polarization. In some embodiments, the polymeric structures are such that they are not phagocytosed by the myeloid cell but can remain attached to the surface. In some embodiments, one or more growth factors may be M1 polarizing factors, such as a cytokine. In some embodiments, one or more growth factors may be an M2 polarizing factor, such as a cytokine. In some embodiments, one or more growth factors may be a macrophage-activating cytokine, such as IFNγ.In some forms, the polymeric structures are capable of sustained release of one or more growth factors in an in vivo environment, such as in a solid tumor.

[0290] In some embodiments, the nucleic acid comprises a sequence that encodes a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in an untranslated region (UTR) of an mRNA. In some embodiments, the sequence in the UTR is a sequence that binds to a Petition 870250099881, dated 10 / 31 / 2025, p. 120 / 364 113 / 178 RNA-binding protein. In some embodiments, translation is inhibited or prevented after the RNA-binding protein binds to the sequence in an untranslated region (UTR). In some embodiments, the nucleic acid is expressed in a bicistronic vector. Manipulated myeloid cells “targeted” to attack diseased cells.

[0291] This disclosure involves compositions and methods for preparing targeted myeloid killer cells; leveraging the innate functional role in immune defense, ranging from properties related to the detection of foreign bodies, particles, diseased cells, cellular debris, inflammatory signaling, chemotactic activity; activating endogenous DAMP and PAMP signaling pathways; triggering myelopoiesis, extravasation; chemotaxis; phagocytosis; pinocytosis; recruitment; engulfment; elimination; activating intracellular oxidative disruption and lysis or death of pathogens, detecting, engulfing and killing diseased or damaged cells; removing unwanted cellular, tissue or acellular debris in vivo; antigen presentation and role in activating innate immunity; activating and modulating an immune response cascade; activating the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis, immune editing to stress response and restoration of tissue homeostasis.In one aspect, this document provides methods and compositions for enhancing one or more functions of a myeloid cell for use in a therapeutic application; one or more functions may include: detecting foreign bodies, particles, diseased cells, cellular debris, inflammatory signaling, chemotactic activity; activating endogenous DAMP and PAMP signaling pathways; triggering myelopoiesis, extravasation; chemotaxis; phagocytosis; pinocytosis; recruitment; trogocytosis; engulfment; elimination; activating intracellular oxidative disruption and intracellular lysis or killing of pathogens; detecting, engulfing, and killing diseased or damaged cells; removing cellular, tissue, or acellular debris in vivo; antigen presentation and role in activating innate immunity. Petition 870250099881, dated 10 / 31 / 2025, page 121 / 364 114 / 178 activate and modulate an immune response cascade; activate the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis, immune editing to stress response and restoration of tissue homeostasis. In one modality, the compositions and methods are also directed to enhance the targeting and kill function of certain myeloid cells, by genetic modification of these cells.The compositions and methods described in this document are aimed at creating engineered myeloid cells, wherein the engineered myeloid cells comprise at least one genetic modification, and may be directed to recognize and induce effector functions against a pathogen, a diseased cell, such as a tumor or cancerous cell, so that the engineered myeloid cell is able to recognize, target, phagocytose, kill and / or eliminate the pathogen or the diseased cell or the cancerous cell, and, furthermore, may activate a specific immune response cascade after phagocytosis, killing and / or eliminating the pathogen or the diseased cell.

[0292] Myeloid cells appear to be the most abundant cells in a tumor. Myeloid cells are also capable of recognizing a tumor cell over a normal, healthy body cell and mounting an immune response to a tumor cell in the body. As sentinels of the innate immune response, myeloid cells can detect non-self or aberrant cell types and eliminate them through a process called phagocytosis. This can be targeted for therapeutic advantage in triggering myeloid cell-mediated phagocytosis and lysis of tumor cells. However, these naturally occurring tumor-infiltrating myeloid cells (TIMs) can be subject to the influence of the tumor microenvironment (TME). TIMs constitute a heterogeneous population of cells. Many TIMs originate from circulating monocytes and granulocytes, which in turn derive from hematopoietic stem cells derived from bone marrow.However, in the presence of persistent stimulation by tumor-derived factors, monocyte and granulocyte progenitors become skewed. Petition 870250099881, dated 10 / 31 / 2025, page 122 / 364 115 / 178 of their intrinsic terminal differentiation pathway into mature macrophages, DCs, or granulocytes and may become tumor-promoting myeloid cell types. Differentiation into alternatively activated immature pathological myeloid cells is favored. These immature myeloid cells include tumor-associated DCs (TADCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Alternatively to this emergent myelopoiesis, TAMs may also originate from tissue-resident macrophages, which in turn may be of embryonic or monocytic origin. These tissue-resident macrophages undergo changes in phenotype and function during carcinogenesis, and proliferation may help maintain TAMs derived from tissue-resident macrophages.A tumor microenvironment can trigger a tumor-infiltrating myeloid cell to become myeloid-derived suppressor cells and acquire the ability to suppress T cells. As a result, innovative methods are needed to create therapeutically effective TIMs that can infiltrate a tumor and target tumor cells for uptake and phagocytic elimination.

[0293] In one aspect, engineered myeloid cells are provided in this document that can target specific target cells, for example, tumor cells or pathogenic cells. In some embodiments, the engineered myeloid cells provided in this document are potent in infiltrating, targeting, and killing tumor cells. An engineered myeloid / phagocytic cell described in this document is designed to comprise a nucleic acid that encodes one or more proteins that help direct the phagocytic cell to a target cell, for example, a tumor cell or a cancerous cell. In one embodiment, the engineered myeloid cell is able to easily infiltrate a tumor. In one embodiment, the engineered myeloid cell has high specificity for the target cell, with no or negligible cross-reactivity to a non-tumor, non-diseased subject cell while in circulation. In one embodiment Petition 870250099881, dated 10 / 31 / 2025, pp. 123 / 364 In one embodiment, the manipulated myeloid / phagocytic cell described in this document is designed to comprehend a nucleic acid that will help the cell overcome / pass the influence of TME and mount a potent antitumor response. In another embodiment, the manipulated myeloid / phagocytic cell described in this document is designed to comprehend a nucleic acid that enhances the phagocytosis of the target cell. In another embodiment, the manipulated myeloid / phagocytic cell described in this document is designed to comprehend a nucleic acid that enhances the reduction or elimination of trogocytosis and / or potentiates phagocytic lysis of the target cell.

[0294] Consequently, in some embodiments, the compositions disclosed in this document comprise a myeloid cell comprising a nucleic acid encoding a chimeric receptor fusion protein (CFP), for example, a phagocytic receptor (PR) fusion protein (PFP). The nucleic acid may comprise a sequence encoding a PR subunit comprising: (i) a transmembrane domain and (ii) an intracellular domain comprising an intracellular PR signaling domain; and an extracellular antigen-binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular antigen-binding domain are operationally linked; wherein the intracellular PR signaling domain is derived from a receptor with a signal transduction domain.The nucleic acid additionally encodes one or more polypeptides that constitute one or more plasma membrane receptors that help to engulf the phagocytic cell in the target cell and enhance its phagocytic activity.

