Inducible promoters for viral vector production

Abstract

The aspects described herein relate to stable cell lines for the production of recombinant viral vectors (e.g., recombinant adeno-associated virus vectors), said stable cell lines comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein. This document further provides methods for preparing stable cell lines and methods for producing viral vectors.

Description

[0001] This application is a divisional application of patent application No. 202180038621.7, filed on March 26, 2021, entitled "Inducible Promoter for Viral Vector Production".

[0002] Cross-reference to related applications

[0003] Pursuant to 35 USC § 119(e), this international application claims the rights of U.S. Provisional Application No. 63 / 000,155, filed March 26, 2020; U.S. Provisional Application No. 63 / 010,330, filed April 15, 2020; and U.S. Provisional Application No. 63 / 148,905, filed February 12, 2021, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to cell lines for the rapid and scalable production of viral vectors, such as adeno-associated virus (AAV). Background Technology

[0005] Recombinant viral vectors (e.g., recombinant adeno-associated virus vectors, lentiviral vectors, and adenocarcinogenic vectors) carrying heterologous DNA (transgenic) are used to deliver genes into cells, which can be expressed in the cells to allow, for example, the production of recombinant proteins in vitro or in vivo, vaccination, or treatment of disease states or genetic defects. Disease states or genetic defects can be treated by, for example, by providing effective levels of normal gene product through viral genes (e.g., replication), by increasing levels of gene product to correct dysfunctional genes, or by blocking the endogenous production of genes (where gene expression is harmful to the cell or organism).

[0006] Methods for delivering exogenous genes into mammalian cells include the use of mammalian viral vectors, such as vectors derived from retroviruses (e.g., lentiviruses), adenoviruses, herpesviruses, vaccinia virus, polioviruses, adeno-associated viruses, hybrid viruses, etc. A limiting factor in the field of gene therapy is identifying efficient and scalable methods for the mass production of such viral vectors.

[0007] Adeno-associated virus (AAV) systems offer numerous advantages for transgenic delivery, making them ideal viral vectors for gene therapy. Regarding AAV production, the viral protein replication (rep) has long been considered essential for AAV genome replication and excision; however, the optimal amount of Rep protein required for efficient rAAV production remains unclear. Rep protein toxicity to cell lines has been shown, leading to difficulty in producing stable cell lines expressing Rep. Further evidence suggests that reduced Rep78 / 68 production during viral replication results in higher levels of rAAV production. Conversely, other existing techniques clearly indicate that high Rep protein expression (resulting from replacing the native rep p5 promoter with a stronger promoter) leads to high levels of rAAV expression. Considering existing techniques, it appears that different levels of Rep may be required for rAAV vector production based on specific virus-producing cells. Similarly, capsid proteins may be toxic to cells. Therefore, it is crucial to obtain fine temporal and / or spatial control over the Rep and / or other toxic genes required for viral production to optimize viral vector production. Summary of the Invention

[0008] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein.

[0009] In any embodiment of any aspect provided herein, the tunable promoter is an inducible or repressive promoter. In any embodiment of any aspect provided herein, the tunable promoter is an inducible promoter.

[0010] In any embodiment provided herein, the toxic protein is a viral protein. Exemplary viral proteins include replication (rep), capsid (cap), envelope (env), and polymerase (pol).

[0011] In any embodiment of any aspect provided herein, the toxic protein is associated with nucleic acid transcription.

[0012] In any embodiment of any aspect provided herein, the toxic protein is associated with the production of a capsid or coating.

[0013] In any embodiment of any aspect provided herein, the inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0014] In any embodiment of any aspect provided herein, the cell comprises at least two inducible promoters, wherein the at least two inducible promoters are induced by different components and the at least two inducible promoters are operatively linked to different heterologous genes encoding different toxic proteins.

[0015] In any embodiment of any aspect provided herein, the cell includes a first inducible promoter operatively linked to a repressive element capable of terminating protein expression.

[0016] In any embodiment of any aspect provided herein, the first inducible promoter further encodes a protein that represses the expression of the first inducible promoter.

[0017] In any embodiment provided herein, the cell contains a first inducible promoter that further encodes a protein that induces expression of a second inducible promoter.

[0018] In any embodiment of any aspect provided herein, the cell is a eukaryotic cell or a prokaryotic cell.

[0019] In any embodiment of any aspect provided herein, the cells are selected from the cell types listed in Table 2. In any embodiment of any aspect provided herein, the cells are derived from a cell type selected from the cell types listed in Table 2.

[0020] In any embodiment of any aspect provided herein, contacting the cells with an inducer causes at least one toxic protein to be expressed.

[0021] In any embodiment of any aspect provided herein, the cells are used for the production of viral particles selected from the group consisting of adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0022] One aspect of the invention described herein is a stable cell line for the production of recombinant AAV vectors, the stable cell line comprising at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous rep gene encoding a rep protein.

[0023] In any embodiment of any aspect provided herein, the inducible promoter is further operatively linked to a heterologous cap gene encoding the cap protein.

[0024] In any embodiment of any aspect provided herein, the stable cell further comprises a second inducible promoter operatively linked to a heterologous cap gene encoding the cap protein, wherein the second inducible promoter is induced by a compound different from the first inducible promoter.

[0025] One aspect of the invention described herein is a stable cell line for the production of recombinant AAV vectors, the stable cell line comprising at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous cap gene encoding a cap protein.

[0026] One aspect of the invention described herein is a method for producing any of the stable cell lines provided herein, the method comprising: (a) transforming a cell population with at least one nucleic acid cassette comprising an inducible promoter operatively linked to a heterologous gene encoding a toxic protein; (b) culturing the cell population of (a) under conditions and for a time sufficient to allow expression of the nucleic acid cassette; (c) selecting cells stably expressing the nucleic acid cassette; and (d) growing the cells of (c) to produce the cell line.

[0027] One aspect of the invention described herein is a method for producing adeno-associated virus (AAV) particles, the method comprising: (a) providing any one of a stable cell line for rAAV production in an AAV expression system; (b) culturing the cells under conditions expressing at least one toxic protein; (c) culturing the cells under conditions for producing AAV particles; and (d) optionally isolating the AAV particles.

[0028] In any embodiment of any aspect provided herein, the cells are cultured in suspension.

[0029] In any embodiment of any aspect provided herein, the cells are cultured under conditions free of animal components.

[0030] In any embodiment of any aspect provided herein, step (c) includes separating AAV particles from the cells.

[0031] In any embodiment of any aspect provided herein, step (c) includes separating AAV particles from the culture medium in which the cells are cultured.

[0032] In any embodiment of any aspect provided herein, the cells are cultured in a shake flask.

[0033] In any embodiment of any aspect provided herein, the cells are cultured in a bioreactor.

[0034] In any embodiment of any aspect provided herein, step (c) occurs after the expression of the toxic protein.

[0035] In any embodiment of any aspect provided herein, if the stable cell contains at least two inducible promoters, then the at least two inducible promoters are induced substantially simultaneously.

[0036] In any embodiment of any aspect provided herein, if the stable cell contains at least two inducible promoters, the at least two inducible promoters are induced at different times and / or induced for different durations.

[0037] In any embodiment of any aspect provided herein, the method is capable of producing all serotypes, chimeras, and heterozygotes of AAV. Exemplary AAVs include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13, or chimeric AAVs composed of AAV1-13 2.5, 218, 9.45, and other chimeric or heterozygous capsids.

[0038] In any embodiment of any aspect provided herein, the AAV particle contains a rational haploid capsid.

[0039] In any embodiment of any aspect provided herein, the AAV expression system comprises at least one of a recombinant AAV plasmid, a plasmid expressing Rep, a plasmid expressing Cap, and an adenovirus helper plasmid.

[0040] In any embodiment of any aspect provided herein, the recombinant AAV plasmid encodes a transgene. In any embodiment of any aspect provided herein, the transgene is a therapeutic transgene.

[0041] In any embodiment of any aspect provided herein, the method provides at least approximately 4 × 10⁴ cells per cell prior to purification. 4 Each particle contains a vector genome. In any embodiment provided herein, the method provides at least about 1 × 10⁻⁶ particles per cell prior to purification. 5 Each particle contains a vector genome. In any embodiment provided herein, the method provides at least about 1 × 10⁻⁶ cells per liter of cell culture. 12 A purified particle containing the vector genome. In any embodiment provided herein, the method provides at least about 1 × 10⁻⁶ cells per liter of cell culture. 13 A purified particle containing the vector genome.

[0042] One aspect of the invention described herein is a method for producing viral particles, the method comprising: (a) providing any of the stable cell lines described herein in a viral expression system; (b) culturing the cells under conditions expressing at least one toxic protein, wherein the at least one toxic protein is operatively linked to at least one inducible promoter; (c) culturing the cells under conditions for producing viral particles; and (d) optionally isolating the viral particles.

[0043] In any embodiment of any aspect provided herein, the viral particles are selected from the group consisting of adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0044] One aspect of the invention described herein provides a cell line for the production of recombinant viral vectors, the cell line comprising transient expression of at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous gene encoding a toxic protein.

[0045] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0046] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one trichodinin-inducible promoter operably linked to at least one heterologous gene encoding a toxic protein.

[0047] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one hypoxia-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0048] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one trichodinin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodinin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0049] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0050] One aspect of the invention described herein provides a stable cell line for the production of a recombinant viral vector, the stable cell line comprising at least one trichodinin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxin protein and at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxin protein.

[0051] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter having a sequence having SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein. Variations of these and other promoters suitable for use in this invention are described below.

[0052] One aspect of the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter having a sequence having any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein. Variations of these and other promoters suitable for use in this invention are described below.

[0053] One aspect of the invention described herein provides a stable cell line for the production of a recombinant viral vector, the stable cell line comprising at least one inducible promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0054] One aspect of the invention described herein provides a stable cell line for the production of a recombinant viral vector, the stable cell line comprising at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0055] One aspect of the invention described herein provides a stable cell line for the production of a recombinant viral vector, the stable cell line comprising at least one inducible promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0056] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0057] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one trichodinin-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0058] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one hypoxia-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0059] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one trichodin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters.

[0060] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0061] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one tuftrin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein.

[0062] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence having SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0063] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence having any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0064] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0065] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0066] One aspect of the invention described herein provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0067] In any embodiment of any aspect provided herein, the stable cell line further comprises at least one repressive element operatively linked to at least one heterologous gene encoding a toxic protein.

[0068] In any embodiment of any aspect provided herein, the stable cell line has at least two inducible promoters, and the at least two inducible promoters are identical.

[0069] In any embodiment of any aspect provided herein, the stable cell line has at least two inducible promoters, and the at least two inducible promoters are different.

[0070] Certain aspects of the techniques described herein relate generally to nucleic acid constructs, methods, and systems for the sequential and / or temporal regulation of gene expression of one or more viral proteins. Such nucleic acid constructs described herein are applicable to viral vector expression systems (e.g., AAV expression systems) and to the generation of stable cell lines for both viral vector expression systems and AAV vector expression systems.

[0071] One aspect of this document provides a nucleic acid construct comprising nucleic acid sequences, said construct including at least one of: a nucleic acid sequence encoding a viral (e.g., E4) protein, a nucleic acid sequence encoding a second viral (e.g., E2A) protein, and a nucleic acid sequence encoding viral RNA (e.g., VA RNA), wherein each nucleic acid sequence encoding any one of E4, E2A, and VA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a regulated transcription activator, wherein the first and second regulated promoters are regulated promoters of different types. In one embodiment, the regulated promoter is an inducible promoter, such as a hypoxia-inducible promoter and / or a trichodin-inducible promoter.

[0072] Exemplary transcription activators include homologous domain transcription activators, zinc finger transcription activators, winged helical (forkhead) transcription activators, leucine zipper transcription activators, and helical-loop-helical transcription activators. In one embodiment, the transcription activator is a zinc finger transcription activator (ZF-TA).

[0073] In one embodiment, the first Rep protein and the second Rep protein are encoded by the nucleic acid sequence, or by one or more nucleic acid sequences. In one embodiment, the first Rep protein is a large Rep (e.g., Rep78), and the second Rep protein is a small Rep (e.g., Rep52). In one embodiment, the nucleic acids encoding the first and second Rep proteins are under the control of a second regulated promoter or a regulated transcription activator. In one embodiment, the individual nucleic acids encoding the first and second Rep proteins are under the control of a regulated transcription activator. In one embodiment, the individual nucleic acids encoding the first and second Rep proteins are under the control of the same regulated transcription activator. In one embodiment, the transcription activator is a zinc finger transcription activator (ZF-TA).

[0074] In one embodiment, the nucleic acid sequence encoding the Rep protein contains a modified start codon. In one embodiment, the nucleic acid sequence encoding the Rep78 protein contains a modified start codon. In one embodiment, the nucleic acid sequence encoding the Rep78 protein contains a modified start codon selected from the following: ACC, AUC, CUG, and AGG. In this type of embodiment, the nucleic acid sequence encoding the Rep52 protein contains a typical start codon. In this type of embodiment, the nucleic acid sequence encoding the Rep52 protein contains the ATG start codon.

[0075] In any embodiment of any aspect, the Rep protein is a modified Rep protein. In any embodiment of any aspect, the Rep protein is a modified Rep78 protein.

[0076] In one embodiment of any aspect, the modified Rep protein has a lysine-to-arginine mutation at amino acid position 84. In one embodiment of any aspect, the modified Rep78 protein has a lysine-to-arginine mutation at amino acid position 84.

[0077] In any embodiment of any aspect, the nucleic acid encoding the Rep protein further includes nucleic acid encoding a ribozyme at its 3' end.

[0078] In any embodiment of any aspect, the second regulated promoter operably linked to the nucleic acid encoding the Rep protein is an inducible promoter or a binding site containing a regulated transcription activator. In any embodiment of any aspect, the first regulated promoter operably linked to the nucleic acid encoding any one of E4, E2A, and VA is an inducible promoter.

[0079] In any embodiment of any aspect, the inducible promoter is selected from the group consisting of: trichodin-inducible promoters, hypoxia-inducible promoters, tetracycline-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, metallothionein-inducible promoters, hormone-inducible promoters, and metal-inducible promoters. In one embodiment, the inducible promoter is a trichodin-inducible promoter or a hypoxia-inducible promoter.

[0080] In any implementation of any aspect, the induced promoter lacks a minimal promoter.

[0081] In any embodiment of any aspect, the inducible promoter further comprises a TATA box sequence, or a p5 replication sequence, or both a TATA box sequence and a p5 replication sequence. In any embodiment of any aspect, the inducible promoter comprises a TATA box sequence, or a p5 replication sequence, or both a TATA box sequence and a p5 replication sequence, without comprising a minimal promoter. In such embodiments, the inducible promoter may be a second tunable promoter.

[0082] Another aspect described herein provides a nucleic acid construct comprising nucleic acids encoding a promoter, a TATA box, and / or p5, wherein the promoter does not contain a minimal promoter.

[0083] Another aspect described herein provides a nucleic acid construct comprising a nucleic acid sequence encoding a modulotropic transcription activator, the nucleic acid sequence encoding the modulotropic transcription activator being operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or other helper gene-responsive promoter. In one embodiment, the helper gene-responsive promoter comprises a sequence bound to the transcription activator, and wherein the expression or activity of the transcription activator can be induced by the expression of one or more helper genes. In some embodiments, the modulotropic transcription activator is a zinc finger transcription activator.

[0084] Another aspect described herein provides a nucleic acid construct comprising a toxic protein operatively linked to a promoter containing a target site for binding a regulated transcriptional activator, such as a zinc finger transcriptional activator (ZF-TA). In one embodiment, the toxic protein is a Rep protein. In one embodiment, the helper gene responsive promoter comprises a sequence that binds to the transcriptional activator, and wherein the expression or activity of the transcriptional activator can be induced by the expression of one or more helper genes.

[0085] Another aspect described herein provides a nucleic acid construct comprising a gene encoding a Rep protein, the gene being operatively linked to a promoter comprising a target site for binding a regulated transcription activator, such as a zinc finger transcription activator (ZF-TA).

[0086] In any embodiment of any aspect, the regulated transcription activator (e.g., zinc finger transcription activator (ZF-TA)) is expressed from a nucleic acid construct encoding a regulated transcription activator (e.g., zinc finger (ZF) transcription activator) operably linked to an inducible promoter, wherein the inducible promoter is an accessory gene responsive promoter (e.g., an E4 responsive promoter or an E2 responsive promoter or other accessory gene responsive promoter).

[0087] In any embodiment of any aspect, the nucleic acid construct further comprises at least one of the following: a nucleic acid sequence encoding at least one accessory protein, wherein each nucleic acid construct is operatively linked to a regulated promoter; a nucleic acid encoding a toxic protein, wherein the toxic protein is under the control of a second regulated promoter or a regulated transcription activator; a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; or a nucleic acid encoding a Rep protein, wherein the Rep protein is under the control of a second regulated promoter or a regulated transcription activator. In one embodiment, the regulated transcription activator is a zinc finger transcription activator.

[0088] Another aspect described herein provides a nucleic acid construct comprising: a nucleic acid sequence encoding a tetracycline-responsive transactivator protein, the nucleic acid sequence encoding the tetracycline-responsive transactivator protein being operatively linked to a promoter (e.g., a constitutive promoter); a nucleic acid sequence comprising at least one of: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; and / or a nucleic acid sequence encoding a regulated transcription activator factor, the nucleic acid sequence encoding the regulated transcription activator factor being operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter or an E2-responsive promoter; and a nucleic acid construct comprising a Rep protein, the nucleic acid construct comprising the Rep protein being operatively linked to a promoter comprising a target site for binding the regulated transcription activator factor. In one embodiment, the regulated transcription activator factor is a zinc finger transcription activator factor.

[0089] Another aspect described herein provides a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and a recombinase recognition sequence (RRS) located at the 3' side of the nucleic acid sequence encoding the Cap protein. Another aspect described herein provides a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and a recombinase recognition sequence (RRS) located at the 5' side of the nucleic acid sequence encoding the Cap protein.

[0090] In any embodiment of any aspect, the nucleic acid sequence encoding the Cap protein is operatively linked to a constitutive promoter.

[0091] In one embodiment of any aspect, the nucleic acid sequence encoding the Cap protein is operatively linked to a regulated promoter. In one embodiment of any aspect, the regulated promoter is an inducible promoter.

[0092] In any embodiment of any aspect, the RRS is a flip enzyme-responsive RRS.

[0093] In any embodiment of any aspect, the nucleic acid construct further comprises a nucleic acid encoding a recombinase protein, said nucleic acid being operatively linked to an inducible promoter.

[0094] Another aspect provided herein is a nucleic acid construct comprising: a first nucleic acid construct comprising a promoter, a termination nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation, a promoter, a termination nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0095] Another aspect provided herein is a nucleic acid construct comprising: a first nucleic acid construct comprising, in a 5' to 3' orientation, a promoter, a terminating nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation, a promoter, a terminating nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0096] In any embodiment of any aspect, the nucleic acid construct further comprises a nucleic acid encoding one or more selectable markers, the nucleic acid encoding one or more selectable markers being side-attached between a third recombinase recognition sequence (RRS) pair, wherein the RRS pairs are oriented in the same direction relative to each other, and wherein the nucleic acid encoding one or more selectable markers is operatively linked to one or more promoters described herein.

[0097] In any embodiment of any aspect, the first RRS pair and the second RRS pair are in the same orientation relative to each other.

[0098] In any embodiment of any aspect, the first RRS pair and the second RRS pair are in opposite directions relative to each other.

[0099] In any embodiment of any aspect, the first RRS pair, the second RRS pair, and the third RRS pair each respond to a different tyrosine recombinase or serine integrase.

[0100] In any embodiment of any aspect, the first RRS pair and the second RRS pair respond to the same tyrosine recombinase or serine integrase.

[0101] In any embodiment of any aspect, the first RRS pair or the second RRS pair or both are Cre-responsive RRS.

[0102] In any embodiment of any aspect, the third RRS pair is a flip enzyme-responsive RRS.

[0103] In any embodiment of any aspect, the cell further comprises a construct containing a nucleic acid encoding a recombinase protein, the nucleic acid encoding the recombinase protein being operatively linked to an inducible promoter. In any embodiment of any aspect, the cell further comprises a nucleic acid encoding a flippant recombinase protein, the nucleic acid encoding the flippant recombinase protein being operatively linked to an inducible promoter.

[0104] In any embodiment of any aspect, the cell further comprises a nucleic acid encoding a Cre recombinase protein, the nucleic acid encoding the Cre recombinase protein being operatively linked to an inducible promoter.

[0105] Another aspect described herein provides cells containing any of the nucleic acid constructs described herein.

[0106] Another aspect described herein provides a cell comprising at least one of any nucleic acid constructs described herein. In one embodiment of any aspect, the cell comprises at least two of any nucleic acid constructs described herein. In one embodiment of any aspect, the cell comprises at least three of any nucleic acid constructs described herein.

[0107] In any embodiment of any aspect, the cell further comprises a construct containing a nucleic acid sequence encoding a tetracycline-responsive transactivator protein, the nucleic acid sequence encoding the tetracycline-responsive transactivator protein being operatively linked to a promoter (e.g., a constitutive promoter).

[0108] In any embodiment of any aspect, the cell further comprises a construct containing a nucleic acid sequence encoding a marker protein. In any embodiment of any aspect, the nucleic acid sequence encoding the marker protein is side-joined with a recombinase recognition sequence (RRS) that is oriented in the same direction as each other.

