RNA-based synthesis pathways for producing engineered immune cells for extracorporeal cell therapy

Abstract

This disclosure relates to a method for producing cells, such as immune cells, for in vitro cell therapy by contacting target cells, such as immune cells, with a synthetic circuit that can be used to selectively express a payload in target cells, such as immune cells. In some embodiments, the synthetic circuit includes a payload sequence including a sensor. In some embodiments, the synthetic circuit further includes a regulator sequence including a sensor. The present invention also provides a method for treating a wide range of diseases or disorders by in vitro cell therapy using such a synthetic circuit.

Description

Technical Field

[0001] Cross - reference to Related Applications This PCT application claims the benefit of priority of U.S. Provisional Application No. 63 / 515,247, filed on July 24, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Reference to a Sequence Listing Submitted Electronically via EFS - WEB The content of the sequence listing was transmitted electronically (Name: 4597_029PC01_SequenceListing_ST26.xml; Size: 4,240 bytes; and Creation Date: July 23, 2024), submitted together with this application, and the entire disclosure is incorporated herein by reference.

Background Art

[0003] Cell therapies, such as chimeric antigen receptor (CAR) - T cell therapy, are an innovative new pillar in cancer treatment. Although treatment with CAR - T cells has led to significant clinical responses in certain subsets of B - cell leukemia or lymphoma, many challenges limit the therapeutic efficacy of CAR - T cells in solid tumors and hematological malignancies. Barriers to effective CAR - T cell therapy include life - threatening severe toxicity, poor antitumor activity, antigen escape, restricted migration, and limited tumor infiltration. In addition, the interaction between the host and tumor microenvironment and CAR - T cells critically alters the function of CAR - T cells. Furthermore, developing and implementing these therapies require a complex workforce. To overcome these significant challenges, innovative strategies and approaches are needed to engineer more potent CAR - T cells with improved antitumor activity and reduced toxicity. See Sterner et al., Blood Cancer Journal. 11:69 (April 6, 2021). Therefore, it is necessary to develop new cell constructs that improve clinical efficacy in both hematological malignancies and solid tumors, as well as strategies to overcome the limitations of cell therapy in both hematological malignancies and solid tumors. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sterner et al.,Blood Cancer Journal.11:69(April 6,2021 [Overview of the Initiative]

[0005] Provided herein is a method for producing engineered immune cells for extracorporeal cell therapy in a subject requiring such therapy, comprising contacting the immune cells obtained from the subject with a synthesis circuit in a closed in-line system connected to the subject, wherein the synthesis circuit is capable of selectively expressing a payload in the immune cells. In some embodiments, the immune cells used in the method are not exposed to the outside of the in-line system for the entire duration of therapy. In some embodiments, the immune cells in the method express a payload on the immune cells as a result of contact. In some embodiments, the immune cells in the method are returned directly from the in-line system to the subject. Furthermore, provided herein are methods for extracorporeally treating a disease or condition in a subject requiring treatment, comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthetic circuit in a closed in-line system connected to the subject, and the synthetic circuit is capable of selectively expressing the payload in the immune cells.

[0006] In some embodiments, the Disclosure provides a method for in vitro treating a disease or condition in a subject requiring treatment, comprising contacting immune cells obtained from the subject with a synthesis circuit in a closed in-line system connected to the subject, and administering immune cells expressing a payload encoded by the synthesis circuit to the subject, wherein the synthesis circuit is capable of selectively expressing the payload in the immune cells.

[0007] In some embodiments, subjects have not undergone lymphocyte apheresis prior to treatment. In some embodiments, the therapy is 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 2 hours.

[0008] In some embodiments, the synthesis circuit includes a nucleotide sequence encoding a payload (payload sequence), the payload sequence includes a sensor that recognizes a marker, and the recognition of the marker by the sensor reduces the expression of the payload.

[0009] In some embodiments, the disclosure provides immune cells expressing a payload encoded by a synthetic circuit for a method of extracorporeal cell therapy, the synthetic circuit capable of selectively expressing the payload in the immune cells. In some embodiments, the payload is expressed on the surface of the immune cells. In some embodiments, the immune cells include T cells, NK cells, B cells, or any combination thereof. In some embodiments, the immune cells include CD8+ T cells, CD4+ T cells, or any combination thereof.

[0010] In some embodiments, the disclosure provides an inline system comprising immune cells expressing a payload encoded by a synthetic circuit for extracorporeal cell therapy. In some embodiments, the inline system is completely closed. In some embodiments, the inline system is connected to a subject requiring therapy. In some embodiments, the immune cells in the inline system are obtained by the subject. In some embodiments, the inline system further comprises cells other than immune cells.

[0011] In some embodiments, payloads useful to this disclosure include chimeric antigen receptors (CARs), T cell receptors (TCRs), or TCR mimics. In some embodiments, CARs include CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, and Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe extracellular components of the following proteins are targeted: hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof. In some embodiments, the TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2)The target is the extracellular component of the ETS fusion gene, NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof.

[0012] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and optionally (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence comprises a sensor capable of specifically recognizing the regulator (Type P sensor), and the regulator sequence comprises a sensor capable of specifically recognizing a marker (Type R sensor), wherein the regulator and the marker recognized by the Type R sensor (Type R marker) are not the same.

[0013] In some embodiments, a synthetic circuit useful to the present disclosure comprises a nucleotide sequence encoding a payload (payload sequence), the payload sequence comprising a sensor (type P sensor) capable of specifically recognizing a marker, the marker being either intrinsically absent in immune cells or intrinsically present only in immune cells.

[0014] In some embodiments, payload sequences useful to this disclosure include multiple type P sensors. In some embodiments, the multiple type P sensors include two type P sensors, three type P sensors, four type P sensors, five type P sensors, six type P sensors, seven type P sensors, or eight or more type P sensors. In some embodiments, each of the type P sensors is the same. In some embodiments, one or more of the type P sensors are different.

[0015] In some embodiments, a payload array useful to the present disclosure includes a spacer array (Type P spacers). In some embodiments, the payload array includes a plurality of Type P spacers. In some embodiments, each of the Type P spacers is the same. In some embodiments, one or more of the Type P spacers are different. In some embodiments, (a) at least one Type P spacer is located upstream of the Type P sensor, (b) at least one Type P spacer is located downstream of the Type P sensor, or (c) both (a) and (b). In some embodiments, a synthesis circuit described herein (e.g., above) includes at least two Type P sensors, and at least one Type P spacer is located between the at least two Type P sensors.

[0016] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence comprises a first sensor capable of specifically recognizing the regulator (first type P sensor) and a second sensor capable of specifically recognizing a marker (second type P sensor), and the regulator sequence comprises a sensor capable of specifically recognizing a marker (type R sensor), wherein the regulator, the marker recognized by the second type P sensor (second type P marker), and / or the marker recognized by the type R sensor (type R marker) are not the same.

[0017] In some embodiments, a payload array useful to the present disclosure includes a plurality of first type P sensors. In some embodiments, the plurality of first type P sensors includes 2 first type P sensors, 3 first type P sensors, 4 first type P sensors, 5 first type P sensors, 6 first type P sensors, 7 first type P sensors, 8 first type P sensors, 9 first type P sensors, 10 first type P sensors, 11 first type P sensors, or 12 first type P sensors. In some embodiments, each of the first type P sensors is the same. In some embodiments, one or more of the first type P sensors are different.

[0018] In some embodiments, payload arrays useful to the present disclosure include a plurality of second type P sensors. In some embodiments, the plurality of second type P sensors include 2 second type P sensors, 3 second type P sensors, 4 second type P sensors, 5 second type P sensors, 6 second type P sensors, 7 second type P sensors, 8 second type P sensors, 9 second type P sensors, 10 second type P sensors, 11 second type P sensors, or 12 second type P sensors. In some embodiments, each of the second type P sensors is the same. In some embodiments, one or more of the second type P sensors are different.

[0019] In some embodiments, the payload array provided herein (e.g., as described above) includes a plurality of type P spacers. In some embodiments, each of the type P spacers is the same. In some embodiments, one or more of the type P spacers are different. In some embodiments, (a) at least one type P spacer is located between a first type P sensor and a second type P sensor, (b) at least one type P spacer is located upstream of both the first type P sensor and the second type P sensor, (c) at least one type P spacer is located downstream of both the first type P sensor and the second type P sensor, or (d) any combination of (a) to (c).

[0020] In some embodiments, when a synthesis circuit useful for the present disclosure includes a plurality of first type P sensors, two or more first type P sensors are separated by type P spacers. In some embodiments, each of the first type P sensors is separated by a type P spacer.

[0021] When the synthetic circuit useful for the present disclosure includes a plurality of second type P sensors, in some embodiments, two or more second type P sensors are separated by a type P spacer. In some embodiments, each of the second type P sensors is separated by a type P spacer.

[0022] In some embodiments, the type P spacer useful for the present disclosure is about 1 to about 50 nucleotides in length. In some embodiments, the type P spacer is at least about 10 nucleotides in length. In some embodiments, the type P spacer is about 10 nucleotides in length, about 20 nucleotides in length, or about 50 nucleotides in length. In some embodiments, the type P spacer comprises, consists essentially of, or consists of tttcctttcccccttccctt (SEQ ID NO: 2) or gcggccgctaaa (SEQ ID NO: 3), or a fragment thereof.

[0023] Regarding any of the synthetic circuits provided herein useful for the present disclosure (such as those described above), in some embodiments, the regulator array includes a plurality of type R sensors. In some embodiments, the plurality of type R sensors includes two type R sensors, three type R sensors, four type R sensors, five type R sensors, six type R sensors, seven type R sensors, or eight or more type R sensors. In some embodiments, each of the type R sensors is the same. In some embodiments, one or more of the type R sensors are different.

[0024] In some embodiments, the regulator array useful for the present disclosure includes a spacer array (type R spacer). In some embodiments, the regulator array includes a plurality of type R spacers. In some embodiments, each of the type R spacers is the same. In some embodiments, one or more of the type R spacers are different.

[0025] When a synthetic circuit useful for the present disclosure includes a plurality of type R sensors, in some embodiments, two or more type R sensors are separated by a type R spacer. In some embodiments, each of the type R sensors is separated by a type R spacer. In some embodiments, at least one type R spacer is upstream of at least one type R sensor.

[0026] In some embodiments, a type R spacer useful for the present disclosure is about 1 to about 50 nucleotides in length. In some embodiments, the type R spacer is at least about 10 nucleotides in length. In some embodiments, the type R spacer is about 10 nucleotides in length, about 20 nucleotides in length, or about 50 nucleotides in length. In some embodiments, the type R spacer comprises, consists essentially of, or consists of the sequence tttcctttcccccttccctttttcctttcctttcctttcccccttccctt (SEQ ID NO: 1), or a fragment thereof. In some embodiments, the type R spacer comprises, consists essentially of, or consists of the sequence tttcctttcccccttccctt (SEQ ID NO: 2). In some embodiments, the type R spacer comprises, consists essentially of, or consists of the sequence gcggccgctaaa (SEQ ID NO: 3).

[0027] With respect to any of the synthetic circuits provided herein (e.g., those described above) useful for the present disclosure, in some embodiments, the first marker, the second marker, or both the first and second markers comprise a microRNA, a protein, a metabolite, or a combination thereof.

[0028] In some embodiments, the marker is a regulator, which includes an RNA-binding protein, siRNA, shRNA, pre-miRNA, ribozyme, or a combination thereof. In some embodiments, the RNA-binding protein includes a ribonuclease. In some embodiments, the ribonuclease includes a Cas protein. In some embodiments, the Cas protein includes a Cas6 protein.

[0029] With respect to any of the synthetic circuits provided herein (e.g., those described above) that are useful for the present disclosure, in some embodiments, the payload sequence, the regulator sequence, or both the payload sequence and the regulator sequence include linear RNA or circular RNA. In some embodiments, the payload sequence is self-replicating RNA and the regulator sequence is non-replicating RNA. In some embodiments, the payload sequence is self-replicating RNA and the regulator sequence is circular RNA. In some embodiments, the payload sequence is self-replicating RNA and the regulator sequence is linear non-replicating RNA. In some embodiments, the payload sequence is circular RNA and the regulator sequence is circular RNA. In some embodiments, the payload sequence is circular RNA and the regulator sequence is linear non-replicating RNA.

[0030] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence comprises a circular RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

[0031] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0032] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence comprises a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

[0033] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, and the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0034] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0035] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence comprises a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

[0036] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a self-replicating RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, and the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0037] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a circular RNA and comprises a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence comprises a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

[0038] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a circular RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is a circular RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, and the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0039] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a circular RNA and comprises a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence comprises a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

[0040] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence is a circular RNA and comprises a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is a non-replicating linear RNA and comprises a sensor (type R sensor) capable of specifically recognizing a marker, and the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0041] With respect to any of the synthesis circuits provided herein (e.g., those described above), in some embodiments, (a) the payload array comprises a plurality of first type P sensors, (b) the payload array comprises a plurality of second type P sensors, (c) the regulator array comprises a plurality of type R sensors, or (d) any combination of (a) to (c).

[0042] In some embodiments, (a) the payload array includes a spacer array (Type P spacers), (b) the regulator array includes a spacer array (Type R spacers), or (c) both (a) and (b). In some embodiments, (a) the Type P spacers are located between a first Type P sensor and a second Type P sensor, (b) the Type P spacers are located upstream of both the first Type P sensor and the second Type P sensor, (c) the Type P spacers are located downstream of both the first Type P sensor and the second Type P sensor, or (d) any combination of (a) to (c).

[0043] In some embodiments, the payload array includes a plurality of first type P sensors, where two or more first type sensors are separated by type P spacers. In some embodiments, the payload array includes a plurality of second type P sensors, where two or more second type sensors are separated by type P spacers. In some embodiments, the regulator array includes a plurality of type R sensors, where two or more type R sensors are separated by type R spacers.

[0044] In some embodiments, the type P spacer, the type R spacer, or both are about 1 to about 50 nucleotides in length. In some embodiments, the type P spacer, the type R spacer, or both contain, essentially consist of, or comprise the sequence tttcctttcccccttccctttttcctttcctttcccccttccctt (SEQ ID NO: 1) or a fragment thereof. In some embodiments, the type P spacer, the type R spacer, or both contain, essentially consist of, or comprise the sequence tttcctttcccccttccctt (SEQ ID NO: 2). In some embodiments, the type P spacer, the type R spacer, or both are the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some embodiments, the type P spacer, the type R spacer, or both are the sequence gcggccgctaaa (SEQ ID NO: 3) or essentially consist of or comprise the sequence gcggccgctaaa (SEQ ID NO: 3).

[0045] In some embodiments, the type P marker, the type R marker, or both include microRNA, a protein, a metabolite, or a combination thereof. In some embodiments, the regulator includes an RNA-binding protein, siRNA, shRNA, pre-miRNA, a ribozyme, or a combination thereof.

[0046] In some embodiments, the regulator is an RNA-binding protein, and the RNA-binding protein comprises a ribonuclease. In some embodiments, the ribonuclease comprises a Cas protein. In some embodiments, the Cas protein comprises a Cas6 protein.

[0047] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein, if the payload sequence and the regulator sequence are present in an immune cell, the payload is expressed in the immune cell for a first expression, and the regulator is expressed in the immune cell for a second expression, with the first expression being greater than the second expression.

[0048] In some embodiments, (a) a payload array includes a first sensor (first type P sensor) capable of specifically recognizing a regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, and (b) a regulator array includes a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0049] In some embodiments, recognition of a type P marker by a second type P sensor inhibits the expression of the payload. In some embodiments, recognition of a type R marker by a type R sensor inhibits the expression of the regulator.

[0050] In some embodiments, (a) immune cells do not express levels of type P markers sufficient to activate a second type P sensor, or (b) immune cells express levels of type R markers sufficient to activate a type R sensor. In some embodiments, (a) non-immune cells express levels of type P markers sufficient to activate a second type P sensor, or (b) non-immune cells do not express levels of type R markers sufficient to activate a type R sensor, or (c) both (a) and (b).

[0051] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein, if the payload sequence and the regulator sequence are present in a non-immune cell, the payload is expressed in the non-immune cell for a first expression, and the regulator is expressed in the non-immune cell for a second expression, the second expression being greater than the first expression.

[0052] In some embodiments, (a) a payload array includes a first sensor (first type P sensor) capable of specifically recognizing a regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, and (b) a regulator array includes a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0053] In some embodiments, binding of a type P marker to a second type P sensor inhibits payload expression. In some embodiments, non-immune cells include (a) a level of type P marker sufficient to activate a second type P sensor, (b) a level of type R marker insufficient to activate a type R sensor, or (c) both (a) and (b). In some embodiments, binding of a type R marker to a type R sensor inhibits regulator expression. In some embodiments, immune cells include (a) a level of type P marker insufficient to activate a second type P sensor, and (b) a level of type R marker sufficient to activate a type R sensor.

[0054] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein when the synthetic circuit comes into contact with a population of cells including immune and non-immune cells, the expression of the payload in the immune cells is higher than the corresponding expression in the non-immune cells. In some embodiments, the expression of the payload in immune cells is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 times higher than the corresponding expression in non-immune cells.

[0055] In some embodiments, (a) a payload array includes a first sensor (first type P sensor) capable of specifically recognizing a regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, and (b) a regulator array includes a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0056] In some embodiments, binding of a type P marker to a second type P sensor inhibits payload expression. In some embodiments, non-immune cells include (a) a level of type P marker sufficient to activate a second type P sensor, (b) a level of type R marker insufficient to activate a type R sensor, or (c) both (a) and (b). In some embodiments, binding of a type R marker to a type R sensor inhibits regulator expression. In some embodiments, immune cells include (a) a level of type P marker insufficient to activate a second type P sensor, and (b) a level of type R marker sufficient to activate a type R sensor.

[0057] In some embodiments, a synthetic circuit useful to the present disclosure comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein when the synthetic circuit comes into contact with a population of cells including immune and non-immune cells, the expression of the payload in the immune cells is higher than the corresponding expression in the non-immune cells. In some embodiments, the expression of the payload in immune cells is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 times higher than the corresponding expression in non-immune cells.

[0058] In some embodiments, (a) a payload array includes a first sensor (first type P sensor) capable of specifically recognizing a regulator and a second sensor (second type P sensor) capable of specifically recognizing a marker, and (b) a regulator array includes a sensor (type R sensor) capable of specifically recognizing a marker, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

[0059] In some embodiments, binding of a type P marker to a second type P sensor inhibits the expression of the payload. In some embodiments, binding of a type R marker to a type R sensor inhibits the expression of the regulator. In some embodiments, (a) immune cells do not contain sufficient levels of type P marker to activate the second type P sensor, or (b) immune cells express sufficient levels of type R marker to activate the type R sensor. In some embodiments, (a) non-immune cells express sufficient levels of type P marker to activate the second type P sensor, or (b) non-immune cells do not express sufficient levels of type R marker to activate the type R sensor, or (c) both (a) and (b).

[0060] With respect to the synthesis circuit described above, in some embodiments, the payload sequence is self-replicating RNA. In some embodiments, the regulator sequence is non-replicating linear RNA. In some embodiments, the payload sequence is circular RNA. In some embodiments, the regulator sequence is circular RNA. In some embodiments, the payload sequence is self-replicating RNA and the regulator sequence is circular RNA. In some embodiments, the payload sequence is self-replicating RNA and the regulator sequence is non-replicating linear RNA. In some embodiments, the payload sequence is circular RNA and the regulator sequence is circular RNA. In some embodiments, the payload sequence is circular RNA and the regulator sequence is non-replicating linear RNA.

[0061] In some embodiments, the composite circuit includes a payload array and a regulator array, wherein (a) the payload array includes a plurality of first type P sensors, (b) the payload array includes a plurality of second type P sensors, (c) the regulator array includes a plurality of type R sensors, or (d) any combination of (a) to (c).

[0062] In some embodiments, (a) the payload array includes a spacer array (type P spacers), (b) the payload array includes a spacer array (type R spacers), or (c) both (a) and (b). In some embodiments, a type P spacer is located between a first type P sensor and a second type P sensor. In some embodiments, a type P spacer is located between an array encoding the payload and (a) a first type P sensor, (b) a second type P sensor, or (c) both (a) and (b). In some embodiments, a type R spacer is located between an array encoding the regulator and a type R sensor. In some embodiments, the payload array includes a plurality of first type P sensors, with two or more first type sensors separated by type P spacers. In some embodiments, the payload array includes a plurality of second type P sensors, with two or more second type sensors separated by type P spacers. In some embodiments, the regulator array includes a plurality of type R sensors, with two or more type R sensors separated by type R spacers.

[0063] In some aspects, the type P spacer, the type R spacer, or both are about 1 to about 50 nucleotides in length. In some aspects, the type P spacer, the type R spacer, or both contain, essentially consist of, or comprise the sequence tttcctttcccccttccctttttcctttcctttcctttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the type P spacer, the type R spacer, or both contain, essentially consist of, or comprise the sequence tttcctttcccccttccctt (SEQ ID NO: 2) or a fragment thereof. In some aspects, the type P spacer, the type R spacer, or both are contained in, essentially consist of, or comprise the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

[0064] In some embodiments, the type P marker, the type R marker, or both include a microRNA. In some embodiments, the regulator includes an RNA-binding protein, siRNA, an aptamer, or a combination thereof. In some embodiments, the RNA-binding protein includes a ribonuclease. In some embodiments, the ribonuclease includes a Cas protein. In some embodiments, the Cas protein includes a Cas6 protein.

[0065] With respect to any of the synthetic circuits provided in this disclosure (e.g., those described above), in some embodiments, the payload includes a therapeutic protein, a reporter protein, an immunomodulatory protein, a chimeric antigen receptor, or a combination thereof. In some embodiments, the payload sequence includes one or more elements that enhance the translation of the encoded protein compared to a regulator sequence. In some embodiments, one or more elements include an aptamer for a translation initiation factor (e.g., eIF4G).

[0066] In some embodiments, the synthetic pathways provided herein (e.g., as described above) further include (1) an intra-sequence ribosome entry site (IRES), (2) a UTR, (3) a sequence encoding a signal peptide, (4) a translation initiation sequence, (5) a poly(A) sequence, (6) a sequence encoding an RNA-binding protein, (7) a sequence encoding a 2A ribosome skipping peptide, or (8) any combination of (1) to (7).

[0067] With respect to any of the synthesis circuits provided herein (e.g., those described above), in some embodiments, the synthesis circuit does not include any sequences derived from a non-human genome.

[0068] Some aspects of this disclosure relate to vectors including synthesis circuits provided herein.

[0069] Also provided herein are nanoparticles comprising (i) any of the synthesis circuits of the Disclosure (e.g., those described above) and (ii) one or more lipids and / or lipid-like materials. In some embodiments, the one or more lipids include ionized lipids, cationic lipids, lipidoids, non-cationic helper lipids, phospholipids, sterols or other structural lipids, or combinations thereof.

[0070] In some embodiments, the ionized lipids are ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 5), di((Z)-nona-2-en-1-yl)9-((4-( Dimethylaminobutanoyl(oxy)heptadecanedioate (L319), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), Nl-[2(didodecylamino)ethyl]-N1,N4,N4-tridodecyl1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimeth L-aminomethyl-[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), 2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12 (Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA), (2R)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,This includes 12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), or combinations thereof.

[0071] In some embodiments, cationic lipids include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTEVI), and 2,3-dioleyloxy-N-[2(sperminecarbocyanide). [Samide)ethyl]-N,N-dimethyl-l-propaneaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(l,2-dioleoyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N, N-Dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-Dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), l,2-Distearoyl-3-trimethylammonium-propane (DSTAP), l,2-Dipalmitoyl-3-trimethylammonium-propane (DPTAP), l,2-Dilinoleyl-3-trimethylammonium-propane (DLTAP), l,2-Dimyristoyl-3-trimethylammonium -Includes propane (DMTAP), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC), l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC), l,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0072] In some embodiments, the lipidoid is 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine 2,5-dione(cKK-E12), tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazandiyl)bis(propane-3,1diyl))bis(azantriyl))tetrapropionate(306Oi 10), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione(OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azantriyl))tetrakis(ethane2,1-diyl)(9Z,9'Z,9''Z,9''' Z,12Z,12'Z,12''Z,12'''Z)-Tetakis(octadeca-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(axantriyl))Tetakis(butane-4,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-Tetakis(O (OF-C4-Deg-Lin), N1,N3,N5-Tris(3-(didodecylamino)propyl)benzene 1,3,5-Tricarboxamide (TT3), Hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((Benzene-1,3,5-tricarbonyl)ris(azandiyl))tris(propane-3,1-diyl)) The compound comprises ris(azantriyl)hexanonanoate (FTT5), PL-1, 98N12-5, ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18(A2)), A12-Iso5-2DC18(A12), or any combination thereof. In some embodiments, the lipidoid is TT3.