[0295] In some embodiments, the myeloid cell described in this document comprises one or more recombinant proteins comprising a chimeric receptor, wherein the chimeric receptor can respond to a first phagocytic signal directed to a target cell, which may be a diseased cell, a tumor cell or a pathogen, and a Petition 870250099881, dated 10 / 31 / 2025, pp. 124 / 364 117 / 178 second signal, which is an inflammatory signal, that increases the phagocytic and killing response to the target initiated by the first signal. In some embodiments, recombinant proteins may comprise one or more of the amino acid sequences represented in Table 1A. Table 1A. Sequences of chimeric fusion proteins and their domains (Respective CDR1, 2 and 3 sequences underlined, Kabat nomenclature) Protein / Domain Sequence Anti-CD5 Heavy Chain Variable Domain EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLE WMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAV YFCTRRGYDWYFDVWGQGTTVTV (SEQ ID NO: 36) Anti-CD5 Light Chain Variable Domain DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLI YRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESP WTFGGGTKLEIK (SEQ ID NO: 37) scFv anti-CD5 EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLE WMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAV YFCTRRGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRAN RLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFG GGTKLEIK (SEQ ID NO: 38) Intracellular signaling domain of the FcRy chain LYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 39) Intracellular signaling domain of the FcRy chain LYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 40) Intracellular signaling domain of the chain FcRy RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 41) Domain ofIntracellular signaling of the FcRy chain RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 41) Recruitment domain PI3K YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM (SEQ ID NO: 42) Intracellular domain CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVT QEDGKESRISVQERQ (SEQ ID NO: 43) Transmembrane domain of the CD8a chain IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 44) Transmembrane domain of the CD8a chain IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 45) Hinge domain of the CD8a chain ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLD (SEQ ID NO: 46) Petition 870250099881, dated 10 / 31 / 2025, page 125 / 364 118 / 178 Protein / Domain Sequence Anti-HER2 Heavy Chain Variable Domain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVE (SEQ ID NO: 47) Anti-HER2 Light Chain Variable Domain LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG FYAMDVWGQGTLVTV (SEQ ID NO: 48) scFv anti-HER2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESSGGGGSGGG GSGGGGSLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDVWGQGTLVTV (SEQ ID NO: 49) Signal peptide GMCSF MWLQSLLLLGTVACSIS (SEQ ID NO: 50) CD28 transmembrane domain FWVLWVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51) CD2 transmembrane domain IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 52) CD68 transmembrane domain ILLPLIIGLILLGLLALVLIAFCII (SEQ ID NO: 53) Intracellular domain of TNFR1QRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTL GFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALA SDPIPNPLQKWEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVR RLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGR VLRDMDLLGCLEDIEEALCGPAALPPAPSLLR (SEQ ID NO: 54) Intracellular domain of TNFR2 PLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASA LDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVT CIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEEC AFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS (SEQ ID NO: 55) Intracellular domain of MDA5 MSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQR TVATSGNMQAVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAARY MNPELTDLPSPSFENAHDEYLQLLNLLQPTLVDKLLVRDVLDKCMEE ELLTIEDRNRIAAAENNGNESGVRELLKRIVQKENWFSAFLNVLRQTG NNELVQELTGSDCSESNAEIEN (SEQ ID NO: 56) CD8a chain hinge domain + transmembrane domain ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 57) CD8a chain hinge domain + transmembrane domainALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 58) CD5-FcRy-PI3K MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAASGYTFTN YGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSK NTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLS WFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYE DFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD IYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYEKSDGVYTGLS TRNQETYETLKHEKPPQGSGSYEDMRGILYAAPQLRSIRGQPGPNH EEDADSYENM (SEQ ID NO: 59) Petição 870250099881, de 31 / 10 / 2025, pág. 126 / 364 119 / 178 Proteína / domínio Sequência HER2-FcRy-PI3K MWLQSLLLLGTVACSISDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQ PEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSE VQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY YCSRWGGDGFYAMDVWGQGTLVTVSSSGGGGSGALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDIYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYEKSDGVYT GLSTRNQETYETLKHEKPPQGSGSYEDMRGILYAAPQLRSIRGQPGP NHEEDADSYENM (SEQ ID NO: 60) CD5-FcRy-CD40 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAASGYTFTN YGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSK NTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLS WFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYE DFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD IYIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDGVYTGLST RNQETYETLKHEKPPQKKVAKKPTNKAPHPKQEPQEINFPDDLPGSN TAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ ID NO: 61)CD5-FcRy-MDA5 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAASGYTFTN YGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSK NTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLS WFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYE DFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD IYIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDGVYTGLST RNQETYETLKHEKPPQGSGSMSNGYSTDENFRYLISCFRARVKMYIQ VEPVLDYLTFLPAEVKEQIQRTVATSGNMQAVELLLSTLEKGVWHLG WTREFVEALRRTGSPLAARYMNPELTDLPSPSFENAHDEYLQLLNLL QPTLVDKLLVRDVLDKCMEEELLTIEDRNRIAAAENNGNESGVRELLK RIVQKENWFSAFLNVLRQTGNNELVQELTGSDCSESNAEIEN (SEQ ID NO: 62) CD5-FcRy-TNFR1 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAASGYTFTN YGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSK NTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLS WFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYE DFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDGVYTGLST RNQETYETLKHEKPPQGSGSQRWKSKLYSIVCGKSTPEKEGELEGT TTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAP RREVAPPYQGADPILATALASDPIPNPLQKWEDSAHKPQSLDTDDPA TLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSML ATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAP SLLR (SEQ ID NO: 63) Petition 870250099881, dated 10 / 31 / 2025, page 127 / 364 120 / 178 Protein / domain Sequence CD5-FcRy-TNFR2 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAASGYTFTN YGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSK NTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLS WFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYE DFGIYYCQQYDESPWTFGGGTKLEIKSGGGGGSGALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD IYIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDGVYTGLST RNQETYETLKHEKPPQGSGSPLCLQREAKVPHLPADKARGTQGPEQ QHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARA STGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDT DSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPD AGMKPS (SEQ ID NO: 64) Table 1B - Ligand sequences Sequence SSGGGGSGGGGSGGGGS (SEQ IP NO: 65) SGGGGSG (SEQ IP NO: 66)_________________________________ SGGG (SEQ IP NO: 67)______________________________________ GSGS (SEQ IP NO: 68)___________________________________________

[0296] In some embodiments, the therapeutically effective myeloid cell comprises, presents, or expresses one or more exogenous or recombinant tumor antigens. In some embodiments, the tumor antigens are tissue-specific antigens. In some embodiments, the tumor antigens are endogenous overexpressed antigens. In some embodiments, the tumor antigens are mutant protein antigens. In some embodiments, the therapeutically effective myeloid cell comprises, presents, or expresses a melanoma antigen, such as a tyrosinase-related protein 2 (TRP2) antigen, such as a TRP2 epitope, such as amino acids 180-188 of TRP2 (SVYDFFVWL (SEQ ID NO: 69)).In some embodiments, the therapeutically effective myeloid cell comprises or presents or expresses a mutant antigen, for example, a glioblastoma antigen, such as an isocitrate dehydrogenase 1 (mlDHI) antigen, such as a mutant IDH1 antigen (R132H), such as GWVKPIIIGHHAYGDQYRATDFVVP (SEQ ID NO: 70). In some embodiments, a method may comprise administering a myeloid cell that comprises, presents, or expresses one or more exogenous or recombinant antigens to treat a cancer, such as a brain cancer, a glioma, or a glioblastoma. Petition 870250099881, dated 10 / 31 / 2025, page 128 / 364 121 / 178 mRNA compositions comprising modifications for prolonged expression of proteins encoded in myeloid cells.

[0297] Modifications to a manipulated polynucleic acid comprising a sequence encoding a protein or peptide, designed for specific and enhanced expression of the protein or peptide encoded therein, are provided in this document. In some embodiments, a manipulated polynucleic acid described in this document is, for example, a recombinant mRNA (in some embodiments, interchangeably referred to as an mRNA construct), comprising a 5' untranslated region (UTR), a coding sequence downstream of the 5' UTR, for example, a therapeutic protein, and followed by a 3' UTR downstream of the coding sequence, wherein the 5' UTR and the 3' UTR are heterologous to the coding sequence and designed to enhance the expression of the sequence encoding the therapeutic protein.

[0298] In some embodiments, UTR 5' and UTR 3' in recombinant or engineered polynuclear acid (mRNA) are from heterologous sources. In some embodiments, UTR 5' and UTR 3' in recombinant or engineered polynuclear acid (mRNA) are from the same gene.

[0299] In some embodiments, the UTR 5' comprises a sequence that has at least 80% identity with the UTR 5' of the bovine growth hormone (BGH) gene. In some embodiments, the UTR 5' comprises a sequence that has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the UTR 5' of BGH.

[0300] In some embodiments, the UTR 3' comprises a sequence that has at least 80% identity with the UTR 3' of the bovine growth hormone (BGH) gene. In some embodiments, the UTR 3' comprises a sequence that has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Petition 870250099881, dated 10 / 31 / 2025, p. 129 / 364 122 / 178 98% or at least 99% sequence identity with the UTR 3' of BGH.

[0301] In some modalities, a 5' UTR may be derived from BGH and may comprise a sequence that is extensively modified. In some modalities, a 3' UTR may be derived from BGH and may comprise a sequence that is extensively modified.

[0302] In some embodiments, the 5' UTR comprises a sequence that has at least 80% identity with the 5' UTR of a globin gene. In some embodiments, the 5' UTR comprises a sequence that has at least 80% identity with the 5' UTR of a human globin gene. In some embodiments, the 5' UTR comprises a sequence that has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the 5' UTR of a human globin gene.

[0303] In some embodiments, the 3' UTR comprises a sequence that has at least 80% identity with the 3' UTR of a bovine globin gene. In some embodiments, the 3' UTR comprises a sequence that has at least 80% identity with the 3' UTR of a human globin gene. In some embodiments, the 3' UTR comprises a sequence that has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the 3' UTR of a human globin gene.

[0304] In some embodiments, a 5' UTR may be derived from the globin gene and may comprise a sequence that is extensively modified. In some embodiments, a 3' UTR may be derived from the globin gene and may comprise a sequence that is extensively modified. In some embodiments, a 5' UTR or a 3' UTR may comprise an artificial sequence. An artificial sequence is a sequence of oligonucleotides or polynucleotides that are not present in nature but can be synthesized. In some embodiments, a 5' UTR comprises a Kozak sequence. In some embodiments, a Petition 870250099881, dated 10 / 31 / 2025, pp. 130 / 364 123 / 178 UTR 5' comprises a 5' cap structure. The 5' cap may be a 1' cap or 0' cap structure, or it may comprise an ARCA cap (ARCA, analog of anti-reverse cap). In some embodiments, UTR 3' additionally comprises a poly A tail. In some embodiments, a poly A tail is enzymatically linked to the UTR 3' of the mRNA. In some embodiments, the poly A tail is incorporated into the mRNA after the mRNA is prepared, for example, by in vitro translation (IVT) and, in some embodiments, after a first round of purification of the IVT-generated mRNA. In some embodiments, the length of the poly A tail is regulated. In some embodiments, there are approximately 80-200 adenosine (A) residues in the poly A tail.

[0305] In some embodiments, a 5' UTR may comprise a sequence of 5'-GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTT3' (SEQ ID NO: 71). As is understood throughout this document, any mRNA sequence may be presented in this document where the T (thymine) residue can be read as U (uracil) residues, for example, the sequence above is 5'-GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUU-3' (SEQ ID NO: 72). In some embodiments, a 5' UTR may comprise a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with 5'GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUU-3' (SEQ ID NO: 72).

[0306] In some modes, UTR 3' comprises a sequence:

[0307] 5'CTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTG CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG Petition 870250099881, dated 10 / 31 / 2025, pp. 131 / 364 124 / 178 GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC-3' (SEQ ID NO: 73). (The underlined region corresponds to the BGH poly A signal).

[0308] As previously indicated, UTR 3' comprises a sequence: 5'CUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUG UGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCC UUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAA AUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCU GGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGA CAAUAGC-3' (SEQ ID NO: 74).

[0309] In some embodiments, the 3' UTR comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with 5'CUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUG UGCCUUCUAGUUGCCAGCCAUCUGUUGUUGCCCCUCCGUGCC UUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAA AUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCU GGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGA CAAUAGC-3' (SEQ ID NO: 74).

