Polynucleotides encoding integrin beta-6 and methods of use thereof

Abstract

The present disclosure deals with lipid nanoparticle (LNP) compositions ITB6 molecules and uses thereof. The LNP compositions of the present disclosure include mRNA therapeutics encoding ITB6 polypeptides. The LNP compositions of the present disclosure can reprogram dendritic cells, suppress T cells, and / or induce immune tolerance in vivo. Also disclosed herein are methods of using LNP compositions comprising polynucleotides (e.g., mRNA) encoding ITB6 molecules to treat diseases associated with abnormal T cell function or inhibit immune responses in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 274,211, filed November 1, 2021. The contents of the aforementioned application are incorporated herein by reference in their entirety. [Background technology]

[0002] Integrin β (ITGB) superfamily members play important roles in multiple biological functions, including immunosuppression.

[0003] Immune cells are believed to contribute to the development and / or progression of a wide variety of diseases, for example, autoimmune disease and / or inflammatory disease.Much effort has been made to develop a therapeutic method for suppressing immune cells.However, there is still an unmet need to develop a therapeutic method that can suppress immune cells for the treatment of autoimmune disease and / or inflammatory disease. Summary of the Invention

[0004] The present disclosure provides, inter alia, lipid nanoparticle (LNP) compositions and uses thereof comprising a polynucleotide (e.g., mRNA) encoding an ITB6 molecule. The LNP compositions of the present disclosure comprise a nucleic acid (e.g., mRNA) therapeutic agent encoding an ITB6 polypeptide. In certain embodiments, the LNP compositions of the present disclosure can reprogram myeloid cells and / or dendritic cells in vivo, suppress T cells, and / or induce immune tolerance. Also disclosed herein are methods of using LNP compositions comprising a polynucleotide (e.g., mRNA) encoding an ITB6 molecule to treat diseases associated with abnormal T cell function or inhibit an immune response in a subject. While both ITG6 and ITB8 have similar functions and can be used in the methods described herein, ITB6 has been found to produce superior results when used in the subject compositions. Additional embodiments of the present disclosure are described in further detail below.

[0005] In one aspect, disclosed herein is a lipid nanoparticle (LNP) composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0006] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition for immunomodulation, e.g., for inducing immune tolerance or reprogramming immune cells, the composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0007] In some embodiments, the ITB6 molecule comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from, the amino acid sequence of ITB6 provided in Table 1A or Table 2A, e.g., any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof.

[0008] In some embodiments, the LNP composition comprises the amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof.

[0009] In some embodiments, the LNP composition comprises a nucleic acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleic acid sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175, or a functional fragment thereof.

[0010] In some embodiments, the LNP composition results in suppression of T cell activity and / or T cell function (e.g., T cell anergy and / or T cell apoptosis) in a population of immune cells, e.g., compared to T cell activity and / or T cell function in an otherwise similar or identical population of immune cells that have not been contacted with an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0011] In some embodiments, the inhibition of T cell activity and / or T cell function is determined by: (i) a reduction in the level and / or activity of an effector cytokine, e.g., IFNg (e.g., secreted IFNg); (ii) reduced proliferation, survival, and / or expansion of T cells; (iii) increased T cell apoptosis; (iv) reducing the expression and / or activity of a T cell transcription factor, e.g., T-bet, and / or (v) increased Treg differentiation; Contains any one, two, three, four, or all of the following:

[0012] In some embodiments, the LNP composition comprises: (i) reduced engraftment of donor cells, e.g., donor immune cells, e.g., T cells, in a subject or host, e.g., a human, rat, or mouse; (ii) a reduction in the level, activity, and / or secretion of IFNg from engrafted donor immune cells, e.g., T cells, in a subject or host, e.g., a human, rat, or mouse; and / or (iii) the absence of, prevention of, or delay in the onset of graft-versus-host disease (GvHD) in a subject or host, e.g., a human, rat, or mouse; results.

[0013] In some embodiments, the LNP composition results in an improvement or reduction in symptoms of GvHD in the subject, e.g., weight loss, host B cell depletion, and / or reduced donor T cell engraftment, e.g., as measured by the assays described in Example 6. In some embodiments, the LNP composition further results in Treg expansion in the subject, e.g., as measured by the assays described in Example 6.

[0014] In certain aspects, disclosed herein are pharmaceutical compositions comprising LNPs that include a polynucleotide comprising mRNA encoding an ITB6 molecule, eg, as described herein.

[0015] In another aspect, the present disclosure provides a method for modulating, e.g., suppressing, an immune response, the method comprising administering to a subject in need thereof an effective amount of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, thereby modulating the immune response.

[0016] More specifically, the subject compositions can increase Treg cell function (e.g., by increasing the number of Treg cells) and reduce T cell proliferation, thereby reducing the symptoms and / or severity of autoimmune disease. In one embodiment, the increase in Treg cells occurs in an antigen-dependent manner.

[0017] In yet another aspect, provided herein is a method for treating or preventing a disease or a symptom thereof, the method comprising administering to a subject in need thereof an effective amount of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, thereby treating or preventing the disease.

[0018] In some embodiments, the disease is an ITB6-associated disease, e.g., a disease associated with ITB6 expression and / or activity. In some embodiments, the disease is a disease associated with abnormal immune cell (e.g., T cell) function, e.g., an autoimmune disease or an inflammatory disease.

[0019] In another aspect, the present disclosure provides a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, for use in a method of modulating, e.g., suppressing, an immune response in a subject.

[0020] In yet another aspect, provided herein is a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, for use in a method of treating or preventing a disease or a symptom thereof.

[0021] In some embodiments, the disease is an ITB6-associated disease, e.g., a disease associated with ITB6 expression and / or activity. In some embodiments, the disease is a disease associated with abnormal immune cell (e.g., T cell) function, e.g., an autoimmune disease or an inflammatory disease.

[0022] In another aspect, the present disclosure provides the use of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, in the manufacture of a medicament for modulating, e.g., suppressing, an immune response in a subject.

[0023] In yet another aspect, provided herein is the use of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, in the manufacture of a medicament for treating or preventing a disease or a symptom thereof.

[0024] In some embodiments, the disease is an ITB6-associated disease, e.g., a disease associated with ITB6 expression and / or activity. In some embodiments, the disease is a disease associated with abnormal immune cell (e.g., T cell) function, e.g., an autoimmune disease or an inflammatory disease.

[0025] In another aspect, the present disclosure provides the use of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, to modulate, e.g., suppress, an immune response in a subject.

[0026] In yet another aspect, provided herein is the use of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, for treating or preventing a disease or a symptom thereof.

[0027] In another aspect, the disclosure provides a method for assessing a subject's responsiveness to a therapy comprising an LNP composition comprising mRNA encoding an ITB6 molecule, the method comprising: (a) measuring the expression level of one or more (e.g., two, three, four, or five) biomarkers (e.g., selected from the group consisting of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI) in a sample from the subject collected after administration of a therapy; (b) comparing the expression level in the sample to a reference expression level; An increase in the expression level of one or more biomarkers is indicative of a response to the therapy.

[0028] In some embodiments, the one or more biomarkers are one or more (eg, two, three, four, or five) of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI.

[0029] In some embodiments, the level of one or more of the one or more biomarkers in the sample from the subject after treatment is at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) higher than a reference expression level of the one or more biomarkers, wherein the reference expression level is (a) the expression level of one or more biomarkers in a sample from a subject before treatment with ITB6 mRNA; or (b) the expression level of one or more biomarkers in a sample from a subject not treated with ITB6 mRNA.

[0030] In some embodiments, if the expression levels of one or more (e.g., two, three, four, or five) biomarkers compared to the reference expression levels indicate responsiveness to a therapy (e.g., a therapy comprising ITB6 mRNA), the method further includes administering one or more additional doses of the therapy to the subject.

[0031] In some embodiments, the disease is an ITB6-associated disease, e.g., a disease associated with ITB6 expression and / or activity. In some embodiments, the disease is a disease associated with abnormal immune cell (e.g., T cell) function, e.g., an autoimmune disease or an inflammatory disease.

[0032] In another aspect, the present disclosure provides the use of a polynucleotide comprising mRNA encoding an ITB6 molecule, or an LNP composition comprising the same, as a pharmaceutical.

[0033] Additional features of any of the LNP compositions, pharmaceutical compositions comprising the LNPs, methods or compositions for use disclosed herein can include the following embodiments.

[0034] In some embodiments of any of the methods disclosed herein, the LNP composition comprises a polynucleotide (e.g., mRNA) described herein. In some embodiments, the ITB6 molecule comprises a native ITB6 molecule, a fragment of a native ITB6 molecule, or a variant thereof. In some embodiments, the ITB6 molecule comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from, the ITB6 amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A, for example, or a functional fragment thereof. In some embodiments, the ITB6 molecule comprises the amino acid sequence of any one of the ITB6 amino acid sequences provided in Table 1A or Table 2A, e.g., SEQ ID NO: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ITB6 molecule comprises the amino acid sequence of SEQ ID NO: 15.

[0035] In some embodiments, the ITB6 molecule comprises an amino acid sequence for a leader sequence and / or an affinity tag. In some embodiments, the ITB6 molecule does not comprise an amino acid sequence for a leader sequence and / or an affinity tag.

[0036] In some embodiments, the ITB6 molecule lacks a leader sequence and / or affinity tag (e.g., a leader sequence and / or affinity tag described in Table 1A or Table 2A) but otherwise comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from, the amino acid sequence of an ITB6 of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A, or a functional fragment thereof.

[0037] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175, or a functional fragment thereof. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175, or a functional fragment thereof.

[0038] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 18 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 18. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 175, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 18, and the 3' UTR sequence of SEQ ID NO: 142.

[0039] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 2 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 160, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 2, and the 3' UTR sequence of SEQ ID NO: 110.

[0040] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 3 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 161, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110.

[0041] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 4 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 162, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 4, and the 3' UTR sequence of SEQ ID NO: 143.

[0042] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 5 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 163, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 110. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 164, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144.

[0043] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 169, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145.

[0044] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 6 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 165, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 166, which comprises, from the 5' to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 167, which comprises, from the 5' to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 145. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 168, which comprises, from the 5' to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 143.

[0045] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 8 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 170, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 8, and the 3' UTR sequence of SEQ ID NO: 110.

[0046] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 10 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 171, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110.

[0047] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 12 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 172, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110.

[0048] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 14 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 14. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 173, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110.

[0049] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the nucleotide sequence of SEQ ID NO: 16 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 174, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110.

[0050] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of any of ITB6 human variant 5.1, human variant 1.1, human variant 1.2, human variant 1.3, human variant 1.4, human variant 1.5, human variant 1.6, human variant 1.7, human variant 1.8, human variant 1.9, human variant 1.10, human variant 2.1, human variant 3.1, or human variant 4.1, rat variant 1.1, or mouse variant 1.1, as set forth in Table 2A.

[0051] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence encoding a leader sequence and / or an affinity tag. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule does not comprise a nucleotide sequence encoding a leader sequence and / or an affinity tag.

[0052] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule lacks a nucleotide sequence encoding a leader sequence and / or affinity tag (e.g., a leader sequence and / or affinity tag described in Table 1A or Table 2A) but otherwise comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, a nucleic acid sequence identical to the nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175, or a functional fragment thereof.

[0053] In some embodiments, a polynucleotide (e.g., an mRNA) comprises at least one chemical modification, in some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is selected from the group consisting of N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, each mRNA in the lipid nanoparticle comprises a fully modified N1-methylpseudouridine.

[0054] In some embodiments of any of the methods disclosed herein, the LNP composition is an LNP composition described herein, hi some embodiments, the LNP composition comprises (i) an ionizable lipid, e.g., an amino lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

[0055] In some embodiments, the LNP composition comprises an ionizable lipid, including an amino lipid. In some embodiments, the ionizable lipid comprises a compound of any of Formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III). In some embodiments, the ionizable lipid comprises a compound of Formula (I). In some embodiments, the ionizable lipid comprises Compound 18. In some embodiments, the ionizable lipid comprises Compound 25.

[0056] In some embodiments, the lipid nanoparticles have the formula (I): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 where: R' 分岐状 but, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl, 1 is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0057] In some embodiments, the compound of formula (I) is [ka] is selected from.

[0058] In some embodiments, the lipid nanoparticles further comprise a phospholipid, a structural lipid, and a PEG lipid.

[0059] In some embodiments, the PEG lipid is Compound I.

[0060] In some embodiments, the lipid nanoparticles comprise: (i) 40 to 50 mol % of a compound of formula (I), 30 to 45 mol % of a structural lipid, 5 to 15 mol % of a phospholipid, and 1 to 5 mol % of a PEG lipid; or (ii) 45 to 50 mol % of the compound of formula (I), 35 to 45 mol % of a structural lipid, 8 to 12 mol % of a phospholipid, and 1.5 to 3.5 mol % of a PEG lipid.

[0061] In some embodiments, the lipid nanoparticles comprise: (i) Compound 18, (ii) cholesterol, and (iii) PEG-DMG or Compound VI, (i) Compound 25, (ii) cholesterol, and (iii) PEG-DMG or Compound VI, (i) Compound 301, (ii) cholesterol, and (iii) PEG-DMG or Compound VI, (i) Compound 357, (ii) cholesterol, and (iii) PEG-DMG or Compound VI, (i) Compound 18, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG-DMG or Compound VI, (i) Compound 25, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG-DMG or Compound VI, (i) Compound 301, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG-DMG or Compound VI, (i) Compound 357, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG-DMG or Compound VI.

[0062] In some embodiments, LNPs comprise about 20 mol% to about 60 mol% ionizable lipids, about 5 mol% to about 25 mol% non-cationic helper lipids or phospholipids, about 25 mol% to about 55 mol% sterol or other structured lipids, and about 0.5 mol% to about 15 mol% PEG lipids. In some embodiments, LNPs comprise about 35 mol% to about 55 mol% ionizable lipids, about 5 mol% to about 25 mol% non-cationic helper lipids or phospholipids, about 30 mol% to about 40 mol% sterol or other structured lipids, and about 0 mol% to about 10 mol% PEG lipids. In some embodiments, LNPs comprise about 50 mol% ionizable lipids, about 10 mol% non-cationic helper lipids or phospholipids, about 38.5 mol% sterol or other structured lipids, and about 1.5 mol% PEG lipids. In some embodiments, the LNP comprises about 49.83 mol% ionizable lipid, about 9.83 mol% non-cationic helper lipid or phospholipid, about 30.33 mol% sterol or other structural lipid, and about 2.0 mol% PEG lipid.

[0063] In some embodiments, a polynucleotide (e.g., RNA, e.g., mRNA) disclosed herein is formulated with a delivery agent, including, for example, a compound having Formula (II). In some embodiments, the delivery agent comprises an ionizable amino lipid, a helper lipid (e.g., DSPC), a sterol (e.g., cholesterol), and a PEG lipid (e.g., Compound VI or PEG-DMG), for example, about (i) 40-50 mol% ionizable amino lipid, optionally 45-50 mol% ionizable amino lipid, e.g., 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, e.g., about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%, (ii) 30-45 mol% sterol (e.g., cholesterol), optionally 35-42 mol% sterol, e.g., 30-31 mol%, 31-32 mol%, , 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%, or 40-42 mol% of a sterol, (iii) 5-15 mol% of a helper lipid (e.g., DSPC), optionally 10-15 mol% of a helper lipid, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, or 10-15 mol% of a sterol ... and (iv) 1-5% PEG lipid (e.g., Compound I or PEG-DMG), optionally 1-5% PEG lipid, e.g., 1.5-2.5%, 1-2%, 2-3%, 3-4%, or 4-5% PEG lipid. In some embodiments, the delivery agent comprises an ionizable amino lipid, cholesterol, DSPC, and Compound I in a molar ratio of 47:39:11:3.

[0064] In some embodiments of any of the LNP compositions, methods for use, or compositions disclosed herein, the LNPs comprise between about 45 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNPs comprise between about 45.5 mol% and about 49.5 mol% ionizable lipids. In some embodiments, the LNPs comprise between about 46 mol% and about 49 mol% ionizable lipids. In some embodiments, the LNPs comprise between about 46.5 mol% and about 48.5 mol% ionizable lipids. In some embodiments, the LNPs comprise between about 47 mol% and about 48 mol% ionizable lipids.

[0065] In some embodiments, the LNP comprises about 45 mol% to about 49.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 49 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 48.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 48 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 47.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 47 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 46.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 46 mol% ionizable lipids. In some embodiments, the LNP comprises about 45 mol% to about 45.5 mol% ionizable lipids.

[0066] In some embodiments, the LNP comprises between about 45.5 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 46 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 46.5 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 47 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 47.5 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 48 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 48.5 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 49 mol% and about 50 mol% ionizable lipids. In some embodiments, the LNP comprises between about 49.5 mol% and about 50 mol% ionizable lipids.

[0067] In some embodiments, the LNP comprises about 45 mol% to about 46 mol% ionizable lipids. In some embodiments, the LNP comprises about 45.5 mol% to about 46.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 46 mol% to about 47 mol% ionizable lipids. In some embodiments, the LNP comprises about 46.5 mol% to about 47.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 47 mol% to about 48 mol% ionizable lipids. In some embodiments, the LNP comprises about 47.5 mol% to about 48.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 48 mol% to about 49 mol% ionizable lipids. In some embodiments, the LNP comprises about 48.5 mol% to about 49.5 mol% ionizable lipids. In some embodiments, the LNP comprises about 49 mol% to about 50 mol% ionizable lipids.

[0068] In some embodiments, the LNPs comprise about 45 mol% ionizable lipids. In some embodiments, the LNPs comprise about 45.5 mol% ionizable lipids. In some embodiments, the LNPs comprise about 46 mol% ionizable lipids. In some embodiments, the LNPs comprise about 46.5 mol% ionizable lipids. In some embodiments, the LNPs comprise about 47 mol% ionizable lipids. In some embodiments, the LNPs comprise about 47.5 mol% ionizable lipids. In some embodiments, the LNPs comprise about 48 mol% ionizable lipids. In some embodiments, the LNPs comprise about 48.5 mol% ionizable lipids. In some embodiments, the LNPs comprise about 49 mol% ionizable lipids. In some embodiments, the LNPs comprise about 49.5 mol% ionizable lipids. In some embodiments, the LNPs comprise about 50 mol% ionizable lipids.

[0069] In some embodiments, the LNPs comprise between about 1 mol% and about 5 mol% PEG-lipids. In some embodiments, the LNPs comprise between about 1.5 mol% and about 4.5 mol% PEG-lipids. In some embodiments, the LNPs comprise between about 2 mol% and about 4 mol% PEG-lipids. In some embodiments, the LNPs comprise between about 2.5 mol% and about 3.5 mol% PEG-lipids.

[0070] In some embodiments, the LNP comprises about 1 mol% to about 4.5 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 4 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 3.5 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 3 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 2.5 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 2 mol% PEG lipid. In some embodiments, the LNP comprises about 1 mol% to about 1.5 mol% PEG lipid.

[0071] In some embodiments, the LNP comprises about 1.5 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 2 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 2.5 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 3 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 3.5 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 4 mol% to about 5 mol% PEG lipid. In some embodiments, the LNP comprises about 4.5 mol% to about 5 mol% PEG lipid.

[0072] In some embodiments, the LNPs comprise about 1 mol% to about 2 mol% PEG lipids. In some embodiments, the LNPs comprise about 1.5 mol% to about 2.5 mol% PEG lipids. In some embodiments, the LNPs comprise about 2 mol% to about 3 mol% PEG lipids. In some embodiments, the LNPs comprise about 3.5 mol% to about 4.5 mol% PEG lipids. In some embodiments, the LNPs comprise about 4 mol% to about 5 mol% PEG lipids.

[0073] In some embodiments, the LNPs comprise about 1 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 1.5 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 2 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 2.5 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 3 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 3.5 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 4 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 4.5 mol% PEG lipids. In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise about 5 mol% PEG lipids.