[0073] In some embodiments, phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 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-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 This includes PE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-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 any combination thereof.

[0074] In some aspects, phospholipids include 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0PC, OSPC), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (16:0-18:1 PE, POPE), 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphoethanolamine (16:0-18:2 PE), 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphoethanolamine (16:0-20:4 PE), 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphoethanolamine (16:0-22:6 PE), 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (18:0-18:1 Selected from the group consisting of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6 PE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof.

[0075] In some embodiments, sterols include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and combinations thereof.

[0076] In some embodiments, one or more lipids and / or lipid-like materials are pegylated.

[0077] In some embodiments, any of the nanoparticles provided herein (e.g., those described above) further comprises a targeted ligand.

[0078] With respect to any of the nanoparticles provided herein (e.g., those described above), in some embodiments, one or more lipids and / or lipid-like materials comprise ionized lipids (e.g., cationic lipids) in a molar ratio of about 10–50%. In some embodiments, one or more lipids and / or lipid-like materials comprise phospholipids in a molar ratio of about 10–40%. In some embodiments, one or more lipids and / or lipid-like materials comprise sterols (e.g., cholesterol) in a molar ratio of about 20–50%. In some embodiments, one or more lipids and / or lipid-like materials comprise pegylated lipids in a molar ratio of about 0–10%.

[0079] Also provided herein are pharmaceutical compositions useful for this disclosure, comprising any of the synthesis circuits, vectors, or nanoparticles described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, lymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. [Brief explanation of the drawing]

[0080] [Figure 1] This is a schematic diagram of an exemplary composite circuit described herein. As shown, the regulator array and / or payload array may be linear or annular. [Figure 2] This is a schematic diagram showing a target site array consisting of target sites (TSs) that all bind to the same siRNA. Each sequence includes a coding region (encoding mVenus-PEST) and a 3'-UTR. As shown, these sequences include no target site ("no TS"), a single target site ("1×TS"), two target sites ("2×TS"), three target sites ("3×TS"), or four target sites ("4×TS"). As shown, some sequences further include one or more spacer sequences ranging in length from 10 to 50 nucleotides. [Figure 3]This figure illustrates the effect of the number of adjacent target sites (TS) immediately following the stop codon of the mVenus-PEST reporter on payload expression. Specifically, the figure provides a graph of the median mVenus fluorescence in arbitrary units (au) for constructs containing either 1×siRNA TS, 2×siRNA TS, 3×siRNA TS, 4×siRNA TS, no TS, or no reporter after administration of siRNA (0, 1, 10, or 100 nM). The data represent median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of the reporter and siRNA. [Figure 4] This figure illustrates the effect of spacer sequences on payload expression. Specifically, it provides graphs showing the median mVenus fluorescence (au) for reporter constructs containing two siRNA target sites that are directly adjacent (2×siRNA2), separated by a 20-nucleotide (nt) spacer sequence (2×siRNA2-20nt), or separated by a 50nt spacer sequence (2×siRNA2-50nt) after administration of siRNA (0, 1, 10, or 100 nM), as well as a control reporter construct without target sites (no TS) and a control condition where the mVenus reporter was not transfected (no reporter). The data represent the median fluorescence intensity of three technical replicates (n=3) measured by flow cytometry 20 hours after electroporation of the reporter and siRNA. [Figure 5]This figure illustrates the effect of increased copy number of target sites on payload expression when a 20nt spacer sequence is present between target sites. Specifically, the figure provides graphs representing median MVenus fluorescence (au) for constructs containing 2, 3, or 4 siRNA target sites (TS) with a 20 nucleotide (nt) spacer sequence between adjacent TSs (2×siRNA2-20nt, 3×siRNA2-20nt, 4×siRNA2-20nt, respectively), no TS, or no reporter after administration of siRNA (0, 1, 10, or 100 nM). The data represent median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of reporter and siRNA. [Figure 6A] This report evaluates the detargeting of payload expression for linear RNA. The graphs show mVenus-PEST fluorescence (au) in hepatocytes (Huh-7) and control (HEK293T) cell lines for a linear modRNA circuit containing an mVenus-PEST reporter and 0-4 miR-b target sites (no miR TS, 1×TS, 2×TS, or 4×TS) in the 3'UTR. Background autofluorescence levels for each cell line are also plotted. Data points represent the average geometric mean fluorescence intensity collected from three technical replicates (n=3) 20 hours after electroporation. [Figure 6B] This shows an evaluation of detargeting of payload expression for circular RNA. The graph shows the median fluorescence intensity (au) in HEK293T and HeLa cell lines for circRNAs containing 1 to 4 miR-a target sites (no TS, 1×TS, 2×TS, or 4×TS) immediately following the stop codon of the mVenus-PEST reporter. The data represent the average of geometric mean fluorescence intensities collected from three technical replicates (n=3) at 4 and 24 hours after electroporation. [Figure 7]Figure A is a schematic diagram showing the locations of regulator target sites in an exemplary circRNA expressing CVB3 IRES, mVenus-PEST, and mVenus-PEST with a regulator (Cas6e) target site at one of five positions. Figure B is a bar graph showing the effect of the presence or absence of the Cas6e regulator on the normalized TS (without) expression rate (%) in the circRNA construct shown in Figure 7A, compared to electroporation alone and knockdown of circRNA without the Cas6e target site. [Figure 8A] This is a schematic diagram showing the N×base pair spacer between the stop codon and the 3'UTR target site array. [Figure 8B] This graph shows the mVenus fluorescence (au) after administration of siRNA (0, 1, 10, 100 nM) for constructs containing a 1× or 2× siRNA target site (TS) and 0, 7, or 12 nt spacers. The data represent the geometric mean fluorescence intensity collected by flow cytometry from three technical replicates (n=3) 6 hours after electroporation of the reporter and siRNA. [Figure 8C] This graph shows the mVenus fluorescence (au) after administration of siRNA (0, 1, 10, 100 nM) for constructs containing a 1× or 2× siRNA target site (TS) and a 0, 7, or 12 nt spacer. The data represent the geometric mean fluorescence intensity collected by flow cytometry from three technical replicates (n=3) 24 hours after electroporation of the reporter and siRNA. [Figure 9] This is a schematic diagram showing target site arrays for multiple input classifiers. [Figure 10]This graph shows the effect of co-electroporation of siRNA(s) with or without a target site within the target site array on the median mVenus fluorescence (au) in HEK293T cells electroporated with an mVenus-PEST reporter containing a target site array immediately after the stop codon, and either no siRNA, siRNA1, siRNA2, or both (3×siRNA1-20nt, 3×siRNA2-20nt, 3×siRNA2-siRNA1 alternating arrangement, 3×siRNA1-siRNA2 alternating arrangement, 3×siRNA2 adjacent to 3×siRNA1, 3×siRNA1 adjacent to 3×siRNA2, no target site, no reporter). [Figure 11] This is a schematic diagram showing the N×miR and regulator target sites located at either the 5'UTR or 3'UTR at positions A, B, and C. [Figure 12] This bar graph shows the expression of circRNA (compared to the absence of miR target sequences (TS)) 4 and 24 hours after transfection in HEK293T cells, Huh-7 cells, and HeLa cells that were electroporated with circRNA. The circular RNA contained either 4×miR-b TS or 4×miR-a TS. [Figure 13] Figures A-C illustrate the downregulation of circRNA by the regulator Cas6e. Figure A is a schematic diagram showing linear and circular regulator RNAs that downregulate target circRNA, containing a Cas6e target site (TS) and encoding the fluorescent protein mVenus-PEST. Figure B is a bar graph showing that mVenus expression is reduced to background levels in BHK-21 cells transfected with target RNA containing Cas6e TS, circRNA, and mRNA regulator. Figure C is a bar graph showing that Cas6e does not affect mCherry expression of RNA (mRNA and circRNA) that does not contain its target site. [Figure 14]Bar graphs A and B show the effects of the regulator on mRNA of a linear non-replicating (non-rep) payload mRNA strand (A) containing the RNA regulator's target sequence and replicon RNA (B) containing the RNA regulator's target sequence. A shows the effect on payload expression (au) of unmodified RNA (unmodRNA) payload or modRNA payload for mRNA transfected into BHK-21 cells with or without co-transfection of modified RNA (modRNA) expressing the RNA regulator. B shows the effect on mVenus-positive cells (%) of replicon RNA containing the RNA regulator's target sequence, transfected into BHK-21 cells at two different doses (20 ng or 40 ng), with or without co-transfection of cells co-transfected with modRNA expressing the RNA regulator. [Figure 15] This bar graph shows the expression (multiplicative change compared to the absence of miR target sequences (TS)) of RNA encoding EGFP-PEST, driven by CVB3 IRES, in HEK293T cells and Huh-7 cells electroporated with circular RNA. The circular RNA either did not contain miR TS or contained a 4×miR-b target site or a 4×miR-a target site immediately after the stop codon. A reporter-less control was also used. The EP-only group (reporter-less control) was normalized to the mean of CVB3 without miR TS. [Figure 16] This bar graph shows the normalized ratio of mVenus-PEST expression levels in HEK293T (non-cancer) cells and HeLa (cancer) cells after electroporation of various human miRNA target sites corresponding to miRNAs that are more active in HEK293T cells than in HeLa cells. The geometric mean of mVenus-PEST expression in each cell type was normalized to the geometric mean of modRNA without the miRNA sensor, after subtracting the background fluorescence level. Flow cytometry data were collected for both cell types (n=3) approximately 24 hours after electroporation. [Figure 17A] This shows the mean geometric mean (GMean) of mVenus fluorescence (au) of non-replicating modRNAs transfected into HEK293T cells or the human liver cell line Huh-7 via electroporation. Non-replicating modRNAs expressing the near-infrared fluorescent reporter protein miRFP720 were co-transfected with each replicon RNA to function as transfection markers. The data represent the mean geometric mean of three technical replicates (n=3) of flow cytometry data collected approximately 24 hours after electroporation. Expression output (A) and the percentage of miRFP720-positive cells (B) that are also mVenus-PEST-positive were calculated. [Figure 17B] This shows the expression of mVenus fluorescence from replicon RNA transfected into HEK293T cells or the human liver cell line Huh-7 via electroporation. Non-replicating modRNA expressing the near-infrared fluorescent reporter protein miRFP720 was co-transfected with each replicon RNA and used as a transfection marker. The data represent the mean geometric mean of three technical replicas (n=3) of flow cytometry data collected approximately 24 hours after electroporation. Expression output (A) and the percentage of miRFP720-positive cells (B) that are also mVenus-PEST positive were calculated. [Figure 18] This bar graph shows the mean radiance (p / s / cm2 / sr) of firefly luciferase in the spleen, lungs, kidneys, lymph nodes (LN), and liver of mice injected with lipid nanoparticles containing a reporter modRNA encoding firefly luciferase with an added sensor for liver-specific microRNA miR-b (gray bars), lipid nanoparticles containing a reporter modRNA encoding firefly luciferase without the added sensor (white bars), or a vehicle control (black bars). The mice were sacrificed after 6 hours. [Figure 19]This bar graph shows the mean radiance (p / s / cm2 / sr) of firefly luciferase in the liver and spleen of mice injected with either a reporter modRNA encoding firefly luciferase with an added sensor for spleen-related miRNA miR-h (gray bars), a reporter modRNA encoding firefly luciferase without the added sensor (white bars), or a vehicle control. Mice were sacrificed after 6 hours. [Figure 20] This bar graph shows the expression of a green fluorescent reporter (Green Object Mean Intensity (GCU)) in Huh7 cells (black bars) and HEK293T cells (gray bars) transfected with an RNA circuit. The RNA circuit consists of (1) a replicon payload strand containing a first type P sensor that expresses a green fluorescent reporter and responds to the regulator protein Cas6e, and (2) a linear non-replicating regulator strand containing a type R sensor that expresses the regulator protein Cas6e and responds to miR-b. Control Huh7 cells and HEK293T cells were transfected in parallel with the same RNA circuit, except that they lacked the type R sensor. The expression of the green fluorescent payload was measured 6 hours after transfection via quantitative imaging. [Figure 21]This bar graph shows the effect of a type R sensor on the expression of the mVenus reporter protein (payload reporter) from a replicon payload RNA sequence. A linear non-replicating regulator sequence was constructed expressing a Cas6e regulator protein linked to the mCherry reporter via a 2A self-cleaving peptide, so that the Cas6e regulator protein and the mCherry reporter (regulator reporter) are co-expressed from the same RNA sequence. Another version of the regulator sequence was constructed, further containing a type R sensor that recognizes miR-i. A549 lung cancer cells expressing high levels of miR-i were either transfected with the payload sequence alone, or co-transfected with the payload sequence and one of the two versions of the regulator sequence (with or without the type R sensor). The "+" after "Regulator" below the graph indicates that the payload sequence was co-transfected with one of the two versions of the regulator sequence. The "+" after "Type R Sensor" below the graph indicates that the payload sequence was co-transfected with the version of the regulator containing the type R sensor that recognizes miR-i. The mean fluorescence intensity (MFI, au) of the mCherry regulator reporter and mVenus payload reporter in A549 lung cancer cells was measured by flow cytometry. [Modes for carrying out the invention]

[0081] This disclosure generally relates to a method for producing engineered immune cells for extracorporeal cell therapy in a subject in need thereof, comprising contacting the immune cells obtained by the subject with a synthesis circuit in a closed in-line system connected to the subject, wherein the synthesis circuit is capable of selectively expressing a payload in the immune cells. Also provided herein is a method for extracorporeal treatment of a disease or condition in a subject in need of treatment, comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthesis circuit in a closed in-line system connected to the subject, wherein the synthesis circuit is capable of selectively expressing the payload in the immune cells. In some embodiments, this disclosure also includes a method for extracorporeal treatment of a disease or condition in a subject in need of treatment, comprising contacting the immune cells obtained by the subject with a synthesis circuit in a closed in-line system connected to the subject, and administering immune cells expressing a payload encoded by the synthesis circuit to the subject, wherein the synthesis circuit is capable of selectively expressing the payload in the immune cells. This disclosure also includes immune cells used in the method and an in-line system containing such cells.

[0082] A synthetic circuit useful to this disclosure is a programmable synthetic circuit that can be used to selectively control gene expression in immune cells. As further described herein, a synthetic circuit useful to this disclosure comprises a first nucleotide sequence encoding a payload (payload sequence) and a second nucleotide sequence encoding a regulator (regulator sequence), both of which include one or more “sensors,” i.e., target sites capable of recognizing and interacting with other molecules. For example, in some embodiments, the payload sequence includes a sensor (also referred herein as a “first type P sensor”) capable of recognizing a regulator (e.g., a regulator encoded by the regulator sequence), and the regulator sequence includes a sensor (also referred herein as a “type R sensor”) capable of recognizing a marker (e.g., miRNA expressed in immune cells). In some embodiments, the payload sequence may further include an additional type P sensor (also referred herein as a “second type P sensor”) capable of recognizing a marker (e.g., miRNA expressed in host cells). As further described herein, Type P and Type R sensors can be activated by recognition of their congeneral regulators or markers, thereby controlling the activity of payload and regulator sequences (e.g., inhibiting the expression of encoded proteins). While not bound by any particular theory, the synthetic circuits described herein, by using such sensors that are specifically programmable based on markers present in immune and / or non-immune cells, enable highly specific gene control and rapid decision-making. Additional aspects of this disclosure are provided throughout this application.

[0083] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains. In case of any conflict, including definitions, this application shall prevail. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0084] Throughout this disclosure, the terms “a” entity or “an” entity refer to one or more such entities, for example, “a polynucleotide” is understood to represent one or more polynucleotides. Accordingly, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

[0085] Furthermore, as used herein, “and / or” shall be considered a specific disclosure of each of two designated features or components, whether one is accompanied by the other or not. Accordingly, the term “and / or” as used herein in expressions such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in expressions such as “A, B, and / or C” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0086] Where a description of an aspect is made using the word “comprising,” it is understood that similar aspects are also provided, described using the terms “comprising” and / or “essentially consisting of.” As used herein, “comprising” is synonymous with “including,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding additional undescribed elements or method steps. As used herein, “comprising” excludes elements, steps, or components not specified in the elements of the claim. As used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claim.

[0087] The term "approximately" is used herein to mean roughly, nearly, around, or within a certain range. When the term "approximately" is used with a numerical range, it modifies the range by extending its boundary above or below the stated numerical value. Generally, unless otherwise stated herein, the term "approximately" is used to add or subtract a given value from a numerical value with a variation of 10 percent above or below (up or down). Unless otherwise stated herein, all numbers used herein, including in the claims, representing amounts of ingredients, reaction conditions, etc., should be understood to be modified by the term "approximately" in all instances. Therefore, unless otherwise stated, numerical parameters are approximations and may vary depending on the desired properties to be obtained. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted with regard to significant figures and customary rounding methods.

[0088] The term "at least" preceding a number or set of numbers is understood to include all subsequent numbers or integers that are logically included from the numbers adjacent to the term "at least" and from the context. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a set of numbers or a range, it is understood that "at least" can modify each number in that set of numbers or range. "At least" is not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, and 5.18%, without considering the number of significant figures).

[0089] The terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “nucleic acid sequence,” “polynucleotide,” and their grammatical variations are used interchangeably and refer to phosphate polymers of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”) in either single-stranded or double-stranded helical form, or any phosphoester analogs thereof, such as phosphorothioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, in particular DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any specific tertiary form. Therefore, this term includes, among other things, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and double-stranded DNA found in chromosomes. When describing the structure of a particular double-stranded DNA molecule, the sequence may be described herein in accordance with the usual convention of providing only the 5' to 3' sequence along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation. Examples of DNA include, but are not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A “nucleic acid composition” of this disclosure comprises one or more nucleic acids as described herein. As described herein, a polynucleotide of this disclosure comprises DNA, RNA, or both.In some embodiments, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) including tRNA, rRNA, shRNA, siRNA, miRNA, and mRNA, whether spliced ​​or not, any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide (normucleotidic) backbone, such as polyamides (e.g., peptide nucleic acids “PNA”) and polymorpholinopolymers, and other sequence-specific synthetic nucleic acid polymers that contain nucleic acid bases in a configuration that enables base pairing and base stacking as found in DNA and RNA.

[0090] As used herein, the term "polypeptide" encompasses both peptides and proteins unless otherwise specified.

[0091] The term "coding region" refers to a DNA or RNA region (transcribed region) that "codes" a specific protein, such as a payload and / or regulator.

[0092] The term “RNA” is used herein to mean a molecule containing at least one ribonucleotide residue. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. This term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinant RNA produced by the addition, deletion, substitution and / or modification of one or more nucleotides, which differs from naturally occurring RNA, e.g., modified RNA. The term “mRNA” refers to “messenger RNA” and refers to a “transcript” produced using a DNA template that codes for a peptide or protein. Typically, mRNA contains a 5'-UTR, a protein-coding region and a 3'-UTR. mRNA has a limited half-life in cells and in vitro. In relation to this disclosure, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. In some embodiments, as further described herein, RNA is linear RNA. In some aspects, RNA is circular RNA. In some aspects, RNA is self-replicating RNA. In some aspects, RNA is non-replicating RNA.

[0093] As used herein, the term “gene circuit” refers to a controllable gene expression system. Gene circuits useful to this disclosure as described herein include synthetic gene circuits (“synthetic circuits”). As used herein, the term “synthetic circuit” refers to an engineered, non-natural gene circuit. As is evident from this disclosure, the synthetic circuits described herein are specifically programmed to selectively express a payload in cells of interest (i.e., immune cells).

[0094] As used herein, the term “self-replicating RNA” refers to RNA (e.g., mRNA) that can direct itself to amplify or replicate within a cell (also referred to herein as “repRNA”). The RNA molecule must encode an enzyme(s) (e.g., alphaviral nonstructural proteins nsP1, nsP2, nsP3, nsP4) necessary for catalyzing RNA amplification, and must also contain a cis-RNA sequence necessary for replication that is recognized and utilized by the encoded enzyme(s). An alphaviral RNA vector replicon must contain elements in the following order: a 5' viral sequence or cell sequence required for amplification mediated by non-structural proteins (also referred to as a 5'CSE, or 5' cis replication sequence, or cis 5' viral sequence required for replication, or a 5' sequence capable of initiating alphaviral transcription); a sequence encoding an alphaviral non-structural protein that is biologically active when expressed (e.g., nsP1, nsP2, nsP3, nsP4); and a 3' viral sequence or cell sequence required for amplification mediated by non-structural proteins (also referred to as a 3'CSE, or cis 3' viral sequence required for replication, or an alphaviral RNA polymerase recognition sequence). In certain embodiments, an alphaviral RNA vector replicon may contain means for expressing one or more heterologous sequences, e.g., an IRES or a viral (e.g., alphaviral) subgenome promoter (e.g., a linkage region promoter), which may be modified to increase or decrease viral transcription of a subgenome fragment, or to reduce homology with a deletion-type helper or structural protein expression cassette, and one or more heterologous sequences to be expressed. The replicon may also contain additional sequences, such as one or more heterologous sequences encoding one or more polypeptides (e.g., protein-coding genes or 3' proximal genes) and / or polyadenylated regions. The replicon should not contain sequences encoding all of the alphavirus structural proteins (capsid, E1, E2).Non-exclusive examples of heterologous sequences that can be expressed by repliconvectors are described, for example, in U.S. Patent No. 6,015,686 (which is incorporated herein by reference in its entirety), and include, for example, antigens, lymphokines, cytokines, and so on.

[0095] As used herein, the term “circular RNA” refers to RNA (e.g., mRNA) that forms a circular structure by covalent bonding. In the context of this disclosure, circular RNA can be prepared by methodologies known to those skilled in the art (e.g., Wesselhoeft, RA et al., 2018, Nature communications, 2018, 9(1), 1-10 (which is incorporated herein by reference in its entirety)). As will be apparent from this disclosure, either the payload sequence and / or the regulator sequence may be in the form of circular RNA. Accordingly, in some embodiments, the synthetic circuits provided herein include a payload sequence which is circular RNA. In some embodiments, the synthetic circuits provided herein include a regulator sequence which is circular RNA. In some embodiments, the synthetic circuits provided herein include a payload sequence and a regulator sequence, wherein the payload sequence is circular RNA and the regulator sequence is circular RNA. Unless otherwise stated, circular RNA is not self-replicating.

[0096] As used herein, the term “payload sequence” refers to the nucleotide sequence that encodes the payload. As used herein, the term “payload” refers to any protein that may be encoded by the payload sequence. In some embodiments, the payload includes a therapeutic protein. Unless otherwise stated, as described herein, the payload does not include a regulator. Non-limiting examples of payloads are provided elsewhere in this disclosure.

[0097] As used herein, the term “regulator sequence” refers to a nucleotide sequence that codes for a regulator. As used herein, the term “regulator” includes any active substance that can control the expression of a payload encoded by a payload sequence. Non-limiting examples of such regulators are provided elsewhere in this disclosure. Furthermore, as described herein, regulators useful to this disclosure can be specifically recognized by a type P sensor on the payload sequence. Furthermore, as described herein, in some embodiments, when a regulator is specifically recognized by a type P sensor, the expression of the payload (coded by the payload sequence) is reduced or inhibited.

[0098] As used herein, the term “sensor” means any part capable of recognizing the markers and / or regulators described herein. As used herein, “recognizing” a marker (or regulator) may include a physical interaction between the marker (or regulator) and the sensor, such as the marker binding to a specific marker recognition site within the sensor.

[0099] As used herein, the term “Type P sensor” refers to a sensor present on a payload sequence. Therefore, a payload sequence useful to this disclosure includes a code region encoding a payload (“Payload Code Region”) and a Type P sensor. In some embodiments, the payload sequence may include multiple Type P sensors. As further described herein, in some embodiments, the payload sequence may include (a) a payload code region, (b) a first Type P sensor capable of recognizing a regulator, and (c) a second Type P sensor capable of recognizing a marker (“Type P Marker”). Unless otherwise stated, recognition of the Type P Marker by the second Type P sensor activates the second Type P sensor, resulting in reduced or inhibited expression of the encoded payload.