[0310] In some embodiments, mRNA constructs comprising sequences encoding one or more polypeptides, such as the CFPs described in this document, are mRNA constructs designed to include specific 5' and 3' UTRs (by convention, read in the 5' to 3' direction for each sequence) that provide enhanced intracellular stability and prolonged expression of the encoded polypeptides. Exemplary UTRs may include:

[0311] UTR 5' A1: Petition 870250099881, dated 10 / 31 / 2025, pp. 132 / 364 125 / 178 5'GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC3' (SEQ ID NO: 75)

[0312] UTR 5' B1: 5'ACUCCUCCCCAUCCUCCCUCUGUCCCUCUGUCCCUCUGA CCCUGCACUGUCCCAGCACC-3' (SEQ ID NO: 76)

[0313] UTR 5' C1: 5'ACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGAC ACC-3' (SEQ ID NO: 77)

[0314] UTR 5' D1: 5'CTTGCCCGCCGATATCTCTGCCGGTGACTAGCTGCTTCCTTTCTCTC TCGCGCGCGGTGTGGTGGCAGCAGGCGCAGCCCAGCCTCGAA-3' (SEQ ID NO: 78)

[0315] The sequence above UTR 5' D1 is alternatively represented below: 5'CUUGCCCGCCGAUAUCUCUGCCGGGUGACUAGCUGCUUCCUUUCUC UCUCGCGCGCGGUGUGGUGGCAGCAGGCGCAGCCCAGCCUCGAA-3' (SEQ ID NO: 79)

[0316] UTR 5' E1: 5'CTTCCTTTTTGTCCGACATCTTGACGAGGCTGCGGTGTCTGCTGCTATT CTCCGAGCTTCGCA-3' (SEQ ID NO: 80)

[0317] The sequence above UTR 5' E1 is alternatively represented below: 5'CUUCCUUUUUGUCCGACAUCUUGACGAGGCUGCGGUGUCUGCUGCU AUUCUCCGAGCUUCGCA-3' (SEQ ID NO: 81) Petition 870250099881, of 31 / 10 / 2025, p. 133 / 364 126 / 178

[0318] UTR 5' F1: 5'AGCAATCCTTTCTTTCAGCTGGAGTGCTCCTCAGGAGCCAGCCCCACC CTTAGAAAAG-3' (SEQ ID NO: 82)

[0319] The sequence upstream of UTR 5' F1 may alternatively be represented as: 5'AGCAAUCCUUUCUUUCAGCUGGAGUGCUCCUCAGGAGCCAGCCCCA CCCUUAGAAAAG-3' (SEQ ID NO: 83)

[0320] UTR 3' A2: 5'UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGG CCUCCCCCCAGCCCCUCCUCCCCUGCACCCGUACCCCCGUGG UCUUUGAAUAAAGUCUGA-3' (SEQ ID NO: 84)

[0321] UTR 3' B2: 5'CAGGACACAGCCUUGGAUCAGGACAGAGACUGGGGGCCAUCCUGC CCCUCCAACCCGACAUGUGUACCUCAGCUUUUCCCUCCACUUGCAUC AAUAAAGCUUCUGUGUUUGGAACAG-3' (SEQ ID NO: 85)

[0322] UTR 3' C2: 5'GCUCGCUUUCUUGUGCAAUUUCUAUUAAAGGUUCUUGUUCC CUAAGUCACUAACUAACUGGGGGAUAUUAUAGGGCCUUGAGCAUCUGGGGGAUAAAA-3' (SEQ ID NO: 86)

[0323] UTR 3' C2-2x: 5'GCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCUUGUUCC CUAAGUCACUACUAACUGGGGAUAUUAUAGGGCCUUGAGCA UAUUAAAGGGCCUGGGGGA UAUUAUAGGGCCUUWAWAWAAAAAAAAA-3' Petition 870250099881, dated 10 / 31 / 2025, p. 134 / 364 127 / 178 (SEQ ID NO: 87).

[0324] UTR 3′ D2: 5′CTGGAGAGAATCACAGATGTGGAATATTTGTCATAAATAATGAAA ACCT-3′ (SEQ ID NO: 88)

[0325] UTR 3' E2: 5'ATAGGTCCAACCAGCTGTACATTTGGAAAAATAAACTTTATTAAATCAA A-3' (SEQ ID NO: 89)

[0326] UTR 3' F2: 5'CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCC ACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTA AGCTATACTAACCCCAGGGTTGGTCAATTTTCGTGCCAGCCACACCCTG GTACTGCATGCACGCAATGCTAGCTGCCCCCTGCCTT CCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACC TGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC-3' (SEQ ID NO: 90).

[0327] The sequence upstream of the 3' F2 UTR is alternatively depicted below: 5'CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACCACCCC C ACGG G AAAC AGC AG UG AU U AACC UUU AGCAAU AAACG AAAG UUU AA CUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACAC CCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUG GGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCAC CUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC-3' (SEQ ID NO: 91)

[0328] UTR 3' G2: 5'Petition 870250099881, of 31 / 10 / 2025, p. 135 / 364 128 / 178 GCUGAAGAAGUGGGAAUGGGAGCACUCUGUCUUCUUUGCUAGAGAA GUGGAGAGAAAAUACAAAAGGUAAAGCAGUUGAGAUUCUCUACAACC UAAAAAAUUCCUAGGUGCUAUUUUCUUAUCCUUUGUACUGUAGCUAA AUGUACCUGAGACAUAUUAGUCUUUGAAAAAUAAAGUUAUGUAAGGU UUUUUUUAUCUUUAAAAUAGCUCUGUGGGUUUUAACAUUUUUAUAAAG AUAUACCAA-3' (SEQ ID NO: 92)

[0329] In some embodiments, a manipulated polynucleic acid comprises a 5' UTR, a coding sequence encoding a therapeutic protein, and followed by a 3' UTR, wherein the 5' UTR and the 3' UTR are heterologous to the coding sequence and designed to enhance the expression of the sequence encoding the therapeutic protein.

[0330] The manipulated nucleic acid can be generated following molecular biology techniques known to those skilled in the art. Methods include, but are not limited to, primer design, generating PCR amplification products, restriction digestion, ligation, cloning, gel purification of the cloned product, bacterial propagation of the cloned DNA, isolation and purification of the cloned plasmid or vector. General guidance can be found in: Molecular Cloning of PCR Products: by Michael Finney, Paul E. Nisson, Ayoub Rashtchian in Current Protocols in Molecular Biology, Volume 56, Issue 1 (First published: November 1, 2001); Recombinational Cloning by Jaehong Park, Joshua LaBaer in Current Protocols in Molecular Biology Volume 74, Issue 1 (First published: May 15, 2006) and others. In some modalities, specific amplification techniques can be used, such as the TAS (Transcription-Based Amplification System) technique, described by Kwoh et al.in 1989; the 3SR technique, which are incorporated into this document by reference. (Self-Sustaining Sequence Replication), described by Guatelli et al. in 1990; the NASBA technique (Nucleic Acid Sequence-Based Amplification), described by Kievitis et al. in 1991; the SDA technique (Strip Displacement Amplification) (Walker et al., 1992); the TMA technique (Amplification. Petition 870250099881, dated 10 / 31 / 2025, pp. 136 / 364 129 / 178 (Mediated by Transcription).

[0331] The manipulated nucleic acid may comprise a sequence for integration within the genome of a target cell. In some embodiments, the manipulated nucleic acid is an mRNA. In some embodiments, therefore, the strategic incorporation of miRNA binding sequences into the mRNA sequence in the 5' or 3' UTR regions may negatively regulate the mRNA half-life and thus contribute to the cell-specific expression of the mRNA when the mRNA is administered in vivo.

[0332] In some embodiments, the manipulated nucleic acid sequence is optimized for expression in humans. In some embodiments, the manipulated nucleic acid sequence is optimized for expression in a type of myeloid cell in a human, where the desired target cell type in this case is myeloid cells.

[0333] In some embodiments, the manipulated nucleic acid is a manipulated mRNA.

[0334] In some embodiments, mRNA isolated from recombinant mRNA comprises a sequence that encodes a therapeutic protein or peptide.

[0335] In some embodiments, recombinant mRNA is designed for expression in specific cell types, preferably with other cell types, for example, in a cell population comprising heterogeneous cell types ex vivo. In some embodiments, recombinant mRNA is designed for expression in certain specific cell types in vitro or in vivo.

[0336] In some embodiments, cell-specific expression can be achieved by incorporating one or more miRNA binding sites into the 3' UTR of recombinant mRNA for downregulation of mRNA in a cell expressing the corresponding miRNA.

[0337] In some embodiments, a recombinant mRNA, such as Petition 870250099881, dated 10 / 31 / 2025, pp. 137 / 364 130 / 178 described in the paragraphs above, is delivered to a cell ex vivo. In some embodiments, a recombinant mRNA, as described in the paragraphs above, is delivered to a cell in vivo. The outer surface of a cell is directly placed in contact with the recombinant mRNA (e.g., an isolated recombinant mRNA or mRNA construct, as previously described) for delivery of the mRNA to a cell ex vivo, where the cell absorbs the recombinant mRNA, where the mRNA is translated, and the encoded protein or peptide is expressed within the cell. The recombinant mRNA can be formulated into a lipid nanoparticle (LNP) delivery vehicle. Such a formulation can be used for in vivo delivery. In some embodiments, such a lipid nanoparticle formulation is designed for systemic in vivo delivery.For example, recombinant mRNA is formulated for delivery to a cell within a living organism by injecting a liquid formulation comprising the mRNA encapsulated in an LNP. In some embodiments, the miRNA targeting sites comprise 10-40 nucleotide residues that can form base pairing with the corresponding contiguous sequences on the miRNA strand. In some embodiments, the recombinant mRNA is designed to comprise 2, 3, 4, or 5 copies of the miRNA target sites, i.e., miRNA binding sites.Therefore, this document provides a method and compositions for expressing an exogenous polypeptide from a recombinant polynucleic acid (e.g., an mRNA that is an isolated recombinant mRNA) in a specific cell, the method comprising placing a heterogeneous population of cells comprising the specific cell and at least one other cell that is not the specific cell in contact with a composition comprising the recombinant polynucleic acid comprising a sequence encoding the exogenous polypeptide, and one or more miRNA target sequences; wherein the exogenous polypeptide is expressed in the specific cell from the heterogeneous cell population, and in... Petition 870250099881, dated 10 / 31 / 2025, pp. 138 / 364 131 / 178 that the exogenous polypeptide is not substantially expressed in the non-specific cell in the heterogeneous cell population. The miRNA target sites are designed so that the corresponding miRNAs are naturally expressed in a cell type that is not the specific cell, and where expression of the exogenous polypeptide encoded by the sequence within the mRNA is not desired; where the naturally occurring miRNA in the non-specific cell binds to the miRNA target site of the mRNA and degrades it. In some embodiments, the specific cell is a myeloid cell. In some embodiments, the non-specific cell type, where expression of the sequence encoding the exogenous polypeptide is not required, is a non-myeloid cell, for example, a neuron, an epithelial cell, a liver cell, a muscle cell, a lymphocyte, etc.In some modalities, a cell that is a non-myeloid cell is a cell of high abundance in the heterogeneous cell population. In some modalities, the non-myeloid cell may be a liver cell. In some modalities, the non-myeloid cell is an epithelial cell.