[0074] In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 18 and about 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 18 and about 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 18 and 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise 50 mol% Compound 18 and 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 49.83 mol% Compound 18, about 9.83 mol% non-cationic helper lipids or phospholipids, about 30.33 mol% sterol or other structured lipid, and about 2.0 mol% PEG lipid.

[0075] In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 25 and about 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 25 and about 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 50 mol% Compound 25 and 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise 50 mol% Compound 25 and 10 mol% non-cationic helper lipids or phospholipids. In some embodiments of the disclosed LNPs or methods, the LNPs comprise about 49.83 mol% Compound 25, about 9.83 mol% non-cationic helper lipids or phospholipids, about 30.33 mol% sterol or other structured lipid, and about 2.0 mol% PEG lipid.

[0076] In some embodiments, the LNPs are formulated for intravenous, subcutaneous, intramuscular, intraocular, intranasal, rectal, or oral delivery. In some embodiments, the LNPs are formulated for intravenous delivery. In some embodiments, the LNPs are formulated for subcutaneous delivery. In some embodiments, the LNPs are formulated for intramuscular delivery. In some embodiments, the LNPs are formulated for intraocular delivery. In some embodiments, the LNPs are formulated for intranasal delivery. In some embodiments, the LNPs are formulated for rectal delivery. In some embodiments, the LNPs are formulated for oral delivery.

[0077] In some embodiments, the disease associated with abnormal immune cell (e.g., T cell) function is an autoimmune disease or a disease involving overactivated immune function. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an autoimmune disease, such as rheumatoid arthritis (RA); graft-versus-host disease (GVHD) (e.g., acute GVHD or chronic GVHD); diabetes, e.g., type 1 diabetes; inflammatory bowel disease (IBD); lupus (e.g., systemic lupus erythematosus (SLE)), multiple sclerosis; autoimmune hepatitis (e.g., type 1 or type 2); primary biliary cholangitis; organ transplant-related rejection; psoriasis; or polymyositis (also known as dermatomyositis).

[0078] In some embodiments, the autoimmune disease is rheumatoid arthritis (RA). In some embodiments, the autoimmune disease is graft-versus-host disease (GVHD) (e.g., acute GVHD or chronic GVHD). In some embodiments, the autoimmune disease is diabetes, e.g., type 1 diabetes. In some embodiments, the autoimmune disease is inflammatory bowel disease (IBD). In some embodiments, IBD comprises colitis, ulcerative colitis, or Crohn's disease. In some embodiments, the autoimmune disease is lupus, e.g., systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the autoimmune disease is autoimmune hepatitis, e.g., type 1 or type 2. In some embodiments, the autoimmune disease is primary biliary cholangitis.

[0079] In some embodiments, the autoimmune disease is organ transplant-associated rejection. In some embodiments, the organ transplant-associated rejection comprises allograft rejection, e.g., kidney transplant rejection, liver transplant rejection, bone marrow transplant rejection, or stem cell transplant rejection. In some embodiments, the stem cell transplant comprises a graft of any one or all of the following types of cells: stem cells, umbilical cord blood stem cells, hematopoietic stem cells, embryonic stem cells, cells derived from or comprising mesenchymal stem cells, and / or induced stem cells (e.g., induced pluripotent stem cells). In some embodiments, the stem cells comprise pluripotent stem cells.

[0080] In some embodiments of any of the methods or compositions for use disclosed herein, the subject is a mammal, for example, a human.

[0081] Additional features of any of the aforementioned polynucleotides, LNP compositions, or methods of using the polynucleotides or LNP compositions include one or more of the following enumerated embodiments. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.

[0082] Other embodiments of the present disclosure E1. A lipid nanoparticle (LNP) composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0083] E2. An LNP composition for immunomodulation, e.g., inhibiting an immune response, said composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0084] E3. The LNP composition of any one of embodiments E1-E2, wherein the ITB6 molecule comprises a native ITB6 molecule, a fragment of a native ITB6 molecule, or a variant thereof.

[0085] E4. The LNP composition of any one of embodiments E1-E3, wherein the ITB6 molecule comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from, an ITB6 amino acid sequence provided in Table 1A or Table 2A, e.g., any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof.

[0086] E5. The LNP composition of any one of embodiments E1-E4, wherein the ITB6 molecule comprises the amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof.

[0087] E6. The LNP composition of any one of embodiments E1-E4, wherein the polynucleotide encoding the ITB6 molecule comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, an ITB6 nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 60-160-175, as provided in Table 1A or Table 2A, or a functional fragment thereof.

[0088] E7. The polynucleotide encoding the ITB6 molecule is: (I) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO:2 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO:2 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO:160, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO:56, the ORF sequence of SEQ ID NO:2, and the 3' UTR sequence of SEQ ID NO:110; (II) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO: 3 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 3 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 161, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110; (III) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO:4 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO:4 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO:162, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO:50, the ORF sequence of SEQ ID NO:4, and the 3' UTR sequence of SEQ ID NO:143; (IV) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO: 5 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) a nucleotide sequence or functional fragment thereof that has from its 5' end to its 3' end the 5' UTR sequence of SEQ ID NO: 50; (d) the nucleotide sequence of SEQ ID NO: 164, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144; or (e) the nucleotide sequence of SEQ ID NO: 169, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145. (V) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 6 or differs therefrom by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) SEQ ID NO: 6 or a functional fragment thereof; (c) the nucleotide sequence of SEQ ID NO: 165, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110. (d) the nucleotide sequence of SEQ ID NO: 166, which comprises, from its 5' to 3' end, the 5'UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3'UTR sequence of SEQ ID NO: 110; (e) the nucleotide sequence of SEQ ID NO: 167, which comprises, from its 5' to 3' end, the 5'UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3'UTR sequence of SEQ ID NO: 145; or (g) the nucleotide sequence of SEQ ID NO: 168, which comprises, from its 5' to 3' end, the 5'UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3'UTR sequence of SEQ ID NO: 143; (VI) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO:8 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO:8 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO:170, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO:50, the ORF sequence of SEQ ID NO:8, and the 3' UTR sequence of SEQ ID NO:110; (VII) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO: 10 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 10 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 171, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110; (VIII) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differs from the nucleotide sequence of SEQ ID NO: 12 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 12 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 172, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110; (IX) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differs from, the nucleotide sequence of SEQ ID NO: 14 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 14 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 173, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110; (X) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differs from the nucleotide sequence of SEQ ID NO: 16 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 16 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 174, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110; or (XI) (a) a nucleotide sequence or functional fragment thereof that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differs from the nucleotide sequence of SEQ ID NO: 18 by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides; (b) the nucleotide sequence of SEQ ID NO: 18 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 175, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 18, and the 3' UTR sequence of SEQ ID NO: 142; The LNP composition of any one of embodiments E1-E6, comprising:

[0089] E8. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 17; (b) a nucleotide sequence or functional fragment thereof having SEQ ID NO: 18; or (c) the nucleotide sequence of SEQ ID NO: 175, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 17, and the 3' UTR sequence of SEQ ID NO: 142.

[0090] E9. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 2; (b) the nucleotide sequence of SEQ ID NO: 2 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 160, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 2, and the 3' UTR sequence of SEQ ID NO: 110.

[0091] E10. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 3; (b) the nucleotide sequence of SEQ ID NO: 3; or (c) the nucleotide sequence of SEQ ID NO: 161, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110.

[0092] E11. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 4; (b) the nucleotide sequence of SEQ ID NO: 4; or (c) the nucleotide sequence of SEQ ID NO: 162, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 4, and the 3' UTR sequence of SEQ ID NO: 143.

[0093] E12. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 5; (b) the nucleotide sequence of SEQ ID NO: 5; or (c) the nucleotide sequence of SEQ ID NO: 163, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 110.

[0094] E13. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 5; (b) the nucleotide sequence of SEQ ID NO: 5; or (c) the nucleotide sequence of SEQ ID NO: 164, comprising, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144.

[0095] E14. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 5; (b) the nucleotide sequence of SEQ ID NO: 5; or (c) the nucleotide sequence of SEQ ID NO: 169, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145.

[0096] E15. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 6; (b) the nucleotide sequence of SEQ ID NO: 6 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 165, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0097] E16. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 6; (b) the nucleotide sequence of SEQ ID NO: 6; or (c) the nucleotide sequence of SEQ ID NO: 166, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0098] E17. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 6; and (b) the nucleotide sequence of SEQ ID NO: 167, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 145.

[0099] E18. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of SEQ ID NO: 6; and (b) the nucleotide sequence of SEQ ID NO: 168, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 143.

[0100] E19. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 8; (b) the nucleotide sequence of SEQ ID NO: 8; or (c) the nucleotide sequence of SEQ ID NO: 170, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 8, and the 3' UTR sequence of SEQ ID NO: 110.

[0101] E20. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 10; (b) the nucleotide sequence of SEQ ID NO: 10 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 171, comprising, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110.

[0102] E21. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 12; (b) the nucleotide sequence of SEQ ID NO: 12 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 172, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110.

[0103] E22. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 14; (b) the nucleotide sequence of SEQ ID NO: 14 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 173, which comprises, from the 5' end to the 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110.

[0104] E23. The LNP composition of any one of embodiments E1-E7, wherein the polynucleotide encoding the ITB6 molecule comprises: (a) a nucleotide sequence or functional fragment thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to or differing by no more than 10, 25, 50, 100, 150, 200, 250, or 300 nucleotides from the nucleotide sequence of SEQ ID NO: 16; (b) the nucleotide sequence of SEQ ID NO: 16 or a functional fragment thereof; or (c) the nucleotide sequence of SEQ ID NO: 174, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110.

[0105] E24. The LNP composition of any one of embodiments E1-E23, wherein the ITB6 molecule comprises an amino acid sequence that does not include a leader sequence and / or an affinity tag.

[0106] E25. The LNP composition of any one of embodiments E1-E24, wherein the ITB6 molecule comprises a half-life extender, e.g., a protein (or fragment thereof) that binds to a serum protein such as albumin; an immunoglobulin domain, e.g., IgG; FcRn or transferrin.

[0107] E26. The LNP composition of embodiment E25, wherein the half-life extending agent is an immunoglobulin Fc region or a variant thereof.

[0108] E27. The LNP composition of any one of embodiments E1-E26, which results in modulation (e.g., suppression) of T cell activity and / or T cell function in a population of immune cells, e.g., compared to T cell activity and / or T cell function in an otherwise similar or identical population of immune cells that have not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0109] E28. The LNP composition of embodiment E27, wherein the population of immune cells comprises T cells (e.g., CD4+ T cells, CD8+ T cells, or regulatory T cells (Tregs)), B cells, dendritic cells, granulocytes, monocytes, and / or macrophages.

[0110] E29. The LNP composition of embodiment E27 or E28, wherein the T cell activity and / or T cell function is the activity and / or function of CD8+ T cells (e.g., antigen-specific CD8+ cells) and / or CD4+ T cells (antigen-specific CD4+ T cells).

[0111] E30. The modulation (e.g., suppression) of T cell activity and / or T cell function is selected from the group consisting of: (i) increased levels of Treg differentiation; (ii) reduced proliferation, survival, and / or expansion of T cells (e.g., reduced proliferation, survival, and / or expansion of CD4+ T cells); (iii) reduced expression, activity, and / or secretion of effector cytokines (e.g., IFNg); and / or (iv) reduced expression and / or activity of T cell transcription factors (e.g., T-bet); The LNP composition of any one of embodiments E27-E29, comprising any one, two, three, or all of:

[0112] E31. The LNP composition of any one of embodiments E27-E30, wherein said suppression of T cell activity and / or T cell function occurs and / or is determined in vitro, eg, in a sample.

[0113] E32. The LNP composition of any one of embodiments E27-E31, wherein said suppression of T cell activity and / or T cell function occurs and / or is determined in vivo, eg, in a subject.

[0114] E33. The LNP composition of any one of embodiments E27-E32, wherein said suppression of T cell activity and / or T cell function comprises (i) an increase in the level of Treg differentiation.

[0115] E34. The LNP composition of embodiment E33, wherein the Treg differentiation level (e.g., from antigen-specific CD4+ T cells) is increased by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference Treg differentiation level, e.g., as determined by the methods described in Examples 2 and 4.

[0116] E35. The LNP composition of embodiment E33 or E34, wherein the reference Treg differentiation level (e.g., from antigen-specific CD4+ T cells) is the Treg differentiation level in an otherwise similar sample or subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0117] E36. The LNP composition of any one of embodiments E27-E35, wherein said suppression of T cell activity and / or T cell function comprises (ii) a reduction in T cell proliferation, survival and / or expansion, e.g., a reduction in CD4+ T cell proliferation, survival and / or expansion.

[0118] E37. The LNP composition of embodiment E36, wherein the proliferation, survival, and / or expansion of the T cells is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of T cell proliferation, survival, and / or expansion, e.g., as determined by the methods described in Examples 3 and 7.

[0119] E38. The LNP composition of embodiment E36 or E37, wherein said reference level of T cell proliferation, survival and / or expansion is said level of T cell proliferation, survival and / or expansion in an otherwise similar sample or subject that has not been contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0120] E39. The LNP composition of any one of embodiments E36-E38, wherein said reduction in T cell proliferation, survival, and / or expansion occurs upon or is determined after (a) co-culture of T cells (e.g., CD4+ T cells) with dendritic cells that have been contacted with an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, and / or (b) contact with a cytokine (e.g., TGF beta).

[0121] E40. The LNP composition of any one of embodiments E27-E39, wherein said suppression of T cell activity and / or T cell function comprises (iii) reducing the expression, activity and / or secretion of an effector cytokine (e.g., IFNg).

[0122] E41. The LNP composition of embodiment E40, wherein the expression, activity, and / or secretion of an effector cytokine is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of expression, activity, and / or secretion of the effector cytokine.

[0123] E42. The LNP composition of embodiment E40 or E41, wherein the reference level of expression, activity, and / or secretion of the effector cytokine is the level of expression, activity, and / or secretion of the effector cytokine in an otherwise similar or identical sample that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or has not been contacted with an immune cell (e.g., T cell) activator or stimulator.

[0124] E43. The LNP composition of any one of embodiments E40-E42, wherein the effector cytokine is IFNg and the expression of IFNg (e.g., produced by antigen-specific CD8+ T cells) is reduced in the sample by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold), e.g., as determined by the methods described in Examples 5 and 9.

[0125] E44. The LNP composition of embodiment E43, wherein the sample has been contacted with an immune cell (eg, T cell) activator or stimulator.

[0126] E45. The LNP composition of any one of embodiments E43 or E44, wherein the T cells (e.g., CD8+ T cells) in the sample have been stimulated and / or activated, e.g., with a peptide or a costimulatory molecule.

[0127] E46. The LNP composition of any one of embodiments E27-E45, wherein said suppression of T cell activity and / or T cell function comprises (iv) reducing the expression and / or activity of a T cell transcription factor (e.g., T-bet).

[0128] E47. The LNP composition of embodiment E46, wherein the expression and / or activity of the T cell transcription factor is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of expression and / or activity of the T cell transcription factor.

[0129] E48. The LNP composition of embodiment E46 or E47, wherein the reference level of expression and / or activity of the T cell transcription factor is the level of expression and / or activity of the T cell transcription factor in an otherwise similar sample or subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0130] E49. The LNP composition of any one of embodiments E46-E48, wherein the T cell transcription factor is T-bet and the expression of T-bet is reduced in a sample or subject by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold), e.g., as determined by a method described in Example 8.

[0131] E50. The LNP composition of embodiment E49, wherein said reduction in the expression and / or activity of T-bet occurs during or is determined after co-culture of T cells with dendritic cells that have been contacted with an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0132] E51. In a subject having or at risk of having graft-versus-host disease (GvHD), (i) reduced donor immune cell (e.g., T cell) proliferation; (ii) weight loss, host B cell depletion, and / or reduced donor immune cell (e.g., T cell) engraftment, optionally with a parallel expansion of Tregs; (iii) reduction of Tbet+ cells in a T cell population (e.g., a CD8+ T cell population); (iv) reducing the expression, activity, and / or secretion of pro-inflammatory cytokines (e.g., IFNg); and / or (v) maintaining or expanding Treg and / or host lymphocyte populations; The LNP composition of any one of embodiments E1-E50, which provides any one, two, three, four, or all of:

[0133] E52.(i) The LNP composition of embodiment E51, which results in reduced donor immune cell (e.g., T cell) proliferation.

[0134] E53. The LNP composition of embodiment E52, wherein the donor immune cell (e.g., T cell) proliferation is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of donor immune cell (e.g., T cell) proliferation, e.g., as determined by a method described in Example 7.

[0135] E54. The LNP composition of embodiment E51 or E52, wherein the reference level of donor cell (e.g., T cell) proliferation is the level of donor immune cell (e.g., T cell) proliferation in the subject before contact with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or in an otherwise similar subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0136] E55.(ii) The LNP composition of any of embodiments E51-E54, which results in weight loss, host B cell depletion, and / or reduced donor immune cell (e.g., T cell) engraftment, optionally with a parallel expansion of Tregs.

[0137] E56. The LNP composition of embodiment E55, wherein the weight loss, host B cell depletion, and / or donor immune cell (e.g., T cell) engraftment is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of weight loss, host B cell depletion, and / or donor immune cell (e.g., T cell) engraftment, e.g., as determined by a method described in Example 6.

[0138] E57. The LNP composition of embodiment E56, wherein the reference level of weight loss, host B cell depletion, and / or donor immune cell (e.g., T cell) engraftment is the level of weight loss, host B cell depletion, and / or donor immune cell (e.g., T cell) engraftment in the subject prior to contact with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or in an otherwise similar subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0139] E58. The LNP composition of any of embodiments E51-E57, wherein the donor immune cells specified in (i) or (ii) comprise T cells, e.g., CD8+ T cells, CD4+ T cells, or regulatory T cells (e.g., CD25+ and / or FoxP3+ T cells).

[0140] E59.(iii) The LNP composition of any of embodiments E51-E58, which results in a reduction of Tbet+ cells in a T cell population (e.g., a CD8+ T cell population).

[0141] E60. The LNP composition of any of embodiments E51-E59, wherein Tbet+ cells in the T cell population (e.g., a CD8+ T cell population) are reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of Tbet+ cells in the T cell population (e.g., a CD8+ T cell population), e.g., as determined by a method described in Example 8.

[0142] E61. The LNP composition of embodiment E60, wherein the reference level of Tbet+ cells in said T cell population (e.g., a CD8+ T cell population) is said level of Tbet+ cells in a T cell population (e.g., a CD8+ T cell population) in said subject prior to contact with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or in an otherwise similar subject that has not been contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0143] E62.(iv) The LNP composition of any of embodiments E51-E61, which results in a reduction in the expression, activity and / or secretion of a pro-inflammatory cytokine (eg, IFNg).

[0144] E63. The LNP composition of any of embodiments E51-E62, wherein the expression, activity, and / or secretion of a proinflammatory cytokine (e.g., IFNg) is reduced by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of proinflammatory cytokine (e.g., IFNg) expression, activity, and / or secretion, e.g., as measured by an assay described in Example 9.

[0145] E64. The LNP composition of embodiment E63, wherein the reference level of inflammatory cytokine (e.g., IFNg) expression, activity, and / or secretion is the level of inflammatory cytokine (e.g., IFNg) expression, activity, and / or secretion in the subject before contact with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or in an otherwise similar subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0146] E65.(v) The LNP composition of any of embodiments E51-E64, which results in the maintenance or expansion of Treg and / or host lymphocyte populations.

[0147] E66. The LNP composition of any of embodiments E51-E65, wherein the Treg and / or the host lymphocyte population is substantially unchanged or is increased by about 1-10 fold (e.g., about 2-8 fold, 3-7 fold, 4-6 fold, 1-8 fold, 1-6 fold, 1-4 fold, 1-2 fold, 8-10 fold, 6-10 fold, 4-10 fold, 2-10 fold, 1-3 fold, 2-4 fold, 3-5 fold, 5-7 fold, 6-8 fold, or 7-9 fold) relative to a reference level of the Treg and / or host lymphocyte population, e.g., as measured by an assay described in Example 10.

