RNA-based synthesis loop for generating engineered immune cells for in vitro cell therapy

Abstract

The present disclosure relates to a method of generating a cell, e.g., an immune cell, for in vitro cell therapy by contacting a synthesis loop useful for selectively expressing a payload in a target cell, e.g., an immune cell, with the target cell, e.g., the immune cell. In some aspects, a synthesis loop includes a sequence of payloads including sensors. In some aspects, the synthesis loop further comprises a sensor-containing modulator sequence. Also provided herein are methods of using such synthetic loops in the treatment of various diseases or conditions using in vitro cell therapy.

Description

Cross-references to related applications

[0001] This PCT application claims priority to U.S. Provisional Application No. 63 / 515,247, filed July 24, 2023, which is incorporated herein by reference in its entirety.

[0002] References to sequence lists submitted electronically via EFS-WEB The contents of the sequence list (filename: 4597_029PC01_SequenceListing_ST26.xml; size: 4,240 bytes; creation date: July 23, 2024), which was submitted electronically and together with the application, are incorporated herein by reference in their entirety. Background Technology

[0003] Cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, represent a revolutionary new pillar in cancer treatment. While CAR-T cell therapy has yielded significant clinical responses in certain subsets of B-cell leukemia or lymphoma, numerous challenges limit its efficacy in solid tumors and hematologic malignancies. Barriers to effective CAR-T cell therapy include severe life-threatening toxicity, limited antitumor activity, antigen escape, transport limitations, and limited tumor invasion. Furthermore, the interaction between the host and tumor microenvironment and CAR-T cells significantly alters CAR-T cell function. In addition, the development and implementation of 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, there is a need to develop new cell constructs to improve clinical efficacy in hematologic malignancies and solid tumors, as well as strategies to overcome the limitations of cell therapy in hematologic malignancies and solid tumors. Summary of the Invention

[0004] This document provides a method for generating engineered immune cells for in vitro cell therapy to a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject, wherein the synthetic circuit is capable of selectively expressing a payload in the immune cells. In some aspects, the immune cells used in the method are not exposed outside the online system throughout the duration of the therapy. In some aspects, the immune cells in the method express a payload on the immune cells due to the contact. In some aspects, the immune cells in the method are administered directly back to the subject from the online system.

[0005] This article also provides a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthetic circuit connected to a closed online system of the subject, and wherein the synthetic circuit is capable of selectively expressing the payload in the cells.

[0006] In some aspects, this disclosure provides a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit in a closed online system connected to the subject, and administering the 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.

[0007] In some respects, the subject does not undergo lymphocyte clearance prior to the treatment. In some respects, the treatment is administered within 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.

[0008] In some aspects, the synthetic circuit includes a nucleotide sequence encoding a payload (payload sequence), wherein the payload sequence includes a sensor for recognizing a marker, and wherein recognizing the marker with the sensor reduces the expression of the payload.

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

[0010] In some aspects, this disclosure provides an online system comprising immune cells expressing a payload encoded by a synthetic circuit for a method of in vitro cell therapy. In some aspects, the online system is completely closed. In some aspects, the online system is connected to a subject requiring the therapy. In some aspects, the immune cells in the online system are obtained through the subject. In some aspects, the online system further comprises cells other than immune cells.

[0011] In some respects, payloads that may be used in this disclosure include chimeric antigen receptors (CARs), T-cell receptors (TCRs), or TCR mimics.In some aspects, CAR targets CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE 1, ETV6-AML, Sperminin 17, XAGE 1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivability protein, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) The extracellular portion of the protein, or any combination thereof, is associated with the following proteins: ETS fusion gene, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, and APRIL.In some aspects, TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE 1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, 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 The extracellular portion 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.

[0012] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 (P-type sensor) capable of specifically recognizing the regulator, wherein the regulator sequence comprises a sensor (R-type sensor) capable of specifically recognizing a marker, and wherein the regulator is different from the marker (R-type marker) recognized by the R-type sensor.

[0013] In some aspects, the synthetic circuits that can be used in this disclosure may include a nucleotide sequence (payload sequence) encoding the payload, wherein the payload sequence includes a sensor (P-type sensor) capable of specifically recognizing a marker, wherein the marker is not endogenously present in the immune cells, or the marker is endogenously present only in the immune cells.

[0014] In some aspects, the payload sequence available in this disclosure may comprise a plurality of the said P-type sensors. In some aspects, the plurality of said P-type sensors may comprise two, three, four, five, six, seven, or eight or more P-type sensors. In some aspects, each of said P-type sensors is identical. In some aspects, one or more of said P-type sensors are different.

[0015] In some aspects, the payload sequence used in this disclosure may comprise a sequence of spacer sub-sequences (P-type spacers). In some aspects, the payload sequence comprises a plurality of P-type spacers. In some aspects, each of the P-type spacers is identical. In some aspects, one or more of the P-type spacers are different. In some aspects, (a) at least one P-type spacer is located upstream of the P-type sensor, (b) at least one P-type spacer is located downstream of the P-type sensor, or (c) both (a) and (b). In some aspects, the synthesis loop described herein (e.g., as described above) comprises at least two P-type sensors, wherein at least one P-type spacer is located between the at least two P-type sensors.

[0016] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker, wherein the regulator sequence includes a sensor (R-type sensor) capable of specifically recognizing a marker, and wherein the regulator, the marker recognized by the second P-type sensor (second P-type marker), and / or the marker recognized by the R-type sensor (R-type marker) are different.

[0017] In some aspects, the payload sequence available in this disclosure may comprise a plurality of the first P-type sensors. In some aspects, the plurality of first P-type sensors may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve first P-type sensors. In some aspects, each of the first P-type sensors is identical. In some aspects, one or more of the first P-type sensors are different.

[0018] In some aspects, the payload sequence available in this disclosure may comprise a plurality of the second P-type sensors. In some aspects, the plurality of second P-type sensors may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve second P-type sensors. In some aspects, each of the second P-type sensors is identical. In some aspects, one or more of the second P-type sensors are different.

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

[0020] When the synthesis circuit available in this disclosure comprises a plurality of first P-type sensors, in some aspects, two or more of the first P-type sensors are separated by P-type spacers. In some aspects, each of the first P-type sensors is separated by a P-type spacer.

[0021] When the synthesis circuit available in this disclosure includes a plurality of second P-type sensors, in some aspects, two or more of the second P-type sensors are separated by P-type spacers. In some aspects, each of the second P-type sensors is separated by a P-type spacer.

[0022] In some aspects, the length of the P-type spacer used in this disclosure is between about 1 and about 50 nucleotides. In some aspects, the length of the P-type spacer is at least about 10 nucleotides. In some aspects, the length of the P-type spacer is about 10 nucleotides, about 20 nucleotides, or about 50 nucleotides. In some aspects, the P-type spacer comprises, substantially comprises, or consists of the sequence tttcctttcccccttccctt (SEQ ID NO: 2) or gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

[0023] For any synthetic circuit provided herein that can be used in this disclosure (e.g., those described above), in some aspects, the modulator sequence comprises a plurality of said R-type sensors. In some aspects, the plurality of said R-type sensors comprises two, three, four, five, six, seven, or eight or more R-type sensors. In some aspects, each of said R-type sensors is identical. In some aspects, one or more of said R-type sensors are different.

[0024] In some aspects, the modulator sequence available in this disclosure may comprise a spacer sequence (R-type spacer). In some aspects, the modulator sequence may comprise a plurality of R-type spacers. In some aspects, each of the R-type spacers is identical. In some aspects, one or more R-type spacers are distinct.

[0025] When the synthesis circuit available in this disclosure includes a plurality of the R-type sensors, in some aspects, two or more of the R-type sensors are separated by R-type spacers. In some aspects, each of the R-type sensors is separated by an R-type spacer. In some aspects, at least one R-type spacer is upstream of at least one R-type sensor.

[0026] In some aspects, the length of the R-type spacer used in this disclosure is between about 1 and about 50 nucleotides. In some aspects, the length of the R-type spacer is at least about 10 nucleotides. In some aspects, the length of the R-type spacer is about 10 nucleotides, about 20 nucleotides, or about 50 nucleotides. In some aspects, the R-type spacer comprises, is substantially composed of, or is composed of the sequence tttcctttcccccttccctttccttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the R-type spacer comprises, is substantially composed of, or is composed of the sequence tttcctttcccccttccctt (SEQ ID NO: 2). In some aspects, the R-type spacer comprises, is substantially composed of, or is composed of the sequence gcggccgctaaa (SEQ ID NO: 3).

[0027] For any synthetic circuit provided herein (e.g., described above) that can be used in this disclosure, in some aspects the first marker, the second marker, or the first marker and the second marker comprise microRNA, protein, metabolite, or combination thereof.

[0028] In some aspects, the marker is a regulator, wherein the regulator includes RNA-binding proteins, siRNA, shRNA, precursor miRNA, ribozymes, or combinations thereof. In some aspects, the RNA-binding protein includes ribonucleases. In some aspects, the ribonuclease includes Cas proteins. In some aspects, the Cas protein includes the Cas6 protein.

[0029] For any synthetic circuit provided herein (e.g., as described above) that can be used in this disclosure, in some aspects, the payload sequence, the regulator sequence, or both the payload sequence and the regulator sequence comprise linear RNA or circular RNA. In some aspects, the payload sequence is a self-replicating RNA, and the regulator sequence is a non-replicating RNA. In some aspects, the payload sequence is a self-replicating RNA, and the regulator sequence is a circular RNA. In some aspects, the payload sequence is a self-replicating RNA, and the regulator sequence is a linear non-replicating RNA. In some aspects, the payload sequence is a circular RNA, and the regulator sequence is a circular RNA. In some aspects, the payload sequence is a circular RNA, and the regulator sequence is a linear non-replicating RNA.

[0030] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 includes a sensor (P-type sensor) capable of specifically recognizing the regulator; wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator is different from the marker recognized by the R-type sensor (R-type marker).

[0031] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0032] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 includes a sensor (P-type sensor) capable of specifically recognizing the regulator; wherein the regulator sequence is a non-replicating circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator is different from the marker (R-type marker) recognized by the R-type sensor.

[0033] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a non-replicating linear RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0034] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0035] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 includes a sensor (P-type sensor) capable of specifically recognizing the regulator; wherein the regulator sequence is a non-replicating circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator is different from the marker (R-type marker) recognized by the R-type sensor.

[0036] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a non-replicating linear RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0037] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 includes a sensor (P-type sensor) capable of specifically recognizing the regulator; wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator is different from the marker recognized by the R-type sensor (R-type marker).

[0038] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0039] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (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 includes a sensor (P-type sensor) capable of specifically recognizing the regulator; wherein the regulator sequence is a non-replicating linear RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator is different from the marker recognized by the R-type sensor (R-type marker).

[0040] In some aspects, the synthetic circuits that can be used in this disclosure include: (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 includes a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing a marker; wherein the regulator sequence is a non-replicating linear RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker; and wherein the regulator, the marker recognized by the second P-type sensor (P-type marker), and the marker recognized by the R-type sensor (R-type marker) are different.

[0041] For any synthetic circuit provided herein (e.g., described above), in some respects, (a) the payload sequence comprises a plurality of the first P-type sensors, (b) the payload sequence comprises a plurality of the second P-type sensors, (c) the modulator sequence comprises a plurality of the R-type sensors, or (d) any combination of (a) to (c).

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

[0043] In some aspects, the payload sequence includes the plurality of first P-type sensors, wherein two or more of the first type sensors are separated by P-type spacers. In some aspects, the payload sequence includes the plurality of second P-type sensors, wherein two or more of the second type sensors are separated by P-type spacers. In some aspects, the regulator sequence includes the plurality of R-type sensors, wherein two or more of the R-type sensors are separated by R-type spacers.

[0044] In some aspects, the length of the P-type spacer, the R-type spacer, or both is between about 1 and about 50 nucleotides. In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence tttcctttcccccttccctttccttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence tttccttcccccttccctt (SEQ ID NO: 2). In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some respects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3).

[0045] In some aspects, the P-type marker, the R-type marker, or both comprise microRNA, protein, metabolite, or a combination thereof. In some aspects, the regulator comprises RNA-binding protein, siRNA, shRNA, precursor miRNA, ribozyme, or a combination thereof.

[0046] In some aspects, the regulator is an RNA-binding protein, wherein the RNA-binding protein includes a ribonuclease. In some aspects, the ribonuclease includes a Cas protein. In some aspects, the Cas protein includes the Cas6 protein.

[0047] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein when the payload sequence and the regulator sequence are present in an immune cell, the payload is expressed as a first expression in the immune cell, the regulator is expressed as a second expression in the immune cell, and wherein the first expression is greater than the second expression.

[0048] In some respects, (a) the payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) the regulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, and wherein the regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

[0049] In some aspects, the recognition of the P-type marker by the second P-type sensor suppresses the expression of the payload. In some aspects, the recognition of the R-type marker by the R-type sensor suppresses the expression of the regulator.

[0050] In some aspects, (a) the immune cells do not express the P-type marker at a level sufficient to activate the second P-type sensor, and (b) the immune cells express the R-type marker at a level sufficient to activate the R-type sensor. In some aspects, (a) non-immune cells express the P-type marker at a level sufficient to activate the second P-type sensor, (b) non-immune cells do not express the R-type marker at a level sufficient to activate the R-type sensor, or (c) both (a) and (b).

[0051] In some aspects, the synthetic circuits that can be used in this disclosure may comprise: (a) a first nucleotide sequence encoding a payload (payload sequence) and (b) a second nucleotide sequence encoding a regulator (regulator sequence), wherein when the payload sequence and the regulator sequence are present in non-immune cells, the payload is expressed as a first expression in the non-immune cells, the regulator is expressed as a second expression in the non-immune cells, and wherein the second expression is greater than the first expression.

[0052] In some respects, (a) the payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) the regulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, wherein the regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

[0054] In some aspects, the synthetic circuits used in this disclosure may comprise: (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 is contacted with a cell population comprising 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. In some aspects, 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.

[0055] In some respects, (a) the payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) the regulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, wherein the regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

[0057] In some aspects, the synthetic circuits used in this disclosure may comprise: (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 is contacted with a cell population comprising 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. In some aspects, 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.

[0058] In some respects, (a) the payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) the regulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, wherein the regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

[0060] For at least the above-described synthetic circuits, in some aspects, the payload sequence is a self-replicating RNA. In some aspects, the regulator sequence is a non-replicating linear RNA. In some aspects, the payload is a circular RNA. In some aspects, the regulator sequence is a circular RNA. In some aspects, the payload sequence is a self-replicating RNA, and the regulator sequence is a circular RNA. In some aspects, the payload sequence is a self-replicating RNA, and the regulator sequence is a non-replicating linear RNA. In some aspects, the payload sequence is a circular RNA, and the regulator sequence is a circular RNA. In some aspects, the payload sequence is a circular RNA, and the regulator sequence is a non-replicating linear RNA.

[0061] In some respects, the above-described synthetic circuit comprises a payload sequence and a regulator sequence, wherein: (a) the payload sequence comprises a plurality of first P-type sensors, (b) the payload sequence comprises a plurality of second P-type sensors, (c) the regulator sequence comprises a plurality of R-type sensors, or (d) any combination of (a) to (c).

[0062] In some aspects, (a) the payload sequence includes a sequence of spacer sub-sequences (P-type spacers), (b) the payload sequence includes a sequence of spacer sub-sequences (R-type spacers), or (c) both (a) and (b). In some aspects, the P-type spacers are located between the first P-type sensor and the second P-type sensor. In some aspects, the P-type spacers are located between the payload encoding sequence and (a) the first P-type sensor, the payload encoding sequence and (b) the second P-type sensor, or (c) both (a) and (b). In some aspects, the R-type spacers are located between the regulator encoding sequence and the R-type sensor. In some aspects, the payload sequence includes the plurality of first P-type sensors, wherein two or more of the first P-type sensors are separated by P-type spacers. In some aspects, the payload sequence includes the plurality of second P-type sensors, wherein two or more of the second P-type sensors are separated by P-type spacers. In some aspects, the regulator sequence includes the plurality of R-type sensors, wherein two or more of the R-type sensors are separated by R-type spacers.

[0063] In some aspects, the length of the P-type spacer, the R-type spacer, or both is between about 1 and about 50 nucleotides. In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence tttcctttcccccttccctttccttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence tttccttcccccttccctt (SEQ ID NO: 2). In some aspects, the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

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

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

[0066] In some respects, the synthetic circuits provided herein (e.g., those described above) further comprise: (1) an internal 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 jumping peptide, or (8) any combination of (1) to (7).

[0067] For any synthetic circuit provided herein (e.g., described above), in some respects, the synthetic circuit does not contain any sequence derived from a non-human genome.

[0068] Some aspects of this disclosure relate to a carrier comprising the synthetic circuit provided herein.

[0069] This document also provides nanoparticles comprising (i) any synthetic circuit of this disclosure (e.g., described above) and (ii) one or more types of lipids and / or lipid-like materials. In some aspects, the one or more types of lipids include ionizable lipids, cationic lipids, lipid-like substances, non-cationic auxiliary lipids, phospholipids, sterols or other structural lipids or combinations thereof.

[0070] In some aspects, the ionizable lipids include ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate heptadecaned-9-yl ester (SM-102), 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate heptadecaned-9-yl ester (lipid 5), 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate di(( Z)-Non-2-en-1-yl) ester (L319), 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tetrazyl)-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy -N,N-Dimethylaminopropane (DLin-DMA), 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DM) A) 1,2-Dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N, N-Dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)) or combinations thereof.

[0071] In some aspects, the cationic lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleoyloxy-N- [2(Speramido)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propionate ammonium (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(1,2-dioleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N ...oleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyloxypropyl-3-yl)-N,N-dioleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyloxypropyl-3-yl)-N,N-dioleoyloxypropyl-3-yl)-N,N-dioleoyloxypropyl-3-yl)-N,N-dioleoyloxypropyl-3-yl)-N,N-dioleo Acyl-N,N-dimethylammonium chloride (DODAC), 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (DLePC), 1,2-distearyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1, 2-Distearatel-sn-glycerol-3-ethylphosphocholine (DSePC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)-sn-glycerol-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0072] In some aspects, the lipids include 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecane-2-ol) (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazin 2,5-dione (cKK-E12), tetra(8-methylnonyl)3,3′,3″,3 -(((methylazonidyl)bis(propane-3,1-diyl))bis(azontriyl))tetrapropionate (306Oi) 10), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecano-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(azanetriyl))tetra(ethane-2,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadec-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadecyl-9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tris(3-(bis(dodecylamino)propyl)phenyl)1,3,5-tricarboxamide (TT3), hexa(octyl-3-yl)9,9′,9″,9 ,9″″,9 "-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexylnonanoate (FTT5), PL-1, 98N12-5, 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidone-1-yl)propyl)-2,5-dihydro-1H-imidazolium-2-carboxylate (A2-Iso5-2DC18(A2)), A12-Iso5-2DC18(A12), or any combination thereof. In some aspects, the lipid is TT3.

[0073] In some aspects, the phospholipids include 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:O diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), and 1-hexadecyl-sn-glycerol-3-phosphate choline (C16). Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(eicosahenoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and any combination thereof.

[0074] In some aspects, the phospholipids are selected from the group consisting of: 1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycerol-3-phosphate choline (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycerol-3-phosphate choline (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycerol-3-phosphate choline (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphate choline (16:0-18:0 PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (16:0-18:1... PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (18:0-18:1) PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (18:0-18:2PC), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (18:0-20:4PC), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (18:0-22:6PC), 1-oleoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC,OSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:1 PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:2 PE), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:1 PE), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonico-sn-glycerol-3-phosphate ethanolamine (18:0-20:4 PE), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (18:0-22:6 PE), 1-oleoyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), and any combination thereof.

[0075] In some respects, sterols include cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, tomatine, ursolic acid, α-tocopherol, and combinations thereof.

[0076] In some respects, the one or more types of lipids and / or lipid-like materials are polyethylene glycol-modified.

[0077] In some respects, any nanoparticles provided herein (e.g., those described above) further include a targeting ligand.

[0078] For any nanoparticles provided herein (e.g., those described above), in some aspects, the one or more types of lipids and / or lipid-like materials comprise about 10%-50% in molar proportion of ionizable lipids (e.g., cationic lipids). In some aspects, the one or more types of lipids and / or lipid-like materials comprise about 10%-40% in molar proportion of phospholipids. In some aspects, the one or more types of lipids and / or lipid-like materials comprise about 20%-50% in molar proportion of sterols (e.g., cholesterol). In some aspects, the one or more types of lipids and / or lipid-like materials comprise about 0-10% in molar proportion of polyethylene glycol-modified lipids.

[0079] This document provides a pharmaceutical composition that can be used in accordance with this disclosure, the pharmaceutical composition comprising any of the synthetic circuits, carriers or nanoparticles described herein, and a pharmaceutically acceptable loading agent. In some aspects, the pharmaceutical composition is formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of an exemplary synthetic circuit described herein. As shown, the modulator sequence and / or payload sequence can be linear or cyclic.

[0081] Figure 2 This is a schematic diagram showing an array of target sites consisting of all target sites (TSs) that bind to the same siRNA. Each sequence contains a coding region (encoding mVenus-PEST) and a 3'-UTR. As shown, sequences contain no target site ("No TS"), a single target site ("1X TS"), two target sites ("2X TS"), three target sites ("3X TS"), or four target sites ("4X TS"). As shown, some sequences also contain one or more spacer subsequences ranging in length from 10 to 50 nucleotides.

[0082] Figure 3 The effect of the number of adjacent target sites (TS) immediately following the stop codon of the mVenus-PEST reporter gene on payload expression is shown. Specifically, the figure provides a graph depicting the median fluorescence (any unit (au)) of mVenus constructs containing 1X siRNA TS, 2X siRNA TS, 3X siRNA TS, 4X siRNA TS, no TS, or no reporter gene after siRNA administration (0, 1, 10, or 100 nM). Data represent the median collected by flow cytometry from three technique replicates (n = 3) 20 h after reporter gene and siRNA electroporation.