[0109] In any embodiment, the cell further comprises a synthetic gene regulation system, wherein the synthetic gene regulation system comprises a nucleic acid sequence or a target DNA-binding protein operatively linked to a promoter; and a nucleic acid sequence operatively linked to a target promoter encoding a gene of interest, wherein the target DNA-binding protein is capable of binding to the target sequence, wherein the target sequence is located within the target promoter and / or within the nucleic acid sequence encoding the gene of interest, thereby attenuating or preventing the expression of the gene of interest.

[0110] In any embodiment of any aspect, the representation of the construct is a stable representation.

[0111] In any embodiment of any aspect, the representation of the construct is transient.

[0112] In any embodiment of any aspect, the cell contains at least two nucleic acid constructs, and the expression of the at least two nucleic acid constructs is stable.

[0113] In any embodiment of any aspect, the cell contains at least two nucleic acid constructs, and the expression of the at least two nucleic acid constructs is transient.

[0114] In any embodiment of any aspect, the cell contains at least two nucleic acid constructs, and the expression of at least one nucleic acid construct is stable.

[0115] Another aspect described herein provides stable cells comprising stable expression of at least one nucleic acid construct described herein.

[0116] Another aspect described herein provides a cell containing transient expression of at least one nucleic acid construct described herein.

[0117] Another aspect described herein provides a method for producing viral particles, the method comprising providing any cell line, any stable cell line, or any transient cell line described herein in a viral expression system; culturing the cells for a sufficient time and under conditions of expression of at least one nucleic acid under the control of an adjustable promoter; culturing the cells under conditions for producing viral particles; and optionally isolating viral particles.

[0118] Another aspect described herein provides a method for producing viral particles, the method comprising: (a) providing a cell line expressing a nucleic acid sequence comprising at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter, wherein the first regulated promoter and the second regulated promoter are different; (b) culturing the cells for a sufficient time and under conditions where at least the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding VA RNA is first expressed in a viral vector production protocol; (c) culturing the cells for a sufficient time and under conditions where the nucleic acid sequence encoding the Rep protein is second expressed in a viral vector production protocol; (d) culturing the cells under conditions for producing viral particles; and (e) optionally isolating viral particles. In one embodiment, the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding VA RNA is expressed between 3 and 4 hours in the viral vector production protocol. In one embodiment, the nucleic acid sequence encoding the Rep protein is expressed between 6 and 8 hours in the viral vector production protocol.

[0119] In any embodiment of any aspect, the culture in step (b) is performed using an inducer of a first tunable promoter. In any embodiment of any aspect, the culture in step (c) is performed using an inducer of a second tunable promoter. In any embodiment of any aspect, the inducer or individual inducers may act directly or indirectly to induce the expression of a given nucleic acid.

[0120] Another aspect described herein provides a method for producing viral particles, the method comprising: (a) providing a cell line expressing at least one of the following: a nucleic acid construct comprising a nucleic acid sequence encoding a modulotropic transcription activator operably linked to an inducible promoter, wherein the inducible promoter is a helper gene responsive promoter (e.g., an E4 responsive promoter or an E2 responsive promoter or other helper gene responsive promoter); a nucleic acid construct comprising a toxic protein operably linked to the promoter, the promoter comprising a target site for binding the modulotropic transcription activator; a nucleic acid construct comprising a Rep protein operably linked to the promoter, the promoter comprising a target site for binding the modulotropic transcription activator; a nucleic acid sequence encoding at least one helper protein, wherein each nucleic acid construct is operably linked to a modulotropic promoter; a nucleic acid encoding a toxic protein, the nucleic acid encoding the toxic protein being under the control of a second modulotropic promoter or a modulotropic transcription activator; a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein E4, E2A, and VA are encoded. Each nucleic acid sequence of any of the RNAs is operatively linked to a regulated promoter; or a nucleic acid encoding a Rep protein, said nucleic acid encoding the Rep protein being under the control of a second regulated promoter or a regulated transcription activator; (b) the cells are cultured for a sufficient time and under conditions where at least the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding the VA protein is first expressed in the viral vector production protocol; (c) the cells are cultured for a sufficient time and under conditions where the nucleic acid sequence encoding the toxin protein or the Rep protein is second expressed in the viral vector production protocol; (d) the cells are cultured under conditions for producing viral particles; and (e) optionally, viral particles are isolated. In one embodiment, the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding the VA RNA is expressed between 3 and 4 hours in the viral vector production protocol. In one embodiment, the nucleic acid sequence encoding the Rep protein is expressed between 6 and 8 hours in the viral vector production protocol. In one embodiment, the regulated transcription activator is a zinc finger transcription activator (ZF-TA). In one implementation, the expression or activity of transcription activators can be induced by the expression of one or more helper genes.

[0121] In any embodiment of any aspect, the culture in step (b) is performed using an inducer of a regulated promoter operatively linked to a nucleic acid encoding at least E4, E2A, or VA RNA. In any embodiment of any aspect, the culture in step (c) is performed using an inducer of a transcriptional activator (e.g., ZF-TA) or a second regulated promoter. In any embodiment of any aspect, the expression of the transcriptional activator (e.g., ZA-TA) is induced by the expression of E4, E2, or VA RNA. In any embodiment of any aspect, the expression of the transcriptional activator (e.g., ZA-TA) is directly or indirectly induced by the expression of E4, E2, or VA RNA.

[0122] Another aspect described herein provides a method for producing viral particles, the method comprising: (a) providing a cell line expressing the following nucleic acid sequences: a nucleic acid sequence encoding a tetracycline-responsive transactivator protein operably linked to a promoter (e.g., a constitutive promoter); a nucleic acid sequence comprising at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operably linked to a regulated promoter; a nucleic acid sequence encoding a regulated transcription activator operably linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or a VA RNA-responsive promoter; and a nucleic acid construct comprising a Rep protein operably linked to a promoter, the promoter comprising a target site for binding the regulated transcription activator; (b) culturing the cells for a sufficient time and expressing at least the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding VA RNA. The RNA nucleic acid sequence is cultured under conditions where it is first expressed in the viral vector production protocol; (c) the cells are cultured for a sufficient time and under conditions where the nucleic acid sequence encoding the toxin protein or Rep protein is second expressed in the viral vector production protocol; (d) the cells are cultured under conditions for producing viral particles; and (e) viral particles are optionally isolated. In one embodiment, the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding VA RNA is expressed between 3 and 4 hours in the viral vector production protocol. In one embodiment, the nucleic acid sequence encoding the Rep protein is expressed between 6 and 8 hours in the viral vector production protocol. In one embodiment, the regulated transcription activator is a zinc finger transcription activator. In one embodiment, the expression or activity of the transcription activator can be induced by the expression of one or more accessory genes.

[0123] In any embodiment of any aspect, the culture in step (b) is performed using an inducer with a tunable promoter operatively linked to a nucleic acid encoding at least E4, E2A, or VA RNA. In any embodiment of any aspect, the culture in step (c) is performed using a transcription activator (e.g., ZF-TA). In any embodiment of any aspect, the expression of the transcription activator (ZF-TA) is induced by the expression of E4, E2, or VA RNA. In any embodiment of any aspect, the expression of the zinc finger transcription activator (ZF-TA) is directly or indirectly induced by the expression of E4, E2, or VA.

[0124] Another aspect described herein provides a method for producing viral particles, the method comprising: (a) providing a cell line expressing a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the Cap protein; (b) culturing the cells for a sufficient time and under conditions where at least the nucleic acid sequence encoding the Cap protein is highly expressed for the first 24 hours in the viral vector production protocol and moderately expressed for the remaining 48 hours in the viral vector production protocol; (c) culturing the cells for a sufficient time and under conditions for producing viral particles; and (d) optionally isolating viral particles.

[0125] In any embodiment of any aspect, the culture in step (b) is performed using an inducer with a tunable promoter operatively linked to the Cap protein.

[0126] Another aspect described herein provides a method for producing viral particles, the method comprising: (a) providing cells expressing: a first nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence side-attached to a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence side-attached to a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA; and (b) culturing the cells for a sufficient time and obtaining at least one nucleic acid sequence encoding the E4 protein, the E2A protein, or the VA protein. The nucleic acid sequence of RNA is cultured under conditions where it is first expressed in the viral vector production protocol (e.g., at hour 10 of production); (c) the cells are cultured for a sufficient time and under conditions where the nucleic acid sequence encoding the toxin protein or Rep protein is second expressed in the viral vector production protocol (e.g., at hour 12 of production); (d) the cells are cultured under conditions for producing viral particles; and (e) viral particles are optionally isolated. In one embodiment, the nucleic acid sequence encoding the E4 protein, the nucleic acid sequence encoding the E2A protein, or the nucleic acid sequence encoding VA RNA is expressed between 3 and 4 hours in the viral vector production protocol. In one embodiment, the nucleic acid sequence encoding the Rep protein is expressed between 6 and 8 hours in the viral vector production protocol.

[0127] In any embodiment of any aspect, the culture in step (b) is a culture of a recombinase specific to a first recombinase recognition sequence (RRS). In any embodiment of any aspect, the culture in step (c) is a culture of a recombinase specific to a second recombinase recognition sequence (RRS).

[0128] Another aspect described herein provides a method for generating stable cell lines for producing viral particles, the stable cell lines containing packaging sequences (sometimes referred to as AAV genomes). Attached Figure Description

[0129] Figure 1A A schematic diagram of hypoxia-induced gene expression is shown. Transcription factor HIF1A (HIF1α) degrades under normal oxygen conditions but is stable under hypoxia, dimerizing with HIF1B (HIF1β) to form HIF1 and translocating to the nucleus. In the nucleus, the HIF1 complex binds to hypoxia-responsive elements and initiates the expression of genes of interest.

[0130] Figure 1B A schematic diagram of the structural organization of HIF1α and HIF1β is shown. Both HIF1α and HIF1β possess a bHLH domain for DNA binding. HIF1β has a Per-ARNT-Sim (PAS) domain for central heterodimerization, while the C-terminal domain of HIF1α (TAD N / TAD C) recruits transcriptional co-regulatory proteins. When HIF1α and HIF1β dimerize, they translocate to the nucleus and, upon binding to hypoxia-responsive elements, initiate the expression of hypoxia-regulated genes.

[0131] Figure 2 A schematic diagram of promoters RTV-015 and Synp-HYP-001 is shown. The RTV-015 promoter contains five HRE1 elements and a synthetic minimum promoter MP1. These elements are separated by spacers (not shown). Synp-HYP-001 contains four HRE2 elements and a CMV minimum promoter. The HRE2 elements are not separated by spacers, but there is a spacer (not shown) between the last HRE2 element and the CMV minimum promoter.

[0132] Figure 3 The temporal progression of luciferase expression from the RTV-015, SYNP-HYP-001, and CMV-IE constructs in HEK293-F cells transiently transduced under hypoxia is shown. Cells were placed in a hypoxic state at 0 h, and luciferase activity was then monitored. Luciferase expression from the CMV minimal promoter (used as a control) remained unchanged, but the other constructs showed an increase in luciferase activity over time.

[0133] Figure 4 Measurements of luciferase expression from the RTV-015 and CMV-IE constructs in HEK293-T after transient transduction under normoxic and hypoxic conditions (24 hours post-hypoxia) are shown. Luciferase expression from the CMV-IE promoter was identical under both normoxic and hypoxic conditions. The RTV-015 construct showed almost no luciferase activity under normoxic conditions but was induced after 24 hours of hypoxia.

[0134] Figure 5 The measurement of luciferase expression from RTV-015 and CMV-IE constructs in the CHO_GS suspension cell line transiently transduced under normoxic and hypoxic conditions (24 hours after hypoxia) is shown. Luciferase expression from CMV-IE was identical under both normoxic and hypoxic conditions. Figure 4 Similar results were shown in the study, where RTV-015 showed almost no luciferase activity under normoxic conditions, but was induced after 24 hours of hypoxia.

[0135] Figure 6This explains the mechanism of action of trichosin and other adenylate cyclase activators.

[0136] Figure 7 The promoter activity was shown after transient transfection into the suspension cell line HEK293-F. Cells were induced with 20 µM tuftin (at time 0 h), and luciferase expression was measured at 0 h, 3 h, 5 h, and 24 h. All constructs showed increased activity (to varying degrees), while CMV-IE activity remained constant.

[0137] Figures 8A-8C A bright-field micrograph of 11th generation C2C12 cells is shown. Figure 8A The image shows cells before transformation (day 2). Figure 8B The image shows C2C12 cells 24 hours after transfection (day 3). Figure 8C The image shows differentiated C2C12 cells after 5.5 days in differentiation medium (day 7.5). Scale bar is 50 µm.

[0138] Figure 9 Exemplary data illustrating the differences in E1A gene expression between Hek293 and Pro10 cells are presented. Results are the average of three (3) experiments, with error bars representing standard deviation. Pro10 cells expressed E1A RNA at approximately half the level of parental Hek293 cells.

[0139] Figure 10 Exemplary data showing the live cell density at the transfection and collection points are presented.

[0140] Figure 11 Exemplary data showing viability at transfection and collection points are presented.

[0141] Figure 12 Exemplary data showing E1A RNA levels at the collection site are presented.

[0142] Figure 13 Exemplary data of vg / cell of rAAV generated by Pro10 at different concentrations of E1 are presented.

[0143] Figure 14 Exemplary data showing the live cell density under various indicative conditions tested are presented.

[0144] Figure 15 Exemplary data on cell viability are presented, illustrating the various indicative conditions tested. The mutated Rep did not significantly affect cell viability or growth during production.

[0145] Figure 16Exemplary data showing the final viral titer from virus production is presented.

[0146] Figure 17 Exemplary data showing viral titers produced by wtAAV and rAAV are presented. wtAAV produces up to 2 log vg / mL more than rAAV.

[0147] Figure 18 Exemplary data showing the levels of E2A gene expression during wt and rAAV production are presented.

[0148] Figure 19 Exemplary data showing the levels of E4 gene expression during wt and rAAV production are presented.

[0149] Figure 20 Exemplary data showing the levels of Rep78, Rep52, and Cap2 expression during wtAAV production are presented.

[0150] Figure 21 Exemplary data showing the levels of Rep78, Rep52, and Cap2 expression during rAAV production are presented.

[0151] Figure 22 A schematic diagram illustrating the mechanism of leaky scanning is presented.

[0152] Figure 23 Exemplary data showing a comparison of vg / mL between wt and mut-Rep are presented. Removal of nine potential ATGs that can be used as translation initiation sites from Rep78 had no effect on its ability to prepare rAAV.

[0153] Figure 24 Exemplary data are presented, showing protein blots from Rep78 and Rep52 protein expression in an rAAV production run using multiple start codons. The % number is the publicly available translation initiation rate compared to ATG.

[0154] Figure 25 Exemplary data showing vg / mL of rAAV produced by using alternative start codons to control the Rep78 / 52 ratio are presented. Only the ACG mutation significantly improved viral titers.

[0155] Figure 26 Exemplary data illustrating Cap2 gene expression induced by adenovirus helper genes are presented.

[0156] Figure 27Exemplary data showing viral titers from C7 and C8 cells induced with an adenovirus helper function plus E1 are presented. The control is standard triple transfection without an inducible cassette. P = generation.

[0157] Figure 28 Exemplary data demonstrating the effect of adenovirus helper function on Cap2 expression in stable cell lines controlled by the trichodin promoter are presented.

[0158] Figure 29 Exemplary data illustrating the effect of tuftin on the design of novel promoters are presented.

[0159] Figure 30 Exemplary data demonstrating the effect of adenovirus helper functions on FORN-pJB42 activity are presented.

[0160] Figure 31 Exemplary data showing the construction of the Rep LoxP build is presented.

[0161] Figure 32 Exemplary data illustrating the Rep construct are presented. The effect of Cre and / or NHK477 treatment on the induction of Rep expression is shown. For both promoters, neither Rep protein was expressed in the untreated samples.

[0162] Figure 33 Exemplary data demonstrating AAV production controlled by cre-recombinase expressed using Rep are presented.

[0163] Figure 34 Exemplary data are presented, illustrating the expected outcomes and results of the experiment. The average value is for n=3.

[0164] Figure 35 Exemplary data are presented to illustrate the concept of cascade gene expression for rAAV production in Pro10 cells.

[0165] Figure 36 Exemplary data illustrating a ZF-TF design are presented.

[0166] Figure 37 Exemplary data are presented to illustrate a comparison between standard Rep2-Cap8 transfection and novel plasmid constructions. Detailed Implementation

[0167] Generally, the invention described herein provides a stable cell line for the production of recombinant viral vectors, the stable cell line expressing at least one toxic protein under the control of at least one regulated promoter (e.g., an inducible promoter); a method for producing such cells; a promoter linked to another sequence (e.g., a zinc finger) that provides regulatory control; and a method for preparing recombinant viral vectors using said promoter. The stable cell line provides temporal and / or spatial control over at least one (e.g., at least one, at least two, at least three) toxic proteins during viral vector production. Toxic proteins (e.g., derived from viral gene expression) (such as replication (rep), capsid (cap), helper gene products, polymerase (pol), reverse polymerase, or envelope (env)) are proteins that are detrimental to cells during expression. However, these viral genes are essential for the preparation of recombinant viral vectors. Current methods for mitigating the negative effects of toxic protein expression include using cells that transiently express toxic proteins during production; however, these methods adversely affect production yield. Our stable cell described herein provides a stable cell line that allows toxic protein expression primarily when needed, thereby limiting the negative effects of its expression. Compared to current methods, our method, which utilizes these stable cell lines, as described in this paper, yields higher production of recombinant viral vectors.

[0168] definition

[0169] For convenience, the meanings of some terms and phrases used in the specification, embodiments, and appended claims are provided below. Unless otherwise stated or implied by the context, the following terms and phrases have the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed technology, as the scope of this technology is defined only by the claims. 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 technology pertains. If there is a significant difference between the use of terminology in the art and the definitions provided herein, the definitions provided in this specification shall prevail.

[0170] Definitions of common terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 19th edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999–2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (ed.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael RichardGreen and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (Ed.), Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (Ed.), John Wileyand Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in ProteinScience (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, DavidH Margulies, Ethan M Shevach, Warren Strobe (ed.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737); their contents are incorporated herein by reference in their entirety.

[0171] As used herein, the terms “nucleotide sequence,” “nucleic acid sequence,” “RNA sequence,” and “DNA sequence” are used interchangeably and refer to sequences of nucleic acids (e.g., circular nucleic acids to be introduced into target cells and encode gene products or polypeptides). A nucleic acid sequence may contain at least a sequence encoding a toxic polypeptide (i.e., a protein). A heterologous nucleic acid sequence is a nucleic acid sequence that is not “naturally present,” i.e., a sequence that is not normally expressed in cells.

[0172] As used herein, the term "promoter" refers to a DNA region typically located upstream of the nucleic acid sequence to be transcribed, which is required for transcription to occur. A promoter allows for the appropriate activation or repression of transcription of the sequence under its control. Promoters typically contain specific sequences (e.g., enhancer sequences) that are recognized and bound by transcription factors. Transcription factors bind to the promoter DNA sequence and induce the recruitment of RNA polymerase (an enzyme that synthesizes RNA from the coding region of a gene). A large number of promoters are known in the art.

[0173] Regulated promoters include inducible promoters and repressive promoters. As used herein, an "inducible promoter" is a promoter characterized by initiating or enhancing transcriptional activity in the presence of an inducer or inducer, under the influence of an inducer or inducer, in contact with an inducer or inducer, or when a suitable inducing condition (e.g., hypoxia) is applied. An "inducer" or "inducer" as defined herein can be an endogenous or generally exogenous compound or protein, provided in a manner that is active in inducing transcriptional activity derived from an inducible promoter. As used herein, the term "inducer" can also refer to the application of suitable conditions that induce transcriptional activity derived from an inducible promoter (e.g., hypoxia). In some embodiments, the inducer or inducer (i.e., a chemical, compound, protein, or suitable condition) may itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module), and may itself be under the control of, for example, an inducible or repressive promoter. In some embodiments, an inducible promoter is induced in the absence of certain reagents (e.g., a repressor). Examples of inducible promoters include, but are not limited to: trichodin-inducible promoters, hypoxia-inducible promoters, temperature-inducible promoters, tetracycline-inducible (Tet-ON) promoters, pH-inducible promoters, osmolarity-inducible promoters, metallothionein-inducible promoters, hormone (e.g., ecdysone)-inducible promoters, carbon source-inducible promoters, alcohol (e.g., ethanol)-inducible promoters, amino acid-inducible promoters, mifepristone (RU-486)-inducible promoters, cumate-inducible promoters, and 4-hydroxytamoxifen (OHT)-inducible promoters. Gas-inducible promoters, riboswitch-inducible promoters, ribozyme-inducible promoters and aptamerase-inducible promoters, rapamycin-inducible promoters, chemically-inducible promoters, proximity-inducible promoters, Rheoswitch® promoters, CRISPR-inducible promoters and inducible promoters derived from mammalian viruses (e.g., adenovirus late promoters; and mouse mammary tumor virus long terminal repeats (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0174] Inducible promoters can be said to drive the expression of the nucleic acid sequences they regulate (e.g., heterologous genes encoding toxic proteins). The phrases "operably / operatively linked" and "under control" indicate that the promoter is in the appropriate functional position and / or orientation relative to the nucleic acid sequence it regulates in order to control the transcription initiation and / or expression of said sequence.