[0148] E67. The LNP composition of embodiment E66, wherein the reference level of Treg and / or host lymphocyte population is the level of Treg and / or host lymphocyte population in the subject prior to contact with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or in an otherwise similar subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0149] E68. The LNP composition of any of embodiments E1-E67, which results in a delay in the onset of GvHD in a subject.

[0150] E69. The LNP composition of E68, wherein the GvHD onset is delayed by at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, or 2 years relative to a reference GvHD onset, e.g., the GvHD onset in an otherwise similar subject not contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0151] E70. The LNP composition of any of embodiments E1-E68, which results in a delay in the onset of GvHD in a subject.

[0152] E71. The LNP composition of E70, wherein said GvHD onset is delayed by at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, or 2 years relative to a reference GvHD onset, e.g., said GvHD onset in an otherwise similar subject not contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0153] E72. The LNP composition of any one of embodiments E1-E71, which results in amelioration of GvHD or symptoms thereof in a subject, e.g., compared to the subject before contact with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, or compared to a subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0154] 1. A method for assessing a subject's responsiveness to a therapy comprising an LNP composition comprising mRNA encoding an E73.ITB6 molecule, the method comprising: (a) measuring the expression level of one or more biomarkers (e.g., selected from the group consisting of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI) in a sample from the subject collected after administration of the therapy; (b) comparing the expression level in the sample to a reference expression level; wherein an increase in the expression level of the one or more biomarkers is indicative of a response to the therapy.

[0155] E74. The method of embodiment E73, wherein said one or more biomarkers is one or more of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI.

[0156] E75. The level of one or more of the one or more biomarkers in the sample from the subject after treatment is at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) higher than a reference expression level of said one or more biomarkers, and said reference expression level is: (a) the expression level of the one or more biomarkers in a sample from the subject prior to treatment with ITB6 mRNA; or (b) the expression level of the one or more biomarkers in a sample from a subject not treated with ITB6 mRNA; The method of embodiment E73 or E74, comprising:

[0157] E76. The LNP composition of any one of the preceding embodiments, wherein the polynucleotide comprising mRNA encoding the ITB6 molecule comprises at least one chemical modification.

[0158] E77. The LNP composition of embodiment E76, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0159] E78. The LNP composition of embodiment E76 or E77, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0160] E79. The LNP composition of any one of embodiments E76-E78, wherein the chemical modification is N1-methylpseudouridine.

[0161] E80. The LNP composition of any one of the preceding embodiments, wherein the mRNA in the lipid nanoparticle comprises fully modified N1-methylpseudouridine.

[0162] E81. The LNP composition of any one of the preceding embodiments, wherein the LNP composition comprises (i) an ionizable lipid, e.g., an amino lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

[0163] E82. The LNP composition of embodiment E81, wherein the ionizable lipid comprises an amino lipid.

[0164] E83. The LNP composition of embodiment E81 or E82, wherein the ionizable lipid comprises a compound of any of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0165] E84. The LNP composition of any one of embodiments E81-E83, wherein the ionizable lipid comprises a compound of formula (I).

[0166] E85. The LNP composition of any one of embodiments E81-E84, wherein the ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0167] E86. The LNP composition of any one of embodiments E81-E85, wherein the LNP comprises a molar ratio of about 20-60% ionizable lipid:5-25% phospholipid:25-55% cholesterol:0.5-15% PEG lipid.

[0168] E87. The LNP composition of embodiment E86, wherein the LNP comprises a molar ratio of about 50% ionizable lipid:about 10% phospholipid:about 38.5% cholesterol:about 1.5% PEG lipid.

[0169] E88. The LNP composition of embodiment E86 or E87, wherein the LNP comprises a molar ratio of about 49.83% ionizable lipid:about 9.83% phospholipid:about 30.33% cholesterol:about 2.0% PEG lipid.

[0170] E89. The LNP composition of any one of embodiments E86-E88, wherein the ionizable lipid comprises a compound of any of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0171] E90. The LNP composition of embodiment E89, wherein the ionizable lipid comprises a compound of formula (I).

[0172] E91. The LNP composition of embodiment E89 or E90, wherein the ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0173] E92. The LNP composition of any one of the preceding embodiments, formulated for intravenous, subcutaneous, intramuscular, intranasal, intraocular, rectal, or oral delivery.

[0174] E93. The LNP composition of any one of the preceding embodiments, further comprising a pharmaceutically acceptable carrier or excipient.

[0175] E94. A pharmaceutical composition comprising the LNP composition of any one of embodiments E1-E93.

[0176] E95. A method of modulating, e.g., suppressing, an immune response in a subject, comprising administering to said subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0177] E96. A method for treating, preventing, or preventing a symptom of a disease associated with abnormal T cell function, e.g., an autoimmune disease or an inflammatory disease, comprising administering to a subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0178] E97. A composition, comprising an LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, for use in a method for treating or preventing a disease involving abnormal T cell function, e.g., an autoimmune disease or an inflammatory disease, or preventing a symptom thereof.

[0179] E98. The method of E96 or the LNP composition for use of embodiment E97, wherein the disease is selected from rheumatoid arthritis (RA); graft-versus-host disease (GVHD) (e.g., acute GVHD or chronic GVHD); diabetes, e.g., type 1 diabetes; inflammatory bowel disease (IBD); lupus (e.g., systemic lupus erythematosus (SLE)), multiple sclerosis; autoimmune hepatitis (e.g., type 1 or type 2); primary biliary cholangitis; organ transplant-associated rejection; or myasthenia gravis.

[0180] E99. The method or LNP composition for use according to any one of E95-E98, wherein the ITB6 molecule comprises a native ITB6 molecule, a fragment of a native ITB6 molecule, or a variant thereof.

[0181] E100. The method of, or LNP composition for use according to E99, wherein the ITB6 molecule comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an ITB6 of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A.

[0182] E101. The method of, or LNP composition for use according to E99, wherein the ITB6 molecule comprises the amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15.

[0183] E102. The method or LNP composition for use according to E99, wherein the polynucleotide encoding the ITB6 molecule comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an ITB6 nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175 as provided in Table 1A or Table 2A.

[0184] E103. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 18; (b) the nucleotide sequence of SEQ ID NO: 18, or (c) the nucleotide sequence of SEQ ID NO: 175, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 18, and the 3' UTR sequence of SEQ ID NO: 142; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0185] E104. The polynucleotide encoding the ITB6 molecule is (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2; (b) the nucleotide sequence of SEQ ID NO: 2, or (c) the nucleotide sequence of SEQ ID NO: 160, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 2, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0186] E105. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3; (b) the nucleotide sequence of SEQ ID NO: 3, or (c) the nucleotide sequence of SEQ ID NO: 161, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0187] E106. The polynucleotide encoding the ITB6 molecule (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:4; (b) the nucleotide sequence of SEQ ID NO: 4, or (c) the nucleotide sequence of SEQ ID NO: 162, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 4, and the 3' UTR sequence of SEQ ID NO: 143; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0188] E107. The polynucleotide encoding the ITB6 molecule is (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:5; (b) the nucleotide sequence of SEQ ID NO: 5, or (c) the nucleotide sequence of SEQ ID NO: 163, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0189] E108. The polynucleotide encoding the ITB6 molecule is (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:5; (b) the nucleotide sequence of SEQ ID NO: 5, or (c) the nucleotide sequence of SEQ ID NO: 164, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0190] E109. The polynucleotide encoding the ITB6 molecule is (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:5; (b) the nucleotide sequence of SEQ ID NO: 5, or (c) the nucleotide sequence of SEQ ID NO: 169, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0191] E110. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:6; (b) the nucleotide sequence of SEQ ID NO: 6, or (c) the nucleotide sequence of SEQ ID NO: 165, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0192] E111. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:6; (b) the nucleotide sequence of SEQ ID NO: 6, or (c) the nucleotide sequence of SEQ ID NO: 166, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0193] E112. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:6; (b) the nucleotide sequence of SEQ ID NO: 6, or (c) the nucleotide sequence of SEQ ID NO: 167, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 145; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0194] E113. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:6; (b) the nucleotide sequence of SEQ ID NO: 6, or (c) the nucleotide sequence of SEQ ID NO: 168, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 143; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0195] E114. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:8; (b) the nucleotide sequence of SEQ ID NO: 8, or (c) the nucleotide sequence of SEQ ID NO: 170, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 8, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0196] E115. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 10; (b) the nucleotide sequence of SEQ ID NO: 10, or (c) the nucleotide sequence of SEQ ID NO: 171, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0197] E116. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 12; (b) the nucleotide sequence of SEQ ID NO: 12, or (c) the nucleotide sequence of SEQ ID NO: 172, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0198] E117. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 14; (b) the nucleotide sequence of SEQ ID NO: 14, or (c) the nucleotide sequence of SEQ ID NO: 173, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0199] E118. The polynucleotide encoding the ITB6 molecule is: (a) a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 16; (b) the nucleotide sequence of SEQ ID NO: 16, or (c) the nucleotide sequence of SEQ ID NO: 174, comprising from the 5' to 3' end the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110; 10. An LNP composition for use according to or in accordance with claim 99, comprising:

[0200] E119. The method or LNP composition for use according to E99, wherein said ITB6 molecule comprises an amino acid sequence that does not include a leader sequence and / or an affinity tag.

[0201] E120. The method or LNP composition for use according to any one of embodiments E95-E119, wherein the subject is a mammal, eg, a human.

[0202] E121. The LNP composition or method for use of any one of embodiments E95-E120, wherein the LNP composition is administered to a subject, eg, according to a dosing interval as described herein.

[0203] E122. The LNP composition or method for use of embodiment E121, wherein the dosing interval comprises an initial dose of the LNP composition and one or more subsequent doses of the same LNP composition (e.g., 1 to 50 doses, 5 to 50 doses, 10 to 50 doses, 15 to 50 doses, 20 to 50 doses, 25 to 50 doses, 30 to 50 doses, 35 to 50 doses, 40 to 50 doses, 45 to 50 doses, 1 to 45 doses, 1 to 40 doses, 1 to 35 doses, 1 to 30 doses, 1 to 25 doses, 1 to 20 doses, 1 to 15 doses, 1 to 10 doses, or 1 to 5 doses).

[0204] E123. The LNP composition or method for use of embodiment E121 or E122, wherein the dosing interval comprises one or more doses of the LNP composition and one or more doses of an additional agent.

[0205] E124. The LNP composition or method for use of any one of embodiments E121-E123, wherein the dosing interval is carried out for at least 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0206] E125. The LNP composition or method for use of any one of embodiments E120-E124, wherein the dosing interval comprises a cycle, eg, a 7-day cycle.

[0207] E126. The LNP composition or method for use of any one of embodiments E120-E125, wherein the dosing interval is repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.

[0208] E127. The LNP composition or method for use of any one of embodiments E120-E126, wherein said repeat dosing interval is carried out over at least 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, or 5 years.

[0209] E128. The LNP composition or method for use of any one of embodiments E120-E127, wherein the LNP composition is administered daily for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year.

[0210] E129. The LNP composition or method for use of any one of embodiments E120-E128, wherein the LNP composition is administered for at least 2, 3, 4, 5, or 6 consecutive days in a 7-day cycle, e.g., the cycle is repeated about 1 to 20 times (e.g., 2 to 15, 5 to 10, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 10 to 15, or 5 to 15 times).

[0211] E130. The LNP composition or method for use of any one of embodiments E120-E129, wherein said LNP composition is administered by a route of administration selected from subcutaneous, intramuscular, intravenous, oral, intraocular, or rectal.

[0212] E131. The LNP composition or method for use of any one of embodiments E120-E130, wherein the LNP composition is administered at a dose of about 0.1-10 mg per kg (e.g., about 0.2-5 mg per kg, 0.5-2 mg per kg, 0.1-5 mg per kg, 0.1-2 mg per kg, 0.1-1 mg per kg, 0.1-0.5 mg per kg, 5-10 mg per kg, 2-10 mg per kg, 1-10 mg per kg, 0.5-10 mg per kg, or 0.2-10 mg per kg), e.g., about 0.2-1 mg per kg (e.g., about 0.5 mg per kg).

[0213] E132. The method or LNP composition for use according to any one of embodiments E95-E131, further comprising administering an additional agent, eg, a standard of care drug.

[0214] E133. The LNP composition or method for use of any one of embodiments E95 to E132, wherein the composition or method results in suppression of the activity and / or function (e.g., T cell anergy and / or T cell apoptosis) of T cells in a sample from the subject, e.g., compared to the activity and / or function of T cells in a sample from an otherwise similar subject that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0215] E134. Suppression of T cell activity and / or function is determined by: (i) reduced donor immune cell (e.g., T cell) proliferation; (ii) weight loss, host B cell depletion, and / or reduced donor immune cell (e.g., T cell) engraftment, optionally with a parallel expansion of Tregs; (iii) reduction of Tbet+ cells in a T cell population (e.g., a CD8+ T cell population); (iv) reducing the expression, activity, and / or secretion of inflammatory cytokines (e.g., IFNg) in the sample; and / or (v) maintaining or expanding Treg and / or host lymphocyte populations; (i) a reduction in the level and / or activity of IFNg, e.g., secreted IFNg, in the sample; (ii) reduced proliferation, survival and / or expansion of T cells; (iii) increased T cell apoptosis; (iv) reducing the expression and / or activity of T cell transcription factors, e.g., T-bet; (v) increasing and / or activating Treg differentiation, and / or (vi) an increase in the expression level of one or more (e.g., two, three, four, or five) of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI; The LNP composition or method for use of embodiment E133, comprising any one, two, three, four, five, or all of:

[0216] E135. The LNP composition or method for use of embodiment E134, wherein the reduction in inflammatory cytokine (e.g., IFNg) expression, activity, and / or secretion is about 1.2-10 fold (e.g., about 2-25 fold, 5-20 fold, 10-15 fold, 5-30 fold, 10-30 fold, 20-30 fold, 2-20 fold, 2-15 fold, or 2-10 fold).

[0217] E136. The LNP composition or method for use of embodiment E134 or E135, wherein the sample comprises immune cells, e.g., T cells, e.g., CD8 T cells.

[0218] E137. The LNP composition for use or method of any one of embodiments E134-E136, wherein the sample has been contacted with an activator or stimulator of immune cells, e.g., T cells.

[0219] E138. The LNP composition or method for use of embodiment E137, wherein the T cells, e.g., CD8 T cells, have been stimulated and / or activated, e.g., with a peptide or a costimulatory molecule.

[0220] E139. The LNP composition or method for use of any one of embodiments E136-E138, wherein said CD8 T cells are antigen-specific.

[0221] E140. The LNP composition or method for use of any one of embodiments E136-E139, wherein said reduction in donor immune cell (e.g., T cell) proliferation is about 1.2-10 fold (e.g., about 2-25 fold, 5-20 fold, 10-15 fold, 5-30 fold, 10-30 fold, 20-30 fold, 2-20 fold, 2-15 fold, or 2-10 fold).

[0222] E141. The LNP composition or method for use of embodiment E140, wherein said reduction in donor immune cell (e.g., T cell) proliferation occurs upon (a) co-culture of T cells with dendritic cells, e.g., CD11C+ cells, that have been contacted with an LNP composition comprising an ITB6 molecule, and / or (b) contact with a cytokine, e.g., TGF beta.

[0223] E142. The LNP composition or method for use of embodiment E140 or E141, wherein the reduction in Tbet+ cells is about 1.2-10 fold (e.g., about 2-25 fold, 5-20 fold, 10-15 fold, 5-30 fold, 10-30 fold, 20-30 fold, 2-20 fold, 2-15 fold, or 2-10 fold).

[0224] E143. The LNP composition or method for use of any one of embodiments E134 to E142, wherein said reduction in T-bet+ cells occurs upon co-culture of T cells with dendritic cells, e.g., CD11C+ cells, that have been contacted with an LNP composition comprising an ITB6 molecule.

[0225] E144. The LNP composition or method for use of any one of embodiments E134-E143, wherein said T cells comprise CD8+ T cells, CD4+ T cells, or regulatory T cells.

[0226] E145. The LNP composition for use or method of any one of embodiments E134-E144, wherein the method or composition reduces the level (e.g., expression) and / or activity of a costimulatory molecule, e.g., CD80, CD86, and / or MHCII, in the sample upon stimulation.

[0227] E146. The LNP composition or method for use of embodiment E145, wherein the reduction in the level and / or activity of the costimulatory molecule is about 1.2-5 fold (e.g., about 2-4 fold, 2-5 fold, 2-3 fold, 3-5 fold, or 4-5 fold).

[0228] E147. The LNP composition or method for use of embodiment E145 or E146, wherein the sample is contacted with a stimulant, eg, LPS, or PolyIC.

[0229] E148. The LNP composition or method for use of any one of embodiments E145-E147, wherein said reduction in the level and / or activity of said costimulatory molecule occurs in vitro or in vivo.

[0230] E149. The LNP composition or method for use of any one of embodiments E95-E133, wherein said disease involving abnormal T cell function is graft-versus-host disease (GvHD).

[0231] E150. The method or composition, (i) reduced engraftment of donor cells, e.g., donor immune cells, e.g., T cells, in a subject or host, e.g., a human, rat, or mouse; (ii) reducing the level, activity and / or secretion of IFNg from engrafted donor immune cells, e.g., T cells, in a subject or host, e.g., a human, rat or mouse; and / or (iii) the absence of, prevention of, or delay in the onset of graft-versus-host disease (GvHD) in a subject or host, e.g., a human, rat, or mouse; The LNP composition or method for use of embodiment E149, wherein the LNP composition or method results in:

[0232] E151. The LNP composition or method for use of embodiment E150, wherein the donor immune cells specified in (i) or (ii) comprise T cells, e.g., CD8+ T cells, CD4+ T cells, or regulatory T cells (e.g., CD25+ and / or FoxP3+ T cells).

[0233] E152. The LNP composition or method for use of embodiment E150 or E151, wherein said reduction in donor cell engraftment is about 1.5 to 30-fold (e.g., about 2 to 25-fold, 5 to 20-fold, 10 to 15-fold, 5 to 30-fold, 10 to 30-fold, 20 to 30-fold, 2 to 20-fold, 2 to 15-fold, or 2 to 10-fold).

[0234] E153. The LNP composition or method for use of any one of embodiments E150 to E152, wherein said reduction in IFNg levels, IFNg activity and / or secretion is about 1.5 to 10 fold (e.g., about 2 to 25 fold, 5 to 20 fold, 10 to 15 fold, 5 to 30 fold, 10 to 30 fold, 20 to 30 fold, 2 to 20 fold, 2 to 15 fold, or 2 to 10 fold).

[0235] E154. The LNP composition or method for use of any one of embodiments E149-E153, wherein said delay in GvHD onset is a delay of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, or 2 years.

[0236] E155. The LNP composition or method for use of any one of embodiments E149 to E154, wherein any one of (i) to (iii) specified in embodiment E146 is compared to an otherwise similar host, e.g., a host that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0237] E156. The LNP composition or method for use of any one of embodiments E95-E133, wherein the disease involving abnormal T cell function is arthritis, e.g., collagen-induced arthritis (CIA).

[0238] E157. The LNP composition for use or method of embodiment E156, wherein the method or composition results in an improvement or reduction in the severity of joint swelling in the subject, e.g., as described herein.

[0239] E158. The LNP composition or method for use of embodiment E156 or E157, wherein swelling is determined, eg, by an arthritis score as described herein.

[0240] E159. The LNP composition or method for use of any one of embodiments E156-E158, wherein said reduction in joint swelling is compared to joint swelling in an otherwise similar subject, for example, a subject not contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0241] E160. The LNP composition or method for use of any one of embodiments E95 to E133, wherein the disease involving abnormal T cell function is colitis, e.g., dextran sulfate sodium (DSS)-induced colitis.