[0100] As used herein, the term “Type R sensor” refers to a sensor present on a regulator array. In some embodiments, a regulator array that may be used to construct the composite circuits described herein includes (a) a code region encoding a regulator ("regulator code region") and a Type R sensor, the Type R sensor capable of recognizing a marker ("Type R marker"). Unless otherwise stated, recognition of a Type R marker by the Type R sensor activates the Type R sensor, resulting in reduced or inhibited expression of the encoded regulator.

[0101] When used to describe a marker (e.g., a type P marker and / or a type R marker), the term “sufficient level” refers to the amount of marker needed to be recognized by a sensor (e.g., a type P sensor and / or a type R sensor) and to mediate downregulation of the sequence containing the sensor. As is evident from this disclosure, downregulation of a sequence can reduce or inhibit the expression of any protein encoded by the sequence (e.g., payload and / or regulator). Thus, in some embodiments, even if a cell expresses a marker, if the cell does not express an sufficient level of the marker, the sensor specific to that marker may remain inactive.

[0102] The term “sequence identity” is used herein to mean the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, determined by comparing sequences. In certain embodiments, sequence identity is calculated based on the total length or a portion thereof of two given sequence numbers. That portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both sequence numbers, or any other specific percentage. The term “identity” may also mean, depending on the context, the degree of sequence relevance between amino acid sequences or nucleic acid sequences, which is determined by the matching of strings of such sequences.

[0103] In some embodiments, the methods for determining identity are designed to obtain the greatest match between the sequences being tested. Methods for determining identity and similarity are systematized in publicly available computer programs.

[0104] As used herein, the terms “effective dose” or “therapeutic effective dose” (e.g., of any synthetic circuit disclosed herein) mean an amount sufficient to produce a beneficial or desired outcome (including clinical outcomes) when administered to a subject (including humans), and therefore, “effective dose” or any synonymous term depends on the context in which it is applied.

[0105] In vitro cell therapy Some aspects of the present disclosure include a method for producing engineered immune cells for extracorporeal cell therapy in a subject requiring such therapy, the method comprising contacting the immune cells obtained from the subject with a synthesis circuit in a closed in-line system connected to the subject. In some aspects, the synthesis circuit can selectively express a payload in immune cells, such as T cells, but cannot express the payload in non-immune cells.

[0106] In some embodiments, immune cells are not exposed to the outside of the in-line system from the beginning to the end of therapy. In some embodiments, immune cells are leached from the target via the in-line system, transfected by a synthetic circuit to express a payload, and then reinjected into the target. In some embodiments, immune cells express the payload on the immune cells as a result of contact. In some embodiments, immune cells are returned directly from the in-line system to the target.

[0107] In some embodiments, the method of the present disclosure includes (1) connecting a closed inline system to a subject requiring treatment, (2) collecting immune cells from the subject within the inline system, (3) bringing the immune cells into contact with a synthesis circuit, and (4) reintroducing the immune cells to the subject.

[0108] The Disclosure also provides a method for in vitro treating a disease or condition in a subject requiring treatment, comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthetic circuit in a closed in-line system connected to the subject, and the synthetic circuit is capable of selectively expressing the payload in the immune cells. In some embodiments, the Disclosure includes a method for in vitro treating a disease or condition in a subject requiring treatment, comprising contacting immune cells obtained from the subject with a synthetic circuit in a closed in-line system connected to the subject, and administering immune cells expressing a payload encoded by the synthetic circuit to the subject, wherein the synthetic circuit is capable of selectively expressing the payload in the immune cells.

[0109] One advantage of this disclosure, though not limited to this, is that the subjects receiving the therapy do not need to undergo lymphocyte depletion before treatment. Another advantage of this disclosure is that the rapid process of programmed payload expression results in less fatigue or exhaustion of immune cells. In some embodiments, the therapy is administered in less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours.

[0110] In some embodiments, the method utilizes a closed, in-line system connected to a target, as described in US20210244871A1, published on August 12, 2021, or WO2022072885A1, published on April 7, 2022 (both of which are incorporated herein by reference).

[0111] Cells, for example, immune cells Some aspects of this disclosure relate to cells expressing a payload, such as a chimeric antigen receptor, via a synthesis circuit in an in-line system connected to a target.

[0112] In some embodiments, the cells are immune cells. In some embodiments, the cells include pluripotent or totuitous cells. In certain embodiments, the pluripotent cells include embryonic stem cells (ESCs). In some embodiments, the cells are hematopoietic stem cells.

[0113] In some embodiments, starting cells, e.g., immune cells, are isolated from a human subject. In some embodiments, starting cells are isolated from a human subject for allogeneic cell therapy. In some embodiments, the cells are T cells. In some embodiments, the cells are NK cells. In some embodiments, the cells are Tregs. In some embodiments, the cells, e.g., T cells and / or NK cells, are isolated from a human subject. In some embodiments, the cells, e.g., T cells and / or NK cells, are engineered to contain a chimeric antigen receptor (CAR) encoded by a synthetic pathway. In some embodiments, the cells, e.g., T cells and / or NK cells, are engineered to contain an engineered T cell receptor (TCR) encoded by a synthetic pathway.

[0114] In some embodiments, the cell comprises a construct expressing an antigen receptor and / or another additional polypeptide. In some embodiments, the antigen receptor comprises an antibody, a modified antibody such as scFv, a CAR, a modified TCR, a TCR mime (e.g., an antibody-T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), or a chimeric signaling receptor (CSR). For example, the TCR may comprise a modified TCR in which the antigen-binding domain of the TCR (e.g., an alpha / beta TCR or a gamma / delta TCR) is replaced by the antigen-binding domain of an antibody (with or without the constant domain of the antibody), such a modified TCR is specific to the antibody antigen while retaining the signaling function of the TCR. Chimeric signaling receptors may include (1) an extracellular binding domain (e.g., native / modified receptor extracellular domain, native / modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or a domain that recruits and / or activates JAK / STAT, kinases, phosphatases, and ubiquitin, SH3, SH2, and PDZ). See, for example, EP340793B1, WO2017 / 070608, WO2018 / 200582, WO2018 / 200583, WO2018 / 200585, and Xu et al., Cell Discovery (2018) 4:62.

[0115] In some embodiments, the antigen receptor targets the antigen of interest (e.g., tumor antigen or pathogen antigen). Antigens include AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, Braf, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, and CD1 38, CD171, CD352, CEA (carcinoembryonic antigen), claudin 18.2, claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast-activating protein α), fetus Fetal AchR (fetal acetylcholine receptor), FBP (folate-binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanylic acid cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma receptor). (Trivalent antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insertion domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat containing 8 family member A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (Melan A), mouse cytomegalovirus (MCMV),MCSP (Melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neuronal cell adhesion molecule), nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, tumor embryo antigen, PD-1, PD-L1, PRAME (melanoma preferential expression antigen), progesterone receptor, PSA (prostate-specific antigen), PSCA Examples of antigens that may be included, but are not limited to, prostate stem cell antigens, PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate 6-transmembrane epithelial antigen 1), Survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens derived from HIV, HBV, HCV, HPV, and other pathogens.

[0116] In certain embodiments, the antigen receptor targets hTERT. In some embodiments, the antigen receptor targets KRAS. In some embodiments, the antigen receptor targets Braf. In some embodiments, the antigen receptor targets TGFβRII. In some embodiments, the antigen receptor targets MAGEA10 / A4. In some embodiments, the antigen receptor targets AFP. In some embodiments, the antigen receptor targets PRAME. In some embodiments, the antigen receptor targets MAGEA1. In some embodiments, the antigen receptor targets WT-1. In some embodiments, the antigen receptor targets NY-ESO. In some embodiments, the antigen receptor targets CD19.

[0117] In some embodiments, the antigen receptor targets BCMA. In some embodiments, the antigen receptor targets CD147. In some embodiments, the antigen receptor targets CD19. In some embodiments, the antigen receptor targets CD19 and CD22. In some embodiments, the antigen receptor targets CD19 and CD28. In some embodiments, the antigen receptor targets CD20. In some embodiments, the antigen receptor targets CD20 and CD19. In some embodiments, the antigen receptor targets CD22. In some embodiments, the antigen receptor targets CD30. In some embodiments, the antigen receptor targets CEA. In some embodiments, the antigen receptor targets DLL3. In some embodiments, the antigen receptor targets EGFRvIII. In some embodiments, the antigen receptor targets GD2. In some embodiments, the antigen receptor targets HER2. In some embodiments, the antigen receptor targets IL-1RAP. In some embodiments, the antigen receptor targets mesothelin. In some embodiments, the antigen receptor targets mesothelin. In some embodiments, the antigen receptor targets NKG2D. In some embodiments, the antigen receptor targets PSMA. In some embodiments, the antigen receptor targets TnMUC1.

[0118] synthesis circuit Provided herein are methods for producing cells for in vitro therapy using a synthetic circuit comprising a plurality of nucleotide sequences (e.g., a first nucleotide sequence and a second nucleotide sequence), wherein one or more of the plurality of nucleotide sequences comprises a sensor capable of controlling the activity and / or expression of one or more of the plurality of nucleotide sequences. Thus, in some embodiments, the disclosure relates to a method using a polynucleotide (e.g., isolated polynucleotide) comprising (a) a nucleotide sequence and (b) at least one sensor, wherein the at least one sensor controls the activity and / or expression of the nucleotide sequence. For example, in some embodiments, the nucleotide sequence encodes a payload (payload sequence), and at least one sensor (type P sensor) can recognize a marker (e.g., a type P marker), and when the sensor recognizes the marker, the sensor is activated and thereby controls the expression of the encoded protein (e.g., payload). In some embodiments, the nucleotide sequence comprises a regulator (regulator sequence), and at least one sensor (type R sensor) can recognize a marker (e.g., a type R marker), and when the sensor recognizes the marker, the sensor is activated and thereby controls the expression of the regulator. As used herein, controlling “regulator expression” may include (i) controlling the amount of regulator expressed in cells, (ii) controlling the activity of the regulator, or (iii) both (i) and (ii). Similarly, controlling “payload expression” may include (i) controlling the amount of payload expressed in cells, (ii) controlling the activity of the payload, or (iii) both (i) and (ii).

[0119] In some embodiments, provided herein are methods utilizing a synthetic circuit for in vitro therapy, wherein the synthetic circuit comprises (a) a first nucleotide sequence encoding a payload (payload sequence) and optionally (b) a second nucleotide sequence encoding a regulator (regulator sequence), the payload sequence comprising a sensor capable of recognizing the regulator (type P sensor), and the regulator sequence comprising a sensor capable of recognizing a marker expressed in a cell (type R sensor). In some embodiments, recognition of the marker by the type R sensor reduces or inhibits the expression of the regulator. Inhibition of regulator expression prevents activation of the type P sensor, thereby enabling the expression of the encoded payload.

[0120] In some embodiments, synthetic circuits useful to this disclosure include a nucleotide sequence encoding a payload (payload sequence). The payload sequence may include a sensor capable of recognizing a marker (e.g., microRNA). In some embodiments, when the sensor recognizes the marker, the expression of the payload is prevented or reduced compared to other cells that do not contain the marker. Non-limiting examples of such synthetic circuits are described in U.S. Patent No. 10,000,757B2 issued June 19, 2018, or PCT Publication WO2007000668A2 published December 21, 2007, which are incorporated herein by reference in their entirety. In some embodiments, synthetic circuits useful to this disclosure include a payload sequence but do not include a regulator sequence. In some embodiments, the synthetic circuit includes a nucleotide sequence encoding a payload (payload sequence) that includes a sensor capable of recognizing a marker (type P sensor). In some embodiments, the type P sensor comprises at least one type P sensor, and the marker (e.g., miRNA) is not endogenously expressed only in target cells, e.g., immune cells, or the marker is endogenously present only in target cells, e.g., immune cells.

[0121] In some embodiments, synthetic circuits useful to the present disclosure include a synthetic RNA circuit comprising: a first nucleotide sequence comprising at least one sensor capable of recognizing a first marker (e.g., a first microRNA) specifically expressed in a first cell type (e.g., immune cells); and a second nucleotide sequence comprising at least one sensor recognized by at least one second marker (e.g., microRNA) that is not expressed in the first cell type (e.g., immune cells) or is expressed at a lower level compared to a second cell type. Exemplary synthetic circuits, but not limited to them, are described in PCT Publication WO2016040395A1, published on March 17, 2016, which is incorporated herein by reference in its entirety.

[0122] Payload array As will be apparent from this disclosure, in some embodiments, a synthetic circuit useful to this disclosure comprises a nucleotide sequence encoding a payload (payload sequence). In some embodiments, the payload sequence may encode any suitable protein known in the art. Non-limiting examples of suitable payloads include chimeric antigen receptors (CARs), T cell receptors (TCRs), TCR mimics, or combinations thereof.

[0123] In some embodiments, the payload sequence is linear (e.g., linear RNA). In some embodiments, the payload sequence is circular (e.g., circular RNA). In some embodiments, the payload sequence is self-replicating (e.g., self-replicating RNA). In some embodiments, the payload sequence is non-replicating (e.g., non-replicating RNA).

[0124] Type P sensor In some embodiments, a payload array useful to the present disclosure includes a sensor capable of recognizing a regulator (e.g., encoded by a regulator array). In some embodiments, the payload array includes a sensor capable of recognizing a marker. In some embodiments, the payload array includes a first sensor (first type P sensor) capable of recognizing a regulator and a second sensor (second type P sensor) capable of recognizing a marker.

[0125] Accordingly, in some embodiments, provided herein is a synthetic circuit comprising (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein the payload sequence comprises a first sensor capable of recognizing the regulator (first type P sensor) and a second sensor capable of recognizing a marker (second type P sensor). In some embodiments, the first type P sensor and the second type P sensor are different (i.e., they do not recognize the same ligand). In some embodiments, the composite circuit provided herein includes a plurality of sensors. For example, in some embodiments, the payload array provided herein includes a plurality of type P sensors. In some embodiments, each of the plurality of sensors on the payload array is the same. In some embodiments, one or more of the plurality of sensors on the payload array are different. If the payload array includes a first type P sensor (e.g., one that recognizes a regulator) and a second type P sensor (e.g., one that recognizes a marker), in some embodiments, the payload array includes a plurality of first type P sensors. For example, in some embodiments, the payload array includes about 2 first type P sensors, about 3 first type P sensors, about 4 first type P sensors, about 5 first type P sensors, about 6 first type P sensors, about 7 first type P sensors, about 8 first type P sensors, about 9 first type sensors, about 10 first type P sensors, about 11 first type P sensors, about 12 first type P sensors, about 13 first type P sensors, about 14 first type P sensors, about 15 first type P sensors, about 16 first type P sensors, about 17 first type P sensors, about 18 first type P sensors, about 19 first type P sensors, or about 20 or more first type P sensors. In some embodiments, the payload array includes at least 2 first type P sensors. In some embodiments, the payload array includes at least 3 first type P sensors. In some embodiments, the payload array includes at least 4 first type P sensors. In some embodiments, the payload array includes at least five first type P sensors. In some embodiments, the payload array includes at least six first type P sensors. In some embodiments, the payload array includes at least seven first type P sensors. In some embodiments, the payload array includes at least eight first type P sensors. In some embodiments, the payload array includes at least nine first type P sensors.In some embodiments, the payload array includes at least 10 first type P sensors. In some embodiments, the payload array includes at least 11 first type P sensors. In some embodiments, the payload array includes at least 13 first type P sensors. In some embodiments, the payload array includes at least 13 first type P sensors. In some embodiments, the payload array includes at least 14 first type P sensors. In some embodiments, the payload array includes at least 15 first type P sensors. In some embodiments, the payload array includes at least 16 first type P sensors. In some embodiments, the payload array includes at least 17 first type P sensors. In some embodiments, the payload array includes at least 18 first type P sensors. In some embodiments, the payload array includes at least 19 first type P sensors. In some embodiments, the payload array includes at least 20 first type P sensors.

[0126] In some embodiments, each of the first type P sensors is the same. For example, in some embodiments, the composite circuit described herein includes a payload array and a regulator array, the payload array including a plurality of first type P sensors, each of the plurality of first type P sensors specifically recognizes the same regulator (for example, each of the first type P sensors includes the same coupling site for the regulator). In some embodiments, one or more of the first type P sensors are different. For example, in some embodiments, one or more of the first type P sensors recognize different regulators. In some embodiments, one or more of the first type P sensors recognize different coupling sites on the same regulator.

[0127] In some embodiments, if the payload array includes a first type P sensor (e.g., one that recognizes a regulator) and a second type P sensor (e.g., one that recognizes a marker), the payload array includes a plurality of second type P sensors. For example, in some embodiments, the payload array includes about 2 second type P sensors, about 3 second type P sensors, about 4 second type P sensors, about 5 second type P sensors, about 6 second type P sensors, about 7 second type P sensors, about 8 second type P sensors, about 9 second type P sensors, about 10 second type P sensors, about 11 second type P sensors, about 12 second type P sensors, about 13 second type P sensors, about 14 second type P sensors, about 15 second type P sensors, about 16 second type P sensors, about 17 second type P sensors, about 18 second type P sensors, about 19 second type P sensors, or about 20 or more second type P sensors. In some embodiments, the payload array includes at least two second type P sensors. In some embodiments, the payload array includes at least three second type P sensors. In some embodiments, the payload array includes at least four second type P sensors. In some embodiments, the payload array includes at least five second type P sensors. In some embodiments, the payload array includes at least six second type P sensors. In some embodiments, the payload array includes at least seven second type P sensors. In some embodiments, the payload array includes at least eight second type P sensors. In some embodiments, the payload array includes at least nine second type P sensors. In some embodiments, the payload array includes at least ten second type P sensors. In some embodiments, the payload array includes at least twelve second type P sensors. In some embodiments, the payload array includes at least thirteen second type P sensors. In some embodiments, the payload array includes at least fourteen second type P sensors.In some embodiments, the payload array includes at least 15 second type P sensors. In some embodiments, the payload array includes at least 16 second type P sensors. In some embodiments, the payload array includes at least 17 second type P sensors. In some embodiments, the payload array includes at least 18 second type P sensors. In some embodiments, the payload array includes at least 19 second type P sensors. In some embodiments, the payload array includes at least 20 second type P sensors.

[0128] In some embodiments, each of the second type P sensors is the same. For example, in some embodiments, the synthesis circuit includes a payload array and a regulator array, the payload array includes a plurality of second type P sensors, and each of the plurality of second type P sensors specifically recognizes the same marker. In some embodiments, one or more of the second type P sensors are different. In some embodiments, one or more of the second type P sensors specifically recognize different markers. In some embodiments, one or more of the second type P sensors recognize different binding sites on the same marker.

[0129] In some embodiments, if the payload array includes a first type P sensor (e.g., for recognizing a regulator) and a second type P sensor (e.g., for recognizing a marker), the payload array includes a plurality of first type P sensors and a plurality of second type P sensors. In some embodiments, the payload array is (a) approximately 2 first type P sensors, approximately 3 first type P sensors, approximately 4 first type P sensors, approximately 5 first type P sensors, approximately 6 first type P sensors, approximately 7 first type P sensors, approximately 8 first type P sensors, approximately 9 first type sensors, approximately 10 first type P sensors, approximately 11 first type P sensors, approximately 12 first type P sensors, approximately 13 first type P sensors, approximately 14 first type P sensors, approximately 15 first type P sensors, approximately 16 first type P sensors, approximately 17 first type P sensors, approximately 18 first type P sensors, approximately 19 first type P sensors, or approximately 20 or more first type P sensors, or approximately 20 or more first type P sensors. (b) a first type P sensor, (b) about two second type P sensors, about three second type P sensors, about four second type P sensors, about five second type P sensors, about six second type P sensors, about seven second type P sensors, about eight second type P sensors, about nine second type P sensors, about ten second type P sensors, about eleven second type P sensors, about twelve second type P sensors, about thirteen second type P sensors, about fourteen second type P sensors, about fifteen second type P sensors, about sixteen second type P sensors, about seventeen second type P sensors, about eighteen second type P sensors, about nineteen second type P sensors, or about twenty or more second type P sensors, or (c) including both (a) and (b). In some embodiments, as further described herein, each of the first type P sensors is the same. In some embodiments, one or more of the first type P sensors are different. In some embodiments, each of the second type P sensors is the same. In some embodiments, one or more of the first type P sensors are different.

[0130] Type P Spacer In some embodiments, payload sequences useful to the present disclosure further include spacer sequences ("Type P Spacers"). In some embodiments, the payload sequence includes a plurality of Type P Spacers. For example, in some embodiments, the payload sequence includes about 2 Type P Spacers, about 3 Type P Spacers, about 4 Type P Spacers, about 5 Type P Spacers, about 6 Type P Spacers, about 7 Type P Spacers, about 8 Type P Spacers, about 9 Type P Spacers, or about 10 or more Type P Spacers. In some embodiments, the payload sequence includes at least 2 Type P Spacers. In some embodiments, the payload sequence includes at least 3 Type P Spacers. In some embodiments, the payload sequence includes at least 4 Type P Spacers. In some embodiments, the payload sequence includes at least 5 Type P Spacers. In some embodiments, the payload sequence includes at least 6 Type P Spacers. In some embodiments, the payload sequence includes at least 7 Type P Spacers. In some embodiments, the payload sequence includes at least 8 Type P Spacers. In some embodiments, the payload array includes at least nine type P spacers. In some embodiments, the payload array includes at least ten type P spacers. In some embodiments, each of the type P spacers is the same. In some embodiments, one or more of the type P spacers are different.

[0131] Accordingly, in some embodiments, the synthesis circuits provided herein include a payload sequence, which includes a type P sensor (e.g., a regulator and / or marker) and a type P spacer. In some embodiments, the type P spacer is located upstream of the type P sensor (e.g., the type P spacer is located closer to the 5' end of the payload sequence compared to the type P sensor). In some embodiments, the type P spacer is located downstream of the type P sensor (e.g., the type P spacer is located closer to the 3' end of the payload sequence compared to the type P sensor). In some embodiments, the type P sensor is located downstream of the coding region of the payload sequence, and the type P spacer is located between the coding region of the payload sequence and the type P sensor (e.g., after the stop codon of the coding region and before the start of the type P sensor). As used herein, the term “coding region of the payload sequence” refers to the portion of the payload sequence that specifically codes for the payload.

[0132] In some embodiments, when the payload array includes multiple type P sensors, the type P spacer is located upstream of one or more of the multiple type P sensors. In some embodiments, the type P spacer is located downstream of one or more of the multiple type P sensors. In some embodiments, the type P spacer is located between at least two of the type P sensors. In some embodiments, each of the multiple type P sensors is separated by a type P spacer. For example, in some embodiments, the synthesis circuit provided herein includes a payload array which includes a first type P sensor (e.g., specifically recognizing a regulator), a second type P sensor (e.g., specifically recognizing a marker), and a type P spacer, which is located between the first type P sensor and the second type P sensor. As described herein, in some embodiments, the payload array includes multiple first type P sensors, each of which is separated by a type P spacer. In some embodiments, the payload array includes multiple second type P sensors, each of which is separated by a type P spacer. In some embodiments, the payload array comprises a plurality of first type P sensors and a plurality of second type P sensors, wherein (a) each of the plurality of first type P sensors is separated by a type P spacer, (b) each of the plurality of second type P sensors is separated by a type P spacer, and (c) both (a) and (b).

[0133] If the payload sequence includes multiple type P spacers, in some embodiments, each of the type P spacers is the same. In some embodiments, one or more of the type P spacers are different. Although not bound by any theory, in some embodiments, a type P spacer useful to the present disclosure is one of a length appropriate for the spacer to assist in the binding of a type P sensor to its ligand (e.g., a regulator and / or marker). In some embodiments, the type P spacer is about 1 to about 100 nucleotides long. In some embodiments, the type P spacer is about 1 nucleotide, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, about 75 nucleotides, about 80 nucleotides, about 85 nucleotides, about 90 nucleotides, about 95 nucleotides, or about 100 nucleotides long. In some embodiments, the type P spacer is about 1 to about 50 nucleotides long. In some embodiments, the type P spacer is about 5 nucleotides long. In some embodiments, the type P spacer is about 10 nucleotides long. In some embodiments, the type P spacer is about 15 nucleotides long. In some embodiments, the type P spacer is about 20 nucleotides long. In some embodiments, the type P spacer is about 25 nucleotides long, in some embodiments, the type P spacer is about 30 nucleotides long, and in some embodiments, the type P spacer is about 35 nucleotides long. In some embodiments, the type P spacer is about 40 nucleotides long. In some embodiments, the type P spacer is about 45 nucleotides long. In some embodiments, the type P spacer is about 50 nucleotides long.

