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JP-7854941-B2 - Cell classification indicator circuit and method of using the same

JP7854941B2JP 7854941 B2JP7854941 B2JP 7854941B2JP-7854941-B2

Inventors

  • ベネンソン，ヤーコブ
  • アンジェリッシ，バルトロメオ

Assignees

  • アイドゲノーシッシェ テヒニッシェ ホッホシューレ チューリッヒ

Dates

Publication Date
20260507
Application Date
20210414
Priority Date
20200414

Claims (20)

  1. It is a continuous polynucleotide molecule, a) a first cassette encoding a first RNA whose expression is operably linked to (i) a transactivator response element and (ii) a transcription factor response element , wherein the first RNA comprises (i) a nucleic acid sequence encoding the output; and (ii ) a target site for the iRNA; and b) a second cassette encoding a second RNA whose expression is operably linked to a transcription factor response element , wherein the second RNA comprises the nucleic acid sequence of the transactivator; Includes, Here, the transactivator of the second cassette, when expressed as a protein, binds to and transactivates the transactivator response element of the first cassette, and, The miRNAs are miR-let7c-5p, miR-22-3p, hsa-miR-26b, hsa-miR-126-5p, hsa-miR-122-5p, Mmu-m iR-322-5p, hsa-miR-424-5p, hsa-miR-208a-3p, hsa-miR-208b-3p, hsa-miR-216a-5p, mmu-m iR-217-5p, hsa-miR-217-5p, hsa-miR-375-3p, hsa-miR-124-3p, hsa-miR-1-3p, hsa-miR-1 33a-3p, hsa-miR-133b, hsa-miR-9-5p, hsa-miR-338-3p, hsa-miR-219a-5p, hsa-miR507, hsa -miR-514a-3p, hsa-miR-509-5p, hsa-miR-7-5p, hsa-miR-205-5p, hsa-miR-142-3p, hsa-mi R-199a-3p, hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-192-5p, hsa-miR-194-5p, hsa-mi R-449a, hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-143, hsa-miR-148a-3p, and any combination thereof selected. The aforementioned unbroken polynucleic acid molecule.
  2. The first RNA is a continuous polynucleic acid molecule according to claim 1, comprising a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR-9 target site, or a combination thereof, wherein optionally, The first RNA comprises a 3'UTR, wherein the 3'UTR comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR-9 target site, or a combination thereof. The first RNA comprises a 5'UTR, wherein the 5'UTR comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR -9 target site, or a combination thereof, wherein the first RNA optionally comprises a miR-122 target site or a miR-1 target site .
  3. The second RNA is miR-let7c-5p, miR-22-3p, hsa-miR-26b, hsa-miR-126-5p, hsa-miR-122-5p, Mmu-miR-322- 5p, hsa-miR-424-5p, hsa-miR-208a-3p, hsa-miR-208b-3p, hsa-miR-216a-5p, mmu-miR-217 -5p, hsa-miR-217-5p, hsa-miR-375-3p, hsa-miR-124-3p, hsa-miR-1-3p, hsa-miR-133a-3p , hsa-miR-133b, hsa-miR-9-5p, hsa-miR-338-3p, hsa-miR-219a-5p, hsa-miR507, hsa-miR-5 14a-3p, hsa-miR-509-5p, hsa-miR-7-5p, hsa-miR-205-5p, hsa-miR-142-3p, hsa-miR-199a -3p, hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-192-5p, hsa-miR-194-5p, hsa-miR-449a An unbroken polynucleic acid molecule according to claim 1 or 2, further comprising a target site for microRN A selected from hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-143, hsa-miR-148a-3p and any combination thereof .
  4. The second RNA further comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR-9 target site, or a combination thereof, the continuous polynucleic acid molecule according to any one of claims 1 to 3, optionally wherein, The second RNA comprises a 3'UTR, where the 3'UTR comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR-9 target site, or a combination thereof. The unbroken polynucleic acid molecule wherein the second RNA comprises a 5'UTR, where the 5'UTR comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, a miR-199 target site, a miR-9 target site, or a combination thereof, wherein the second RNA optionally comprises a miR-122 target site or a miR-1 target site .
  5. The seamless polynucleic acid molecule according to claim 4, wherein at least one miRNA target site of the first cassette and at least one miRNA target site of the second cassette are either the same nucleic acid sequence or different sequences regulated by the same miRNA, optionally wherein the first RNA and the second RNA each contain a miR-199a-3p target site or a miR-9-5p target site.
  6. A seamless polynucleic acid molecule according to any one of claims 1 to 5, wherein the transactivator response element of the first cassette includes a nucleic acid sequence represented by any one of sequence numbers 139 to 198 or a combination thereof.