[0242] E161. The method or composition, (i) an increase in colon length in a subject; and / or (ii) resulting in weight maintenance in a subject; The LNP composition or method for use of embodiment E160, wherein the subject has or is identified as having colitis, e.g., DSS-induced colitis.

[0243] E162. The LNP composition or method for use of embodiment E160 or E161, wherein the length of the colon is increased by about 1.2 to 5 times (e.g., about 2 to 4 times, 2 to 5 times, 2 to 3 times, 3 to 5 times, or 4 to 5 times).

[0244] E163. The LNP composition or method for use of embodiment E160 or E161, wherein said change in colon length or body weight is compared with an otherwise similar subject, for example, a subject not contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0245] E164. The LNP composition or method for use of any one of embodiments E95-E133, wherein the disease involving abnormal T cell function is diabetes, eg, type 1 diabetes (T1D).

[0246] E165. The LNP composition for use or method of embodiment E164, wherein said method or said composition results in a reduction of blood glucose levels in a sample, e.g., a sample from a subject.

[0247] E166. The LNP composition or method for use of embodiment E164 or E165, wherein said reduction in blood glucose is at least 1.2-10 fold (e.g., about 2-25 fold, 5-20 fold, 10-15 fold, 5-30 fold, 10-30 fold, 20-30 fold, 2-20 fold, 2-15 fold, or 2-10 fold).

[0248] E167. The LNP composition or method for use of embodiment E164 or E165, wherein said reduction in blood glucose is compared to an otherwise similar subject, for example, a subject not contacted with said LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

[0249] E168. The LNP composition or method for use of any one of embodiments E95-E167, wherein the polynucleotide comprising mRNA encoding the ITB6 molecule comprises at least one chemical modification.

[0250] E169. The LNP composition or method for use according to E168, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0251] E170. The LNP composition or method for use according to E168, wherein said chemical modification is selected from the group consisting of N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0252] E171. The LNP composition or method for use according to E170, wherein said chemical modification is N1-methylpseudouridine.

[0253] E172. The LNP composition or method for use of any one of the preceding embodiments, wherein the mRNA in the lipid nanoparticle comprises fully modified N1-methylpseudouridine.

[0254] E173. The LNP composition or method for use of any one of the preceding embodiments, wherein the LNP composition comprises (i) an ionizable lipid, e.g., an amino lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

[0255] E174. The LNP composition or method for use of embodiment E173, wherein the ionizable lipid comprises an amino lipid.

[0256] E175. The LNP composition or method for use of embodiment E173 or E174, wherein the ionizable lipid comprises a compound of any of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0257] E176. The LNP composition or method for use of any one of embodiments E173-E175, wherein said ionizable lipid comprises a compound of formula (I).

[0258] E177. The LNP composition or method for use of any one of embodiments E173-E176, wherein the ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0259] E178. The LNP composition or method for use of any one of embodiments E173-E177, wherein the LNP comprises a molar ratio of about 20-60% ionizable lipid: 5-25% phospholipid: 25-55% cholesterol; and 0.5-15% PEG lipid.

[0260] E179. The LNP composition or method for use of embodiment E178, wherein the LNP comprises a molar ratio of about 50% ionizable lipid: about 10% phospholipid: about 38.5% cholesterol; and about 1.5% PEG lipid.

[0261] E180. The LNP composition or method for use of embodiment E178 or E179, wherein the LNP comprises a molar ratio of about 49.83% ionizable lipid: about 9.83% phospholipid: about 30.33% cholesterol; and about 2.0% PEG lipid.

[0262] E181. The LNP composition or method for use of any one of embodiments E173-E180, wherein the ionizable lipid comprises a compound of any of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0263] E182. The LNP composition or method for use of embodiment E181, wherein the ionizable lipid comprises a compound of formula (I).

[0264] E183. The LNP composition or method for use of embodiment E181 or E182, wherein the ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0265] E184. The LNP composition or method for use of any one of embodiments E95-E183, wherein the LNP composition or method is formulated for intravenous, subcutaneous, intramuscular, intranasal, intraocular, rectal, or oral delivery.

[0266] E185. The LNP composition or method for use of any one of embodiments E95-E184, further comprising a pharmaceutically acceptable carrier or excipient.

[0267] E186. A kit comprising a container containing a lipid nanoparticle (LNP) composition described in any one of embodiments E1-E93, or a pharmaceutical composition described in embodiment E91, and a package insert containing instructions for administering the lipid nanoparticle or the pharmaceutical composition to treat or delay a disease associated with abnormal T cell function in an individual.

[0268] E187. The kit of embodiment E186, wherein the lipid nanoparticle composition comprises a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0269] [Figure 1]Figures A-C illustrate ITB6 expression in a mouse bone marrow-derived dendritic cell line (JAWS II). The construct was tagged with the V5 epitope for easy detection. Figure A shows ITB6 expression in mock-transfected cells. Figure B shows ITB6 expression in cells transfected with a mouse ITB6 mRNA construct. Figure C shows ITB6 expression in cells transfected with a human ITB6 mRNA construct. [Figure 2A] Representative flow cytometry graphs illustrating the frequency of FOXP3-GFP-expressing cells in CD4+ T cells cocultured with a murine bone marrow-derived dendritic cell line (JAWSII) transfected with LNPs formulated with control mRNA (NTFIX, dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA, with or without latent TGFβ, are provided. [Figure 2B] Graphs are provided illustrating the frequency of GFP+ cells in live CD4+ cells co-cultured with mouse bone marrow-derived dendritic cells (JAWSII) transfected with LNPs formulated with control mRNA (NTFIX, dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA, with or without latent TGFβ. [Figure 3A] 1 illustrates the frequency of OTII (CD45.2+CD4+) cells in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) without OVA treatment, with OVA treatment alone, or with LNP formulated with OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. 1 is a graph showing the frequency of OTII (CD45.2+CD4+) cells in the spleen. [Figure 3B]1 illustrates the absolute cell counts of OTII (CD45.2+CD4+) cells in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) without OVA treatment, with OVA treatment alone, or with LNP formulated with OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. 1 is a graph showing the absolute cell counts of OTII (CD45.2+CD4+) cells in the spleens. [Figure 3C] 1 illustrates the frequency of OTII regulatory Tregs (CD25+Fox3+CD45.2+CD4+) in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) treated with no OVA treatment, OVA treatment alone, or OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA formulated with LNPs. This figure shows the frequency of OTII Tregs (CD25+Fox3+CD45.2+CD4+) in the spleens. [Figure 3D] 1 shows the absolute number of OTII regulatory Treg (CD25+Fox3+CD45.2+CD4+) cells in the spleen of mice (adoptively transferred with CFSE-labeled OTII cells) treated with no OVA treatment, OVA treatment alone, or OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA-coated LNPs. 1 shows the absolute number of Treg (CD25+Fox3+CD45.2+CD4+) cells in the spleen. [Figure 3E-1] CFSE dilution in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) without OVA treatment, treated with OVA alone, or treated with LNP formulated with OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA is shown. Representative histograms showing CFSE dilution as a measure of OTII cell proliferation in the spleen are provided. [Figure 3E-2]CFSE dilution in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) without OVA treatment, treated with OVA alone, or treated with LNP formulated with OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA is shown. Representative histograms showing CFSE dilution as a measure of OTII cell proliferation in the spleen are provided. [Figure 3F] 1 illustrates the frequency of CFSE-low cells in the spleens of mice (adoptively transferred with CFSE-labeled OTII cells) treated without OVA treatment, with OVA alone, or with LNP formulated with OVA plus control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. This graph shows the frequency of CFSE-low cells (cells that have undergone proliferation and diluted CFSE) in the spleen. [Figure 4A] Illustrates the frequency of OTII (CD45.2+CD4+) cells in the spleen of mice (adoptively transferred with CFSE-labeled RagKO-OTII cells) treated with no OVA treatment, OVA treatment alone, or OVA plus control mRNA (dOXL40) or mouse ITB6 mRNA formulated LNP. Provides a graph showing the total frequency of OTII (CD45.2+CD4+) cells in the spleen. [Figure 4B] Figure 1 shows the frequency of Treg (CD25+FoxP3+) in the spleen of mice (adoptively transferred with CFSE-labeled RagKO-OTII cells) treated with no OVA treatment, OVA treatment only, or OVA plus LNP formulated with control mRNA (dOXL40) or mouse ITB6 mRNA. Graphs are provided showing the frequency of OTII Treg (CD25+Fox3+CD45.2+CD4+) in the spleen. [Figure 4C]CFSE dilution in the spleens of mice (adoptively transferred with CFSE-labeled RagKO-OTII cells) treated with LNPs formulated with no OVA treatment, OVA alone, or OVA and control mRNA (dOXL40) or mouse ITB6 mRNA is shown. Representative histograms showing CFSE dilution as a measure of OTII cell proliferation in the spleen are provided. [Figure 4D] 1 illustrates the frequency of CFSE-low cells in the spleens of mice (adoptively transferred with CFSE-labeled RagKO-OTII cells) treated with LNPs formulated with no OVA treatment, OVA alone, or OVA and control mRNA (dOXL40) or mouse ITB6 mRNA. A graph showing the frequency of CFSE-low cells (cells that have undergone proliferation and diluted CFSE) in the spleens is provided. [Figure 5A] 1 illustrates the response of OTI CD8+ T cells in OTI (CD45.2+) cells upon restimulation with peptide. Figure 1 is a representative graph showing the frequency of IFNγ+ OTI CD8+ T cells after ex vivo restimulation with peptide (SIINFEKL) from mice treated with OVA alone or with LNP formulated with mouse ITB6 mRNA in addition to OVA. [Figure 5B] 1 illustrates the frequency of IFNγ+ cells in OTI (CD45.2+) cells upon restimulation with peptide. Graph showing the frequency of IFNγ+ cells in OTI (CD45.2+) cells from mice treated with no OVA treatment, OVA only treatment, or rapamycin, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 6A] 1 illustrates the weight change in GvHD mice after treatment. Graphs from one study show the weight change in naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40) or mouse ITB6 mRNA. [Figure 6B]1 illustrates the weight change in GvHD mice after treatment. Graphs from another study show the weight change in naive mice or mice treated with LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 6C] 1 depicts the frequency of B cells, CD4+ T cells, and CD8+ T cells in GvHD mice on day 8 of treatment. 2 depicts graphs showing the total frequency of B cells, CD4+ T cells, and CD8+ T cells in the blood of naive mice or mice receiving donor cells and treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 6D] 1 depicts the frequency of B cells, CD4+ T cells, and CD8+ T cells in GvHD mice on day 8 of treatment. 2 depicts graphs showing the frequency of donor (H2-Kb+) B cells, CD4+ T cells, and CD8+ T cells in the blood of naive mice or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 7A] 1 depicts the frequencies of B cells, CD4+ T cells, and CD8+ T cells in GvHD mice on day 13 of treatment. 2 depicts graphs showing the total frequencies of B cells, CD4+ T cells, and CD8+ T cells in the blood of naive mice or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 7B] 1 depicts the frequency of B cells, CD4+ T cells, and CD8+ T cells in GvHD mice on day 13 of treatment. 2 depicts graphs showing the frequency of donor (H2-Kb+) B cells, CD4+ cells, and CD8+ cells in the blood of naive mice or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 7C]1 illustrates the frequency of Tregs in GvHD mice on day 8 of treatment. Graphs show the frequency of Tregs (CD25+Foxp3+) in the CD4+ T cell population of naive mice or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA on day 8. [Figure 7D] 1 illustrates the frequency of Tregs in GvHD mice on day 13 of treatment.

[0023] Figure 1 is a graph showing the frequency of Tregs (CD25+Foxp3+) in the CD4+ T cell population in naive mice or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA on day 13. [Figure 8A] 1 depicts the frequency of B cells, CD4+ cells, and CD8+ cells in the spleens of GvHD mice on day 15 of treatment. 2 depicts graphs showing the frequency of B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 8B] 1 depicts the absolute cell counts of B cells, CD4+ cells, and CD8+ cells in the spleens of GvHD mice on day 15 of treatment. 2 depicts graphs showing the absolute cell counts of B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 8C] 1 illustrates the frequency of B cells, CD4+ cells, and CD8+ cells in the spleens of GvHD mice on day 15 of treatment. 2 is a graph showing the frequency of donor (H2-Kb+) B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 8D]1 illustrates the frequency of Tregs in the spleens of GvHD mice on day 15 of treatment. 2 is a graph showing the frequency of Tregs (CD25+Foxp3+) in the spleens of naive mice, or mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 8E] Figure 1 illustrates the frequency of Tregs in the spleens of GvHD mice on day 15 of treatment.Figure 2 is a graph showing the absolute number of Treg (CD25+Foxp3+) cells in the spleens of naive mice, or mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 9A] Figure 1 illustrates the proliferation of donor and host cells in the spleens of GvHD mice on day 15 of treatment, as measured by Ki67 staining. Figure 2 shows the overall frequency of Ki67 B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 9B] 1 is a representative graph showing the relative proliferation of donor and host cells in mice treated with PBS or LNPs formulated with mouse ITB6 mRNA, as measured by Ki67 staining. [Figure 9C] Graph showing the frequency of Ki67 donor and host B cells in naive mice, or in mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 9D]10 is a graph showing the frequency of Ki67 donor and host CD4+ T cells in naive mice, or in mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 9E] 1 is a graph showing the frequency of Ki67 donor and host CD8+ T cells in naive mice, or in mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 10] Figures A-B show the frequency of Tbet cells in the spleens of GvHD mice. A is a graph showing the frequency of Tbet cells in the CD8+ T cell population in naive mice, or in mice treated with PBS or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. B is a graph showing the frequency of Tbet cells in the Tconv cell (CD25-Foxp3-CD4+) population in naive mice, or in mice treated with PBS or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 11] Illustrates serum levels of CXCL1, IFNγ, TNFα, MCP1, M1P1α, IL10, IL13, IL5, IL9, and M1P1β on day 15 in naive mice, or in GvHD mice treated with PBS, or LNP formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 12]Figures A-D show the frequency and proliferation of B cells, CD4+ cells, and CD8+ cells, as well as the frequency of Tregs, in the spleens of GvHD mice on day 22 of treatment. (A) Graphs showing the frequency of B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, mice treated with PBS, or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. (B) Graphs showing the frequency of donor (H2-Kb+) B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, mice treated with PBS, or mice treated with LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. (C) Graph showing the frequency of Treg (CD25+Foxp3+) in the CD4+ cell population in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. (D) Graph showing the frequency of Ki67+ B cells, CD4+ cells, and CD8+ cells in the spleens of naive mice, or mice treated with PBS, or LNPs formulated with control mRNA (dOXL40), mouse ITB6 mRNA, or human ITB6 mRNA. [Figure 13A] 1 is a table illustrating the experimental groups for the NOD-scid-gamma (NSG) mouse model of GvHD experiments. [Figure 13B] 1 is a graph showing survival curves after treatment, with the time of LNP treatment indicated by the vertical arrow at the top of the graph. [Figure 13C] 1 is a graph showing changes in body weight over time. [Figure 14A] 1 is a graph showing OTII% in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14B] 1 is a graph showing the % Tregs in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14C] 1 is a graph showing proliferation in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14D] 1 is a graph showing proliferation in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14E] 1 is a graph showing proliferation in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14F-1] 1 is a series of graphs showing the proliferation of OTII (CD45.2+) and % of CFSE-low populations in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14F-2] 1 is a series of graphs showing the proliferation of OTII (CD45.2+) and % of CFSE-low populations in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 14F-3] 1 is a series of graphs showing the proliferation of OTII (CD45.2+) and % of CFSE-low populations in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 15] 1 is a pair of graphs showing KLH-specific IgG in mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 16] 1 is a pair of graphs showing KLH-specific IgM in mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 17] 1 is a pair of graphs showing KLH-specific IgM in mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 18] 1 is a pair of graphs showing KLH-specific IgG in mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 19] 1 is a pair of graphs showing KLH-specific IgG in mice after treatment with LNPs formulated with human ITB6 mRNA. [Figure 20]1 is a schematic diagram and a pair of graphs showing the experimental design, % B cells in the spleens of mice after treatment with LNPs formulated with human ITB6 mRNA, and % H2-Kb+ donor cells in the spleens. [Figure 21] 1 is a schematic diagram and table showing the experimental design. [Figure 22] 1 is a heatmap showing genes altered after exposure to LNPs formulated with ITB6 mRNA compared to LNPs formulated with dOX40L. [Figure 23] 1 is a summary table showing the fold change and peak expression of several biomarker genes following treatment with LNPs formulated with ITB6 mRNA in mouse, rat, NHP, and human PBMCs. [Figure 24] A and B are a pair of graphs showing the mean clinical score (A) and mean days to disease onset (B) in a mouse model of EAE after treatment with vehicle, LNP formulated with dmOX40L control, or LNP formulated with ITB6 mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0270] Without wishing to be bound by theory, in some embodiments, it is believed that administration of LNPs comprising a polynucleotide comprising mRNA encoding an ITB6 molecule can result in T cell suppression, e.g., reduced T cell expansion, reduced T cell proliferation, T cell anergy, and / or T cell apoptosis, e.g., by the induction and / or proliferation of Treg cells. Exemplary inhibitory effects of the LNP compositions disclosed herein on T cells in vitro and in vivo are provided in at least Examples 3-5, and effects on the induction and / or proliferation of Treg cells are provided in at least Example 12. Exemplary in vivo protective effects of LNPs comprising an ITB6 molecule are provided in at least Examples 6-11 (in a GvHD model) and Example 16 (in an EAE model).

[0271] Thus, the present disclosure provides, inter alia, LNP compositions comprising an ITB6-encoding nucleic acid (e.g., mRNA) therapeutic. Also disclosed herein are methods of using such LNP compositions to inhibit an immune response in a subject or to treat or prevent a disease associated with abnormal immune cell function.

[0272] definition Obtaining: As used herein, "obtaining" refers to obtaining a physical entity (e.g., a sample, polypeptide, or nucleic acid) or value (e.g., a numerical value) by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change in a physical substance (e.g., a starting material). Exemplary changes include separating or purifying a substance, combining two or more separate entities into a mixture, or performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Obtaining a value directly includes performing a process involving a physical change in a sample or another substance, e.g., performing an analytical process involving a physical change in a substance, e.g., a sample, an analyte, or a reagent; performing an analytical method, e.g., a method comprising one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or a fragment or other derivative thereof with another substance, e.g., a buffer, solvent, or reactant; or altering the structure of an analyte, or a fragment or other derivative thereof, e.g., by severing or forming a covalent or non-covalent bond between a first and second atom of the analyte; or altering the structure of a reagent, or a fragment or other derivative thereof, e.g., by severing or forming a covalent or non-covalent bond between a first and second atom of the reagent.

[0273] Administering: As used herein, "administering" refers to a method of delivering a composition to a subject or patient. The administration method can be selected to target (e.g., specifically deliver) delivery to a particular region or system of the body. For example, administration can be parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable injection technique), oral, transdermal or intradermal, interdermal, rectal, intravaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosal, nasal, buccal, enteral, intravitreal, intratumoral, sublingual, intranasal; intratracheal instillation, bronchial instillation, and / or by inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or via a portal vein catheter. Preferred administration means are intravenous or subcutaneous.

[0274] Approximately, about: As used herein, the term "approximately" or "about," as applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, unless otherwise specified or otherwise clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of an LNP, "about" can mean + / - 5% of the recited value. For example, an LNP containing a lipid component having about 40% of a given compound may contain 30-50% of that compound.

[0275] Biomarker: As used herein, the term "biomarker" refers to an indicator, e.g., a predictive and / or prognostic indicator, that can be detected in a sample (e.g., a gene) or can be derived from one or more indicators detected in a sample. A biomarker can serve as an indicator of the effectiveness of a therapy (e.g., a therapy comprising ITB6 mRNA) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, a biomarker is a gene. In other embodiments, a biomarker is a collection of genes. Biomarkers include, but are not limited to, polynucleotide (e.g., DNA and / or RNA), changes in polynucleotide copy number (e.g., DNA copy number), polypeptide, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate, and / or glycolipid-based molecular markers.