[0134] Unless otherwise stated, a type P spacer useful in this disclosure is not limited to any particular nucleotide sequence, as long as the type P spacer is long enough to perform its intended function (e.g., assisting in the binding of a type P sensor to its ligand). Therefore, in some embodiments, a type P spacer useful in this disclosure includes a randomly generated nucleotide sequence. In some embodiments, when multiple type P spacers are used to separate multiple type P sensors, one or more of the multiple type P spacers have different sequences, and as a result, the multiple type P spacers do not include a randomly generated repeating nucleotide sequence. In some embodiments, a type P spacer useful in this disclosure includes, is essentially derived from, or consists of the sequence tttcctttcccccttccctttttcctttcctttcctttcccccttccctt (SEQ ID NO: 1) or a fragment thereof. In some embodiments, a type P spacer includes the sequence described in SEQ ID NO: 1. In some embodiments, a type P spacer is essentially derived from the sequence described in SEQ ID NO: 1. In some embodiments, a type P spacer consists of the sequence described in SEQ ID NO: 1. In some embodiments, a type P spacer useful for the present disclosure includes, is essentially derived from, or consists of the sequence ttcctttcccccttccctt (SEQ ID NO: 2) or a fragment thereof. In some embodiments, the type P spacer includes the sequence described in SEQ ID NO: 2. In some embodiments, the type P spacer is essentially derived from the sequence described in SEQ ID NO: 2. In some embodiments, the type P spacer consists of the sequence described in SEQ ID NO: 2. In some embodiments, a type P spacer useful for the present disclosure includes, is essentially derived from, or consists of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some embodiments, the type P spacer includes the sequence described in SEQ ID NO: 3. In some embodiments, the type P spacer is essentially derived from the sequence described in SEQ ID NO: 3. In some embodiments, the type P spacer consists of the sequence described in SEQ ID NO: 3.In some embodiments, a Type P spacer is the same as a Type R spacer (as further described elsewhere in this disclosure). In some embodiments, a Type P spacer is different from a Type R spacer. [Table 1]

[0135] Regulator array As described herein, in some embodiments, the synthesis circuit includes a regulator or a nucleotide sequence encoding it ("regulator sequence"). Thus, in some embodiments, the synthesis circuit of the present disclosure includes a payload sequence (e.g., any of the payload sequences described above) and a regulator sequence. Non-limiting examples of regulators that can be used in the present disclosure are provided elsewhere in the present disclosure.

[0136] In some embodiments, the regulator sequence is linear (e.g., linear RNA). In some embodiments, the regulator sequence is circular (e.g., circular RNA). In some embodiments, the regulator sequence is non-replicating (e.g., non-replicating RNA).

[0137] Type R sensor In some embodiments, the regulator array includes sensors capable of specifically recognizing markers. Accordingly, some aspects of the present disclosure relate to a composite circuit comprising a payload array and a regulator array, wherein the payload array includes sensors (Type P sensors) and the regulator array includes sensors ("Type R sensors"). In some embodiments, the present disclosure provides a composite circuit comprising a payload array and a regulator array, wherein the payload array includes a first Type P sensor (e.g., specifically recognizing a regulator) and a second Type P sensor (e.g., specifically recognizing a marker), and the regulator array includes a Type R sensor (e.g., specifically recognizing a marker). Where the composite circuit described herein includes both Type P sensors and Type R sensors, in some embodiments, the Type P sensors and Type R sensors are not the same (e.g., they do not specifically recognize the same ligand).

[0138] In some embodiments, a regulator array useful to this disclosure includes a plurality of sensors. For example, in some embodiments, a regulator array includes about 2 Type R sensors, about 3 Type R sensors, about 4 Type R sensors, about 5 Type R sensors, about 6 Type R sensors, about 7 Type R sensors, about 8 Type R sensors, about 9 Type R sensors, or about 10 or more Type R sensors. In some embodiments, a regulator array includes at least 2 Type R sensors. In some embodiments, a regulator array includes at least 3 Type R sensors. In some embodiments, a regulator array includes at least 4 Type R sensors. In some embodiments, a regulator array includes at least 5 Type R sensors. In some embodiments, a regulator array includes at least 6 Type R sensors. In some embodiments, a regulator array includes at least 7 Type R sensors. In some embodiments, a regulator array includes at least 8 Type R sensors. In some embodiments, a regulator array includes at least 9 Type R sensors. In some embodiments, a regulator array includes at least 10 Type R sensors.

[0139] In some embodiments, each of the multiple sensors on the regulator array is the same. For example, in some embodiments, the synthesis circuit described herein includes a payload array and a regulator array, the regulator array including multiple type R sensors, each of the multiple type R sensors recognizing the same marker (for example, each type R sensor includes the same binding site for the marker). In some embodiments, one or more of the multiple sensors on the regulator array are different. For example, in some embodiments, one or more of the multiple type R sensors recognize different markers. In some embodiments, one or more of the multiple type R sensors recognize different binding sites for the same marker.

[0140] Type R Spacer In some embodiments, the regulator array provided herein further includes a spacer array ("Type R spacer"). Thus, in some embodiments, the composite circuit provided herein includes a payload array and a regulator array, the payload array including a Type P sensor (e.g., a first Type P sensor and / or a second Type P sensor) and a Type P spacer, and the regulator array including a Type R sensor and a Type R spacer. In some embodiments, the Type R spacer and the Type P spacer are not the same. In some embodiments, the Type R spacer and the Type P spacer are the same.

[0141] In some embodiments, the regulator array includes a plurality of Type R spacers. In some embodiments, the regulator array includes about 2 Type R spacers, about 3 Type R spacers, about 4 Type R spacers, about 5 Type R spacers, about 6 Type R spacers, about 7 Type R spacers, about 8 Type R spacers, about 9 Type R spacers, or about 10 or more Type R spacers. In some embodiments, the regulator array includes at least 2 Type R spacers. In some embodiments, the regulator array includes at least 3 Type R spacers. In some embodiments, the regulator array includes at least 4 Type R spacers. In some embodiments, the regulator array includes at least 5 Type R spacers. In some embodiments, the regulator array includes at least 6 Type R spacers. In some embodiments, the regulator array includes at least 7 Type R spacers. In some embodiments, the regulator array includes at least 8 Type R spacers. In some embodiments, the regulator array includes at least 9 Type R spacers. In some embodiments, the regulator array includes at least 10 type R spacers. In some embodiments, each of the type R spacers is identical. In some embodiments, one or more of the type R spacers are different.

[0142] In some embodiments, the type R spacer is located upstream of the type R sensor in the regulator array. In some embodiments, the type R spacer is located downstream of the type R sensor. In some embodiments, if the regulator array codes for a regulator, the type R spacer is located between the coding region of the regulator array and the type R sensor (e.g., after the stop codon of the coding region and before the start of the type R sensor). As used herein, the term “coding region of the regulator array” refers to the portion of the regulator array that specifically codes for a regulator.

[0143] In some embodiments, when the regulator array includes multiple type R sensors, the type R spacer is located upstream of one or more of the multiple type R sensors. In some embodiments, the type R spacer is located downstream of one or more of the multiple type R sensors. In some embodiments, the type R spacer is located between at least two of the multiple type R sensors. In some embodiments, each of the multiple type R sensors is separated by a type R spacer.

[0144] If the regulator sequence includes multiple type R spacers, in some embodiments, each of the type R spacers is the same. In some embodiments, one or more of the type R spacers are different. In some embodiments, the type R spacers may be of any length appropriate for the type R spacer to assist in the binding of the type R sensor to its ligand (e.g., a marker). In some embodiments, the type R spacers are about 1 to about 100 nucleotides long. In some embodiments, the type R spacers are about 1 nucleotide, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, about 75 nucleotides, about 80 nucleotides, about 85 nucleotides, about 90 nucleotides, about 95 nucleotides, or about 100 nucleotides long. In some embodiments, the type R spacers are about 1 to about 50 nucleotides long. In some embodiments, the type R spacer is approximately 5 nucleotides long. In some embodiments, the type R spacer is approximately 10 nucleotides long. In some embodiments, the type R spacer is approximately 15 nucleotides long. In some embodiments, the type R spacer is approximately 20 nucleotides long. In some embodiments, the type R spacer is approximately 25 nucleotides long. In some embodiments, the type R spacer is approximately 30 nucleotides long. In some embodiments, the type R spacer is approximately 35 nucleotides long. In some embodiments, the type R spacer is approximately 40 nucleotides long. In some embodiments, the type R spacer is approximately 45 nucleotides long. In some embodiments, the type R spacer is approximately 50 nucleotides long.

[0145] Unless otherwise stated, a type R spacer useful in this disclosure is not limited to any particular nucleotide sequence, as long as the type R spacer is long enough to perform its intended function (e.g., assisting in the binding of a type R sensor to its ligand). Therefore, in some embodiments, a type R spacer useful in this disclosure includes a randomly generated nucleotide sequence. In some embodiments, when multiple type R spacers are used to separate multiple type R sensors, one or more of the multiple type R spacers have different sequences, and as a result, the multiple type R spacers do not include a randomly generated repeating nucleotide sequence. In some embodiments, a type R spacer useful in this disclosure includes, is essentially derived from, or consists of the sequence tttcctttcccccttccctttttcctttcctttcctttcccccttccctt (SEQ ID NO: 1) or a fragment thereof. In some embodiments, a type R spacer includes the sequence described in SEQ ID NO: 1. In some embodiments, a type R spacer is essentially derived from the sequence described in SEQ ID NO: 1. In some embodiments, a type R spacer consists of the sequence described in SEQ ID NO: 1. In some embodiments, a type R spacer useful for the present disclosure includes, is essentially derived from, or consists of the sequence ttcctttcccccttccctt (SEQ ID NO: 2) or a fragment thereof. In some embodiments, the type R spacer includes the sequence described in SEQ ID NO: 2. In some embodiments, the type R spacer is essentially derived from the sequence described in SEQ ID NO: 2. In some embodiments, the type R spacer consists of the sequence described in SEQ ID NO: 2. In some embodiments, a type R spacer useful for the present disclosure includes, is essentially derived from, or consists of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some embodiments, the type R spacer includes the sequence described in SEQ ID NO: 3. In some embodiments, the type R spacer is essentially derived from the sequence described in SEQ ID NO: 3. In some embodiments, the type R spacer consists of the sequence described in SEQ ID NO: 3.In some aspects, the Type R spacer is the same as the Type P spacer. In some aspects, the Type R spacer is different from the Type P spacer. [Table 2]

[0146] marker As is evident from this disclosure, the synthetic circuits described herein can be programmed to selectively control the expression of specific genes (or the proteins they encode) in immune cells. While not bound by any particular theory, in some embodiments, the payload sequence and / or regulator sequence include a sensor (e.g., a type P sensor or a type R sensor) programmed to recognize a specific marker, so that the sensor "works" (i.e., becomes active) only in cells containing sufficient levels of the marker for the sensor to recognize it. If the cell does not contain sufficient levels of the marker, the sensor does not specifically recognize the marker, and therefore the sensor "does not work" (i.e., is inactive). The following table summarizes possible scenarios with respect to marker levels and payload expression. Regulator expression states are also listed in the table. Unless otherwise stated, markers useful to this disclosure do not include the regulators described herein. [Table 3]

[0147] To supplement the explanation, in some embodiments, the synthetic circuits provided herein include a payload sequence, the payload sequence includes a type P sensor capable of specifically recognizing a marker expressed in non-immune cells, and the recognition of the marker by the type P sensor reduces or inhibits the expression of the encoded payload in non-immune cells. For such a synthetic circuit, when introduced into non-immune cells (i.e., expressing a sufficient level of the marker to be recognized by the type P sensor), the type P sensor is activated (i.e., binds to the marker), thereby inhibiting or reducing the expression of the encoded payload in non-immune cells. However, when introduced into immune cells (i.e., not expressing a sufficient level of the marker to be recognized by the type P sensor), the type P sensor remains inactive (i.e., does not bind to the ligand), and therefore the payload is expressed in the immune cells. As is evident from this disclosure, the payload can be selectively expressed if both of the following are true: (1) none of the second type P sensors are activated, and (2) one or more type R sensors are activated. Payload expression may be inhibited if either or both of the following two conditions are not met: (1) All type P markers are at a low level. (2) At least one of the type R markers is at a high level.

[0148] As used herein, “payload expression” (or its grammatical equivalent) means any of the following: (a) the amount of payload expressed in a cell, (b) the rate at which the payload is expressed in a cell, (c) the duration of payload expression, or (d) any combination of (a) to (c). Not bound by any theory, in some embodiments, the synthetic circuits provided herein enable selective expression of payloads in immune cells by controlling the expression of payloads and regulator sequences. For example, as is evident from this disclosure, when introduced into immune cells (i.e., cells that do not express sufficient levels of type P markers to be recognized by type P sensors, but express sufficient levels of type R markers to be recognized by at least one type R sensor), the expression of payloads in immune cells is increased compared to the expression of regulators in immune cells. In some embodiments, payload expression in immune cells increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to regulator expression in immune cells. In some embodiments, payload expression in immune cells increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times compared to regulator expression. In some embodiments, regulator expression in immune cells decreases in immune cells compared to payload expression.In some embodiments, regulator expression in immune cells is reduced by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 100% compared to payload expression in immune cells.

[0149] Unless otherwise stated, a marker includes any molecule that is expressed in a cell and can be specifically recognized by a sensor provided herein (e.g., a type P sensor and / or a type R sensor). As described herein, in some embodiments, a marker is selectively expressed (or expressed to a sufficient level) in certain cells but not in other cells. For example, in some embodiments, a marker is expressed to a sufficient level in a first cell to be recognized by a sensor (e.g., a type P sensor or a type R sensor) but not in a second cell. In such embodiments, when the synthetic circuit described herein is introduced into the first cell, the sensor (e.g., a type P sensor and / or a type R sensor) specifically recognizes the marker and is activated. When such a synthetic circuit is introduced into the second cell, the sensor (e.g., a type P sensor and / or a type R sensor) remains inactive (i.e., does not bind to the marker).

[0150] Non-limiting examples of markers that may be used in this disclosure include microRNAs (miRNAs), proteins, metabolites, or combinations thereof. In some embodiments, the markers include miRNAs. In some embodiments, the markers include proteins. In some embodiments, the markers include metabolites.

[0151] As further described elsewhere in this disclosure, the synthetic circuits provided herein may include multiple sensors. For example, in some embodiments, the synthetic circuit includes a payload sequence, the payload sequence includes multiple sensors (e.g., multiple first type P sensors and / or multiple second type P sensors). In some embodiments, the synthetic circuit includes a regulator sequence, the regulator sequence includes multiple sensors. In some embodiments, the synthetic circuit includes a payload sequence and a regulator sequence, the payload sequence includes multiple sensors, and the regulator sequence includes multiple sensors. When the synthetic circuit includes multiple sensors, in some embodiments, each of the multiple sensors may specifically recognize the same marker. For example, in some embodiments, the payload sequence of the synthetic circuit provided herein includes multiple sensors, each of the multiple sensors recognizes the same miRNA. When the synthetic circuit includes multiple sensors, in some embodiments, one or more of the multiple sensors may specifically recognize different markers. For example, in some embodiments, the synthesis circuit comprises a payload sequence and a regulator sequence, each comprising a sensor, wherein the sensor of the payload sequence specifically recognizes a first marker (e.g., miRNA), and the sensor of the regulator sequence specifically recognizes a second marker (e.g., a metabolite or a different miRNA).

[0152] In some embodiments, the payload is a chimeric antigen receptor (CAR). In some embodiments, the CAR is CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe extracellular components of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof are targeted.

[0153] In some embodiments, the payload includes TCR. In some embodiments, TCR includes AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe extracellular components of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof are targeted.

[0154] Regulator As described herein, in some embodiments, the synthetic circuit described herein includes a payload array, which includes a sensor capable of specifically recognizing a regulator. Unless otherwise stated, specific recognition of the regulator by a type P sensor (e.g., a first type P sensor) reduces or inhibits the expression of the payload encoded by the payload array. Therefore, if the payload array includes both a sensor capable of specifically recognizing a regulator (e.g., a first type P sensor) and a sensor capable of specifically recognizing a marker (e.g., a second type P sensor), the expression of the encoded payload may be controlled by at least two different ways. Although not bound by any particular theory, in some embodiments, such a dual approach to control allows for greater selectivity in the expression of the payload.

[0155] In some embodiments, when the synthetic circuitry described herein is introduced into non-immune cells (i.e., cells that do not express a level of type R marker sufficient to be recognized by a type R sensor), and as a result, the expression of the regulator increases, the regulator can be specifically recognized by a type P sensor (e.g., a first type P sensor), thereby activating the type P sensor. As further described herein, the activation of the type P sensor reduces or inhibits the expression of the payload encoded by the payload sequence. Thus, in some embodiments, when the synthetic circuitry provided herein comes into contact with a cell population including both immune and non-immune cells, the expression of the payload in non-immune cells (i.e., cells with increased regulator expression) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to immune cells (i.e., cells with decreased regulator expression). In some embodiments, when the synthetic circuit provided herein comes into contact with a cell population including both immune and non-immune cells, the expression of the payload in immune cells (i.e., reduced regulator expression) increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the corresponding expression in non-immune cells. In some embodiments, compared to non-immune cells, the expression of the payload in immune cells increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

[0156] The regulators that can be used in the synthesis circuits described herein include any suitable regulators in the art. Non-limiting examples of such regulators include RNA-binding proteins, siRNA, shRNA, pri-miRNA, ribozymes, or combinations thereof. In some embodiments, the regulator is siRNA. In some embodiments, the regulator is shRNA. In some embodiments, the regulator is pri-miRNA. In some embodiments, the regulator is a ribozyme. In some embodiments, the regulator is an RNA-binding protein. In some embodiments, the RNA-binding protein comprises a ribonuclease. In some embodiments, the ribonuclease comprises a Cas protein. In some embodiments, the Cas protein comprises a Cas6 protein.

[0157] Modality As further described herein, the synthetic circuits of this disclosure include certain properties (e.g., structural and / or functional) that are particularly useful for selectively controlling the expression of a gene (or the protein it encodes) in a cell of interest (e.g., an immune cell). For example, in some embodiments, the synthetic circuits provided herein include a payload sequence and a regulator sequence, which are programmed such that, when both are present in an immune cell, the payload (encoded by the payload sequence) is strongly expressed while the expression of the regulator (encoded by the regulator sequence) is strongly reduced or inhibited. As previously stated in this disclosure, in some embodiments, the synthetic circuits described herein include a sensor (e.g., a type P sensor and / or a type R sensor), which may be programmed to enable the selective expression of the payload or regulator in a particular cell of interest. In some embodiments, in addition to such a sensor, the synthetic circuits provided herein include a payload sequence and a regulator sequence, which have certain modalities that help facilitate the selective expression of the payload and / or regulator.

[0158] Unless otherwise stated, the payload sequence comprises one or more of the following RNA modalities: linear RNA, circular RNA, self-replicating RNA, and non-replicating RNA. Unless otherwise stated, the regulator sequence comprises one or more of the following RNA modalities: linear RNA, circular RNA, and non-replicating RNA. For example, in some embodiments, the synthesis circuit provided herein comprises a payload sequence, and the payload sequence is self-replicating RNA. In some embodiments, the synthesis circuit provided herein comprises a regulator sequence, and the regulator sequence is non-replicating RNA. Therefore, in some embodiments, the synthesis circuit provided herein comprises a payload sequence and a regulator sequence, where the payload sequence is self-replicating RNA and the regulator sequence is non-replicating RNA. In some embodiments, the synthesis circuit provided herein comprises a payload sequence and a regulator sequence, where the payload sequence is self-replicating RNA and the regulator sequence is circular RNA (i.e., not self-replicating).

[0159] The performance of an RNA circuit is improved by using self-replicating RNA to express the payload sequence while using non-replicating RNA to express the regulator sequence.

[0160] While we do not wish to be bound by any particular theory, expressing the payload sequence from repRNA under marker conditions that allow the payload sequence to be "on" improves circuit performance because the repRNA replicates over a long period and strongly expresses high levels of the payload protein. This may be due to the self-replicating properties of the self-replicating RNA.

[0161] While we do not wish to be bound by any particular theory, expressing the regulator sequence from linear RNA instead of repRNA improves the performance of the following circuit.

[0162] (1) Non-replicating linear RNA can rapidly express regulator proteins at levels sufficient to effectively inhibit payload protein expression (from repRNA), while expression from repRNA is slower (e.g., requires replication), potentially allowing the payload repRNA to initiate replication, which could result in "leaky expression" of the payload in non-immune cells.

[0163] (2) Once repRNA begins replication, it becomes more difficult to knock it down (e.g., miRNA). Therefore, compared to expressing regulator proteins from linear RNA, "leak expression" of regulator proteins may occur in immune cells, which may significantly reduce expression from payload repRNA.

[0164] Therefore, the combination of a repRNA payload sequence and a linear RNA regulator sequence enables highly potent expression of the payload in immune cells while minimizing the expression of the transgene (payload) in non-immune cells. Similar to controlling the repRNA payload using linear RNA, controlling the circular RNA payload using a linear RNA regulator strand may also yield advantageous results due to the greater durability of circular RNA compared to linear RNA.

[0165] As will be apparent from this disclosure, synthetic circuits useful in this disclosure may include various combinations of RNA modalities, insofar as the payload can be selectively expressed in the cells of interest (i.e., immune cells).

[0166] For example, in some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is self-replicating RNA and includes a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence is non-replicating RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker. In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is self-replicating RNA and includes (i) a first sensor (first type P sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is non-replicating RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker.

[0167] In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is self-replicating RNA and includes a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence is circular RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker. In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is self-replicating RNA and includes (i) a first sensor (first type P sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is circular RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker.

[0168] In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is circular RNA and includes a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence is circular RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker. In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is circular RNA and includes (i) a first sensor (first type P sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is circular RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker. In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is circular RNA and includes a sensor (type P sensor) capable of specifically recognizing the regulator, and the regulator sequence is non-replicating RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker. In some embodiments, the synthetic circuit provided herein comprises (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence), wherein the payload sequence is non-replicating RNA and includes (i) a first sensor (first type P sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second type P sensor) capable of specifically recognizing a marker, wherein the regulator sequence is non-replicating RNA and includes a sensor (type R sensor) capable of specifically recognizing a marker.

[0169] Additional components In some embodiments, the synthesis circuits described herein include one or more additional components that assist in the function of the synthesis circuit. For example, in some embodiments, the synthesis circuits described herein include a payload array, the payload array including a type P sensor and one or more additional components described herein. In some embodiments, the synthesis circuits described herein include a regulator array, the regulator array including a type R sensor and one or more additional components described herein. In some embodiments, the synthesis circuits described herein include a payload array and a regulator array, each of which includes one or more additional components described herein.