  7. A seamless polynucleic acid molecule according to any one of claims 1 to 6, wherein the transcription factor response elements of the first cassette and/or the second cassette include a nucleic acid sequence represented by any one of sequence numbers 199 to 237 or a combination thereof.
  8. A seamless polynucleic acid molecule according to any one of claims 1 to 7, wherein the first cassette and/or the second cassette comprises a promoter element, optionally wherein the promoter element comprises a nucleic acid sequence represented by any one of sequence numbers 238 to 268 or a combination thereof.
  9. The first cassette comprises, from 5' to 3', (i) an upstream regulatory component including a transactivator response element and a transcription factor response element; (ii) a nucleic acid sequence encoding the output; and (iii) a downstream component including a miR-199a-3p target site or a miR-9-5p target site; and the second cassette comprises, from 5' to 3', (i) an upstream regulatory component including a promoter element; (ii) a nucleic acid sequence encoding the transactivator; and (iii) a downstream component including a miR-199a-3p target site or a miR-9-5p target site, The seamless polynucleic acid molecule according to claim 8 .
  10. A seamless polynucleic acid molecule according to any one of claims 7 to 9, wherein the first cassette and/or the second cassette comprises two or more transcription factor response elements, optionally wherein the first cassette and/or the second cassette comprises two different transcription factor response elements.
  11. The seamless polynucleic acid molecule according to claim 9 or 10, wherein the regulatory component upstream of the first cassette includes a promoter element.
  12. A seamless polynucleic acid molecule according to any one of claims 1 to 11, wherein the first cassette and the second cassette are in a dispersed orientation.
  13. The transactivator of the second cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA, or A seamless polynucleic acid molecule according to any one of claims 1 to 12, wherein the transactivator of the second cassette comprises a nucleic acid sequence represented by any one of sequence numbers 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and 111, a nucleic acid encoding a protein represented by any one of sequence numbers 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, and 112-138, or a nucleic acid sequence encoding a protein having at least 90 % identity with the transactivator represented by any one of sequence numbers 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, and 112-138.
  14. A seamless polynucleic acid molecule according to any one of claims 1 to 13, wherein the output is a therapeutic agent.
  15. The seamless polynucleic acid molecule according to claim 14, wherein the output is a sequence encoding a fluorescent protein, a cytotoxin, an enzyme catalyzing prodrug activity, an immunomodulatory protein and/or RNA, a DNA modifying factor, a cell surface receptor, a gene expression regulator, a kinase, an epigenetic modifier, and/or a factor necessary for vector replication, and/or a pathogen antigen polypeptide.
  16. The seamless polynucleic acid molecule according to claim 15, wherein the immunomodulatory protein and/or RNA is a cytokine or colony-stimulating factor.
  17. An uninterrupted polynucleic acid molecule according to any one of claims 1 to 16, wherein the uninterrupted polynucleic acid molecule comprises a nucleic acid sequence represented by any one of sequence numbers 269 to 304 .
  18. A seamless polynucleic acid molecule according to any one of claims 1 to 17, wherein the (i) transactivator response element and (ii) transcription factor response element of the first cassette, the transcription factor response element of the second cassette, and the target site for miRNA are all parts of a logical gene circuit that regulates the expression of the output.
  19. A vector comprising a continuous polynucleic acid molecule as described in any one of claims 1 to 18.
  20. An engineered viral genome comprising an uninterrupted polynucleic acid molecule as described in any one of claims 1 to 18, wherein optionally, The manipulated viral genome is an adeno-associated virus (AAV) genome, a lentivirus genome, an adenovirus genome, a herpes simplex virus (HSV) genome, a vaccinia virus genome, a poxvirus genome, a Newcastle disease virus (NDV) genome, a coxsackievirus genome, a reovirus genome, a measles virus genome, a varicella stomatitis virus (VSV) genome, a parvovirus genome, a Seneca Valley virus genome, a maraba virus genome, or a common cold virus genome.