[0276] Such biomarkers include, but are not limited to, PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI.

[0277] PMEPA1: As used herein, unless otherwise indicated, "PMEPA1" refers to any native prostate transmembrane protein, androgen-induced (Prostate Transmembrane Protein, Androgen Induced) 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed PMEPA1 as well as any form of PMEPA1 resulting from processing in cells. The term also encompasses naturally occurring variants of PMEPA1, such as splice variants or allelic variants. PMEPA1 is also referred to in the art as STAG1, TMEPAI, solid tumor-associated 1 protein, and transmembrane prostate androgen-induced protein. The nucleic acid sequence of an exemplary human PMEPA1 is set forth under NCBI Reference Sequence: NG_031951.1. The amino acid sequence of an exemplary protein encoded by human PMEPA1 is set forth under UniProt Accession No. Q969W9-1.

[0278] ITGAE: As used herein, "ITGAE" and "CD103" refer to any native integrin subunit alpha E from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed ITGAE as well as any form of ITGAE resulting from processing in cells. The term also encompasses naturally occurring variants of ITGAE, such as splice variants or allelic variants. ITGAE is also referred to in the art as HUMINAE, mucosal lymphocyte 1 antigen, integrin alpha-IEL, HML-1 antigen, and CD103 antigen. The nucleic acid sequence of an exemplary human ITGAE is set forth under NCBI Reference Sequence: NC_000017.11. The amino acid sequence of an exemplary protein encoded by human ITGAE is set forth under UniProt Accession No. P38570.

[0279] SMAD7: As used herein, "SMAD7," unless otherwise indicated, refers to any naturally occurring SMAD family member 7 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed SMAD7 as well as any form of SMAD7 resulting from processing in cells. The term also encompasses naturally occurring variants of SMAD7, such as splice variants or allelic variants. SMAD7 is also referred to in the art as SMAD family member 7, MADH7, MADH8, Mothers Against Decapentaplegic Homolog 7, MAD homolog 7, MAD homolog 8, HSMAD7, and CRCS3. An exemplary nucleic acid sequence of human SMAD7 is set forth under NCBI Reference Sequence: NM_005904.4. The amino acid sequence of an exemplary protein encoded by human SMAD7 is shown under UniProt accession number O15105.

[0280] SKIL: As used herein, unless otherwise indicated, "SKIL" refers to any naturally occurring SKI-like proto-oncogene from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed SKIL as well as any form of SKIL resulting from processing in a cell. The term also encompasses naturally occurring variants of SKIL, such as splice variants or allelic variants. SKIL is also referred to in the art as SKI-like proto-oncogene, SNO, SnoN, SnoA, Ski-like protein, Ski-related oncogene, SnoN, and SnoI. The nucleic acid sequence of an exemplary human SKIL is set forth under NCBI Reference Sequence: NM_005414.5. The amino acid sequence of an exemplary protein encoded by human SKIL is set forth under UniProt Accession Number P12757.

[0281] SKI: As used herein, unless otherwise indicated, "SKI" refers to any naturally occurring SKI proto-oncogene from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed SKI as well as any form of SKI resulting from processing in cells. The term also encompasses naturally occurring variants of SKI, such as splice variants or allelic variants. SKI is also referred to in the art as V-Ski avian sarcoma viral oncogene homolog, Sloan-Kettering Institute proto-oncogene, proto-oncogene C-Ski, Ski oncogene, SGS, and SKV. The nucleic acid sequence of an exemplary human SKI is set forth under NCBI Reference Sequence: NM_003036.4. The amino acid sequence of an exemplary protein encoded by human SKI is set forth under UniProt Accession Number P12755.

[0282] Conjugated: As used herein, the term "conjugated," when used in reference to two or more moieties, means that the moieties are physically associated or joined to one another, either directly or through one or more additional moieties that serve as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. In some embodiments, two or more moieties may be conjugated by a direct covalent chemical bond. In other embodiments, two or more moieties may be conjugated by ionic or hydrogen bonding.

[0283] Contacting: As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell with an mRNA or lipid nanoparticle composition means that the cell and the mRNA or lipid nanoparticle share a physical connection. Methods for contacting a cell with an external entity both in vivo, in vitro, and ex vivo are well known in the field of biology. In exemplary embodiments of the present disclosure, the step of contacting a mammalian cell with a composition (e.g., a nanoparticle of the present disclosure or a pharmaceutical composition) is performed in vivo. For example, contacting a lipid nanoparticle composition with a cell (e.g., a mammalian cell) that may be located within an organism (e.g., a mammal) may be performed by any suitable administration route (e.g., parenteral administration to the organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For cells present in vitro, the composition (e.g., lipid nanoparticle) and the cell may be contacted, for example, by adding the composition to the cell's culture medium, which may involve or result in transfection. Furthermore, more than one cell may be contacted with the nanoparticle composition.

[0284] Delivering: As used herein, the term "delivering" means providing an entity to a target location. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering LNPs containing the therapeutic and / or prophylactic agent to a subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Administering LNPs to a mammal or mammalian cells can involve contacting one or more cells with lipid nanoparticles.

[0285] Detecting: The term "detecting" is used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of a target molecule in a sample, as well as determining whether the target molecule is present in a sample at detectable levels. Detecting can be direct or indirect.

[0286] Encapsulate: As used herein, the term "encapsulate" means to enclose, surround, or surround. In some embodiments, a compound, polynucleotide (e.g., mRNA), or other composition may be fully encapsulated, partially encapsulated, or substantially encapsulated. For example, in some embodiments, the mRNA of the present disclosure may be encapsulated in a lipid nanoparticle, e.g., a liposome.

[0287] Encapsulation efficiency: As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the LNP relative to the original total amount of therapeutic and / or prophylactic agent used in preparing the LNP. For example, if 97 mg of therapeutic and / or prophylactic agent are encapsulated in the LNP out of a total of 100 mg of therapeutic and / or prophylactic agent originally provided in the composition, the encapsulation efficiency may be determined as 97%. As used herein, "encapsulation" may refer to complete, substantial, or partial entrapment, entrapment, surrounding, or envelopment.

[0288] Effective amount: As used herein, the term "effective amount" of a drug refers to an amount sufficient to achieve a beneficial or desired result, e.g., a clinical result; therefore, "effective amount" depends on the context in which it is applied. For example, in the context of the amount of target cell delivery-enhancing lipid in a lipid composition (e.g., LNP) of the present disclosure, an effective amount of the target cell delivery-enhancing lipid is an amount sufficient to achieve a beneficial or desired result compared to a lipid composition (e.g., LNP) lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results achieved by a lipid composition (e.g., LNP) include an increase in the percentage of transfected cells and / or an increase in the expression level of a protein encoded by a nucleic acid associated with / encapsulated by the lipid composition (e.g., LNP). In the context of administering lipid nanoparticles containing target cell delivery-enhancing lipids to a subject such that an effective amount of the lipid nanoparticles is taken up by target cells, an effective amount of the LNPs containing target cell delivery-enhancing lipid is an amount sufficient to achieve a beneficial or desired result compared to an LNP lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results in a subject include an increased percentage of transfected cells, an increased expression level of a protein encoded by a nucleic acid associated with / encapsulated by an LNP containing a target cell delivery-enhancing lipid, and / or an increased in vivo prophylactic or therapeutic effect of a nucleic acid or its encoded protein associated with / encapsulated by an LNP containing a target cell delivery-enhancing lipid, compared to an LNP lacking the target cell delivery-enhancing lipid. In some embodiments, a therapeutically effective amount of LNPs containing a target cell delivery-enhancing lipid is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition. In another embodiment, an effective amount of lipid nanoparticles is sufficient to result in expression of a desired protein in at least about 5%, 10%, 15%, 20%, 25%, or more of the target cells.For example, an effective amount of an LNP containing a target cell delivery-enhancing lipid can be an amount that results in transfection of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of target cells after a single intravenous injection.

[0289] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0290] Expression level: The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. The expression levels of one or more genes of interest may be determined by counting methods known to those skilled in the art and disclosed herein, including, for example, calculating the median or mean expression levels of all of the genes of interest. Prior to counting, the expression level of each gene of interest may be normalized using statistical methods known to those skilled in the art and disclosed herein, including, for example, normalizing to the expression level of one or more housekeeping genes, or normalizing to the total library size, or normalizing to the median or mean expression level across all measured genes. In some cases, prior to counting across multiple genes of interest, the normalized expression level of each gene of interest may be standardized using statistical methods known to those skilled in the art and disclosed herein, including, for example, calculating a Z-score of the normalized expression level of each gene of interest.

[0291] Reference expression level: As used herein, the terms "reference expression level" and "reference level" are used interchangeably to refer to an expression level against which another expression level, e.g., the expression level of one or more genes described herein (e.g., any gene or combination of genes selected from PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI) in a sample from an individual is compared, e.g., to make a diagnostic (e.g., predictive and / or prognostic) and / or therapeutic decision. For example, the reference expression level may be derived from an expression level in a reference population (e.g., the median expression level in a reference population, e.g., a population of patients with an autoimmune or inflammatory disease who have not been treated with ITB6 mRNA therapy), an expression level in a reference sample, and / or a pre-assigned value (e.g., a cut-off value previously determined to significantly (e.g., statistically significantly) separate a first subset of individuals who have experienced disease progression from a second subset of individuals who have not experienced disease progression), where the reference expression level significantly separates the first and second subsets of individuals based on a significant difference between the expression level in the first subset of individuals compared to that in the second subset of individuals. In some embodiments, the cut-off value may be the median or average expression level in the reference population. In other embodiments, the reference level may be the top 40%, top 30%, top 20%, top 10%, top 5%, or top 1% of expression levels in the reference population. In certain embodiments, the cut-off value may be the median expression level in the reference population. Those skilled in the art will understand that the numerical value of the reference expression level may vary depending on the indication or disorder, the technique used to detect the expression level (e.g., RNA-seq, microarray analysis, or RT-qPCR), and / or the particular combination of genes tested (e.g., any combination of genes selected from PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI).

[0292] Ex vivo: As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event may occur in an environment that is minimally altered from the native (e.g., in vivo) environment.

[0293] Fragment: As used herein, "fragment" refers to a portion. For example, a fragment of a protein may include a polypeptide obtained by digesting a full-length protein isolated from cultured cells or obtained by recombinant DNA techniques. A fragment of a protein may be, for example, a portion of a protein that includes one or more functional domains such that the fragment retains the functional activity of the protein.

[0294] GC-rich: As used herein, the term "GC-rich" refers to the nucleobase composition of a polynucleotide (e.g., mRNA), or any portion thereof (e.g., RNA element), that comprises guanine (G) and / or cytosine (C) nucleobases, or derivatives or analogs thereof, and that has a GC content of greater than about 50%. The term "GC-rich" refers to all or a portion of a polynucleotide, including but not limited to a gene, non-coding region, 5' UTR, 3' UTR, open reading frame, RNA element, sequence motif, or any individual sequence, fragment, or segment thereof, that comprises a GC content of about 50%. In some embodiments of the present disclosure, the GC-rich polynucleotide, or any portion thereof, is composed exclusively of guanine (G) and / or cytosine (C) nucleobases.

[0295] GC content: As used herein, the term "GC content" refers to the percentage of nucleobases in a polynucleotide (e.g., mRNA), or portion thereof (e.g., RNA element), that are either guanine (G) and cytosine (C) nucleobases, or derivatives or analogs thereof (out of the total number of possible nucleobases, which includes adenine (A) and thymine (T) or uracil (U), and derivatives or analogs thereof, in DNA and RNA). The term "GC content" refers to all or a portion of a polynucleotide, including, but not limited to, a gene, a non-coding region, a 5' UTR or 3' UTR, an open reading frame, an RNA element, a sequence motif, or any individual sequence, fragment, or segment thereof.

[0296] ITB6 molecule: As used herein, the term "ITB6 molecule" refers to a full-length native ITB6 (e.g., a mammalian ITB6, e.g., human ITB6, e.g., related to UniProt: P18564, NCBI Gene ID: 3694), a fragment (e.g., a functional fragment) of ITB6, or a variant of ITB6 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to native wild-type ITB6 or a fragment (e.g., a functional fragment) thereof. In some embodiments, the ITB6 molecule is an ITGB6 gene product, e.g., an ITB6 polypeptide. In some embodiments, the variant, e.g., an active variant, is a derivative, e.g., a mutant, of the wild-type polypeptide. In some embodiments, an ITB6 variant, e.g., an active ITB6 variant, has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of a wild-type ITB6 polypeptide. In some embodiments, an ITB6 molecule comprises a portion of ITB6 (e.g., the extracellular portion of ITB6) and a heterologous sequence, e.g., a sequence other than that of native ITB6. In some embodiments, an ITB6 molecule comprises a soluble ITB6.

[0297] Heterologous: As used herein, "heterologous" indicates that a sequence (e.g., an amino acid sequence or a non-coding region of a polynucleotide or nucleic acid molecule encoding an amino acid sequence) is not normally present in nature in a given polypeptide or polynucleotide. For example, an amino acid sequence corresponding to a domain or motif of one protein may be heterologous to a second protein.

[0298] Isolated: As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances can have various levels of purity relative to the substances with which they are associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, an isolated agent is more than about 80%, more than about 85%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0299] Liposome: As used herein, "liposome" refers to a structure comprising a lipid-containing membrane enclosing an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes include single-layered liposomes (also known in the art as unilamellar liposomes) and multi-layered liposomes (also known in the art as multilamellar liposomes).

[0300] Modified: As used herein, "modified" or "modification" refers to an altered state of a polynucleotide (e.g., mRNA) or a change in its composition or structure. Polynucleotides can be modified in various ways, including chemically, structurally, and / or functionally. For example, a polynucleotide can be structurally modified by the incorporation of one or more RNA elements that contain sequence and / or RNA secondary structure(s) that provide one or more functions (e.g., translational control activity). Thus, polynucleotides of the present disclosure can be composed of one or more modifications (e.g., can include one or more chemical, structural, or functional modifications, including any combination thereof).

[0301] Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the present disclosure. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one embodiment, an mRNA molecule of the present disclosure is modified by the introduction of non-natural nucleosides and / or nucleotides (e.g., as they relate to natural ribonucleotides A, U, G, and C). Non-classical nucleotides, such as cap structures, although different in chemical structure from A, C, G, U ribonucleotides, are not considered "modified."

[0302] mRNA: As used herein, "mRNA" refers to messenger ribonucleic acid. mRNA may be natural or non-natural. For example, mRNA may contain modified and / or non-natural components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. mRNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. mRNA may have a nucleotide sequence encoding a polypeptide. Translation of mRNA, for example, in vivo translation of mRNA inside a mammalian cell, can produce a polypeptide. By convention, the basic components of an mRNA molecule include at least a coding region, a 5'-untranslated region (5'-UTR), a 3'UTR, a 5'-cap, and a polyA sequence.

[0303] Nanoparticle: As used herein, "nanoparticle" refers to a particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties compared to a bulk sample of the same material. Typically, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about 100 nm. Also typically, nanoparticles have any one structural feature on a scale of about 50 nm to about 500 nm, about 50 nm to about 200 nm, or about 70 nm to about 120 nm. In exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 1 to 1000 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 10 to 500 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 50 to 200 nm. Spherical nanoparticles may have diameters of, for example, about 50 to 100 or 70 to 120 nanometers. Nanoparticles most often behave as units in terms of their transport and properties. It should be noted that the novel properties that differentiate nanoparticles from corresponding bulk materials typically manifest themselves on size scales below 1000 nm, or at sizes around 100 nm, although nanoparticles can be of larger sizes, e.g., in the case of particles that are oval, tubular, etc. Although the size of most molecules would fit within the above outline, individual molecules are not typically referred to as nanoparticles.

[0304] Nucleic Acid: As used herein, the term "nucleic acid" is used in the broadest sense to encompass any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), or hybrids thereof.

[0305] Nucleic Acid Structure: As used herein, the term "nucleic acid structure" (used interchangeably with "polynucleotide structure") refers to the arrangement or organization of atoms, chemical components, elements, motifs, and / or sequences of linked nucleotides, or derivatives or analogs thereof, that comprise a nucleic acid (e.g., mRNA). The term also refers to the two-dimensional or three-dimensional state of a nucleic acid. Accordingly, the term "RNA structure" refers to the arrangement or organization of atoms, chemical components, elements, motifs, and / or sequences of linked nucleotides, or derivatives or analogs thereof, that comprise an RNA molecule (e.g., mRNA), and / or refers to the two-dimensional and / or three-dimensional state of an RNA molecule. Nucleic acid structures can be further divided into four organizational categories, referred to herein as "molecular structure," "primary structure," "secondary structure," and "tertiary structure," based on increasing organizational complexity.

[0306] Nucleobase: As used herein, the term "nucleobase" (alternatively, "nucleotide base" or "nitrogenous base") refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivative or analog of naturally occurring purines and pyrimidines, that confers improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases found predominantly in natural nucleic acids. Other natural, unnatural, and / or synthetic nucleobases, as known in the art and / or described herein, can be incorporated into nucleic acids.

[0307] Nucleoside / Nucleotide: As used herein, the term "nucleoside" refers to a compound containing a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA), or a derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as a "nucleobase"), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term "nucleotide" refers to a nucleoside covalently linked to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof, that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0308] Open reading frame: As used herein, the term "open reading frame" (abbreviated "ORF") refers to a segment or region of an mRNA molecule that encodes a polypeptide. An ORF contains a contiguous string of non-overlapping, in-frame codons beginning with an initiation codon and ending with a termination codon, and is translated by the ribosome.

[0309] Patient: As used herein, "patient" refers to a subject who may seek or need treatment, who is in need of treatment, who is undergoing treatment, who will be undergoing treatment, or who is under the care of a trained professional for a particular disease or condition. In certain embodiments, the patient is a human patient. In some embodiments, the patient is, for example, a patient suffering from an autoimmune disease as described herein.

[0310] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0311] Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in a patient. Excipients may include anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0312] Pharmaceutically acceptable salts: As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound has been modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Included are sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or in a mixture of the two (generally, non-aqueous solvents such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred).Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418; Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0313] Polypeptide: As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically joined by peptide bonds, which may be naturally (e.g., isolated or purified) or synthetically produced.

[0314] Responsiveness: "Responsiveness" or "effective response" may be assessed using any endpoint that indicates benefit to an individual, including, but not limited to, (i) some inhibition of disease progression, including slowing and complete prevention; (ii) a reduction in the number of disease episodes and / or symptoms; (iii) a reduction in lesion size; (iv) an inhibition (i.e., a reduction, slowing, or complete halt) of disease cell infiltration into adjacent peripheral organs and / or tissues; (v) an inhibition (i.e., a reduction, slowing, or complete halt) of disease spread; (vi) a reduction in the autoimmune response (which may, but need not, result in regression or disappearance of disease lesions); (vii) some alleviation of one or more symptoms associated with the disorder; (viii) an increase in the disease-free presentation period after treatment; and / or (ix) a reduction in mortality at a given time point after treatment.

[0315] RNA: As used herein, "RNA" refers to a ribonucleic acid, which can be natural or non-natural. For example, RNA may contain modified and / or non-natural components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, e.g., in vivo translation of the mRNA inside a mammalian cell, can produce the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (lncRNA), and mixtures thereof.