[0170] In some embodiments, the payload sequence useful to the present disclosure comprises one or more additional components that enhance the expression of the encoded payload. In some embodiments, the regulator sequence does not contain one or more additional components that enhance the expression of the encoded regulator. Thus, in some embodiments, when such a synthetic circuit is introduced into immune cells, the expression of the payload increases compared to the expression of the regulator. In some embodiments, the expression of the payload increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the expression of the regulator. In some embodiments, payload expression increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

[0171] In some embodiments, a payload sequence useful to the present disclosure includes one or more additional components that enhance the stability of the payload sequence. In some embodiments, a regulator sequence does not include one or more additional components that enhance the stability of the payload sequence. In some embodiments, the increased stability increases the expression of the encoded protein. In some embodiments, when such a synthetic circuit is introduced into immune cells, the expression of the payload increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, payload expression increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

[0172] In some embodiments, one or more additional components that may be included in the synthetic circuit provided herein include aptamers for translation initiation factors. For example, in some embodiments, the synthetic circuit provided herein includes a payload sequence, which is a circular RNA and includes an aptamer for translation initiation factors. Including such additional components can assist in the expression of the encoded payload when the synthetic circuit is introduced into immune cells, particularly when the payload sequence is a circular RNA. See, for example, Prats et al., Int J Mol Sci 21(22):8591 (Nov. 2020). Non-limiting examples of additional components useful to this disclosure include (1) intra-sequence ribosome entry sites (IRES), (2) untranslated regions (UTR), (3) sequences encoding signal peptides, (4) translation initiation sequences, (5) poly(A) sequences, (6) sequences encoding RNA-binding proteins, (7) sequences encoding 2A ribosome skipped peptides, (8) 5' caps, (9) translation enhancer elements, or (10) any combination of (1) to (10). Further disclosures relating to such additional components are provided below.

[0173] Terminal structural modification: Untranslated region (UTR) In some embodiments, the synthesis circuit described herein includes a UTR. For example, in some embodiments, the synthesis circuit described herein includes a payload array, and the payload array includes a UTR. In some embodiments, the UTR is a 5'-UTR. In some embodiments, the UTR is a 3'-UTR. In some embodiments, the UTR includes both a 5'-UTR and a 3'-UTR.

[0174] The uncoding region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR, on the other hand, begins immediately after the stop codon and continues to the transcription termination signal. Much evidence has been presented regarding the regulatory role that UTRs play in the stability and translation of nucleic acid molecules. Therefore, when a payload sequence described herein contains a UTR, its stability is improved, for example, compared to a sequence that does not contain a UTR. In some embodiments, as described herein, this improved stability increases the expression of the encoded protein.

[0175] 5'UTR and translation begins Natural 5'UTRs have a function that plays a role in translation initiation. They have signatures like Kozak sequences, which are commonly known to be involved in the process by which ribosomes initiate translation of many genes. Kozak sequences have a common CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5'UTRs are also known to form secondary structures involved in the binding of elongation factors.

[0176] The 5'UTR secondary structure involved in elongation factor binding can interact with other RNA-binding molecules in the 5'UTR or 3'UTR to regulate gene expression. For example, microRNA-mediated repression requires the elongation factor EIF4A2 to bind to a secondary structured element of the 5'UTR (Meijer HA et al., Science, 2013, 340, 82-85, the entire work is incorporated herein by reference). By incorporating different secondary structures of the 5'UTR into the flanking region, it is possible to stabilize or selectively destabilize mRNA in specific tissues or cells.

[0177] By incorporating features typically found in genes abundantly expressed in specific target organs, the stability of nucleic acid sequences (e.g., payload sequences of the synthetic circuits provided herein) and protein production can be enhanced. For example, introducing the 5'UTR of liver-expressed mRNA such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII can be used to enhance the expression of nucleic acid molecules such as mRNA in hepatocyte lines or the liver. Similarly, by using the 5'UTR of other tissue-specific mRNAs, it is possible to improve the expression in muscle (MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (Tie-1, CD36), bone marrow cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0178] Other non-UTR sequences can also be incorporated into UTRs (e.g., 5'-UTR and / or 3'-UTR). For example, an intron or a portion of an intron sequence can be incorporated into a flanking region of a nucleic acid sequence (e.g., the payload sequence of the synthesis circuit provided herein).

[0179] In some embodiments, one or more nucleotides within the UTR (e.g., 5'-UTR and / or 3'-UTR) can be mutated, substituted, and / or removed. For example, one or more nucleotides upstream of the start codon can be replaced with other nucleotides. The nucleotide(s) to be replaced may be 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, 30, 35, 40, 45, 50, 55, 60, or 60 or more nucleotides upstream of the start codon. As another example, one or more nucleotides upstream of the start codon can be removed from the UTR.

[0180] 3'UTR and AU Rich Elements The 3'UTR is known to contain embedded adenosine and uridine stretches. These AU-rich signatures are particularly common in genes with high turnover rates. Based on sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995). Class I AREs contain several copies of the AUUUA motif dispersed within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) notamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples within this class. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, particularly HuR, have been shown to enhance mRNA stability. HuR binds to all three classes of AREs. By incorporating a HuR-specific binding site into the 3'UTR of a nucleic acid molecule, HuR binding can occur, thereby stabilizing the message in vivo.

[0181] In some embodiments, the introduction, removal, or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of nucleic acid sequences. When manipulating specific nucleic acid sequences (e.g., payload sequences and / or regulator sequences described herein), one or more copies of AREs can be introduced to reduce the stability of the nucleic acid sequence, thereby reducing translation and consequently decreasing protein production. Similarly, to enhance intracellular stability, AREs can be identified, removed, or mutated, thereby increasing translation and consequently increasing protein production.

[0182] Translation Enhancer Element (TEE) In some embodiments, the synthetic circuits provided herein include a translational enhancer element (TEE). As used herein, the term “translational enhancer element” refers to a cis-acting sequence that increases the expression of a protein encoded by a nucleotide sequence. Non-limiting examples of TEEs that may be used herein are known in the art. See, for example, US20130177581A, which is incorporated herein by reference in its entirety. In some embodiments, the synthetic circuits provided herein include a payload sequence and a regulator sequence, wherein the payload sequence includes a TEE. When such a synthetic circuit is introduced into immune cells, the expression of the payload is increased compared, for example, to a corresponding synthetic circuit in which the payload sequence does not include a TEE.

[0183] In some embodiments, the TEE is located between the transcription promoter and the start codon of the sequence (e.g., the payload sequence). In some embodiments, TEEs useful to this disclosure are U.S. Publication Nos. US20140147454, US20090226470, US20070048776, US20130177581, US20110124100, WO1999024595, WO2012009644, WO2009075886, WO2007025008, U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7, It has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with any of the TEEs provided in U.S. Patent No. 456,273 or U.S. Patent No. 7,183,395 (each of which is incorporated herein by reference in whole).

[0184] In some embodiments, the synthesis circuits provided herein include multiple TEEs. For example, in some embodiments, the synthesis circuits provided herein include a payload sequence which includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In some embodiments, the TEE sequences in the 5'UTR of RNA (e.g., modified RNA) are the same or different TEE sequences. In some embodiments, the TEE sequences are a pattern such as ABABAB, AABBAABBAABB, or ABCABCABC or a variation thereof, which are repeated once, twice, or three or more times. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0185] RNA-binding protein (RBP) In some embodiments, the synthetic pathways provided herein include sequences encoding RNA-binding proteins. RNA-binding proteins (RBPs) can control numerous aspects of co-transcription and post-transcriptional gene expression, such as, but not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, modification, transport, and localization. RNA-binding domains (RBDs), such as, but not limited to, RNA recognition motifs (RRs) and hnRNP K homology (KH) domains, typically control sequence association between the RBP and its RNA target (Ray et al., Nature 2013.499:172-177; the whole is incorporated herein by reference). In some embodiments, a standard RBD binds to a short RNA sequence. In some embodiments, a standard RBD recognizes an RNA structure.

[0186] Non-limiting examples of RNA-binding proteins and related nucleic acids and protein sequences are described in US2014 / 0147454, which is incorporated herein by reference in its entirety.

[0187] 5' capping In some embodiments, the synthetic circuits described herein include a 5'-cap structure. For example, in some embodiments, the synthetic circuits provided herein include a payload sequence, the payload sequence including a 5'-cap structure. The 5'-cap structure of mRNA is involved in nuclear export, enhances mRNA stability, and binds to mRNA cap-binding proteins (CBPs). CBPs associate with poly(A)-binding proteins to form mature cyclic mRNA species, thereby contributing to in-cellular mRNA stability and translational capacity. The cap also assists in the removal of 5'-adjacent introns during mRNA splicing.

[0188] The RNA modifications described herein may generate a non-hydrolyzable cap structure that increases the mRNA half-life by preventing capping removal. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, the modified nucleotides can be used during the capping reaction. For example, a phosphorothioate bond can be generated in the 5'-ppp-5' cap by using the vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) together with α-thio-guanosine nucleotides according to the manufacturer's instructions. Additional modified guanosine nucleotides, such as α-methylphosphonic acid and seleno-phosphate nucleotides, may also be used.

[0189] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5' end and / or pre-5' nucleotide of mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Multiple different 5'-cap structures can be used to generate 5'-caps for nucleic acid molecules such as mRNA molecules.

[0190] In this specification, cap analogs, also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, have a different chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5'-caps, but retain their capping function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and can be bound to nucleic acid molecules.

[0191] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, where one guanine contains an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; this can also be expressed as 3'O-Me-m7G(5')ppp(5')G)). The 3'-O atom of the other unmodified guanine is linked to the 5' terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). The N7- and 3'-O-methylated guanines provide the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0192] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-β-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0193] In some embodiments, the cap is a dinucleotide cap analog. In some embodiments, the dinucleotide cap analog is modified at different phosphate positions with a borano phosphate group or a phosphoroseleno acid group, as described in U.S. Patent No. 8,519,110 (the contents of which are incorporated herein by reference in their entirety).

[0194] In some embodiments, the cap is a cap analogue, which is an N7-(4-chlorophenoxyethyl)-substituted dicube form of a cap analogue known in the art and / or described herein. Non-limiting examples of cap analogues in N7-(4-chlorophenoxyethyl)-substituted dinucleotide form include the N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogues (see, for example, Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574, which contains various cap analogues and methods for synthesizing them; the entire contents of which are incorporated herein by reference). In some embodiments, the cap analogues of this disclosure are 4-chloro / bromophenoxyethyl analogues.

[0195] While cap analogs can simultaneously cap nucleic acid molecules in in vitro transcription reactions, up to 20% of the transcript remains uncapped. This is because the structure of the cap analog differs from the endogenous 5' cap structure of nucleic acids produced by the endogenous cellular transcription mechanism, and can lead to reduced translational capacity and decreased cellular stability.

[0196] In some embodiments, the provision of RNA containing a 5'-cap or a 5'-cap analog is achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is co-transcribed into the resulting RNA strand.

[0197] In some embodiments, RNA may be produced, for example, by in vitro transcription, and the 5' cap may be attached to the RNA post-transcriptionally using a capping enzyme, for example, a vaccinia virus capping enzyme. In some embodiments, the nucleotide sequence encoding IL-12 is post-transcriptionally capped using an enzyme to produce a more authentic 5'-cap structure. As used herein, the expression “more authentic” refers to a feature that more closely reflects or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a “more authentic” feature is one that better represents an endogenous, wild-type, natural or physiological cellular function and / or structure compared to a synthetic feature or analogue of the prior art, or that surpasses the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5' cap structures of this disclosure include, among others, those that enhance the binding of cap-binding proteins, extend the half-life, reduce sensitivity to 5' endonucleases, and / or reduce 5' capping removal compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can produce a standard 5'-5'-triphosphate linkage between the 5' terminal nucleotide of mRNA and the guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5' terminal nucleotide of mRNA contains a 2'-O-methylation. This cap results in higher translational capacity and cellular stability, as well as reduced activation of pro-inflammatory cytokines, compared to, for example, other 5' cap analogs known in the art. The cap structures include 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2mp, and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up.

[0198] In some embodiments, the 5' terminal cap comprises an endogenous cap or a cap analog. In some embodiments, the 5' terminal cap comprises a guanine analog. Useful guanine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0199] In some embodiments, the 5' cap includes a 5'-5' triphosphate linkage. In some embodiments, the 5' cap includes a 5'-5' triphosphate linkage with thiophosphate modification. In some embodiments, the 5' cap includes a 2'-O or 3'-O-ribose-methylated nucleotide. In some embodiments, the 5' cap includes a modified guanosine nucleotide or a modified adenosine nucleotide. In some embodiments, the 5' cap includes 7-methylguanylate. Exemplary cap structures include m7G(5')ppp(5')G, m7,2'O-mG(5')ppSp(5')G, m7G(5')ppp(5')2'O-mG, and m7,3'O-mG(5')ppp(5')2'O-mA.

[0200] In some embodiments, the synthesis circuits described herein include a modified 5' cap. For example, in some embodiments, the payload sequence of the synthesis circuit includes a modified 5' cap. Modifications on the 5' cap can increase mRNA stability, increase mRNA half-life, and improve mRNA translation efficiency. In some embodiments, the modified 5' cap includes one or more of the following modifications: modification at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), substitution of the methylene moiety (CH2) of the sugar ring oxygen (forming the carbocyclic ring), modification of the triphosphate crosslinking portion of the cap structure, or modification of the nucleoside (G) portion.

[0201] Modifiable 5' cap structures include, but are not limited to, the caps described in U.S. Patent Application No. 2014 / 0147454 and WO2018 / 160540 (which are incorporated herein by reference in their entirety).

[0202] IRES array In some embodiments, the synthetic pathways provided herein include intra-sequence ribosome entry sites (IRESs). For example, in some embodiments, the synthetic pathways provided herein include a payload sequence, the payload sequence including an IRES. IRESs, initially identified as features of picornavirus RNA, play a crucial role in the initiation of protein synthesis in the absence of a 5' cap structure. IRESs can function independently as ribosome-binding sites of mRNA, or they can function as one of several ribosome-binding sites. Nucleic acids or mRNAs containing multiple functional ribosome-binding sites can encode several peptides or polypeptides that are independently translated by ribosomes ("multicistronic nucleic acid molecules"). If the nucleic acid or mRNA comprises an IRES, a second translatable region is optionally provided. Examples of IRES sequences that can be used in accordance with this disclosure include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), swine cholera virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0203] Poly A Tail In some embodiments, the synthesis circuit provided herein includes a poly-A tail. In some embodiments, the synthesis circuit provided herein includes a payload array, the payload array including a poly-A tail.

[0204] In some embodiments, the length of the poly-A tail is longer than about 30 nucleotides. In some embodiments, the poly-A tail is longer than about 35 nucleotides. In some embodiments, the length is at least about 40 nucleotides. In some embodiments, the length is at least about 45 nucleotides. In some embodiments, the length is at least about 55 nucleotides. In some embodiments, the length is at least about 60 nucleotides. In some embodiments, the length is at least 70 nucleotides. In some embodiments, the length is at least about 80 nucleotides. In some embodiments, the length is at least about 90 nucleotides. In some embodiments, the length is at least about 100 nucleotides. In some embodiments, the length is at least about 120 nucleotides. In some embodiments, the length is at least about 140 nucleotides. In some embodiments, the length is at least about 160 nucleotides. In some embodiments, the length is at least about 180 nucleotides. In some embodiments, the length is at least about 200 nucleotides. In some embodiments, the length is at least about 250 nucleotides. In some embodiments, the length is at least about 300 nucleotides. In some embodiments, the length is at least about 350 nucleotides. In some embodiments, the length is at least about 400 nucleotides. In some embodiments, the length is at least about 450 nucleotides. In some embodiments, the length is at least about 500 nucleotides. In some embodiments, the length is at least about 600 nucleotides. In some embodiments, the length is at least about 700 nucleotides. In some embodiments, the length is at least about 800 nucleotides. In some embodiments, the length is at least about 900 nucleotides. In some embodiments, the length is at least about 1000 nucleotides. In some embodiments, the length is at least about 1100 nucleotides. In some embodiments, the length is at least about 1200 nucleotides.In some embodiments, the length is at least about 1300 nucleotides. In some embodiments, the length is at least about 1400 nucleotides. In some embodiments, the length is at least about 1500 nucleotides. In some embodiments, the length is at least about 1600 nucleotides. In some embodiments, the length is at least about 1700 nucleotides. In some embodiments, the length is at least about 1800 nucleotides. In some embodiments, the length is at least about 1900 nucleotides. In some embodiments, the length is at least about 2000 nucleotides. In some embodiments, the length is at least about 2500 nucleotides. In some embodiments, the length is at least about 3000 nucleotides.

[0205] In some embodiments, the poly-A tail contains a poly-AG quartet. The G quartet is a sequence of four guanine nucleotides cyclically hydrogen-bonded, and can be formed by G-rich sequences in both DNA and RNA. In some embodiments, the G quartet is incorporated into the end of the poly-A tail. The resulting nucleic acid or mRNA can be assayed for other parameters, including stability, protein production, and half-life at various time points. The poly-AG quartet has been found to yield protein production equivalent to at least 75% of the protein production observed when using a 120-nucleotide poly-A tail alone.

[0206] Modified nucleoside In some embodiments, the synthetic pathway provided herein comprises one or more modified nucleosides. In some embodiments, the synthetic pathway provided herein comprises a payload sequence, the payload sequence comprising one or more modified nucleosides. In some embodiments, the one or more modified nucleosides are 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-o This includes xo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methylcytidine, N4-acetylcytidine, 5-methyluridine, 5-iodocytidine, and combinations thereof.

[0207] In some embodiments, the synthetic pathways provided herein include one or more uridines replaced by modified nucleosides. In some embodiments, the modified nucleosides that replace the uridines are pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), or 5-methyl-uridine (m5U).

[0208] Nanoparticles and delivery systems In some embodiments, this disclosure relates to the delivery of synthetic circuits (e.g., those described herein) to cells. In some embodiments, delivery may be carried out in vivo (e.g., by administering the synthetic circuits described herein to a target) or ex vivo (e.g., by culturing the synthetic circuits described herein with cells in vitro). In some embodiments, delivery of the synthetic circuits described herein may be carried out using any suitable delivery system known in the art. In certain embodiments, the delivery system is a vector. Thus, in some embodiments, this disclosure provides a vector containing one of the synthetic circuits provided herein. Suitable vectors that can be used are known in the art. See, for example, Sung et al., Biomator Res 23(8)(2019) (the entire work is incorporated herein by reference).

[0209] In some embodiments, the synthetic circuit is delivered using nanoparticles (e.g., lipid nanoparticles or lipid-like nanoparticles). Accordingly, in some embodiments, the disclosure relates to synthetic circuits encapsulated within nanoparticles (e.g., as described herein), compositions comprising such nanoparticles, and the use of such nanoparticles for treating diseases or disorders in subjects requiring treatment. More specifically, in some embodiments, provided herein are nanoparticles comprising (i) any of the synthetic circuits described herein and (ii) one or more nanoparticle components.

[0210] Nanoparticles (NP) As used herein, “nanoparticles” (NPs) refer to particles such as vesicles having characteristic dimensions measured in nanometers (nm). Nanoparticles can be used in methods for delivering therapeutic drugs to target sites. Non-limiting examples of NPs include lipid nanoparticles (LNPs), lipid-like nanoparticles (LLNs), polymer nanoparticles (PNPs), and inorganic nanoparticles.

[0211] Lipid nanoparticles (LNPs) As used herein, “lipid nanoparticles” (LNPs) refer to nanoparticles composed of lipids. Lipid nanoparticles can be used in methods for delivering therapeutic agents to target sites. Non-limiting examples of LNPs include cationic lipid nanoparticles, ionized lipid nanoparticles, liposomes, bora amphiphilic materials, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and monolayer membrane structures (e.g., archasomes and micelles).

[0212] As used herein, “cationic lipid nanoparticles” refers to nanoparticles containing cationic lipids. As used herein, “ionized lipid nanoparticles” refers to nanoparticles containing ionized lipids. In embodiments of this disclosure, LNPs include one or more of the following lipids: “noncationic helper lipids,” “phospholipids,” “sterols and other structural lipids,” and “PEG / PEGylated lipids.”

[0213] An example LNP includes one or more of the following components:

[0214] (i) Ionized / cationic lipids,

[0215] (ii) Phospholipids or noncationic helper lipids,

[0216] (iii) Sterols or other structural lipids

[0217] (iv) PEG / PEGylated lipids, and

[0218] (v) Targeted delivery molecule (lipid composition / targeted ligand).

[0219] Lipid-like nanoparticles (LLNs) As used herein, “lipid-like nanoparticles” (LLNs) refer to nanoparticles comprising lipids and lipid-like materials or lipidoids as described herein. In embodiments of this disclosure, LLNs comprise one or more of the following lipids: “noncationic helper lipids,” “phospholipids,” “sterols and other structural lipids,” and “PEG / PEGylated lipids.” Lipid-like nanoparticles can be used in methods for delivering therapeutic agents to a target site.

[0220] An example LLN includes one or more of the following components:

[0221] (i) Ionizable / cationic lipid-like materials or lipidoids,

[0222] (ii) Phospholipids or noncationic helper lipids,

[0223] (iii) Sterols or other structural lipids

[0224] (iv) PEG / PEGylated lipids, and

[0225] (v) Targeted delivery molecule (lipid composition / targeted ligand).

[0226] Ionized lipids Non-limiting examples of ionized lipids include ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 5), di((Z)-nona-2-en-1-yl)9-((4-(dimeth (L319) Dilinoleyl(butanoyl)oxy(heptadecanedioate), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), Nl-[2(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethyl Aminomethyl-[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), 2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12 (Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA), (2R)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,Examples include 12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), or any combination thereof.

[0227] Cationic lipids Non-limiting examples of cationic lipids include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTEVI), and 2,3-dioleyloxy-N-[2(sperminecarboxamyl [DOSPA]-N,N-dimethyl-l-propaneaminium trifluoroacetate, N,N-distearyl-N,N-dimethylammonium bromide, N-(l,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide, N-(l,2-dioleoyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide, N,N -Dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), l,2-dilinoleyl-3-trimethylammonium-propane (DLTAP), l,2-dimyristoyl-3-trimethylammonium- Examples include propane (DMTAP), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC), l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC), l,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0228] In some aspects of this disclosure, LNPs primarily comprise cationic lipids together with other lipid components. These typically include, but are not limited to, hophatidylcholine (PC) classes (e.g., 1,s-distearoyl-sn-glycero-3-johocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-johoethanolamine (DOPE)), sterols (e.g., cholesterol), and other lipid molecules belonging to polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folic acid (polyethylene glycol)-2000 (DSPE-PEG2000) and C14-PEG2000).

[0229] DOTAP In embodiments of this disclosure, the cationic lipid is DOTAP. DOTAP can be used to efficiently transfect DNA containing yeast artificial chromosomes (YACs) into eukaryotic cells to result in transient or stable gene expression, and is also suitable for efficiently introducing other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins, into mammalian cell research samples.

[0230] Lipofectamine In aspects of this disclosure, the cationic lipid is lipofectamine. As used herein, lipofectamine is a common transfection reagent used in molecular and cell biology and manufactured and marketed by Invitrogen. Lipofectamine is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine contains lipid subunits that can form liposomes or lipid nanoparticles in an aqueous environment and encapsulate transfection payloads, such as modRNA. A neutral copolymer mediates the fusion of the liposome with the cell membrane, allowing the RNA-containing liposome (with a positively charged surface) to fuse with the negatively charged plasma membrane of a living cell, thereby enabling the nucleic acid cargo molecule to translocate into the cytoplasm for replication or expression.

[0231] Lipid-like material or lipidoid Unless otherwise stated, "lipid-like materials" and "lipidoids" may be used interchangeably. Non-limiting examples of lipid-like materials and / or lipidoids include 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine 2,5-dione(cKK-E12), tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazandiyl)bis(propane-3,1diyl))bis(azantriyl))tetrapropionate(306Oi 10), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(axantryyl))tetrakis(ethane2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(axantryyl) Tetrakis(butane-4,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-Tetrakis(octadeca-9,12-dienoate)(OF-C4-Deg-Lin), N1,N3,N5-Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), Hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)lis(azandiyl))tris(propane-3,1-diyl))tris(azantriyl))hexanonaate (FTT5), PL-1 [Nature Examples include [disclosed in Communications, 12-7264 (2021), incorporated herein by reference], 98N12-5 [disclosed in Molecular Therapy vol.17 no.5 May 2009, incorporated herein by reference], ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18(A2)) and A12-Iso5-2DC18(A12), or any combination thereof.

[0232] As used herein, TT3 can form nanoparticles for delivering various biological activators into cells. In addition, this disclosure also shows that unencapsulated TT3-LLN can induce immunogenic cell death (ICD) in cancer cells in vivo and in vitro. Immunogenic cell death, as described herein, refers to a form of cell death that can induce an effective immune response through the activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. In some aspects of this disclosure, the cells undergoing immunogenic cell death are tumor cells. Immunogenic tumor cell death can induce an effective anti-tumor immune response.

[0233] In some aspects, lipidoids are TT3.