[0175] As used herein, a “repressed promoter” is characterized by terminating or preventing transcriptional activity such that the activity is downregulated in the presence, influence, or contact with a repressor or inhibitor. A “repressor” or “inhibitor” as defined herein can be an endogenous, or generally exogenous, compound or protein, provided in a manner that is active in preventing transcriptional activity derived from a repressed promoter. In some embodiments, the repressor or inhibitor (i.e., a chemical, compound, or protein) itself may be the result of transcription or expression of a nucleic acid sequence (i.e., the repressor may be a repressor protein expressed by another component or module), and may itself be under the control of, for example, an inducible or repressed promoter. In some embodiments, the repressed promoter is repressed in the absence of certain reagents (e.g., an inducer). Examples of repressive promoters include, but are not limited to, tetracycline OFF, glucose, copper ion, L-methionine, low phosphate / ester, ADH1, Gal80, and MET25.

[0176] In some implementations, the promoter can be both repressive and inducible, for example, by using different repressors or inducers (e.g., agonists and agonists of related transcriptional regulators or pathways) or by different conditions of repressive or induced expression. Thus, for example, an inducible promoter can be given when an inducer is administered, while a repressor can be given when a repressor is administered.

[0177] As used herein, “introduction” broadly refers to placing a synthetic nucleic acid, expression vector, or plasmid into a viral expression system (e.g., a cell or viral vector) such that it is present in, for example, the cell or viral vector expression system. More narrowly, introduction refers to any suitable manner of placing a synthetic nucleic acid, expression vector, or plasmid into the viral expression system described herein. Introduction can be carried out by means of appropriately transporting the synthetic nucleic acid, expression vector, or plasmid into the interior of a cell or viral expression system, thereby generating the synthetic nucleic acid, expression vector, or plasmid, for example, by host cellular mechanisms. Such introduction may involve, for example, transformation, transfection, electroporation, or lipid transfection.

[0178] As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral. The term "vector" encompasses any genetic element that, when bound to appropriate control elements, is capable of replicating and can transfer a gene sequence into a cell. Vectors may include, but are not limited to: cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, artificial chromosomes, viruses, viral particles, etc.

[0179] As used herein, an "expression vector" refers to a nucleic acid that contains an open reading frame (ORF) and, when introduced into a cell, contains all the nucleic acid components necessary to allow the expression of mRNA containing the ORF. The "expression vector" of this invention also contains elements necessary for vector replication and propagation in the host cell. Specifically, as used herein, an "expression vector" refers to a vector that directs the expression of a heterologous nucleic acid as described herein. The expressed sequence is generally, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms (e.g., for expression in human cells and for cloning and amplification in a prokaryotic host). The term "expression" refers to cellular processes involving the production of RNA and proteins, and, where applicable, the secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing.

[0180] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding polypeptides as described herein, replacing non-essential viral genes. Vectors and / or particles can be used for the purpose of transferring the synthetic nucleic acids described herein into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0181] As used herein, the term "cap protein" refers to a capsid protein, appropriately an AAV capsid protein (e.g., one or more of the AAV VP capsid proteins). For example, AAV viral particles typically contain capsid proteins VP1, VP2, and VP3. As is well known in the art, capsid proteins can be naturally occurring or modified.

[0182] As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to: AAV 1, AAV 2, AAV 3 (including 3A and 3B), AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, AAV 13, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, and any other AAV now known or later discovered. See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th edition, Lippincott-Raven). Many relatively new AAV serotypes and clades have been identified (see, for example, Gao et al., J. Virol. 78:6381 (2004); Moris et al., Virol. 33:375 (2004); and Table 1). The “rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) containing one or more heterologous nucleic acid sequences. rAAV vectors typically require only a 145-base cis-ITR to generate the virus. All other viral sequences are optional and can be provided trans-. (Muzyczka, Curr. Topics Microbiol. Immunol. 158:97 (1992)). Typically, the rAAV vector genome will retain only one or more ITR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and non-structural protein-coding sequences can be provided trans-. (e.g., from the vector, such as a plasmid, or by stable integration of the sequence into the packaging cell). In embodiments of the present invention, the rAAV vector genome contains at least one ITR sequence (e.g., an AAV ITR sequence) and optionally two ITRs (e.g., two AAV ITRs), which are typically located at the 5' and 3' ends of the vector genome and side-mounted with heterologous nucleic acids, but not necessarily adjacent to them. The ITRs may be the same as or different from each other.

[0183]

[0184] Genomic sequences of various AAV serotypes, as well as sequences of the native ITR, Rep protein, and capsid subunit, are known in the art. Such sequences can be found in literature or public databases (e.g., GenBank). See, for example, GenBank accessions NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, ...62, NC_000883, NC_001701, NC_002077, NC_001401, NC_001729, NC_001863, NC_001862, NC_000883, NC_001701, NC_002077, NC_001401, NC_001729, NC_001863, NC_001862, NC_000883, NC_001701, NC_001863, NC_001862, NC_000883, NC_001701, NC_001863, NC_001862, NC_001862, NC 001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358, NC_001540, AF513851, AF513852 and AY530579; the contents of which are incorporated herein by reference to teach AAV nucleic acid and amino acid sequences. See also, for example, Bantel-Schaal et al., J Virol. 73:939 (1999); Chiorini et al., J Virol. 71:6823 (1997); Chiorini et al., J. Virol. 73:1309 (1999); Gao et al., Proc. Nat. Acad. Sci. USA 99:11854 (2002); Moris et al., Virology, 33:375 (2004); Mori et al., Virology, 330:375 (2004); Muramatsu et al., Virology, 221:208 (1996); Ruffing et al., J. Gen. Virol. 75:3385 (1994); Rutledge et al., J Virol. 72:309 (1998); Schmidt et al., Virol. 82:8911 (2008); Shade et al., J. Virol. 58:921 (1986); Srivastava et al., J Virol. 45:555 (1983); Xiao et al., J. Virol. 73:3994 (1999); International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244 and U.S. Patent No. 6,156,303, the disclosures of which are incorporated herein by reference to teach AAV nucleic acids and amino acid sequences. See also Table 1.Early descriptions of the AAV1, AAV2, and AAV3 ITR sequences were provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (which is incorporated herein by reference in its entirety).

[0185] As used herein, the term “expression” refers to cellular processes involved in the production of RNA and proteins, including (if applicable) but not limited to, for example, transcription, transcript processing, translation and protein folding, post-translational modifications and other types of processing.

[0186] The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operatively linked to a suitable regulatory sequence. A gene may or may not include regions before and after the coding region, such as a 5' untranslated (5'UTR) or "leader" sequence and a 3' UTR or "trailer" sequence, as well as intercalation sequences (introns) between the individual coding segments (exons).

[0187] As used herein, unless otherwise stated, the term "polypeptide" encompasses both peptides and proteins. A "polypeptide" or "protein" is a sequence of nucleotide bases and may be an RNA, DNA, or DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), and may be either a single-stranded or double-stranded DNA sequence.

[0188] As used herein, the term "cell culture" refers to a cluster of proliferating cells that may be in either an undifferentiated or differentiated state.

[0189] A "viral vector expression system" is a system of one or more polynucleotides that, when introduced into a suitable host cell, is sufficient to support the production of a viral vector. Viral vector expression systems typically include polynucleotides encoding appropriate viral proteins (e.g., envelope and polymerase genes for the production of lentiviruses or adenoviruses).

[0190] An AAV expression system is a system of one or more polynucleotides that, when introduced into a suitable host cell, is sufficient to support the production of recombinant AAV (rAAV). An AAV expression system typically includes polynucleotides encoding AAV rep and cap, a helper gene, and the rAAV genome.

[0191] The AAV genome has palindromic sequences at both its 5' and 3' ends. The palindromic nature of these sequences allows for the formation of hairpin structures, which are stabilized by hydrogen bonds between complementary base pairs. These hairpin structures are thought to adopt a "Y" or "T" shape. See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 and 70 (4th ed., Lippincott-Raven).

[0192] The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired function, such as replication, viral packaging, integration, and / or proviral rescue). ITRs can be AAV ITRs or non-AAV ITRs. For example, non-AAV ITR sequences (e.g., non-AAV ITR sequences of other parvoviruses, such as canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19) or SV40 hairpins that serve as the origin of SV40 replication can be used as ITRs, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Furthermore, ITRs can be partially or fully synthetic, such as the "double-D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al.

[0193] “AAV inverted terminal repeat” or “AAV ITR” can be derived from any AAV, including but not limited to serotype 1, serotype 2, serotype 3a, serotype 3b, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 or serotype 13, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, or any other AAV now known or later discovered (see, for example, Table 1). An AAV ITR does not need to have a naturally occurring terminal repeat sequence (e.g., a naturally occurring AAV ITR sequence can be altered by insertion, deletion, truncation, and / or missense mutations), as long as the terminal repeat mediates the desired function (e.g., replication, viral packaging, integration, and / or proviral rescue, etc.).

[0194] As used herein, “a / an” or “the” can be singular or plural, depending on the context of such use. For example, “a cell” can refer to a single cell or multiple cells.

[0195] As used herein, “and / or” means and covers any and all possible combinations of one or more of the items listed, and when interpreted as an alternative (“or”), it means the absence of a combination.

[0196] Furthermore, as used herein, when referring to measurable values ​​(e.g., the amount, dosage, time, temperature, etc. of the compositions of the present invention), the term “about” is intended to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0197] As used herein, the term “comprising / including / containing” is used to refer to compositions, methods and their respective components that are essential to a method or composition, but the inclusion of unspecified elements (whether essential or not) remains open.

[0198] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of elements that do not materially affect the basic, novel, or functional characteristics of that embodiment. The term "consistent of" refers to compositions, methods, and their respective components as described herein, excluding any elements not listed in the description of that embodiment.

[0199] Nucleic acid constructs

[0200] One aspect provided herein is a nucleic acid construct comprising at least a nucleic acid sequence encoding at least one accessory protein, wherein each nucleic acid construct is operatively linked to a tunable promoter. Exemplary accessory genes (e.g., for AAV viral particles) include, but are not limited to, E1, E2A, E4, and VA. Furthermore, accessory genes may comprise one or more of the elements known in the Pro10 cell line. Accessory genes for generating other viral particles (e.g., lentiviral or adenoviral particles) may be further used in the nucleic acid constructs described herein. In one embodiment, the nucleic acid construct encoding at least one accessory protein comprises a nucleic acid sequence operatively linked to a constitutive promoter encoding a tetracycline-responsive transactivator protein. In one embodiment, the nucleic acid construct encoding at least one accessory protein further comprises a nucleic acid sequence encoding a biomarker protein. In one embodiment, the nucleic acid construct encoding at least one accessory protein comprises a nucleic acid sequence operatively linked to a constitutive promoter encoding a tetracycline-responsive transactivator protein and a nucleic acid sequence encoding a biomarker protein.

[0201] In one embodiment, the tunable promoter is a repressive promoter (e.g., a tetracycline-repressive promoter). In one embodiment, the repressive promoter is repressed by a tetracycline-responsive trans-activator, and the repression is reversed by contact with tetracycline or doxycycline.

[0202] In one embodiment, the expression of at least one accessory gene is responsive to an inducer of a regulated promoter. In one embodiment, the expression of at least one accessory gene is responsive to an inducer of a repressive promoter. In one embodiment, the expression of at least one accessory gene is responsive to tetracycline or doxycycline.

[0203] Another aspect provided in this article is a nucleic acid construct encoding a tetracycline-responsive transactivator protein that can be operatively linked to a constitutive promoter.

[0204] Another aspect provided in this article is the nucleic acid sequence encoding the biomarker protein.

[0205] Another aspect provided herein is a nucleic acid construct comprising at least a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is operatively linked to a tunable promoter. In one embodiment, the nucleic acid encoding the toxic protein further encodes a ribozyme (e.g., a small self-cleaving ribozyme) on the 3' side of the nucleic acid encoding the toxic protein.

[0206] In one embodiment, the regulated promoter is an inducible promoter or a binding site containing a regulated transcription activator. In one embodiment, the inducible promoter contains a TATA box sequence, or a p5 replication sequence, or both a TATA box sequence and a p5 replication sequence. In one embodiment, the inducible promoter contains a TATA box sequence, or a p5 replication sequence, or both a TATA box sequence and a p5 replication sequence, without containing a minimal promoter. In one embodiment, the regulated promoter contains a binding site for a zinc finger transcription activator (ZF-TA).

[0207] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding at least one accessory protein, wherein the at least one accessory gene is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is under the control of a second regulated promoter or transcription activator.

[0208] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence containing at least one of nucleic acid sequences encoding accessory genes, such as a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a tunable promoter. In some embodiments, any accessory protein (e.g., for generating lentiviral particles, adenovirus particles) can be used. For example, nucleic acids encoding Gag, Pol, etc.

[0209] Another aspect provided in this article is a nucleic acid construct encoding the Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a regulated promoter or zinc finger transcription activator (ZF-TA).

[0210] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence containing at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a transcription activator, wherein the first and second regulated promoters are different types of regulated promoters (e.g., the first regulated promoter may be a hypoxic promoter, while the second promoter is of another type). In one embodiment, the regulated promoter is an inducible promoter, or an inducible promoter containing a TATA box sequence or a p5 replication sequence, or both a TATA box sequence and a p5 replication sequence. In one embodiment, the regulated promoter is an inducible promoter lacking a minimal promoter but containing a TATA box sequence and / or a p5 replication sequence.

[0211] Exemplary transcription activators include homologous domain transcription activators, zinc finger transcription activators, winged helical (forkhead) transcription activators, leucine zipper transcription activators, and helical-loop-helical transcription activators. In one embodiment, the transcription activator is a zinc finger transcription activator (ZF-TA).

[0212] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a zinc finger (ZF) transcription activator operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter. In various embodiments, this construct is used to induce the expression of zinc finger transcription activators (ZF-TA) from other nucleic acid constructs described herein.

[0213] Another aspect provided herein is a nucleic acid construct containing a nucleic acid sequence encoding a zinc finger (ZF) transcription activator, the nucleic acid sequence encoding the zinc finger (ZF) transcription activator being operatively linked to a constitutive promoter.

[0214] Another aspect provided in this paper is a nucleic acid construct containing a toxic protein operatively linked to a promoter containing a target site for binding zinc finger transcription activator (ZF-TA).

[0215] Another aspect provided in this article is a nucleic acid construct containing the Rep protein, which is operatively linked to a promoter containing a target site for binding zinc finger transcription activator (ZF-TA).

[0216] In various embodiments, any nucleic acid construct described herein further comprises: at least one of a nucleic acid sequence encoding at least one accessory protein, wherein each nucleic acid construct is operatively linked to a regulated promoter; a nucleic acid encoding a toxic protein, which is under the control of a second regulated promoter or a zinc finger transcription activator; a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; or a nucleic acid encoding a Rep protein, which is under the control of a second regulated promoter or a zinc finger transcription activator.

[0217] Another aspect provided herein is a nucleic acid construct comprising: a nucleic acid sequence encoding a tetracycline-responsive transactivator protein operatively linked to a constitutive promoter; a nucleic acid sequence containing at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA, or any combination thereof, is operatively linked to a regulated promoter; and a nucleic acid sequence encoding a zinc finger (ZF) transcription activator operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter or an E2-responsive promoter; and a nucleic acid construct comprising a Rep protein operatively linked to a promoter containing a target site for binding a zinc finger transcription activator (ZF-TA).

[0218] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a toxic protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the toxic protein. By way of example only, when expressed by cells, the toxic protein or polypeptide causes reduced cell viability, or reduced protein production or synthesis. Exemplary toxic proteins include, but are not limited to, Cap, Rep, or proteins encoded by helper genes. In one embodiment, the toxic protein is operatively linked to a constitutive promoter. In another embodiment, the toxic protein is operatively linked to a regulated promoter (e.g., an inducible promoter). In one embodiment, the nucleic acid construct comprises a nucleic acid encoding a recombinase protein operatively linked to an inducible promoter.

[0219] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the Cap protein. In one embodiment, the Cap protein is operatively linked to a constitutive promoter. In another embodiment, the Cap protein is operatively linked to a tunable promoter (e.g., an inducible promoter). In one embodiment, the nucleic acid construct comprises a nucleic acid encoding a recombinase protein, the nucleic acid encoding the recombinase protein being operatively linked to an inducible promoter.

[0220] In one implementation, the RRS is a flip enzyme responsive RRS.

[0221] Another aspect provided herein is a nucleic acid construct comprising a nucleic acid encoding a recombinase protein, the nucleic acid encoding the recombinase protein being operatively linked to an inducible promoter.

[0222] Another aspect provided herein is a nucleic acid construct comprising: a first nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a terminating nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a terminating nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0223] In one implementation, the RRS flanked by the nucleic acid encoding Rep and the RRS flanked by the accessory gene (e.g., E2A, E4, or VA) are identical, such that they are controlled by the same inducer. In another implementation, the RRS flanked by the nucleic acid encoding Rep and the RRS flanked by the accessory gene (e.g., E2A, E4, or VA) are different. Different flanked RRSs allow for time-dependent control of the Rep protein and the accessory gene.

[0224] In one embodiment, the nucleic acid construct further comprises a nucleic acid encoding one or more selectable markers, the nucleic acid encoding one or more selectable markers being side-attached between a third recombinase recognition sequence (RRS) pair, wherein the RRS pairs are oriented in the same direction relative to each other, and wherein the nucleic acid encoding one or more selectable markers is operatively linked to one or more promoters from another construct provided herein.

[0225] When a nucleic acid sequence (e.g., a target gene) is flanked by recombinase recognition sequence (RRS) pairs in the same orientation, the nucleic acid sequence can be excised upon recognition of the appropriate recombinase RRS. Alternatively, when a nucleic acid sequence is flanked by recombinase recognition sequence (RRS) pairs in opposite orientations relative to each other, the nucleic acid sequence can be inverted upon recognition of the appropriate recombinase RRS. In some embodiments, the inversion and / or excision reaction places the nucleic acid sequence (e.g., a target gene) in a new position and / or orientation and depends on elements other than the RRS site to operatively link the nucleic acid to a promoter, thereby driving the expression of the gene encoded by the nucleic acid sequence.

[0226] Depending on the location of the recombinase recognition sequence, the recombinase excision response can result in either activation or inhibition of gene expression. For example, to activate gene expression, transcription termination sequences flanking the recombination site can be placed upstream of the target gene of interest. In the presence of the recombinase, the transcription termination sequences are removed or excised, allowing gene expression to occur. Conversely, to inhibit gene expression, the recombination site can be engineered to flank the gene of interest. In such implementations, the gene is expressed in the absence of the recombinase. Once the recombinase is induced, the gene is excised, thus completely and irreversibly inhibiting gene expression. The determining factors controlling the gene's on / off state are the presence of the recombinase and the location of the RRS. Recombinase expression can be regulated by signal-inducible promoters, thereby restricting recombinase expression to a specific cell subpopulation that experiences that particular signal. For example, by controlling the expression of Cre recombinase with a neuron-specific promoter, the gene can be turned on or off only in neurons, while leaving the same gene unaffected in other tissues. These enzymes are engineered to be highly active in a wide range of organisms, including bacteria, mammals, insects, plants, and fish.

[0227] In one embodiment, the first RRS pair, the second RRS pair, and the third RRS pair each respond to a different tyrosine recombinase or serine integrase. In one embodiment, the first RRS pair and the second RRS pair respond to the same tyrosine recombinase or serine integrase. In one embodiment, the first RRS pair, the second RRS pair, or both are Cre-responsive RRS. In one embodiment, the third RRS pair is a flippant enzyme-responsive RRS.

[0228] In various embodiments, any nucleic acid construct described herein further comprises a nucleic acid sequence encoding a tetracycline-responsive transactivator protein operatively linked to a constitutive promoter.

[0229] In various embodiments, any nucleic acid construct described herein further comprises a nucleic acid sequence encoding a biomarker protein. Exemplary biomarker proteins include fluorescent proteins (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), or luciferase); molecular tags (e.g., Myc tags, Flag tags, or His tags); and molecular barcodes.

[0230] In various embodiments, any nucleic acid construct described herein further comprises a nucleic acid sequence operatively linked to a constitutive promoter encoding a tetracycline-responsive transactivator protein and a nucleic acid sequence encoding a biomarker protein.

[0231] In one embodiment, the nucleic acid sequence encoding the marker protein is removed from the construct. In another embodiment, the nucleic acid sequence encoding the marker protein is flanked by a recombinase recognition sequence (RRS) that is oriented in the same direction as each other, for example, to facilitate its removal from the construct.

[0232] In some embodiments, the trans-activator is a CRISPR trans-activator or a CAS trans-activator. As used herein, “CRISPR trans-activator” or “CAS trans-activator” refers to a transcription activator protein domain or whole protein associated with, for example, dCas9 or sgRNA, which facilitates the recruitment of co-factors or RNA polymerases for transcription of genes targeted by the system. Transcription activators, for example, have at least a DNA-binding domain and a domain for transcriptional activation. The activation domain can recruit general transcription factors or RNA polymerases to gene sequences, or function by promoting transcription by stalled RNA polymerases, and in eukaryotes can act as a mover nucleosome on DNA or a modifier of histones to increase gene expression. These activators can be introduced into the system by linking to dCas9 or sgRNA. CRISPR trans-activators or CAS trans-activators are known in the art and readily recognized by those skilled in the art.

[0233] Recombinase recognition sequence (RRS)

[0234] As used herein, “recombinase” is a site-specific enzyme that recognizes short DNA sequences, referred to herein as recombinase recognition sequences or RRS, which are typically between about 30 base pairs (bp) and 40 bp and mediate recombination between these recombinase recognition sequences, causing excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences.

[0235] Exemplary recombinases include, but are not limited to: And HbiF.

[0236] Exemplary recombinase recognition sequences (RRS) include, but are not limited to: And B2RT.