[0316] RNA element: As used herein, the term "RNA element" refers to a portion, fragment, or segment of an RNA molecule that provides a biological function and / or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by incorporation of one or more RNA elements, such as those described herein, provides the modified polynucleotide with one or more desirable functional properties. RNA elements as described herein can be naturally occurring, non-naturally occurring, synthetic, engineered, or any combination thereof. For example, naturally occurring RNA elements that provide regulatory activity include elements found throughout the transcriptomes of viruses, prokaryotes, and eukaryotes (e.g., humans). RNA elements in certain eukaryotic mRNAs and translated viral RNAs have been shown to be involved in mediating many functions within cells.Exemplary natural RNA elements include translation initiation elements (e.g., internal ribosome entry sites (IRES), see Kieft et al., (2001) RNA7(2):194-206), translation enhancer elements (e.g., APP mRNA translation enhancer elements, see Rogers et al., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev Mol Cell Biol 8(2):113-126), translation repression elements (see, e.g., Blumer et al., (2002) Mech Dev 110(1-2):97-112), protein-binding RNA elements (e.g., iron-responsive elements, see Selezneva et al., (2013) J Mol Biol 425(18):3301-3310), cytoplasmic polyadenylation elements (Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and catalytic RNA elements (e.g., ribozymes, see Scott et al. (2009) Biochim Biophys Acta 1789(9-10):634-641).

[0317] Specific delivery: As used herein, the terms "specific delivery," "specifically deliver," or "specifically delivering" refer to greater (e.g., at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) delivery of a therapeutic and / or prophylactic agent by a nanoparticle to intended target cells (e.g., mammalian target cells) compared to off-target cells (e.g., non-target cells). The level of nanoparticle delivery to a particular cell can be measured by comparing the amount of protein produced in target cells versus non-target cells (e.g., by mean fluorescence intensity using flow cytometry), comparing the percentage of target cells versus non-target cells expressing the protein (e.g., by quantitative flow cytometry), comparing the amount of protein produced in target cells versus non-target cells to the total amount of protein in the target cells versus non-target cells, or comparing the amount of therapeutic and / or prophylactic agent in target cells versus non-target cells to the total amount of therapeutic and / or prophylactic agent in the target cells versus non-target cells. It will be understood that the ability of nanoparticles to specifically deliver to target cells need not be determined in the subject being treated, but may be determined in a surrogate such as an animal model (e.g., a mouse or NHP model).

[0318] Substantially: As used herein, the term "substantially" refers to the qualitative condition indicating the total extent or degree or nearly the total extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach and / or proceed to perfection or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of perfection that is inherently present in many biological and chemical phenomena.

[0319] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0320] Targeting moiety: As used herein, a "targeting moiety" is a compound or agent that can target a nanoparticle to a particular cell, tissue, and / or organ type.

[0321] Therapeutic Agent: The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0322] Transfection: As used herein, the term "transfection" refers to a method of introducing a species (e.g., a polynucleotide such as mRNA) into a cell.

[0323] Subject: As used herein, the term "subject" refers to any living organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, a subject may be a patient.

[0324] Treating: As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, palliating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0325] Preventing: As used herein, the term "preventing" refers to partially or completely inhibiting the onset of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. In some embodiments, a treatment intended to prevent a disease may be given prophylactically (e.g., administered before the onset of one or more symptoms).

[0326] Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule before it has been changed in any way. Unmodified may, but does not always, refer to the wild-type or native form of a biomolecule. A molecule may undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

[0327] Variant: As used herein, the term "variant" refers to a molecule that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the wild-type molecule, e.g., as measured by art-recognized assays.

[0328] ITB6 molecule ITB6 (also known as integrin beta 6) is a subunit of the heterodimeric integrin αvβ6, a transmembrane glycoprotein. In certain aspects, the present disclosure provides LNP compositions comprising, e.g., a polynucleotide (e.g., mRNA) encoding an ITB6 molecule, e.g., as described herein. In certain embodiments, the ITB6 molecule comprises a native ITB6 molecule, a fragment of a native ITB6 molecule, or a variant thereof. In certain embodiments, the ITB6 molecule comprises a variant of a native ITB6 molecule (e.g., an ITB6 variant, e.g., as described herein) or a fragment thereof. In certain embodiments, an LNP composition comprising a polynucleotide (e.g., mRNA) encoding an ITB6 molecule can be administered alone or in combination with an additional agent, e.g., an LNP composition comprising a polynucleotide (e.g., mRNA) encoding a different ITB6 variant or fragment thereof, or an LNP composition comprising a polynucleotide (e.g., mRNA) encoding a different molecule.

[0329] In certain aspects, the LNP compositions disclosed herein comprise a polynucleotide (e.g., mRNA) encoding an ITB6 molecule. In certain embodiments, the ITB6 molecule comprises a native ITB6 molecule, a fragment of a native ITB6 molecule, or a variant thereof. In certain embodiments, the ITB6 molecule comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the ITB6 amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A, or a functional fragment thereof. In certain embodiments, the ITB6 molecule comprises the amino acid sequence of the ITB6 amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A, or a functional fragment thereof. In certain embodiments, the ITB6 molecule comprises the amino acid sequence of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15, or a functional fragment thereof.

[0330] In some embodiments, the ITB6 molecule lacks a leader sequence and / or affinity tag (e.g., a leader sequence and / or affinity tag described in Table 1A or Table 2A) but otherwise comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from, the amino acid sequence of an ITB6 of any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15 provided in Table 1A or Table 2A, or a functional fragment thereof.

[0331] In one embodiment, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 nucleotides from, the nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175 provided in Table 1A or Table 2A, or a functional fragment thereof. In one embodiment, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 18. In one embodiment, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 14. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 16.

[0332] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule lacks a nucleotide sequence encoding a leader sequence and / or affinity tag (e.g., a leader sequence and / or affinity tag described in Table 1A or Table 2A), but otherwise comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to, or differing by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 nucleotides from, a nucleic acid sequence identical to the nucleotide sequence of any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, 16, or 160-175 provided in Table 1A or Table 2A, or a functional fragment thereof.

[0333] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 18 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 18. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 175, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 18, and the 3' UTR sequence of SEQ ID NO: 142.

[0334] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., m 7 GpppG 2’OMe pA, (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 18; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 142, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0335] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 2 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 160, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 2, and the 3' UTR sequence of SEQ ID NO: 110.

[0336] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 56; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 2; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0337] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or differs from the sequence of SEQ ID NO: 3 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 161, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110.

[0338] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 3; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0339] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 4 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 162, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 4, and the 3' UTR sequence of SEQ ID NO: 143.

[0340] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 4; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 143, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0341] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 5 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 163, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 110.

[0342] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 5; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0343] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 164, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144.

[0344] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 5; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 144, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0345] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 169, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145.

[0346] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap II-b; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 5; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 145, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0347] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 6 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 165, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0348] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 6; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0349] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 166, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0350] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap II-b; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 6; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0351] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 167, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 145.

[0352] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap II-b; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 6; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 145, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0353] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 168, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 143.

[0354] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap II-b; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 6; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 143, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0355] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 8 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 170, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 8, and the 3' UTR sequence of SEQ ID NO: 110.

[0356] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 8; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0357] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 10 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 171, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110.

[0358] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 10; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0359] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 12 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 172, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110.

[0360] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 12; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0361] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or differs from, the sequence of SEQ ID NO: 14 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 14. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 173, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110.

[0362] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap II-b; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 50; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 14; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0363] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or differs from the sequence of SEQ ID NO: 16 by no more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 174, which comprises, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110.

[0364] In some embodiments, the polynucleotide encoding the ITB6 molecule comprises, from the 5' to the 3' end: (i) a 5' cap as provided herein, e.g., cap C1; (ii) a 5'UTR of a sequence provided herein, such as SEQ ID NO: 56; (iii) an open reading frame encoding an ITB6 polypeptide, for example, a sequence-optimized nucleic acid sequence encoding ITB6 set forth as SEQ ID NO: 16; (iv) at least one stop codon (if not present at the 5' end of the 3'UTR); (v) a 3'UTR of a sequence provided herein, such as SEQ ID NO: 110, and (vi) comprises a poly-A tail provided herein (e.g., SEQ ID NO: 502).

[0365] In some embodiments, the polynucleotide (e.g., mRNA) encoding the ITB6 molecule further comprises one or more elements, such as a 5'UTR and / or a 3'UTR. In some embodiments, the 5'UTR and / or 3'UTR comprises one or more microRNA (mIR) binding sites, e.g., as disclosed herein. Exemplary 5'UTRs and 3'UTRs are disclosed herein in the section entitled "5'UTRs and 3'UTRs."

[0366] In some embodiments, a polynucleotide encoding an ITB6 molecule comprises the nucleotide sequence of any of human variant 5.1, human variant 1.1, human variant 1.2, human variant 1.3, human variant 1.4, human variant 1.5, human variant 1.6, human variant 1.7, human variant 1.8, human variant 1.9, human variant 1.10, human variant 2.1, human variant 3.1, human variant 4.1, rat variant 1.1, or mouse variant 1.1, as set forth in Table 2A. In some embodiments, a polynucleotide encoding an ITB6 molecule comprises a chemical modification(s) set forth in Table 2A for any of human variant 5.1, human variant 1.1, human variant 1.2, human variant 1.3, human variant 1.4, human variant 1.5, human variant 1.6, human variant 1.7, human variant 1.8, human variant 1.9, human variant 1.10, human variant 2.1, human variant 3.1, human variant 4.1, rat variant 1.1, or mouse variant 1.1. In some embodiments, a polynucleotide encoding an ITB6 molecule does not comprise a chemical modification(s) shown in Table 2A for any of human variant 5.1, human variant 1.1, human variant 1.2, human variant 1.3, human variant 1.4, human variant 1.5, human variant 1.6, human variant 1.7, human variant 1.8, human variant 1.9, human variant 1.10, human variant 2.1, human variant 3.1, human variant 4.1, rat variant 1.1, or mouse variant 1.1.

[0367] In certain aspects, the LNP compositions disclosed herein comprise a polynucleotide (e.g., mRNA) encoding an ITB6 molecule, e.g., as described herein. In certain embodiments, the ITB6 molecule comprises a half-life extender, e.g., a protein (or fragment thereof) that binds to a serum protein such as albumin, IgG, FcRn, or transferrin. In certain embodiments, the half-life extender is an immunoglobulin Fc region or a variant thereof, e.g., IgG1 Fc.

[0368] In some embodiments, the LNP compositions described herein comprise a polynucleotide (e.g., mRNA) encoding an ITB6 molecule. In some embodiments, the ITB6 molecule further comprises a targeting moiety. In some embodiments, the targeting moiety comprises an antibody molecule (e.g., a Fab or scFv), a receptor molecule (e.g., a receptor, a receptor fragment, or a functional variant thereof), a ligand molecule (e.g., a ligand, a ligand fragment, or a functional variant thereof), or a combination thereof.

[0369] [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4] [Table 1A-5] [Table 1A-6] [Table 1A-7] [Table 1A-8] [Table 1A-9] [Table 1A-10] [Table 1A-11]

[0370] In some embodiments, a polynucleotide of the present disclosure, e.g., a polynucleotide comprising an mRNA nucleotide sequence encoding an ITB6 polypeptide, comprises (1) a 5' cap, e.g., as disclosed herein, e.g., as provided in Table 2A, (2) a 5' UTR, e.g., as provided in Table 2A, (3) a nucleotide sequence ORF provided in Table 2A, (4) a stop codon, (5) a 3' UTR, e.g., as provided in Table 2A, and (6) a tail (e.g., a poly-A tail), e.g., as disclosed herein, e.g., a poly-A tail of about 100 residues (e.g., SEQ ID NO: 502).

[0371] In some embodiments, the polynucleotide comprises an mRNA nucleotide sequence encoding an ITB6 polypeptide.

[0372] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 175, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 18, and the 3' UTR sequence of SEQ ID NO: 142.

[0373] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 160, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 2, and the 3' UTR sequence of SEQ ID NO: 110.

[0374] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 161, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 3, and the 3' UTR sequence of SEQ ID NO: 110.

[0375] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 162, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 4, and the 3' UTR sequence of SEQ ID NO: 143.

[0376] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 163, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 110.

[0377] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 164, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 144.

[0378] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 169, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 5, and the 3' UTR sequence of SEQ ID NO: 145.

[0379] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 165, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0380] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 166, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 110.

[0381] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 167, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 145.

[0382] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 168, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 6, and the 3' UTR sequence of SEQ ID NO: 143.

[0383] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 170, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 8, and the 3' UTR sequence of SEQ ID NO: 110.

[0384] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 171, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 10, and the 3' UTR sequence of SEQ ID NO: 110.

[0385] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 172, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 12, and the 3' UTR sequence of SEQ ID NO: 110.

[0386] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 173, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 50, the ORF sequence of SEQ ID NO: 14, and the 3' UTR sequence of SEQ ID NO: 110.

[0387] In some embodiments, a polynucleotide (e.g., mRNA) encoding an ITB6 molecule comprises the nucleotide sequence of SEQ ID NO: 174, which includes, from the 5' to 3' end, the 5' UTR sequence of SEQ ID NO: 56, the ORF sequence of SEQ ID NO: 16, and the 3' UTR sequence of SEQ ID NO: 110.

[0388] In some embodiments, all of the 5'UTR, ORF, and / or 3'UTR sequences comprise a modification(s) set forth in Table 2A. In some embodiments, one, two, or all of the 5'UTR, ORF, and / or 3'UTR sequences do not comprise a modification(s) set forth in Table 2A. In some embodiments, a 5'UTR set forth in Table 2A additionally comprises a first nucleotide that is "A" or "G." [Table 2A-1] [Table 2A-2] [Table 2A-3] [Table 2A-4] [Table 2A-5] [Table 2A-6] [Table 2A-7] [Table 2A-8]

Table 2A-9

Table 2A-10

Table 2A-11

Table 2A-12

Table 2A-13

Table 2A-14

Table 2A-15

Table 2A-16

Table 2A-17

Table 2A-18

Table 2A-19

Table 2A-20

Table 2A-21

Table 2A-22

Table 2A-23

Table 2A-24

Table 2A-25

Table 2A-26

Table 2A-27

Table 2A-28

Table 2A-29

Table 2A-30

Table 2A-31

Table 2A-32

Table 2A-33

Table 2A-34

Table 2A-35

Table 2A-36

Table 2A-37

Table 2A-38

Table 2A-39

Table 2A-40

Table 2A-41

Table 2A-42

Table 2A-43

Table 2A-44

Table 2A-45

Table 2A-46

Table 2A-47

Table 2A-48

Table 2A-49

Table 2A-50

[0389] Lipid content of LNP As noted above, with respect to lipids, the LNPs disclosed herein comprise (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG-lipid, along with a nucleic acid cargo of interest. The lipid nanoparticles of the present invention can be produced using components, compositions, and methods generally known in the art. For example, see PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2016 / 014280, PCT / US2017 / 038426, See PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491, all of which are incorporated by reference in their entireties.

[0390] The nucleic acids of the present disclosure (e.g., ITB6 mRNA) are typically formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0391] In some embodiments, the lipid nanoparticles comprise 20-60% molar ratio of ionizable cationic lipid. For example, the lipid nanoparticles may comprise 40-50 mol%, optionally 45-50 mol%, e.g., 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, e.g., about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol% molar ratio of ionizable cationic lipid.

[0392] In some embodiments, the lipid nanoparticles comprise 5-25% molar non-cationic lipid. For example, the lipid nanoparticles may comprise 5-15% molar, optionally 10-12% molar, e.g., 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-11%, 11-12%, 12-13%, 13-14%, or 14-15% molar non-cationic lipid.

[0393] In some embodiments, the lipid nanoparticles comprise a molar ratio of sterol of 25-55%. For example, the lipid nanoparticles may comprise a molar ratio of sterol of 30-45 mol%, optionally 35-40 mol%, e.g., 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol%.

[0394] In some embodiments, the lipid nanoparticles comprise 0.5-15% molar ratio of PEG-modified lipids. For example, the lipid nanoparticles may comprise 1-5%, optionally 1-3 mol%, e.g., 1.5-2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol% molar ratio of PEG-modified lipids.

[0395] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0396] In some embodiments, the lipid nanoparticles comprise a molar ratio of 40-50% ionizable cationic lipid, 5-15% non-cationic lipid, 30-45% sterol, and 1-5% PEG-modified lipid.

[0397] In some embodiments, the lipid nanoparticles comprise a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1-3% PEG-modified lipid.

[0398] In some embodiments, the lipid nanoparticles comprise a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1.5-2.5% PEG-modified lipid.

[0399] Ionizable amino lipids The lipid nanoparticles of the present disclosure comprise one or more ionizable lipids. In certain embodiments, the ionizable lipids of the present disclosure comprise a central amine moiety and at least one biodegradable group. The ionizable lipids described herein can be advantageously used in the lipid nanoparticles of the present disclosure to deliver nucleic acid molecules to mammalian cells or organs. The structures of the ionizable lipids described below include the prefix I to distinguish them from other lipids of the present invention.

[0400] In some embodiments, the present disclosure provides a compound of formula (I): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 where: R' 分岐状 but, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl, 1 is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0401] In some embodiments of the compounds of Formula (I), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aα , R aβ , R aγ , and R aδ are H and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0402] In some embodiments of the compounds of Formula (I), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aα , R aβ , R aγ , and R aδ are H and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 3, and m is 7.

[0403] In some embodiments of the compounds of Formula (I), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aα is C 2-12 alkyl, and R aβ , R aγ , and R aδ are H and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), n2 is 2, and R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0404] In some embodiments of the compounds of Formula (I), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aα , R aβ , and R aδ are H and R aγ is C 2-12 alkyl, and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0405] In some embodiments, the compound of formula (I) is [ka] is selected from.

[0406] In some embodiments, the compound of formula (I) is [ka] is.

[0407] In some embodiments, the compound of formula (I) is [ka] is.

[0408] In some embodiments, the compound of formula (I) is [ka] is.

[0409] In some embodiments, the compound of formula (I) is [ka] is.

[0410] In some embodiments, the present disclosure provides a compound of formula (Ia): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 where: R'分岐状 but, [ka] where: [ka] represents the attachment point, R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl, 1 is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0411] In some embodiments, the present disclosure provides a compound of formula (Ib): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 where: R' 分岐状 but, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; Each R 5 independently, C 1-3 Alkyl, C 2-3selected from the group consisting of alkenyl, and H; Each R 6 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl, 1 is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0412] In some embodiments of formula (I) or (Ib), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0413] In some embodiments of formula (I) or (Ib), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 3, and m is 7.

[0414] In some embodiments of formula (I) or (Ib), R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aβ and R aδ are H and R aγ is C 2-12 alkyl, and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 is -(CH2) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0415] In some embodiments, the present disclosure provides a compound of formula (Ic): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 where: R' 分岐状 but, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 but, [ka] and where: [ka] represents the attachment point, R 10 is N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl, 1 is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0416] In some embodiments, R' a is R' 分岐状 and R' 分岐状 teeth, [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R aα is C 2-12 alkyl, and R 2 and R 3 are respectively, C 1-14 alkyl, and R 4 teeth, [ka] and [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), n2 is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0417] In some embodiments, the compound of Formula (Ic) is [ka] is.

[0418] In some embodiments, the present disclosure provides a compound of formula (II): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' 環状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ and R aδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, wherein R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R bγ and R bδ are each independently H, C1-12 Alkyl, and C 2-12 alkenyl, wherein R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl, Y a But C 3-6 is a carbocyclic ring, R*” a But C 1-15 Alkyl and C 2-15 alkenyl, s is 2 or 3, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0419] In some embodiments, the present disclosure provides a compound of formula (II-a): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ and R aδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, wherein R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R bγ and R bδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, wherein R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0420] In some embodiments, the present disclosure provides a compound of formula (II-b): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ and R bγ are each independently, C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0421] In some embodiments, the present disclosure provides a compound of formula (II-c): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' aBut R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ But C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R' is C 1-12 Alkyl or C 2-12 is alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0422] In some embodiments, the present disclosure provides a compound of formula (II-d): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ and R bγ are each independently, C 1-12 Alkyl and C 2-12 alkenyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where, [ka] represents the point of attachment, R 10 is N(R)2, where each R is independently C 1-6 Alkyl, C 2-3alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0423] In some embodiments, the present disclosure provides a compound of formula (II-e): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ But C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently, C 1-14 Alkyl and C 2-14 alkenyl, R 4 But -(CH2) nOH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 Alkyl or C 2-12 is alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0424] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6. In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5.