[0234] Phospholipids or other noncationic helper lipids Unless otherwise specified, "phospholipids" and "other noncationic helper lipids" can be used without distinction. Non-limiting examples include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero -3-phosphocholine (DSPC), 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 dietherPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Examples include PE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-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 any combination thereof.

[0235] In some embodiments, the phospholipids are 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0PC, OSPC), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (16:0-18:1 PE, POPE), 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphoethanolamine (16:0-18:2 PE), 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphoethanolamine (16:0-20:4 PE), 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphoethanolamine (16:0-22:6 PE), 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (18:0-18:1 Selected from the group consisting of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6 PE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof.

[0236] In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0237] Sterols or other structural lipids As used herein, “sterols or other structural lipids” refers to cholesterol or cholesterol analogs that may be used to fill gaps in the packing of lipid membranes and to provide structural integrity.

[0238] Non-limiting examples of sterols include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and combinations thereof. In some embodiments, the sterol is cholesterol.

[0239] PEG / PEGylated lipids In this specification, "PEG lipids" and "PEGylated lipids" are used interchangeably.

[0240] Non-limiting examples of PEG lipids include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 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-dipalmetrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In some embodiments, the lipid portion of the PEG lipid includes those having a length of approximately C14 to approximately C22.

[0241] In some embodiments, the PEG portion has a size of approximately 1,000, 2,000, 5,000, 10,000, 15,000, or 20,000 Daltons. In certain embodiments, C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), or both.

[0242] In some embodiments, PEG-lipids can be embedded in LNPs before encapsulation of polynucleotides. In some embodiments, PEG-lipids (or other lipid components disclosed herein) can be added to LNPs after encapsulation of polynucleotides. For example, in some embodiments, a synthesis pathway is encapsulated in an LNP, and then, for example, micelles are used to attach PEG-lipids (or other lipid components disclosed herein) to the LNP.

[0243] In some embodiments, the nanoparticles do not contain any PEGylated lipids. In some embodiments, the lipid nanoparticles do not contain any PEGylated lipids.

[0244] Targeted delivery molecules (lipid composition / targeted ligand) In embodiments of this disclosure, the nanoparticles described herein (e.g., LNPs and LLNs) include a targeted delivery molecule (lipid composition / targeted ligand). As used herein, “targeted delivery molecule (lipid composition / targeted ligand)” and “targeted delivery molecule” are used without distinction, and in some embodiments, the targeted delivery molecule may be an additional lipid or lipid-like component described herein. In some embodiments, the targeted delivery molecule may alter the overall charge of the nanoparticles. In some embodiments, the targeted delivery molecule may be a non-covalent or covalent ligand to the nanoparticles. In some embodiments, the targeted delivery ligand may be a small molecule or a large molecule.

[0245] Non-limiting examples of targeted delivery molecules are disclosed in Pharmaceuticals (Basel). Jul 20;15(7):897.(2022), Nat Rev Drug Discov 20,101-124(2021) and Advanced Drug Delivery Reviews, Volume 188,(2022) (as incorporated herein by reference). Non-limiting examples of targeted delivery molecules include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18:1PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14:0PA), bis(monoleoylglycero)phosphate (S,R isomer) (ammonium salt) (18BMP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethylethyl phosphate (DOCPe), folic acid, N-acetylgalactosamine (GalNAc), and anti-CD3 antibodies.

[0246] Mole ratio In some embodiments, the nanoparticles described herein contain the lipids described herein (e.g., ionized lipids, cationic lipids, non-cationic helper lipids, phospholipids, sterols or other structural lipids, or PEG lipids) and / or lipidoids in a lipid and / or lipid-like composition in a molar ratio of about 10% to about 50%. In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a lipid composition in a molar ratio of about 10%, about 20%, about 30%, about 40%, or about 50%.

[0247] In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a molar ratio of about 10% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a molar ratio of about 20% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a molar ratio of about 30% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a molar ratio of about 40% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain the lipids and / or lipidoids described herein in a molar ratio of about 40% in the lipid and / or lipid-like composition.

[0248] In some embodiments, the nanoparticles described herein contain pegylated lipids in a molar ratio of about 0% to about 10% in the lipid and / or lipid-like composition. In some embodiments, the lipid nanoparticles contain pegylated lipids in a molar ratio of about 0%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% in the lipid and / or lipid-like composition.

[0249] In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 0.25% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 0.5% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 0.75% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 1.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 2.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 3.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 4.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 5.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 6.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 7.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 8.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 9.0% in the lipid and / or lipid-like composition. In some embodiments, the nanoparticles provided herein contain pegylated lipids in a molar ratio of about 10.0% in the lipid and / or lipid-like composition.

[0250] For example, in some embodiments, the pegylated lipids include C14-PEG2000. In some embodiments, C14-PEG2000 is present in lipid nanoparticles at a molar ratio of about 0.25% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present at a molar ratio of about 0.5% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present at a molar ratio of about 0.75% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present at a molar ratio of about 1% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present at a molar ratio of about 2% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present at a molar ratio of about 3% in the lipid and / or lipid-like composition. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 4%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 5%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 6%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 7%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 8%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 9%. In some embodiments, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 10%.

[0251] particle size The particle size of nanoparticles can affect drug release rate, biodistribution, mucosal adhesion, intracellular water uptake and buffer exchange into the nanoparticles, and protein diffusion. In some aspects of this disclosure, the diameter of the NPs is in the range of about 30 to about 500 nm. In some aspects of this disclosure, the diameter of the NPs is in the range of about 30 to about 500 nm, about 50 to about 400 nm, about 70 to about 300 nm, about 100 to about 200 nm, about 100 to about 175 nm, or about 100 to about 160 nm. In some aspects of this disclosure, the diameter of the NPs is in the range of 100 to 160 nm. In some embodiments of this disclosure, the diameter of the NP may be about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm, about 117 nm, about 118 nm, about 119 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, or about 160 nm. In certain embodiments, the lipid nanoparticles have a diameter of about 140 nm.

[0252] Zeta potential As used herein, “zeta potential” refers to a measure of the effective charge on the surface of a nanoparticle. The magnitude of the zeta potential provides information about the stability of the particle. In some embodiments of this disclosure, the zeta potential of the nanoparticle is in the range of about -20 to about 20 mV.In some aspects of this disclosure, the zeta potentials of NP are approximately -6.0mv, approximately -5.9mv, approximately -5.8mv, approximately -5.7mv, approximately -5.6mv, approximately -5.5mv, approximately -5.4mv, approximately -5.3mv, approximately -5.2mv, approximately -5.1mv, approximately -5.0mv, approximately -4.9mv, approximately -4.8mv, approximately -4.7mv, approximately -4.6mv, approximately -4.5mv, approximately -4.4mv, approximately -4.3mv, approximately -4.2mv, approximately -4.1mv, approximately -4.0mv, approximately -3.9mv, approximately -3.8mv, approximately -3.7mv, approximately -3.6mv, approximately -3.5mv, approximately -3.4mv, approx. -3.3mv, approx. -3.2mv, approx. -3.1mv, approx. -3.0mv, approx. -2.9mv, approx. -2.8mv, approx. -2.7mv, approx. -2.6mv, approx. -2.5mv, approx. -2.4mv, approx. -2.3mv, approx. -2.2mv, approx. -2.1mv, approx. -2.0mv, approx. -1.9mv, approx. -1.8mv, approx. -1.7mv, approx. -1.6mv, approx. -1.5mv, approx. -1.4mv, approx. -1.3mv, approx. -1.2mv, approx. -1.1mv, approx. -1.0mv, approx. -0.9mv, approx. -0.8mv, approx. -0.7mv, approx. -0.6mv, approx. -0. 5mv, approx. -0.4mv, approx. -0.3mv, approx. -0.2mv, approx. -0.1mv, approx. 0.0mv, approx. 0.1mv, approx. 0.2mv, approx. 0.3mv, approx. 0.4mv, approx. 0.5mv, approx. 0.6mv, approx. 0.7mv, approx. 0.8mv, approx. 0.9mv, approx. 1.0mv, approx. 1.1mv, approx. 1.2mv, approx. 1.3mv, approx. 1.4mv, approx. 1.5mv, approx. 1.6mv, approx. 1.7mv, approx. 1.8mv, approx. 1.9mv, approx. 2.0mv, approx. 2.1mv, approx. 2.2mv, approx. 2.3mv, approx. 2.4mv, approx. 2.5mv, approx. 2.6mv, approx. 2.7mv, approx. 2. It could be 8mv, approximately 2.9mv, approximately 3.0mv, approximately 3.1mv, approximately 3.2mv, approximately 3.3mv, approximately 3.4mv, approximately 3.5mv, approximately 3.6mv, approximately 3.7mv, approximately 3.8mv, approximately 3.9mv, approximately 4.0mv, approximately 4.1mv, approximately 4.2mv, approximately 4.3mv, approximately 4.4mv, approximately 4.5mv, approximately 4.6mv, approximately 4.7mv, approximately 4.8mv, approximately 4.9mv, approximately 5.0mv, approximately 5.1mv, approximately 5.2mv, approximately 5.3mv, approximately 5.4mv, approximately 5.5mv, approximately 5.6mv, approximately 5.7mv, approximately 5.8mv, approximately 5.9mv, or approximately 6.0mv.

[0253] mass ratio In some embodiments, the mass ratio of lipids to the synthesis pathway in LNPs or LLNs ranges from approximately 1:2 to approximately 15:1. In some embodiments, the mass ratio of lipids to the synthesis pathway is approximately 1:2, approximately 1:1.9, approximately 1:1.8, approximately 1:1.7, approximately 1:1.6, approximately 1:1.5, approximately 1:1.4, approximately 1:1.3, approximately 1:1.2, approximately 1:1.1, approximately 1:1, approximately 1.1:1, approximately 1.2:1, approximately 1.3:1, approximately 1.4:1, approximately 1.5:1, approximately 1.6:1, approximately 1.7:1, approximately 1.8:1, approximately 1.9:1, approximately 2:1, and approximately 2.5. The ratio may be approximately 1:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1. In some aspects of this disclosure, the mass ratio of lipids to the synthesis pathway is approximately 10:1.

[0254] Pharmaceutical composition In some embodiments, this disclosure relates to pharmaceutical compositions comprising synthesis circuits, vectors, and / or nanoparticles described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier (excipient). "Acceptable" means that, as used herein, the carrier must be compatible with the active ingredient of the composition and must not be harmful to the subject being treated. In some embodiments, the carrier may stabilize the active ingredient. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkoins, Ed. KE. Hoover.

[0255] The pharmaceutical composition used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. The nanoparticles can be placed in a container with a sterile access port, for example, in an intravenous fluid bag or vial having a stopper that can be pierced with a hypodermic needle.

[0256] In some embodiments, the pharmaceutical composition can be formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. In some embodiments of the present disclosure, the pharmaceutical composition can be formulated for intratumoral injection. Intratumoral injection, as used herein, refers to direct injection into the tumor. A high concentration of the composition can be obtained in situ while using a small amount of the drug. Local delivery of immunotherapy enables multiple combination therapies while avoiding significant systemic exposure and off-target toxicity. <()

[0257] In some embodiments, the pharmaceutical composition can be formulated for intramuscular injection, intravenous injection, or subcutaneous injection.

[0258] In some embodiments, the pharmaceutical composition is in the form of a lyophilized formulation or an aqueous solution and contains a pharmaceutically acceptable carrier, buffer, excipient, salt, or stabilizer. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. The acceptable carriers and excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG).

[0259] In some embodiments, the pharmaceutical compositions described herein include nanoparticles that can be prepared by methods known in the art, for example, Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and the methods described in U.S. applications No. 4,485,045 and 4,544,545, which are incorporated herein by reference in their entirety. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556, which is incorporated herein by reference in its entirety. In some embodiments, liposomes can be prepared by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). Liposomes of a desired diameter are obtained by extruding them through a filter of a predetermined pore size.

[0260] In some embodiments, pharmaceutical compositions are formulated in a sustained-release form. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing nanoparticles, the matrix of which is in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include, but are not limited to, polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPROM DEPOT (microspheres for injection consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0261] In some embodiments, preferred surfactants include, but are not limited to, nonionic agents such as polyoxyethylene sorbitan (e.g., TWEEN® 20, 40, 60, 80, or 85) and other sorbitans (e.g., SPAN® 20, 30, 60, 80, or 85). In some embodiments, the surfactant-containing composition contains 0.05 to 5% of the surfactant. In some embodiments, the composition contains 0.1 to 2.5%. It will be understood that other components, such as mannitol or other pharmaceutically acceptable vehicles, may be added if necessary.

[0262] In some embodiments, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, granules, liquids or suspensions, or suppositories for oral, parenteral, or rectal administration, or administration by inhalation or inhalation.

[0263] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutically acceptable non-toxic salt thereof to form a solid pre-formulation composition containing a homogeneous mixture of the compound of the Disclosure or a pharmaceutically acceptable non-toxic salt thereof. When these pre-formulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, thereby allowing the composition to be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. The solid pre-formulation composition is then subdivided into the above-mentioned unit dosage forms containing about 0.1 to about 500 mg of the active ingredient of the Disclosure. Tablets or pills of the novel compositions may be coated or otherwise formulated to provide a dosage form that offers the advantage of sustained action. For example, a tablet or pill may contain an inner and outer dosing component, the latter in the form of a coating covering the former. The two components can be separated by an enteric coating, which functions to withstand disintegration in the stomach and allows the inner component to pass intact into the duodenum or be released with delay. Various materials can be used for such enteric coatings or coatings, and such materials include several polymer acids, as well as mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0264] Suitable emulsions can be prepared using commercially available fat emulsions, such as INTRALIPID®, LIPOSYN®, INFONUTROL®, LIPOFUNDIN®, and LIPIPHYSAN®. The active ingredient can be dissolved in a pre-mixed emulsion composition, or dissolved in oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., egg phospholipid, soybean phospholipid, or soybean lecithin) and water to form the emulsion. It will be understood that other components, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to about 20% oil, for example, about 5 to about 20%. The fat emulsion may contain fat droplets of a suitable size and may have a pH in the range of about 5.5 to about 8.0.

[0265] Pharmaceutical compositions for inhalation or inhalation comprise liquid and suspension formulations and powder formulations in pharmaceutically acceptable aqueous solvents or organic solvents, or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered orally or via the respiratory route for topical or systemic effects.

[0266] Compositions in pharmaceutically acceptable solvents can be atomized using gas. The atomized solution may be inhaled directly from the atomizing device, or the atomizing device may be attached to a face mask, tent, or intermittent positive airway pressure (PAP) device. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in an appropriate manner.

[0267] Therapeutic applications In some aspects of this disclosure, the synthetic circuits, vectors, nanoparticles, and / or pharmaceutical compositions described herein (collectively, the “Compositions”) are used to treat diseases or disorders. As is evident from this disclosure, any of the compositions provided herein can be used to treat a wide range of diseases or disorders. Any suitable disease or disorder can be coded by the payload sequence of the synthetic circuits provided herein, whether in therapeutic agents or otherwise. Accordingly, some aspects of this disclosure relate to methods for treating a disease or disorder in a subject requiring treatment, which include administering any of the compositions provided herein (e.g., synthetic circuits) to the subject.

[0268] In some embodiments, any of the compositions described herein is administered to a subject requiring it via a suitable route, e.g., intratumoral administration, intravenous administration (e.g., as a bolus or continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-sacral, intrathecal, oral, inhalation, or topical route. Commercially available liquid formulation nebulizers, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be sprayed, and lyophilized powders can be sprayed after reconstitution. In some embodiments, the pharmaceutical compositions described herein are aerosolized using fluorocarbon formulations and metered-dose inhalers, or inhaled as lyophilized pulverized powders. In some embodiments, the pharmaceutical compositions described herein are formulated for intratumoral injection. In some embodiments, the pharmaceutical compositions described herein are administered to a subject via a topical route, e.g., by injection into a local site such as a tumor site or an infection site. In some embodiments, the subject is a human.

[0269] As will be apparent from this disclosure, in some embodiments, the compositions described herein are administered to a subject in an effective amount that imparts a therapeutic effect, either alone or in combination with one or more other activators. In some embodiments, the compositions are administered to a subject suffering from cancer, and the therapeutic effect includes a reduction in tumor tissue volume, a reduction in cancer cells, an increase in immune activity, or a combination thereof. Whether the administered composition (e.g., nanoparticles such as LNP or LLN) has achieved a therapeutic effect can be determined using any suitable method known in the art (e.g., measurement of tumor volume and / or T cell activity). The effective amount will vary depending on the specific condition being treated, the severity of the condition, the parameters of the individual patient, e.g., age, physical condition, build, sex and weight, duration of treatment, the nature of the combination therapy (if any), the specific route of administration, and similar factors within the expertise of the healthcare professional, as will be recognized by those skilled in the art.

[0270] Empirical considerations such as half-life generally indicate that the frequency of administration, which contributes to dose determination, can be determined and adjusted over the course of treatment, generally based on the treatment and / or suppression and / or improvement and / or delay of the target disease / impairment, but not necessarily. Alternatively, a sustained continuous release formulation of the compositions described herein (e.g., nanoparticles such as LNP or LLN) may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0271] In some embodiments of this disclosure, the treatment is a single injection of the composition disclosed herein. In some embodiments, the single injection is administered intratumorally to a subject requiring it.

[0272] In some embodiments of this disclosure, the dosage of the compositions described herein can be determined empirically in an individual who has received one or more doses of the compositions (e.g., nanoparticles described herein). In some embodiments, the individual is administered a gradually increasing dose of the compositions described herein. To evaluate the efficacy of the compositions described herein, indicators of disease / disorder may be tracked. In the case of repeated administration over several days or more, in some embodiments, the treatment is continued, depending on the condition, until the desired suppression of symptoms occurs or until a sufficient therapeutic level is achieved to alleviate the target disease or disorder or its symptoms.

[0273] In some embodiments of this disclosure, the method includes administering one or more doses of the compositions described herein to a subject requiring it.

[0274] As described herein, the synthetic circuits of this disclosure are particularly useful for selectively expressing payloads in immune cells of interest. Accordingly, several aspects of this disclosure relate to methods for inducing selective expression of payloads in cells, comprising contacting a population of cells with one of the compositions provided herein (e.g., a synthetic circuit). In some embodiments, the payload is expressed in cells if the cells satisfy the following conditions: (i) contain a sufficient level of type R marker, as a result of the type R marker being specifically recognized by the type R sensor, thereby inducing activation of the type R sensor and reducing or inhibiting the expression of the regulator; (ii) not contain a sufficient level of type P marker, as a result of the type P marker not being recognized by the type P sensor, and the type P sensor remaining in an inactive form; or (iii) both (i) and (ii). If the cells satisfy such conditions, the expression of the payload is increased in the cells compared to a reference cell (e.g., a corresponding cell that does not satisfy any of the above conditions). In some embodiments, after contact, intracellular payload expression increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to reference cells. In some embodiments, after contact, payload expression increases intracellularly by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times compared to reference cells.

[0275] In some embodiments, the compositions described herein are co-administered with at least one additional preferred therapeutic agent. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered sequentially to the subject, i.e., each therapeutic agent is administered at a different time. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered to the subject almost simultaneously.

[0276] In some embodiments, the therapeutic application of the synthetic circuits described herein involves producing an encoded payload in immune cells. Therefore, in some embodiments, this disclosure relates to a method for selectively producing a payload in immune cells. In some embodiments, the method involves contacting immune cells with any of the compositions described herein (e.g., synthetic circuits, vectors, and / or nanoparticles) under conditions suitable for producing the encoded IL-12 protein. In some embodiments, the method further includes purifying the produced payload. In some embodiments, the contact occurs in vivo (e.g., by administering the synthetic circuits, vectors, and / or nanoparticles to a target). In some embodiments, the contact occurs ex vivo (e.g., by culturing cells in vitro with the synthetic circuits, vectors, and / or nanoparticles). Cells containing the synthetic circuits, vectors, and / or nanoparticles (e.g., host cells) are incorporated herein. Non-exclusive examples of cells that can be used include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0277] kit for use in treatment This disclosure also provides kits for use in therapeutic applications. In some embodiments, the kit comprises one or more containers containing compositions described herein.

[0278] In some embodiments, the kit includes instructions for use according to any of the methods described herein. For example, the instructions included may include instructions regarding the administration of the pharmaceutical compositions described herein for treating, delaying the onset of, or alleviating an immune disorder. In some embodiments, the instructions include instructions for administering the compositions described herein to a subject at risk of an immune disorder.

[0279] In some embodiments, the instructions include dosage information, dosing schedule, and route of administration. In some embodiments, the container is a unit dose, bulk package (e.g., a multi-dose package), or a split unit dose. In some embodiments, the instructions are instructions written on a label or package insert (e.g., a paper sheet included in the kit). In some embodiments, the instructions are instructions that can be read by a machine (e.g., instructions stored on a magnetic or optical storage disk).

[0280] In some embodiments, the kits described herein are housed in suitable packaging. In some embodiments, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), or combinations thereof. In some embodiments, the packaging includes a package for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device such as a mini pump. In some embodiments, the kit includes a sterile access port (e.g., the container may be an intravenous fluid bag or vial having a stopper that can be pierced with a hypodermic needle). In some embodiments, the container may also have a sterile access port (e.g., the container may be an intravenous fluid bag or vial having a stopper that can be pierced with a hypodermic needle). In some embodiments, at least one active agent is a composition described herein.

[0281] In some embodiments, the kit further includes additional components such as buffers and explanatory information. In some embodiments, the kit includes a container and a label or accompanying information on or attached to the container. In some embodiments, this disclosure provides a product comprising the contents of a kit described herein.

[0282] general technology Unless otherwise stated, the implementation of this disclosure will utilize prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which is within the scope of the art of those skilled in the art. Molecular Cloning:A Laboratory Manual,second edition(Sambrook,et al.,1989)Cold Spring Harbor Press;Oligonucleotide Synthesis(MJGait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney,ed.1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGiffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Method of Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology(DMWeir and CCBlackwell,eds.);Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.,1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989);Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999);The Antibodies (M. Zanette and JDCapra, eds., Harwood Academic Publishers, 1995). It is assumed that those skilled in the art will be able to make the most of this disclosure based on the above description without further explanation. All publications listed herein (including those listed above and elsewhere in this disclosure) are incorporated by reference in their entirety. . [Examples]

[0283] The following experiments (Examples 1-7) describe optimized RNA-based logic circuits for better reaction rates. The optimized circuit includes, but is not limited to, a polynucleotide sequence encoding a payload (payload sequence), a polynucleotide sequence encoding a regulator (regulator sequence), a regulator sequence sensor capable of specifically recognizing a marker (Type R sensor), a payload sequence sensor capable of specifically recognizing the regulator (First Type P sensor), and a payload sequence sensor capable of specifically recognizing a marker (Second Type P sensor), as illustrated in Figure 1. As shown in Figure 1, one or more proteins encoded by the regulator sequence interact with the payload to positively or negatively regulate it.

[0284] Furthermore, as shown in Figure 1, the regulator sequence and payload sequence may include linear (self-replicating or non-replicating) or circular RNA.

[0285] Key features of the optimized payload sequence within the circuit include, but are not limited to, multiple miRNA classifiers that detarget expression in multiple organs to facilitate systemic delivery, and an optimized number and arrangement of miRNA sensors for a strong OFF switch. Key features of the optimized regulator sequence within the circuit include, but are not limited to, highly specific and sensitive binding that rapidly engages with the target (e.g., immune cells) to avoid expression in non-immune cells, expression levels adjusted to ensure better switching behavior, multiple miRNA classifiers that enable payload expression in immune cells, and purpose-appropriateness based on gene therapy applications.

[0286] Example 1: Suppression of a single sensor As shown in Figure 2, a single-sensor 3'UTR target site array was tested for optimal suppression.

[0287] Number of target sites. First, the effect of the number of target sites on suppression was evaluated and measured by arbitrary units (au) of median mVenus fluorescence for constructs containing either 1×siRNA2, 2×siRNA2, 3×siRNA2, 4×siRNA2, no target site (TS), or no reporter after administration of siRNA (0, 1, 10, or 100 nM) (Figure 3). As shown in Figure 3, modRNA constructs containing 1–4×siRNA2 target sites immediately after the mVenus-PEST reporter stop codon showed preferential knockdown in HEK293T cells with increasing co-administration doses of siRNA2 siRNA. Incorporating more than two identical adjacent target sites did not significantly increase suppression, regardless of siRNA dose. Data represent median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of reporter and siRNA. The data represents the median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of the reporter and siRNA.