[0083] Figure 4The effect of spacer sequences on payload expression is shown. Specifically, the figure provides a graph depicting the median mVenus fluorescence (au) after siRNA administration (0, 1, 10, or 100 nM) for the following: reporter gene constructs containing two siRNA target sites (2X siRNA2), separated by a 20 nucleotide (nt) spacer sequence (2X siRNA2 - 20 nt), or separated by a 50 nt spacer sequence (2X siRNA2 - 50 nt); a control reporter gene construct without target sites (no TS); and a control condition untransfected with mVenus reporter genes (no reporter gene). Data represent median fluorescence intensity from three technical replicates (n=3) as measured by flow cytometry 20 hours after electroporation of the reporter gene and siRNA.

[0084] Figure 5 The effect of increasing target site copy number on payload expression is shown when a 20 nt spacer sequence is included between target sites. Specifically, the figure provides a graph depicting the median mVenus fluorescence (au) of constructs containing two, three, or four siRNA target sites (TSs) and 20 nucleotide (nt) spacer sequences between adjacent TSs (2X siRNA² - 20 nt, 3X siRNA² - 20 nt, 4X siRNA² - 20 nt, respectively), without TSs, or without a reporter gene, after siRNA administration (0, 1, 10, or 100 nM). Data represent the median collected by flow cytometry from three technical replicates (n = 3) 20 hours after reporter gene and siRNA electroporation.

[0085] Figures 6A-6B This shows the linear ( Figure 6A ) and cyclic ( Figure 6B RNA, assessment of detargeting payload expression. Figure 6A This is a graph depicting mVenus-PEST fluorescence (au) in linear modRNA loops containing the mVenus-PEST reporter gene and 0 to four miR-b target sites (no miR TS, 1X TS, 2X TS, or 4X TS) in hepatocyte (Huh-7) and control (HEK293T) cell lines. Background autofluorescence levels for each cell line were also plotted. Data points represent the mean geometric mean fluorescence intensity collected from three technical replicates (n = 3) 20 hours after electroporation. Figure 6BThis is a graph depicting the median fluorescence intensity (au) of circRNA constructs containing 1–4 miR-a target sites (without TS, 1X TS, 2X TS, or 4X TS) immediately following the stop codon of the mVenus-PEST reporter gene in HEK293T and HeLa cell lines. Data represent the geometric mean fluorescence intensity collected from three technical replicates (n = 3) at 4 and 24 hours post-electroporation.

[0086] Figure 7A This is a schematic diagram showing the location of the regulator target site in an exemplary circRNA that expresses mVenus-PEST and contains CVB3 IRES, mVenus-PEST, and a regulator (Cas6e) target site in one of five locations. Figure 7B This describes the effect of the presence or absence of the Cas6e regulator on circRNAs compared to those with electroporation only and those without Cas6e target site knockdown. Figure 7A The bar chart shows the effect of the percentage of normalized (no)TS expression in the circRNA construct.

[0087] Figure 8A This is a schematic diagram showing the Nx base pair spacers between the stop codon and the array of 3' UTR target sites. Figures 8B-8C This is a graph depicting mVenus fluorescence (any unit) of constructs containing 1X or 2X siRNA target sites (TS) and 0, 7, or 12 nt spacers after siRNA administration (0, 1, 10, 100 nM). Data represent the geometric mean fluorescence intensity collected by flow cytometry from three technique replicates (n = 3) at 6 h and 24 h after reporter gene and siRNA electroporation.

[0088] Figure 9 This is a schematic diagram showing the target site array of multiple input classifiers.

[0089] Figure 10 It is a graph depicting the effect of cotransduction of one or more siRNAs with or without target sites in the target site array on the median fluorescence (any unit) of mVenus in HEK293T cells electroporated with an mVenus-PEST reporter gene containing a target site array immediately following a stop codon, and without siRNA, siRNA1, siRNA2, or both (3X siRNA1 - 20nt, 3X siRNA2 - siRNA1 interleaved, 3X siRNA1 - siRNA2 interleaved, 3X siRNA2 3X siRNA1 adjacent, 3X siRNA1 3X siRNA2 adjacent, no target site, no reporter gene).

[0090] Figure 11 This is a schematic diagram showing the Nx miR and modulator target sites placed in the 5' or 3' UTR at positions A, B, and C.

[0091] Figure 12 This is a bar graph depicting the expression of circRNA (fold change compared to the absence of miR target sequences (TS)) in HEK293T, Huh-7, and HeLa cells electroporated with circRNA at 4 and 24 hours post-transfection. Circular RNA contains either 4x miR-b TS or 4x miR-a TS.

[0092] Figures 13A-13C The study showed that the regulator Cas6e downregulated circRNA. Figure 13A This is a schematic diagram showing how linear and circular regulator RNAs downregulate target circRNAs containing the Cas6e target site (TS) and encoding the fluorescent protein mVenus-PEST. Figure 13B This is a bar graph showing that the expression of circRNA and the mRNA regulator mVenus is reduced to background levels in BHK-21 cells transfected with target RNA containing Cas6e TS. Figure 13C This is a bar graph showing that Cas6e has no effect on mCherry expression of RNAs (mRNA and circRNA) that do not contain its target sites.

[0093] Figures 14A-14B It is a linear non-replicating (non-rep) payload mRNA strand that shows the effect of regulators on target sequences containing RNA regulators. Figure 14A ) and replicon RNAs containing target sequences of RNA regulators ( Figure 14B Bar graph showing the effect of mRNA. Figure 14A The effects of mRNA transfection into BHK-21 cells with or without modRNA expression regulators on payload expression were shown for both unmodified (unmodRNA) and modified (modRNA) payloads. Figure 14B The effect of co-transfection with or without modRNA expressing RNA regulator on the percentage of mVenus positive cells was shown for BHK-21 cells with replicon RNA containing the target sequence of RNA regulator (transfected at two different doses (20 ng or 40 ng)).

[0094] Figure 15This is a bar graph depicting the expression (fold change relative to miR-target sequence (TS)-free) of CVB3 IRES-driven RNA encoding EGFP-PEST in HEK293T and Huh-7 cells electroporated with circular RNA. Circular RNA is immediately following a stop codon in cells with no miR TS, TS with 4x miR-b target sites, or 4x miR-a target sites. A reporter-free control was also used. The mean of the EP-only group (reporter-free control) relative to CVB3 cells without miR TS was normalized.

[0095] Figure 16 This is a bar graph depicting the ratio of normalized mVenus-PEST expression levels in HEK293T (non-cancer) cells to HeLa (cancer) cells after electroporation with multiple human miRNA target sites (corresponding to miRNAs with higher activity in HEK293T cells than in HeLa cells). The geometric mean of mVenus-PEST expression in each cell type was normalized relative to the geometric mean of modRNAs without miRNA sensors after subtracting background fluorescence levels. Flow cytometry data for both cell types (n = 3) were collected approximately 24 hours after electroporation.

[0096] Figure 17A The mean geometric mean (GMean) of mVenus fluorescence (au) of non-replicating modRNA transfected into HEK293T cells or the human hepatocyte cell line Huh-7 via electroporation is shown. Figure 17B The expression of mVenus fluorescence of replicon RNA transfected into HEK293T cells or the human hepatocyte cell line Huh-7 via electroporation is shown. Non-replicating modRNA expressing the near-infrared fluorescent reporter protein miRFP720 was co-transfected with each replicon RNA as a transfection marker. Data represent the geometric mean of flow cytometry data from three technical replicates (n = 3) collected approximately 24 hours after electroporation. Expression output of miRFP720-positive cells that were also mVenus-PEST positive was calculated. Figure 17A ) and percentage ( Figure 17B ).

[0097] Figure 18 This is a bar graph depicting the mean radiance (p / s / cm² / sr) of firefly luciferase from the spleen, lung, kidney, lymph node (LN), and liver of mice injected with lipid nanoparticles containing a reporter gene modRNA encoding firefly luciferase, with or without liver-specific microRNA miR-b (gray bars) (white bars), or a mediator control (black bars). Mice were sacrificed 6 hours later.

[0098] Figure 19 This is a bar graph depicting the mean radiance (p / s / cm2 / sr) of firefly luciferase from the liver and spleen of mice injected with lipid nanoparticles containing a reporter gene modRNA encoding firefly luciferase, with or without spleen-associated miRNA (miR-h) added (gray bars) or without added (white bars), or as a mediator control. Mice were sacrificed 6 hours later.

[0099] Figure 20 This is a bar graph depicting the expression (average intensity of green target object, GCU) of green fluorescent reporter in Huh7 (black bar) and HEK293T (gray bar) cells transfected with an RNA circuit consisting of: (1) a replicon payload strand expressing the green fluorescent reporter and containing a first P-type sensor responsive to the regulator protein Cas6e, and (2) a linear non-replicating regulator strand expressing the regulator protein Cas6e and containing an R-type sensor responsive to miR-b. Control Huh7 and HEK293T cells were transfected in parallel with the same RNA circuit but lacking the R-type sensor. The expression of the green fluorescent payload was measured by quantitative imaging 6 hours after transfection.

[0100] Figure 21 This is a bar graph depicting the effect of the R-type sensor on the expression of the mVenus reporter protein (payload reporter) of the replicon payload RNA sequence. A linear non-replicating regulator sequence was constructed that expresses the Cas6e regulator protein linked to the mCherry reporter via a 2A self-cleaving peptide, such that the Cas6e regulator protein and the mCherry reporter (regulator reporter) are co-expressed by the same RNA sequence. Another regulator sequence form was constructed that also contains an R-type sensor that recognizes miR-i. A549 lung cancer cells expressing high levels of miR-i were transfected with the payload sequence alone, or with the payload sequence and one of the two regulator sequence forms (with or without the R-type sensor). At the bottom of the graph, a "+" after "Regulator" indicates co-transfection of the payload sequence with one of the two regulator sequence forms. A "+" after "R-type Sensor" at the bottom of the graph indicates co-transfection of the payload sequence with a regulator form containing an R-type sensor that recognizes miR-i. The mean fluorescence intensity (MFI, au) of mCherry regulator reporter and mVenus payload reporter in A549 lung cancer cells was measured by flow cytometry. Detailed Implementation

[0101] This disclosure generally relates to a method for generating engineered immune cells for in vitro cell therapy of a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject, wherein the synthetic circuit is capable of selectively expressing a payload in the immune cells. This document also provides a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthetic circuit connected to a closed online system of the subject, and wherein the synthetic circuit is capable of selectively expressing the payload in the cells. In some aspects, this disclosure further includes a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject, and administering the 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. This disclosure also includes the immune cells used in the method and an online system comprising the cells.

[0102] The synthetic circuits available for use in this disclosure are programmable synthetic circuits that can be used to selectively regulate gene expression in immune cells. As further described herein, the synthetic circuits available for use in this disclosure comprise a first nucleotide sequence encoding a payload (payload sequence) and a second nucleotide sequence encoding a regulator (regulator sequence), wherein both the payload sequence and the regulator sequence comprise one or more “sensors,” i.e., target sites capable of recognizing and interacting with other molecules. For example, in some aspects, the payload sequence comprises a sensor (also referred to herein as a “first P-type sensor”) capable of recognizing a regulator (e.g., a regulator encoded by the regulator sequence), and the regulator sequence comprises a sensor (also referred herein as an “R-type sensor”) capable of recognizing a marker (e.g., miRNA expressed in immune cells). In some aspects, the payload sequence may further comprise an additional P-type sensor (also referred herein as a “second P-type sensor”) capable of recognizing a marker (e.g., miRNA expressed in host cells). As further described herein, the P-type and R-type sensors can be activated by recognizing their homologous regulators or markers, thereby modulating the activity of the payload sequence and the regulator sequence (e.g., inhibiting the expression of the encoded protein). Unbound by any single theory, the synthetic circuits described herein allow for highly specific gene regulation and rapid decision-making by using sensors that can be specifically programmed based on markers present in immune and / or non-immune cells. Further aspects of this disclosure are provided throughout this application.

[0103] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the event of any conflict, this application (including the definitions) shall prevail. Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms.

[0104] Throughout this disclosure, the term "an" or "a" entity refers to one or more of the said entities; for example, "a polynucleotide" should be understood to mean one or more polynucleotides. Therefore, the terms "an" or "a," "one or more," and "at least one" are used interchangeably herein.

[0105] Furthermore, when used herein, “and / or” will be considered as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: 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).

[0106] It should be understood that wherever the term "comprising" is used to describe an aspect herein, other similar aspects described as "composed of" and / or "substantially composed of" are also provided. As used herein, "comprising" is synonymous with "including," "containing," or "characterized in," and is inclusive or open-ended, and does not exclude additional, unlisted elements or method steps. As used herein, "composed of" does not include any element, step, or component not specified in the elements of the claim. As used herein, "substantially composed of" does not exclude materials or steps that do not substantially affect the basic and novel features of the claim.

[0107] The term “about” is used herein to mean approximately, roughly, about, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated value. Generally, unless otherwise indicated, the term “about” is used herein to modify numerical values ​​by a variation of around 10% above and below the value (increase or decrease). Unless otherwise indicated, all figures, including the quantities of expressed components, reaction conditions, etc., used in the description of the claims, should be understood to be modified by the term “about” in all cases. Therefore, unless otherwise indicated to the contrary, numerical parameters are approximate values ​​and can vary depending on the desired properties sought to be obtained. At least, and not as an attempt to limit the application of the doctrine of equivalence to the claims, each numerical parameter should be interpreted according to the number of significant figures and ordinary rounding methods.

[0108] The term "at least" preceding a number or series of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as clearly can be seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or ranges, it should be understood that "at least" can modify every number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant figures).

[0109] The terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “nucleic acid sequence,” “polynucleotide,” and their grammatical variations are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”) or any phosphate ester analogues thereof, such as thiophosphates and thioesters, in single-stranded or double-stranded helical form. 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, particularly DNA or RNA molecules, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Therefore, this term includes double-stranded DNA, especially found in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a specific double-stranded DNA molecule, the sequence may be described herein according to the normal convention of giving the sequence only along the non-transcribed DNA strand (i.e., the strand having a sequence homologous to mRNA) in a 5' to 3' orientation. A “recombinant DNA molecule” is a DNA molecule manipulated through molecular biology. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The “nucleic acid compositions” of this disclosure comprise one or more nucleic acids as described herein. As described herein, the polynucleotides of this disclosure comprise DNA, RNA, or both. In some respects, 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 unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleosides in a configuration that allows base pairing and base stacking, as found in DNA and RNA.

[0110] Unless otherwise indicated, as used herein, the term "polypeptide" encompasses both peptides and proteins.

[0111] The term “coding region” refers to a region of DNA or RNA (transcriptional region) that “encodes” a specific protein (e.g., a payload and / or regulator).

[0112] 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 the β-D-furanose group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or completely purified RNA, substantially pure RNA), synthetic RNA, and recombinant RNA (such as modified RNA) (which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides). The term “mRNA” means “messenger RNA” and refers to a “transcription” that is generated using a DNA template and encodes a peptide or protein. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In the context of this disclosure, mRNA can be generated by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various commercially available in vitro transcription kits are available. As further described herein, in some aspects, RNA is linear RNA. In some aspects, RNA is circular RNA. In some aspects, RNA is self-replicating RNA. In some respects, RNA is non-replicating RNA.

[0113] As used herein, the term "genetic circuit" refers to a controllable gene expression system. As described herein, gene circuits that can be used in this disclosure include synthetic gene circuits ("synthetic circuits"). As used herein, the term "synthetic circuit" refers to an engineered, non-natural gene circuit. It is clear from this disclosure that the synthetic circuits described herein have been specifically programmed to selectively express payloads in target cells (i.e., immune cells).

[0114] As used herein, the term “self-replicating RNA” refers to RNA (e.g., mRNA) capable of directing its own amplification or replication within a cell (also referred to herein as “repRNA”). To direct its own amplification, the RNA molecule should encode an enzyme necessary to catalyze RNA amplification (e.g., alphavirus non-structural proteins nsP1, nsP2, nsP3, nsP4) and also contain cis-RNA sequences required for replication, which are recognized and utilized by the encoded enzymes. The alphavirus RNA vector replicon should contain the following ordered elements: a 5' viral or cellular sequence required for non-structural protein-mediated amplification (also referred to as 5'CSE, or 5' cis-replication sequence, or cis-5' viral sequence required for replication, or a 5' sequence capable of initiating alphavirus transcription); a sequence encoding biologically active alphavirus non-structural proteins (e.g., nsP1, nsP2, nsP3, nsP4) when expressed; and a 3' viral or cellular sequence required for non-structural protein-mediated amplification (also referred to as 3'CSE, or cis-3' viral sequence required for replication, or alphavirus RNA polymerase recognition sequence). The alphavirus RNA vector replicon may contain means for expressing one or more heterologous sequences, such as, for example, IRES or a viral (e.g., alphavirus) subgenomic promoter (e.g., a linker promoter), which may be modified in some respects to increase or decrease viral transcription of subgenomic segments, or to reduce homology with defective accessory or structural protein expression cassettes, as well as 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 polyadenylate bundles. The replicon should not contain sequences encoding all alphavirus structural proteins (capsid, E1, E2). Non-limiting examples of heterologous sequences that can be expressed by the replicon vector are described, for example, in U.S. Patent No. 6,015,686 (incorporated herein by reference in its entirety), and include, for example, antigens, lymphokines, cytokines, etc.

[0115] As used herein, the term "circular RNA" refers to RNA (e.g., mRNA) that forms a circular structure by covalent bonds. In the context of this disclosure, circular RNA can be generated by methods known to those skilled in the art (e.g., Wesselhoeft, RA et al., 2018, Nature communications, 2018, 9(1), 1-10, which are incorporated herein by reference in their entirety). It is clear from this disclosure that any payload sequence and / or regulator sequence can be in the form of circular RNA. Therefore, in some aspects, the synthetic circuits provided herein contain a payload sequence as a circular RNA. Therefore, in some aspects, the synthetic circuits provided herein contain a regulator sequence as a circular RNA. In some aspects, the synthetic circuits provided herein contain a payload sequence and a regulator sequence, wherein the payload sequence is a circular RNA and the regulator sequence is a circular RNA. Unless otherwise indicated, circular RNA is not self-replicating.

[0116] As used herein, the term "payload sequence" refers to a nucleotide sequence encoding a payload. As used herein, the term "payload" refers to any protein that can be encoded by a payload sequence. In some aspects, payloads include therapeutic proteins. As stated herein, unless otherwise indicated, payloads do not include regulators. Non-limiting examples of payloads are provided elsewhere in this disclosure.

[0117] As used herein, the term "regulator sequence" refers to a nucleotide sequence encoding a regulator. As used herein, the term "regulator" includes any agent capable of regulating 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 usable in this disclosure can be specifically recognized by a P-type sensor on the payload sequence. Also as described herein, in some aspects, when a regulator is specifically recognized by a P-type sensor, the expression of the payload (encoded by the payload sequence) is reduced or suppressed.

[0118] As used herein, the term "sensor" refers to any part capable of recognizing the markers and / or modifiers described herein. As used herein, "recognizing" a marker (or modifier) ​​may include a physical interaction between the marker (or modifier) ​​and the sensor (e.g., the marker binding to a specific marker recognition site within the sensor).

[0119] As used herein, the term "P-type sensor" refers to a sensor present in a payload sequence. Therefore, a payload sequence usable in this disclosure comprises an encoding region ("payload encoding region") encoding the payload and a P-type sensor. In some aspects, the payload sequence comprises multiple P-type sensors. As further described herein, in some aspects, the payload sequence may comprise: (a) a payload encoding region, (b) a first P-type sensor capable of recognizing a modulator, and (c) a second P-type sensor capable of recognizing a marker ("P-type marker"). Unless otherwise indicated, recognition of the P-type marker by the second P-type sensor activates the second P-type sensor, such that the expression of the encoded payload is reduced or suppressed.

[0120] As used herein, the term "R-type sensor" refers to a sensor present on a regulator sequence. In some aspects, a regulator sequence that can be used to construct the synthetic circuit described herein comprises: (a) a coding region encoding the regulator ("regulator coding region") and an R-type sensor, wherein the R-type sensor is capable of recognizing a marker ("R-type marker"). Unless otherwise indicated, the recognition of the R-type marker by the R-type sensor activates the R-type sensor, causing the expression of the encoded regulator to be reduced or suppressed.

[0121] When used to describe markers (e.g., P-type markers and / or R-type markers), the term "sufficient level" refers to the amount of marker required for recognition by a sensor (e.g., a P-type sensor and / or an R-type sensor) and to mediate the downregulation of the sequence containing the sensor. It is clear from this disclosure that downregulation of a sequence can lead to a reduction or inhibition of the expression of any protein encoded by the sequence (e.g., a payload and / or regulator). Therefore, in some respects, cells can express markers, but when cells do not express sufficient levels of the marker, the sensor specific to the marker may remain inactive.

[0122] The term "sequence identity" is used herein to mean a relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. In some respects, sequence identity is calculated based on the full length or a portion thereof of two given SEQ ID NOs. A portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any other specified percentage, of the two SEQ ID NOs. The term "identity" may also mean sequence correlation between amino acid or nucleic acid sequences, as determined by matching between such sequence strings.

[0123] In some respects, methods for determining identity are designed to give the maximum match between the tested sequences. Methods for determining identity and similarity have been incorporated into publicly available computer programs.

[0124] As used herein, the term “effective amount” or “therapeutic effective amount” for example, the synthetic circuits disclosed herein, means an amount sufficient to achieve a beneficial or desired outcome (including clinical outcomes) when administered to subjects, including humans, and thus, “effective amount” or its synonyms depend on the context in which they are used.

[0125] In vitro cell therapy Some aspects of this disclosure include a method for generating engineered immune cells for in vitro cell therapy to a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject. In some aspects, the synthetic circuit is capable of selectively expressing a payload in immune cells (e.g., T cells) but not in non-immune cells.