[0338] In some embodiments, the mRNA comprises a sequence encoding an exogenous therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is designed to be preferentially expressed in myeloid cells, not in non-myeloid cells. For example, systemically administered nucleic acids, such as a nucleic acid therapeutic, are frequently absorbed by liver cells or epithelial cells. Where expression of the therapeutic is desired in myeloid cells and expression is desired to be avoided in epithelial cells, for example, the mRNA is designed to comprise a miRNA target site for a miRNA that is readily present in an epithelial cell and not expressed in a myeloid cell.

[0339] For example, miRNA miR-10a is highly enriched in epithelial cells, muscle cells, and sinusoidal endothelial cells. Petition 870250099881, dated 10 / 31 / 2025, pp. 139 / 364 132 / 178 liver and myeloid cells do not express miR-10a. For example, miR227b is highly enriched in liver cells (hepatocytes), and myeloid cells are relatively devoid of miR-27b. Similarly, miR192 is enriched in hepatocytes, and its expression is almost absent in myeloid cells. For example, miR-122 is highly expressed in hepatocytes with very little expression in myeloid cells. By "expression" of a miRNA, it is understood that miRNAs are readily formed and present in such cells. By "not expressed," "not readily expressed," or "almost absent" in the case of a miRNA, it can be understood that the miRNA is undetectable, or its effect on an mRNA with a target site for the miRNA is undetectable, or both.In some embodiments, the expression of the exogenous polypeptide (or protein) encoded by recombinant mRNA comprising the target miRNA sequence for a miRNA expressed in a cell other than the specific cell where expression is desired is reduced in the non-specific cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 95% or more compared to the specific cell where expression is desired.

[0340] In some embodiments, miRNA-122 binding sites (miR122) are incorporated within the 3' UTR of mRNA for expression of a polypeptide encoded by a sequence in mRNA specifically in a myeloid cell. A human miR-122 may comprise a sequence (NR_029667): ccttagcagagctgtggagtgtgacaatgg tgtttgtgtctaaactatcaaacgccattatcacactaaatagctactgctaggc (SEQ ID NO: 93).

[0341] In some embodiments, miRNA-192 binding sites (miR192) are incorporated within the 3' UTR of mRNA for expression of a polypeptide encoded by a sequence in the mRNA specifically in a myeloid cell. A human miR-192 may comprise a sequence (NR_029578): qccqaqaccqaqtQcacaQQQCtctgacctataaattaacaqccaqtq ctctcg tctcccctctg g ctg Petition 870250099881, dated 10 / 31 / 2025, pp. 140 / 364 133 / 178 ccaattccataggtcacaggtatgttcgcctcaatgccagc (SEQ ID NO: 1)

[0342] For example, a representative target sequence for miRNA is: ggcugucaauucauaggucag (SEQ ID NO: 4), which can be included in the 3' UTR of the recombinant mRNA construct. The reverse sequence complementary to the target sequence is shown in SEQ ID NO: 1 in bold and underlined.

[0343] In some embodiments, miRNA-27b binding sites are incorporated within the 3' UTR of the mRNA for expression of a polypeptide encoded by a sequence in the mRNA specifically in a myeloid cell. In some embodiments, a human miR27b comprises a sequence NR_029665: acctctctaacaaqqtqcaaaacttagctqattqqtqaacaqtqattqqtttccqctttqttcacaqtqqct aagttctgcacctgaagagaaggtg (SEQ ID NO: 2). For example, a representative target sequence for miRNA is: guucaccaaucagcuaagcucu (SEQ ID NO: 6), which can be included in the 3' UTR of the recombinant mRNA construct. The reverse sequence complementary to the target sequence is shown in SEQ ID NO: 2 in bold and underlined.

[0344] In some embodiments, miR-10a binding sites are incorporated within the 3' UTR of mRNA for expression of a polypeptide encoded by a sequence in the mRNA specifically in a myeloid cell. In some embodiments, a human miR-10a comprises a sequence NR_029608: qatctqtctqtcttctqtatataccctqtaqatccqaatttqtqtaaqqaattttqtqqtcacaaattcqtatct aggggaatatgtagttgacataaacactccgctct(SEQ ID NO: 3). For example, a representative target sequence for miRNA is: cacaaauucggaucuacagggua (SEQ ID NO: 8), which can be included in UTR 3' of the recombinant mRNA construct. The reverse sequence complementary to the target sequence is shown in SEQ ID NO: 3 in bold and underlined.

[0345] In one embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-122 target sequences into an mRNA encoding an exogenous therapeutic polypeptide when in contact with a population Petition 870250099881, dated 10 / 31 / 2025, pp. 141 / 364 134 / 178 cellular comprising myeloid cells and non-myeloid cells, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell of the non-myeloid cells. In one embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-122 target sequences into an mRNA encoding an exogenous therapeutic polypeptide in a formulation comprising a nanoparticle, for example, a lipid nanoparticle when distributed systemically, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell.

[0346] In one embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-192b target sequences into an mRNA encoding an exogenous therapeutic polypeptide, when in contact with a cell population comprising myeloid cells and non-myeloid cells, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell of the non-myeloid cells. In another embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-192b target sequences into an mRNA encoding an exogenous therapeutic polypeptide in a formulation comprising a nanoparticle, for example, a lipid nanoparticle, when distributed systemically, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell.

[0347] In one embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-27b target sequences into an mRNA encoding an exogenous therapeutic polypeptide, when in contact with a cell population comprising myeloid cells and non-myeloid cells, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell of the non-myeloid cells. In one embodiment, the incorporation of Petition 870250099881, dated 10 / 31 / 2025, page 142 / 364 135 / 178, 2, 3, 4 or more copies of miR-27b target sequences in an mRNA encoding an exogenous therapeutic polypeptide in a formulation comprising a nanoparticle, for example, a lipid nanoparticle, when distributed systemically, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell.

[0348] In one embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-1 Oa target sequences into an mRNA encoding an exogenous therapeutic polypeptide, when in contact with a cell population comprising myeloid cells and non-myeloid cells, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell of the non-myeloid cells. In another embodiment, the incorporation of 1, 2, 3, 4 or more copies of miR-1 Oa target sequences into an mRNA encoding an exogenous therapeutic polypeptide in a formulation comprising a nanoparticle, for example, a lipid nanoparticle, when distributed systemically, results in the expression of the therapeutic polypeptide substantially in at least one myeloid cell and not expressed in an epithelial cell or a liver cell.

[0349] In some embodiments, incorporating a miRNA sequence as described above into the mRNA construct reduces the expression of the miRNA-encoded polypeptide in a non-myeloid cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. For example, the non-myeloid cell is a liver cell or an epithelial cell and the target miRNA sequence in the mRNA binds to miR-192b. For example, the non-myeloid cell is a liver cell or an epithelial cell and the target miRNA sequence in the mRNA binds to miR-122. For example, the non-myeloid cell is a liver cell or an epithelial cell and the target miRNA sequence in the mRNA binds to miR-27b. For example, the non-myeloid cell is a liver cell or an epithelial cell. Petition 870250099881, dated 10 / 31 / 2025, pp. 143 / 364 136 / 178 epithelial and the target miRNA sequence in the mRNA binds to miR-10a.

[0350] In some embodiments, an mRNA construct can be designed to comprise at least two miRNA target sequences described in this document, where the miRNA target sequences are non-identical. In some embodiments, at least two miRNA target sequences can hybridize with different regions of the same miRNA.

[0351] In some embodiments, at least two miRNA target sequences can hybridize with at least two different miRNA sequences.

[0352] In one embodiment, a miRNA target sequence is incorporated into the 3' UTR of an mRNA construct comprising a 3' UTR derived from a human alpha globin gene. In another embodiment, a miRNA target sequence is incorporated into the 3' UTR of an mRNA construct comprising a 3' UTR derived from BGH.

[0353] A recombinant mRNA is provided in this document, for example, an isolated recombinant mRNA comprising a sequence encoding a TROP2-binding CFP and a 5' UTR and a 3' UTR; wherein the 3' UTR comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to the 3' UTR of a globin gene comprising at least one copy of any of the miRNA target sequences of any of the SEQ ID NOs: 4-9 or miRNA target sequences highlighted in SEQ ID NO: 10. In some embodiments, the 5' UTR is GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGÇÇAÇÇ (SEQ ID NO: 94) (Kozak sequence underlined). In some embodiments, the 5' UTR comprises an ARCA cap. In some modalities, the UTR 3' is from a BGH UTR 3', which has a sequence that is at least 70%, at least 80%, or at least 90% identical to the UTR 3' CTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTG CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG Petition 870250099881, dated 10 / 31 / 2025, pp. 144 / 364 137 / 178 AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC (SEQ ID NO: 73) (underlined, BGH poly A sequence). In some embodiments, a poly A tail is enzymatically added. In some embodiments, the 3' UTR disclosed above additionally comprises 2 or more, 3 or more, or 4 or more miRNA target sites selected from SEQ IDs 4-9 or miRNA target sequences highlighted in SEQ ID NO: 10 (none shown in the UTR sequence here). In some embodiments, the 3' UTR disclosed above is additionally modified with the addition of 2 or more, 3 or more, or 4 or more miRNA target sites selected from SEQ IDs 4-9 or miRNA target sequences highlighted in SEQ ID NO: 10 (none shown in the UTR sequence here).