[0288] Spacing between target sites. Next, the effect of spacing between target sites on suppression was evaluated and measured by median mVenus fluorescence (au) for constructs containing either 2×siRNA2, 2×siRNA2-20nt, 2×siRNA2-50nt, no TS, or no reporter after administration of siRNA (0, 1, 10, or 100 nM) (Figure 4). Data represent median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of reporter and siRNA. As shown in Figure 4, modRNA constructs containing 2×siRNA2 target sites with varying spacings immediately after the mVenus-PEST reporter stop codon show preferential knockdown in HEK293T cells with increasing co-administered doses of siRNA2. Incorporating spacing between target sites does not significantly increase knockdown for low, medium, or high doses of siRNA.

[0289] The effect of the spacing between target sites (2–20 nucleotides) on suppression was evaluated and measured by median mVenus fluorescence (au) for constructs containing either 2×siRNA2-20nt, 3×siRNA2-20nt, 4×siRNA2-20nt, no TS, or no reporter after administration of siRNA (0, 1, 10, or 100 nM) (Figure 5). Data represent median values ​​collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation of reporter and siRNA. As shown in Figure 5, modRNA constructs containing 2×~4×siRNA2 sites with a 20nt spacer between target sites positioned immediately after the stop codon of the mVenus-PEST reporter show preferential knockdown in HEK293T cells with increasing co-administered doses of siRNA2. Constructs with a 4×siRNA2 target site and 20nt showed slightly lower expression than constructs with 2×-20nt or 3×-20nt, which is thought to be due to a decrease in maximum expression rather than increased downregulation. This trend applies to low-dose, medium-dose, and high-dose siRNAs.

[0290] Optimization of miRNA sensors in various RNA modalities. Next, detargeting of payload expression was evaluated for linear RNA (Figure 6A) and circular RNA (Figure 6B) modalities. Figure 6A shows that the mVenus-PEST reporter and a linear modRNA circuit containing N×miR-b target sites (1-4×TS) at the 3'UTR show preferential knockdown in hepatocytes (Huh-7) compared to the control (HEK293T) cell line (Figure 6A). This detargeting of payload expression in hepatocytes is consistent with miR-b being a liver-specific mRNA. Data represent mean geometric mean collected from three technical replicates (n=3) 20 hours after electroporation. Figure 6B shows that a circRNA construct containing 1-4 miR-a target sites immediately after the stop codon of the mVenus-PEST reporter shows preferential knockdown in the HEK293T cell line compared to the HeLa cell line. This is consistent with the observation that miR-α is highly expressed in HEK293 cells but not in HeLa cells. Incorporating more than two identical adjacent target sites did not significantly increase suppression. The degree of suppression increased over time, approaching baseline at 24 hours in HEK293T cells. Data represent the mean geometric mean collected from three technical replicates (n=3) at 4 and 24 hours after electroporation.

[0291] Optimization of Cas6e target site location. This includes an example demonstrating that the location of the target site in circRNA determines the expression level in the absence of the corresponding regulator. Specifically, BHK-21 cells were transfected with circRNA expressing mVenus-PEST, either without the Cas6e target site or with the Cas6e target site located at one of the five positions shown in Figure 7A, and modRNA expressing the Cas6e regulator. Expression in the absence of the regulator varied depending on the location of the target site, but in the presence of the regulator, complete knockdown was observed in all constructs (Figure 7B).

[0292] Optimization of the spacing between the stop codon and the target site. Figures 8B–8C show the effect of the N× nucleotide spacer shown in Figure 8A. Specifically, Figures 8B–8C provide graphs representing mVenus fluorescence (au) after administration of siRNA (0, 1, 10, 100 nM) for constructs containing 1× or 2× siRNA target sites (TS) and 0, 7, 12 nucleotide spacers. The data represent geometric mean collected by flow cytometry from three technical replicates (n=3) at 6 and 24 hours after electroporation of the reporter and siRNA.

[0293] Example 2: Suppression of multiple sensors Next, as shown in Figure 9, target site arrays for multiple input classifiers were tested for optimal suppression.

[0294] As shown in Figure 10, HEK293T cells were electroporated with an mVenus-PEST reporter containing a target site array immediately following the stop codon, and either no siRNA, siRNA1, siRNA2, or both. Knockdown was observed for all tested constructs (3×siRNA1-20nt, 3×siRNA2-20nt, 3×siRNA2-siRNA1 alternating, 3×siRNA1-siRNA2 alternating, 3×siRNA2 3×siRNA1 adjacent, 3×siRNA1 3×siRNA2 adjacent, no target site, no reporter) when the co-electroporated siRNA(s) contained a target site within the target site array. Since siRNA2 alone reduces expression to background levels, the combined sensor responded to both siRNAs, but there was no visible synergistic effect. The data represent the mean geometric mean collected by flow cytometry from three technical replicates (n=3) 20 hours after electroporation.

[0295] The test defines the operating range of a circuit with N×miR and regulator target sites positioned at either the 5'UTR or 3'UTR at positions A, B, and C outlined in Figure 11. The test evaluates, in particular, the following: (1) the types and number of target sites that can be positioned at A, B, and C without compromising maximal payload expression in the on-target cell type; (2) the effect of the order of target sites at A, B, and C on payload knockdown in different cell types; and (3) the effect of combinations of miR target sites at both the 5'UTR and 3'UTR on payload knockdown.

[0296] Example 3: Behavior of synthetic RNA-based gene circuits CircRNAs containing miRNA target sites are degraded by the RISC complex. As shown in Figure 12, total RNA was extracted 4 and 24 hours after transfection from HEK293T cells, HeLa cells, and Huh-7 cells transfected with circular RNA containing either miR-b TS or miR-a TS. The amount of transfected circRNA was measured using RT-qPCR with a probe spanning the circRNA splice site. HEK cells contained high levels of miR-a but not miR-b. Huh-7 cells contained high levels of both miR-a and miR-b, while HeLa cells had low expression levels of both miR-a and miR-b. CircRNAs containing the miR-b target site had minimal effect in HEK and HeLa cells, but were significant in Huh-7 cells up to 4 hours after transfection. Similarly, circRNAs containing the miR-a target site are downregulated at a rate consistent with their relative expression levels in all cell types, most rapidly in HEK cells, followed by Huh-7 and HeLa. CircRNAs without the miR sensor are not targeted for degradation and are used as a control against circRNAs containing the miR sensor.

[0297] Downregulation of circRNA by Cas6e. The regulatory protein Cas6e efficiently downregulates the expression of circRNA molecules containing a protein-coding sequence followed by a Cas6e target site. BHK-21 cells were transfected with either circular or linear RNA encoding the fluorescent protein mVenus-PEST. Each of these either lacked the Cas6e target site or contained the Cas6e target site after a stop codon. Furthermore, other cells were co-transfected with either a Cas6e regulator, a P2A self-cleaving peptide sequence, and modified linear mRNA or circRNA encoding the fluorescent protein mCherry-PEST. See Figure 13A for schematic diagrams showing linear and circular regulator RNAs containing Cas6e TS and encoding the fluorescent protein mVenus, which downregulate the target circRNA. Downregulation of circRNA containing the target site in the presence of the Cas6e regulator is consistent with the results observed for linear mRNA. When both the target site and regulator are present, mVenus expression is reduced to background levels (Figure 13B). Cas6e does not affect the expression of circRNAs that do not contain its target site (Figure 13C).

[0298] RNA regulators targeting rep and non-rep payloads. In this experiment, endribonuclease was used as an RNA regulator to control the mRNA strand expressing the mVenus fluorescent reporter payload protein. As shown in Figure 14A, a linear non-replicating (non-rep) payload mRNA strand containing the RNA regulator's target sequence was synthesized from either an unmodified base (unmodRNA payload) or a base in which uridine was replaced with N1-methylpseudridine (modRNA payload). The payload mRNA was transfected into BHK-21 cells with or without co-transfection of modRNA expressing the RNA regulator. After 24 hours, the cells were assayed by flow cytometry to evaluate payload expression. The RNA regulator downregulated the payload mRNA synthesized from the unmodified base but did not downregulate the modRNA payload. Furthermore, as shown in Figure 14B, BHK-21 cells were transfected with replicon RNA containing the target sequence of the RNA regulator at two different doses (20 ng or 40 ng), either co-transfected with modRNA expressing the RNA regulator or without co-transfection. Almost all cells transfected with the replicon alone expressed the payload, but co-transfection with the replicon and RNA regulator reduced the percentage of payload-positive cells to less than 10%.

[0299] Example 4. Expression of circular RNA homology to cell-type-specific miRNAs is downregulated in those cell types. In this example, HEK293T cells and Huh-7 cells were electroporated with circular RNA encoding EGFP-PEST driven by coxsackievirus B3 (CVB3)IRES. These circular RNAs either did not contain a miR TS or contained a 4×miR-b target site or a 4×miR-a target site immediately after the stop codon. Fluorescence of individual cells was measured using flow cytometry 24 hours after transfection. The data are shown in Figure 15. In HEK cells containing high levels of miR-a but lacking miR-b, translation of circRNAs with a 4×miR-a target site was found to be downregulated to autofluorescence levels, while circRNAs with a 4×miR-b target site were not downregulated to autofluorescence levels. In Huh-7 cells containing levels of both miR-a and miR-b, translation of circRNAs with either TS was found to be downregulated to autofluorescence levels. In Huh-7 cells, the expression of circRNAs containing miR-b target sites was downregulated, but this was not the case in HEK293T cells, indicating that this downregulation is a result of miRNA-mediated RNA degradation.

[0300] Example 5. miRNA classifier in vitro In this example, non-replicating modRNAs containing various human miRNA target sites corresponding to miRNAs that express the mVenus-PEST fluorescent protein and are more active in HEK293T (non-cancer) cells than in HeLa (cancer) cells were electroporated into both cell types. Flow cytometry data were collected for both cell types (n=3) approximately 24 hours after electroporation. The geometric mean of mVenus-PEST expression in each cell type was normalized to the geometric mean of modRNA without the miRNA sensor after subtracting the background fluorescence level. Figure 16 shows the ratio of these normalized expression levels in HEK293T to the normalized expression levels in HeLa.

[0301] Figures 17A and 17B show that non-replicating modRNA (Figure 17A) and replicon RNA (Figure 17B) reporter constructs containing an N×miR-b target site in the 3'UTR were designed and constructed to express the mVenus-PEST fluorescent protein. Each construct was transfected into HEK293T cells or the human liver cell line Huh-7 via electroporation. Non-replicating modRNA expressing the near-infrared fluorescent reporter protein miRFP720 was co-transfected with each replicon RNA to function as a transfection marker. The data represent the mean geometric mean of three technical replicates (n=3) of flow cytometry data collected approximately 24 hours after electroporation. The miRFP720-positive cell population in each cell type (HEK293T or Huh7) was considered to have been successfully transfected. Expression levels (Figure 17A) and the percentage of miRFP720-positive cells that are also mVenus-PEST-positive (Figure 17B) were calculated. mVenus-PEST expression was used as a substitute for circuit activity.

[0302] 1× target sites result in near-complete knockdown of non-replicating modRNA, while 2× or higher sites result in complete knockdown reaching autofluorescence levels in transfected Huh-7 cell lines. Furthermore, the spacing between miR target sites has minimal effect on knockdown efficiency (see Figure 17A).

[0303] In Huh-7 cells expressing high levels of miR-b, almost all cells containing more than 0× target sites showed knockdown of the mVenus-PEST reporter, while in miR-b non-expressing HEK293T cells, no number of miR-b target sites caused knockdown (see Figure 17B).

[0304] Therefore, miRNA sensors result in efficient knockdown in vitro.

[0305] Example 6. miRNA sensing in vivo Detargeting of the liver. In this example, mice were injected with lipid nanoparticles containing a reporter modRNA encoding firefly luciferase, or with a vehicle control. After 6 hours, the mice were sacrificed, and their organs (i.e., spleen, lungs, kidneys, lymph nodes, and liver) were evaluated for luciferase activity. Adding a liver-specific microRNA miR-b sensor to the reporter modRNA resulted in a 59-fold reduction in luciferase expression in the liver compared to reporter modRNA lacking the sensor. On the other hand, luciferase expression in the spleen, lungs, kidneys, and lymph nodes was not significantly affected by the addition of the miR-b sensor. Therefore, the miR-b sensor specifically detargets reporter expression in the liver (see Figure 18).

[0306] Spleen detargeting. In this example, mice were injected with lipid nanoparticles containing a reporter modRNA encoding firefly luciferase, or with a vehicle control. After 6 hours, the mice were sacrificed, and their organs (i.e., liver and spleen) were evaluated for luciferase activity. Adding a spleen-associated miRNA miR-h sensor to the reporter modRNA resulted in a 30-fold reduction in luciferase expression in the spleen compared to reporter modRNA lacking the sensor. On the other hand, the addition of the miR-h sensor had only a minimal effect on luciferase expression in the liver. The miR-h sensor detargets reporter expression in the spleen (see Figure 19).

[0307] Example 7. Type R Sensor Type R sensors enable payload expression when their homologous markers are abundant. In this example, an RNA circuit was transfected via electroporation into Huh7 cells expressing high levels of miR-b and HEK293T cells expressing low levels of miR-b. The RNA circuit consisted of (1) a replicon payload strand containing a first type P sensor expressing a green fluorescent reporter and responding to the regulator protein Cas6e, and (2) a linear non-replicating regulator strand containing a type R sensor expressing the regulator protein Cas6e and responding to miR-b.

[0308] As a control, Huh7 cells and HEK293T cells were transfected in parallel with the same RNA pathway except for the lack of a type R sensor. Expression of the green fluorescent payload was measured via quantitative imaging.

[0309] Six hours after transfection, payload expression from RNA circuits with the miR-b type R sensor is turned on in miR-b rich Huh7 cells, but remains off in HEK293T cells. In fact, payload expression in HEK293T cells was reduced to one-seventh compared to Huh7 cells (see Figure 20). Payload expression from RNA circuits lacking the type R sensor remains off in both cell types (see Figure 20).

[0310] Type R sensors enable payload expression when their congener markers are abundant. A549 lung cancer cells expressing high levels of miR-i were transfected via electroporation with a replicon payload sequence containing a first type P sensor responsive to a Cas6e regulator protein, and expressing the mVenus reporter. Some cells were co-transfected with a linear non-replicating regulator sequence expressing a Cas6e regulator protein linked to the mCherry reporter via a 2A self-cleaving peptide. This resulted in suppression of the payload sequence, as evidenced by decreased mVenus expression. However, some cells were co-transfected with a version of the regulator sequence containing a type R sensor for miR-i. In these cells, the type R sensor was activated, resulting in downregulation of the regulator chain, as indicated by the suppression of mCherry, and enabling the expression of the payload sequence, as evidenced by a significant increase in mVenus expression (see Figure 21).

[0311] Example 7. Extracorporeal cell therapy Blood is collected from the subject via a catheter connected to an inline extracorporeal system. A synthesis circuit containing chimeric antigen receptors (CARs) is added to the system containing the blood. After sufficient incubation time for effective transfection of the synthesis circuit, the mononuclear cells are returned to the subject via venous access along with the remaining blood components; all of this takes place within a closed-loop system.

[0312] It should be understood that the section describing embodiments for carrying out the invention, rather than the section describing the summary and abstract of the invention, is intended to be used in interpreting the claims. The section describing the summary and abstract of the invention may describe one or more exemplary embodiments of the present disclosure as contemplated by the inventor(s) of this disclosure, but not all, and is therefore not intended to limit the scope of this disclosure and the attached claims.

[0313] This disclosure has been described above using functional foundational elements that exemplify the implementation of specific functions and their relationships. The boundaries of these functional foundational elements are arbitrarily defined herein for the sake of explanation. Different boundaries may be defined, as long as the specific functions and their relationships are adequately implemented.

[0314] The foregoing descriptions of specific embodiments are sufficient to illustrate the general nature of the disclosure and can be easily modified and / or adapted for various uses by applying the knowledge of a person skilled in the art without excessive experimentation and without departing from the general concepts of the disclosure. Such adaptations and modifications are therefore intended to be within the meaning and scope of equivalent embodiments of the embodiments of the disclosure, based on the teachings and guidance provided herein. It should be understood that the expressions and terms used herein are for illustrative purposes only, not limiting purposes, and that they are to be interpreted by a person skilled in the art in light of these teachings and guidance.

[0315] The breadth and scope of this disclosure are not limited by any of the exemplary embodiments described above, but are defined solely by the following claims and their equivalents.

Claims

1. A method for producing engineered immune cells for extracorporeal cell therapy in a subject requiring such treatment, comprising contacting the immune cells obtained from the subject with a synthesis circuit in a closed in-line system connected to the subject, wherein the synthesis circuit is capable of selectively expressing a payload in the immune cells.

2. The method according to claim 1, wherein the immune cells are not exposed to the outside of the inline system for the entire duration of the therapy.

3. The method according to claim 1 or 2, wherein the immune cells express a payload on the immune cells as a result of the contact.

4. The method according to any one of claims 1 to 3, wherein the cells are returned directly to the target from the inline system.

5. A method for extracorporeally treating a disease or condition in a subject requiring treatment, comprising administering cells, for example, immune cells, to the subject, wherein the cells express a payload encoded by a synthetic circuit in a closed in-line system connected to the subject, and the synthetic circuit is capable of selectively expressing the payload in the cells.

6. A method for in vitro treating a disease or condition in a subject requiring treatment, comprising: contacting cells obtained from the subject, such as immune cells, with a synthesis circuit in a closed in-line system connected to the subject; and administering the cells expressing a payload encoded by the synthesis circuit to the subject, wherein the synthesis circuit can selectively express the payload in the immune cells.

7. The method according to any one of claims 1 to 6, wherein the subject has not undergone lymphocyte removal before the treatment.

8. The method according to any one of claims 1 to 7, wherein the therapy is less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours.

9. The aforementioned synthesis circuit (a) A first nucleotide sequence (payload sequence) that codes for the payload and (b) Optionally, a second nucleotide sequence (regulator sequence) encoding a regulator is included, The payload array includes a sensor (Type P sensor) capable of specifically recognizing the regulator. The regulator array includes a sensor (Type R sensor) capable of specifically recognizing the marker, The method according to any one of claims 1 to 8, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same.

10. The method according to any one of claims 1 to 8, wherein the synthesis circuit comprises a nucleotide sequence encoding the payload (payload sequence), the payload sequence comprises a sensor (type P sensor) capable of specifically recognizing the marker, and the marker is either intrinsically absent in the immune cells or is intrinsically present only in the immune cells.

11. The method according to claim 9 or 10, wherein the payload array includes a plurality of the type P sensors.

12. The method according to claim 11, wherein the plurality of type P sensors include two type P sensors, three type P sensors, four type P sensors, five type P sensors, six type P sensors, seven type P sensors, or eight or more type P sensors.

13. The method according to any one of claims 10 to 12, wherein each of the aforementioned type P sensors is the same.

14. The method according to any one of claims 10 to 12, wherein one or more of the Type P sensors are different.

15. The method according to any one of claims 9 to 14, wherein the payload array includes a spacer array (type P spacer).

16. The method according to claim 15, wherein the payload array includes a plurality of type P spacers.

17. The method according to claim 16, wherein each of the aforementioned type P spacers is the same.

18. The method according to claim 16, wherein one or more of the Type P spacers are different.

19. The method according to any one of claims 15 to 18, wherein (a) at least one type P spacer is located upstream of the type P sensor, (b) at least one type P spacer is located downstream of the type P sensor, or (c) both (a) and (b).

20. The method according to any one of claims 15 to 19, comprising at least two type P sensors, wherein at least one type P spacer is positioned between the at least two type P sensors.

21. Immune cells in a closed inline system for in vitro therapy, wherein the immune cells express a payload encoded by a synthesis circuit for the method according to any one of claims 1 to 20, and the synthesis circuit can selectively express the payload in the immune cells.

22. The immune cell according to claim 21, wherein the payload is expressed on the surface of the immune cell.

23. An inline system comprising immune cells according to claim 21 or 22, or immune cells expressing a payload encoded by a synthetic circuit for the method according to any one of claims 1 to 20.

24. The inline system according to claim 23, which is completely closed.

25. The inline system according to claim 23 or 24, which is connected to a subject requiring the aforementioned therapy.

26. The inline system according to claim 25, wherein the immune cells are obtained by the subject.

27. The inline system according to any one of claims 23 to 26, further comprising cells other than immune cells.

28. The method according to any one of claims 1 to 20, wherein the payload is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a TCR mimetic; the immune cell according to claim 21 or 22; or the inline system according to any one of claims 23 to 27.

29. The aforementioned CARs are CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, Globo H, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, Regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe method, cells, or in-line system according to claim 28, which targets the extracellular components of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof.

30. The aforementioned TCRs are AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, and Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl OO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, Globo H, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-AI, Regmine, HPV E6, E7, MAGE AI, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivorbin, telomerase, PCTA-1 / galectin 8, melan A / MARTI, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin BI, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe method according to claim 28, an immune cell, or an inline system, which targets the extracellular components of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL protein, or any combination thereof.

31. The aforementioned synthesis circuit (a) A first nucleotide sequence (payload sequence) that codes for the payload and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The payload array includes a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing the marker. The regulator array includes a sensor (Type R sensor) capable of specifically recognizing the marker, The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator, the marker recognized by the second type P sensor (second type P marker), and / or the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22, and 28 to 30; or the inline system according to any one of claims 23 to 30.

32. The method according to claim 31, an immune cell, or an inline system, wherein the payload sequence comprises a plurality of the first type P sensors.

33. The method according to claim 32, an immune cell, or an inline system, wherein the plurality of first type P sensors include two first type P sensors, three first type P sensors, four first type P sensors, five first type P sensors, six first type P sensors, seven first type P sensors, eight first type P sensors, nine first type P sensors, ten first type P sensors, eleven first type P sensors, or twelve first type P sensors.

34. The method according to claim 32 or 33, wherein each of the first type P sensors is the same, an immune cell, or an inline system.

35. The method according to claim 32 or 33, an immune cell, or an inline system, wherein one or more of the first type P sensors are different.

36. The method according to any one of claims 31 to 35, comprising a payload sequence comprising a plurality of the second type P sensors, immune cells, or an inline system.

37. The method according to claim 36, immune cells, or inline system, wherein the plurality of second type P sensors include two second type P sensors, three second type P sensors, four second type P sensors, five second type P sensors, six second type P sensors, seven second type P sensors, eight second type P sensors, nine second type P sensors, ten second type P sensors, eleven second type P sensors, or twelve second type P sensors.

38. The method according to claim 36 or 37, wherein each of the second type P sensors is the same, immune cells, or inline system.

39. The method according to claim 36 or 37, an immune cell, or an inline system, wherein one or more of the second type P sensors are different.

40. The method according to any one of claims 31 to 39, an immune cell, or an inline system, wherein the payload sequence includes a spacer sequence (type P spacer).

41. The method according to claim 40, an immune cell, or an inline system, wherein the payload sequence comprises a plurality of type P spacers.

42. The method according to claim 41, an immune cell, or an inline system, wherein each of the aforementioned type P spacers is the same.

43. The method according to claim 41, an immune cell, or an inline system, wherein one or more of the type P spacers are different.

44. (a) at least one type P spacer is positioned between the first type P sensor and the second type P sensor; (b) at least one type P spacer is positioned upstream of both the first type P sensor and the second type P sensor; (c) at least one type P spacer is positioned downstream of both the first type P sensor and the second type P sensor; or (d) any combination of (a) to (c), the method, immune cells, or inline system according to any one of claims 41 to 43.

45. The method according to any one of claims 41 to 44, comprising the plurality of first type P sensors, wherein two or more of the first type P sensors are separated by type P spacers, an immune cell, or an inline system.

46. The method according to claim 45, immune cells, or inline system, wherein each of the first type P sensors is separated by a type P spacer.

47. The method according to any one of claims 40 to 46, comprising the plurality of the second type P sensors, wherein two or more of the second type P sensors are separated by a type P spacer, an immune cell, or an inline system.

48. The method according to claim 47, an immune cell, or an inline system, wherein each of the second type P sensors is separated by a type P spacer.