[0126] In some respects, immune cells remain unexposed outside the online system from the start to the end of treatment. In some respects, immune cells flow from the subject via the online system, are transfected with a synthetic circuit to express a payload, and are then infused back into the subject. In some respects, immune cells express a payload due to the contact. In some respects, immune cells are administered directly back to the subject from the online system.

[0127] In some respects, the methods disclosed herein include (1) connecting a closed online system to a subject in need, (2) extracting immune cells from the subject in the online system, (3) contacting the immune cells with a synthetic circuit, and (4) reintroducing the immune cells into the subject.

[0128] This disclosure also provides a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising administering immune cells to the subject, wherein the immune cells express a payload encoded by a synthetic circuit connected to a closed online system of the subject, and wherein the synthetic circuit is capable of selectively expressing the payload in the cells. In some aspects, this disclosure includes a method for in vitro treatment of a disease or ailment of a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject, and administering the 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.

[0129] One advantage of this disclosure includes, but is not limited to, the fact that subjects receiving treatment do not need to undergo lymph node dissection prior to treatment. Another advantage of this disclosure is that immune cells are less likely to become fatigued or depleted due to the rapid, programmed expression of the payload. In some respects, the therapy is administered within 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.

[0130] In some respects, the method utilizes a closed online system connected to the subject, as described in the following documents: US20210244871A1, published August 12, 2021, which is incorporated herein in its entirety, or WO2022072885A1, published April 7, 2022.

[0131] Cells, such as immune cells Some aspects of this disclosure relate to cells that express payloads (e.g., chimeric antigen receptors) through synthetic circuits connected to an online system of a subject.

[0132] In some respects, cells are immune cells. In some respects, cells include pluripotent or multipotent cells. In some respects, pluripotent cells include embryonic stem cells (ESCs). In some respects, cells are hematopoietic stem cells.

[0133] In some aspects, the initiating cells (e.g., immune cells) are isolated from a human subject. In some aspects, the initiating cells are isolated from a human subject for use in allogeneic cell therapy. In some aspects, the cells are T cells. In some aspects, the cells are NK cells. In some aspects, the cells are Tregs. In some aspects, the cells (e.g., T cells and / or NK cells) are isolated from a human subject. In some aspects, the cells (e.g., T cells and / or NK cells) are engineered to contain a chimeric antigen receptor (CAR) encoded by a synthetic circuit. In some aspects, the cells (e.g., T cells and / or NK cells) are engineered to contain an engineered T cell receptor (TCR) encoded by a synthetic circuit.

[0134] In some respects, the cell contains a construct expressing an antigen receptor and / or another additional polypeptide. In some respects, the antigen receptor includes antibodies, engineered antibodies (such as scFv), CARs, engineered TCRs, TCR mimics (e.g., antibody-T cell receptors (abTCRs) or chimeric antibody-T cell receptors (caTCRs)), or chimeric signal transduction receptors (CSRs). For example, a TCR may comprise an engineered TCR in which the antigen-binding domain of the TCR (e.g., α / β TCR or γ / δ TCR) has been replaced by the antigen-binding domain of an antibody (with or without the constant domain of the antibody); the engineered TCR then becomes specific to the antigen of the antibody while maintaining the signal transduction function of the TCR. Chimeric signal transduction receptors may include (1) extracellular binding domains (e.g., natural / modified receptor extracellular domains, natural / modified ligand extracellular domains, scFv, nanobodies, Fab, DARPin, and affinity compounds), (2) transmembrane domains, and (3) intracellular signal transduction domains (e.g., domains that activate transcription factors or recruit and / or activate JAK / STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ). See, for example, EP340793B1, WO 2017 / 070608, WO 2018 / 200582, WO 2018 / 200583, WO2018 / 200585, and Xu et al., Cell Discovery (2018) 4:62.

[0135] In some respects, antigen receptors target target antigens (e.g., tumor antigens or pathogen antigens). Antigens may include, but are not limited to, 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, CD138, CD171, CD352, CEA (carcinoembryonic antigen), and sealing protein 18.2. Sealing protein 6, c-MET, DLL3 (δ-like protein 3), DLL4, ENPP3 (extracellular nucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, liver glycoside B2, EPHa2 (liver glycoside receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate-binding protein), FCRL5, FR-α (folate receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (phosphatidylinositol proteoglycan 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-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Igκ, Igλ, IL-22Ra (IL-22 receptor α), IL-13Ra2 (IL-13 receptor α2), KDR (kinase insertion domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (member A of the protein family 8 containing leucine-rich repeat sequences), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melanin A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complex (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer cell family 2 member D) ligand, NY-ESO, oncoemulsification antigen, PD-1, PD-L1, PR AME (melanoma preferentially expressed antigen), progesterone receptor, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate six-transmembrane epithelial antigen 1), susceptin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens.

[0136] In some respects, antigen receptors target hTERT. In some respects, antigen receptors target KRAS. In some respects, antigen receptors target Braf. In some respects, antigen receptors target TGFβRII. In some respects, antigen receptors target MAGEA10 / A4. In some respects, antigen receptors target AFP. In some respects, antigen receptors target PRAME. In some respects, antigen receptors target MAGE A1. In some respects, antigen receptors target WT-1. In some respects, antigen receptors target NY-ESO. In some respects, antigen receptors target CD19.

[0137] In some respects, antigen receptors target BCMA. In some respects, antigen receptors target CD147. In some respects, antigen receptors target CD19. In some respects, antigen receptors target both CD19 and CD22. In some respects, antigen receptors target both CD19 and CD28. In some respects, antigen receptors target CD20. In some respects, antigen receptors target both CD20 and CD19. In some respects, antigen receptors target CD22. In some respects, antigen receptors target CD30. In some respects, antigen receptors target CEA. In some respects, antigen receptors target DLL3. In some respects, antigen receptors target EGFRvIII. In some respects, antigen receptors target GD2. In some respects, antigen receptors target HER2. In some respects, antigen receptors target IL-1RAP. In some respects, antigen receptors target mesothelin. In some respects, antigen receptors target mesothelin. In some respects, antigen receptors target NKG2D. In some respects, antigen receptors target PSMA. In some respects, antigen receptors target TnMUC1.

[0138] Synthetic circuit This document provides a method for generating cells for in vitro therapies utilizing 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 comprise a sensor capable of modulating the activity and / or expression of one or more of the plurality of nucleotide sequences. Therefore, in some aspects, this disclosure relates to a method using a polynucleotide (e.g., an isolated polynucleotide) comprising: (a) a nucleotide sequence and (b) at least one sensor, wherein the at least one sensor modulates the activity and / or expression of the nucleotide sequence. For example, in some aspects, the nucleotide sequence encodes a payload (payload sequence), and the at least one sensor (P-type sensor) is capable of recognizing a marker (e.g., a P-type marker), wherein when the sensor recognizes the marker, the sensor is activated, thereby modulating the expression of the encoded protein (e.g., the payload). In some aspects, the nucleotide sequence encodes a regulator (regulator sequence), and the at least one sensor (R-type sensor) is capable of recognizing a marker (e.g., an R-type marker), wherein when the sensor recognizes the marker, the sensor is activated, thereby modulating the expression of the regulator. As used herein, regulating “regulatory expression” can include: (i) regulating the amount of regulatory agent expressed in cells, (ii) regulating the activity of the regulatory agent, or (iii) both of (i) and (ii). Similarly, regulating “payload expression” can include: (i) regulating the amount of payload expressed in cells, (ii) regulating the activity of the payload, or (iii) both of (i) and (ii).

[0139] In some aspects, this article provides a method 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); wherein the payload sequence comprises a sensor (P-type sensor) capable of recognizing the regulator; and wherein the regulator sequence comprises a sensor (R-type sensor) capable of recognizing a marker expressed in cells. In some aspects, the recognition of the marker by the R-type sensor reduces or inhibits the expression of the regulator. Inhibition of regulator expression prevents activation of the P-type sensor; thereby allowing the expression of the encoded payload.

[0140] In some aspects, the synthetic circuits available for use in this disclosure may comprise a nucleotide sequence encoding a payload (payload sequence). The payload sequence may comprise a sensor capable of recognizing a marker (e.g., microRNA). In some embodiments, when the sensor recognizes the marker, payload expression is suppressed or reduced relative to other cells that do not contain the marker. Non-limiting examples of such synthetic circuits are described in U.S. Patent No. 10,000,757 B2, published June 19, 2018, or PCT Publication No. WO2007000668A2, published December 21, 2007, which are incorporated herein by reference in their entirety. In some aspects, the synthetic circuits available for use in this disclosure may comprise a payload sequence but not a modulator sequence. In some aspects, the synthetic circuit comprises a nucleotide sequence encoding a payload (payload sequence), said nucleotide sequence (payload sequence) comprising a sensor (P-type sensor) capable of recognizing a marker. In some aspects, a P-type sensor includes at least one P-type sensor, wherein 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).

[0141] In some aspects, synthetic circuits that can be used in this disclosure include synthetic RNA circuits comprising a first nucleotide sequence containing 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 containing at least one sensor recognized by at least one second marker (e.g., microRNA) not expressed in the first cell type (e.g., immune cells) or expressed at a low level relative to a second cell type. Without limitation, exemplary synthetic circuits are described in PCT Publication No. WO2016040395A1, published March 17, 2016, which is incorporated herein by reference in its entirety.

[0142] Payload sequence As will be clearly seen from this disclosure, in some aspects, the synthetic circuits that can be used in this disclosure may comprise a nucleotide sequence encoding a payload (payload sequence). In some aspects, 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.

[0143] In some respects, the payload sequence is linear (e.g., linear RNA). In some respects, the payload sequence is circular (e.g., circular RNA). In some respects, the payload sequence is self-replicating (e.g., self-replicating RNA). In some respects, the payload sequence is non-replicating (e.g., non-replicating RNA).

[0144] P-type sensor In some aspects, the payload sequence used in this disclosure includes a sensor capable of identifying a regulator (e.g., encoded by a regulator sequence). In some aspects, the payload sequence includes a sensor capable of identifying a marker. In some aspects, the payload sequence includes a first sensor (first P-type sensor) capable of identifying a regulator and a second sensor (second P-type sensor) capable of identifying a marker.

[0145] Therefore, in some aspects, this paper provides 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 (first P-type sensor) capable of recognizing the regulator and a second sensor (second P-type sensor) capable of recognizing a marker. In some aspects, the first P-type sensor and the second P-type sensor are different (i.e., they do not recognize the same ligand).

[0146] In some respects, the synthesis circuit provided herein includes multiple sensors. For example, in some respects, the payload sequence provided herein includes multiple P-type sensors. In some respects, each of the multiple sensors in the payload sequence is identical. In some respects, one or more of the multiple sensors in the payload sequence are different. When the payload sequence includes a first P-type sensor (e.g., a modulator) and a second P-type sensor (e.g., a marker), in some respects, the payload sequence includes multiple first P-type sensors. For example, in some aspects, the payload sequence comprises about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten, about eleven, about twelve, about thirteen, about fourteen, about fifteen, about sixteen, about seventeen, about eighteen, about eighteen, about nineteen, or about twenty or more first-P type sensors. In some aspects, the payload sequence comprises at least two first-P type sensors. In some aspects, the payload sequence comprises at least three first-P type sensors. In some aspects, the payload sequence comprises at least four first-P type sensors. In some aspects, the payload sequence comprises at least five first-P type sensors. In some aspects, the payload sequence comprises at least six first-P type sensors. In some aspects, the payload sequence includes at least seven first P-type sensors. In some aspects, the payload sequence includes at least eight first P-type sensors. In some aspects, the payload sequence includes at least nine first P-type sensors. In some aspects, the payload sequence includes at least ten first P-type sensors. In some aspects, the payload sequence includes at least eleven first P-type sensors. In some aspects, the payload sequence includes at least thirteen first P-type sensors. In some aspects, the payload sequence includes at least thirteen first P-type sensors. In some aspects, the payload sequence includes at least fourteen first P-type sensors. In some aspects, the payload sequence includes at least fifteen first P-type sensors. In some aspects, the payload sequence includes at least sixteen first P-type sensors. In some aspects, the payload sequence includes at least seventeen first P-type sensors. In some aspects, the payload sequence includes at least eighteen first P-type sensors. In some aspects, the payload sequence includes at least nineteen first P-type sensors. In some aspects, the payload sequence includes at least twenty first P-type sensors.

[0147] In some aspects, each of the first P-type sensors is identical. For example, in some aspects, the synthetic circuit described herein comprises a payload sequence and a modulator sequence, wherein the payload sequence comprises a plurality of first P-type sensors, and wherein each of the plurality of first P-type sensors specifically recognizes the same modulator (e.g., each of the first P-type sensors contains the same modulator binding site). In some aspects, one or more first P-type sensors are different. For example, in some aspects, one or more first P-type sensors recognize different modulators. In some aspects, one or more first P-type sensors recognize different binding sites on the same modulator.

[0148] In some aspects, when the payload sequence includes a first P-type sensor (e.g., an identification modulator) and a second P-type sensor (e.g., an identification marker), the payload sequence includes multiple second P-type sensors. For example, in some aspects, the payload sequence includes about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten, about eleven, about twelve, about thirteen, about fourteen, about fifteen, about sixteen, about seventeen, about eighteen, about nineteen, or about twenty or more second P-type sensors. In some aspects, the payload sequence includes at least two second P-type sensors. In some aspects, the payload sequence includes at least three second P-type sensors. In some aspects, the payload sequence includes at least four second-P type sensors. In some aspects, the payload sequence includes at least five second-P type sensors. In some aspects, the payload sequence includes at least six second-P type sensors. In some aspects, the payload sequence includes at least seven second-P type sensors. In some aspects, the payload sequence includes at least eight second-P type sensors. In some aspects, the payload sequence includes at least nine second-P type sensors. In some aspects, the payload sequence includes at least ten second-P type sensors. In some aspects, the payload sequence includes at least twelve second-P type sensors. In some aspects, the payload sequence includes at least thirteen second-P type sensors. In some aspects, the payload sequence includes at least fourteen second-P type sensors. In some aspects, the payload sequence includes at least fifteen second-P type sensors. In some aspects, the payload sequence includes at least sixteen second-P type sensors. In some aspects, the payload sequence includes at least seventeen second-P type sensors. In some aspects, the payload sequence includes at least eighteen second-P type sensors. In some aspects, the payload sequence includes at least nineteen second-P type sensors. In some respects, the payload sequence contains at least 20 second P-type sensors.

[0149] In some respects, each of the second P-type sensors is identical. For example, in some respects, the synthetic circuit comprises a payload sequence and a modulator sequence, wherein the payload sequence comprises a plurality of second P-type sensors, and wherein each of the plurality of second P-type sensors specifically recognizes the same marker. In some respects, one or more second P-type sensors are different. In some respects, one or more second P-type sensors specifically recognize different markers. In some respects, one or more second P-type sensors recognize different binding sites on the same marker.

[0150] In some aspects, when the payload sequence includes a first P-type sensor (e.g., an identification modulator) and a second P-type sensor (e.g., an identification marker), the payload sequence includes a plurality of first P-type sensors and a plurality of second P-type sensors. In some aspects, the payload sequence includes: (a) about two first P-type sensors, about three first P-type sensors, about four first P-type sensors, about five first P-type sensors, about six first P-type sensors, about seven first P-type sensors, about eight first P-type sensors, about nine first P-type sensors, about ten first P-type sensors, about eleven first P-type sensors, about twelve first P-type sensors, about thirteen first P-type sensors, about fourteen first P-type sensors, about fifteen first P-type sensors, about sixteen first P-type sensors, about seventeen first P-type sensors, about eighteen first P-type sensors, about nineteen first P-type sensors, or about twenty or more first P-type sensors. (a) a first type P sensor; (b) approximately two second type P sensors, approximately three second type P sensors, approximately four second type P sensors, approximately five second type P sensors, approximately six second type P sensors, approximately seven second type P sensors, approximately eight second type P sensors, approximately nine second type P sensors, approximately ten second type P sensors, approximately eleven second type P sensors, approximately twelve second type P sensors, approximately thirteen second type P sensors, approximately fourteen second type P sensors, approximately fifteen second type P sensors, approximately sixteen second type P sensors, approximately seventeen second type P sensors, approximately eighteen second type P sensors, approximately nineteen second type P sensors, or approximately twenty or more second type P sensors; or (c) both (a) and (b). As further described herein, in some respects, each first type P sensor is identical. In some respects, one or more first type P sensors are different. In some respects, each second type P sensor is identical. In some respects, one or more first type P sensors are different.

[0151] P-type spacer In some aspects, the payload sequence used in this disclosure may further include a sequence of spacer sub-sequences (“P-type spacers”). In some aspects, the payload sequence includes multiple P-type spacers. For example, in some aspects, the payload sequence includes about two P-type spacers, about three P-type spacers, about four P-type spacers, about five P-type spacers, about six P-type spacers, about seven P-type spacers, about eight P-type spacers, about nine P-type spacers, or about ten or more P-type spacers. In some aspects, the payload sequence includes at least two P-type spacers. In some aspects, the payload sequence includes at least three P-type spacers. In some aspects, the payload sequence includes at least four P-type spacers. In some aspects, the payload sequence includes at least five P-type spacers. In some aspects, the payload sequence includes at least six P-type spacers. In some aspects, the payload sequence includes at least seven P-type spacers. In some aspects, the payload sequence includes at least eight P-type spacers. In some aspects, the payload sequence includes at least nine P-type spacers. In some respects, the payload sequence contains at least 10 P-type spacers. In some respects, each P-type spacer is identical. In some respects, one or more P-type spacers are different.

[0152] Therefore, in some aspects, the synthetic circuit provided herein comprises a payload sequence, wherein the payload sequence comprises a P-type sensor (e.g., a specific recognition modulator and / or marker) and a P-type spacer. In some aspects, the P-type spacer is upstream of the P-type sensor (e.g., the P-type spacer is located closer to the 5' end of the payload sequence than the P-type sensor). In some aspects, the P-type spacer is downstream of the P-type sensor (e.g., the P-type spacer is located closer to the 3' end of the payload sequence than the P-type sensor). In some aspects, the P-type sensor is located downstream of the coding region of the payload sequence, and the P-type spacer is located between the coding region of the payload sequence and the P-type sensor (e.g., after the stop codon of the coding region and before the start of the P-type sensor). As used herein, the term "coding region of the payload sequence" refers to the portion of the payload sequence specifically encoding the payload.

[0153] When a payload sequence comprises multiple P-type sensors, in some aspects, the P-type spacer is upstream of one or more of the multiple P-type sensors. In some aspects, the P-type spacer is downstream of one or more of the multiple P-type sensors. In some aspects, the P-type spacer is located between at least two P-type sensors. In some aspects, each of the multiple P-type sensors is separated by a P-type spacer. For example, in some aspects, the synthesis circuit provided herein comprises a payload sequence comprising a first P-type sensor (e.g., a specific recognition modulator), a second P-type sensor (e.g., a specific recognition marker), and a P-type spacer, wherein the P-type spacer is located between the first P-type sensor and the second P-type sensor. As described herein, in some aspects, the payload sequence comprises multiple first P-type sensors, wherein each of the multiple first P-type sensors is separated by a P-type spacer. In some aspects, the payload sequence comprises multiple second P-type sensors, wherein each of the multiple second P-type sensors is separated by a P-type spacer. In some respects, the payload sequence comprises a plurality of first P-type sensors and a plurality of second P-type sensors, wherein: (a) each of the plurality of first P-type sensors is separated by a P-type spacer, (b) each of the plurality of second P-type sensors is separated by a P-type spacer, and (c) both cases of (a) and (b).

[0154] In cases where the payload sequence contains multiple P-type spacers, in some aspects, each P-type spacer is identical. In some aspects, one or more P-type spacers are different. Without being bound by any theory, in some aspects, the P-type spacers used in this disclosure have suitable lengths such that the spacers facilitate the binding of the P-type sensor to its ligands (e.g., modulators and / or labels). In some aspects, the length of the P-type spacers is between about 1 and about 100 nucleotides. In some aspects, the length of the P-type spacers 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. In some respects, the length of the P-type spacer is between about 1 and about 50 nucleotides. In some respects, the length of the P-type spacer is about 5 nucleotides. In some respects, the length of the P-type spacer is about 10 nucleotides. In some respects, the length of the P-type spacer is about 15 nucleotides. In some respects, the length of the P-type spacer is about 20 nucleotides. In some respects, the length of the P-type spacer is about 25 nucleotides. In some respects, the length of the P-type spacer is about 30 nucleotides. In some respects, the length of the P-type spacer is about 35 nucleotides. In some respects, the length of the P-type spacer is about 40 nucleotides. In some respects, the length of the P-type spacer is about 45 nucleotides. In some respects, the length of the P-type spacer is about 50 nucleotides.

[0155] Unless otherwise indicated, the P-type spacers that can be used in this disclosure are not limited to any particular nucleotide sequence, provided that the P-type spacer is of sufficient length to perform its intended function (e.g., to facilitate the binding of a P-type sensor to its ligand). Therefore, in some aspects, the P-type spacers that can be used in this disclosure include randomly generated nucleotide sequences. In some aspects, where multiple P-type spacers are used to separate multiple P-type sensors, one or more of the multiple P-type spacers have differential sequences such that the multiple P-type spacers do not include randomly generated repetitive nucleotide sequences. In some aspects, the P-type spacers that can be used in this disclosure comprise, are substantially composed of, or consist of the sequence tttcctttcccccttcccttttcctttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the P-type spacer comprises the sequence shown in SEQ ID NO: 1. In some aspects, the P-type spacer consists substantially of the sequence shown in SEQ ID NO: 1. In some aspects, the P-type spacer consists of the sequence shown in SEQ ID NO: 1. In some aspects, the P-type spacer used in this disclosure may comprise, consist substantially of, or consist of the sequence tttcctttcccccttccctt (SEQ ID NO: 2) or a fragment thereof. In some aspects, the P-type spacer comprises the sequence shown in SEQ ID NO: 2. In some aspects, the P-type spacer consists substantially of the sequence shown in SEQ ID NO: 2. In some aspects, the P-type spacer consists of the sequence shown in SEQ ID NO: 2. In some aspects, the P-type spacer used in this disclosure may comprise, consist substantially of, or consist of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some aspects, the P-type spacer comprises the sequence shown in SEQ ID NO: 3. In some aspects, the P-type spacer consists substantially of the sequence shown in SEQ ID NO: 3. In some aspects, the P-type spacer consists of the sequence shown in SEQ ID NO: 3. In some aspects, the P-type spacer is identical to the R-type spacer (further described elsewhere in this disclosure). In some respects, P-type spacers differ from R-type spacers.