[0237] Based on their different biochemical properties, recombinases can be divided into two distinct families: serine recombinases (e.g., dissociative enzymes and invertases) and tyrosine recombinases (e.g., integrases). Serine and tyrosine recombinases are further classified into bidirectional and unidirectional recombinases. Examples of bidirectional serine recombinases include, but are not limited to: β-6, CinH, ParA, and γδ; examples of unidirectional serine recombinases include, but are not limited to: Bxbl, φC31 (phiC31), TP901, TGI, φBTI, R4, cpRVl, cpFCl, MRU, A118, U153, and gp29. Examples of bidirectional tyrosine recombinases include, but are not limited to: Cre, FLP, and R; examples of unidirectional tyrosine recombinases include, but are not limited to: λ, HK101, HK022, and pSAM2. The names of serine and tyrosine recombinases derive from conserved nucleophilic amino acid residues that the recombinases utilize to attack DNA and are covalently linked to DNA during strand exchange. Recombinases have been used for many standard biological purposes, including creating gene knockouts and solving classification problems.

[0238] In some embodiments, the recombinase used in this invention is an orthogonal recombinase. When the first recombinase and the second recombinase are orthogonal, it means that the second recombinase does not recognize the RRS that is specific to the first recombinase, and the first recombinase does not recognize the RRS that is specific to the second recombinase.

[0239] The outcome of recombination depends in part on the position and orientation of the two short, repetitive DNA sequences (e.g., RRS) to be recombinated, which are typically less than 30 bp in length. Site-specific recombinases bind to these repetitive sequences, which are specific to each recombinase and are referred to herein as “recombinase recognition sequences” or “recombinase recognition sites.” Thus, as used herein, a recombinase is “specific” to a recombinase recognition site when it can mediate an inversion or excision between repetitive DNA sequences. As used herein, a recombinase may also be referred to as recognizing its “cognate recombinase recognition site,” which is flanked by an intervening genetic element (e.g., a promoter, terminator, or target gene). When a genetic element is located between and adjacent to two repetitive DNA sequences, the genetic element is said to be “flanked” by a recombinase recognition site. In some embodiments, recombinase recognition sites do not overlap. However, in other embodiments, recombinase recognition sites overlap as described below, which allows for a significant increase in the complexity of recombination.

[0240] Inversion recombination occurs between two short, inverted repeat DNA sequences. Unwilling to be bound by theory, DNA looping, assisted by DNA-bending proteins, brings the two repeat sequences together, at which point DNA cleavage and ligation occur. This reaction is ATP-independent and requires supercoiled DNA. The end result of this type of inversion recombination event is an inversion of the DNA between the repeat sites (i.e., the DNA reverses direction between the two RRSs), so that the coding strand is now the non-coding strand, and vice versa. In this type of reaction, DNA is conserved; there is no net increase or loss of DNA.

[0241] Conversely, excision (integration) recombination occurs between two short, repetitive DNA sequences oriented in the same direction. In this case, the intervening DNA is excised / removed. For example, an AND gate can be assembled by placing terminators between two different sets of recombinase sites oriented for excision, with a promoter and an output (e.g., a GFP coding sequence) flanked by them. In this example, both terminators must be excised via the input-dependent action of the recombinase to allow readthrough from the promoter to the GFP coding sequence. Therefore, two inputs are required to excise the two terminators to generate the output.

[0242] Recombinases can also be classified as irreversible or reversible. As used herein, an "irreversible recombinase" is one that catalyzes recombination between two complementary recombination sites, but cannot catalyze recombination between heterozygous sites formed by that recombination without the assistance of additional factors. Therefore, an "irreversible recognition site" is a recombinase recognition site that serves as the first of two DNA recognition sequences for an irreversible recombinase, and is modified to a heterozygous recognition site after recombination at that site. A "complementary irreversible recognition site" is a recombinase recognition site that serves as the second of two DNA recognition sequences for an irreversible recombinase, and is modified to a heterozygous recombination site after homologous recombination at that site. For example, attB and attP, described below, are irreversible recombination sites for the Bxbl and phiC31 recombinases—attB is a complementary irreversible recombination site of attP, and vice versa. Recently, it has been shown that mutating the attB / attP sites can create orthogonal B / P pairs that interact only with each other and not with other mutants.

[72] This allows a single recombinase to control the excision, integration, or inversion of multiple orthogonal B / P pairs.

[0243] For example, the phiC31 (φC31) integrase catalyzes only the attB × attP reaction in the absence of additional factors (not found in eukaryotic cells). Recombinases cannot mediate recombination between attL and attR heterozygous recombination sites formed during recombination between attB and attP. Because recombinases (e.g., phiC31 integrase) cannot catalyze the reverse reaction alone, phiC31atB × attP recombination is stable.

[0244] Irreversible recombinases and nucleic acids encoding irreversible recombinases are described in the art and can be obtained using conventional methods. Examples of irreversible recombinases include, but are not limited to: phiC31 (φC31) recombinase, *E. coli* phage P4 recombinase, *E. coli* phage λ integrase, *Listeria A118* phage recombinase and *Actinomyces* phage R4 Sre recombinase, HK101, HK022, pSAM2, Bxbl, TP901, TGI, φBTI, cpRVl, cpFCl, MRU, U153, and gp29. Conversely, a "reversible recombinase" is a recombinase that catalyzes recombination between two complementary recombinase recognition sites and, without the aid of additional factors, reverses recombination between sites formed by an initial recombination event. The product site generated by recombination is itself a substrate for subsequent recombination. Examples of reversible recombinase systems include, but are not limited to: the Cre-lox and Flp-frt systems, R, β-6, CinH, ParA, and γδ.

[0245] The recombinases described herein are not intended to be exclusive examples of recombinases that can be used in embodiments of the present invention. Other examples of recombinases useful in the invention described herein are known to those skilled in the art, and any new recombinases discovered or produced are expected to be usable in different embodiments of the present invention.

[0246] In some embodiments, the recombinase is a serine recombinase. Therefore, in some embodiments, the recombinase is considered irreversible. For some serine recombinases, the initial recombination event can be reversed in the presence of a recombinase directionality factor (RDF). RDFs are a diverse group of proteins involved in controlling the directionality of integrase-mediated site-specific recombination reactions. Typically, RDFs are small DNA-binding proteins that act as cofactors influencing the selection of substrates for recombination by their homologous recombinases. See Lewis and Hatfull, Nucleic Acids Res, 1 June 2001; 29(11): 2205-2216. For example, when recombination sites attB and attP are placed in an antiparallel orientation, the presence of the recombinase will stably invert the DNA sequence between the two sites and generate attL and attR sites (“BP reaction”). This inversion remains stable unless the RDF is also expressed with bxb1 or phiC, which will invert the sequence between attL and attR and regenerate attB and attP sites (“LR reaction”). Examples of RDFs include, but are not limited to, gp47 for bxb1, gp3 for phiC31, gp3 for PhiBT1, ORF7 for TP901-1, gp25 for TG1, and gp3 for PhiRv1.

[0247] In some embodiments, the recombinase is a tyrosine recombinase. Therefore, in some embodiments, the recombinase is considered reversible.

[0248] In some embodiments, all recombinases used in the AAV expression system described herein may be of the same type (e.g., serine or tyrosine). In some embodiments, tyrosine recombinases and serine recombinases may be used together in the same nucleic acid construct described herein.

[0249] In some embodiments, the recombinase comprises the sequence of Bxbl recombinase and the corresponding recombinase recognition sequences are Bxbl attB and Bxbl attP.

[0250] In some embodiments, the recombinase comprises the sequence of phiC31 (φC31) recombinase and the corresponding recombinase recognition sequence comprises phiC31 attB and phiC31 attP.

[0251] The recombinase can recognize multiple RRS pairs. In some embodiments, the recombinase contains a Cre sequence and the corresponding recombinase recognition sequence contains loxP. In some embodiments, the recombinase contains a Cre sequence and the corresponding recombinase recognition sequence contains lox2272. In some embodiments, the recombinase contains a Cre sequence and the corresponding recombinase recognition sequence contains loxN.

[0252] In some implementations, the recombinase contains the sequence of Dre and the corresponding recombinase recognition sequence contains rox.

[0253] In some embodiments, the recombinase contains the sequence VCr and the corresponding recombinase recognition sequence contains VloxP.

[0254] In some embodiments, the recombinase contains the sequence VCr and the corresponding recombinase recognition sequence contains VloxP.

[0255] In some embodiments, the recombinase contains the Flp sequence and the corresponding recombinase recognition sequence contains FRT.

[0256] In some embodiments, the recombinase contains the sequence of SCre and the corresponding recombinase recognition sequence contains SloxM1.

[0257] In some implementations, the recombinase contains the sequence of Vika and the corresponding recombinase recognition sequence contains vox.

[0258] In some embodiments, the recombinase contains the sequence B3 and the corresponding recombinase recognition sequence contains B3RT.

[0259] In some embodiments, the recombinase contains the sequence of KD and the corresponding recombinase recognition sequence contains KDRT.

[0260] The sequences of some recombinases are shown below:

[0261] hPGK (SEQ ID NO: 10):

[0262]

[0263]

[0264] EF1α (SEQ ID NO: 11):

[0265]

[0266] SFFV (SEQ ID NO: 12):

[0267]

[0268] CAG(SEQ ID NO:13):

[0269]

[0270]

[0271]

[0272] NLS-iCre(SEQ ID NO:14):

[0273]

[0274]

[0275] NLS-FlpO(SEQ ID NO:15):

[0276]

[0277] NLS-DreO(SEQ ID NO:16):

[0278]

[0279] NLS-SCre(SEQ ID NO:17):

[0280]

[0281]

[0282] NLS-VCre(SEQ ID NO:18):

[0283]

[0284]

[0285] NLS-VikaO(SEQ ID NO:19):

[0286]

[0287]

[0288] NLS-B3(SEQ ID NO:20):

[0289]

[0290]

[0291] NLS-KD (SEQ ID NO: 21):

[0292]

[0293]

[0294] NLS-B2 (SEQ ID NO: 22):

[0295]

[0296]

[0297] NLS-R (SEQ ID NO: 23):

[0298]

[0299]

[0300] NLS-PhiC31 (SEQ ID NO: 24):

[0301]

[0302]

[0303] NLS-bxb1 (SEQ ID NO: 25):

[0304]

[0305]

[0306] The sequences of some recombinase recognition sequences (RRS) are shown below:

[0307] loxP (SEQ ID NO: 26):

[0308]

[0309] lox2272 (SEQ ID NO: 27):

[0310]

[0311] loxN (SEQ ID NO: 28):

[0312]

[0313] FRT (SEQ ID NO: 29):

[0314]

[0315] F3(SEQ ID NO:30):

[0316]

[0317] F14(SEQ ID NO:31):

[0318]

[0319] Rox(SEQ ID NO:32):

[0320]

[0321] VloxP(SEQ ID NO:33):

[0322]

[0323] Vlox2272(SEQ ID NO:34):

[0324]

[0325] SloxP(SEQ ID NO:35):

[0326]

[0327] SloxM1(SEQ ID NO:36):

[0328]

[0329] Slox2272(SEQ ID NO:37):

[0330]

[0331] Vox(SEQ ID NO:38):

[0332]

[0333] B3RT(SEQ ID NO:39):

[0334]

[0335] KDRT(SEQ ID NO:40):

[0336]

[0337] B2RT(SEQ ID NO:41):

[0338]

[0339] RSRT (SEQ ID NO: 42):

[0340]

[0341] PhiC31 attB (SEQ ID NO: 43):

[0342]

[0343] PhiC31 attP (SEQ ID NO: 44):

[0344]

[0345] Bxb1 attB (SEQ ID NO: 45):

[0346]

[0347] Bxb1 attP (SEQ ID NO: 46):

[0348]

[0349] cell lines

[0350] This article provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein.

[0351] One aspect of this application provides a stable cell line for the production of recombinant viral vectors (e.g., AAV vector production), the stable cell line comprising at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous rep or pol gene encoding a rep protein or a polymerase protein, respectively. In one embodiment, the inducible promoter is further operatively linked to a heterologous cap or env gene encoding a cap protein or an env protein, respectively. Alternatively, in one embodiment, the stable cell line further comprises a second inducible promoter operatively linked to a heterologous cap or env gene encoding a cap protein or an env protein, wherein the second inducible promoter is induced by an inducer different from the first inducible promoter.

[0352] Another aspect of this application provides a stable cell line for the production of recombinant AAV vectors, the stable cell line comprising at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous cap gene encoding a cap protein.

[0353] Another aspect of this application provides a stable cell line for the production of recombinant AAV vectors, the stable cell line comprising at least one tunable promoter (e.g., an inducible promoter), wherein the inducible promoter is operatively linked to a heterologous helper gene encoding a helper gene product. Helper genes are commonly used in the production of AAV vectors. Exemplary helper genes commonly used in AAV production include E1 (E1A and E1B), E2A, E4, and VA RNA.

[0354] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-induced promoter, hypoxia-induced promoter, tetracycline-induced promoter, alcohol-induced promoter, steroid-induced promoter, RU486-induced promoter, ecdysone-induced promoter, rapamycin-induced promoter, metallothionein-induced promoter, hormone-induced promoter, and metal-induced promoter.

[0355] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one trichodinin-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0356] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one hypoxia-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0357] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one trichodinin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodinin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0358] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0359] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one trichodin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein.

[0360] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising at least one inducible promoter having a sequence having SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0361] Another aspect of this application provides a stable cell line for the production of a recombinant viral vector, the stable cell line comprising at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0362] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising: at least one inducible promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0363] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising: at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0364] Another aspect of this application provides a stable cell line for the production of recombinant viral vectors, the stable cell line comprising: at least one inducible promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0365] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-induced promoter, hypoxia-induced promoter, tetracycline-induced promoter, alcohol-induced promoter, steroid-induced promoter, RU486-induced promoter, ecdysone-induced promoter, rapamycin-induced promoter, metallothionein-induced promoter, hormone-induced promoter, and metal-induced promoter.

[0366] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one trichodinin-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0367] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one hypoxia-inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein.

[0368] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising: at least one trichodin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0369] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising: at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, and at least one inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0370] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one trichoin-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein and at least one hypoxia-inducible promoter operably linked to at least one heterologous gene encoding at least one toxic protein.

[0371] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence having SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0372] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0373] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising: at least one inducible promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0374] Another aspect of this application provides a stable cell line for rAAV production, the stable cell line comprising: at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter operatively linked to at least one heterologous gene encoding a toxic protein, wherein the at least one inducible promoter is selected from the group consisting of: trichodin-inducible promoter, hypoxia-inducible promoter, tetracycline-inducible promoter, alcohol-inducible promoter, steroid-inducible promoter, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, metallothionein-inducible promoter, hormone-inducible promoter, and metal-inducible promoter.

[0375] A stable cell line for rAAV production, the stable cell line comprising: at least one inducible promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein; and at least one inducible promoter having a sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 9, the inducible promoter being operatively linked to at least one heterologous gene encoding a toxic protein.

[0376] In one embodiment, the stable cell further comprises at least one repressive element operatively linked to at least one heterologous gene encoding a toxic protein.

[0377] In one embodiment, the stable cell line has at least two inducible promoters, and the at least two inducible promoters are identical; for example, the stable cell contains two trichodin promoters. In another embodiment, the stable cell line has at least two inducible promoters, and the at least two inducible promoters are different; for example, the stable cell contains a trichodin promoter and a hypoxia promoter.

[0378] The stable cell lines described herein may contain at least one, two, three, four, five, or more inducible promoters operatively linked to different toxic genes. Alternatively, an inducible promoter may be operatively linked to at least two, three, four, five, or more genes. For example, an inducible promoter may be operatively linked to at least two different toxic genes (e.g., rep and cap), such that induced expression (e.g., by contacting cells with an inducer used for the inducible promoter) will result in the expression of at least two different toxic genes. An inducible promoter may be operatively linked to at least two different accessory genes, such that induced expression (e.g., by contacting cells with an inducer used for the inducible promoter) will result in the expression of at least two different accessory genes. An inducible promoter may be operatively linked to at least one toxic protein and at least one different accessory gene, such that induced expression (e.g., by contacting cells with an inducer used for the inducible promoter) will result in the expression of at least one toxic protein and at least one different accessory gene.

[0379] In one embodiment, the cell contains at least two separate inducible promoters, wherein the at least two inducible promoters are induced by different inducers (e.g., by hypoxia and laurin), and the at least two inducible promoters are operatively linked to different heterologous genes encoding different toxic proteins. For example, the cell contains a first inducible promoter operatively linked to a first toxic gene and a second inducible promoter operatively linked to a second toxic gene.

[0380] In one embodiment, the cell contains at least two inducible promoters, wherein the at least two inducible promoters are induced by different inducers, or the at least two inducible promoters are induced by the same inducer. For example, the cell contains a first inducible promoter operatively linked to a first toxic gene and a second inducible promoter operatively linked to a second toxic gene.

[0381] In one embodiment, the cell comprises at least two inducible promoters, wherein the at least two inducible promoters are induced by different inducers, and each of the at least two inducible promoters is operatively linked to at least one different heterologous gene encoding at least one toxic protein. For example, the cell comprises a first inducible promoter operatively linked to a first toxic gene and a second toxic gene, and a second inducible promoter operatively linked to a third toxic gene.

[0382] In one embodiment, the stable cell line comprises at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein incorporated into the genome. In another embodiment, the stable cell line comprises at least one inducible promoter operatively linked to a heterologous gene encoding a toxic protein in a stable, free form (e.g., as a stable plasmid).

[0383] In one embodiment, the cell line for recombinant viral vector production (e.g., AAV vector production) containing at least one inducible promoter need not be a stable cell line. For example, a vector containing at least one inducible promoter can be transiently introduced (e.g., transiently transfected) into cells. One aspect of this application provides a cell line for recombinant viral vector production containing the transient introduction of at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous gene encoding a toxic protein. Another aspect of this application provides a cell line for rAAV vector production containing the transient introduction of at least one inducible promoter, wherein the inducible promoter is operatively linked to a heterologous gene (e.g., rep or cap) encoding a toxic protein required for rAAV production. Transient expression can be achieved, for example, by introducing a synthetic nucleic acid, expression vector, or plasmid for expressing at least one inducible promoter into cells, wherein the inducible promoter is operatively linked to a heterologous gene encoding a toxic protein. Such introduction may involve, for example, transformation, transfection, electroporation, or lipid transfection. Those skilled in the art can determine whether a cell has had a transient introduction of the synthetic nucleic acid, vector, or plasmid by, for example, using PCR-based assays or Western blotting, to assess the mRNA or protein levels of the synthetic nucleic acid, expression vector, or plasmid.

[0384] Another aspect provided in this article is a cell expressing a nucleic acid construct encoding a tetracycline-responsive transactivator protein that is operatively linked to a constitutive promoter.

[0385] Another aspect presented in this article is cells that express nucleic acid sequences encoding biomarker proteins.

[0386] Another aspect provided herein is a cell expressing a nucleic acid construct, which at least contains a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is operatively linked to a tunable promoter.

[0387] Another aspect provided herein is a cell expressing a nucleic acid construct comprising: a nucleic acid sequence encoding at least one accessory protein, wherein at least one accessory gene is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is under the control of a second regulated promoter or a zinc finger transcription activator (ZF-TA).

[0388] Another aspect provided herein is a cell expressing a nucleic acid construct comprising a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter.

[0389] Another aspect provided in this article is a cell expressing a nucleic acid construct encoding the Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a regulated promoter or zinc finger transcription activator (ZF-TA).

[0390] Another aspect provided herein is a cell expressing a nucleic acid construct comprising: a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a transcriptional activator, wherein the first and second regulated promoters are different. In one embodiment, the transcriptional activator is a zinc finger transcriptional activator (ZF-TA).

[0391] Another aspect provided herein is a cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding a zinc finger (ZF) transcription activator, the nucleic acid sequence encoding the zinc finger (ZF) transcription activator being operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter. In various embodiments, the zinc finger transcription activator (ZF-TA) is expressed by this construct.

[0392] Another aspect provided in this article is a cell expressing a nucleic acid construct containing a target site for binding zinc finger transcription activator (ZF-TA).

[0393] Another aspect provided in this article is a cell expressing a nucleic acid construct containing a Rep protein operatively linked to a promoter containing a target site for binding zinc finger transcription activator (ZF-TA).

[0394] Another aspect provided herein is a cell expressing the following nucleic acid construct: comprising: a nucleic acid sequence encoding a tetracycline-responsive transactivator protein operatively linked to a constitutive promoter; a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; and a nucleic acid sequence encoding, for example, a zinc finger (ZF) transcription activator operatively linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter or an E2-responsive promoter; and a nucleic acid construct containing a Rep protein operatively linked to a promoter, the promoter containing a target site for binding the zinc finger transcription activator (ZF-TA).

[0395] Another aspect provided herein is a cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding a toxic protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the toxic protein.

[0396] Another aspect provided herein is a cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and recombinase recognition sequences (RRS) located on the 5' and 3' sides of the nucleic acid sequence encoding the Cap protein, such that the RRS are adjacent to the nucleic acid sequence encoding the Cap protein.

[0397] Another aspect provided herein is a cell expressing a nucleic acid construct comprising: a first nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0398] Expression of the construct can be stable, i.e., integrated into the genome of a cell. One aspect of this document provides stable cells expressing at least one of the nucleic acid constructs described herein. In one embodiment, the stable cells express at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of the nucleic acid constructs described herein.

[0399] One aspect provided in this article is a stable cell expressing a nucleic acid construct, which contains at least one nucleic acid sequence encoding at least one accessory protein, wherein each nucleic acid construct is operatively linked to a tunable promoter.