Description

Disclosed herein are contiguous DNA sequences encoding highly compact multi-input gene logic gates for precise in vivo cell targeting, and methods for treating diseases using combinations of in vivo delivery with such contiguous DNA sequences. Background Gene therapy is emerging as a next-generation treatment option for genetic diseases and cancer. However, current gene therapy vectors suffer from low efficiency, high toxicity, and long development timelines for producing therapeutic lead compounds. One reason for these weaknesses is the inadequately strict control of therapeutic gene expression in gene therapy vectors, which results in (i) gene expression in unintended cell types and tissues, or (ii) insufficient or excessively high drug doses. In other words, precise control of gene expression, both in terms of the dosage of gene products (i.e., the number of protein molecules per cell) and cell type-dependent expression, remains an unresolved challenge in gene therapy. Abstract Research in biomolecular computing and synthetic biology has long strived to enable new types of therapeutic approaches based on: (i) multi-input sensing of molecular disease indicators; (ii) molecular-level calculations to determine the intensity of therapeutic responses; and (iii) in situ augmentation of therapy in a highly accurate and coordinated manner. Described herein is a cell classification indicator gene circuit that enables the precise identification of heterogeneous cell types through the complex logical integration of inputs from multiple cells. Also described herein are methods for treating diseases using the classification indicator gene circuit. Cancer has been considered a class of diseases that would greatly benefit from cell classification indicator approaches due to the similarity of tumors to healthy cells, tumor heterogeneity, and its dissemination in both primary and secondary sites. The research described herein supports the concept that multi-input gene circuits for precise cell targeting are an ideal means for next-generation gene therapy. Therefore, in some respects, this disclosure relates to inseparable polynucleic acid molecules. In some embodiments, inseparable polynucleic acid molecules are a) A first cassette encoding a first RNA whose expression is operably linked to a transactivator response element, wherein the first RNA comprises (i) the nucleic acid sequence of the output; and (ii) a target site for the miRNAs listed in Table 1 or a combination thereof; b) a second cassette encoding a second RNA, wherein the second RNA contains the nucleic acid sequence of the transactivator; Includes, Here, the transactivator of the second cassette, when expressed as a protein, binds to and transactivates the transactivator response element of the first cassette. In some embodiments, the first RNA includes the let-7c target site, let-7a target site, let-7b target site, let-7d target site, let-7e target site, let-7f target site, let-7g target site, let-7i target site, miR-22 target site, miR-26b target site, miR-122 target site, miR-208a target site, miR-208b target site, miR-1 target site, miR-217 target site, miR-216a target site, or a combination thereof. In some embodiments, the first RNA comprises a 3'UTR, where the 3'UTR includes the let-7c target site, let-7a target site, let-7b target site, let-7d target site, let-7e target site, let-7f target site, let-7g target site, let-7i target site, miR-22 target site, miR-26b target site, miR-122 target site, miR-208a target site, miR-208b target site, miR-1 target site, miR-217 target site, miR-216a target site, or a combination thereof. In some embodiments, the first RNA comprises a 5'UTR, where the 5'UTR includes the let-7c target site, let-7a target site, let-7b target site, let-7d target site, let-7e target site, let-7f target site, let-7g target site, let-7i target site, miR-22 target site, miR-26b target site, miR-122 target site, miR-208a target site, miR-208b target site, miR-1 target site, miR-217 target site, miR-216a target site, or a combination thereof. In some embodiments, the second RNA further comprises a target site for the microRNAs listed in Table 1 or combinations thereof. In some embodiments, the second RNA further comprises a let-7c target site, a let-7a target site, a let-7b target site, a let-7d target site, a let-7e target site, a let-7f target site, a let-7g target site, a let-7i target site, a miR-22 target site, a miR-26b target site, a miR-122 target site, a miR-208a target site, a miR-208b target site, a miR-1 target site, a miR-217 target site, a miR-216a target site, or a combination thereof. In some embodiments, the second RNA comprises a 3'UTR, where the 3'UTR includes the let-7c target site, let-7a target site, let-7b target site, let-7d target site, let-7e target site, let-7f target site, let-7g target site, let-7i target site, miR-22 target site, miR-26b target site, miR-122 target site, miR-208a target site, miR-208b target