[0156] Table 1. Exemplary P-type spacers

[0157] regulator sequence As described herein, in some aspects, the synthetic circuit comprises a nucleotide sequence (“regulator sequence”) that contains or encodes a regulator. Therefore, in some aspects, the synthetic circuits of this disclosure comprise a payload sequence (e.g., any of the payload sequences described above) and a regulator sequence. Non-limiting examples of regulators that may be used with this disclosure are provided elsewhere in this disclosure.

[0158] In some respects, the regulatory sequence is linear (e.g., linear RNA). In some respects, the regulatory sequence is circular (e.g., circular RNA). In some respects, the regulatory sequence is non-replicating (e.g., non-replicating RNA).

[0159] R-type sensor In some aspects, the modulator sequence includes a sensor capable of specifically recognizing a marker. Therefore, some aspects of this disclosure relate to a synthetic circuit comprising a payload sequence and a modulator sequence, wherein the payload sequence includes a sensor (“P-type sensor”), and wherein the modulator sequence includes a sensor (“R-type sensor”). In some aspects, this disclosure provides a synthetic circuit comprising a payload sequence and a modulator sequence, wherein the payload sequence includes a first P-type sensor (e.g., specifically recognizing a modulator) and a second P-type sensor (e.g., specifically recognizing a marker), and wherein the modulator sequence includes an R-type sensor (e.g., specifically recognizing a marker). When the synthetic circuit described herein includes both a P-type sensor and an R-type sensor, in some aspects, the P-type sensor and the R-type sensor are different (e.g., do not specifically recognize the same ligand).

[0160] In some aspects, the modulator sequence used in this disclosure may comprise multiple sensors. For example, in some aspects, the modulator sequence comprises about two R-type sensors, about three R-type sensors, about four R-type sensors, about five R-type sensors, about six R-type sensors, about seven R-type sensors, about eight R-type sensors, about nine R-type sensors, or about ten or more R-type sensors. In some aspects, the modulator sequence comprises at least two R-type sensors. In some aspects, the modulator sequence comprises at least three R-type sensors. In some aspects, the modulator sequence comprises at least four R-type sensors. In some aspects, the modulator sequence comprises at least five R-type sensors. In some aspects, the modulator sequence comprises at least six R-type sensors. In some aspects, the modulator sequence comprises at least seven R-type sensors. In some aspects, the modulator sequence comprises at least eight R-type sensors. In some aspects, the modulator sequence comprises at least nine R-type sensors. In some aspects, the modulator sequence comprises at least ten R-type sensors.

[0161] In some aspects, each of the plurality of sensors on the modulator sequence is identical. For example, in some aspects, the synthetic circuit described herein comprises a payload sequence and a modulator sequence, wherein the modulator sequence comprises a plurality of R-type sensors, and wherein each of the plurality of R-type sensors recognizes the same marker (e.g., each R-type sensor contains the same binding site of the marker). In some aspects, one or more of the plurality of sensors on the modulator sequence are different. For example, in some aspects, one or more of the plurality of R-type sensors recognize different markers. In some aspects, one or more of the plurality of R-type sensors recognize different binding sites of the same marker.

[0162] R-type spacer In some aspects, the modulator sequence provided herein further comprises a spacer sequence (“R-type spacer”). Therefore, in some aspects, the synthetic circuit provided herein comprises a payload sequence and a modulator sequence, wherein the payload sequence comprises a P-type sensor (e.g., a first P-type sensor and / or a second P-type sensor) and a P-type spacer, and wherein the modulator sequence comprises an R-type sensor and an R-type spacer. In some aspects, the R-type spacer and the P-type spacer are different. In some aspects, the R-type spacer and the P-type spacer are the same.

[0163] In some aspects, the modulator sequence contains multiple R-type spacers. In some aspects, the modulator sequence contains about two R-type spacers, about three R-type spacers, about four R-type spacers, about five R-type spacers, about six R-type spacers, about seven R-type spacers, about eight R-type spacers, about nine R-type spacers, or about ten or more R-type spacers. In some aspects, the modulator sequence contains at least two R-type spacers. In some aspects, the modulator sequence contains at least three R-type spacers. In some aspects, the modulator sequence contains at least four R-type spacers. In some aspects, the modulator sequence contains at least five R-type spacers. In some aspects, the modulator sequence contains at least six R-type spacers. In some aspects, the modulator sequence contains at least seven R-type spacers. In some aspects, the modulator sequence contains at least eight R-type spacers. In some aspects, the modulator sequence contains at least nine R-type spacers. In some aspects, the modulator sequence contains at least ten R-type spacers. In some aspects, each R-type spacer is identical. In some respects, one or more R-type spacers are different.

[0164] In some respects, the R-type spacer is located upstream of the R-type sensor within the regulator sequence. In some respects, the R-type spacer is located downstream of the R-type sensor. In some respects, where the regulator sequence encodes the regulator, the R-type spacer is located between the coding region of the regulator sequence and the R-type sensor (e.g., after the stop codon of the coding region and before the start of the R-type sensor). As used herein, the term "coding region of the regulator sequence" refers to the portion of the regulator sequence specifically encoding the regulator.

[0165] When the modulator sequence comprises multiple R-type sensors, in some aspects, the R-type spacer is upstream of one or more of the multiple R-type sensors. In some aspects, the R-type spacer is downstream of one or more of the multiple R-type sensors. In some aspects, the R-type spacer is located between at least two of the multiple R-type sensors. In some aspects, each of the multiple R-type sensors is separated by an R-type spacer.

[0166] In cases where the regulator sequence contains multiple R-spacers, in some respects, each R-spacer is identical. In some respects, one or more R-spacers are different. In some respects, the R-spacers can have any suitable length, allowing them to facilitate the binding of the R-sensor to its ligand (e.g., a label). In some respects, the length of the R-spacers is between about 1 and about 100 nucleotides. In some respects, the length of the R-spacers 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. In some respects, the length of the R-spacers is between about 1 and about 50 nucleotides. In some respects, the length of an R-type spacer is approximately 5 nucleotides. In some respects, the length of an R-type spacer is approximately 10 nucleotides. In some respects, the length of an R-type spacer is approximately 15 nucleotides. In some respects, the length of an R-type spacer is approximately 20 nucleotides. In some respects, the length of an R-type spacer is approximately 25 nucleotides. In some respects, the length of an R-type spacer is approximately 30 nucleotides. In some respects, the length of an R-type spacer is approximately 35 nucleotides. In some respects, the length of an R-type spacer is approximately 40 nucleotides. In some respects, the length of an R-type spacer is approximately 45 nucleotides. In some respects, the length of an R-type spacer is approximately 50 nucleotides.

[0167] Unless otherwise indicated, the R-spacers that can be used in this disclosure are not limited to any particular nucleotide sequence, provided that the R-spacer is of sufficient length to perform its intended function (e.g., to facilitate the binding of the R-sensor to its ligand). Therefore, in some aspects, the R-spacers that can be used in this disclosure include randomly generated nucleotide sequences. In some aspects, where multiple R-spacers are used to separate multiple R-sensors, one or more of the multiple R-spacers have differential sequences such that the multiple R-spacers do not include randomly generated repetitive nucleotide sequences. In some aspects, the R-spacers that can be used in this disclosure comprise, are substantially composed of, or consist of the sequence tttcctttcccccttcccttttcctttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof. In some aspects, the R-spacers comprise the sequence shown in SEQ ID NO: 1. In some aspects, the R-spacers are substantially composed of the sequence shown in SEQ ID NO: 1. In some aspects, the R-spacers are composed of the sequence shown in SEQ ID NO: 1. In some aspects, the R-type spacer used in this disclosure comprises, is substantially composed of, or is composed of the sequence tttcctttcccccttccctt (SEQ ID NO: 2) or a fragment thereof. In some aspects, the R-type spacer comprises the sequence shown in SEQ ID NO: 2. In some aspects, the R-type spacer is substantially composed of the sequence shown in SEQ ID NO: 2. In some aspects, the R-type spacer is composed of the sequence shown in SEQ ID NO: 2. In some aspects, the R-type spacer used in this disclosure comprises, is substantially composed of, or is composed of the sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof. In some aspects, the R-type spacer comprises the sequence shown in SEQ ID NO: 3. In some aspects, the R-type spacer is substantially composed of the sequence shown in SEQ ID NO: 3. In some aspects, the R-type spacer is composed of the sequence shown in SEQ ID NO: 3. In some aspects, the R-type spacer is the same as the P-type spacer. In some aspects, the R-type spacer is different from the P-type spacer.

[0168] Table 2. Exemplary R-type spacers

[0169] markers As is clear from this disclosure, the synthetic circuits described herein can be programmed to selectively regulate the expression of specific genes (or the proteins they encode) in immune cells. Without being bound by any theory, in some respects, because the payload sequence and / or regulator sequence contain sensors (e.g., P-type or R-type sensors) programmed to recognize specific markers, the sensors are “turned on” (i.e., in an active form) only in cells containing sufficient levels of the marker to be recognized by the sensor. When cells do not contain sufficient levels of the marker, the sensors are “turned off” (i.e., in an inactive form) because the sensors cannot specifically recognize the marker. The table below summarizes the possible scenarios relating to marker levels and payload expression. The state of regulator expression is also listed in the table. Unless otherwise indicated, the markers available for use in this disclosure do not contain regulators as described herein.

[0170] Table 3.

[0171] To aid illustration, in some respects, the synthetic circuits provided herein include a payload sequence comprising a P-type sensor capable of specifically recognizing a marker expressed in non-immune cells, and wherein recognition of the marker by the P-type sensor reduces or inhibits the expression of the payload encoded in the non-immune cells. For such a synthetic circuit, when introduced into non-immune cells (i.e., expressing a level of the marker sufficient for recognition by the P-type sensor), the P-type sensor becomes active (i.e., binds to the marker), thereby inhibiting or reducing the expression of the payload encoded in the non-immune cells. However, when introduced into immune cells (i.e., not expressing a level of the marker sufficient for recognition by the P-type sensor), the P-type sensor remains inactive (i.e., does not bind to the ligand), and thus the payload is expressed in the immune cells. It is clear from this disclosure that the payload can be selectively expressed when both of the following conditions are true: (1) no second P-type sensor is activated, and (2) one or more R-type sensors are activated. Payload expression can be inhibited when one or both of the following conditions are not met: (1) All P-type markers are low.

[0172] (2) At least one R-type marker is high.

[0173] As used herein, “payload expression” (or its syntactic equivalent) means any of the following: (a) the amount of payload expressed in a cell, (b) the rate of payload expression in a cell, (c) the duration of payload expression, or (d) any combination of (a) to (c). Without being bound by any theory, in some aspects, the synthetic circuits provided herein allow for the selective expression of a payload in immune cells by modulating the expression of the payload sequence and the modulator sequence. For example, it is clear from this disclosure that when introduced into immune cells (i.e., without expressing a level of P-type marker sufficient to be recognized by a P-type sensor, and expressing a level of R-type marker sufficient to be recognized by at least one R-type sensor), the expression of the payload in the immune cells increases, as compared to the expression of the modulator in the immune cells. In some aspects, the expression of the payload in the 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%, as compared to the expression of the modulator in the immune cells. In some respects, such as compared with the expression of the modulator, the expression of the payload in immune cells is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold. In some respects, such as compared with the expression of the payload, the expression of the modulator in immune cells is decreased. In some respects, such as compared with the expression of the payload in immune cells, the expression of the modulator in immune cells is decreased 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%.

[0174] Unless otherwise indicated, a marker includes any molecule expressed in a cell and specifically recognizable by sensors (e.g., P-type and / or R-type sensors) provided herein. As described herein, in some aspects, a marker is selectively expressed (or expressed to a sufficient level) in some cells, but not in others. For example, in some aspects, a marker is expressed to a level sufficient to be recognized by sensors (e.g., P-type or R-type sensors) in a first cell but not in a second cell. In these aspects, when the synthetic circuit described herein is introduced into the first cell, the sensor (e.g., P-type and / or R-type sensor) specifically recognizes the marker and becomes active. When such a synthetic circuit is introduced into the second cell, the sensor (e.g., P-type and / or R-type sensor) remains inactive (i.e., does not bind to the marker).

[0175] Non-limiting examples of markers that may be used with this disclosure include microRNAs (miRNAs), proteins, metabolites, or combinations thereof. In some aspects, the markers include miRNAs. In some aspects, the markers include proteins. In some aspects, the markers include metabolites.

[0176] As further explained elsewhere in this disclosure, the synthetic circuits provided herein may include multiple sensors. For example, in some aspects, the synthetic circuit includes a payload sequence, wherein the payload sequence includes multiple sensors (e.g., multiple first P-type sensors and / or multiple second P-type sensors). In some aspects, the synthetic circuit includes a modulator sequence, wherein the modulator sequence includes multiple sensors. In some aspects, the synthetic circuit includes a payload sequence and a modulator sequence, wherein the payload sequence includes multiple sensors, and wherein the modulator sequence includes multiple sensors. When the synthetic circuit includes multiple sensors, in some aspects, each of the multiple sensors may specifically recognize the same marker. For example, in some aspects, the payload sequence of the synthetic circuit provided herein includes multiple sensors, wherein each of the multiple sensors recognizes the same miRNA. When the synthetic circuit includes multiple sensors, in some aspects, one or more of the multiple sensors specifically recognize different markers. For example, in some aspects, the synthetic circuit includes a payload sequence and a modulator sequence, wherein the payload sequence and the modulator sequence each include a sensor, wherein the sensor of the payload sequence specifically recognizes a first marker (e.g., miRNA), and wherein the sensor of the modulator sequence specifically recognizes a second marker (e.g., a metabolite or a different miRNA).

[0177] In some respects, the payload is a chimeric antigen receptor (CAR).In some aspects, CAR targets CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, 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 The extracellular portion 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.

[0178] In some respects, the payload includes a TCR.In some aspects, TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE 1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, 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 The extracellular portion 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.

[0179] regulator As described herein, in some aspects, the synthetic circuit described herein comprises a payload sequence, wherein the payload sequence includes a sensor capable of specifically recognizing a modulator. Unless otherwise indicated, the specific recognition of the modulator by a P-type sensor (e.g., a first P-type sensor) reduces or inhibits the expression of the payload encoded by the payload sequence. Therefore, when the payload sequence includes a sensor capable of specifically recognizing a modulator (e.g., a first P-type sensor) and a sensor capable of specifically recognizing a marker (e.g., a second P-type sensor), the expression of the encoded payload can be modulated in at least two different ways. Not bound by any one theory, in some aspects, this dual modulation approach allows for greater selectivity in the expression of the payload.

[0180] In some aspects, when the synthetic circuit described herein is introduced into non-immune cells (i.e., those not expressing R-type markers at levels sufficient for recognition by an R-type sensor) to increase the expression of the regulator, the regulator can be specifically recognized by a P-type sensor (e.g., a first P-type sensor), thereby leading to activation of the P-type sensor. As further described herein, activation of the P-type sensor reduces or inhibits the expression of the payload encoded by the payload sequence. Thus, in some aspects, when the synthetic circuit provided herein comes into contact with a cell population comprising both immune cells and non-immune cells, the expression of the payload in non-immune cells (i.e., those 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., those with reduced regulator expression). In some aspects, when the synthetic circuit provided herein comes into contact with a cell population comprising both immune cells and non-immune cells, the expression of the payload in immune cells (i.e., with 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 aspects, the expression of the payload in immune cells increases by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to non-immune cells.

[0181] Regulators that can be used with the synthetic circuits described herein include any suitable regulator in the art. Non-limiting examples of such regulators include RNA-binding proteins, siRNA, shRNA, premiRNA, ribozymes, or combinations thereof. In some aspects, the regulator is siRNA. In some aspects, the regulator is shRNA. In some aspects, the regulator is premiRNA. In some aspects, the regulator is a ribozyme. In some aspects, the regulator is an RNA-binding protein. In some aspects, RNA-binding proteins include ribonucleases. In some aspects, ribonucleases include Cas proteins. In some aspects, Cas proteins include Cas6 proteins.

[0182] form As further described herein, the synthetic circuits of this disclosure include certain properties (e.g., structural and / or functional properties) that make them particularly suitable for selectively regulating the expression of genes (or their encoded proteins) in target cells (e.g., immune cells). For example, in some aspects, the synthetic circuits provided herein include a payload sequence and a regulator sequence, which are programmed such that, when both are present in immune cells, the payload (encoded by the payload sequence) is robustly expressed, while the expression of the regulator (encoded by the regulator sequence) is robustly reduced or suppressed. As described foregoing in this disclosure, in some aspects, the synthetic circuits described herein include sensors (e.g., P-type sensors and / or R-type sensors) that can be programmed to allow selective expression of the payload or regulator in specific target cells. In some aspects, in addition to such sensors, the synthetic circuits provided herein also include a payload sequence and a regulator sequence, wherein the payload sequence and / or regulator sequence have a specific morphology that facilitates the selective expression of the payload and / or regulator.

[0183] Unless otherwise indicated, the payload sequence comprises one or more of the following RNA morphologies: linear RNA, circular RNA, self-replicating RNA, and non-replicating RNA. Unless otherwise indicated, the regulator sequence comprises one or more of the following RNA morphologies: linear RNA, circular RNA, and non-replicating RNA. For example, in some aspects, the synthetic circuits provided herein comprise a payload sequence in which the payload sequence is a self-replicating RNA. In some aspects, the synthetic circuits provided herein comprise a regulator sequence in which the regulator sequence is a self-replicating RNA. Thus, in some aspects, the synthetic circuits provided herein comprise a payload sequence and a regulator sequence in which the payload sequence is a self-replicating RNA and the regulator sequence is a non-replicating RNA. In some aspects, the synthetic circuits provided herein comprise a payload sequence and a regulator sequence in which the payload sequence is a self-replicating RNA and the regulator sequence is a circular RNA (i.e., non-self-replicating).

[0184] The performance of RNA circuits is improved by using self-replicating RNA to express payload sequences and non-replicating RNA to express regulatory sequences.

[0185] Unbound by any specific theory, under marker conditions that keep the payload sequence "on," the performance of the loop is improved by expressing the payload sequence via repRNA, given that the repRNA will replicate and robustly express high levels of the payload protein over a long period. This is likely due to the self-replicating nature of self-replicating RNA.

[0186] Unbound by any particular theory, the performance of the circuit is improved by linear RNA rather than repRNA expression regulator sequences, given the following: (1) Non-replicating linear RNAs can rapidly express sufficient levels of regulatory proteins to effectively suppress payload protein expression (expressed by repRNA), while repRNA expression is slower (e.g., requires replication) and may allow payload repRNAs to initiate replication, leading to “leakage expression” of the payload in non-immune cells; and (2) Once repRNA begins to replicate, it becomes more difficult to knock down (e.g., using miRNA); therefore, “leakage expression” of regulatory proteins may exist in immune cells compared to expression of regulatory proteins by linear RNA, and this may significantly reduce the expression of the payload repRNA.

[0187] Therefore, the combination of the repRNA payload sequence and the linear RNA regulator sequence allows for very strong expression of the payload in immune cells while minimizing transgene (payload) expression in non-immune cells. Similar to the use of linear RNA to regulate repRNA payloads, regulating circular RNA payloads using linear RNA regulator chains may also yield favorable results, as circular RNAs are more persistent than linear RNAs.

[0188] As is clearly seen from this disclosure, the synthetic circuits that can be used in this disclosure can contain a variety of combinations of RNA morphologies, as long as the payload can be selectively expressed in the target cell (i.e., immune cell).

[0189] For example, in some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a self-replicating RNA and includes a sensor (P-type sensor) capable of specifically recognizing the regulator; and wherein the regulator sequence is a non-replicating RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker. In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a self-replicating RNA and includes (i) a sensor (first P-type sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second P-type sensor) capable of specifically recognizing a marker; and wherein the regulator sequence is a non-replicating RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker.

[0190] In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a self-replicating RNA and includes a sensor (P-type sensor) capable of specifically recognizing the regulator; and wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker. In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a self-replicating RNA and includes (i) a sensor (first P-type sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second P-type sensor) capable of specifically recognizing a marker; and wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker.

[0191] In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a circular RNA and includes a sensor (P-type sensor) capable of specifically recognizing the regulator; and wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker. In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a circular RNA and includes (i) a sensor (first P-type sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second P-type sensor) capable of specifically recognizing a marker; and wherein the regulator sequence is a circular RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker.

[0192] In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a circular RNA and includes a sensor (P-type sensor) capable of specifically recognizing the regulator; and wherein the regulator sequence is a non-replicating RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker. In some aspects, this document provides a synthetic circuit comprising: (a) a first sequence encoding a payload (payload sequence) and (b) a second sequence encoding a regulator (regulator sequence); wherein the payload sequence is a non-replicating RNA and includes (i) a sensor (first P-type sensor) capable of specifically recognizing the regulator and (ii) a second sensor (second P-type sensor) capable of specifically recognizing a marker; and wherein the regulator sequence is a non-replicating RNA and includes a sensor (R-type sensor) capable of specifically recognizing a marker.

[0193] Other components In some aspects, the synthesis circuit described herein includes one or more additional components that aid in the functionality of the synthesis circuit. For example, in some aspects, the synthesis circuit described herein includes a payload sequence, wherein the payload sequence includes a P-type sensor and one or more additional components described herein. In some aspects, the synthesis circuit described herein includes a regulator sequence, wherein the regulator sequence includes an R-type sensor and one or more additional components described herein. In some aspects, the synthesis circuit described herein includes a payload sequence and a regulator sequence, wherein each of the payload sequence and the regulator sequence includes one or more additional components described herein.