[0400] Another aspect provided in this article is a stable cell expressing a nucleic acid construct encoding a tetracycline-responsive transactivator protein that is operatively linked to a constitutive promoter.

[0401] Another aspect presented in this article is stable cells that express nucleic acid sequences encoding biomarker proteins.

[0402] Another aspect provided in this article is a stable cell expressing a nucleic acid construct, which at least contains a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is operatively linked to a regulated promoter.

[0403] Another aspect provided in this article is a stable cell expressing a nucleic acid construct comprising: a nucleic acid sequence encoding at least one accessory protein, wherein at least one accessory gene is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is under the control of a second regulated promoter or a zinc finger transcription activator (ZF-TA).

[0404] Another aspect provided herein is a stable cell expressing a nucleic acid construct comprising a nucleic acid sequence containing at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter.

[0405] Another aspect presented in this article is a stable cell expressing a nucleic acid construct encoding the Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a regulated promoter or zinc finger transcription activator (ZF-TA).

[0406] Another aspect provided herein is a stable cell expressing a nucleic acid construct comprising: a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a transcription activator, wherein the first regulated promoter and the second regulated promoter are distinct.

[0407] Another aspect provided herein is a stable cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding a zinc finger (ZF) transcription activator operably linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter. In various embodiments, the zinc finger transcription activator (ZF-TA) is expressed from this construct.

[0408] Another aspect provided in this article is a stable cell expressing a nucleic acid construct containing a toxic protein operatively linked to a promoter containing a zinc finger transcription activator (ZF-TA).

[0409] Another aspect provided in this article is a stable cell expressing a nucleic acid construct containing a Rep protein operatively linked to a promoter containing a target site for binding transcription activators, such as zinc finger transcription activators (ZF-TA).

[0410] Another aspect provided herein is a stable cell expressing the following nucleic acid construct: comprising: a nucleic acid sequence operatively linked to a constitutive promoter encoding a tetracycline-responsive transactivator protein; a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; and a nucleic acid sequence operatively linked to an inducible promoter encoding a zinc finger (ZF) transcription activator, wherein the inducible promoter is an E4-responsive promoter or an E2-responsive promoter; and a nucleic acid construct comprising a Rep protein operatively linked to a promoter containing a target site for binding the zinc finger transcription activator (ZF-TA).

[0411] Another aspect provided in this article is a stable cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding a toxic protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the toxic protein.

[0412] Another aspect provided in this article is a stable cell expressing a nucleic acid construct comprising a nucleic acid sequence encoding the Cap protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the Cap protein.

[0413] Another aspect provided herein is a stable cell expressing a nucleic acid construct comprising: a first nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0414] The expression of the construct can be transient, i.e., not integrated into the cellular genome. One aspect of this document provides a cell having transient expression of at least one nucleic acid construct described herein. In one embodiment, the cell has transient expression of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of the nucleic acid constructs described herein.

[0415] One aspect provided herein is a cell with transient expression of nucleic acid constructs, said nucleic acid constructs comprising at least one nucleic acid sequence encoding at least one accessory protein, wherein each nucleic acid construct is operatively linked to a tunable promoter.

[0416] Another aspect provided in this article is the transient expression of a nucleic acid construct encoding a tetracycline-responsive transactivator protein that is operatively linked to a constitutive promoter in cells.

[0417] Another aspect presented in this article is the transient expression of nucleic acid sequences encoding marker proteins in cells.

[0418] Another aspect provided herein is a cell with transient expression of a nucleic acid construct, the nucleic acid construct containing at least a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is operatively linked to a tunable promoter.

[0419] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising: a nucleic acid sequence encoding at least one accessory protein, wherein at least one accessory gene is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a toxic protein, wherein the nucleic acid encoding the toxic protein is under the control of a second regulated promoter or a zinc finger transcription activator (ZF-TA).

[0420] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising a nucleic acid sequence containing at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter.

[0421] Another aspect provided in this article is a cell with transient expression of a nucleic acid construct encoding the Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a regulated promoter or zinc finger transcription activator (ZF-TA).

[0422] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising: a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a first regulated promoter; and a nucleic acid sequence encoding a Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a transcriptional activator, wherein the first regulated promoter and the second regulated promoter are distinct.

[0423] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising a nucleic acid sequence encoding a zinc finger (ZF) transcription activator operably linked to an inducible promoter, wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter. In various embodiments, the zinc finger transcription activator (ZF-TA) is expressed from this construct.

[0424] Another aspect provided in this article is a cell with transient expression of a nucleic acid construct containing a toxic protein operatively linked to a promoter containing a zinc finger transcription activator (ZF-TA).

[0425] Another aspect provided in this article is a cell with transient expression of a nucleic acid construct comprising a Rep protein operatively linked to a promoter containing a target site for binding transcription activators, such as zinc finger transcription activators (ZF-TA).

[0426] Another aspect provided herein is a cell with transient expression having the following nucleic acid construct: comprising: a nucleic acid sequence operatively linked to a constitutive promoter encoding a tetracycline-responsive transactivator protein; a nucleic acid sequence containing at least one of a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding a VA protein, wherein each nucleic acid sequence encoding any one of E4, E2A, and VA RNA is operatively linked to a regulated promoter; and a nucleic acid sequence operatively linked to an inducible promoter encoding a zinc finger (ZF) transcription activator, wherein the inducible promoter is an E4-responsive promoter or an E2-responsive promoter; and a nucleic acid construct comprising a Rep protein operatively linked to a promoter containing a target site for binding the zinc finger transcription activator (ZF-TA).

[0427] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising a nucleic acid sequence encoding a toxic protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the toxic protein.

[0428] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising a nucleic acid sequence encoding a Cap protein and a recombinase recognition sequence (RRS) located 3' to the side of the nucleic acid sequence encoding the Cap protein.

[0429] Another aspect provided herein is a cell with transient expression of a nucleic acid construct comprising: a first nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a first recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding a Rep protein, wherein the promoter is operatively linked to the nucleic acid encoding the Rep protein; and a second nucleic acid construct comprising, in a 5' to 3' orientation: a promoter, a termination nucleic acid sequence flanked by a second recombinase recognition sequence (RRS) pair, and a nucleic acid sequence encoding one or more of E2A, E4, and VA RNA, wherein the promoter is operatively linked to the nucleic acid encoding one or more of E2A, E4, and VA RNA.

[0430] If a cell expresses at least two nucleic acid constructs, the type of expression (i.e., stable or transient) for each of the at least two nucleic acid constructs can be the same or different. For example, if a cell expresses at least two nucleic acid constructs, the expression of both of the at least two nucleic acid constructs can be stable. If a cell expresses at least two nucleic acid constructs, the expression of both of the at least two nucleic acid constructs can be transient. Alternatively, if a cell expresses at least two nucleic acid constructs, the expression of at least one of the two nucleic acid constructs is stable (e.g., a cell can simultaneously have both stable expression of a nucleic acid construct and transient expression of a different nucleic acid construct).

[0431] Cell culture systems used for viral vector propagation (e.g., viral vector production cells) (including primary cells, semi-continuous cell lines, and continuous cell lines) can express any of the inducible promoters described herein to control the gene products required for viral vector propagation. Primary cell lines are derived from animal tissues (e.g., human, mouse, dog, monkey, etc.) and can be passaged once or twice to generate secondary cultures (i.e., subcultures of primary cultures), but only for a limited period. Secondary cultures are morphologically and virally susceptible to the primary culture. Semi-continuous cell lines (e.g., human diploid cells) are derived from fetal tissue and can be passaged approximately 50 times. Continuous cell lines are cancer cell lines or other immortalized cell lines that proliferate rapidly and can be cultured indefinitely. These cells can become heteroploid during continuous passage. Compared to primary and semi-continuous cell lines, continuous cell lines generally have a narrower range of viral susceptibility but are readily adapted for viral propagation.

[0432] Cell lines used for propagating viral vectors are known in the art, including but not limited to the exemplary cell lines presented in Table 2. In one embodiment, the cell line used for viral propagation is selected from Table 2; in another embodiment, the cell line used for viral propagation is derived from the cell lines selected from Table 2.

[0433]

[0434] In one embodiment, the cell line is a HEK293 cell line that has been modified to no longer be an adherent cell line. In one embodiment, the cell line is a suspension-grown HEK293 cell line. In one embodiment, the cell line is the Pro10 cell line. The Pro10 cell line is described, for example, in U.S. Patent No. 9,441,206, which is incorporated herein by reference.

[0435] This document further provides a method for generating any of the stable cell lines described herein, the method comprising: (a) transforming a cell population with at least one nucleic acid cassette or a nucleic acid described herein, the nucleic acid cassette comprising an inducible promoter operatively linked to a heterologous gene encoding a toxic protein; (b) culturing the cell population of (a) under conditions and for a time sufficient to allow expression of the nucleic acid cassette or construct; (c) selecting cells stably expressing the nucleic acid cassette; and (d) growing the cells of (c) to generate a cell line.

[0436] Techniques and methods for preparing stable cell lines are known in the art and readily recognized by those skilled in the art. In one embodiment, a vector containing a gene conferring antibiotic resistance to the cells is co-introduced into the parental cells. For example, the bsr, bls, or BSD gene confers resistance to blast fungicide; the Shble gene confers resistance to Zeocin™; the pac gene confers resistance to puromycin; the neo gene confers resistance to G418 (Geneticin); the hph gene confers resistance to hygromycin B; and the Shble gene confers resistance to humicin. For initial selection, cells expressing the antibiotic-conferring gene are cultured in the presence of antibiotics. For example, cells expressing the neo gene are cultured in a medium containing genimycin. After initial selection, surviving cells are isolated, cultured under conditions allowing nucleic acid cassette expression, harvested, and assayed to confirm cassette expression.

[0437] In one embodiment, the method further includes culturing cells under conditions and for a duration sufficient to induce the expression of at least one toxic protein (e.g., Rep, Cap) or accessory protein. Western blotting or other suitable assays can be used to assess the expression of the nucleic acid cassette or at least one toxic protein. Suitable induction of expression involves applying at least one inducer to the cells. The inducer is applied for a suitable period of time to induce expression from an inducible promoter. As discussed herein, the inducer can be a reagent applied to the cells or can be the conditions the cells are subjected to. Suitable inducers are discussed herein.

[0438] The clone that produces the nucleic acid cassette is amplified to produce an innovative, stable cell line (the term "cell line" is intended to include the progeny (subclones) of the original line). In one implementation, stability is confirmed by the ability to produce at least one toxic protein over at least about 12 months and more than about 50 generations. In one implementation, stability is determined by at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more, and by more than about 2 generations, 3 generations, 4 generations, 5 generations, 6 generations, 7 generations, 8 generations, 9 generations, 10 generations, 11 generations, 12 generations, 13 generations, 14 generations, 15 generations, 16 generations, 17 generations, 18 generations, 19 generations, 20 generations, 21 generations, 22 generations, 23 generations, 24 generations, 25 generations, 26 generations, 27 generations, 28 generations, 29 generations, 30 generations, 31 generations, 32 generations, 33 generations, 34 generations, 35 generations, 36 generations, 37 generations. Generations 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 The ability to produce at least one toxic protein is confirmed by generations 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more.

[0439] Regulatory transcription activators

[0440] The said or individual nucleic acid sequences encoding the Rep protein are under the control of a second regulated promoter or a regulated transcriptional activator. In any embodiment of any aspect, the second regulated promoter includes a binding site for the regulated transcriptional activator, and the second regulated promoter is operatively linked to the said or individual nucleic acid sequences encoding the said or individual Rep protein.

[0441] Exemplary transcriptional activators include homologous domain transcriptional activators, zinc finger transcriptional activators, winged helical (forkhead) transcriptional activators, leucine zipper transcriptional activators, and helical-loop-helical transcriptional activators. In one embodiment, the regulated transcriptional activator is a zinc finger transcriptional activator (ZF-TA). In any embodiment of any aspect, the second regulated promoter includes a binding site for the zinc finger transcriptional activator (ZF-TA).

[0442] In any embodiment of any aspect, the zinc finger transcription activator (ZF-TA) is expressed from a nucleic acid construct encoding a zinc finger (ZF) transcription activator operably linked to an inducible promoter, optionally wherein the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter.

[0443] In any embodiment of any aspect, the second regulated promoter includes a binding site for a zinc finger (ZF) transcription activator, and the second regulated promoter is operatively linked to the nucleic acid encoding the said or each Rep protein.

[0444] In any embodiment of any aspect, the zinc finger transcription activator (ZF-TA) is encoded by the following nucleic acid sequence:

[0445]

[0446]

[0447] In one implementation, the inducible promoter is an E4-responsive promoter, an E2-responsive promoter, or another helper gene-responsive promoter.

[0448] In one implementation, the binding site of the zinc finger transcription activator (ZF-TA) comprises the following nucleic acid sequence:

[0449] In one embodiment, a second tunable promoter containing a ZF-TA binding site (operably linked to the nucleic acid encoding the said or respective Rep protein) comprises the following nucleic acid sequence, wherein the ZF-TA binding site is indicated in bold:

[0450]

[0451]

[0452] tunable promoter

[0453] At least one regulated promoter is an inducible promoter. An inducible promoter can be a promoter induced by the presence of an inducer, the absence of a repressor, or any other suitable physical or chemical conditions that induce transcription by an inducible promoter. The terms “inducer”, “induction conditions”, etc., should be understood accordingly.

[0454] As a non-limiting example, the inducible promoter used in embodiments of the present invention may be a trichodinin-inducible promoter, a hypoxia-inducible promoter, a small molecule-inducible promoter, a tetracycline-regulated (e.g., inducible or repressive) promoter, an alcohol-inducible promoter, a steroid-inducible promoter, a mifepristone (RU486)-inducible promoter, a ecdysone-inducible promoter, a rapamycin-inducible promoter, a metallothionein-inducible promoter, a hormone-inducible promoter, a cumate-inducible promoter, a temperature-inducible promoter, a pH-inducible promoter, and a metal-inducible promoter. In one embodiment, the inducible promoter used in embodiments of the present invention is a trichodinin-inducible promoter or a hypoxia-inducible promoter.

[0455] As will be further discussed below, various suitable inducible promoters have been described in the art, and other suitable inducible promoters are discussed herein. Those skilled in the art will be able to select one or more suitable inducible promoters for use in various embodiments of the invention. Furthermore, based on the teachings herein or those existing in the prior art, those skilled in the art will be able to induce expression by the promoters as needed.

[0456] tunable introns

[0457] In some embodiments, the present invention may utilize at least one tunable intron. At least one tunable intron can be manipulated to control the expression of one or more proteins. At least one tunable intron may be used alone or in combination with one or more tunable promoters described herein.

[0458] Appropriately, regulated introns control expression during the translation phase. By combining the regulated promoters (e.g., inducible promoters) of the present invention with regulated introns, dual-level expression (i.e., control at both the transcriptional and translational levels) can be achieved. This allows for very tight control of gene expression, for example, to avoid any expression “leakage” (i.e., expression in unintended locations, such as tissues or organs).

[0459] In one embodiment, the tunable intron is an intron containing a resectable sequence that can be spliced ​​from a transcript produced from a nucleic acid sequence via the cell's unfolded protein response (UPR) system to produce a transcript encoding a functional protein. The unfolded protein response (UPR) is a cellular response mechanism to endoplasmic reticulum stress that is highly conserved in all eukaryotes.

[0460] In one embodiment, the nucleic acid construct of the present invention may comprise a sequence encoding a tunable intron, suitably a nucleic acid sequence encoding a tunable intron. In one embodiment, the nucleic acid sequence of the present invention encoding a protein may comprise a sequence encoding a tunable intron. In one embodiment, the nucleic acid construct of the present invention may comprise a nucleic acid sequence encoding a protein, wherein the nucleic acid sequence encoding the protein comprises a sequence encoding a tunable intron.

[0461] The nucleic acid sequence encoding a modulotropic intron may be present in any nucleic acid sequence encoding any protein described herein. In one embodiment, a nucleic acid sequence encoding any of the following may include a sequence encoding a modulotropic intron: E4 protein, E2A protein, Rep protein, and Cap protein. In one embodiment, a nucleic acid sequence encoding a Rep protein may include a sequence encoding a modulotropic intron. In one embodiment, a nucleic acid sequence encoding a Rep protein is provided, wherein the nucleic acid sequence encoding the Rep protein includes a sequence encoding a modulotropic intron.

[0462] In one embodiment, the cell of the present invention may comprise a nucleic acid sequence or nucleic acid construct containing a sequence encoding a tunable intron as described above. In one embodiment, the cell is a stable cell as described herein.

[0463] In one embodiment, the method for producing viral particles according to the present invention may include providing and culturing cells containing a nucleic acid sequence or nucleic acid construct comprising a sequence encoding a tunable intron as described above. In one embodiment, the method further includes the step of treating the cells to induce UPR, thereby inducing the splicing of a resectable sequence from the tunable intron. In one embodiment, treating the cells to induce UPR includes applying stress to the cells. For example, by administering chemical agents to the cells (e.g., trichodin, dithiothreitol (DTT), tunicamycin, carotenoids, saturated fatty acids, agents capable of downregulating the activity of stearoyl-CoA desaturase) or by applying hypoxia, carbohydrate deprivation, etc. to the cells.

[0464] Advantageously, due to the presence of regulated introns, unspliced ​​transcripts generated from nucleic acid sequences encoding proteins encode truncated or otherwise defective forms of proteins. However, when the transcripts are processed in the cell via the UPR mechanism, the excisable sequences of the introns are spliced ​​out, and functional proteins can be generated from the transcripts. Therefore, regulated intron splicing produces functional mRNAs encoding functional protein expression products.

[0465] In one implementation, the tunable intron can be spliced ​​from an IRE1 protein already present in the cell or its homologs or orthologs (IRE1 homologs or orthologs are present in all eukaryotes, including fungi, plants and mammals).

[0466] In some implementations, the tunable intron is the XBP1 intron, the Hac1 intron, the bZIP60 intron, or a homolog thereof. This means that the intron can be the wild-type form of the XBP1, Hac1, or bZIP60 intron or a naturally occurring homolog thereof.

[0467] In one embodiment, the tunable intron comprises the sequence CNG / CNG-Xn-CNG / CNG, where Xn represents a sequence of length n bases, / represents a cleavage site, and the sequence CNG-Xn-CNG is excised from the transcript. Thus, in other words, the tunable intron appropriately comprises a central sequence (Xn) flanked by two splice site target sequences, each having the sequence CNG / CNG, where / represents a cleavage site.

[0468] CNG / CNG is a shared splice site sequence in eukaryotic cells that is targeted in a highly conserved manner by the UPR system. As is known in the art, when the UPR response is induced, the IRE1 protein (whose homologs or orthologs are present in all eukaryotes, including fungi, plants, and mammals) targets this shared splice site sequence.

[0469] In various embodiments, the tunable intron or Xn can be 10 to 500 nucleotides in length, 15 to 350 nucleotides in length, 15 to 100 nucleotides in length, 15 to 35 nucleotides in length, or 20 to 25 nucleotides in length. Therefore, the excisable sequence of the tunable intron appropriately has a length of 16 to 506 nucleotides, 21 to 356 nucleotides, 21 to 106 nucleotides, 21 to 41 nucleotides, or 26 to 31 nucleotides.

[0470] There is considerable freedom for a specific sequence of Xn. Examples are listed below, but many other variants can certainly be used, provided that the tunable introns retain their function (i.e., they are spliced ​​from the transcript at an appropriate level by the UPR system).

[0471] In some embodiments, the tunable intron comprises the sequence CNG / CNG-Xn-CNG / CNG[CG] (SEQ ID NO: 50), wherein Xn represents a sequence of length n nucleotides, and / represents a cleavage site such that the excisable sequence CNG-Xn-CNG (SEQ ID NO: 51) is excised from the transcript after splicing, and wherein the nucleotide at the 5' end of sequence Xn is C or G.

[0472] In some implementations, Xn comprises or consists of the following sequences: (SEQ ID NO: 52), or a sequence that is at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95%, 96%, 97%, 98% or 99% identical.

[0473] In some implementations, Xn comprises one or consists of one of the following sequences:

[0474] as well as

[0475] .

[0476] In other embodiments, Xn comprises or consists of the following sequences: (SEQ ID NO: 58), or a sequence that is at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95%, 96%, 97%, 98% or 99% identical.

[0477] In one implementation, the tunable intron comprises or is composed of the following sequences: (SEQ ID NO: 59), or a sequence that is at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, at least 95%, 96%, 97%, 98%, or 99% identical, wherein / denotes a cleavage site. In variant sequences according to the above sequence identity levels, the splice site target sequence may remain CNG / CNGC, while sequence variations occur in other regions.

[0478] In one implementation, the tunable intron comprises or is composed of the following sequences: (SEQ ID NO: 60), or a sequence that is at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, at least 95%, 96%, 97%, 98%, or 99% identical, wherein / denotes a cleavage site. In variant sequences according to the above sequence identity levels, the splice site target sequence may remain CAG / CUGC, while sequence variations occur in other regions.

[0479] In some implementations, the tunable intron comprises or consists of one of the following sequences:

[0480] as well as

[0481] .

[0482] In other embodiments, the tunable intron comprises or consists of one of the following sequences:

[0483] as well as

[0484] .

[0485] In another embodiment, the intron-containing sequence can be regulated. (SEQ ID NO: 71) or (SEQ ID NO: 72), where / denotes a cleavage site. This sequence was generated by adding the trinucleotide CUG to the mammalian XBP1 intron sequence. It is believed that the addition of this trinucleotide slightly de-optimizes intron splicing to reduce any unwanted splicing in the cell (and thus reduce background expression of the expressed product).