[0354] In some embodiments, the TROP2-binding CFP comprises a leader sequence MWLQSLLLLGTVACSIS (SEQ ID NO: 50); an extracellular scFv-ligand-scFv-VL VH domain and a CD89 transmembrane domain. The TROP2-binding CFP comprises a sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 95) (scFv VH; CDR1, 2, and 3 underlined sequentially); linked to the VL domain via a linker with the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 96), wherein the VL domain comprises a sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSA SYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGT KVEIKR (SEQ ID NO: 97); (CDR1, 2, and 3 underlined sequentially), which is associated with a CD89 sequence comprising the TMD via a linker with the sequence, wherein the CD89 sequence comprising the TMD has a sequence Petition 870250099881, dated 10 / 31 / 2025, pp. 145 / 364 138 / 178 DSIHQDYTTQNL / R / WAVAGLVLVALM / LVENWHSHTALNKEASADVAEPS WSQQMCQPGLTFARTPSVCK (SEQ ID NO: 14) (TMD in italics).

[0355] A recombinant mRNA is provided in this document, for example, an isolated recombinant mRNA comprising a sequence encoding a GPC3-binding CFP and a 5' UTR and a 3' UTR; wherein the 3' UTR comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to the 3' UTR of a globin gene comprising at least one copy of any of the miRNA target sequences of any of the SEQ ID NOs: 4-9 or miRNA target sequences highlighted in SEQ ID NO: 10.

[0356] In some embodiments, the 5' UTR is AGGAGACCCAAGCTGGCTAGCGCCACC; (SEQ ID NO: 98) (Kozak sequence, underlined). In some embodiments, the 5' UTR comprises a Cap 1. In some embodiments, the 3' UTR is a 3' UTR of the globin gene, with multiple miR-27b sequences directed towards binding); where the 3' UTR has a sequence CTCGAGTAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGC CTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTTÇAÇÇA ATCAGCTAAGCTCTCGATTTCACCAATCAGCTAAGCTCTTCTAGATTCA CCAATCAGCTAAGCTCTTCACTTCACCAATCAGCTAAGCTCTGTGGTCT TTGAATAAAGTCTGAGTGGGCGGCGAAGACAATAGC (SEQ ID NO: 99) (miR-27b binding sequence underlined). In some embodiments, a poly-A tail is enzymatically added. In some embodiments, the UTR is additionally incorporated with 2 or more, 3 or more, or 4 or more additional miRNA target sites selected from SEQ ID Nos. 4-9.

[0357] In some embodiments, the GPC3-binding CFP comprises an MWLQSLLLLGTVACSIS leader sequence (SEQ ID NO: 50); an extracellular scFv-ligand-scFv-VL VH domain and a CD89 transmembrane domain. The GPC3-binding CFP comprises a Petition 870250099881, dated 10 / 31 / 2025, pp. 146 / 364 139 / 178 sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEW MGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCT RFYSYTYWGQGTLVTVSS (SEQ ID NO: 16) (scFv VH, CDR1, 2, and 3 sequences sequentially underlined); linked to the VL domain by a linker with the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 96), wherein the VL domain comprises a sequence DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSP QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHV PPTFGQGTKLEIK (SEQ ID NO: 13); (CDR1, 2, and 3 sequences sequentially underlined) that are associated with a CD89 sequence comprising TMD by means of a peptide linker, wherein the CD89 sequence comprising TMD has a sequence DSIHQDYTTQNL / R / WAV4GLVLV4Z.M / ÍVENWHSHTALNKEASADVAEPS WSQQMCQPGLTFARTPSVCK (SEQ ID NO: 14) (TMD in italics).

[0358] DNA, mRNA, and Circular RNA: In some embodiments, naked DNA or messenger RNA (mRNA) can be used to introduce the nucleic acid into the cell. In some embodiments, DNA or mRNA encoding PFP is introduced into the phagocytic cell by encapsulation with lipid nanoparticles (LNPs). The mRNA is single-stranded and can be codon-optimized. In some embodiments, the mRNA may comprise one or more modified or unnatural bases, such as 5'-Methylcytosine or Pseudouridine. The mRNA can be 50-10,000 bases in length. In one aspect, the transgene is distributed as an mRNA. mRNA can comprise more than approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 bases. In some embodiments, mRNA can be more than 10,000 bases long. In some embodiments, mRNA can be about 11,000 bases long.In some forms, mRNA can have around 12,000 chromosomes. Petition 870250099881, dated 10 / 31 / 2025, pp. 147 / 364 140 / 178 bases in length. In some embodiments, the mRNA comprises a transgene sequence encoding a fusion protein. The DNA or RNA encapsulated in LNPs can be used to transfect myeloid cells, such as macrophages, or can be administered directly to a subject.

[0359] In some embodiments, circular RNAs (circRNAs) encoding PFP are used. In circular RNAs (circRNAs), the 3' and 5' ends are covalently linked; they constitute a class of RNA. CircRNA can be distributed within a cell or subject using LNPs. Distribution of nucleic acid in a cell:

[0360] Nucleic acids encoding CFP or PFP, as described in this document, can be introduced into a cell, for example, a myeloid cell, through different delivery approaches. A manipulated nucleic acid, as described in this document, can be introduced into a cell in vitro, ex vivo, or in vivo. In some embodiments, a nucleic acid is introduced into a myeloid cell in the form of a plasmid or vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an expression vector, for example, a vector comprising one or more promoters and other regulatory components, including enhancer binding sequence, initiation and termination codons, a 5' UTR, a 3' UTR comprising a transcript stabilizing element, optional conserved regulatory protein binding sequences, and others. In some embodiments, the vector is a phage, a cosmid, or an artificial chromosome.

[0361] In some embodiments, a vector is introduced or incorporated into the cell by known transfection methods, such as the use of lipofectamine or calcium phosphate, or by physical means, such as electroporation or nucleofection. In some embodiments, the vector is introduced or incorporated into the cell by infection, a process commonly known as viral transduction. Petition 870250099881, dated 10 / 31 / 2025, pp. 148 / 364 141 / 178

[0362] In some embodiments, the vector for CFP expression is of viral origin. In some embodiments, the manipulated nucleic acid is encoded by a viral vector capable of replicating in non-dividing cells. In some embodiments, the nucleic acid encoding the manipulated nucleic acid is encoded by a lentiviral vector, for example, HIV- and FIV-based vectors. In some embodiments, the lentiviral vector is prepared in-house and manufactured on a large scale for the purpose. In some embodiments, commercially available lentiviral vectors are used, as is known to those skilled in the art. In some embodiments, the manipulated nucleic acid is encoded by a herpes simplex virus vector, a vaccinia virus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.

[0363] In some embodiments, stable integration of transgenes into myeloid cells, such as macrophages, and other phagocytic cells can be achieved through the use of a transposase and transposable elements, in particular, mRNA-encoded transposase. In one embodiment, Long Intercalated Element-1 (L1) RNAs can be contemplated for transgene retrotransposition and stable integration into myeloid cells, such as macrophages or phagocytic cells. The retrotransposon can be used for stable integration of a manipulated nucleic acid encoding a phagocytic receptor fusion protein (PFP) or binding protein (PR).

[0364] In some embodiments, the myeloid cell can be modified by the expression of a transgene through the incorporation of the transgene into a transient expression vector. In some embodiments, transgene expression can be temporally regulated by a regulator from outside the cell. Examples include the Tet-on Tet-off system, where transgene expression is regulated by the presence or absence of tetracycline.

[0365] In some forms, the manipulated nucleic acid Petition 870250099881, dated 10 / 31 / 2025, pp. 149 / 364 The RNA described in this document is a circular RNA (circRNA). A circular RNA comprises an RNA molecule where the 5' end and the 3' end of the RNA molecule are joined. Without wishing to be bound to any theory, circRNAs have no free ends and may have a longer half-life compared to some other forms of RNA or nucleic acid and may be resistant to digestion with RNase R exonuclease and transform more slowly than their corresponding linear RNA in vivo. In some embodiments, the half-life of a circRNA is greater than 20 hours. In some forms, the half-life of a circRNA is greater than 30 hours. In some forms, the half-life of a circRNA is greater than 40 hours. In some forms, the half-life of a circRNA is greater than 48 hours. In certain embodiments, a circRNA comprises an internal ribosome entry site (IRES) element that engages a eukaryotic ribosome and an RNA sequence element that encodes a polypeptide operationally linked to the IRES for insertion into cells in order to produce a polypeptide of interest.