[0194] In some aspects, the payload sequence used in this disclosure may include one or more additional components, said one or more additional components enhancing the expression of the encoded payload. In some aspects, the regulator sequence does not include one or more additional components enhancing the expression of the encoded regulator. Thus, in some aspects, 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 aspects, 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 respects, the expression of the payload increased 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.

[0195] In some aspects, the payload sequence used in this disclosure may include one or more additional components that increase the stability of the payload sequence. In some aspects, the modulator sequence may not include one or more additional components that increase the stability of the payload sequence. In some aspects, the increased stability leads to increased expression of the encoded protein. In some aspects, 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 respects, the expression of the payload increased 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.

[0196] In some aspects, the one or more additional components that may be included in the synthetic circuit provided herein include aptamers targeting translation initiation factors. For example, in some aspects, the synthetic circuit provided herein includes a payload sequence, wherein the payload sequence is a circular RNA and includes aptamers targeting translation initiation factors. Including such additional components, particularly when the payload sequence is a circular RNA, can aid in the expression of the encoded payload when the synthetic circuit is introduced into immune cells. See, for example, Prats et al. Int J Mol Sci21(22): 8591 (November 2020). Non-limiting examples of other components that may be used in this disclosure include: (1) an internal ribosome entry site (IRES), (2) an untranslated region (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 jumping peptide, (8) a 5'-cap, (9) a translation enhancer element, or (10) any combination of (1) to (10). Further disclosures relating to such additional components are provided below.

[0197] End-of-line architecture modification: Untranslated Region (UTR) In some aspects, the synthesis loop described herein includes a UTR. For example, in some aspects, the synthesis loop described herein includes a payload sequence, wherein the payload sequence includes a UTR. In some aspects, the UTR is a 5'-UTR. In some aspects, the UTR is a 3'-UTR. In some aspects, the UTR includes both a 5'-UTR and a 3'-UTR.

[0198] The untranslated 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; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Increasing evidence suggests the regulatory role of the UTR in the stability and translation of nucleic acid molecules. Therefore, for example, when the payload sequence described herein contains a UTR, the stability of the payload sequence increases compared to sequences without a UTR. As described herein, in some respects, this increased stability leads to increased expression of the encoded protein.

[0199] 5'-UTR and Translation Start Natural 5'-UTRs possess features that play a role in translation initiation. They contain features like the Kozak sequence, which is well-known for its involvement in ribosome-initiated translation of many genes. The Kozak sequence has a consistent CCR(A / G)CCAUGG pattern, 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 elongation factor binding.

[0200] The 5'-UTR secondary structures 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, the binding of the elongation factor EIF4A2 to secondary structural elements in the 5'-UTR is essential for microRNA-mediated repression (Meijer HA et al., Science, 2013, 340, 82-85, which is incorporated herein by reference in its entirety). Different secondary structures in the 5'-UTR can be incorporated into flanking regions to stabilize or selectively destabilize mRNA in specific tissues or cells.

[0201] By engineering features found in genes that are typically abundantly expressed in specific target organs, humans can enhance the stability of nucleic acid sequences (e.g., the payload sequences of the synthetic circuits provided herein) and protein production. For example, introducing the 5'-UTR of liver-expressed mRNAs (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, it is possible to improve expression in a tissue by using the 5'-UTR of other tissue-specific mRNAs—for muscle (MyoD, myosin, myoglobin, myopoietin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and for lung epithelial cells (SP-A / B / C / D).

[0202] Other non-UTR sequences can also be incorporated into the UTR (e.g., 5'-UTR and / or 3'-UTR). For example, introns or portions of intron sequences can be incorporated into the flanking regions of nucleic acid sequences (e.g., the payload sequences of the synthetic circuits provided herein).

[0203] In some respects, one or more nucleotides within the UTR (e.g., the 5'-UTR and / or the 3'-UTR) can be mutated, substituted, and / or removed. For example, one or more nucleotides upstream of the start codon can be substituted with another nucleotide. The one or more nucleotides to be substituted can 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 more than 60 nucleotides upstream of the start codon. As another example, one or more nucleotides upstream of the start codon can be removed from the UTR.

[0204] 3'UTR and rich AU components 3'-UTRs are known to contain embedded adenosine and uridine segments. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif 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) nonamers. Molecules containing this class of AREs include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myopoietin are two well-studied examples in this class. Most proteins that bind to AREs are known to destabilize messengers, while members of the ELAV family, most notably HuR, have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering HuR-specific binding sites into the 3′-UTR of nucleic acid molecules can lead to HuR binding, thereby stabilizing in vivo information.

[0205] In some respects, the introduction, removal, or modification of AU-rich elements (AREs) in the 3′-UTR can be used to modulate the stability of nucleic acid sequences. When a particular nucleic acid sequence (e.g., the payload sequence and / or modulator sequence described herein) is engineered, one or more copies of an ARE can be introduced to make the nucleic acid sequence less stable, thereby limiting translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein.

[0206] Translation Enhancer Element (TEE) In some aspects, the synthetic circuits provided herein include translation enhancer elements (TEEs). As used herein, the term "translation 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 with this disclosure are known in the art. See, for example, US20130177581A, which is incorporated herein by reference in its entirety. In some aspects, 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, for example, expression of the payload increases compared to a corresponding synthetic circuit whose payload sequence does not include a TEE.

[0207] In some aspects, the TEE is located between the transcription promoter and the start codon of the sequence (e.g., the payload sequence). In some aspects, the TEEs available for use in this disclosure have 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 TEE provided in the following patents: US Patent Nos. US 20140147454, US 20090226470, US20070048776, US 20130177581, US 20110124100, WO 1999024595, WO 2012009644, WO2009075886, WO U.S. Patent Nos. 2007025008, 6,310,197, 6,849,405, 7,456,273, and 7,183,395, each of which is incorporated herein by reference in its entirety.

[0208] In some respects, the synthetic circuits provided herein contain multiple TEEs. For example, in some respects, the synthetic circuits provided herein contain a payload sequence, wherein the payload sequence contains 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 respects, the TEE sequences in the 5' UTR of RNA (e.g., modified RNA) are the same or different TEE sequences. In some respects, the TEE sequences are in patterns repeated once, twice, or more than three times, such as ABABAB or AABBBAABBAABB or ABCABCABC or variants thereof. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0209] RNA-binding protein (RBP) In some respects, the synthetic circuits presented in this paper contain sequences encoding RNA-binding proteins. RNA-binding proteins (RBPs) can regulate many aspects of co-transcriptional and post-transcriptional gene expression, such as, but not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, modification, export, and localization. RNA-binding domains (RBDs), such as, but not limited to, RNA recognition motifs (RR) and hnRNP K-homology (KH) domains, typically regulate sequence associations between RBPs and their RNA targets (Ray et al., Nature 2013. 499:172-177; incorporated herein by reference in its entirety). In some respects, typical RBDs bind short RNA sequences. In some respects, typical RBDs recognize RNA structures.

[0210] Non-limiting examples of RNA-binding proteins and related nucleic acid and protein sequences are described in US 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0211] 5' with a hat In some respects, the synthetic circuits described herein include a 5'-cap structure. For example, in some respects, the synthetic circuits described herein include a payload sequence, wherein the payload sequence includes a 5'-cap structure. The 5' cap structure of mRNA participates in nuclear export, thereby increasing mRNA stability and binding to mRNA cap-binding proteins (CBPs), which associate with poly(A)-binding proteins to form mature circular mRNA species, said CBPs being responsible for mRNA stability and translational capacity in the cell. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0212] Modification of the RNA disclosed herein can generate a non-hydrolyzable cap structure, thereby preventing decapping and thus increasing the mRNA half-life. Since cap structure hydrolysis requires cleavage of the 5'-ppp-5' phosphodiester linker, modified nucleotides can be used during the capping reaction. For example, a vaccine capping enzyme from New England Biolabs (Ipswich, Mass.) can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to generate a thiophosphate linker in the 5'-ppp-5' cap. Other modified guanosine nucleotides include α-methylphosphonic acid and selenophosphate nucleotides.

[0213] Other modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminal and / or 5'-preterminal nucleotides of the mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Several different 5'-cap structures can be used to generate 5'-caps for nucleic acid molecules (such as mRNA molecules).

[0214] Cap analogs (also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs) differ chemically from the natural (i.e., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to nucleic acid molecules.

[0215] For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which 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; which can be equivalently named 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 guanine provides the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0216] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-β-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0217] In some respects, the cap is a dinucleotide cap analog. In some respects, the dinucleotide cap analog is modified at different phosphate positions with a boron phosphate group or a selenophosphate group, as described in U.S. Patent No. 8,519,110, the contents of which are incorporated herein by reference in their entirety.

[0218] In some aspects, the cap is a cap analog, which is an N7-(4-chlorophenoxyethyl)-substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide forms of cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, for example, the various cap analogs and the methods of synthesizing cap analogs, described in Kor et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574; the contents of the referenced are incorporated herein by reference in their entirety). In some aspects, the cap analogs of this disclosure are 4-chloro / bromophenoxyethyl analogs.

[0219] Although cap analogs allow nucleic acid molecules to be capped during in vitro transcription reactions, up to approximately 20% of transcripts remain uncapped. This, along with the structural differences between cap analogs and the endogenous 5'-cap structure of nucleic acids produced by endogenous cellular transcription mechanisms, can lead to reduced translational capacity and decreased cell stability.

[0220] In some respects, providing RNA having a 5'-cap or a 5'-cap analogue is achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analogue, wherein the 5'-cap is co-transcribed into the resulting RNA strand.

[0221] In some respects, RNA can be generated, for example, through in vitro transcription, and a 5'-cap can be attached to RNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme for vaccinia virus). In some respects, enzymes are used to post-transcribe capping of nucleotide sequences encoding IL-12 to generate more realistic 5'-cap structures. As used herein, the phrase "more realistic" means a feature that structurally or functionally closely reflects or mimics an endogenous or wild-type characteristic. That is, a "more realistic" feature better represents an endogenous, wild-type, natural, or physiological cellular function and / or structure compared to prior art synthetic features or analogs, or is superior in one or more respects to the corresponding endogenous, wild-type, natural, or physiological characteristic. Non-limiting examples of more realistic 5'-cap structures disclosed herein are those structures that, compared to synthetic 5'-cap structures (or wild-type, natural, or physiological 5'-cap structures) known in the art, exhibit enhanced binding to cap-binding proteins, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' uncapping. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can generate a typical 5'-5'-triphosphate link between the 5'-terminal nucleotide and the guanine cap nucleotide of mRNA, wherein the cap guanine contains N7 methylation and the 5'-terminal nucleotide of mRNA contains 2'-O-methyl. This cap leads to higher translational efficiency and cellular stability, as well as reduced activation of pro-inflammatory cytokines, compared to, for example, other 5'-cap analog structures known in the art. 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.

[0222] In some respects, the 5' terminal cap includes an endogenous cap or cap analogue. In some respects, the 5' terminal cap contains a guanine analogue. Useful guanine analogues include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-dezo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0223] In some aspects, the 5' cap includes a 5' to 5' triphosphate linker. In some aspects, the 5' cap includes a 5' to 5' triphosphate linker, including thiophosphate modification. In some aspects, the 5' cap includes a 2'-O or 3'-O-ribose methylated nucleotide. In some aspects, the 5' cap includes a modified guanosine nucleotide or a modified adenosine nucleotide. In some aspects, the 5' cap includes 7-methylguanylic acid. 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.

[0224] In some respects, the synthetic circuits described herein include a modified 5' cap. For example, in some respects, the payload sequence of the synthetic circuit includes a modified 5' cap. Modifications to the 5' cap can increase the stability of the mRNA, increase the half-life of the mRNA, and increase the translation efficiency of the mRNA. In some respects, the modified 5' cap includes one or more of the following modifications: modification at the 2' and / or 3' position of the capped guanosine triphosphate (GTP), substitution of the sugar epoxide with a methylene moiety (CH2) (which produces a carbocyclic ring), modification at the triphosphate bridge portion of the cap structure, or modification at the nucleobase (G) portion.

[0225] Modifiable 5' cap structures include, but are not limited to, the caps described in U.S. Application Nos. 2014 / 0147454 and WO2018 / 160540, which are incorporated herein by reference in their entirety.

[0226] IRES sequence In some respects, the synthetic circuits provided herein include an internal ribosome entry site (IRES). For example, in some respects, the synthetic circuits provided herein include a payload sequence in which the payload sequence contains an IRES. IRES were initially identified as characteristic small RNA viral RNAs and play an important role in initiating protein synthesis in the absence of a 5' cap structure. An IRES may serve as a single ribosome binding site or as one of multiple ribosome binding sites for mRNA. Nucleic acids or mRNAs containing more than one functional ribosome binding site may encode several peptides or polypeptides that are independently translated by ribosomes (“polycistronic nucleic acid molecules”). When an IRES is provided to a nucleic acid or mRNA, a second translatable region is optionally further provided. Examples of IRES sequences that may be used under this disclosure include, but are not limited to, those from piconemaviruses (e.g., FMDV), insect pest virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukosis virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0227] A-tail In some respects, the synthetic circuits provided herein include poly-A tails. In other respects, the synthetic circuits provided herein include payload sequences, wherein the payload sequences include poly-A tails.

[0228] In some respects, the length of the poly-A tail is greater than about 30 nucleotides. In some respects, the length of the poly-A tail is greater than about 35 nucleotides. In some respects, the length is at least about 40 nucleotides. In some respects, the length is at least about 45 nucleotides. In some respects, the length is at least about 55 nucleotides. In some respects, the length is at least about 60 nucleotides. In some respects, the length is at least 70 nucleotides. In some respects, the length is at least about 80 nucleotides. In some respects, the length is at least about 90 nucleotides. In some respects, the length is at least about 100 nucleotides. In some respects, the length is at least about 120 nucleotides. In some respects, the length is at least about 140 nucleotides. In some respects, the length is at least about 160 nucleotides. In some respects, the length is at least about 180 nucleotides. In some respects, the length is at least about 200 nucleotides. In some respects, the length is at least about 250 nucleotides. In some respects, the length is at least about 300 nucleotides. In some respects, the length is at least about 350 nucleotides. In some aspects, the length is at least about 400 nucleotides. In some aspects, the length is at least about 450 nucleotides. In some aspects, the length is at least about 500 nucleotides. In some aspects, the length is at least about 600 nucleotides. In some aspects, the length is at least about 700 nucleotides. In some aspects, the length is at least about 800 nucleotides. In some aspects, the length is at least about 900 nucleotides. In some aspects, the length is at least about 1000 nucleotides. In some aspects, the length is at least about 1100 nucleotides. In some aspects, the length is at least about 1200 nucleotides. In some aspects, the length is at least about 1300 nucleotides. In some aspects, the length is at least about 1400 nucleotides. In some aspects, the length is at least about 1500 nucleotides. In some aspects, the length is at least about 1600 nucleotides. In some aspects, the length is at least about 1700 nucleotides. In some aspects, the length is at least about 1800 nucleotides. In some respects, the length is at least approximately 1900 nucleotides. In some respects, the length is at least approximately 2000 nucleotides. In some respects, the length is at least approximately 2500 nucleotides. In some respects, the length is at least approximately 3000 nucleotides.

[0229] In some respects, the poly-A tail comprises a poly-AG tetrad. A G-tetrad is a cyclic array of four guanine nucleotides bound by hydrogen bonds, which can be formed from G-rich sequences in DNA and RNA. In some respects, the G-tetrad is incorporated at the end of the poly-A tail. The stability, protein production, and other parameters, including half-life, of the resulting nucleic acid or mRNA can be determined at different time points. It has been found that protein production resulting from the poly-AG tetrad is equivalent to at least 75% of the protein production seen with a 120-nucleotide poly-A tail alone.

[0230] Modified nucleosides In some aspects, the synthetic circuits provided herein comprise one or more modified nucleosides. In some aspects, the synthetic circuits provided herein comprise a payload sequence, wherein the payload sequence comprises one or more modified nucleosides. In some aspects, the one or more modified nucleosides include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudo-uridine, 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-guanosine, O6-methyl-guanosine, 7-deazo-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deazo-adenosine, pyrrole-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

[0231] In some respects, the synthetic circuits provided herein contain one or more uridines that have been substituted with modified nucleosides. In some respects, the modified nucleosides that substitute for uridines are pseudouridines (…). ), N1-methyl-pseuuridine (m1) ) or 5-methyluridine (m5U).

[0232] Nanoparticles and delivery systems In some aspects, this disclosure relates to the delivery of synthetic circuits (e.g., those described herein) to cells. In some aspects, delivery may occur in vivo (e.g., by administering the synthetic circuits described herein to a subject) or ex vivo (e.g., by culturing the synthetic circuits described herein in vitro with cells). In some aspects, delivery of the synthetic circuits described herein can be performed using any suitable delivery system known in the art. In some aspects, the delivery system is a carrier. Therefore, in some aspects, this disclosure provides a carrier comprising any synthetic circuit described herein. Suitable carriers that can be used are known in the art. See, for example, Sung et al. Biomater Res 23(8)(2019), which is incorporated herein by reference in its entirety.

[0233] In some aspects, nanoparticles (e.g., lipid nanoparticles or lipid-like nanoparticles) are used to deliver synthetic circuits. Therefore, in some aspects, this disclosure relates to a synthetic circuit encapsulated within nanoparticles (e.g., as described herein), compositions comprising such nanoparticles, and the use of such nanoparticles for treating diseases or conditions in subjects in need. More particularly, in some aspects, this document provides a nanoparticle comprising (i) any synthetic circuit described herein and (ii) one or more types of nanoparticle components.

[0234] Nanoparticles (NP) As used herein, a “nanoparticle” (NP) refers to a particle, such as a vesicle, having a characteristic size measured in nanometers (nm). Nanoparticles can be used in methods for delivering drug therapies to target sites. Non-limiting examples of NPs include lipid nanoparticles (LNPs), lipid-like nanoparticles (LLNs), polymer nanoparticles (PNPs), and inorganic nanoparticles.

[0235] Lipid nanoparticles (LNP) As used herein, “lipid nanoparticles” (LNPs) refer to nanoparticles composed of lipids. Lipid nanoparticles can be used in methods for delivering drug therapies to target sites. Non-limiting examples of LNPs include cationic lipid nanoparticles, ionizable lipid nanoparticles, liposomes, solid lipid nanoparticles (SLNs), bolaamphihile molecules, nanostructured lipid carriers (NLCs), and monolayer membrane structures (e.g., archaea and micelles).

[0236] As used herein, “cationic lipid nanoparticles” refers to nanoparticles containing cationic lipids. As used herein, “ionizable lipid nanoparticles” refers to nanoparticles containing ionizable lipids. In various aspects of this disclosure, LNPs comprise one or more of the following lipids: “non-cationic auxiliary lipids”, “phospholipids”, “sterols or other structural lipids”, and “PEG / polyethylene glycol-modified lipids”.

[0237] An exemplary LNP contains one or more of the following components: (i) Ionizable / cationic lipids; (ii) Phospholipids or non-cationic auxiliary lipids; (iii) Sterols or other structural lipids; (iv) PEG / PEGylated lipids and (v) Targeted delivery of molecules (lipid components / targeting ligands).

[0238] Lipid-like nanoparticles (LLN) As used herein, "lipid-like nanoparticles" (LLN) refer to nanoparticles comprising lipids and lipid-like materials or lipid-like substances as described herein. In various aspects of this disclosure, LLNs comprise one or more of the following lipids: "non-cationic assist lipids," "phospholipids," "sterols or other structural lipids," and "PEG / PEGylated lipids." Lipid-like nanoparticles can be used in methods for delivering drug therapies to target sites.

[0239] An exemplary LLN comprises one or more of the following components: (i) Ionizable / cationic lipid-like materials or lipids; (ii) Phospholipids or non-cationic auxiliary lipids; (iii) Sterols or other structural lipids; (iv) PEG / PEGylated lipids; and (v) Targeted delivery of molecules (lipid components / targeting ligands).

[0240] Ionizable lipids Non-limiting examples of ionizable lipids include: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate heptadecaned-9-yl ester (SM-102), 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate heptadecaned-9-yl ester (lipid 5), 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate di(( Z)-Non-2-en-1-yl) ester (L319), 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tetrazyl)-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy -N,N--Dimethylaminopropane (DLin-DMA), 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DM) A) 1,2-Dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N -dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)) or any combination thereof.

[0241] cationic lipids Non-limiting examples of cationic lipids include: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), liposome transfection reagents, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleoyloxy-N-[2-(spermineformylamino)ethyl... N,N-dimethyl-1-trifluoroacetic acid propanediamine (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(1,2-dioleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyl-N,N-di Methylammonium chloride (DODAC), 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (DLePC), 1,2-distearyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-di... Stearoyl-sn-glycerol-3-ethylphosphocholine (DSePC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)-sn-glycerol-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0242] In some aspects of this disclosure, LNPs primarily comprise cationic lipids as well as other lipid components. These typically include other lipid molecules belonging to, but not limited to, the phosphatidylcholine (PC) class (e.g., 1,S-distearyl-sn-glycerol-3-phosphate choline (DSPC)) and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), sterols (e.g., cholesterol), and polyethylene glycol (PEG) lipid conjugates (e.g., 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[folic acid (PEG)-2000 (DSPE-PEG2000) and C14-PEG2000)).

[0243] DOTAP In all respects of this disclosure, the cationic lipid is DOTAP. DOTAP can be used to efficiently transfect DNA, including yeast artificial chromosomes (YAC), into eukaryotic cells for transient or stable gene expression, and is also suitable for efficiently transferring other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins, into research samples of mammalian cells.