[0486] Therefore, in some implementations, Xn includes (SEQ ID NO: 73) or composed of it.

[0487] In another embodiment, the intron-containing sequence can be tunable: (SEQ ID NO: 74), or a sequence that is at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, at least 95%, 96%, 97%, 98%, or 99% identical, wherein / denotes a cleavage site. In variant sequences according to the above sequence identity levels, the splice site target sequence may remain CNG / CNG, while sequence variations occur in other regions.

[0488] In some embodiments, the splice site target sequence (i.e., the CNG / CNG-containing sequence) in the transcript may be side-joined with sequences capable of interacting to form a stem-loop structure. Therefore, the splice site target sequence is preferably side-joined with complementary sequences such that they hybridize to form a stem-loop structure, wherein the splice site target sequence is at least partially or entirely located within the loop region of the stem-loop structure formed in the transcript.

[0489] In some embodiments, when a stem-loop structure is to be formed, the stem-loop structure formed from the transcript preferably comprises a loop containing 6 to 9 nucleotides and a stem of 3 to 10 nucleotides in length. In some embodiments, the stem-loop structure comprises a loop containing 7 to 8 nucleotides and a stem of 4 to 8 nucleotides in length.

[0490] In some implementations, when a stem-loop structure is to be formed, the intron may appropriately contain the following sequence at the splicing target site:

[0491] -Yn-CNG / CNG-A-Zn-

[0492] Wherein, A is a sequence having a length of 0 to 3 nucleotides, and in some embodiments, 1 or 2 nucleotides.

[0493] Where / represents the cutting site,

[0494] Furthermore, Yn and Zn represent sequences that are complementary in the nucleotide sequence when read in opposite directions, and thus can hybridize to form the stem of a stem-loop structure. Yn and Zn are preferably 3 to 10 nucleotides in length, and in some embodiments are 4 to 8 nucleotides in length.

[0495] In some implementations, the intron may suitably contain the following sequence at the splicing target site:

[0496] -Zn-CNG / CNG[CG]-A-Yn-, wherein the components have the same meaning as above. In this case, A preferably has a length of 0, 1, or 2 nucleotides.

[0497] It is evident that providing suitable complementary sequences (such as Yn and Zn in the above structure) to provide a stem structure can be achieved by adjusting the sequence of introns to provide suitable regions that are complementary to the corresponding sequences in adjacent coding (i.e., exon) sequences.

[0498] Further information regarding tunable introns that can be used in this invention can be found in PCT / GB2018 / 052387, which is incorporated herein by reference.

[0499] Hypoxia-inducible promoters:

[0500] In some embodiments, the hypoxia-inducible promoter is a synthetic hypoxia-inducible promoter. In some embodiments, the synthetic hypoxia-inducible promoter includes at least one hypoxia-responsive element (HRE) capable of being bound to and activated by hypoxia-inducible factor (HIF).

[0501] HIFs are a family of transcription factors activated by a decrease in cellular oxygen levels. Under normal oxygen conditions, HIFs degrade after hydroxylation. Hypoxic conditions stabilize HIFs and prevent their degradation. This allows HIFs to translocate to the nucleus, bind to HREs, and activate HRE-responsive genes.

[0502] Hypoxia-inducible promoters typically contain an HRE that can be activated by HIF binding operably linked to the minimal promoter. However, in some cases, the HRE that can be activated by HIF binding is operably linked to a promoter other than the minimal promoter (e.g., a proximal promoter, such as a tissue-specific proximal promoter). The specific promoter associated with the HRE can be selected depending on the situation, but the minimal promoter is generally preferred, especially when minimizing background expression levels is desired.

[0503] HREs are typically composed of polymers of short, conserved sequences called HIF binding sites (HBSs). As the name suggests, HIF binds to HBSs, thus activating the HRE to drive transcription. Therefore, the HRE of the present invention contains multiple HBSs, preferably three or more, more preferably three to ten, more preferably three to eight, and even more preferably four to eight. In some preferred embodiments of the present invention, the HRE contains five, six, or eight HBSs.

[0504] The core common sequence of HBS has been identified. The core common sequence is NCGTG (SEQ ID NO: 75, where N represents any nucleotide). It has been shown that A or G is optimal in the first position, therefore the generally preferred common sequence is [AG]CGTG (SEQ ID NO: 76). It should be noted that HBS is functional when it is present in either strand of the double-stranded DNA (i.e., in either orientation). Thus, for example, HBS can be represented by the reverse complementary common sequence CACG[CT] (SEQ ID NO: 77) in one strand, indicating the presence of the sequence [AG]CGTG (SEQ ID NO: 76) on the corresponding complementary strand (in this case, HBS can be described as being in "reverse orientation" or "opposite orientation").

[0505] Each HBS included in the HRE preferably contains a shared sequence NCGTG (SEQ ID NO: 75) and an optional shared sequence [AG]CGTG (SEQ ID NO: 76). Additional sequences sidebanding the shared sequence may exist, and these additional sequences may have some influence on the affinity of HIF for the HBS. Preferred HBSs for some embodiments of the present invention are discussed below.

[0506] Adjacent HBSs are typically, but not always, separated by spacer sequences. The spacing between HBSs in an HRE can have a significant impact on promoter inducibility and / or overall control. In some cases, it may be desirable to optimize the spacing between adjacent HBSs to maximize promoter inducibility and control. In other cases, it may be desirable to use suboptimal spacings to provide promoters with lower promoter inducibility and / or control. Specific spacings between HBSs present in preferred embodiments of the invention will be discussed below. However, in general, it is typically preferred that the spacing between adjacent core common sequences in adjacent HBSs be 3 to 50 nucleotides. For high levels of expression, it is typically preferred that the spacing between adjacent core common sequences in adjacent HBSs be 7 to 25 nucleotides, preferably about 8 to 22 nucleotides. For intermediate levels of expression, it is typically preferred that the spacing between adjacent core common sequences in adjacent HBSs be 5 to 6 nucleotides or 26 to 32 nucleotides. For low levels of expression, it is typically preferred that the spacing between adjacent core common sequences in adjacent HBSs be 2 to 4 nucleotides or 33 to 50 nucleotides. It should be understood that there is a range of possibilities for changing the interval between adjacent HBSs and thereby adjusting the characteristics of HRE.

[0507] HREs are typically spaced apart from promoters (e.g., minimal promoters), but not necessarily. This spacing can affect the inducibility and / or overall control of the promoter. Generally, a spacing of 0 to 200 nucleotides is preferred between the core common sequence in the final HBS (i.e., the sequence closest to the minimal promoter) and the TATA box of the minimal promoter (or an equivalent sequence if the TATA box is absent), more preferably 10 to 100 nucleotides, even more preferably 20 to 70 nucleotides, even more preferably 20 to 50 nucleotides, and even more preferably 20 to 30 nucleotides. For high levels of expression, a spacing of 20-30 nucleotides is generally preferred between the final HBS and the TATA box of the minimal promoter (or an equivalent sequence if the TATA box is absent); spacings significantly higher or lower than this result in weaker expression levels. It should be understood that there is a range within which the spacing between the final HBS and the MP can be varied, thereby adjusting the characteristics of the HRE.

[0508] In some embodiments, the HRE capable of binding to and being activated by HIF comprises at least one HBS, said HBS comprising or consisting of an HRE1 sequence. The HRE1 HBS sequence is ACGTGC (SEQ ID NO: 78). Of course, HRE1 can be present on either strand of the nucleic acid, so in such cases, the reverse HRE1 will be represented by the presence of the reverse complementary sequence GCACGT (SEQ ID NO: 79).

[0509] In some embodiments, all HBSs present in the HRE contain or consist of HRE1 sequences. The HRE1 sequences present in the HRE can each exist independently in either orientation. In some embodiments, it is preferred that all HRE1 sequences present in the HRE are in the same orientation.

[0510] In some embodiments, the HRE capable of being bound and activated by HIF comprises at least one HBS, said HBS comprising or consisting of the HRE2 sequence. The sequence of HRE2 is CTGACGTA (SEQ ID NO: 80). In HRE2, the HBS is present in the reverse direction compared to HRE1, therefore the HRE2 sequence comprises the reverse complementary sequence of the HRE1 sequence. HRE2 can be present on either strand of the nucleic acid, therefore in such cases, the reverse-directed HRE2 can be represented by the presence of the reverse complementary sequence TACGTGCAG (SEQ ID NO: 81).

[0511] The HRE2 sequence contains additional side sequences and is considered an optimized HBS, which binds to HIF more strongly than HRE1. Therefore, HRE2 can be considered superior to HRE1 when high levels of promoter inducibility and control are desired.

[0512] In some implementations, all HBSs present in the HRE contain or consist of HRE2 sequences. Since the HRE2 sequence effectively contains the HRE1 sequence, it is obvious that when HRE2 is provided, HRE1 will inevitably also be present. The HRE2 sequences present in the HRE can each exist independently in either direction. In some implementations, it is preferred that all HRE2 sequences present in the HRE are in the same direction.

[0513] In some implementations, the HRE that can be bound and activated by HIF includes at least one HBS, which contains an HRE3 sequence or a functional variant thereof, or is composed of an HRE3 sequence or a functional variant thereof.

[0514] The HRE3 sequence is (SEQ ID NO: 82, HBS is underlined). HRE3 represents a composite HBS containing two separate HBSs (i.e., HIF binding sites, underlined) separated by spacer regions, with additional spacer regions at each end. It can be seen that HRE3 contains one HBS in each direction (one containing HRE1 and one containing HRE2, with the HRE1 sequence located on the 5' side relative to the HRE2 sequence). Given that each HRE3 sequence contains two separate HBSs, for the purposes of this invention, each HRE3 sequence or its functional variant contributes two separate HBSs to the total number of HBSs present in the HRE.

[0515] HRE3 or its functional variants can be present on either strand of a nucleic acid; therefore, in such cases, the reverse HRE3 can be represented by the presence of the following reverse complementary sequence: (SEQ ID NO: 83).

[0516] As mentioned above, functional variants of HRE3 also form embodiments of the present invention. Such variants are functional if they retain the ability to be activated by HIF binding. Preferred functional variants of HRE3 retain the same HBS as HRE3 in substantially the same position and orientation, but contain different spacer region sequences. Therefore, in some preferred embodiments, functional variants of HRE3 appropriately have the following sequence:

[0517] (SEQ ID NO: 84);

[0518] Wherein, S1 is an interval region with a length of 8-10, preferably 9,

[0519] S2 is a spacing region with a length of 4-6, preferably 5.

[0520] S3 is an interval with a length of 1 to 3, preferably 2.

[0521] In some embodiments of the present invention, functional variants of HRE3 include sequences (SEQ ID NO: 85).

[0522] In some embodiments, the functional variants of HRE3 have at least 80%, preferably at least 90%, more preferably at least 95% of the same total sequence identity to HRE3 as HRE3, and wherein the HBS sequence is exactly the same as HRE3.

[0523] HRE3 is considered a particularly optimized sequence that binds strongly to HIF. Therefore, the presence of HRE3 or a functional variant thereof that maintains similar properties is preferred when high levels of promoter induction and control are desired.

[0524] In some embodiments, all HBSs present in the HRE contain or consist of HRE3 sequences or functional variants thereof. The HRE3 sequences or functional variants present in the HRE may each exist independently in either orientation. In some embodiments, it is preferred that all HRE3 sequences or functional variants present in the HRE are in the same orientation.

[0525] In some implementations, the HRE may include a combination of two or more of HRE1, HRE2 and / or HRE3.

[0526] In some implementations, the HREs that can be bound to and activated by HIF appropriately include the following sequences:

[0527] (SEQ ID NO: 86);

[0528] Where S is the interval region, and n is 2 to 9, preferably 3 to 7. It should be noted that the sequence of the interval regions can be changed; that is, the individual repeating units [ACGTGC-S] can be modified. n The spacer region in (SEQ ID NO: 87) may or may not have the same sequence or length.

[0529] The length of the spacer can be varied depending on the desired inductive and controllable force of the promoter.

[0530] Therefore, in embodiments where maximizing promoter inducibility and control is desired, a spacer region is provided such that the spacing between the core common sequences in adjacent HBSs is 7 to 18 nucleotides, preferably about 8 to 12 nucleotides, more preferably about 10 nucleotides. While maximizing promoter inducibility and control is generally desired, in some cases, lower levels of inducibility and control may be desired. In embodiments where lower levels of inducibility and control are desired, a spacer region can be provided such that adjacent HBSs are separated by fewer or more amounts (e.g., 4 to 6 nucleotides or 19 to 50 nucleotides). It is evident that the HRE1 HBS contains a nucleotide flanking the core common sequence (underlined -ACGTG). C (SEQ ID NO: 78), therefore the spacing regions in these embodiments take this into account to provide the desired spacing.

[0531] In some implementations, the HREs that can be bound to and activated by HIF appropriately include the following sequences:

[0532] (SEQ ID NO: 88)

[0533] Where S is the interval region.

[0534] The appropriate length of the spacer regions has been discussed above. In some embodiments of the invention, each spacer region has a length of 30-50 nucleotides. In this case, an exemplary but non-limiting spacer region has the following sequence:

[0535] (SEQ ID NO: 89).

[0536] In one implementation, the HREs that can be bound and activated by HIF appropriately include the following sequences:

[0537] (SEQ ID NO: 90, HBS is underlined) or a functional variant that is at least 80% identical, preferably at least 85%, 90%, 95% or 99% identical. Typically, in such functional variants, the HRE1 sequence is substantially identical or completely identical to the reference sequence, and substantially all sequence variations occur in the spacer region sequence.

[0538] This type of HRE typically exhibits very low levels of inducibility and low expression upon induction. This can be desirable when background expression needs to be minimized and high levels of expression are not required during induction. Of course, optimized HBS intervals can produce higher levels of inducibility and post-induction expression.

[0539] In some preferred embodiments, the HREs that can be bound and activated by HIF appropriately include the following sequences:

[0540] (SEQ ID NO: 91);

[0541] Where S is an optional interval region, and n is 2 to 9, preferably 3 to 7.

[0542] It should be noted that the sequence of the spacer region (if it exists) can be changed; that is, the individual repeating units [CTGCACGTA-S] can be modified. n The spacer region in (SEQ ID NO: 92) may or may not have the same sequence or length.

[0543] The details of suitable spacing between core common sequences in adjacent HBSs have been discussed above for the aforementioned embodiments, and these considerations apply equally to these embodiments. It is evident that the HRE2 HBS contains four nucleotides flanking the core common sequence (underlined - CTG CACGT A (SEQ ID NO: 80), therefore the spacing regions in these embodiments take this into account to provide the desired spacing.

[0544] In some embodiments of the present invention, the HREs that can be bound and activated by HIF appropriately include the following sequences:

[0545] (SEQ ID NO: 93); where S is the spacer region. The appropriate length of the spacer region was discussed above.

[0546] In some embodiments of the invention, each spacer region has a length of 20 nucleotides. In this case, an exemplary but non-limiting spacer region has the following sequence: (SEQ ID NO: 94).

[0547] In one embodiment of the invention, the HREs that can be bound and activated by HIF appropriately include the following sequences:

[0548] (SEQ ID NO: 95, HBS is underlined) or a functional variant containing at least 80% identical, preferably at least 85%, 90%, 95%, or 99% identical, sequence. Typically, in such functional variants, the HRE2 sequence is substantially or completely identical to the reference sequence, and substantially all sequence variations occur in the spacer region sequence. Such HREs typically exhibit moderate levels of inducibility and low expression levels upon induction. This can be desirable when background expression is minimized and a moderate level of expression is required upon induction. Of course, further optimization of the HBS spacer can yield higher levels of inducibility and post-induction expression. Similarly, de-optimization can yield lower levels of inducibility and post-induction expression.

[0549] In some implementations, the HREs that can be bound to and activated by HIF appropriately include the following sequences:

[0550] (SEQ ID NO: 96, HBS is underlined) or a functional variant containing at least 80% identical, preferably at least 85%, 90%, 95% or 99% identical, sequence.

[0551] It can be seen that the HRE does not contain additional spacer regions between adjacent HRE2 elements. However, given the four side nucleotides surrounding the core common sequence of HRE2, the core common sequence has an effective spacer of 4 nucleotides.

[0552] This type of HRE typically exhibits moderate inducibility and low expression levels upon induction. This is desirable when background expression is minimized and a moderate level of expression is required during induction. Of course, further optimization of the HBS interval can yield higher levels of inducibility and post-induction expression. Similarly, de-optimization can produce even lower levels of inducibility and post-induction expression.

[0553] In some preferred embodiments, the HRE capable of being bound and activated by HIF suitably comprises 3 to 6 HRE3 sequences, preferably 3 to 5, more preferably 4 HRE3 sequences or functional variants thereof, wherein adjacent HRE3 sequences or functional variants thereof are separated from each other by spacer regions having a length of 4 to 20 nucleotides, preferably 6 to 15 nucleotides, more preferably 9 nucleotides.

[0554] In a preferred embodiment, the HRE capable of being bound and activated by HIF appropriately comprises the following sequence:

[0555]

[0556] Where S is an optional interval region, and n is 2 to 5, preferably 2 to 4, and preferably 3.

[0557] It should be noted that the sequence of the intervals (if they exist) can be changed; that is, the individual repeating units can be altered. The spacer region in (SEQ ID NO: 98) may or may not have the same sequence or length. It should be understood that in some or all cases, the functional variant of the HRE3 sequence described above may be substituted.

[0558] The details of suitable spacing between core shared sequences in adjacent HBSs have been discussed above for the aforementioned embodiments, and these considerations apply equally to these embodiments. It is evident that the HBS of HRE3 composites includes side-mounted sequences containing two core shared sequences (underlined - The spacer region in these embodiments contains 11 nucleotides of the region (SEQ ID NO: 99), and therefore the spacer region S takes this into account to provide the desired spacing. In some embodiments, the spacer region S suitably has a length of 4 to 20 nucleotides, preferably 7 to 15 nucleotides, and more preferably 9 nucleotides.

[0559] In some embodiments of the present invention, the HREs that can be bound and activated by HIF appropriately include the following sequences:

[0560] (SEQ ID NO: 100); where S is the spacer region. The appropriate length of the spacer region has been discussed above. It should be understood that, in some or all cases, functional variants of the HRE3 sequence described herein may be substituted.

[0561] In some implementations, the HREs that can be bound to and activated by HIF appropriately include the following sequences:

[0562] SEQ ID NO: 101); where S is the spacer region. The appropriate length of the spacer region has been discussed above. It should be understood that in some or all cases, functional variants of the HRE3 sequence described herein may be substituted.

[0563] In some embodiments of the invention, each spacer region has a length of 9 nucleotides. In this case, an exemplary but non-limiting spacer region has the following sequence: GCGATTAAG (SEQ ID NO: 102).

[0564] In a preferred embodiment of the invention, the HRE capable of being bound and activated by HIF appropriately comprises the following sequence:

[0565] (SEQ ID NO: 103, HBS is underlined) or a functional variant containing at least 80% identical, preferably at least 85%, 90%, 95% or 99% identical, sequences. Typically, preferably in such functional variants, the HRE1 and HRE2 sequences present in the HRE3 sequence are substantially or identical to the reference sequence, and substantially all sequence variations occur in the spacer region sequence.

[0566] This type of HRE typically exhibits high levels of inducibility and high expression upon induction. This is desirable when high levels of expression are required during induction. Of course, further optimization of the HBS interval could potentially yield even higher levels of inducibility and post-induction expression. Similarly, de-optimization could result in lower levels of inducibility and post-induction expression.

[0567] As mentioned above, hypoxia-inducible promoters typically contain an HRE that can be bound to and activated by HIF, and the HRE is operatively linked to a minimal promoter or a proximal promoter. Preferably, the promoter operatively linked to the HRE is a minimal promoter.

[0568] The minimum promoter can be any suitable minimum promoter. A wide range of minimum promoters are known in the art. Without limitation, suitable minimum promoters include the CMV minimum promoter (CMV-MP), the YB-TATA minimum promoter (YB-TABA), the HSV thymidine kinase minimum promoter (MinTK), and the SV40 minimum promoter (SV40-MP). The minimum promoter can be a synthetic minimum promoter. Particularly preferred minimum promoters are the CMV minimum promoter (CMV-MP) and the YB-TATA minimum promoter (YB-TABA).

[0569] The CMV-MP sequence is:

[0570] (SEQ ID NO: 104).

[0571] The YB-TATA sequence is:

[0572] (SEQ ID NO: 105).

[0573] However, in some preferred embodiments of the invention, a longer sequence comprising YB-TATA MP is used. This longer sequence of YB-TATA MP (referred to herein as long YB-TATA) is... (SEQ ID NO: 106). Therefore, as long as YB-TATA is provided as a component of the inducible promoter described herein, a substantially equivalent sequence replacing YB-TATA with a long YB-TATA is also considered an embodiment of the invention. The space between the final HBS and the TATA cassette of MP is preferably preserved.

[0574] The MinTK sequence is:

[0575] CCCGCTTAA (SEQ ID NO: 107).

[0576] The sequence of SV40-MP is:

[0577] (SEQ ID NO: 108).

[0578] The sequence of MP1 is:

[0579] (SEQ ID NO: 109).

[0580] Therefore, preferred embodiments include an HRE capable of binding to and activating HIF, said HRE being operatively linked to one of the aforementioned minimal promoters, more preferably to CMV-MP or YB-TATA, and most preferably to CMV-MP. When combined with the HRE of the present invention, CMV-MP has shown to provide extremely high levels of inducibility and high promoter strength. Low background expression levels have also been observed.