[0366] circRNAs can be prepared by methods known to those skilled in the art. For example, circRNAs can be chemically synthesized and / or enzymatically synthesized, for example, by enzymatic synthesis of RNA followed by chemical joining of the RNA ends to form the circRNA. In some embodiments, a linear primary construct or linear mRNA can be cyclized or concatemerized to create a circRNA. The cyclization or concatemerization mechanism can occur through methods such as, but not limited to, chemical, enzymatic, or ribozyme-catalyzed methods. The newly formed 5'- / 3' linkage can be an intramolecular linkage or an intermolecular linkage. In some embodiments, a linear primary construct or linear mRNA can be cyclized or concatemerized using the chemical method to form a circRNA. In the chemical method, the 5' end and the 3' end of the nucleic acid (e.g., primary construct) are joined. Petition 870250099881, dated 10 / 31 / 2025, pp. 150 / 364 143 / 178 linear or linear mRNA) contain chemically reactive groups that, when brought together, form a new covalent bond between the 5' and 3' ends of the molecule. The 5' end may contain a reactive NHS ester group and the 3' end may contain a 3'-amino-terminated nucleotide so that in an organic solvent the 3'-amino-terminated nucleotide at the 3' end of a linear RNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'- / 3'amide bond. In some embodiments, a DNA or RNA ligase, for example, a T4 ligase, can be used to enzymatically link a 5'-phosphorylated nucleic acid molecule (e.g., a primary linear construct or linear mRNA) to the 3'-hydroxyl group of a nucleic acid that forms a new phosphodiester bond. In some embodiments, a primary linear construct or linear mRNA can be cyclized or concatemerized using at least a non-nucleic acid moiety.For example, at least a fraction of non-nucleic acid may react with regions or features near the 5' and / or near the 3' end of the primary linear construct or linear mRNA in order to cyclize or concatemerize the primary linear construct or linear mRNA. In some embodiments, a primary linear construct or linear mRNA may be cyclized or concatemerized due to a fraction of non-nucleic acid that causes an attraction between atoms, molecular surfaces, near or attached to the 5' and 3' ends of the primary linear construct or linear mRNA. For example, a primary linear construct or linear mRNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces. In some embodiments, a primary linear construct or linear mRNA may comprise a ribozyme RNA sequence near the 5' and near the 3' end.In some embodiments, a circRNA can be synthesized by inserting DNA fragments into a plasmid containing sequences with the capacity for spontaneous cleavage and autocircularization. In some embodiments, a circRNA is produced by the production of a construct of... Petition 870250099881, dated 10 / 31 / 2025, pp. 151 / 364 144 / 178 DNA encoding an RNA cyclase ribozyme, expressing the DNA construct as RNA, and then allowing the RNA to self-rewind, which produces an intron-free circRNA in vitro. In some embodiments, a circRNA is produced by synthesizing a linear polynucleotide, combining the linear nucleotide with a complementary linking oligonucleotide under hybridization conditions, and ligating the linear polynucleotide.

[0367] circRNA can be modified or unmodified. In some embodiments, circRNA is chemically modified. For example, an A, G, U, or C ribonucleotide of a circRNA may comprise chemical modifications. In some embodiments, any region of a circRNA, for example, the coding region of CFP or PFP, the flanking regions, and / or the terminal regions may contain one, two, or more nucleosidic or nucleotide modifications (optionally different). In some embodiments, a modified circRNA introduced into a cell may exhibit reduced degradation in the cell compared to an unmodified circRNA. Modifications such as the sugar, nucleobase, or internucleosidic linkage (e.g., to a phosphate linkage / to a phosphodiester bond / to the phosphodiester backbone) are also included.In some embodiments, one or more nucleobase atoms, for example, a pyrimidine nucleobase, may be replaced by optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chlorine or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleosidic linkage. Additional modifications to circRNAs are described in US20170204422, the full content of which is incorporated herein by reference.

[0368] In some embodiments, circRNA is conjugated with other polynucleotides, dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texafirin, sapfirin), polycyclic aromatic hydrocarbons (e.g., Petition 870250099881, dated 10 / 31 / 2025, pp. 152 / 364 145 / 178 phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptans (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules with a specific affinity for a ligand, or antibodies, e.g., an antibody that binds to a specified cell type, such as a cancer cell, endothelial cell, or bone cells, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, or cofactors.

[0369] In some embodiments, circRNA is delivered directly to a subject's tissues. Further description of circRNAs in U.S. Patent Nos. 5,766,903, 5,580,859, 5,773,244, 6,210,931, PCT Publication No. WO1992001813, Hsu et al., Nature (1979) 280:339-340, Harland & Misher, Development (1988) 102:837-852, Memczak et al. Nature (2013) 495: 333-338, Jeck et al., and RNA (2013) 19: 141-157, each of which is incorporated herein by reference in its entirety.

[0370] In some embodiments, a nucleic acid is introduced into a myeloid cell with a nanoparticle (NP). A nanoparticle can be of various shapes or sizes and can house the nucleic acid encoding CFP or PFP. In some embodiments, the NP is a lipid nanoparticle (LNP). In some embodiments, the NP comprises poly(amino acids), polysaccharides and poly(alpha-hydroxy acids), gold, silver, carbon, iron, silica, or any combination thereof. In some embodiments, the NP comprises a polylactide-co-glycolide (PGLA) particle. In some embodiments, the nucleic acid is encapsulated in the NP, for example, by means of a water / oil emulsion or a water-oil-water emulsion. In some embodiments, the nucleic acid is conjugated in the NP.

[0371] Nanoparticles (NPs) can be delivered to a cell in Petition 870250099881, dated 10 / 31 / 2025, pp. 153 / 364 146 / 178 vitro, ex vivo, or in vivo. In some embodiments, an NP is delivered to a phagocytic cell ex vivo. In some embodiments, an NP is delivered to a phagocytic cell in vivo. In some embodiments, the NP is less than 100 nm in diameter. In some embodiments, the NP is more than 100 nm in diameter. In some embodiments, the NP is a column-shaped NP. In some embodiments, the NP is a spherical NP. In particular embodiments, the NP is a spherical NP for delivery to a phagocytic cell. In additional embodiments, the NP is at least 100 nm in diameter and does not trigger or triggers reduced toxicity when delivered to a cell.

[0372] In some modalities, the NP is positively charged. In some modalities, the NP is negatively charged. In some modalities, the NP is neutral. In some modalities, the NP is a cationic NP that is distributed and absorbed by a myeloid cell ex vivo or in vivo.

[0373] Rigidity can affect the biological impact of NPs. NPs made of rigid materials may be associated with an increased potential for embolism, while flexible polymer-based NPs that can deform more easily may gain better access to tissues during the complex vascular changes associated with inflammation. The fluidity of NPs also affects the ability of the antigen-laden NP to stimulate immune responses. Thus, intramuscular immunization with solid-phase antigen-containing liposomes may elicit a more robust Th1 / Th17 response than similarly administered fluid-phase liposomes.Without wishing to be bound to any particular theory, solid-phase particles may result from the formation of a deposit of immobilized antigen particles and may result in a prolonged supply of antigen to APCs, also associated with the upregulation of positive co-stimulatory molecules, such as CD80, which support the efficient preparation of T cells. Petition 870250099881, dated 10 / 31 / 2025, pp. 154 / 364 147 / 178

[0374] In some embodiments, a protein corona can form around NPs. A protein corona can form in a two-step process. In the first step, high-affinity proteins rapidly bind to NPs to form a primary corona. In the second step, lower-affinity proteins bind directly to the NP or to proteins in the primary corona, forming a secondary corona. The constituents of the protein corona can thus be impacted by the serum protein content and thus by the homeostatic or immune responses that regulate it. In some embodiments, proteins with high abundance, such as albumin, comprise a significant proportion of the primary corona. In some embodiments, NPs with different charges bind to significant amounts of less abundant proteins in particular environments, for example, in plasma with a certain antigen or antibody.The in vivo formation of a protein corona can alter the NP charge or mask functional groups important for NP targeting to certain receptors and / or increase NP clearance by phagocytes. In some modalities, NPs are manipulated to reduce changes in NP charges or masking of functional groups and / or increase the serum half-life of NPs. In some modalities, the NP comprises lipid-anchored PEG fractions. In some modalities, NP surface coatings are designed to modulate opsonization events. For example, the NP surface may be coated with polymeric ethylene glycol (PEG) or its low molecular weight polyethylene oxide derivative (PEO). Without wishing to be bound to any theory, PEG increases surface hydrophilicity and may prevent NPs from fusing with aqueous solutions.In some embodiments, PEG- or PEO-coated NPs are manipulated to result in reduced toxicity or increased biocompatibility of the NPs. Additional NP design and NP targeting to myeloid cells are described in Getts et al., Trends Immunol. 36(7): 419-427 (2015), the entirety of which is incorporated herein by reference. In some. Petition 870250099881, dated 10 / 31 / 2025, pp. 155 / 364 In 148 / 178 embodiments, the NP comprises an ionizable lipid, a sterol. In some embodiments, the NP comprises a PEG lipid, a phospholipid, and cholesterol.

[0375] The NPs described in this document can be used to introduce the manipulated nucleic acid into a cell in cell culture in vitro / ex vivo or administered in vivo. In some embodiments, the NP is modified for in vivo administration. For example, the NP may comprise surface modification or binding moieties to bind to toxins, proteins, specific ligands, or any combination thereof, before being absorbed by liver or spleen phagocytes. In recent proof-of-concept studies in rodents, highly loaded infused “immune-modifying NPs” (IMPs) can absorb certain blood proteins, including the S100 family and heat shock proteins, before ultimately being removed and destroyed by cells of the mononuclear phagocyte system. Furthermore, this mechanism can also be used to capture and concentrate certain circulating proteins. IMPs have been shown to bind to annexin 1.The accumulation of annexin 1 and its presentation to particular leukocyte subsets can have broad immune outcomes. For example, annexin 1-loaded NPs can reduce neutrophils by inducing apoptosis and / or promote T cell activation. In some modalities, the NP is designed to target a cell surface receptor, for example, a scavenger receptor. In some modalities, an NP is a negatively charged surface particle.

[0376] In some embodiments, the NP encapsulates nucleic acid in which the nucleic acid is a naked DNA molecule. In some embodiments, the NP encapsulates nucleic acid in which the nucleic acid is an mRNA molecule. In some embodiments, the NP encapsulates nucleic acid in which the nucleic acid is a circular RNA (circRNA) molecule. In some embodiments, the NP encapsulates nucleic acid in which the nucleic acid is a vector, a plasmid, or a portion or fragment thereof. Petition 870250099881, dated 10 / 31 / 2025, pp. 156 / 364 149 / 178

[0377] In some embodiments, the NP is a lipid nanoparticle (LNP). LNPs may comprise a polar and / or nonpolar lipid. In some embodiments, cholesterol is present in LNPs for efficient delivery. LNPs are 100-300 nm in diameter and provide efficient mRNA delivery means to various cell types, including myeloid cells such as macrophages. In some embodiments, the LNP can be used to introduce nucleic acids into a cell in in vitro cell culture. In some embodiments, the LNP encapsulates nucleic acid where the nucleic acid is a naked DNA molecule. In some embodiments, the LNP encapsulates nucleic acid where the nucleic acid is an mRNA molecule. In some embodiments described in this document, lipid nanoparticles are formed by associating with or encapsulating complete recombinant (manipulated) mRNA.In some embodiments, the number of mRNA molecules per LNP is regulated for optimal mRNA distribution within the cell. In some embodiments, the LNP is used to deliver mRNA systemically, which can be taken up by myeloid cells in vivo. In some embodiments, the LNP may comprise target fractions.