[0425] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R′ is independently C 1-12 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R' is independently C 2-5 It is alkyl.

[0426] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' b teeth, [ka] and R 2 and R 3 are each independently, C 1-14 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. b teeth, [ka] and R2 and R 3 are each independently, C 6-10 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. b teeth, [ka] and R 2 and R 3 are each C8 alkyl.

[0427] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 1-12 alkyl, and R 2 and R 3 are each independently, C 6-10 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each independently, C 6-10In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0428] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are respectively, C 1-12 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are respectively, C 2-6 It is alkyl.

[0429] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5 and each R' is independently C 2-5 It is alkyl.

[0430] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C 2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 It is alkyl.

[0431] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 alkyl, and R aγ is C 1-12 alkyl, and R 2 and R 3 are each independently, C 6-10 In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0432] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 teeth, [ka] where R 10 is NH(C 1-6alkyl), and n2 is 2. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 teeth, [ka] where R 10 is NH(CH3) and n2 is 2.

[0433] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12 alkyl, and R 4 teeth, [ka] where R 10 is NH(C 1-6 alkyl) and n2 is 2. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 alkyl, and R 4 teeth, [ka] where R 10 is NH(CH3) and n2 is 2.

[0434] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 alkyl, and R 2 and R 3 are each independently, C 6-10 alkyl, and R aγ is C 1-12 alkyl, and R 4 teeth, [ka] where R 10 is NH(C 1-6 alkyl) and n2 is 2. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl, and R 4 teeth, [ka] where R 10 is NH(CH3) and n2 is 2.

[0435] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 is -(CH2) n OH and n is 2, 3, or 4. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 is -(CH2) n OH and n is 2.

[0436] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12 alkyl, and R 4 is -(CH2) nOH and n is 2, 3, or 4. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C 2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 alkyl, and R 4 is -(CH2) n OH and n is 2.

[0437] In some embodiments, the present disclosure provides a compound of formula (II-f): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a But R' 分岐状 or R' 環状 where: R' 分岐状 but, [ka] and R' b but, [ka] and where: [ka] represents the attachment point, R aγ But C 1-12 is alkyl, R2 and R 3 are each independently, C 1-14 is alkyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 is alkyl, m is selected from 4, 5, and 6; 1 is selected from 4, 5, and 6.

[0438] In some embodiments of compounds of Formula (II-f), m and l are each 5; and n is 2, 3, or 4.

[0439] In some embodiments of compounds of Formula (II-f), R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are respectively, C 6-10 It is alkyl.

[0440] In some embodiments of compounds of Formula (II-f), m and l are each 5; n is 2, 3, or 4; and R′ is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are respectively, C 6-10 It is alkyl.

[0441] In some embodiments, the present disclosure provides a compound of formula (II-g): [ka] In the compound of formula R aγ But C 2-6 is alkyl, R' is C 2-5 is alkyl, R 4 But -(CH2)n OH, where n is selected from the group consisting of 3, 4, and 5, and [ka] (where, [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0442] In some embodiments, the present disclosure provides a compound of formula (II-h): [ka] In the compound of formula R aγ and R bγ are each independently, C 2-6 is alkyl, Each R' is independently C 2-5 is alkyl, R 4 But -(CH2) n OH, where n is selected from the group consisting of 3, 4, and 5, and [ka] (where, [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0443] In some embodiments of compounds of Formula (II-g) or (II-h), R 4 teeth, [ka] where: R 10 is NH(CH3) and n2 is 2.

[0444] In some embodiments of compounds of Formula (II-g) or (II-h), R 4 is -(CH2)2OH.

[0445] In some embodiments, the present disclosure provides a compound of formula (III): [ka] or a salt or isomer thereof, wherein R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X 1 , X 2 , and X 3 are independently a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, selected from the group consisting of —C(O)O—CH—, —OC(O)—CH—, —CH—C(O)O—, —CH—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independent, C 3-6 is a carbocyclic ring, Each R* is independent, C 1-12 Alkyl and C 2-12 alkenyl, Each R is independent, C 1-3 Alkyl and C 3-6is selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 selected from the group consisting of alkenyl, and H; Each R” is independent, C 3-12 Alkyl and C 3-12 alkenyl, wherein i)X 1 , X 2 , and X 3 is not -CH2-, and / or ii) At least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0446] In some embodiments, R1, R2, R3, R4, and R5 are each C 5-20 is alkyl, and X 1 is -CH2- and X 2 and X 3 are each -C(O)-.

[0447] In some embodiments, the compound of formula (III) is [ka] is.

[0448] The central amine moiety of a lipid according to any of the formulas herein, for example, a compound having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), can be protonated at physiological pH. Thus, the lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino) lipids. The lipid may also be zwitterionic, i.e., a neutral molecule having both positive and negative charges.

[0449] In some embodiments, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), ranges from about 1 mol% to 99 mol% in the lipid composition.

[0450] In one embodiment, the amount of ionizable amino lipids of the present invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), in the lipid composition is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 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, 96, 97, 98, 99, 100, 101, 3, 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, 96, 97, 98, or 99 mole percent.

[0451] In one embodiment, the amount of ionizable amino lipids of the present invention, e.g., a compound having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), in the lipid composition ranges from about 30 mol% to about 70 mol%, from about 35 mol% to about 65 mol%, from about 40 mol% to about 60 mol%, and from about 45 mol% to about 55 mol%.

[0452] In one specific embodiment, the amount of ionizable amino lipids of the present invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 45 mol % in the lipid composition.

[0453] In one specific embodiment, the amount of ionizable amino lipids of the present invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 40 mol % in the lipid composition.

[0454] In one specific embodiment, the amount of ionizable amino lipids of the present invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 50 mol % in the lipid composition.

[0455] In addition to the ionizable amino lipids disclosed herein, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), the lipid-based compositions (e.g., lipid nanoparticles) disclosed herein can include additional components such as cholesterol and / or cholesterol analogs, non-cationic helper lipids, structural lipids, PEG-lipids, and any combination thereof.

[0456] Additional ionizable lipids of the present invention include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin -DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA) , (13Z,165Z)-N,N-dimethyl-3-nonyldocosa-13-16-dien-1-amine (L608), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N, The ionizable amino lipid may be selected from the non-limiting group consisting of N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition, the ionizable amino lipid may also be a lipid containing a cyclic amine group.

[0457] The ionizable lipids of the present invention can also be compounds disclosed in International Publication No. WO2017 / 075531A1, which is incorporated herein by reference in its entirety.

[0458] The ionizable lipids of the present invention can also be compounds disclosed in International Publication No. WO2015 / 199952A1, which is incorporated herein by reference in its entirety.

[0459] In any of the foregoing or related embodiments, the ionizable lipids of the LNPs of the present disclosure include, for example, compounds contained within any compound having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity).

[0460] In any of the foregoing or related embodiments, the ionizable lipid of the LNP of the present disclosure comprises a compound including any of Compound Nos. 18, 25, 301, and 357.

[0461] In any of the foregoing or related aspects, the ionizable lipid of the LNPs of the present disclosure comprises at least one compound selected from the group consisting of Compound Nos. 18, 25, 301, and 357. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises a compound selected from the group consisting of Compound Nos. 18, 25, 301, and 357. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises Compound 18. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises Compound 25.

[0462] In any of the foregoing or related aspects, synthesis of compounds of the invention, including any of Compound Nos. 18, 25, 301, and 357, follows the synthesis instructions in U.S. Provisional Patent Application No. 62 / 733,315, filed September 19, 2018.

[0463] Representative synthesis routes: Compound I-182: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate 3-Methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione [ka] To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in 100 mL of diethyl ether was added a 2 M solution of methylamine in THF (3.8 mL, 7.6 mmol), resulting in the formation of a precipitate. The mixture was stirred at room temperature for 24 hours and then filtered to collect the solid. The solid was washed with diethyl ether, air-dried, and then dissolved in hot EtOAc and filtered. The filtrate was allowed to cool to room temperature and then cooled to 0° C. to give a precipitate, which was isolated by filtration, washed with cold EtOAc, air-dried, and then dried under vacuum to give 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.70 g, 5 mmol, 73%) as a solid. 1 H NMR (300 MHz, DMSO-d6) δ: ppm 8.50 (br. d, 1H, J = 69 Hz);4.27 (s, 3H);3.02 (sdd, 3H, J = 42 Hz, 4.5 Hz).

[0464] Heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate [ka] To a solution of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.28 mmol) in 10 mL of ethanol was added 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (39 mg, 0.28 mmol). The reaction mixture was stirred at room temperature for 20 hours and then concentrated in vacuo to give a residue. The residue was purified by silica gel chromatography (0-100% in dichloromethane (mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (138 mg, 0.17 mmol, 60%) as a solid. UPLC / ELSD: RT = 3 min. MS (ES): C 51 H 95 m / z (MH + ) 833.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 7.86 (br. s., 1H);4.86 (quintet, 1H, J = 6 Hz);4.05 (t, 2H, J = 6 Hz);3.92 (d, 2H, J = 3 Hz);3.20 (s, 6H);2.63 (br. s, 2H);2.42 (br. s, 3H);2.28 (m, 4H);1.74 (br. s, 2H);1.61 (m, 8H);1.50 (m, 5H);1.41 (m, 3H);1.25 (br. m, 47H);0.88 (t, 9H, J = 7.5 Hz).

[0465] Compound I-301: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate [ka] Compound I-301 was prepared similarly to compound 182, except that heptadecan-9-yl 8-((3-aminopropyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (500 mg, 0.66 mmol) was used instead of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate. After aqueous workup, the residue was purified by silica gel chromatography (0-50% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (180 mg, 32%) as a solid. HPLC / UV (254 nm): RT = 6.77 min. MS (CI): C 52 H 97 m / z (MH + ) 860.7. 1 H NMR (300 MHz, CDCl3): δ ppm 4.86-4.79 (m, 2H);3.66 (bs, 2H);3.25 (d, 3H, J = 4.9 Hz);2.56-2.52 (m, 2H);2.42-2.37 (m, 4H);2.28 (dd, 4H, J = 2.7 Hz, 7.4 Hz);1.78-1.68 (m, 3H);1.64-1.50 (m, 16H);1.48-1.38 (m, 6H);1.32-1.18 (m, 43H);0.88-0.84 (m, 12H).

[0466] Cholesterol / Structural Lipids The LNPs described herein comprise one or more structured lipids.

[0467] As used herein, the term "structured lipid" refers to sterol and also to lipids containing a sterol moiety. The incorporation of structured lipids into lipid nanoparticles can help reduce aggregation of other lipids in the particles. Structured lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, basicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

[0468] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol. Examples of structured lipids include, but are not limited to, the following: [ka]

[0469] The target cell delivery LNPs described herein comprise one or more structural lipids.

[0470] As used herein, the term "structured lipid" refers to a sterol and also to a lipid containing a sterol moiety. The incorporation of a structured lipid into a lipid nanoparticle can help reduce aggregation of other lipids in the particle. In certain embodiments, the structured lipid comprises cholesterol. In other embodiments, the structured lipid can comprise cholesterol and another molecule, as well as a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

[0471] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids that consists of steroid alcohols. Structured lipids can include, but are not limited to, sterols (e.g., plant sterols or animal sterols).

[0472] In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog.

[0473] Non-cationic helper lipids / phospholipids In some embodiments, the lipid-based compositions (e.g., LNPs) described herein comprise one or more non-cationic helper lipids. In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a phospholipid substitute or replacement.

[0474] As used herein, the term "non-cationic helper lipid" refers to a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In one embodiment, the helper lipid is not phosphatidylcholine (PC). In one embodiment, the non-cationic helper lipid is a phospholipid or phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0475] The phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0476] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0477] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0478] In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0479] In some embodiments, the non-cationic helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-i77-glycero-3-phosphocholine (DSPC) substitute.

[0480] phospholipids phospholipids The lipid composition of the lipid nanoparticle compositions disclosed herein can include one or more phospholipids, such as one or more saturated or (poly)unsaturated phospholipids, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0481] The phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0482] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0483] Certain phospholipids can facilitate fusion with membrane.For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membrane (for example, cell membrane or intracellular membrane).By fusion of phospholipid with membrane, one or more elements (for example, therapeutic agent) of lipid-containing composition (for example, LNP) may be able to pass through the membrane, for example, one or more elements can be delivered to target tissue.

[0484] Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with or crosslinked to one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cell recognition, or for conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0485] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0486] In some embodiments, the phospholipids of the present invention are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DL ...linoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinoleoyl- Toyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-Glycero-3-phosphocholine (C16LysoPC), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0487] In certain embodiments, the phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC. In certain embodiments, the phospholipids useful or potentially useful in the present invention are represented by formula (IV): [ka] or a salt thereof, wherein Each R 1 are independently optionally substituted alkyl, or optionally, two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a compound of the formula: [ka] It is of L 2 Each occurrence of independently represents a bond, or optionally a substituted C 1-6 alkylene, wherein optionally substituted C 1-6 The methylene units of alkylene are O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ), R 2 where each occurrence of is independently optionally replaced by C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, wherein optionally R 2one or more methylene units of are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or -N(R N )S(O)2O, R N is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; p is 1 or 2, However, the compound has the following formula: [ka] wherein R 2 Each occurrence of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl.

[0488] In some embodiments, the phospholipid may be one or more of the phospholipids described in US Application No. 62 / 520,530.

[0489] Modification of phospholipid head groups In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, the phospholipid with the modified head group is DSPC or an analog thereof with a modified quaternary amine. For example, in certain embodiments of formula (IV), R 1 At least one of R is not methyl. 1 In certain embodiments, the compound of formula (IV) has the following formula: [ka] or a salt thereof, wherein each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each v is independently 1, 2, or 3.

[0490] In certain embodiments, the compound of formula (IV) has the formula (IV-a): [ka] or a salt thereof.

[0491] In certain embodiments, the phospholipid that is useful or may be useful in the present invention comprises cyclic moiety instead of glyceride moiety.In certain embodiments, the phospholipid that is useful in the present invention is DSPC or its analogue that has cyclic moiety instead of glyceride moiety.In certain embodiments, the compound of formula (IV) is represented by formula (IV-b): [ka] or a salt thereof.

[0492] Modification of phospholipid tails In certain embodiments, the phospholipid that is or may be useful in the present invention comprises modified tail.In certain embodiments, the phospholipid that is or may be useful in the present invention is DSPC or its analog with modified tail.As described herein, "modified tail" can be a tail with a shorter or longer aliphatic chain, a branched aliphatic chain, a substituted aliphatic chain, an aliphatic chain with one or more methylenes replaced by cyclic or heteroatom groups, or any combination thereof.For example, in certain embodiments, the compound of (IV) is a compound of formula (IV-a) or its salt, wherein R 2 At least one occurrence of R 2 where each occurrence of is optionally replaced by C 1-30 alkyl, where R 2 one or more methylene units of are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NRN C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), -S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), -N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O.

[0493] In certain embodiments, the compound of formula (IV) has formula (IV-c): [ka] or a salt thereof, wherein each x is independently an integer between 0 and 30 (endpoints included); Each occurrence of G is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), -OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O. Each possibility represents a separate embodiment of the present invention.

[0494] In certain embodiments, the phospholipids useful or potentially useful in the present invention comprise a modified phosphocholine moiety, wherein the alkyl chain connecting the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2).Thus, in certain embodiments, the phospholipids useful or potentially useful in the present invention are compounds of formula (IV), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10.For example, in certain embodiments, the compounds of formula (IV) have the following formula: [ka] or a salt thereof.

[0495] alternative lipids In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phosphocholine moiety, in which the alkyl chain connecting the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids are useful.

[0496] In certain embodiments, alternative lipids are used in place of the phospholipids of the present disclosure.

[0497] In certain embodiments, the lipid substitute of the present invention is oleic acid.

[0498] In certain embodiments, the replacement lipid is one of the following: [ka]

[0499] PEG lipids The lipid composition of the pharmaceutical compositions disclosed herein may include one or more polyethylene glycol (PEG) lipids.

[0500] As used herein, the term "PEG lipid" refers to a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0501] In some embodiments, PEG lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0502] In one embodiment, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0503] In some embodiments, the lipid portion of the PEG-lipid is about C 14 ~About C 22 , preferably about C 14 ~About C 16 In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG lipid is 2k -DMG.

[0504] In one embodiment, the lipid nanoparticles described herein can include a PEG-lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[0505] PEG lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584A2, which are incorporated herein by reference in their entireties.

[0506] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein may be synthesized as described in International Patent Application No. PCT / US2016 / 000129, entitled "Compositions and Methods for Delivery of Therapeutic Agents," filed December 10, 2016, which is incorporated by reference in its entirety.

[0507] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0508] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. PEG-DMG has the following structure: [ka]

[0509] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety. Any of these exemplary PEG lipids described herein may be modified to include a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0510] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (V): [ka] or a salt thereof, wherein R 3 But, -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100 (endpoints included); L 1 is optionally replaced by C 1-10 alkylene, wherein optionally substituted C 1-10 At least one methylene of the alkylene is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN ), NR N C(O)O, or NR N C(O)N(R N ) and D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a compound of the formula: [ka] It is of L 2 Each occurrence of independently represents a bond, or optionally a substituted C 1-6 alkylene, wherein optionally substituted C 1-6 The methylene units of alkylene are O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ), R 2 where each occurrence of is independently optionally replaced by C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, wherein optionally R 2 one or more methylene units of are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), NR NC(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O, R N is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; p is 1 or 2.

[0511] In certain embodiments, the compound of formula (V) is a PEG-OH lipid (i.e., R 3 -OR O and R O is hydrogen). In certain embodiments, the compound of formula (V) has the formula (V-OH): [ka] or a salt thereof.

[0512] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VI). As used herein, the formula (VI): [ka] or a salt thereof, wherein R 3 But, -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100 (endpoints included); R 5 is optionally replaced by C 10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl, and optionally R 5 one or more methylene groups in N ), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), -NR NC(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), -S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), -N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O, R N Each occurrence of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0513] In certain embodiments, the compound of formula (VI) has the formula (VI-OH): [ka] or a salt thereof. In some embodiments, r is 45. In another of the above or related aspects, there is provided a PEG lipid of the invention, wherein r is 40-50.

[0514] In still other embodiments, the compound of formula (VI) is [ka] or a salt thereof.

[0515] In one embodiment, the compound of formula (VI) is [ka] is.

[0516] In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG lipid.

[0517] In some embodiments, the PEG lipid can be one or more of the PEG lipids described in US Application No. 62 / 520,530.

[0518] In some embodiments, the PEG-modified lipids of the present invention include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also known as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0519] In certain embodiments, the LNPs provided herein exhibit increased PEG detachment compared to existing LNP formulations containing PEG-lipids. As used herein, "PEG detachment" refers to the cleavage of PEG groups from PEG-lipids. Often, cleavage of PEG groups from PEG-lipids occurs via serum-driven esterase cleavage or hydrolysis. In certain embodiments, the PEG-lipids provided herein are designed to control the rate of PEG detachment. In certain embodiments, the LNPs provided herein exhibit greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 50% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 60% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 70% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs exhibit greater than 80% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs exhibit greater than 90% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 90% PEG detachment after about 6 hours in human serum.

[0520] In other embodiments, the LNPs provided herein exhibit less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 60% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 70% PEG detachment after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 80% PEG detachment after about 6 hours in human serum.

[0521] In addition to the PEG-lipids provided herein, LNPs may contain one or more additional lipid components. In certain embodiments, the PEG-lipids are present in the LNPs at a molar ratio of 0.15-15% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 0.15-5% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 1-5% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 0.15-2% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 1-2% relative to other lipids. In certain embodiments, the PEG-lipids are present at approximately 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of approximately 1.5% relative to other lipids.