[0581] The HRE is preferably separated from the minimum promoter (or other type of promoter, if used) by a spacer sequence. The space between the HRE and the minimum promoter affects the inducibility and control of the hypoxia-inducible promoter. Typically, the space between the core common sequence in the last HBS (i.e., the sequence closest to the minimum promoter) and the TATA box (or equivalent sequence, if the TATA box is absent) of the minimum promoter is preferably 10 to 100 nucleotides, more preferably 20 to 70 nucleotides, even more preferably 20 to 50 nucleotides, and even more preferably 20 to 30 nucleotides. In embodiments where optimization of the inducibility and control of the hypoxia-inducible promoter is desired, the space between the last HBS and the TATA box (or equivalent sequence, if the TATA box is absent) of the minimum promoter is preferably 20 to 30 nucleotides. In embodiments where a slightly lower level of inducibility and control is desired, the space between the last HBS and the TATA box (or equivalent sequence, if the TATA box is absent) can be smaller or larger, for example, 0 to 10 nucleotides or 31 to 100 nucleotides. While maximizing promoter induction and control is generally desirable, in some cases, lower levels of induction and control may be desired.

[0582] In an exemplary, non-limiting embodiment, the hypoxia-inducible promoter comprises one of the following sequences (HBS sequences are underlined, and the minimum promoter sequence is shown in bold):

[0583] (Synp-RTV-015; SEQ ID NO: 1), or a functional variant containing at least 80% identical, preferably at least 85%, 90%, 95%, or 99% identical, sequence thereof; and

[0584] HYP-001 (SEQ ID NO: 3), or a functional variant containing at least 80% identical, preferably at least 85%, 90%, 95% or 99% identical, sequence.

[0585] Typically, in such functional variants, the HRE1, HRE2, and MP sequences are substantially identical to the reference sequence, and virtually all sequence variations occur in the spacer region sequence.

[0586] In some preferred embodiments, after induction by hypoxia of the cells (e.g., after the cells were exposed to 5% oxygen for 5 hours, previously normoxic (e.g., exposed to 20% oxygen)), the expression level of the transgene increases by at least 5-fold, more preferably 10-fold, 15-fold, 20-fold, 30-fold, or 50-fold.

[0587] In some preferred embodiments, the expression level of the transgene after induction (e.g., after cells have been exposed to 5% oxygen for 5 hours, previously normoxic (e.g., exposed to 20% oxygen)) is at least 50% of the expression level provided by the CMV-IE promoter (i.e., another identical vector in the same cells under the same conditions, but in which the expression of the transgene is controlled by a CMV-IE rather than a hypoxia-inducible promoter). More preferably, the expression level of the transgene is at least 75%, 100%, 150%, 200%, 300%, 400%, or 500% of the expression level provided by the CMV-IE promoter.

[0588] Other naturally occurring hypoxia-inducible promoters are described in US20110158947A1, which is incorporated herein by reference. Therefore, in some embodiments, the hypoxia-inducible promoter may be selected from the group consisting of: adenosine A2B receptor (A2BR) promoter, plasminogen activator receptor (uPAR), VEGF receptor (VEGFR1 and VEGFR2) promoter, platelet-derived endothelial growth factor / thymidine phosphorylase (PDECGF / TP) promoter, nitric oxide synthase (NOS) promoter, phosphoglycerate kinase-1 (PGK-1) promoter, pyruvate kinase M (PK-M) promoter, glucose transporter 1 (GLUT1) promoter, hypoxia-inducible factor (HIF-1) promoter, early growth response factor 1 (Egr-1) promoter, nuclear factor kb (NFkB) promoter, and hepatocyte growth factor activator (HGF) promoter. A) Promoters, including vascular endothelial growth factor (VEGF) promoter, CXCL8 promoter, CCL11 promoter, transforming growth factor-β (TGF-β) promoter, procollagen promoter, integrin-linked kinase (ILK) promoter, K1PDC1 promoter, erythropoietin (EPO) promoter, serine / threonine kinase-15 (STK15) promoter, histone demethylase 1A containing a Jumonji domain (JMJD1A) promoter, endothelin-2 (EDN2) promoter, choline kinase (Chk) promoter, sphingosine kinase 1 promoter, carcinoembryonic antigen (CEA, ceacam5) promoter, monocyte chemoattractant protein 1 (MCP-1 / CCL2) promoter, and MCP-5 (Ccl1) promoter. 2) Promoters, prostate-specific antigen (PSA) promoter, c-Met promoter, matrix metalloproteinase class III β-tubulin (TUBB3) promoter, glutamine:fructose-6-phosphate aminotransferase (GFAT) promoter, protein phosphatase 1 nuclear targeting subunit β-secretase (BACE1) promoter, and plasminogen activator inhibitor 1 (PAI-1) promoter.

[0589] Other naturally occurring hypoxia-inducible promoters are described in WO2016 / 146819, which is incorporated herein by reference. See, for example, Table 4.

[0590] Hypoxia-responsive elements are described in L. Marignol, M. Lawler, M. Coffey & D. Hollywood (2005), Achieving hypoxia-inducible gene expression in tumors, Cancer Biology & Therapy, 4:4, 365-370; US 6218179; Madan et al., PNAS 90: 3928, 1993; JP2005095173A; US 2006 / 0099709; and WO1999 / 048916. Hypoxia-responsive elements in mice are disclosed in US5942434.

[0591] The induction of a hypoxia-inducible promoter can be achieved by hypoxia of the cells (i.e., treating the cell population to induce hypoxia in the cells), thereby inducing the expression of a transgene linked to the hypoxia-inducible promoter and producing an expression product. Suitable methods for any particular cell type will be apparent to those skilled in the art. Typically, eukaryotic cells are cultured under aerobic conditions, and many methods are known in the art to achieve this for various cell and culture types. Hypoxic conditions can be achieved by reducing the amount of oxygen supplied to the cells. For example, cells can be grown under normoxic conditions (e.g., approximately 20% oxygen) before switching to a gas mixture containing less or no oxygen to induce hypoxia. For example, a gas containing 5% oxygen can be used to induce hypoxia in the cells. An exemplary suitable gas mixture for inducing hypoxic conditions in cell culture is 5% oxygen, 10% carbon dioxide, and 85% nitrogen, but other gas mixtures may also be used. In an alternative approach, hypoxia in cell culture can be induced by introducing a reagent that can induce hypoxia in the cells. For example, CoCl2 (e.g., a final concentration of approximately 100 μM in the cell culture medium) can be used at a suitable concentration to induce hypoxia. Typically, in this invention, it is preferred to achieve hypoxia without adding such reagents, as this would increase costs, and in many cases the reagents are undesirable and may be difficult to remove.

[0592] In some cases, it may be desirable to alter the amount of oxygen supplied to cells when they are under hypoxic conditions to optimize or otherwise modulate the expression of a desired product. For example, it may be desirable to first establish a high-hypoxic condition to strongly induce hypoxia, and then culture the cells for a period of time under a lower-hypoxic condition that is less harmful to cell health and viability. Therefore, the methods disclosed herein may include altering the level of hypoxia experienced by the cells.

[0593] Expression of hypoxia-inducible promoters, as discussed in this article, can be modulated (e.g., uninduced, inhibited, or regulated) by altering the oxygen level to which the cell containing the promoter is exposed. For example, hypoxia-inducible expression can be shut down (uninduced) by exposing the cell to normoxic conditions (e.g., exposure to 20% oxygen).

[0594] In one embodiment, the synthetic hypoxia-inducible promoter does not contain or is not composed of one of the following structures:

[0595]

[0596] Here, S x This represents a spacer region of length X nucleotides.

[0597] In one implementation, the synthetic hypoxia-inducible promoter does not contain one of the following sequences or is not composed of one of the following sequences:

[0598]

[0599] Trichorein-inducible promoters:

[0600] In some implementations, the inducible promoter is the trichodin-inducible promoter.

[0601] In some embodiments, the tricholin-inducible promoter is a synthetic tricholin-inducible promoter. In some embodiments, the synthetic tricholin-inducible promoter includes a synthetic tricholin-inducible cis-regulatory element (CRE) capable of binding to CREB and / or AP1.

[0602] In one embodiment, the trichoin responsiveness enhancer element has a sequence comprising the following: (SEQ ID NO: 110).

[0603] In the sequence of SEQ ID NO: 107, cAMPRE is present. underlined text The text indicates that the AP-3 site is located at... Bold underline textThe sequence indicates that the spaces between the sites are neutral DNA. As can be seen from this sequence, the new enhancer element consists of 7 CRE sites and 6 AP-3 sites, with a 5 bp spacer between the elements. This enhancer is combined with the following minimal promoters, with a 5 bp spacer between the enhancer and the minimal promoter: YB-Tata (FORNYB-REP), CMV (FORNCMV), CMV53 (FORNCMV53), MinTK (FORNMinTK), MLP (FORNMLP), SV40 (FORNSV40), and pJB42 (FORNJB42). The sequences of the trichodin-responsive enhancer elements combined with the minimal promoters are provided herein, for example, in Example 7.

[0604] The enhancer element is further combined with the minimum promoter TATA-m6A. The minimum promoter TATA-m6A has a sequence including (SEQ ID NO: 111):

[0605] (SEQ ID NO: 111).

[0606] SEQ ID NO: 111 contains underlined text Highlighted common sequence TATA boxes and Bold and underlined text Book The m6a sequence is highlighted. The TATA box is the minimal sequence required to stabilize transcription derived from enhancers, while the m6A sequence is the signal used to methylate mRNA. This and other chemical modifications of mRNA (of which at least 160 are known) are thought to create another layer of posttranscriptional control during gene expression. Among these, m6a is the most well understood, and studies have shown it is involved in a wide range of mRNA functions, such as splicing, export, translation, and stability. Approximately one-quarter of all eukaryotic mRNAs have been observed to have at least one m6a site, making it the most prevalent form of mRNA modification (Han et al., 2020). In one such study on m6a methylation, it was shown that placing the methylation sequence at the 5' end of the mRNA (before the ATG) can enhance transcript translation (Meyer et al., 2015). We modified these findings by adding the m6a sequence to our TATA minimal promoter to improve translation efficiency derived from a weak but very small minimal promoter. This should allow us to achieve high expression and generate new minimal promoters while reducing the overall size of the promoter.

[0607] One aspect provided herein is a synthetic tuftin-inducible promoter comprising the sequence according to SEQ ID NO: 110 or a functional variant thereof.

[0608] In one embodiment, the synthetic tuftin-inducible promoter contains at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the same sequence as SEQ ID NO: 110.

[0609] In one embodiment, the functional variant of the synthetic tuftin-inducible promoter retains at least 25%, 50%, 75%, 80%, 85%, 80%, 95%, or 100% of the activity of the reference promoter.

[0610] In one implementation, a synthetic trichodinin-inducible promoter is operatively linked to a nucleic acid sequence to drive the expression of said nucleic acid sequence (e.g., the nucleic acid sequence encoding a toxic gene described herein). However, the nucleic acid need not be the nucleic acid sequence described herein; any sequence can be operatively linked to the synthetic trichodinin-inducible promoter.

[0611] Although the CRE / promoter is referred to as the trichodin-inducible type, it can also be induced by other agents, as discussed in more detail below. The trichodin-inducible mechanism involves the activation of adenylate cyclase, thereby increasing intracellular cAMP. Therefore, the CRE / promoter can also be induced by other activators of adenylate cyclase or factors that increase intracellular cAMP.

[0612] Preferably, the CRE contains at least two, more preferably at least three transcription factor binding sites (TFBSs) for CREB and / or AP1 (as used herein, the term "TFBS for X" means a TFBS that can be bound by transcription factor X).

[0613] Preferably, the CRE contains at least four TFBSs for CREB and / or AP1. Suitablely, the CRE contains three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen TFBSs for CREB and / or AP1.

[0614] While there is no specific upper limit on the number of TFBSs used for CREB and / or AP1, it is generally preferred that the CRE contains 15 or fewer TFBSs for CREB and / or AP1, and optionally 10 or fewer TFBSs for CREB and / or AP1.

[0615] In some implementations, the CRE includes at least one TFBS for each of CREB and AP1. In some implementations, the CRE includes at least two, three, four, five, six, or seven TFBS for each of CREB and AP1.

[0616] TFBS used for CREB typically contains or consists of the highly conserved consortium sequence TGACGTCA (SEQ ID NO: 112). This sequence is referred to as a cAMP responsive element (or cAMPRE or CRE; the abbreviation cAMPRE will be used herein to avoid confusion with the abbreviation for cis-regulatory elements). Other cAMPREs that may be used will be discussed below.

[0617] TFBS used for AP1 typically contains or consists of the common sequence TGA[GC]TCA (SEQ ID NO: 113). In specific embodiments of the present invention, sequences TGAGTCA (named AP1(1), SEQ ID NO: 114), TGACTCAG (named AP1(2), SEQ ID NO: 115), and TGACTCA (named AP1(3), SEQ ID NO: 116) are used, therefore AP1(1), AP1(3), and AP1(2) can be considered as preferred TFBSs for AP1. The general term AP1 for TFBS refers to a TFBS containing the above-mentioned common sequence, which covers AP1(1), AP1(3), and AP1(2).

[0618] In some preferred embodiments, the CRE contains at least one TFBS for transcription factors other than CREB and / or AP1. In some preferred embodiments of the invention, the CRE contains at least one TFBS for ATF6 and / or hypoxia-inducible factor (HIF). The CRE may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 TFBSs for transcription factors other than CREB and / or AP1 (e.g., for ATF6 and / or HIF). In some embodiments of the invention, the CRE contains at least one TFBS for each of ATF6 and HIF.

[0619] The TFBS for HIF comprises or consists of the concordant sequence NCGTG (SEQ ID NO: 75), more preferably comprises or consists of [AG]CGTG (SEQ ID NO: 76). This sequence is referred to as the HIF binding sequence (HBS). In a specific embodiment of the invention, the HBS sequence CTGCACGTA (named HRE1, SEQ ID NO: 80) is used, therefore HRE1 can be considered a preferred TFBS for HIF. However, other TFBSs for HIF are known and can be used in the invention, such as ACGTGC (SEQ ID NO: 78) or... (SEQ ID NO: 82).

[0620] The TFBS for ATF6 comprises or consists of the common sequence TGACGT (SEQ ID NO: 117), more preferably comprises or consists of TGACGTG (SEQ ID NO: 118). In a specific embodiment of the invention, the TFBS sequence TGACGTGCT (SEQ ID NO: 119) is used, and this sequence can be considered a preferred TFBS for ATF6. However, in general, any sequence comprising the common sequence TGACGT (SEQ ID NO: 117), more preferably TGACGTG (SEQ ID NO: 118), can be used.

[0621] Each of the TFBSs discussed above can exist in either direction (i.e., they can be functional when present on either strand of the double-stranded DNA). Therefore, it is evident that any of the TFBSs can be represented by a reverse complementary concordant sequence on one strand, indicating the presence of a TFBS sequence on the corresponding complementary strand (in such cases, the TFBS can be described as being in the "reverse direction" or "opposite direction"). Generally, whether referring to TFBSs by name or by listing their sequences, it should be considered as meaning that the TFBS exists in either direction. When listing TFBS sequences, it should be understood that the directions shown represent the specifically disclosed and generally preferred embodiments.

[0622] In some implementations, the CRE includes:

[0623] - 5 TFBS for CREB and 3 TFBS for AP1;

[0624] - 5 TFBS for CREB and 4 TFBS for AP1;

[0625] - 8 TFBS for AP1;

[0626] - 3 TFBS for ATF6, 4 TFBS for AP1, and 3 TFBS for HIF; or

[0627] - 7 TFBS for CREB and 6 TFBS for AP1;

[0628] The adjacent TFBSs are optionally, but preferably, separated by a sequence of interval regions.

[0629] The spacer sequence can be of any suitable length. Typically, spacer lengths are 2 to 100 nucleotides, 5 to 50 nucleotides, 6 to 40 nucleotides, 7 to 30 nucleotides, 8 to 25 nucleotides, or 10 to 20 nucleotides. In some embodiments of the invention, spacer lengths of 5, 10, and 20 nucleotides have been used and function well, but other lengths can also be used. In some embodiments, spacer lengths that are multiples of 5 nucleotides are preferred. Those skilled in the art can readily determine a suitable length for the spacer.

[0630] It should be noted that the sequence and length of the spacers can be changed; that is, the individual spacers in the sequence do not necessarily have the same sequence or length as any other spacers. For convenience, some or all of the spacers between TFBSs in a CRE do indeed have the same sequence and length, so while this may be preferred, it is not necessary.

[0631] TFBSs can be in any appropriate order, but in a preferred embodiment, they are provided in the order in which they are listed, i.e., in the first embodiment listed above, there are 4 TFBSs for cAMPRE in the direction from upstream to downstream, followed by 3 TFBSs for AP1.

[0632] In some implementations, the CRE consists of the following:

[0633] - 5 TFBS for CREB and 3 TFBS for AP1;

[0634] - 5 TFBS for CREB and 4 TFBS for AP1;

[0635] - 8 TFBS for AP1;

[0636] - 3 TFBS for ATF6, 4 TFBS for AP1, and 3 TFBS for HIF; or

[0637] - 7 TFBS for CREB and 6 TFBS for AP1;

[0638] The adjacent TFBSs are optionally, but preferably, separated by a sequence of interval regions.

[0639] The appropriate length of the interval zone was discussed above.

[0640] Similarly, TFBSs may be in any appropriate order, but in the preferred embodiment, they are provided in the order in which they are listed.

[0641] In some implementations, the CRE includes one of the following structures:

[0642] (Includes 5×cAMPRE and 3×AP1 TFBS CRE);

[0643] (CRE containing 5×cAMPRE and 3×AP1(2) TFBS);

[0644] (Includes 5×cAMPRE and 4×AP1 TFBS CRE);

[0645] S-AP1(2) (CRE containing 5×cAMPRE and 4×AP1(2) TFBS);

[0646] (Includes 8×AP1 TFBS CRE);

[0647] (CRE containing 8×AP1(1) TFBS);

[0648] (Including CREs with 3×ATF6, 4×AP1, and 3×HIF TFBS); and

[0649] (CRE containing 3×ATF6, 4×AP1(1) and 3×HRE1 TFBS);

[0650] (Including 5×cAMPRE, 4×AP1 TFBS CRE); and

[0651] S-AP1(1) (containing 5×cAMPRE, 4×AP1(1) TFBS CRE); and

[0652] (CRE containing 7×cAMPRE and 6×AP1(3)TFBS);

[0653] Here, S represents an optional but preferred sequence of interval regions. The appropriate length of the interval regions was discussed above.

[0654] In these structures, a reference to a TF indicates the existence of a TFBS used for that TF. cAMPRE is used to refer to a TFBS used for CREB.

[0655] In some specific implementations, the CRE includes one of the following structures:

[0656] AP1 (containing 5×cAMPRE and 3×AP1 TFBS CRE).

[0657] -S 10 -AP1(2) (CRE containing 5×cAMPRE and 3×AP1(2) TFBS);

[0658] AP1-S 10 -AP1 (containing 5×cAMPRE and 4×AP1 TFBS CRE).

[0659] (CRE containing 5×cAMPRE and 4×AP1(2) TFBS);

[0660] (Includes 8×AP1 TFBS CRE);

[0661] (CRE containing 8×AP1(1) TFBS);

[0662] (Including CREs with 3×ATF6, 4×AP1, and 3×HIF TFBS); and

[0663] (CRE containing 3×ATF6, 4×AP1(1) and 3×HRE1 TFBS);

[0664] AP1-S 10 -AP1 (containing 5×cAMPRE and 4×AP1 TFBS CRE); and

[0665] (Including 5×cAMPRE and 4×AP1(1) TFBS CRE); and

[0666] (Including CREs with 7×cAMPRE and 6×AP1(3) TFBS);

[0667] Among them, S x This represents a spacer sequence of length X nucleotides.

[0668] It has been found that the length of the intervals detailed above is effective in the specific examples discussed below. While other lengths of intervals are also expected to be functional, these represent preferred interval lengths; this applies to all aspects and embodiments of the invention that incorporate these TFBSs as described below.

[0669] In some specific implementations, CRE includes one of the following sequences:

[0670] (SEQ ID NO: 120, containing 5×cAMPRE and 3×AP1 TFBS CRE);

[0671] (SEQ ID NO: 121, containing 5×cAMPRE and 4×AP1 TFBS CRE);

[0672] (SEQ ID NO: 122, containing 8×AP1 TFBS CRE); and

[0673] (SEQ ID NO: 123, containing CRE3×ATF6, 4×AP1 and 3×HIF TFBS);

[0674] Here, S represents an optional but preferred sequence of interval regions. The appropriate length of the interval regions was discussed above.

[0675] In some specific implementations, CRE includes one of the following sequences:

[0676] (SEQ ID NO: 124, CRE from Synp-FORCSV-10 containing 5×cAMPRE and 3×AP1(2) TFBS);

[0677] (SEQ ID NO: 125, CRE from Synp-FORCMV-09, containing 5×cAMPRE and 4×AP1(2) TFBS);

[0678] (SEQ ID NO: 126, CRE from Synp-FMP-02 and Synp-FLP-01, containing 8×AP1(1) TFBS);

[0679] (SEQ ID NO: 127, containing CREs of 3×ATF6, 4×AP1(1) and 3×HRE1 TFBS); and

[0680] (SEQ ID NO: 128, containing 5×cAMPRE and 4×AP1(1) TFBS CRE); and

[0681] (SEQ ID NO: 129, containing a CRE of 7×cAMPRE and 6×AP1 TFBS); and

[0682] (SEQ ID NO: 130, CRE from FORNEW, FORNCMV, FORNCMV53, FORNMinTK, FORNMLP, FORNSV40, FORNpJB42, FORNTATAM6a, containing 7×cAMPRE and 6×AP1(3) TFBS).