[0378] In some embodiments, the LNP does not comprise myeloid cell targeting fractions on the surface, but the mRNA is designed for specific myeloid cell expression. In some embodiments, the LNP encapsulates nucleic acid where the nucleic acid is inserted into a vector, such as a plasmid vector. In some embodiments, the LNP encapsulates nucleic acid where the nucleic acid is a circRNA molecule.

[0379] In some embodiments, mRNA can be encapsulated within mammalian retroviral PEG10 packages that distribute the mRNA within a cell. Specific fusogens can be used for cell targeting with PEG10 distribution to the organ, tissue, or cells, for example, myeloid cells. PEG10 is known to bind to its own mRNA and distribute it within a cell. UTR regions Petition 870250099881, dated 10 / 31 / 2025, pp. 157 / 364 150 / 178 PEG10 can be incorporated by flanking the coding region of mRNA, to facilitate PEG10 encapsulation. (Segei et al., Science (2021), 373: 6557, p882-889).

[0380] In some embodiments, LNP is used to deliver nucleic acid to the subject. LNP can be used to deliver nucleic acid systemically in a subject. It can be delivered by injection. In some embodiments, the LNP comprising the nucleic acid is injected intravenously. In some embodiments, the LNP is injected subcutaneously. In some embodiments, the LNP is injected intramuscularly. Pharmaceutical Composition

[0381] A pharmaceutical composition is provided in this document, comprising engineered myeloid cells, such as macrophages, comprising an engineered nucleic acid encoding CFP and a pharmaceutically acceptable excipient.

[0382] Also provided in this document is a pharmaceutical composition comprising a nucleic acid encoding the CFP and a pharmaceutically acceptable excipient. The pharmaceutical composition may comprise DNA, mRNA or circRNA or an LNP or a liposomal composition comprising any of these.

[0383] Also provided in this document is a pharmaceutical composition comprising a vector comprising the engineered nucleic acid encoding the CFP and a pharmaceutically acceptable excipient. The pharmaceutical composition may comprise DNA, mRNA or circRNA inserted into a plasmid vector or a viral vector.

[0384] In some embodiments, manipulated myeloid cells, such as macrophages, are grown in a cell culture sufficient for a therapeutic dose and washed and resuspended in a pharmaceutical composition.

[0385] In some forms, the excipient comprises a Petition 870250099881, dated 10 / 31 / 2025, pp. 158 / 364 151 / 178 sterile buffer (e.g., HEPES or PBS) at neutral pH. In some embodiments, the pH of the pharmaceutical composition is 7.5. In some embodiments, the pH may vary within an acceptable range. In some embodiments, the manipulated cells may be comprised in a sterile enriched cell suspension medium comprising synthetic or complement-deactivated serum. In some embodiments, the pharmaceutical composition additionally comprises cytokines, chemokines, or growth factors for cell preservation and function.

[0386] In some embodiments, the pharmaceutical composition may comprise additional therapeutic agents, co-administered with the manipulated cells. Treatment methods

[0387] Methods are provided in this document for treating cancer in a subject using a pharmaceutical composition comprising engineered myeloid cells, such as phagocytic cells (e.g., macrophages), expressing an engineered nucleic acid encoding a CFP, such as a phagocytic receptor (PR) fusion protein (PFP), to target, attack, and kill cancer cells directly or indirectly. The engineered myeloid cells, such as phagocytic cells, are also referred to as CAR-P cells in the descriptions in this document.

[0388] Cancers include, but are not limited to, T-cell lymphoma, cutaneous lymphoma, B-cell cancer (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (such as, for example, alpha-chain disease, gamma-chain disease, and mu-chain disease), benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, cancer of Petition 870250099881, dated 10 / 31 / 2025, pp. 159 / 364 152 / 178 esophageal, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and similar cancers. Other, non-limiting examples of cancer types applicable to the methods covered by this disclosure include human sarcomas and carcinomas, for example, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, for example, acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); Chronic leukemia (chronic myeloid leukemia (granulocytic) and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia and heavy chain disease. In some forms, the cancer is an epithelial cancer. Petition 870250099881, dated 10 / 31 / 2025, pp. 160 / 364 153 / 178 such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other modalities, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other modalities, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. Epithelial cancers may be characterized in several other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, or undifferentiated.In some modalities, this disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered proliferative diseases.

[0389] In some respects, any gene of interest can be expressed in a myeloid cell, so that the cell can be used to treat a disease that requires, for example, an active phagocytic cell, such as an infection, where the myeloid cell can be specifically manipulated to target, engulf and destroy the pathogen.

[0390] In general, cellular immunotherapy involves providing the patient with a drug comprising living cells. In some respects, a patient or subject with cancer is treated with autologous cells, the method comprising isolating myeloid cells derived from PBMCs, such as macrophages, modifying the cells ex vivo to generate phagocytic myeloid cells capable of tumor lysis by introducing into the cells a manipulated nucleic acid that encodes a CFP and administering the modified myeloid cells to the subject.

[0391] In some respects, a subject is administered with one or more doses of a pharmaceutical composition comprising cells Petition 870250099881, dated 10 / 31 / 2025, pp. 161 / 364 154 / 178 therapeutic myeloid cells, such as phagocytic cells, where the cells are allogeneic. An HLA can be matched for compatibility with the subject so that the cells do not lead to graft-versus-host disease, GVHD. A subject arriving at the clinic undergoes HLA typing to determine the HLA antigens expressed by the subject.

[0392] HLA typing is conventionally performed by serological methods using antibodies or by PCR-based methods, such as sequence-specific oligonucleotide probe hybridization (SSOP) or sequence-based typing (SBT).

[0393] Sequence information can be identified by sequencing methods or methods employing mass spectrometry, such as liquid chromatography-mass spectrometry (LC-MS or LC-MS / MS, or alternatively HPLC-MS or HPLC-MS / MS). These sequencing methods may be well known to those skilled in the art and are reviewed in Medzihradszky KF and Chalkley RJ. Mass Spectrom Rev. 2015 Jan-Feb;34(1):43-63.

[0394] In some embodiments, the phagocytic cell is derived from the subject, electroporated, transfected or transduced with the nucleic acid manipulated in vitro, expanded in cell culture in vitro to achieve an adequate number for administration, and then administered to the subject. In some embodiments, the steps of electroporation, transfection or transduction with the nucleic acid manipulated in vitro, expanded in cell culture in vitro to achieve an adequate number for administration take 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10 days.

[0395] In some embodiments, sufficient quantities of electroporated, transfected, or transduced myeloid cells, such as macrophages, comprising the manipulated nucleic acid, are aseptically preserved and administered to the subject as “ready-to-use” products after HLA typing and matching of the product to the recipient’s HLA subtypes. In some embodiments, phagocytes Petition 870250099881, dated 10 / 31 / 2025, pp. 162 / 364 155 / 178 manipulated cells are cryopreserved. In some modalities, the manipulated phagocytes are cryopreserved in a suitable medium to withstand thawing without significant loss of cell viability.

[0396] In some embodiments, the subject is administered a pharmaceutical composition comprising DNA, or mRNA or circRNA in a vector, or in a pharmaceutically acceptable excipient described above.

[0397] In some embodiments, administration of ready-to-use cell products may be instantaneous or may require 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more before administration. The pharmaceutical composition comprising cell or nucleic acid may be preserved over time from preparation to use in frozen condition. In some embodiments, the pharmaceutical composition may be thawed once. In some embodiments, the pharmaceutical composition may be thawed more than once. In some embodiments, the pharmaceutical composition is stabilized after a freeze-thaw cycle before administration to the subject. In some embodiments, the pharmaceutical composition is tested for final quality control after thawing before administration.

[0398] In some embodiments, a composition comprising 10⁶ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 10⁷ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 5 x 10⁷ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 10⁸ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 2 x 10⁸ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 5 x 10⁸ manipulated cells is administered per administration dose. Petition 870250099881, dated 10 / 31 / 2025, pp. 163 / 364 156 / 178 administration. In some embodiments, a composition comprising 10⁹ manipulated cells is administered per administration dose. In some embodiments, a composition comprising 10¹⁰ manipulated cells is administered per administration dose.

[0399] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered once.

[0400] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered more than once.

[0401] In some modalities, manipulated myeloid cells, such as phagocytic cells, are applied two, three, four, five, six, seven, eight, nine, or ten times or more to a subject over a period of time comprising a few months, a year, or more.

[0402] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered twice a week.

[0403] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered once a week.

[0404] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered once every two weeks.

[0405] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered once every three weeks.

[0406] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered once a month.

[0407] In some modalities, the manipulated phagocytic cells are administered once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

[0408] In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered by injection. In some Petition 870250099881, dated 10 / 31 / 2025, pp. 164 / 364 In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered by infusion. In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered by intravenous infusion. In some modalities, manipulated myeloid cells, such as phagocytic cells, are administered by subcutaneous infusion.

[0409] The pharmaceutical composition comprising the manipulated nucleic acid or the manipulated cells may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, enteral infusion (into the intestine), gastroenteral, epidural (into the dura mater), oral (through the mouth), transdermal, peridural, intracerebral (into the brain), intracerebroventricular (into the cerebral ventricles), epicutaneous (application to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous (into the bone marrow), intrathecal infusion (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical, intravitreal (through the eye), intracavernous injection (into a pathological cavity), intracavitary administration (at the base of the penis).intravaginal administration, intrauterine administration, extra-amniotic administration, transdermal administration (diffusion through intact skin for systemic distribution), transmucosal administration (diffusion through a mucous membrane), transvaginal administration, insufflation (breathing), sublingual administration, sublabial administration, enema administration, eye drops (in the conjunctiva), ear drops, auricular administration (in or through the ear), buccal administration (directed towards the cheek), conjunctival administration, cutaneous administration, dental administration (for a tooth or teeth), electro-osmosis, endocervical administration, endosinusal administration, endotracheal administration, extracorporeal administration, hem...