[0244] Liposome transfection reagent In all respects of this disclosure, cationic lipids are liposome transfection reagents. The liposome transfection reagents used herein are common transfection reagents manufactured and marketed by Invitrogen for molecular and cell biology. They are used to improve the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures via liposome transfection. Liposome transfection reagents contain lipid subunits that can form liposomes or lipid nanoparticles in an aqueous environment, which carry a transfection payload (e.g., modRNA). Because neutral colipids mediate the fusion of liposomes with the cell membrane, RNA-containing liposomes (with a positively charged surface) can fuse with the negatively charged plasma membrane of living cells, thereby allowing nucleic acid cargo molecules to cross the cytoplasm for replication or expression.

[0245] lipid-like materials or lipids Unless otherwise indicated, “lipid-like material” and “lipid-like substance” may be used interchangeably. Non-limiting examples of lipid-like materials and / or lipid-like substances include: 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecane-2-ol) (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazin 2,5-dione (cKK-E12), tetra(8-methylnonyl)3,3′,3″,3 -(((methylazonidyl)bis(propane-3,1-diyl))bis(azontriyl))tetrapropionate (306Oi) 10 ), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecano-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(azanetriyl))tetra(ethane-2,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadec-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadecyl-9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tris(3-(bis(dodecylamino)propyl)phenyl)1,3,5-tricarboxamide (TT3), hexa(octyl-3-yl)9,9′,9″,9 ,9″″,9 "-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexylnonanoate (FTT5), PL-1 [published in Nature Communications, 12-7264 (2021), which is incorporated herein by reference], 98N12-5 [published in Molecular Therapy, Vol. 17, No. 5, May 5, 2009, which is incorporated herein by reference], ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazolium-2-carboxylate (A2-Iso5-2DC18(A2)), A12-Iso5-2DC18(A12) or any combination thereof.

[0246] As used herein, TT3 can form nanoparticles for the delivery of various bioactive agents into cells. Furthermore, this disclosure demonstrates that unloaded TT3-LLN can induce immunogenic cell death (ICD) in cancer cells both in vivo and in vitro. As described herein, immunogenic cell death refers to a form of cell death that can induce an effective immune response by activating dendritic cells (DCs) and subsequently activating a specific T cell response. In some aspects of this disclosure, the cells undergoing immunogenic cell death are tumor cells. Immunogenic tumor cell death can trigger an effective antitumor immune response.

[0247] In some respects, lipids are TT3.

[0248] Phospholipids or other non-cationic auxiliary lipids Unless otherwise indicated, "phospholipids" and "other non-cationic co-lipids" may be used interchangeably and non-limiting examples include: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DPPC), and 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC). 3-Phosphocholine (DSPC), 1,2-Diundecanoyl-sn-glycerol-choline (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-Di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:O diether PC), 1-Oleoyl-2-cholesterol hemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-Hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(eicosahenoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and any combination thereof.

[0249] In some embodiments, the phospholipid is selected from the group consisting of: 1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycerol-3-phosphate choline (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycerol-3-phosphate choline (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycerol-3-phosphate choline (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphate choline (16:0-18:0 PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (16:0-18:1... PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (18:0-18:1) PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (18:0-18:2 PC), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (18:0-20:4 PC), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC,OSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:1 PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:2 PE), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:1 PE), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonico-sn-glycerol-3-phosphate ethanolamine (18:0-20:4PE), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (18:0-22:6PE), 1-oleoyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), and any combination thereof.

[0250] In some respects, phospholipids are DSPC. In other respects, phospholipids are DOPE.

[0251] Sterols or other structural lipids As used in this article, "sterols or other structural lipids" refers to cholesterol or cholesterol analogues that can be used to fill lipid membrane stacking defects and provide structural integrity.

[0252] Non-limiting examples of sterols include: cholesterol, coccidosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, tomatine, ursolic acid, α-tocopherol, and combinations thereof. In some respects, sterols are cholesterol.

[0253] PEG-modified lipids As used in this article, “PEG lipid” and “polyethylene glycol-modified lipid” are used interchangeably.

[0254] Non-limiting examples of PEG lipids include: 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dispalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). In some aspects, the lipid portion of PEG-lipids includes those with a length of about C14 to about C22.

[0255] In some respects, the PEG portion has a size of about 1,000, 2,000, 5,000, 10,000, 15,000, or 20,000 Daltons. In some respects, C14-PEG2000 includes 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristic-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000) or both.

[0256] In some respects, PEG-lipids can be embedded in the LNP prior to polynucleotide encapsulation. In other respects, PEG lipids (or other lipid components disclosed herein) can be added to the LNP after polynucleotide encapsulation. For example, in some respects, a synthetic circuit is encapsulated in an LNP, and then PEG lipids (or other lipid components disclosed herein) are attached to the LNP using, for example, micelles.

[0257] In some respects, the nanoparticles do not contain any polyethylene glycol-modified lipids. In other respects, the lipid nanoparticles do not contain any polyethylene glycol-modified lipids.

[0258] Targeted delivery of molecules (lipid compositions / targeting ligands) In various aspects of this disclosure, the nanoparticles (e.g., LNP and LLN) described herein comprise a targeted delivery molecule (lipid composition / targeting ligand). As used herein, “targeting delivery molecule (lipid composition / targeting ligand)” and “targeting delivery molecule” are used interchangeably, and in some aspects, the targeting delivery molecule may be another lipid or lipid-like component, as described herein. In some aspects, the targeting delivery molecule may alter the total charge of the nanoparticle. In some aspects, the targeting delivery molecule may be a ligand that is non-covalently or covalently bound to the nanoparticle. In some aspects, the targeting delivery ligand may be a small molecule or a large molecule.

[0259] Non-limiting examples of targeted delivery molecules are disclosed in Pharmaceuticals (Basel). July 20;15(7):897.(2022), Nat Rev Drug Discov 20, 101-124(2021) and Advanced Drug Delivery Reviews, Vol. 188,(2022), which are incorporated herein by reference. Non-limiting examples of targeted delivery molecules include: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearate-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycerol-3-phosphate (sodium salt) (18:1 PA), 1,2-dimyristoyl-sn-glycerol-3-phosphate (sodium salt) (14:0 PA), bis(monoleoylglycerol) phosphate (S,R isomer) (ammonium salt) (18BMP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)ethyl phosphate (DOCPe), folic acid, N-acetylgalactosamine (GalNAc), and anti-CD3 antibodies.

[0260] Mole ratio In some aspects, the nanoparticles described herein comprise lipids (e.g., ionizable lipids, cationic lipids, non-cationic cofactor lipids, phospholipids, sterols or other structural lipids or PEG lipids) and / or lipid-like substances as described herein, in a molar ratio of about 10% to about 50% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 10%, about 20%, about 30%, about 40%, or about 50% in the lipid composition.

[0261] In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 10% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 20% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 30% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 40% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise lipids and / or lipid-like substances as described herein, in a molar ratio of about 40% in the lipid and / or lipid-like composition.

[0262] In some aspects, the nanoparticles described herein comprise polyethylene glycol-modified lipids in a molar ratio of about 0% to about 10% in the lipid and / or lipid-like composition. In some aspects, the lipid nanoparticles comprise polyethylene glycol-modified 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.

[0263] In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 0.25% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 0.5% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 0.75% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 1.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 2.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 3.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 4.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 5.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 6.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 7.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 8.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 9.0% in the lipid and / or lipid-like composition. In some aspects, the nanoparticles provided herein comprise polyethylene glycol-modified lipids in a molar ratio of about 10.0% in the lipid and / or lipid-like composition.

[0264] For example, in some aspects, the polyethylene glycol-modified lipids include C14-PEG2000. In some aspects, C14-PEG2000 is present in the lipid nanoparticles at a molar ratio of about 0.25% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present at a molar ratio of about 0.5% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present at a molar ratio of about 0.75% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present at a molar ratio of about 1% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present at a molar ratio of about 2% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present at a molar ratio of about 3% in the lipid and / or lipid-like composition. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 4%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 5%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 6%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 7%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 8%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 9%. In some aspects, C14-PEG2000 is present in the lipid and / or lipid-like composition at a molar ratio of about 10%.

[0265] granularity The particle size of nanoparticles can affect drug release rate, biodistribution, mucosal adhesion, cellular water uptake, buffer exchange within the nanoparticles, and protein diffusion. In some aspects of this disclosure, the diameter of the nanoparticles ranges from about 30 to about 500 nm. In some aspects of this disclosure, the diameter of the nanoparticles ranges from 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 nanoparticles ranges from 100 to 160 nm. In some aspects of this disclosure, the diameter of the NPs can 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 some aspects, the lipid nanoparticles have a diameter of about 140 nm.

[0266] ζ potential As used herein, “zeta potential” is a measure of the effective charge on the surface of lipid nanoparticles. The magnitude of the zeta potential provides information about particle stability. In some aspects of this disclosure, the zeta potential of the nanoparticles ranges from about -20 to about 20 mV. In some aspects of this disclosure, the zeta potential of the NP can be approximately -6.0 mV, approximately -5.9 mV, approximately -5.8 mV, approximately -5.7 mV, approximately -5.6 mV, approximately -5.5 mV, approximately -5.4 mV, approximately -5.3 mV, approximately -5.2 mV, approximately -5.1 mV, approximately -5.0 mV, approximately -4.9 mV, approximately -4.8 mV, approximately -4.7 mV, approximately -4.6 mV, approximately -4.5 mV, approximately -4.4 mV, approximately -4.3 mV, approximately -4.2 mV, approximately -4.1 mV, approximately -4.0 mV, approximately -3.9 mV, approximately -3.8 mV, approximately -3.7 mV, approximately -3.6 mV, approximately -3.5 mV, approximately -3.4 mV, approximately -3.3 mV, approximately -3.2 mV. mv, approximately -3.1 mv, approximately -3.0 mv, approximately -2.9 mv, approximately -2.8 mv, approximately -2.7 mv, approximately -2.6 mv, approximately -2.5 mv, approximately -2.4 mv, approximately -2.3 mv, approximately -2.2 mv, approximately -2.1 mv, approximately -2.0 mv, approximately -1.9 mv, approximately -1.8 mv, approximately -1.7 mv, approximately -1.6 mv, approximately -1.5 mv, approximately -1.4 mv, approximately -1.3 mv, approximately -1.2 mv, approximately -1.1 mv, approximately -1.0 mv, approximately -0.9 mv, approximately -0.8 mv, approximately -0.7 mv, approximately -0.6 mv, approximately -0.5 mv, approximately -0.4 mv, approximately -0.3 mv, approximately -0.2 mv, approximately -0.1 mv, approximately 0.0 mv, 0.1 mv, approximately 0.2 mv, approximately 0.3 mv, approximately 0.4 mv, approximately 0.5 mv, approximately 0.6 mv, approximately 0.7 mv, approximately 0.8 mv, approximately 0.9 mv, approximately 1.0 mv, approximately 1.1 mv, approximately 1.2 mv, approximately 1.3 mv, approximately 1.4 mv, approximately 1.5 mv, approximately 1.6 mv, approximately 1.7 mv, approximately 1.8 mv, approximately 1.9 mv, approximately 2.0 mv, approximately 2.1 mv, approximately 2.2 mv, approximately 2.3 mv, approximately 2.4 mv, approximately 2.5 mv, approximately 2.6 mv, approximately 2.7 mv, approximately 2.8 mv, approximately 2.9 mv, approximately 3.0 mv, approximately 3.1 mv, approximately 3.2 mv mv, approximately 3.3 mv, approximately 3.4 mv, approximately 3.5 mv, approximately 3.6 mv, approximately 3.7 mv, approximately 3.8 mv, approximately 3.9 mv, approximately 4.0 mv, approximately 4.1 mv, approximately 4.2 mv, approximately 4.3 mv, approximately 4.4 mv, approximately 4.5 mv, approximately 4.6 mv, approximately 4.7 mv, approximately 4.8 mv, approximately 4.9 mv, approximately 5.0 mv, approximately 5.1 mv, approximately 5.2 mv, approximately 5.3 mv, approximately 5.4 mv, approximately 5.5 mv, approximately 5.6 mv, approximately 5.7 mv, approximately 5.8 mv, approximately 5.9 mv, or approximately 6.0 mv.

[0267] mass ratio In some respects, the mass ratio between the lipids and the synthetic circuit of LNP or LLN ranges from about 1:2 to about 15:1. In some respects, the mass ratio between lipids and synthetic circuits can be approximately 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, or 5.5. The mass ratios are approximately 10:1, 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 between lipids and the synthetic circuit is approximately 10:1.

[0268] Pharmaceutical Composition In some aspects, this disclosure relates to a pharmaceutical composition comprising the synthetic circuit, carrier, and / or nanoparticles described herein. In some aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier (excipient). As used herein, “acceptable” means that the carrier must be compatible with the active ingredient of the composition and harmless to the subject to be treated. In some aspects, the carrier is capable of stabilizing 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 Edition (2000), Lippincott Williams and Wilkoins, editor: KE Hoover.

[0269] The pharmaceutical composition intended for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane. Nanoparticles can be placed in a container with a sterile access port (e.g., an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle).

[0270] In some aspects, the pharmaceutical composition can be formulated for intratumoral, intrathecal, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. In some aspects of this disclosure, the pharmaceutical composition can be formulated for intratumoral injection. As used herein, intratumoral injection refers to direct injection into the tumor. High concentrations of the composition can be obtained in situ while using small amounts of the drug. Local delivery of immunotherapy allows for multiple combination therapies while preventing significant systemic exposure and off-target toxicity.

[0271] In some respects, the pharmaceutical composition can be formulated for intramuscular, intravenous, or subcutaneous injection.

[0272] In some respects, pharmaceutical compositions comprise pharmaceutically acceptable carriers, buffers, excipients, salts, or stabilizers in the form of lyophilized formulations or aqueous solutions. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition (2000), Lippincott Williams and Wilkins, ed. KE Hoover. Acceptable carriers and excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins Substances 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 dextrin; 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™, PLURONICS™, or polyethylene glycol (PEG).

[0273] In some aspects, the pharmaceutical compositions described herein comprise nanoparticles that can be prepared by methods known in the art, such as those described in the following literature: Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545, which are incorporated herein by reference in their entirety. Liposomes with enhanced cycle times are disclosed in U.S. Patent No. 5,013,556, which is incorporated herein by reference in its entirety. In some aspects, liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a defined pore size to produce liposomes with a desired diameter.

[0274] In some respects, pharmaceutical compositions are formulated in a sustained-release form. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing nanoparticles, said matrices being in the form of molded articles, such as membranes 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 (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid esters, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPROM DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0275] In some aspects, suitable surfactants include, but are not limited to, nonionic agents such as polyoxyethylene sorbitol (e.g., TWEEN™ 20, 40, 60, 80, or 85) and other sorbitols (e.g., SPAN™ 20, 30, 60, 80, or 85). In some aspects, compositions containing surfactants contain between 0.05% and 5% of the surfactant. In some aspects, compositions contain 0.1% and 2.5%. It should be understood that, if desired, other ingredients, such as mannitol or other pharmaceutically acceptable mediators, may be added.

[0276] In some respects, the pharmaceutical composition is in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions, suspensions, or suppositories, for oral, parenteral, or rectal administration, or for administration by inhalation or blowing.

[0277] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a drug carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum) and other drug diluents (e.g., water) to form a solid preformed composition containing a homogeneous mixture of the compound of this disclosure or a non-toxic, pharmaceutically acceptable salt thereof. When referring to these homogeneous preformed mixtures, it means that the active ingredient is uniformly dispersed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformed composition is then subdivided into unit dosage forms of the type described above containing about 0.1 to about 500 mg of the active ingredient of this disclosure. Tablets or pills of novel compositions can be coated or otherwise compounded to provide dosage forms that offer the advantage of prolonged action. For example, tablets or pills may comprise an internal dose component and an external dose component, the latter being a coating on the former. These two components can be separated by an enteric coating, which resists disintegration in the stomach and allows the inner components to be delivered intact into the duodenum or released with a delay. A variety of materials can be used for such enteric coatings or coatings, including many polymeric acids and mixtures of polymeric acids with materials such as shellac, hexadecyl alcohol, and cellulose acetate.

[0278] 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 the premixed emulsion composition, or alternatively, the active ingredient can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and in an emulsion formed by mixing it with phospholipids (e.g., lecithin, soybean phospholipids, or soybean lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the emulsion's tension. A suitable emulsion will typically contain up to about 20% (e.g., between about 5% and about 20%) of oil. Fat emulsions can contain fat droplets of suitable size and can have a pH in the range of about 5.5 to about 8.0.

[0279] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some aspects, the composition is administered via oral or nasal inhalation routes to produce local or systemic effects.

[0280] Compositions in pharmaceutically acceptable solvents can be nebulized using a gas. The nebulized solution can be inhaled directly from the nebulizer, or the nebulizer can be attached to a mask plug or intermittent positive pressure ventilator. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in an appropriate manner.

[0281] Therapeutic applications In some aspects of this disclosure, the synthetic circuits, carriers, nanoparticles, and / or pharmaceutical compositions (collectively, also referred to herein as “compositions”) described herein are intended for the treatment of diseases or conditions. It is clear from this disclosure that any composition provided herein can be used to treat a variety of diseases or conditions. Any suitable disease or condition, whether in a therapeutic agent, can be encoded by the payload sequence of the synthetic circuit provided herein. Therefore, some aspects of this disclosure relate to a method of treating a disease or condition in a subject of need, said method comprising administering to the subject any composition (e.g., a synthetic circuit) provided herein.

[0282] In some aspects, any of the compositions described herein may be administered to a subject in need via a suitable route, such as intratumoral administration, intravenous administration (e.g., as a bolus or via continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebrospinal fluid, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or local route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, may be used for administration. Liquid formulations may be nebulized, and lyophilized powders may be nebulized after reconstitution. In some aspects, the pharmaceutical compositions described herein may be nebulized using fluorocarbon formulations and metered-dose inhalers, or inhaled as lyophilized and ground powders. In some aspects, the pharmaceutical compositions described herein are formulated for intratumoral injection. In some aspects, the pharmaceutical compositions described herein may be administered to a subject via a local route, such as injection at a local site, like a tumor site or site of infection. In some aspects, the subject is a human being.

[0283] As will be clearly apparent from this disclosure, in some aspects, the compositions described herein are administered to a subject in an effective amount, alone or in combination with one or more other active agents, to impart a therapeutic effect. In some aspects, the compositions are administered to a subject with cancer, and the therapeutic effect includes a reduction in tumor burden, a reduction in cancer cells, an increase in immune activity, or a combination thereof. Any suitable method known in the art (e.g., measuring tumor volume and / or T cell activity) can be used to determine the administered composition (e.g., nanoparticles, such as…). LNP or LLNWhether a therapeutic effect is achieved. As those skilled in the art will recognize, the effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, duration of treatment, nature of concurrent therapies (if any), specific route of administration, and similarity factors within the professional skills and knowledge of the healthcare practitioner.

[0284] Empirical considerations (such as half-life) often help determine the dosage. The frequency of administration can be determined and adjusted during treatment and is usually, but not necessarily, based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / symptom. Alternatively, the compositions described herein (e.g., nanoparticles, such as...) LNP or LLN Sustained-release formulations may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0285] In some aspects of this disclosure, treatment is a single injection of the compositions disclosed herein. In some aspects, a single injection is administered intratumorally to a subject in need.

[0286] In some aspects of this disclosure, the dosage of the compositions described herein can be empirically determined in individuals who have been given the composition (e.g., the nanoparticles described herein) once or multiple times. In some aspects, individuals are given escalating doses of the compositions described herein. To assess the efficacy of the compositions described herein, indicators of the disease / symptom can be tracked. For repeated administration over several days or longer, depending on the condition, in some aspects, treatment is continued until desired symptom suppression occurs or until a therapeutic level sufficient to alleviate the target disease or symptom or its symptoms is reached.

[0287] In some aspects of this disclosure, the method includes administering one or more doses of the composition described herein to a subject in need.

[0288] As described herein, the synthetic circuits of this disclosure are specifically designed for the selective expression of payloads in target immune cells. Therefore, some aspects of this disclosure relate to a method for inducing selective expression of a payload in cells, the method comprising contacting a cell population with any composition (e.g., the synthetic circuit) provided herein. In some aspects, the payload is expressed in cells when the cells meet the following conditions: (i) containing sufficient levels of an R-type marker such that the R-type marker is specifically recognized by an R-type sensor and induces activation of the R-type sensor, thereby reducing or inhibiting the expression of the regulator; (ii) not containing sufficient levels of a P-type marker such that the P-type marker is not recognized by a P-type sensor, thereby keeping the P-type sensor in an inactive form; or (iii) both (i) and (ii). When cells meet such conditions, the expression of the payload in the cells is increased compared to reference cells (e.g., corresponding cells that do not meet any of the above conditions). In some respects, such as compared with reference cells, after contact, the expression of the payload in 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%. In some respects, such as compared with reference cells, after contact, the expression of the payload in cells increases by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12.5-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold.

[0289] In some aspects, the compositions described herein are co-administered with at least one other suitable therapeutic agent. In some aspects, the compositions described herein and at least one other therapeutic agent are administered to the subject sequentially, i.e., each therapeutic agent is administered at different times. In some aspects, the compositions described herein and at least one other therapeutic agent are administered to the subject substantially simultaneously.

[0290] In some aspects, therapeutic applications of the synthetic circuits described herein include generating an encoded payload in immune cells. Therefore, in some aspects, this disclosure relates to a method for selectively generating a payload in immune cells. In some aspects, the method includes contacting immune cells with any of the compositions described herein (e.g., synthetic circuits, carriers, and / or nanoparticles) under conditions suitable for generating the encoded IL-12 protein. In some aspects, the method further includes purifying the generated payload. In some aspects, contact occurs in vivo (e.g., by administering the synthetic circuit, carrier, and / or nanoparticles to a subject). In some aspects, contact occurs ex vivo (e.g., by administering the synthetic circuit, carrier, and / or nanoparticles to a subject). This document covers cells (e.g., host cells) that contain synthetic circuits, carriers, and / or nanoparticles. Non-limiting examples of cells that may be used include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0291] kits for use in therapy This disclosure also provides a kit for use in therapeutic applications. In some aspects, the kit comprises one or more containers containing the compositions described herein.