[0522] In one embodiment, the amount of PEG lipid in the lipid composition of the pharmaceutical composition disclosed herein is from about 0.1 mol% to about 5 mol%, from about 0.5 mol% to about 5 mol%, from about 1 mol% to about 5 mol%, from about 1.5 mol% to about 5 mol%, from about 2 mol% to about 5 mol%, from about 0.1 mol% to about 4 mol%, from about 0.5 mol% to about 4 mol%, from about 1 mol% to about 4 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 4 mol%, from about The range is 0.1 mol % to about 3 mol %, about 0.5 mol % to about 3 mol %, about 1 mol % to about 3 mol %, about 1.5 mol % to about 3 mol %, about 2 mol % to about 3 mol %, about 0.1 mol % to about 2 mol %, about 0.5 mol % to about 2 mol %, about 1 mol % to about 2 mol %, about 1.5 mol % to about 2 mol %, about 0.1 mol % to about 1.5 mol %, about 0.5 mol % to about 1.5 mol %, or about 1 mol % to about 1.5 mol %.

[0523] In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 2 mol%. In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 1.5 mol%.

[0524] In one embodiment, the amount of PEG lipid in the lipid compositions disclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol%.

[0525] Exemplary Synthesis: Compound:HO-PEG 2000 -Ester-C18 [ka] Into a nitrogen-filled flask containing palladium on carbon (10 wt%, 74 mg, 0.070 mmol) was added benzyl-PEG 2000 -ester-C18 (822 mg, 0.35 mmol) and MeOH (20 mL) were added. The flask was evacuated and backfilled with H2 three times and allowed to stir at room temperature and 1 atm of H2 for 12 h. The mixture was filtered through Celite, rinsed with DCM, and the filtrate was concentrated in vacuo to give the desired product (692 mg, 88%). Using this procedure, n = 40-50. In one embodiment, n for the resulting polydispersed mixture is referred to as 45 on average.

[0526] For example, the value of r can be determined based on the molecular weight of the PEG moiety in the PEG-lipid. For example, a molecular weight of 2,000 (e.g., PEG2000) corresponds to an n value of approximately 45. Because polymers are often found as a distribution of different polymer chain lengths, for a given composition, the value of n can encompass a distribution of values ​​within a range accepted in the art. For example, those skilled in the art who understand the polydispersity of such polymer compositions will understand that an n value of 45 (e.g., in a structural formula) can represent a distribution of values ​​of 40 to 50 in an actual PEG-containing composition, such as a PEG-lipid composition of DMG-PEG200.

[0527] In some embodiments, the target cell delivery lipids of the pharmaceutical compositions disclosed herein do not comprise PEG-lipids.

[0528] In one embodiment, the target cell delivery LNP of the present disclosure comprises a PEG-lipid. In one embodiment, the PEG-lipid is not PEG-DMG. In some aspects, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some aspects, the PEG-lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE lipids. In other aspects, the PEG-lipid is PEG-DMG.

[0529] In one embodiment, the target cell delivery LNP of the present disclosure comprises a PEG lipid having a chain length greater than about 14 or greater than about 10 when branched.

[0530] As used herein, the terms "alkyl," "alkyl group," or "alkylene" mean a straight- or branched-chain saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms), which is optionally substituted. 14 The term "alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon containing from 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0531] As used herein, the terms "alkenyl," "alkenyl group," or "alkenylene" mean a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one double bond, which is optionally substituted. 14 The term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may contain one, two, three, four, or more carbon-carbon double bonds. For example, C 18 Alkenyl may contain one or more double bonds. C containing two double bonds 18 The alkenyl group may be a linoleyl group. Unless otherwise specified, alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

[0532] As used herein, the terms "alkynyl," "alkynyl group," or "alkynylene" mean a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. 2-14 The term "alkynyl" refers to an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may contain one, two, three, four, or more carbon-carbon triple bonds. For example, C 18 An alkynyl may contain one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0533] As used herein, the term "carbocycle" or "carbocyclic group" means an optionally substituted monocyclic or polycyclic ring system containing one or more rings of carbon atoms. The ring may be 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-membered. 3-6 The term "carbocycle" refers to a carbocycle containing a single ring having 3 to 6 carbon atoms. A carbocycle may contain one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., a cycloalkyl group or an aryl group). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2 dihydronaphthyl groups. As used herein, the term "cycloalkyl" refers to a non-aromatic carbocycle, which may or may not contain any double or triple bonds. Unless otherwise specified, carbocycles described herein refer to both unsubstituted and substituted carbocycle groups, i.e., carbocycles that are optionally substituted.

[0534] As used herein, the term "heterocycle" or "heterocyclic group" means an optionally substituted monocyclic or polycyclic ring system containing one or more rings, at least one of which contains at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The ring may be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring. The heterocycle may contain one or more double or triple bonds and may be non-aromatic or aromatic (e.g., a heterocycloalkyl group or a heteroaryl group). Examples of heterocyclic rings include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. As used herein, the term "heterocycloalkyl" refers to a non-aromatic heterocyclic ring, and may or may not contain any double or triple bonds. Unless otherwise specified, the heterocyclic rings described herein refer to both unsubstituted and substituted heterocyclic rings, i.e., optionally substituted heterocyclic rings.

[0535] As used herein, the terms "heteroalkyl," "heteroalkenyl," or "heteroalkynyl" refer to an alkyl, alkenyl, or alkynyl group, respectively, as defined herein, which further contains one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus), wherein one or more heteroatoms are inserted between adjacent carbon atoms within a parent carbon chain and / or one or more heteroatoms are inserted between a carbon atom and the parent molecule, i.e., between the points of attachment. Unless otherwise specified, the heteroalkyl, heteroalkenyl, or heteroalkynyl described herein refers to both unsubstituted and substituted heteroalkyl, heteroalkenyl, or heteroalkynyl, i.e., optionally substituted heteroalkyl, heteroalkenyl, or heteroalkynyl.

[0536] As used herein, a "biodegradable group" is a group that can promote more rapid metabolism of lipids in mammalian organisms. The biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group. As used herein, an "aryl group" is an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a "heteroaryl group" is an optionally substituted heterocyclic group containing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. For example, M and M' may be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M' may be independently selected from the list of biodegradable groups above. Unless otherwise specified, the aryl or heteroaryl groups described herein refer to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0537] Alkyl, alkenyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. Optional substituents include halogen atoms (e.g., chloride, bromide, fluoride, or iodide groups), carboxylic acids (e.g., C(O)OH), alcohols (e.g., hydroxyl, OH), esters (e.g., C(O)OR OC(O)R), aldehydes (e.g., C(O)H), carbonyls (e.g., C(O)R, alternatively represented by C=O), acyl halides (e.g., C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., OC(O)OR), alkoxy (e.g., OR), acetals (e.g., C(OR)R"", where each OR is an alkoxy group, which may be the same or different, and R"" is an alkyl or alkenyl group), phosphates (e.g., P(O) 3- ), thiol (e.g., SH), sulfoxide (e.g., S(O)R), sulfinic acid (e.g., S(O)OH), sulfonic acid (e.g., S(O)2OH), thial (e.g., C(S)H), sulfate (e.g., S(O)4 2-), sulfonyl (e.g., S(O)), amido (e.g., C(O)NR, or N(R)C(O)R), azido (e.g., N), nitro (e.g., NO), cyano (e.g., CN), isocyano (e.g., NC), acyloxy (e.g., OC(O)R), amino (e.g., NR, NRH, or NH), carbamoyl (e.g., OC(O)NR, OC(O)NRH, or OC(O)NH), sulfonamide (e.g., S(O)NR, S(O)NRH, S(O)NH, N(R)S(O)R, N(H)S(O)R, N(R)S(O)H, or N(H)S(O)H), alkyl group, alkenyl group, and cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted, for example, with 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6 Alkyl groups may be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0538] Nitrogen-containing compounds of the present disclosure can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperbenzoic acid (mCPBA) and / or hydrogen peroxide) to yield other compounds of the present disclosure. Thus, all nitrogen-containing compounds shown and claimed are considered to include both the compound as shown and its N-oxide derivative (which may be written as N→O or N+-O-), where valence and structure permit. Furthermore, in other cases, nitrogen in compounds of the present disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as mCPBA. All nitrogen-containing compounds shown and claimed are also considered to include both the compounds as shown and their N-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, where R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3- to 14-membered carbocyclic, or 3- to 14-membered heterocyclic) derivatives, where valence and structure permit.

[0539] Exemplary Additional LNP Components The lipid composition of the pharmaceutical composition disclosed herein may contain one or more components in addition to those described above. For example, the lipid composition may contain one or more permeation enhancer molecules, carbohydrates, polymers, surface modifiers (e.g., surfactants), or other components. For example, the permeation enhancer molecules may be molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0540] A polymer can be included and / or used to encapsulate or partially encapsulate the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions in lipid nanoparticle form). The polymer can be biodegradable and / or biocompatible. The polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0541] LNPs containing polynucleotides encoding ITB6 molecules Disclosed herein are LNP compositions comprising a polynucleotide encoding an ITB6 polypeptide for use in suppressing T cells, inter alia, to treat a disease associated with abnormal T cell function in a subject or to inhibit an immune response. In another embodiment, the invention relates to LNPs comprising a polynucleotide comprising mRNA encoding an ITB6 molecule. The LNP compositions of the present disclosure can be used to reprogram dendritic cells in vivo or ex vivo, suppress T cells, and / or induce immune tolerance.

[0542] In some embodiments, an LNP composition comprising a polynucleotide encoding ITB6 comprises (i) an ionizable lipid, e.g., an amino lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

[0543] In some embodiments, an LNP composition comprising a polynucleotide encoding ITB6 comprises (i) an ionizable lipid, e.g., an amino lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

[0544] In another embodiment, the LNP compositions of the present disclosure are used in a method of treating a disease associated with abnormal T cell function in a subject or a method of inhibiting an immune response in a subject, e.g., as described herein.

[0545] In certain embodiments, an LNP composition comprising a polynucleotide encoding an ITB6 molecule can be administered together with an additional agent, eg, as described herein.

[0546] Nanoparticle Composition In some embodiments, the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNPs).Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent, such as a compound as described herein, and (ii) a polynucleotide encoding a polypeptide of the present invention.In such nanoparticle compositions, the lipid composition disclosed herein can encapsulate the polynucleotide encoding the polypeptide of the present invention.

[0547] Nanoparticle compositions typically have a size of approximately micrometers or less and may contain a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions may be liposomes having a lipid bilayer with a diameter of 500 nm or less.

[0548] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, the nanoparticle compositions are vesicles comprising one or more lipid bilayers. In certain embodiments, the nanoparticle compositions comprise two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0549] In one embodiment, the lipid nanoparticles comprise an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNPs comprise an ionizable amino lipid, a PEG-modified lipid, a sterol, and a structural lipid. In some embodiments, the LNPs have a molar ratio of about 40-50% ionizable amino lipid, about 5-15% structural lipid, about 30-45% sterol, and about 1-5% PEG-modified lipid.

[0550] In some embodiments, the LNPs have a polydispersity value of less than 0.4. In some embodiments, the LNPs have a net neutral charge at neutral pH. In some embodiments, the LNPs have an average diameter of 50-150 nm. In some embodiments, the LNPs have an average diameter of 80-100 nm....

Claims

1. A lipid nanoparticle (LNP) composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule, the LNP composition comprising (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

2. 2. The LNP composition of claim 1, wherein the ITB6 molecule comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of ITB6 provided in Table 1A or Table 2A.

3. 3. The LNP composition of claim 2, wherein the ITB6 molecule comprises an amino acid sequence comprising any one of SEQ ID NOs: 17, 1, 7, 9, 11, 13, or 15.

4. 2. The LNP composition of claim 1, wherein the mRNA comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a nucleic acid sequence comprising any one of SEQ ID NOs: 18, 2-6, 8, 10, 12, 14, and 16.

5. The mRNA, (a) a 5'UTR sequence comprising any one of SEQ ID NOs: 50 or 56, and / or (b) a 3'UTR sequence comprising any one of SEQ ID NOs: 142, 110, 143, 144, or 145; 2. The LNP composition of claim 1, comprising:

6. An LNP composition comprising a polynucleotide including mRNA encoding an ITB6 molecule, wherein the mRNA comprises, from the 5' end to the 3' end, a nucleotide sequence comprising the sequence of SEQ ID NO: 175, which comprises a 5'UTR sequence comprising the sequence of SEQ ID NO: 50, an ORF sequence comprising the sequence of SEQ ID NO: 18, and a 3'UTR sequence comprising the sequence of SEQ ID NO: 142, and the LNP composition comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

7. An LNP composition comprising a polynucleotide comprising an mRNA encoding an ITB6 molecule, wherein the mRNA comprises, from the 5' end to the 3' end: (a) a 5' cap comprising m7GpppG2'Ome; (b) a 5'UTR sequence comprising the sequence of SEQ ID NO: 50; (c) an ORF sequence encoding an amino acid sequence comprising the sequence of SEQ ID NO: 17; (d) a 3'UTR sequence comprising the sequence of SEQ ID NO: 142; and (e) a poly A tail sequence comprising the sequence of SEQ ID NO:

502. wherein the LNP composition further comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

8. An LNP composition comprising a polynucleotide comprising an mRNA encoding an ITB6 molecule, wherein the mRNA comprises a nucleotide sequence comprising the sequence of SEQ ID NO: 175; The mRNA has, from the 5' end to the 3' end: (a) a 5' cap comprising m7GpppG2'Ome; (b) a 5'UTR sequence comprising the sequence of SEQ ID NO: 50; (c) an ORF sequence comprising the sequence of SEQ ID NO: 18; (d) a 3'UTR sequence comprising the sequence of SEQ ID NO: 142; and (e) a poly A tail sequence comprising the sequence of SEQ ID NO:

502. wherein the LNP composition further comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

9. An LNP composition described in any one of claims 1 to 8, which results in suppression of T cell activity and / or function in a population of immune cells compared to the activity and / or function of T cells in an otherwise similar cell population that has not been contacted with the LNP composition comprising a polynucleotide comprising mRNA encoding an ITB6 molecule.

10. The suppression of T cell activity and / or function is characterized by: (i) increased levels of Treg differentiation; (ii) increasing the frequency and absolute number of Treg cells; (iii) reduced proliferation, survival and / or expansion of T cells; (iv) a reduction in the expression, activity and / or secretion of effector cytokines; and / or (v) reducing the expression and / or activity of the T cell transcription factor T-bet; 10. The LNP composition of claim 9, comprising any one, two, three, or all of:

11. The inhibition of T cell activity and / or function is achieved by: (i) reduced proliferation, survival and / or expansion of antigen-specific CD4+ T cells; (ii) an increase in the frequency and absolute number of antigen-specific Treg cells; and / or (iii) reducing the expression, activity and / or secretion of IFNg; 10. The LNP composition of claim 9, comprising one or more of:

12. (i) reduced donor immune cell proliferation; (ii) weight loss, host B cell depletion, and / or reduced donor immune cell engraftment, optionally with a parallel expansion of Tregs; (iii) reduction of Tbet+ cells in the T cell population; (iv) reducing the expression, activity and / or secretion of pro-inflammatory cytokines; (v) maintaining or increasing Treg and / or host lymphocyte populations; and / or (vi) a reduction in clinical scores and / or a delay in disease onset The LNP composition of any one of claims 1 to 8, wherein (i) the donor immune cells are T cells; (ii) the reduced Tbet+ cells are in the CD8+ T cell population; and / or (iii) the inflammatory cytokine is selected from one or more of CXCL1, IFNγ, TNFα, MCP1, M1P1α, IL10, IL13, IL5, IL9, and M1P1β.

14. An LNP composition described in any one of claims 1 to 8, which improves or reduces graft-versus-host disease.

15. The LNP composition of any one of claims 1 to 8, wherein the polynucleotide comprising the mRNA encoding the ITB6 molecule comprises at least one chemical modification.

16. 16. The LNP composition of claim 15, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

17. The LNP composition of any one of claims 1 to 8, wherein the ionizable lipid comprises compound 18.

18. The LNP composition of any one of claims 1 to 8, wherein the ionizable lipid comprises compound 25.

19. A pharmaceutical composition for use in modulating an immune response in a subject in need thereof, comprising an effective amount of an LNP composition described in any one of claims 1 to 8.

20. Use of an effective amount of the LNP composition of any one of claims 1 to 8 in the manufacture of a medicament for modulating an immune response in a subject in need thereof.

21. A pharmaceutical composition for use in treating a disease associated with abnormal T cell function or preventing symptoms thereof, comprising an effective amount of an LNP composition described in any one of claims 1 to 8.

22. Use of an effective amount of the LNP composition described in any one of claims 1 to 8 in the manufacture of a medicament for the treatment of a disease associated with abnormal T cell function or the prevention of symptoms thereof.

23. 22. The pharmaceutical composition for use according to claim 21, wherein the disease is selected from rheumatoid arthritis (RA); graft-versus-host disease (GVHD); diabetes; inflammatory bowel disease (IBD); lupus, multiple sclerosis; autoimmune hepatitis; primary biliary cholangitis; organ transplant-associated rejection; or myasthenia gravis. (a) The GVHD is acute GVHD or chronic GVHD. (b) the diabetes is type 1 diabetes; (c) the lupus is systemic lupus erythematosus (SLE); or (d) the autoimmune hepatitis is type 1 or type 2 autoimmune hepatitis,

25. 20. The pharmaceutical composition for use according to claim 19, wherein the LNP composition comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

26. The pharmaceutical composition for use according to claim 21, wherein the LNP composition comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

27. 26. The pharmaceutical composition for use according to claim 25, wherein the ionizable lipid comprises compound 18.

28. 26. The pharmaceutical composition for use according to claim 25, wherein the ionizable lipid comprises compound 25.

29. The pharmaceutical composition for use according to claim 26, wherein the ionizable lipid comprises compound 18.

30. The pharmaceutical composition for use according to claim 26, wherein the ionizable lipid comprises compound 25.

31. 1. An in vitro or ex vivo method for assessing a subject's responsiveness to a therapy comprising an LNP composition comprising mRNA encoding an ITB6 molecule, the method comprising: (a) measuring the expression level of one or more biomarkers in a sample from the subject collected after administration of the therapy; (b) comparing the expression level in the sample to a reference expression level; wherein an increase in the expression level of the one or more biomarkers is indicative of a response to the therapy.

32. 32. The method of claim 31 , wherein the one or more biomarkers are one or more of PMEPA1, ITGAE / CD103, SMAD7, SKIL, and SKI.

33. the level of one or more of the one or more biomarkers in the sample from the subject after treatment is at least two-fold higher than a reference expression level of the one or more biomarkers, and the reference expression level is: (a) the expression level of the one or more biomarkers in a sample from the subject before treatment with ITB6 mRNA; or 33. The method of claim 31 or 32, comprising (b) the expression level of the one or more biomarkers in a sample from a subject not treated with ITB6 mRNA.

34. The use of claim 22, wherein the disease is selected from rheumatoid arthritis (RA); graft-versus-host disease (GVHD); diabetes; inflammatory bowel disease (IBD); lupus, multiple sclerosis; autoimmune hepatitis; primary biliary cholangitis; organ transplant-associated rejection; or myasthenia gravis. (a) The GVHD is acute GVHD or chronic GVHD. (b) the diabetes is type 1 diabetes; (c) the lupus is systemic lupus erythematosus (SLE); or (d) the use of claim 34, wherein the autoimmune hepatitis is type 1 or type 2 autoimmune hepatitis.

36. The use of claim 20, wherein the LNP composition comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

37. The use of claim 22, wherein the LNP composition comprises (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG lipid.

38. The use described in claim 36, wherein the ionizable lipid comprises compound 18.

39. The use described in claim 36, wherein the ionizable lipid comprises compound 25.

40. The use described in claim 37, wherein the ionizable lipid comprises compound 18.

41. The use described in claim 37, wherein the ionizable lipid comprises compound 25.

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