Claims

1. A method for expressing an exogenous polypeptide from a recombinant polynucleic acid in a myeloid cell, the method characterized in that it comprises placing a heterogeneous population of cells comprising a myeloid cell and a non-myeloid cell in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences; wherein the exogenous polypeptide is expressed in the myeloid cell of the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the non-myeloid cell of the heterogeneous cell population.

2. Method for expressing an exogenous polypeptide from a recombinant polynucleic acid in a myeloid cell, the method characterized in that it comprises placing a heterogeneous population of cells comprising a myeloid cell and a non-myeloid cell in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the exogenous polypeptide is expressed in the myeloid cell of the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the non-myeloid cell of the heterogeneous cell population.

3. Method, according to claim 1 or 2, characterized by the fact that the non-myeloid cell of the heterogeneous cell population expresses a microRNA (miRNA) comprising a sequence that is reverse complementary to at least one or more miRNA target sequences, or a fragment thereof.

4. Method of expressing an exogenous polypeptide from a recombinant polynucleic acid in a first cell type, the method characterized in that it comprises placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences; and wherein the recombinant polynucleic acid lacks a sequence encoding a viral protein or portion thereof, wherein the exogenous polypeptide is expressed in the cell that is the first cell type of the heterogeneous cell population, and wherein the exogenous polypeptide is not substantially expressed in the cell that is the second cell type of the heterogeneous cell population.

5. Method for expressing an exogenous polypeptide from a recombinant polynucleic acid in a first cell type, the method characterized in that it comprises placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; Petition 870250080320, dated 08 / 09 / 2025, p.24 / 32 3 / 9 in which the composition lacks a sequence encoding a viral protein or portion thereof, and in which the exogenous polypeptide is expressed in the cell that is the first cell type in the heterogeneous cell population, and in which the exogenous polypeptide is not substantially expressed in the cell that is the second cell type in the heterogeneous cell population.

6. Method for expressing a chimeric fusion protein from a recombinant polynucleic acid in a first cell type, the method characterized in that it comprises placing a heterogeneous population of cells comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the chimeric fusion protein, and (b) one or more miRNA target sequences; and wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous population of cells, and wherein the chimeric fusion protein is not substantially expressed in the cell that is the second cell type of the heterogeneous population of cells.

7. Method for expressing a chimeric fusion protein from a recombinant polynucleic acid in a first cell type from a heterogeneous cell population, the method characterized in that it comprises placing the heterogeneous cell population comprising a cell that is the first cell type and a cell that is a second cell type in contact with a composition comprising the recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises: (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more sequences Petition 870250080320, dated 08 / 09 / 2025, page.25 / 32 4 / 9 miRNA target and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide; wherein the chimeric fusion protein is expressed in the cell that is the first cell type of the heterogeneous cell population, and wherein the chimeric fusion protein is not substantially expressed in the cell that is the second cell type of the heterogeneous cell population.

8. A method according to any one of claims 4-7, characterized in that the first cell type of the heterogeneous cell population is a myeloid cell and the second cell type is a non-myeloid cell of the heterogeneous population; wherein the non-myeloid cell of the heterogeneous cell population expresses a microRNA (miRNA) comprising a sequence that is reverse complementary to at least one of one or more miRNA target sequences, or a fragment thereof.

9. Method, according to any one of claims 1-3 or 8, characterized in that the non-myeloid cell of the heterogeneous cell population comprises any one of a hepatocyte, an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell and a skeletal cell.

10. A method, according to any one of claims 1-3, 8 or 9, characterized in that the myeloid cell from the heterogeneous cell population does not substantially express a miRNA that is reverse complementary to at least one of one or more miRNA target sequences.

11. Method according to claim 1, characterized in that the recombinant polynucleic acid is an mRNA.

12. Method, according to any one of claims 1-11, characterized in that the exogenous polypeptide is a recombinant polypeptide that activates a myeloid cell when expressed in the myeloid cell ex vivo.

13. Method, according to claim 12, characterized in that activating the myeloid cell comprises activating or increasing the chemotaxis of the myeloid cell compared to a cell that does not express the recombinant polypeptide.

14. Method, according to claim 12 or 13, characterized in that the activation of the myeloid cell comprises the activation of phagocytosis of the myeloid cell.

15. A method according to any one of claims 1-14, characterized in that the exogenous polypeptide comprises a recombinant receptor protein, a recombinant cytokine, chemokine, growth factor, or a portion thereof.

16. A method according to any one of claims 1-15, characterized in that the composition comprising the recombinant polynucleic acid further comprises a nucleic acid delivery vehicle.

17. Method according to claim 16, characterized in that the nucleic acid delivery vehicle comprises a lipid molecule.

18. Method according to claim 16 or 17, characterized in that the nucleic acid delivery vehicle comprises a cationic lipid.

19. Method, according to any one of claims 16-18, characterized in that the nucleic acid delivery vehicle comprises a lipid nanoparticle.

20. Method according to claim 16, characterized in that the nucleic acid delivery vehicle is a polymer.

21. Composition characterized in that it comprises one or more recombinant polynucleic acids modified to express a sequence encoding an exogenous polypeptide in a myeloid cell, the recombinant polynucleic acid comprising: (a) the sequence encoding the exogenous polypeptide, and (b) one or more miRNA target sequences, wherein one or more miRNA target sequences comprise a sequence that is reverse complementary to at least one miRNA sequence or fragment thereof that is expressed in a non-myeloid cell and is absent in a myeloid cell.

22. Composition according to claim 21, characterized in that one or more recombinant polynucleic acids comprise (a) a sequence encoding the exogenous polypeptide, (b) a sequence comprising (i) one or more miRNA target sequences and (ii) a sequence encoding a polypeptide required for the expression of the exogenous polypeptide.

23. Composition, according to claims 21 or 22, characterized in that one or more recombinant polynucleic acids comprise a vector comprising the sequence encoding the human polypeptide and one or more miRNA target sequences.

24. Composition according to claim 21 or 22, characterized in that one or more recombinant polynucleic acids are an mRNA molecule.

25. Composition according to claim 21 or 22, characterized in that the mRNA comprises a 5'-UTR and a 3'-UTR.

26. Composition according to claim 25, characterized in that one or more miRNA target sequences are incorporated into the 5'-UTR and the 3'-UTR.

27. Composition, according to any one of claims 21-26, characterized in that each of the one or more miRNA target sequences comprises a target sequence for a miRNA expressed in a non-myeloid cell and in which the expression of the miRNA is absent in a myeloid cell.

28. Composition, according to any one of claims 21-27, characterized in that the miRNA target sequence comprises a target sequence for a miRNA that is expressed in a hepatocyte, an alveolar cell, a cardiac cell, a renal cell, a stromal cell, an epithelial cell, a muscle cell, an ocular cell, a nerve cell, a brain cell or a skeletal cell.

29. Composition, according to any one of claims 21-28, characterized in that the miRNA target sequence comprises a target sequence for a miRNA that is expressed in an epithelial cell and a hepatocyte.

30. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

4.

31. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

5.

32. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

6.

33. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

7.

34. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

8.

35. Composition, according to any one of claims 21-29, characterized in that the miRNA target sequence comprises the sequence presented in SEQ ID NO:

9. Petition 870250080320, dated 08 / 09 / 2025, p. 29 / 32 8 / 9 36. Composition, according to any one of claims 21-29, characterized in that the 3' UTR of the mRNA comprises one or more sequences selected from SEQ ID NOs: 5, 7 and 9.

37. Composition, according to any one of claims 21-29, characterized in that the 5' UTR of the mRNA comprises one or more sequences selected from SEQ ID NOs: 4, 6 and 8.

38. Composition, according to any one of claims 21-37, characterized in that the composition further comprises a nucleic acid delivery vehicle.

39. Composition according to claim 38, characterized in that the nucleic acid delivery vehicle comprises a lipid molecule.

40. Composition according to claim 38 or 39, characterized in that the nucleic acid delivery vehicle comprises a cationic lipid.

41. Composition, according to any one of claims 38-40, characterized in that the nucleic acid delivery vehicle comprises a lipid nanoparticle.

42. Composition according to claim 38, characterized in that the nucleic acid delivery vehicle is a polymer.

43. Composition, according to any one of claims 21-42, characterized in that the exogenous polypeptide is a therapeutic polypeptide.

44. Composition, according to any one of claims 21-43, characterized in that the exogenous polypeptide comprises a receptor protein, a cytokine, chemokine, growth factor or a portion thereof.

45. Composition, according to any one of claims 21-44, characterized in that the exogenous polypeptide comprises a chimeric antigen receptor. Petition 870250080320, dated 08 / 09 / 2025, pp. 30 / 32 9 / 9 46. ​​A method for expressing an exogenous polypeptide in a myeloid cell of a subject, the method characterized in that it comprises administering to the subject the composition as defined in any one of claims 20 to 36; wherein, after administration, the exogenous polypeptide is expressed in the myeloid cell of the subject and substantially not expressed in a non-myeloid cell of the subject.

47. A method for producing a composition to selectively express an exogenous polypeptide in a myeloid cell, the method characterized in that it comprises: (i) synthesizing a polynucleotide sequence that is a reverse complement of a miRNA that is expressed in a non-myeloid cell, wherein the expression of the miRNA is absent in a non-myeloid cell; (ii) synthesizing a modified polynucleic acid comprising a sequence encoding the exogenous polypeptide and a miRNA target sequence comprising the polynucleotide sequence that is the reverse complement of the miRNA in (i), wherein the sequence encoding the exogenous polypeptide and the miRNA target sequence are operationally linked.

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