[0292] In some aspects, the kit includes instructions for use according to any of the methods described herein. For example, the included instructions may include a description of administering the pharmaceutical composition described herein to treat, delay the onset of, or alleviate an immune disease. In some aspects, the instructions include a description of administering the composition described herein to a subject at risk of an immune disease.

[0293] In some respects, the instructions include dosage information, dosing schedule, and route of administration. In some respects, the container is a unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. In some respects, the instructions are written instructions on a label or packaging insert (e.g., a piece of paper included in the kit). In some respects, the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk).

[0294] In some aspects, the kits described herein are packaged in a suitable manner. In some aspects, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), or combinations thereof. In some aspects, the packaging includes packaging for use in conjunction with a specific device such as an inhaler, a nasal application device (e.g., a nebulizer), or an infusion device such as a micropump. In some aspects, the kit includes a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). In some aspects, the container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). In some aspects, at least one active agent is a composition as described herein.

[0295] In some aspects, the kit further includes additional components, such as buffer solutions and explanatory information. In some aspects, the kit includes a container and a label or packaging insert on or associated with said container. In some aspects, this disclosure provides articles of manufacture containing the contents of the kit described herein.

[0296] General Technology Unless otherwise stated, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JE Cellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Giffiths, and DG Newell, 1993-8) J. Wiley and Sons; Method of Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by JM Miller and MPCalos, 1987); Current Protocols in Molecular Biology (edited by FM Ausubel et al., 1987): PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (edited by CA Janeway and P.).Travers (1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanette and JD Capra, Harwood Academic Publishers, 1995). Without further elaboration, it is believed that those skilled in the art will be able to make the most of this disclosure based on the foregoing description. All publications cited herein (including those listed above and elsewhere in this disclosure) are incorporated herein by reference in their entirety.

[0297] Example The following experiments (Examples 1-7) describe RNA-based logic circuits optimized for better kinetics. The optimized circuits include, but are not limited to: polynucleotide sequences encoding a payload (payload sequence), polynucleotide sequences encoding a regulator (regulator sequence), a regulator sequence sensor capable of specifically recognizing a marker (R-type sensor), a payload sequence sensor capable of specifically recognizing a regulator (Type I P-type sensor), and a payload sequence sensor capable of specifically recognizing a marker (Type II P-type sensor), such as... Figure 1 exemplified in . like Figure 1 As shown, one or more proteins encoded by the regulator sequence interact with the payload, thereby positively or negatively regulating it.

[0298] In addition, such as Figure 1 As shown, the regulator sequence and payload sequence may include linear (self-replicating or non-replicating) or circular RNA.

[0299] Key features of the payload sequence within the optimized loop include, but are not limited to, multiple miRNA classifiers that deexpress in multiple organs to facilitate systemic delivery, and the optimized number and location of miRNA sensors to achieve a powerful on / off switch. Key features of the regulator sequence within the optimized loop include, but are not limited to, highly specific and sensitive binding to rapid targets (i.e., immune cells) to avoid expression in non-immune cells, adjustment of expression levels to ensure better on / off behavior, multiple miRNA classifiers to achieve payload expression in immune cells, and its suitability for gene therapy applications.

[0300] Example 1: Single sensor suppression like Figure 2 As shown, the optimal suppression of the 3'UTR target site array of a single sensor was tested.

[0301] The number of target sites. First, the effect of the number of target sites on inhibition was determined, as assessed by any unit (au) of median mVenus fluorescence in constructs containing 1X siRNA 2, 2X siRNA 2, 3X siRNA 2, 4X siRNA 2, no target site (TS), or no reporter gene after administration of siRNA (0, 1, 10, or 100 nM). Figure 3 ).like Figure 3 As shown, modRNA constructs containing a 1–4X siRNA 2 target site immediately following the stop codon of the mVenus-PEST reporter gene exhibited preferential knockdown in HEK293T cells with increasing dose of co-administered siRNA 2. Incorporation of >2 identical adjacent target sites did not significantly increase repression, regardless of siRNA dose. Data represent the median collected by flow cytometry from three technical replicates (n = 3) 20 h after reporter gene and siRNA electroporation.

[0302] Target site interval. Next, the effect of target site spacing on inhibition was determined, as assessed by median mVenus fluorescence (au) of constructs containing 2X siRNA 2, 2X siRNA 2 - 20 nt, 2X siRNA 2 - 50 nt, without TS, or without a reporter gene, after administration of siRNA (0, 1, 10, or 100 nM). Figure 4 The data represent the median collected by flow cytometry from three technique replicates (n = 3) 20 hours after electroporation of the reporter gene and siRNA. Figure 4As shown, modRNA constructs containing 2X siRNA 2 target sites (with varying intervals between them) immediately following the stop codon of the mVenus-PEST reporter gene exhibit preferential knockdown in HEK293T cells with increasing doses of co-administered siRNA 2. For low, medium, or high doses of siRNA, incorporating intervals between target sites did not significantly increase knockdown.

[0303] The effect of target site spacing (2–20 nucleotides) on repression was determined, as assessed by median mVenus fluorescence (au) of constructs containing 2X siRNA 2–20 nt, 3X siRNA 2–20 nt, 4X siRNA 2–20 nt, without TS, or without a reporter gene after administration of siRNA (0, 1, 10, or 100 nM). Figure 5 The data represent the median collected by flow cytometry from three technique replicates (n = 3) 20 hours after electroporation of the reporter gene and siRNA. Figure 5 As shown, modRNA constructs containing a 2X-4X siRNA 2 site (with a 20 nt spacer between the target sites) immediately following the mVenus-PEST reporter gene exhibited preferential knockdown in HEK293T cells with increasing doses of co-administered siRNA 2. Constructs with a 4X siRNA 2 target site and 20 nt showed slightly lower expression than constructs with 2X-20 nt or 3X-20 nt; however, this is likely not due to increased downregulation, but rather to a decrease in maximal expression. This trend was observed for low, medium, and high doses of siRNA.

[0304] Optimization of miRNA sensors for various RNA morphologies. Then, the linearity was evaluated. Figure 6A ) and cyclic ( Figure 6B The deexpression of the RNA morphology payload. Figure 6A Linear modRNA circuits containing the mVenus-PEST reporter gene and the NxmiR-b target site (1-4X TS) in the 3' UTR were shown to exhibit preferential knockdown in hepatocyte (Huh-7) cell lines compared to the control (HEK293T) cell line. Figure 6A The deexpression of the payload in hepatocytes was consistent with miR-b as a liver-specific mRNA. Data represent the mean geometric mean collected from three technique replicates (n = 3) 20 hours after electroporation. Figure 6BcircRNA constructs containing 1–4 miR-a target sites immediately following the stop codon of the mVenus-PEST reporter gene exhibited preferential knockdown in the HEK293T cell line relative to the HeLa cell line. This is consistent with the high expression of miR-a in HEK293 cells and its absence in HeLa cells. Incorporation of >2 identical adjacent target sites did not significantly increase repression. The degree of repression did increase over time, approaching baseline in HEK293T cells by 24 hours. Data represent the mean geometric mean collected from three technical replicates (n = 3) at 4 and 24 hours post-electroporation.

[0305] Optimization of Cas6e target site localization. This section includes an example demonstrating that, in the absence of a matching regulator, the location of the target site in a circRNA determines the expression level. Specifically, BHK-21 cells were transfected with a circRNA expressing mVenus-PEST and a modRNA expressing a Cas6e regulator, wherein mVenus-PEST either does not contain a Cas6e target site or contains a Cas6e target site at one of five locations, as shown below. Figure 7A As shown in the figure. In the absence of a modulator, expression varies depending on the location of the target site, while in the presence of a modulator, all constructs are observed to be completely knocked down. Figure 7B ).

[0306] Optimize the spacing between the stop codon and the target site. Figures 8B-8C Showing Figure 8A The influence of the Nx nucleotide spacer described in the text. In particular, Figures 8B-8C A graph depicting mVenus fluorescence (any unit) of constructs containing 1X or 2X siRNA target sites (TS) and 0, 7, or 12 nucleotide spacers after siRNA administration (0, 1, 10, 100 nM) is provided. Data represent geometric mean collected by flow cytometry from three technique replicates (n=3) at 6 h and 24 h after reporter gene and siRNA electroporation.

[0307] Example 2: Multi-sensor suppression Next, as Figure 9 As shown, the optimal suppression of the target site array of the multi-input sensor was tested.

[0308] like Figure 10As shown, HEK293T cells were electroporated with the mVenus-PEST reporter gene containing an array of target sites immediately following a stop codon, and with no siRNA, siRNA 1, siRNA 2, or both. Knockdown was observed for all tested constructs (3X siRNA1 - 20 nt, 3X siRNA2 - 20 nt, 3X siRNA2 - siRNA1 interleaved, 3X siRNA1 - siRNA2 interleaved, 3X siRNA2 3X siRNA1 adjacent, 3X siRNA1 3X siRNA2 adjacent, no target site, no reporter gene) when one or more siRNAs co-electroplated had target sites in the target site array. The combined sensor responded to both siRNAs, but there was no apparent synergistic effect because siRNA2 itself reduced expression to background. Data represent the mean geometric mean collected by flow cytometry from three technique replicates (n = 3) 20 hours after electroporation.

[0309] The study will define Figure 11 The operational range of circuits with Nx miR and regulator target sites placed in 5' or 3' UTRs at locations A, B, and C is outlined in this paper. The study will specifically evaluate (1) the types and number of target sites that can be placed in A, B, and C without affecting maximum payload expression in the target cell types, (2) how the order of target sites in A, B, and C affects payload expression knockdown in different cell types, and (3) the effect of combining miR target sites in both 5' and 3' UTRs on payload knockdown.

[0310] Example 3: RNA-based synthetic genetic circuit behavior CircRNAs with miRNA target sites are degraded by the RISC complex. like Figure 12As shown, total RNA was extracted from HEK293T, HeLa, and Huh-7 cells transfected with circular RNA containing miR-b or miR-a TS at 4 and 24 hours post-transfection. The number of transfected circRNAs was measured using RT-qPCR with probes spanning the splicing sites of the circRNAs. HEK cells contained high levels of miR-a but no miR-b. Huh-7 cells contained high levels of both miR-a and miR-b, and HeLa cells expressed lower amounts of both. In Huh-7 cells, circRNAs containing miR-b target sites were significantly affected at 4 hours post-transfection, while in HEK and HeLa cells, the effect was minimal. Similarly, circRNAs containing miR-a target sites were downregulated at a rate consistent with their relative expression levels in all cell types, occurring most rapidly in HEK cells, followed by Huh-7 and HeLa. circRNAs without miR sensors were not targeted and were used as controls for circRNAs containing miR sensors.

[0311] circRNA is downregulated by Cas6e. The regulatory protein Cas6e effectively downregulates the expression of circRNA molecules containing a protein-coding sequence followed by a Cas6e target site. BHK-21 cells were transfected with circular or linear RNA encoding the fluorescent protein mVenus-PEST, each RNA either lacking a Cas6e target site or containing a Cas6e target site after a stop codon. Other cells were co-transfected with modified linear mRNAs or circRNAs encoding a Cas6e regulator, a P2A self-cleaving peptide sequence, and the fluorescent protein mCherry-PEST. For a schematic diagram showing the downregulation of target circRNAs containing Cas6e TS and encoding the fluorescent protein mVenus-PEST by linear and circular regulator RNAs, please refer to [link to schematic diagram]. Figure 13A The downregulation of circRNAs containing the target site in the presence of the Cas6e regulator is consistent with the results observed for linear mRNAs. When both the target site and the regulator are present, mVenus expression decreases to background levels. Figure 13B Cas6e has no effect on the expression of circRNAs that do not contain their target sites. Figure 13C ).

[0312] RNA regulators that target both replication and non-replication payloads. In this experiment, endonuclease was used as an RNA regulator to control the mRNA chain of the mVenus fluorescent reporter expressing the payload protein. For example... Figure 14AAs shown, the linear non-replicating (non-rep) payload mRNA chain containing the target sequence of the RNA regulator is synthesized from unmodified bases (unmodRNA payload) or from bases in which N1-methylpseudouridine is replaced with uridine (modRNA payload). The payload mRNA was transfected into BHK-21 cells with or without co-transfecting with modRNA expressing the RNA regulator. Cells were analyzed by flow cytometry after 24 hours to evaluate payload expression. The RNA regulator downregulated the payload mRNA synthesized from unmodified bases but did not downregulate the modRNA payload. Furthermore, as... Figure 14B As shown, replicon RNA containing the target sequence of the RNA regulator was transfected into BHK-21 cells with or without co-transfection with modRNA expressing the RNA regulator at two different doses (20 ng or 40 ng). Almost all cells transfected with the replicon alone expressed the payload, while co-transfection with the replicon and the RNA regulator reduced the percentage of payload-positive cells to <10%.

[0313] Example 4. In which cell types is the expression of circular RNA homologous to cell type-specific miRNAs downregulated? In this embodiment, HEK293T and Huh-7 cells were electroporated using circular RNA encoding EGFP-PEST driven by Coxsackievirus B3 (CVB3) IRES. These circular RNAs, immediately following a stop codon, contained either miR-TS, 4x miR-b target sites, or 4x miR-a target sites. Twenty-four hours post-transfection, the fluorescence of individual cells was measured by flow cytometry, and the data are shown below. Figure 15 In HEK cells containing high levels of miR-a but not miR-b, the translation of circRNAs with 4x miR-a target sites was downregulated to autofluorescence levels, while that of circRNAs with 4x miR-b target sites was not. In Huh-7 cells containing levels of both miR-a and miR-b, the translation of circRNAs with either miR-a or miR-b target sites was downregulated to autofluorescence levels. The downregulation of circRNAs with miR-b target sites in Huh-7 cells but not in HEK293T cells suggests that the downregulation is a result of miRNA-mediated RNA degradation.

[0314] Example 5. In vitro miRNA classifier In this embodiment, non-replicating modRNAs expressing the mVenus-PEST fluorescent protein and containing multiple human miRNA target sites (corresponding to miRNAs with higher activity in HEK293T (non-cancer) cells than in HeLa (cancer) cells) were electroporated into two cell types. Flow cytometry data for both cell types (n = 3) were collected approximately 24 hours after electroporation. After subtracting the background fluorescence level, the geometric mean of mVenus-PEST expression in each cell type was normalized relative to the geometric mean of modRNAs without miRNA sensors. Figure 16 The ratio of these normalized expression levels in HEK293T to the normalized expression levels in HeLa is shown.

[0315] Figures 17A-17B This demonstrates the design and construction of a non-replicating modRNA containing the Nx miR-b target site in the 3' UTR. Figure 17A ) and replicon RNA ( Figure 17B Reporter gene constructs were used to express the mVenus-PEST fluorescent protein. Each construct was transfected into HEK293T cells or the human hepatocyte cell line Huh-7 via electroporation. A non-replicating modRNA expressing the near-infrared fluorescent reporter protein miRFP720 was co-transfected with each replicon RNA as a transfection marker. Data represent the geometric mean of flow cytometry data from three technical replicates (n = 3) collected approximately 24 hours after electroporation. A miRFP720-positive cell population for each cell type (HEK293T or Huh7) was considered successfully transfected. The expression output of miRFP720-positive cells that were also mVenus-PEST-positive was calculated. Figure 17A ) and percentage ( Figure 17B mVenus-PEST expression is used as a representative of circuit activity.

[0316] In the transfected Huh-7 cell line, 1x target sites provided near-complete knockdown of non-replicating modRNAs, while 2x or more sites produced complete knockdown, reaching autofluorescence levels. Furthermore, the spacing between miR target sites had minimal impact on knockdown efficiency (see [link to relevant documentation]). Figure 17A ).

[0317] Almost all cells containing >0x target sites showed mVenus-PEST reporter gene knockdown in Huh-7 cells expressing high levels of miR-b, and in HEK293T cells that do not express miR-b, no amount of miR-b target sites caused knockdown (see [link]). Figure 17B ).

[0318] Therefore, the miRNA sensor produces an effective knockdown in vitro.

[0319] Example 6. In vivo miRNA sensing The liver expression was relieved. In this embodiment, mice were injected with either lipid nanoparticles or a mediator control containing a reporter gene modRNA encoding firefly luciferase. Six hours later, the mice were sacrificed, and luciferase activity in their organs (i.e., spleen, lung, kidney, lymph nodes, and liver) was assessed. Adding a sensor targeting the liver-specific microRNA miR-b to the reporter gene modRNA resulted in a 59-fold reduction in luciferase expression in the liver compared to the absence of the sensor. However, adding the miR-b sensor did not significantly affect luciferase expression in the spleen, lung, kidney, and lymph nodes. Therefore, the miR-b sensor specifically desensitized reporter gene expression in the liver (see [link to relevant documentation]). Figure 18 ).

[0320] The spleen's expression is relieved. In this embodiment, mice were injected with either lipid nanoparticles or a mediator control containing a reporter gene modRNA encoding firefly luciferase. Six hours later, the mice were sacrificed, and luciferase activity in their organs (i.e., liver and spleen) was assessed. The addition of a sensor targeting the spleen-associated miRNA, miR-h, to the reporter gene modRNA resulted in a 30-fold reduction in luciferase expression in the spleen compared to the absence of the sensor in the reporter gene modRNA. Simultaneously, the addition of the miR-h sensor had only a minimal effect on luciferase expression in the liver. Therefore, the miR-h sensor desensitized reporter gene expression in the spleen (see [link to relevant documentation]). Figure 19 ).

[0321] Example 7. R-type sensor When there are abundant homologous markers for the R-type sensor, the R-type sensor can achieve payload expression. In this embodiment, Huh7 cells expressing high levels of miR-b and HEK293T cells expressing low levels of miR-b were transfected via an RNA circuit using electroporation. The RNA circuit consisted of: (1) a replicon payload strand expressing a green fluorescent reporter and containing a first P-type sensor responsive to the regulator protein Cas6e, and (2) a linear non-replicating regulator strand expressing the regulator protein Cas6e and containing an R-type sensor responsive to miR-b.

[0322] As a control, Huh7 and HEK293T cells were transfected in parallel with the same RNA loop but lacking the R-type sensor. The expression of the green fluorescent payload was measured by quantitative imaging.

[0323] Six hours post-transfection, payload expression of the RNA circuit with the miR-b R-type sensor was turned on in miR-b-rich Huh7 cells, while payload expression remained off in HEK293T cells. In fact, payload expression was reduced 7-fold in HEK293T cells compared to Huh7 cells (see [link to article]). Figure 20 The payload expression of RNA circuits lacking R-type sensors remains switched off in both cell types (see [link]). Figure 20 ).

[0324] When there are abundant homologous markers for the R-type sensor, the R-type sensor can achieve payload expression. A549 lung cancer cells expressing high levels of miR-i were transfected via electroporation with a replicon payload sequence expressing the mVenus reporter gene and containing a first P-type sensor responsive to a Cas6e regulatory protein. Some cells were co-transfected with a linear, non-replicating regulatory sequence expressing the Cas6e regulatory protein linked to an mCherry reporter via a 2A self-cleaving peptide. This resulted in suppression of the payload sequence, as demonstrated by decreased mVenus expression. However, some cells were co-transfected with a regulatory sequence containing an R-type sensor of miR-i. In these cells, the R-type sensor was activated, leading to downregulation of the regulatory chain, as shown by suppression of mCherry, and enabling the payload sequence to be expressed, as demonstrated by a significant increase in mVenus expression (see [link to original text]). Figure 21 ).

[0325] Example 7. In vitro cell therapy Blood is drawn from the subject via a catheter connected to an online in vitro system. A synthetic circuit containing a chimeric antigen receptor (CAR) is added to the blood-containing system. After sufficient incubation time for effective transfection of the synthetic circuit, mononuclear cells, along with the remaining blood components, are returned to the subject via a venous route—all within a closed-loop system.

[0326] It should be understood that the claims are intended to be interpreted using the detailed description section rather than the summary and abstract section. The summary and abstract section may illustrate one or more, but not all, exemplary aspects of this disclosure as conceived by the inventors, and therefore is not intended to limit this disclosure and the appended claims in any way.

[0327] This disclosure has been described above using functional structural units that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have not been defined herein. Alternative boundaries may also be defined, provided that the specified functions and their interrelationships are properly performed.

[0328] The foregoing description of specific aspects will fully reveal the general characteristics of this disclosure, enabling others to readily modify and / or alter such specific aspects for various applications by applying knowledge in the art, without excessive experimentation and without departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such changes and modifications are intended within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification should be interpreted by a skilled person based on the teachings and guidance.

[0329] The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the following claims and their equivalents.

Claims

1. A method for generating engineered immune cells for in vitro cell therapy on a subject in need, the method comprising contacting immune cells obtained from the subject with a synthetic circuit connected to a closed online system of the subject, wherein the synthetic circuit is capable of selectively expressing a payload in the immune cells.

2. The method of claim 1, wherein the immune cells are not exposed outside the online system throughout the entire duration of the therapy.

3. The method of claim 1 or 2, wherein the immune cell expresses a payload on the immune cell due to the contact.

4. The method of any one of claims 1 to 3, wherein the cells are administered directly back to the subject from the online system.

5. A method for in vitro treatment of a disease or ailment of a subject in need, the method comprising administering cells, such as immune cells, to the subject, wherein the cells express a payload encoded by a synthetic circuit connected to a closed online system of the subject, and wherein the synthetic circuit is capable of selectively expressing the payload in the cells.

6. A method for in vitro treatment of a disease or ailment of a subject in need, the method comprising contacting cells, such as immune cells, obtained from the subject with a synthetic circuit in a closed online system connected to the subject, and administering the 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.

7. The method of any one of claims 1 to 6, wherein the subject does not undergo lymphocyte clearance prior to the treatment.

8. The method of any one of claims 1 to 7, wherein the therapy is administered within 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 method of any one of claims 1 to 8, wherein the synthesis circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The optional second nucleotide sequence encoding the regulator (regulator sequence), The payload sequence includes a sensor (P-type sensor) capable of specifically identifying the modulator. The modulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, and The regulator and the marker (R-type marker) identified by the R-type sensor are different.