49. The method according to any one of claims 40 to 48, wherein the type P spacer is about 1 to about 50 nucleotides in length, an immune cell, or an inline system.

50. The method according to claim 49, an immune cell, or an inline system, wherein the type P spacer is at least about 10 nucleotides long.

51. The method according to claim 49, an immune cell, or an inline system, wherein the type P spacer is approximately 10 nucleotides long, approximately 20 nucleotides long, or approximately 50 nucleotides long.

52. The method according to any one of claims 40 to 51, an immune cell, or an inline system, wherein the type P spacer includes, is essentially, or consists of the sequence tttccttccccttccttccttccttccttccttccttccttcctttcctttcctttcctttccttt (SEQ ID NO: 1), tttccttccccccttcctttccttt (SEQ ID NO: 2), or gcggccgctaaa (SEQ ID NO: 3), or a fragment thereof.

53. The method according to any one of claims 41 to 52, comprising a plurality of the Type R sensors in the regulator array, immune cells, or in-line system.

54. The method according to claim 53, an immune cell, or an inline system, wherein the plurality of type R sensors include two type R sensors, three type R sensors, four type R sensors, five type R sensors, six type R sensors, seven type R sensors, or eight or more type R sensors.

55. The method according to claim 53 or 54, wherein each of the aforementioned Type R sensors is the same, immune cells, or inline system.

56. The method according to claim 53 or 54, an immune cell, or an inline system, wherein one or more of the Type R sensors are different.

57. The method according to any one of claims 31 to 56, an immune cell, or an inline system, wherein the regulator array includes a spacer array (type R spacer).

58. The method according to claim 57, an immune cell, or an in-line system, wherein the regulator array comprises a plurality of type R spacers.

59. The method according to claim 58, an immune cell, or an inline system, wherein each of the aforementioned Type R spacers is the same.

60. The method according to claim 58, immune cells, or inline system, wherein one or more of the Type R spacers are different.

61. The method according to any one of claims 57 to 60, comprising the plurality of the type R sensors, wherein two or more of the type R sensors are separated by a type R spacer, an immune cell, or an inline system.

62. The method according to claim 61, an immune cell, or an inline system, wherein each of the Type R sensors is separated by a Type R spacer.

63. The method according to any one of claims 57 to 62, an immune cell, or an inline system, wherein at least one type R spacer is located upstream of at least one type R sensor.

64. The method according to any one of claims 57 to 63, an immune cell, or an inline system, wherein the type R spacer is about 1 to about 50 nucleotides in length.

65. The method according to claim 64, an immune cell, or an inline system, wherein the type R spacer is at least about 10 nucleotides long.

66. The method according to claim 64, an immune cell, or an inline system, wherein the type R spacer is approximately 10 nucleotides long, approximately 20 nucleotides long, or approximately 50 nucleotides long.

67. The method, immune cells, or inline system according to any one of claims 57 to 66, wherein the type R spacer comprises, is essentially, or consists of the sequence tttccttcccccccccccccccccccccccccccccccccccccccccccccccccccc (Sequence ID 1) or a fragment thereof.

68. The method according to claim 67, an immune cell, or an inline system, wherein the type R spacer comprises, is essentially, or consists of the sequence tttccttcccccctttccttt (Sequence ID 2).

69. The method according to any one of claims 57 to 64, an immune cell, or an inline system, wherein the type R spacer comprises, is essentially derived from, or consists of the sequence gcggccgctaaa (sequence number 3).

70. The method according to any one of claims 40 to 69, an immune cell, or an inline system, wherein the first marker, the second marker, or the first and second markers include a microRNA, a protein, a metabolite, or a combination thereof.

71. The method according to any one of claims 31 to 70, an immune cell, or an inline system, wherein the regulator comprises an RNA-binding protein, siRNA, shRNA, pre-miRNA, ribozyme, or a combination thereof.

72. The method according to claim 71, an immune cell, or an inline system, wherein the RNA-binding protein comprises a ribonuclease.

73. The method according to claim 72, an immune cell, or an inline system, wherein the ribonuclease comprises a Cas protein.

74. The method according to claim 73, an immune cell, or an in-line system, wherein the Cas protein comprises the Cas6 protein.

75. The method according to any one of claims 31 to 74, an immune cell, or an inline system, wherein the payload sequence, the regulator sequence, or both the payload sequence and the regulator sequence include circular RNA.

76. The method according to any one of claims 31 to 75, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA and the regulator sequence is a non-replicating RNA.

77. The method according to claim 75 or 76, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA and the regulator sequence is a circular RNA.

78. The method according to claim 75 or 76, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA and the regulator sequence is a linear non-replicating RNA.

79. The method according to claim 75 or 76, an immune cell, or an inline system, wherein the payload sequence is a circular RNA and the regulator sequence is a circular RNA.

80. The method according to claim 75 or 76, an immune cell, or an inline system, wherein the payload sequence is circular RNA and the regulator sequence is linear non-replicating RNA.

81. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The payload sequence is a self-replicating RNA, and the system includes a sensor (Type P sensor) that can specifically recognize the regulator. The regulator sequence is circular RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

82. The synthesis circuit includes (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), The payload sequence is self-replicating RNA and includes a first sensor (first type P sensor) capable of specifically recognizing the regulator, and a second sensor (second type P sensor) capable of specifically recognizing the marker. The regulator sequence is circular RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22, and 28 to 30; or the inline system according to any one of claims 23 to 30.

83. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The payload sequence is a self-replicating RNA, and the system includes a sensor (Type P sensor) that can specifically recognize the regulator. The regulator sequence is a non-replicating linear RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

84. The synthesis circuit includes (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), The payload sequence is self-replicating RNA and includes a first sensor (first type P sensor) capable of specifically recognizing the regulator, and a second sensor (second type P sensor) capable of specifically recognizing the marker. The regulator sequence is a non-replicating linear RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22, and 28 to 30; or the inline system according to any one of claims 23 to 30.

85. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The payload sequence is circular RNA, and the system includes a sensor (Type P sensor) capable of specifically recognizing the regulator. The regulator sequence is circular RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

86. The synthesis circuit includes (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), The payload sequence is circular RNA and includes a first sensor (first type P sensor) capable of specifically recognizing the regulator, and a second sensor (second type P sensor) capable of specifically recognizing the marker. The regulator sequence is circular RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22, and 28 to 30; or the inline system according to any one of claims 23 to 30.

87. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The payload sequence is circular RNA, and the system includes a sensor (Type P sensor) capable of specifically recognizing the regulator. The regulator sequence is a non-replicating linear RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

88. The synthesis circuit includes (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), The payload sequence is circular RNA and includes a first sensor (first type P sensor) capable of specifically recognizing the regulator, and a second sensor (second type P sensor) capable of specifically recognizing the marker. The regulator sequence is a non-replicating linear RNA and includes a sensor (Type R sensor) that can specifically recognize a marker. The method according to any one of claims 1 to 20 and 28 to 30, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same; the immune cells according to any one of claims 21, 22, and 28 to 30; or the inline system according to any one of claims 23 to 30.

89. (a) The payload array includes a plurality of the first type P sensors, (b) The payload array includes a plurality of the second type P sensors, (c) The regulator array includes a plurality of the Type R sensors, or (d) Any combination of (a) to (c) the method, immune cells, or inline system according to any one of claims 81 to 88.

90. (a) The payload array includes a spacer array (Type P spacer), (b) The regulator array includes a spacer array (Type R spacer), or (c) The method, immune cells, or in-line system according to any one of claims 81 to 89, which is both (a) and (b).

91. (a) the type P spacer is positioned between the first type P sensor and the second type P sensor; (b) the type P spacer is positioned upstream of both the first type P sensor and the second type P sensor; (c) the type P spacer is positioned downstream of both the first type P sensor and the second type P sensor; or (d) any combination of (a) to (c), the method, immune cells, or inline system according to claim 90.

92. The method according to claim 90 or 91, an immune cell, or an inline system, wherein the payload array comprises the plurality of first type P sensors, and two or more of the first type sensors are separated by type P spacers.

93. The method according to any one of claims 90 to 92, comprising the payload array comprising the plurality of second type P sensors, wherein two or more of the second type sensors are separated by type P spacers, an immune cell, or an inline system.

94. The method according to any one of claims 90 to 93, comprising the regulator array including the plurality of type R sensors, wherein two or more of the type R sensors are separated by type R spacers, an immune cell, or an inline system.

95. The method according to any one of claims 90 to 94, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both are about 1 to about 50 nucleotides in length.

96. The method, immune cells, or inline system according to any one of claims 90 to 95, wherein the type P spacer, the type R spacer, or both include, essentially, or consist of the sequence tttccttcccccccccccccccccccccccccccccccccccccccccccc (Sequence ID 1) or a fragment thereof.

97. The method according to claim 96, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, are essentially, or consist of the sequence tttccttcccccctttccttt (Sequence ID 2).

98. The method according to any one of claims 90 to 97, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, are essentially, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

99. The method according to claim 98, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, are essentially, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3).

100. The method according to any one of claims 81 to 99, an immune cell, or an inline system, wherein the type P marker, the type R marker, or both comprises a microRNA, a protein, a metabolite, or a combination thereof.

101. The method according to any one of claims 81 to 100, an immune cell, or an inline system, wherein the regulator comprises an RNA-binding protein, siRNA, shRNA, pre-miRNA, ribozyme, or a combination thereof.

102. The method according to claim 101, an immune cell, or an inline system, wherein the RNA-binding protein comprises a ribonuclease.

103. The method according to claim 102, an immune cell, or an inline system, wherein the ribonuclease comprises a Cas protein.

104. The method according to claim 102, an immune cell, or an in-line system, wherein the Cas protein comprises the Cas6 protein.

105. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, When the payload sequence and the regulator sequence are present in an immune cell, the payload is expressed in the immune cell for first expression, and the regulator is expressed in the immune cell for second expression. The method according to any one of claims 1 to 20 and 28 to 30, wherein the first expression is greater than the second expression; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

106. (a) The payload array includes a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing the marker, (b) The regulator array includes a sensor (Type R sensor) that can specifically recognize the marker, The method according to claim 105, an immune cell, or an inline system, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

107. The method according to claim 106, an immune cell, or an inline system, wherein the recognition of the type P marker by the second type P sensor inhibits the expression of the payload.

108. The method according to any one of claims 105 to 107, an immune cell, or an inline system, wherein the recognition of the type R marker by the type R sensor inhibits the expression of the regulator.

109. (a) The immune cells do not express the type P marker at a level sufficient to activate the second type P sensor, and (b) The immune cells express the type R marker at a level sufficient to activate the type R sensor, the method, immune cells, or in-line system according to any one of claims 106 to 108.

110. (a) the non-immune cells express the type P marker at a level sufficient to activate the second type P sensor, (b) the non-immune cells do not express the type R marker at a level sufficient to activate the type R sensor, or (c) both (a) and (b), the method, immune cells, or in-line system according to any one of claims 106 to 109.

111. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, If the payload sequence and the regulator sequence are present in a non-immune cell, the payload is expressed in the non-immune cell for a first expression, and the regulator is expressed in the non-immune cell for a second expression. The method according to any one of claims 1 to 20 and 28 to 30, wherein the second expression is greater than the first expression; the immune cells according to any one of claims 21, 22 and 28 to 30; or the inline system according to any one of claims 23 to 30.

112. (a) The payload array includes a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing the marker, (b) The regulator array includes a sensor (Type R sensor) that can specifically recognize the marker, The method according to claim 111, an immune cell, or an inline system, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same.

113. The method according to claim 111 or 112, an immune cell, or an inline system, wherein the binding of the type P marker to the second type P sensor inhibits the expression of the payload.

114. The method, immune cells, or inline system according to claim 111, wherein the non-immune cells include (a) a level of the type P marker sufficient to activate the second type P sensor, (b) a level of the type R marker insufficient to activate the type R sensor, or (c) both of (a) and (b).

115. The method according to any one of claims 111 to 114, an immune cell, or an inline system, wherein the binding of the type R marker to the type R sensor inhibits the expression of the regulator.

116. The method, immune cells, or in-line system according to any one of claims 111, 114, and 115, wherein the immune cells include (a) a level of the type P marker insufficient to activate the second type P sensor, and (b) a level of the type R marker insufficient to activate the type R sensor.

117. The aforementioned synthesis circuit (a) A first nucleotide sequence that codes for the payload (payload sequence) and (b) comprising a second nucleotide sequence (regulator sequence) encoding the regulator, The method according to any one of claims 1 to 20 and 28 to 30, wherein when the synthesis circuit comes into contact with a population of cells including immune cells and non-immune cells, the expression of the payload in the immune cells is higher than the corresponding expression in the non-immune cells, the immune cells according to any one of claims 21, 22 and 28 to 30, or the inline system according to any one of claims 23 to 30.

118. The method, immune cells, or inline system according to claim 117, wherein the expression of the payload in the immune cells is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 times higher than the corresponding expression in the non-immune cells.

119. (a) The payload array includes a first sensor (first type P sensor) capable of specifically recognizing the regulator and a second sensor (second type P sensor) capable of specifically recognizing the marker, (b) The regulator array includes a sensor (Type R sensor) that can specifically recognize the marker, The method according to claim 117 or 118, wherein the regulator, the marker recognized by the second type P sensor (type P marker), and the marker recognized by the type R sensor (type R marker) are not the same, an immune cell, or an inline system.

120. The method according to any one of claims 117 to 119, an immune cell, or an inline system, wherein the binding of the type P marker to the second type P sensor inhibits the expression of the payload.

121. The method according to any one of claims 117 to 120, an immune cell, or an inline system, wherein the binding of the type R marker to the type R sensor inhibits the expression of the regulator.

122. (a) The immune cells do not contain the type P marker in a level sufficient to activate the second type P sensor, and (b) The immune cells express the type R marker in a level sufficient to activate the type R sensor, the method, immune cells, or in-line system according to any one of claims 117 to 121.

123. (a) the non-immune cells express the type P marker in a level sufficient to activate the second type P sensor, (b) the non-immune cells do not express the type R marker in a level sufficient to activate the type R sensor, or (c) both (a) and (b), the method, immune cells, or in-line system according to any one of claims 117 to 122.

124. The method according to any one of claims 105 to 123, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA.

125. The method according to any one of claims 105 to 124, an immune cell, or an inline system, wherein the regulator sequence is a non-replicating linear RNA.

126. The method according to any one of claims 105 to 125, an immune cell, or an inline system, wherein the payload sequence is circular RNA.

127. The method according to any one of claims 105 to 126, an immune cell, or an inline system, wherein the regulator sequence is circular RNA.

128. The method according to any one of claims 105 to 127, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA and the regulator sequence is a circular RNA.

129. The method according to any one of claims 105 to 123, an immune cell, or an inline system, wherein the payload sequence is a self-replicating RNA and the regulator sequence is a non-replicating linear RNA.

130. The method according to any one of claims 105 to 123, an immune cell, or an inline system, wherein the payload sequence is circular RNA and the regulator sequence is circular RNA.

131. The method according to any one of claims 105 to 123, an immune cell, or an inline system, wherein the payload sequence is circular RNA and the regulator sequence is non-replicating linear RNA.

132. (a) The payload array includes a plurality of the first type P sensors, (b) The payload array includes a plurality of the second type P sensors, (c) The regulator array includes a plurality of the Type R sensors, or (d) Any combination of (a) to (c) the method, immune cells, or inline system according to any one of claims 105 to 131.

133. (a) The payload array includes a spacer array (Type P spacer), (b) The payload array includes a spacer array (Type R spacer), or (c) The method, immune cells, or in-line system according to any one of claims 105 to 132, which is both (a) and (b).

134. The method according to claim 133, an immune cell, or an inline system, wherein the type P spacer is positioned between the first type P sensor and the second type P sensor.

135. The method according to claim 133 or 134, an immune cell, or an inline system, wherein the type P spacer is positioned between the array encoding the payload and (a) the first type P sensor, (b) the second type P sensor, or (c) both (a) and (b).

136. The method according to any one of claims 133 to 135, an immune cell, or an inline system, wherein the type R spacer is positioned between the array encoding the regulator and the type R sensor.

137. The method according to any one of claims 133 to 136, comprising the payload array comprising the plurality of first type P sensors, wherein two or more of the first type sensors are separated by type P spacers, an immune cell, or an inline system.

138. The method according to any one of claims 133 to 137, comprising the payload array comprising the plurality of second type P sensors, wherein two or more of the second type sensors are separated by type P spacers, an immune cell, or an inline system.

139. The method according to any one of claims 133 to 138, comprising the regulator array including the plurality of type R sensors, wherein two or more of the type R sensors are separated by type R spacers, an immune cell, or an inline system.

140. The method according to any one of claims 133 to 139, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both are about 1 to about 50 nucleotides in length.

141. The method according to any one of claims 133 to 140, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, essentially, or consist of the sequence tttccttcccccccccccccccccccccccccccccccccccccccccccc (Sequence ID 1) or a fragment thereof.

142. The method according to claim 141, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, are essentially, or consist of the sequence tttccttcccccctttccttt (Sequence ID 2).

143. The method according to any one of claims 133 to 142, an immune cell, or an inline system, wherein the type P spacer, the type R spacer, or both include, are essentially, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

144. The method according to any one of claims 106 to 143, an immune cell, or an inline system, wherein the type P marker, the type R marker, or both comprises a microRNA.

145. The method according to any one of claims 103 to 144, an immune cell, or an inline system, wherein the regulator comprises an RNA-binding protein, siRNA, an aptamer, or a combination thereof.

146. The method according to claim 145, an immune cell, or an inline system, wherein the RNA-binding protein comprises a ribonuclease.

147. The method according to claim 146, an immune cell, or an inline system, wherein the ribonuclease comprises a Cas protein.

148. The method according to claim 147, an immune cell, or an in-line system, wherein the Cas protein comprises the Cas6 protein.

149. The method according to any one of claims 1 to 148, an immune cell, or an in-line system, wherein the payload comprises a therapeutic protein, a reporter protein, an immunomodulatory protein, a chimeric antigen receptor (CAR), or a combination thereof.

150. The method according to any one of claims 1 to 149, an immune cell, or an inline system, wherein the payload sequence comprises one or more elements that enhance the translation of the encoded protein compared to the regulator sequence.

151. The method according to claim 150, an immune cell, or an inline system, wherein one or more of the elements comprises an aptamer for a translation initiation factor (e.g., eIF4G).

152. The aforementioned synthesis circuit (1) Intra-sequence ribosome entry sites (IRES), (3) UTR, (4) Sequence encoding a signal peptide, (5) Translation start sequence, (6) Poly-A sequence, (7) Sequences encoding RNA-binding proteins, (8) A sequence encoding the 2A ribosome skipped peptide, or (9) The method according to any one of claims 1 to 20 and 28 to 151, further comprising any combination of (1) to (8), the immune cells according to any one of claims 21, 22 and 28 to 151, or the inline system according to any one of claims 23 to 151.

153. The method according to any one of claims 1 to 20 and 28 to 152, wherein the synthesis circuit does not include any sequences derived from a non-human genome; the immune cells according to any one of claims 21, 22 and 28 to 152; or the inline system according to any one of claims 23 to 152.

154. The method according to any one of claims 1 to 20 and 28 to 153, wherein the synthesis circuit is delivered by a delivery agent, the immune cells according to any one of claims 21, 22 and 28 to 153, or the inline system according to any one of claims 23 to 153.

155. The method according to claim 154, immune cells, or in-line system, wherein the delivery agent is a nanoparticle comprising (i) the synthesis circuit and (ii) one or more lipids and / or lipid-like materials.

156. The method according to claim 155, immune cells, or an in-line system, wherein the one or more lipids include ionized lipids, cationic lipids, lipidoids, non-cationic helper lipids, phospholipids, sterols, or other structural lipids, or a combination thereof.

157. The ionized lipids are ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 5), di((Z)-nona-2-en-1-yl)9-((4-(dimethylamino) Butanoyl(oxy)heptadecanedioate (L319), 3-(didodecylamino)-N1,N1,4tridodecyl-1-piperazineethaneamine (KL10), Nl-[2(didodecylamino)ethyl]-N1,N4,N4-tridodecyl1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylamino Tyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z) -Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA), (2R)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,The method according to claim 156, immune cells, or an in-line system comprising 12-dien-1-yloxy]propan-1-amine (octyl-ClinDMA(2S)), or a combination thereof.

158. The cationic lipids include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTEVI), and 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl] -N,N-dimethyl-l-propaneaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(l,2-dioleoyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyl Il-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), l,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), l,2-dimyristoyl-3-trimethylammonium-propane The method according to claim 156, comprising pan(DMTAP), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC), l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC), l,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof, an immune cell, or an in-line system.

159. The aforementioned lipidoid is 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine 2,5-dione(cKK-E12), tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazandiyl)bis(propane-3,1diyl))bis(azantriyl))tetrapropionate(306Oi １０ ), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione(OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azantriyl))tetrakis(ethane2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12 'Z,12''Z,12'''Z)-Tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin),(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(axantriyl))Tetrakis(butane-4,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-Tetrakis(octadeca-9,12-dienoate (T) (OF-C4-Deg-Lin), N1,N3,N5-Tris(3-(didodecylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), Hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)lis(azandiyl))tris(propane-3,1-diyl))tris(azantriyl))hexanonaate ( A method, cell, or in-line system according to claim 156, comprising FTT5), PL-1,98N12-5, ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18(A2)), A12-Iso5-2DC18(A12), or any combination thereof.

160. The method, cells, or in-line system according to claim 159, wherein the lipidoid is TT3.

161. The phospholipids mentioned above are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-diundecane. Noyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2-cholesterylhemisuxinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 A method, cell, or in-line system according to any one of claims 156 to 160, selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-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 any combination thereof.

162. The phospholipids are 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC, OSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2 PE), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 A method, cell, or in-line system according to any one of claims 156 to 161, selected from the group consisting of PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6 PE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof.

163. The method, cells, or in-line system according to any one of claims 156 to 162, wherein the sterols include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and combinations thereof.

164. The method, cells, or in-line system according to any one of claims 155 to 163, wherein the one or more lipids and / or lipid-like materials are pegylated.

165. A method, immune cells, or in-line system according to any one of claims 155 to 164, further comprising an immune ligand.

166. The method according to any one of claims 155 to 165, an immune cell, or an in-line system, wherein the one or more lipids and / or lipid-like materials comprise ionized lipids (e.g., cationic lipids) in a molar ratio of about 10 to 50%.

167. The method according to any one of claims 155 to 166, an immune cell, or an in-line system, wherein the one or more lipids and / or lipid-like materials comprise phospholipids in a molar ratio of about 10 to 40%.

168. The method according to any one of claims 155 to 167, an immune cell, or an in-line system, wherein the one or more lipids and / or lipid-like materials comprise sterols (e.g., cholesterol) in a molar ratio of about 20 to 50%.

169. The method according to any one of claims 155 to 168, an immune cell, or an in-line system, wherein the one or more lipids and / or lipid-like materials comprise pegylated lipids in a molar ratio of about 0 to 10%.

170. The method according to any one of claims 1 to 20 and 28 to 169, wherein the synthesis circuit, the delivery agent, or the nanoparticles are formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier; the immune cells according to any one of claims 21, 22, and 28 to 169; the in-line system according to any one of claims 23 to 169; or the nanoparticles according to any one of claims 155 to 169.

171. The method according to claim 170, immune cells, or an inline system formulated for intratumor, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, lymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration.

172. The method according to any one of claims 1 to 20 and 28 to 171, wherein the cells include T cells, NK cells, B cells, or any combination thereof; the immune cells according to any one of claims 21, 22 and 28 to 171; the inline system according to any one of claims 23 to 171; or the nanoparticles according to any one of claims 155 to 171.

173. The method according to any one of claims 1 to 20 and 28 to 171, wherein the cells include CD8+ T cells, CD4+ T cells, or any combination thereof; the immune cells according to any one of claims 21, 22 and 28 to 171; the inline system according to any one of claims 23 to 171; or the nanoparticles according to any one of claims 155 to 171.

174. The aforementioned synthesis circuit It includes a nucleotide sequence that codes for the payload (payload sequence), The payload array includes a sensor that recognizes a marker, The method according to any one of claims 1 to 8, wherein the recognition of the marker by the sensor reduces the expression of the payload.

Images