10. The method of any one of claims 1 to 8, wherein the synthetic circuit comprises a nucleotide sequence (payload sequence) encoding the payload, wherein the payload sequence comprises a sensor (P-type sensor) capable of specifically recognizing a marker, wherein the marker is not endogenously present in the immune cells, or the marker is endogenously present only in the immune cells.

11. The method of claim 9 or 10, wherein the payload sequence comprises a plurality of the P-type sensors.

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

13. The method of any one of claims 10 to 12, wherein each of the P-type sensors is identical.

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

15. The method of any one of claims 9 to 14, wherein the payload sequence comprises a spacer sequence (P-type spacers).

16. The method of claim 15, wherein the payload sequence comprises a plurality of P-type spacers.

17. The method of claim 16, wherein each of the P-type spacers is identical.

18. The method of claim 16, wherein one or more of the P-type spacers are different.

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

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

21. Immune cells in a closed online system for in vitro therapy, wherein the immune cells express a payload encoded by a synthetic circuit for the method of any one of claims 1 to 20, wherein the synthetic circuit is capable of selectively expressing the payload in the immune cells.

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

23. An online system comprising the immune cells of claim 21 or 22, or comprising immune cells expressing a payload encoded by a synthetic circuit used in any one of claims 1 to 20.

24. The online system as described in claim 23, wherein the online system is completely closed.

25. The online system of claim 23 or 24, wherein the online system is connected to a subject who requires the therapy.

26. The online system of claim 25, wherein the immune cells are obtained through the subject.

27. The online system of any one of claims 23 to 26, wherein the online system further comprises cells other than immune cells.

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

29. The method, cell, or online system of claim 28,The CAR targets CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE 1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, 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 The extracellular portions of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL proteins, or any combination thereof.

30. The method of claim 28, immune cells, or online system.The TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1. lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, H MWMAA, o-acetyl-GD2, folate receptor β, 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-1a, MAGE-1A1, asparagine endopeptidase, HPV E6, E7, MAGE1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galactagogue 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, 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 The extracellular portions of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, APRIL proteins, or any combination thereof.

31. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence), The payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the modulator and a second sensor (second P-type sensor) capable of specifically identifying the marker. The modulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker, and The regulator, the marker identified by the second P-type sensor (second P-type marker), and / or the marker identified by the R-type sensor (R-type marker) are different.

32. The method, immune cells, or online system of claim 31, wherein the payload sequence comprises a plurality of the first P-type sensors.

33. The method, immune cells, or online system of claim 32, wherein the plurality of first P-type sensors comprises two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve first P-type sensors.

34. The method, immune cells, or online system of claim 32 or 33, wherein each of the first P-type sensors is identical.

35. The method, immune cells, or online system of claim 32 or 33, wherein one or more of the first P-type sensors are different.

36. The method, immune cell, or online system of any one of claims 31 to 35, wherein the payload sequence comprises a plurality of the second P-type sensors.

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

38. The method, immune cells, or online system of claim 36 or 37, wherein each of the second P-type sensors is identical.

39. The method, immune cells, or online system of claim 36 or 37, wherein one or more of the second P-type sensors are distinct.

40. The method, immune cell, or online system of any one of claims 31 to 39, wherein the payload sequence comprises a spacer sequence (P-type spacer).

41. The method, immune cell, or online system of claim 40, wherein the payload sequence comprises a plurality of P-type spacers.

42. The method, immune cells, or online system of claim 41, wherein each of the P-type spacers is identical.

43. The method, immune cells, or online system of claim 41, wherein one or more of the said P-type spacers are different.

44. The method, immune cells, or online system according to any one of claims 41 to 43, wherein: (a) At least one P-type spacer is located between the first P-type sensor and the second P-type sensor; (b) At least one P-type spacer is located upstream of both the first P-type sensor and the second P-type sensor; (c) At least one P-type spacer is located downstream of both the first P-type sensor and the second P-type sensor; or (d) Any combination of (a) to (c).

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

46. ​​The method, immune cell, or online system of claim 45, wherein each of the first P-type sensors is separated by P-type spacers.

47. The method, immune cell, or online system of any one of claims 40 to 46, wherein the method, the immune cell, or the online system comprises the plurality of the second P-type sensors, wherein two or more of the second P-type sensors are separated by P-type spacers.

48. The method, immune cell, or online system of claim 47, wherein each of the second P-type sensors is separated by P-type spacers.

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

50. The method, immune cell, or online system of claim 49, wherein the length of the P-type spacer is at least about 10 nucleotides.

51. The method, immune cell, or online system of claim 49, wherein the length of the P-type spacer is about 10 nucleotides, about 20 nucleotides, or about 50 nucleotides.

52. The method, immune cell, or online system of any one of claims 40 to 51, wherein the P-type spacer comprises, substantially comprises, or comprises the following: The sequence tttcctttcccccttcccttttcctttcctttccttcccttccctt (SEQ ID NO: 1), tttcctttcccccttccctt (SEQ ID NO: 2), or gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

53. The method, immune cell, or online system of any one of claims 41 to 52, wherein the modulator sequence comprises a plurality of the R-type sensors.

54. The method, immune cells, or online system of claim 53, wherein the plurality of R-type sensors comprises two R-type sensors, three R-type sensors, four R-type sensors, five R-type sensors, six R-type sensors, seven R-type sensors, or eight or more R-type sensors.

55. The method, immune cells, or online system of claim 53 or 54, wherein each of the R-type sensors is identical.

56. The method, immune cells, or online system of claim 53 or 54, wherein one or more of the R-type sensors are different.

57. The method, immune cell, or online system of any one of claims 31 to 56, wherein the modulator sequence comprises a spacer sequence (R-type spacer).

58. The method, immune cell, or online system of claim 57, wherein the regulator sequence comprises a plurality of R-type spacers.

59. The method, immune cells, or online system of claim 58, wherein each of the R-type spacers is identical.

60. The method, immune cells, or online system of claim 58, wherein one or more of the R-type spacers are distinct.

61. The method, immune cell, or online system of any one of claims 57 to 60, wherein the method, the immune cell, or the online system comprises the plurality of said R-type sensors, wherein two or more of said R-type sensors are separated by R-type spacers.

62. The method, immune cell, or online system of claim 61, wherein each of the R-type sensors is separated by R-type spacers.

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

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

65. The method, immune cell, or online system of claim 64, wherein the length of the R-type spacer is at least about 10 nucleotides.

66. The method, immune cell, or online system of claim 64, wherein the length of the R-type spacer is about 10 nucleotides, about 20 nucleotides, or about 50 nucleotides.

67. The method, immune cell, or online system of any one of claims 57 to 66, wherein the R-type spacer comprises, substantially comprises, or comprises the following: The sequence tttcctttcccccttcccttttcctttcctttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof.

68. The method, immune cells, or online system of claim 67, wherein the R-type spacer comprises, substantially comprises, or comprises the following: Sequence tttcctttcccccttccctt (SEQ ID NO: 2).

69. The method, immune cells, or online system of claims 57 to 64, wherein the R-type spacer comprises, substantially comprises, or comprises the following: Sequence gcggccgctaaa (SEQ ID NO: 3).

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

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

72. The method, immune cell, or online system of claim 71, wherein the RNA-binding protein comprises a ribonuclease.

73. The method, immune cell, or online system of claim 72, wherein the ribonuclease comprises a Cas protein.

74. The method, immune cell, or online system of claim 73, wherein the Cas protein includes the Cas6 protein.

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

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

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

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

79. The method, immune cell, or online system of claim 75 or 76, wherein the payload sequence is a circular RNA and the regulator sequence is a circular RNA.

80. The method, immune cell, or online system of claim 75 or 76, wherein the payload sequence is a circular RNA and the regulator sequence is a linear non-replicating RNA.

81. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence); The payload sequence is a self-replicating RNA and contains a sensor (P-type sensor) capable of specifically recognizing the regulator; The regulator sequence is a circular RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator and the marker (R-type marker) identified by the R-type sensor are different.

82. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (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 a self-replicating RNA and contains a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing the marker. The regulator sequence is a circular RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

83. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence); The payload sequence therein is a self-replicating RNA containing a sensor (P-type sensor) capable of specifically recognizing the regulator; The regulator sequence is a non-replicating linear RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator and the marker (R-type marker) identified by the R-type sensor are different.

84. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (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 a self-replicating RNA and contains a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing the marker. The regulator sequence is a non-replicating linear RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

85. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence); The payload sequence is a circular RNA and contains a sensor (P-type sensor) capable of specifically recognizing the regulator; The regulator sequence is a circular RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator and the marker (R-type marker) identified by the R-type sensor are different.

86. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (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 a circular RNA and contains a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing the marker. The regulator sequence is a circular RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

87. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence); The payload sequence is a circular RNA and contains a sensor (P-type sensor) capable of specifically recognizing the regulator; The regulator sequence is a non-replicating linear RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator and the marker (R-type marker) identified by the R-type sensor are different.

88. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (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 a circular RNA and contains a first sensor (first P-type sensor) capable of specifically recognizing the regulator and a second sensor (second P-type sensor) capable of specifically recognizing the marker. The regulator sequence is a non-replicating linear RNA and contains a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

89. The method, immune cells, or online system according to any one of claims 81 to 88, wherein: (a) The payload sequence comprises a plurality of the first P-type sensors. (b) The payload sequence comprises a plurality of the second P-type sensors. (c) The modulator sequence comprises a plurality of the R-type sensors, or (d) Any combination of (a) to (c).

90. The method, immune cells, or online system according to any one of claims 81 to 89, wherein: (a) The payload sequence includes a spacer subsequence (P-type spacers). (b) The regulator sequence contains a spacer sequence (R-type spacer), or (c) Cases (a) and (b) are both valid.

91. The method, immune cells, or online system of claim 90, wherein: (a) The P-type spacer is located between the first P-type sensor and the second P-type sensor; (b) The P-type spacer is located upstream of both the first P-type sensor and the second P-type sensor; (c) The P-type spacer is located downstream of both the first P-type sensor and the second P-type sensor; or (d) any combination of (a) to (c).

92. The method, immune cells, or online system of claim 90 or 91, wherein the payload sequence comprises the plurality of the first P-type sensors, wherein two or more of the first type sensors are separated by P-type spacers.

93. The method, immune cell, or online system of any one of claims 90 to 92, wherein the payload sequence comprises the plurality of the second P-type sensors, wherein two or more of the second type sensors are separated by P-type spacers.

94. The method, immune cell, or online system of any one of claims 90 to 93, wherein the modulator sequence comprises the plurality of said R-type sensors, wherein two or more said R-type sensors are separated by R-type spacers.

95. The method, immune cell, or online system of any one of claims 90 to 94, wherein the length of the P-type spacer, the R-type spacer, or both is about 1 to about 50 nucleotides.

96. The method, immune cells, or online system of any one of claims 90 to 95, wherein the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the following: The sequence tttcctttcccccttcccttttcctttcctttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof.

97. The method, immune cells, or online system of claim 96, wherein the P-type spacer, R-type spacer, or both comprise, substantially consist of, or consist of the following: Sequence tttcctttcccccttccctt (SEQ IDNO: 2).

98. The method, immune cells, or online system of any one of claims 90 to 97, wherein the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the following: The sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

99. The method, immune cells, or online system of claim 98, wherein the P-type spacer, R-type spacer, or both comprise, substantially consist of, or consist of the following: Sequence gcggccgctaaa (SEQ ID NO: 3).

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

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

102. The method, immune cell, or online system of claim 101, wherein the RNA-binding protein comprises a ribonuclease.

103. The method, immune cell, or online system of claim 102, wherein the ribonuclease comprises a Cas protein.

104. The method, immune cell, or online system of claim 102, wherein the Cas protein includes the Cas6 protein.

105. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence), Wherein, when the payload sequence and the regulator sequence are present in immune cells, the payload is expressed as a first expression in the immune cells, and the regulator is expressed as a second expression in the immune cells. The first expression is greater than the second expression.

106. The method, immune cells, or online system of claim 105, wherein: (a) The payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker. (b) The modulator sequence includes a sensor (R-type sensor) capable of specifically recognizing the marker, and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

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

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

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

111. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence), Wherein, when the payload sequence and the regulator sequence are present in non-immune cells, the payload is expressed as a first expression in the non-immune cells, and the regulator is expressed as a second expression in the non-immune cells. The second expression is greater than the first expression.

112. The method, immune cells, or online system of claim 111, wherein: (a) The payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) The modulator sequence includes a sensor (R-type sensor) capable of specifically identifying the marker; Furthermore, the regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

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

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

116. The method, immune cell, or online system of any one of claims 111, 114, and 115, wherein the immune cell comprises: (a) a level of the P-type marker insufficient to activate the second P-type sensor, and (b) a level of the R-type marker sufficient to activate the R-type sensor.

117. The method of any one of claims 1 to 20 and 28 to 30, the immune cells of any one of claims 21, 22 and 28 to 30, or the online system of any one of claims 23 to 30, wherein the synthetic circuit comprises: (a) The first nucleotide sequence encoding the payload (payload sequence) and (b) The second nucleotide sequence encoding the regulator (regulator sequence), When the synthetic circuit is brought into contact with a cell population containing 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.

118. The method, immune cell, or online system of claim 117, wherein the expression of the payload in the immune cell 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. The method, immune cells, or online system of claim 117 or 118, wherein: (a) The payload sequence includes a first sensor (first P-type sensor) capable of specifically identifying the regulator and a second sensor (second P-type sensor) capable of specifically identifying the marker, and (b) The modulator sequence includes a sensor (R-type sensor) capable of specifically recognizing the marker; and The regulator, the marker identified by the second P-type sensor (P-type marker), and the marker identified by the R-type sensor (R-type marker) are different.

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

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

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

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

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

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

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

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

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

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

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

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

132. The method, immune cells, or online system as claimed in claims 105 to 131, wherein: (a) The payload sequence comprises a plurality of the first P-type sensors. (b) The payload sequence comprises a plurality of the second P-type sensors. (c) The modulator sequence comprises a plurality of the R-type sensors, or (d) Any combination of (a) to (c).

133. The method, immune cells, or online system as claimed in claims 105 to 132, wherein: (a) The payload sequence includes a spacer subsequence (P-type spacers). (b) The payload sequence includes a spacer subsequence (R-type spacers), or (c) Cases (a) and (b) are both valid.

134. The method, immune cell, or online system of claim 133, wherein the P-type spacer is located between the first P-type sensor and the second P-type sensor.

135. The method, immune cell, or online system of claim 133 or 134, wherein the P-type spacer is located between the payload coding sequence and (a) the first P-type sensor, the payload coding sequence and (b) the second P-type sensor, or (c) both (a) and (b).

136. The method, immune cell, or online system of any one of claims 133 to 135, wherein the R-type spacer is located between the regulator coding sequence and the R-type sensor.

137. The method, immune cell, or online system of any one of claims 133 to 136, wherein the payload sequence comprises the plurality of the first P-type sensors, wherein two or more of the first type sensors are separated by P-type spacers.

138. The method, immune cell, or online system of any one of claims 133 to 137, wherein the payload sequence comprises the plurality of second P-type sensors, wherein two or more second type sensors are separated by P-type spacers.

139. The method, immune cell, or online system of any one of claims 133 to 138, wherein the modulator sequence comprises the plurality of said R-type sensors, wherein two or more said R-type sensors are separated by R-type spacers.

140. The method, immune cell, or online system of any one of claims 133 to 139, wherein the length of the P-type spacer, the R-type spacer, or both is about 1 to about 50 nucleotides.

141. The method, immune cells, or online system of any one of claims 133 to 140, wherein the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the following: The sequence tttcctttcccccttcccttttcctttcctttccttcccttccctt (SEQ ID NO: 1) or a fragment thereof.

142. The method, immune cells, or online system of claim 141, wherein the P-type spacer, R-type spacer, or both comprise, substantially consist of, or consist of the following: Sequence tttcctttcccccttccctt (SEQ IDNO: 2).

143. The method, immune cells, or online system of any one of claims 133 to 142, wherein the P-type spacer, the R-type spacer, or both comprise, substantially consist of, or consist of the following: The sequence gcggccgctaaa (SEQ ID NO: 3) or a fragment thereof.

144. The method, immune cell, or online system of any one of claims 106 to 143, wherein the P-type marker, the R-type marker, or both comprise microRNA.

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

146. The method, immune cell, or online system of claim 145, wherein the RNA-binding protein comprises a ribonuclease.

147. The method, immune cell, or online system of claim 146, wherein the ribonuclease comprises a Cas protein.

148. The method, immune cell, or online system of claim 147, wherein the Cas protein includes the Cas6 protein.

149. The method, immune cell, or online system of any one of claims 1 to 148, 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, immune cell, or online system of any one of claims 1 to 149, wherein the payload sequence comprises one or more elements that enhance the translation of the encoded protein compared to the modulator sequence.

151. The method, immune cell, or online system of claim 150, wherein one or more elements comprise an aptamer of a translation initiation factor (e.g., eIF4G).

152. The method of any one of claims 1 to 20 and 28 to 151, the immune cells of any one of claims 21, 22 and 28 to 151, or the online system of any one of claims 23 to 151, wherein the synthetic circuit further comprises (1) Internal ribosome entry site (IRES), (3) UTR, (4) The sequence encoding the signal peptide, (5) Translate the starting sequence. (6) Poly-A sequences, (7) The sequence encoding an RNA-binding protein, (8) The sequence encoding the 2A ribosomal jumping peptide, or (9) Any combination of (1) to (8).

153. The method of any one of claims 1 to 20 and 28 to 152, the immune cells of any one of claims 21, 22 and 28 to 152, or the online system of any one of claims 23 to 152, wherein the synthetic circuit does not contain any sequence derived from a non-human genome.

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

155. The method, immune cell, or online system of claim 154, wherein the delivery agent is a nanoparticle comprising (i) the synthetic circuit and (ii) one or more types of lipids and / or lipid-like materials.

156. The method, immune cell, or online system of claim 155, wherein the one or more types of lipids include ionizable lipids, cationic lipids, lipid-like substances, non-cationic auxiliary lipids, phospholipids, sterols, or other structural lipids or combinations thereof.

157. The method, immune cells, or online system of claim 156, wherein the ionizable lipid comprises ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate heptadecaned-9-yl ester (SM-102), 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoate heptadecaned-9-yl ester (lipid 5), 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate di(( Z )-Non-2-en-1-yl) ester (L319), 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tetrazyl)-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy -N,N--Dimethylaminopropane (DLin-DMA), 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-D) MA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)) or combinations thereof.

158. The method, immune cells, or online system of claim 156, wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), liposome transfection reagent, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleoyloxy-N-[2-(sperminecarbamoylamino)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propane (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1 1,2-Dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(1,2-dioleoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (DLePC), 1,2-distearyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dilinoleoyl-3 - Trimethylammonium-propane (DLTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-distearate-sn-glycerol-3-ethylphosphocholine (DSePC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMePC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)-sn-glycerol-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

159. The method, cell, or online system of claim 156, wherein the lipids comprise 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecane-2-ol) (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazin 2,5-dione (cKK-E12), tetra(8-methylnonyl)3,3′,3″,3 -(((methylazonidyl)bis(propane-3,1-diyl))bis(azontriyl))tetrapropionate (306Oi) 10 ), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecano-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(azanetriyl))tetra(ethane-2,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadec-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9 Z,12Z,12′Z,12″Z,12 Z)-Tetra(octadecyl-9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tris(3-(bis(dodecylamino)propyl)phenyl)1,3,5-tricarboxamide (TT3), hexa(octyl-3-yl)9,9′,9″,9 ,9″″,9 "-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexylnonanoate (FTT5), PL-1, 98N12-5, 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazolium-2-carboxylate (A2-Iso5-2DC18(A2)), A12-Iso5-2DC18(A12) or any combination thereof.

160. The method, cell, or online system of claim 159, wherein the lipid is TT3.

161. The method, cell, or online system of any one of claims 156 to 160, wherein the phospholipid is selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC). Ole-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoic acid)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and any combination thereof.

162. The method, cell, or online system of any one of claims 156 to 161, wherein the phospholipid is selected from the group consisting of: 1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycerol-3-phosphate choline (14:0-18:0 PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycerol-3-phosphate choline (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycerol-3-phosphate choline (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphate choline (16:0-18:0 PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (16:0-18:1... PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (18:0-18:1) PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (18:0-18:2 PC), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (18:0-20:4 PC), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycerol-3-phosphate choline (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline (18:1-18:0 PC,OSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:1 PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:2 PE), 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:1 PE), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonico-sn-glycerol-3-phosphate ethanolamine (18:0-20:4 PE), 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine (18:0-22:6 PE), 1-oleoyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), and any combination thereof.

163. The method, cell, or online system of any one of claims 156 to 162, wherein the sterols include cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, campesterol, tomatine, tomatine, ursolic acid, α-tocopherol, and combinations thereof.

164. The method, cell, or online system of any one of claims 155 to 163, wherein the one or more types of lipids and / or lipid-like materials are polyethylene glycol-modified.

165. The method, immune cell, or online system of any one of claims 155 to 164, wherein the method, the immune cell, or the online system comprises an immune ligand.

166. The method, immune cell, or online system of any one of claims 155 to 165, wherein the one or more types of lipids and / or lipid-like materials comprise about 10% to 50% of ionizable lipids (e.g., cationic lipids).

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

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

169. The method, immune cell, or online system of any one of claims 155 to 168, wherein the one or more types of lipids and / or lipid-like materials comprise polyethylene glycol-modified lipids in a molar ratio of about 0-10%.

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

171. The method, immune cells, or online system of claim 170, wherein the method, immune cells, or online system is formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration.

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

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

174. The method of any one of claims 1 to 8, wherein the synthesis circuit comprises: The nucleotide sequence encoding the payload (payload sequence), The payload sequence includes sensors for identifying markers, and The sensor is used to identify the markers to reduce the expression of the payload.

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