[0683] Here, S represents an optional but preferred sequence of interval regions. The appropriate length of the interval regions was discussed above.

[0684] In some specific preferred embodiments, the CRE includes one of the following sequences:

[0685] (SEQ ID NO: 131, CRE from Synp-FORCSV-10 containing 5×cAMPRE and 3×AP1(2) TFBS);

[0686] (SEQ ID NO: 132, CRE from Synp-FORCMV-09, containing 5×cAMPRE and 4×AP1(2) TFBS);

[0687] (SEQ ID NO: 133, CRE from Synp-FMP-02 and Synp-FLP-01; containing 8×AP1(1) TFBS);

[0688]

[0689] CRE containing 3×ATF6, 4×AP1(1) and 3×HRE1 TFBS); and

[0690] (SEQ ID NO: 135, containing 5×cAMPRE and 4×AP1(1) TFBS CRE);

[0691]

[0692] NO: 136, CRE from FORNEW, FORNCMV, FORNCMV53, FORNMinTK, FORNMLP, FORNSV40, FORNpJB42, FORNTATAM6a, containing 7×cAMPRE and 6×AP1(3) TFBS).

[0693] Or a functional variant of the sequence that contains at least 80% of the same sequence, preferably at least 85%, 90%, 95% or 99% of the same sequence.

[0694] Typically, the TFBS sequence present in such functional variants is the same as the reference sequence, and virtually all variations occur in the spacer sequence located between them.

[0695] It has been shown that, when combined with minimal promoters to form inducible promoters, the aforementioned CREs provide good levels of inducibility and strong expression, as well as low levels of background expression, after induction. Therefore, they are all useful in providing inducible promoters for tuyerein. The CREs exhibit some degree of variation in inducibility and expression levels after induction, which allows for the selection of promoters with desired properties.

[0696] It has been shown that CREs with the following structure, when coupled to a minimal promoter, offer excellent properties in terms of inducibility and expression: Therefore, such a CRE represents a particularly preferred embodiment of the present invention.

[0697] It has been shown that CREs with the following structure, when coupled to a minimal promoter, offer superior properties in terms of inducibility and expression: Therefore, such a CRE represents a particularly preferred embodiment of the invention. Surprisingly, such a CRE performs so well because it contains several TFBSs that are unknown or unexpectedly induced by tuftrin, and the number of TFBSs induced by tuftrin is less than in some other CREs that are less inducible and less potent. Given the combination of TFBSs present in the CRE, an unexpected synergistic effect appears to emerge.

[0698] The structure cAMPRE-S- has been shown. The synthetic trichodin-inducible promoters offer superior properties in terms of inducibility and expression. Therefore, such promoters represent a particularly preferred embodiment of the present invention. Given the presence of TFBS combinations in CRE, a particular synergistic effect appears to emerge.

[0699] In another embodiment, the promoter may include a cis-regulation module (CRM), which includes a CRE according to the first aspect of the invention. Other CREs in the CRM may be tuftrin-inducible CREs, or they may have any other function.

[0700] Preferably, the synthetic tuftin-inducible promoter comprises a CRE (or CRM) as discussed above, which is linked to a minimum promoter or a proximal promoter (preferably a minimum promoter).

[0701] The minimal promoter can be any suitable minimal promoter. A wide range of minimal promoters are known in the art. Suitable minimal promoters, without limitation, include the CMV minimal promoter (CMV-MP), the YB-TATA minimal promoter (YB-TABA), the HSV thymidine kinase minimal promoter (MinTK), the SV40 minimal promoter (SV40-MP), or G6PC-MP (which is a liver-derived non-TATA box MP). The minimal promoter can be a synthetic minimal promoter.

[0702] The CMV-MP sequence is:

[0703]

[0704] The YB-TATA sequence is:

[0705]

[0706] However, shorter versions of YB-TATA MP are known in the art, providing an efficient alternative to the YB-TATAMP sequences listed above. The sequence of this shorter YB-TATA MP (referred to as sYB-TATA) is... (SEQ ID NO: 105). Therefore, as long as YB-TATA is referred to as a component of the inducible promoter described herein, replacing the equivalent sequence of YB-TATA with sYB-TATA is also considered an alternative embodiment of the invention. In other words, in such alternatives, the sequence of sYB-TATA is retained, while the remainder of YB-TATA can be replaced with other sequences (typically spacer sequences).

[0707] The MinTK MP sequence is:

[0708]

[0709] The sequence of SV40-MP is:

[0710]

[0711] The sequence of G6PC-MP is:

[0712]

[0713] In some embodiments, the synthetic tuftin-inducible promoter comprises any of the above-described CRE sequences operatively linked to a minimal promoter or a proximal promoter (preferably a minimal promoter). The CRE is preferably coupled to the MP via a spacer region, but in some cases, another CRE may be provided between them. The CRE may also be operatively linked to the MP without a spacer region.

[0714] The spacer sequence between the CRE and the minimum promoter can be of any suitable length. Typically, the spacer is 5 to 100 nucleotides long, 20 to 80 nucleotides long, or 30 to 70 nucleotides long. For example, spacers of 5, 10, 18, 20, 21, 42, 50, 59, 65, and 66 nucleotides have been used in specific non-limiting examples of the invention, and these spacers function well. However, other lengths of spacers can be used, and those skilled in the art can readily determine suitable lengths.

[0715] In some preferred embodiments, the synthetic trichodin-inducible promoter comprises one of the following structures:

[0716] (That is, CRE and MP containing 5×cAMPRE and 3×ATF6 TFBS);

[0717] MP (i.e., CRE and MP containing 5×cAMPRE and 4×AP1 TFBS).

[0718] (That is, CRE and MP containing 8×AP1 TFBS);

[0719] (That is, CRE and MP containing 3×ATF6, 4×AP1 and 3×HIF TFBS);

[0720] MP (i.e., CRE and MP containing 5×cAMPRE, 4×AP1 TFBS); and

[0721] (Including CRE and MP of 7×cAMPRE and 6×AP1(3) TFBS);

[0722] Here, S represents an optional but preferred sequence of spacers, and MP represents the minimum promoter. The appropriate length of the spacers has been discussed above.

[0723] In a particularly preferred embodiment, the synthetic trichodin-inducible promoter comprises the following structure:

[0724] Where S represents an optional but preferred interval sequence, and MP represents the minimum promoter. More preferably, MP is CMV-MP.

[0725] In some preferred embodiments, the synthetic trichodin-inducible promoter comprises one of the following structures:

[0726] MP (i.e., CRE and SV40-MP containing 5×cAMPRE and 3×AP1 TFBS).

[0727] CMV-MP (i.e., CRE and CMV-MP containing 5×cAMPRE and 4×AP1 TFBS);

[0728] (Min-TK or G6PC MP or CMV-MP) (i.e., CRE containing 8×AP1 TFBS and Min-TK or G6PC MP or CMV-MP).

[0729] (That is, CRE and CMV-MP containing 3×ATF6, 4×AP1 and 3×HIF TFBS);

[0730] YB-TATA (i.e., CRE and YB-TATA containing 5×cAMPRE, 4×AP1 TFBS); and

[0731] (YB TATA or CMV-MP or CMV53 or MinTK or MLP or SV40 or pJV42 or TATAM6a) (i.e., CRE containing 7×cAMPRE and 6×AP1(3) TFBS and YB TATA or CMV-MP or CMV53 or MinTK or MLP or SV40 or pJV42 or TATAM6a),

[0732] Here, S represents an optional but preferred sequence of interval regions. The appropriate length of the interval regions was discussed above.

[0733] In some preferred embodiments, the synthetic trichodin-inducible promoter comprises one of the following sequences:

[0734] (SEQ ID NO: 138, containing 5×cAMPRE and 3×AP1TFBS CRE and SV40-MP);

[0735] (SEQ ID NO: 139, containing 5×cAMPRE and 4×AP1 TFBS CRE and CMV-MP);

[0736]

[0737] (SEQ ID NO: 141, containing CRE and CMV-MP of 3×ATF6, 4×AP1 and 3×HIF TFBS); and

[0738] (SEQ ID NO: 142, containing 5×cAMPRE and 4×AP1 TFBS CRE and YB-TATA);

[0739] Here, S represents an optional but preferred sequence of interval regions. The appropriate length of the interval regions was discussed above.

[0740] In some preferred embodiments, the synthetic tuftin-inducible promoter comprises one of the following sequences (TFBS sequences are underlined, and the smallest promoter sequence is shown in bold):

[0741]

[0742] (SEQ ID NO: 6; core sequence of Synp-FORCSV-10);

[0743]

[0744] (SEQ ID NO: 7; core sequence of Synp-FORCMV-09);

[0745] (SEQ ID NO: 8; Synp-FMP-02); and

[0746]

[0747] Or a functional variant of the sequence that contains at least 80% of the same sequence, preferably at least 85%, 90%, 95% or 99% of the same sequence.

[0748] Typically, the TFBS and MP sequences present in such functional variants are identical to the reference sequence, and virtually all sequence variations occur in the spacer sequences located between them.

[0749] The aforementioned tuftin-inducible promoters have been shown to provide good levels of inducibility and strong expression, as well as low levels of background expression, after induction. The promoters exhibit some degree of variation in inducibility and expression levels after induction, which allows for the selection of promoters with desired properties.

[0750] The included structure has been shown. Synthetic tuftin-inducible promoters (preferably where MP is CMV-MP) offer superior properties in terms of inducibility and expression. Therefore, such promoters represent a particularly preferred embodiment of the invention. As mentioned above, it is surprising that such a CRE performs so well because it contains several TFBSs that are unknown or unexpectedly induced by tuftin, and the number of TFBSs induced by tuftin is less than that of some other CREs that are less inducible and less potent. Given the combination of TFBSs present in the CRE, an unexpected synergistic effect appears to emerge.

[0751] In a preferred embodiment of the invention, the inducibility of the promoter causes the expression level of the transgene under promoter control to increase by at least 3-fold, more preferably 5-fold, 10-fold, 15-fold, 20-fold, 30-fold or 50-fold after induction (e.g., 5 hours after cells (e.g. HEK293 cells) are exposed to 18 μM salvin).

[0752] In some embodiments of the invention, after induction (e.g., 5 hours after cells (e.g., HEK293 cells) are exposed to 18 μM trichodin), the expression level of the transgene under promoter control is at least 50% of the expression level provided by the CMV-IE promoter (i.e., another identical vector in the same cells under the same conditions, but in which the expression of the transgene is controlled by a CMV-IE rather than a trichodin-inducible promoter). More preferably, the expression level of the transgene is at least 75%, 100%, 150%, 200%, 300%, 400%, 500%, 750%, or 1000% of the expression level provided by the CMV-IE promoter.

[0753] The cAMP reaction element TGACGTCA is described in JBC, Vol. 272, No. 31, August 1, 1997, pp. 19158-19164, which is incorporated herein by reference. The novel cAMP reaction element CACTTGATC is described in J. Neurochem, 63(1), 28-40 Jul 1994, which is incorporated herein by reference; this element can be used in the trichomoniasis-inducible promoters discussed above.

[0754] cAMP response elements (CRE) / cAMP self-regulating response elements (CARE) are discussed in Molecular Endocrinology, Vol. 13, No. 7, July 1, 1999, pp. 1207-1217.

[0755] cAMP reaction elements (e.g., in Gut, May 3, 2005, 54(9): 1309-1317) are also discussed. Figure 2 (in the middle). Such elements can be used in the tuftin-inducible promoters discussed above.

[0756] Another cAMP-inducible promoter is described in US6596508 (which is incorporated herein by reference), see, for example, SEQ ID NO: 3 of US659650. Such promoters can be used in this invention. cAMP reaction elements from VIP promoters (e.g., SEQ ID NO: 1 and SEQ ID NO: 2 of US659650) are also disclosed.

[0757] Other cAMP response elements are also described in US8986937, which are incorporated herein by reference. Exemplary naturally occurring cAMP-inducible promoters described therein include the PEPCK promoter (Roesler et al. (1998) The Journal of Biological Chemistry, 273, 14950-14957); a promoter containing a cAMP responsive element (CRE) located at position 294 relative to the translation initiation site of the human cyclin D2 promoter (Muñiz et al. (2006) Biology of Reproduction 75(2): 279-288); and a promoter containing a cAMP responsive element (CRE) of the lactate dehydrogenase A subunit promoter (Welfeld et al. (1989) J. Biol. Chem.). 264(12):6941-7. An exemplary cAMP-inducible promoter comprises a 236-nucleotide glycoprotein hormone α-subunit promoter as described in U.S. Patent Application Publication No. US2008-0187942, published August 7, 2008 (which is incorporated herein by reference), said promoter comprising a cyclic AMP (cAMP) regulatory element (CRE) (AF401991). Such elements can be used in the trichodin-inducible promoter as described above.

[0758] US9060310 (which is incorporated herein by reference) describes further cAMP response elements, such as various CRE palindromes and hairpins of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 of US9060310. Such cAMP response elements can be used in the trichomoniasis-inducible promoter as described above.

[0759] US20070036810 and Mayr B, Montminy M., Nat Rev Mol Cell Biol 2001 Aug;2(8):599-609 (both are incorporated herein by reference) disclose cAMP response elements comprising a palindromic or asymmetric variant of TGACGTCA containing a CRE half-site with the core sequence TGAC. Such elements can be used in the trichomoniasis-inducible promoter as described above.

[0760] Several cAMP-inducible genes and well-known cAMP targets are also discussed in US20070036810 (incorporated herein by reference), such as TSHalpha, phosphoenolpyruvate carboxykinase (PEPCK), lens protein α-B, and EGF-like bimodal proteins. Promoters from such genes may also be relevant in the context of this application.

[0761] Suitable inducers that can activate adenylate cyclase include, but are not limited to:

[0762] - Trichosanthesin (a potent adenylate cyclase activator (CAS No. 66575-29-9)).

[0763] -NKH 477 (water-soluble analogue of fenestrated ...

[0764] -PACAP-27 (a neuropeptide that stimulates adenylate cyclase (CAS No. 127317-03-7)).

[0765] -PACAP-38 (a neuropeptide that stimulates adenylate cyclase (CAS No. 137061-48-4)).

[0766] -Pertussis toxin (CAS No. 70323-44-3); and

[0767] - Cholera toxin (CAS No. 9012-63-9).

[0768] All of the above are available commercially from Sigma-Aldrich, Inc. (now part of Merck KGaA).

[0769] In some preferred embodiments of the present invention, the inducer includes trichodin or NKH477.

[0770] Classifying coleonol as generally regarded as safe (GRAS) is generally desirable from a safety perspective. Coleonol (also known as cyclohexane) is a hemispheric diterpenoid produced by the Indian violet cress (Plectranthus barbatus). Coleonol is commonly used in materials research to enhance cyclic AMP levels. Coleonol is also used in traditional medicine. As coleonol is GRAS, it is a preferred inducer for use in gene therapy applications for the promoters according to the present invention.

[0771] NKH 477 is a water-soluble analogue of keratoside, and therefore may be particularly advantageous in terms of ease of use in cell culture. Because NKH477 is water-soluble, it is a preferred inducer.

[0772] The inducer can be administered to the cells in any suitable manner. For example, the inducer can be added to the culture medium (if necessary, along with a suitable carrier, surfactant, etc.).

[0773] Those skilled in the art can readily determine the appropriate dose rate for any given inducer. Therefore, for any inducer, those skilled in the art can readily determine the appropriate manner of delivery of the inducer to cells and the appropriate concentration to be used. Generally, the inducer can be administered at any appropriate concentration in the range of 1 nM to 1000 μM (optionally in the range of 0.1 μM to 100 μM).

[0774] The cells can be administered tuftin at a concentration of 0.1 μM to 1000 μM, more preferably 1 μM to 100 μM, and even more preferably 5 μM to 30 μM. For example, it has been determined that administering tuftin to cells at a concentration of about 18 μM is optimal for inducing expression in HEK-293 cells.

[0775] NKH 477 can be administered to cells at appropriate concentrations from 0.1 μM to 1000 μM, more preferably from 1 μM to 100 μM, and even more preferably from 2 μM to 20 μM. For example, it has been determined that administering a concentration of about 8 μM to cells is optimal for inducing expression in HEK-293 cells.

[0776] The method may suitably include stopping the administration of the inducer. Stopping the administration of the inducer will at least cause a reduction in the expression of the expression product. Typically, the expression of the expression product will recover to baseline levels over time.

[0777] The method may suitably include varying the concentration of the inducer administered to the cells over time. This can be used to regulate the expression level of the expression product.

[0778] In some embodiments, the methods disclosed herein may involve administering an inhibitor of adenylate cyclase to cells, which reduces or shuts down the expression of the product. Adenylate cyclase inhibitors include, but are not limited to:

[0779] NB001—An inhibitor of adenylate cyclase 1 (AC1);

[0780] 9-Cyclopentyladenine monomethylsulfonate—a stable, cell-permeable, non-competitive adenylate cyclase inhibitor;

[0781] SQ 22,536—Inhibitor of cell permeable adenylate cyclase;

[0782] MDL-12,330A hydrochloride-adenylate cyclase inhibitor;

[0783] 2',5'-Dideoxyadenosine—a cell permeable adenylate cyclase inhibitor;

[0784] A potent inhibitor of 2',5'-dideoxyadenosine 3'-tetrasodium triphosphate—adenylate cyclase;

[0785] MANT-GTPγS—a potent and competitive inhibitor of adenylate cyclase;

[0786] 2',3'-Dideoxyadenosine—a specific adenylate cyclase inhibitor;

[0787] NKY80—a selective adenylate cyclase-V inhibitor; and

[0788] KH7—a selective inhibitor of soluble adenylate cyclase.

[0789] All of the above are available commercially from Sigma-Aldrich, Inc. (now part of Merck KGaA).

[0790] Therefore, administration of adenylate cyclase inhibitors can be used to shut down or reduce the expression of toxic proteins.

[0791] In one embodiment, the synthetic tuftin-inducible promoter does not contain or is not composed of one of the following structures:

[0792] (That is, the structure of RTV-17 containing 8×AP1 TFBS and CMV-MP).

[0793] (That is, the structure of Synp-RTV-019 containing 3×ATF6, 4×AP1 and 3×HIF TFBS and CMV-MP).

[0794] (That is, the structure of FORCYB1 containing 5×cAMPRE and 4×AP1 TFBS and YB-TATA-MP).

[0795] Here, S x This represents a spacer region with a length of X nucleotides.

[0796] In one embodiment, the synthetic tuftin-inducible promoter does not contain or is not composed of one of the following structures:

[0797]

[0798] Temperature-induced promoters:

[0799] Inducible promoters can be induced by lowering the temperature, such as cold shock responsive promoters. In some embodiments, the inducible promoter is a synthetic cold shock responsive promoter derived from the S1006a gene (calccyclin) of CHO cells. The temperature sensitivity of the S1006a gene (calccyclin) promoter was identified by Thaisuchat et al., 2011 (Thaisuchat, H. et al., (2011) 'Identification of a novel temperature sensitive promoter in cho cells', BMC Biotechnology, 11. doi: 10.1186 / 1472-6750-11-51), which is incorporated herein by reference. In some embodiments, the inducible promoter is the one described by Thaisuchat et al., 2011. Figure 2 One of the synthetic cold shock responsive promoters shown. These promoters are induced by lowering the temperature, such as those by Thaisuchat et al., 2011. Figure 3 As shown. Most of these synthetic promoter constructs exhibited expression similar to the known promoter SV40 at 37°C, while induction was 2-3 times higher when the temperature was reduced to 33°C. In some embodiments, the inducible promoter was from Thaisuchat et al., 2011. Figure 2 The SPS5 promoter. In some preferred embodiments, the inducible promoter is from Thaisuchat et al., 2011. Figure 2 SPS8.

[0800] pH-inducible promoters:

[0801] Inducible promoters can be induced by decreasing or increasing the pH of the cell containing the promoter. Suitablely, the inducible promoter can be induced by decreasing the pH, i.e., a promoter that is inducible under acidic conditions. Suitable acid-inducible promoters are described in Hou et al., 2016 (Hou, J. et al. (2016) 'Isolation and functional validation of salinity and osmotic stress inducible promoter from the maizetype-II H+-pyrophosphatase gene by deletion analysis in transgenic tobaccoplants', PLoS ONE, 11(4), pp. 1-23. doi: 10.1371 / journal.pone.0154041), which is incorporated herein by reference.

[0802] In some embodiments, the inducible promoter is a synthetic promoter derived from the YGP1 or CCW14 gene that is inducible under acidic conditions. Rajkumar et al., 2016 (Rajkumar, AS et al., (2016) 'Engineering of synthetic, stress-responsive yeast promoters', 44(17). doi: 10.1093 / nar / gkw553) studied and improved the inducibility of the YGP1 or CCW14 gene under acidic conditions by modifying transcription factor binding sites, which is incorporated herein by reference. In some embodiments, the inducible promoter is the one derived from Rajkumar et al., 2016. Figure 1A , Figure 2 A, Figure 3 A and Figure 4 One of the synthetic promoters that can be induced under acidic conditions in group A. For example, Rajkumar et al., 2016. Figure 1B , Figure 2 B Figure 3 B and Figure 4 As shown in B, these promoters are induced by lowering the pH. Most of these synthetic promoters are induced up to 10-15 times when the pH is lowered from pH 6 to pH 3. In some preferred embodiments, the inducible promoter is YGP1pr from Figure 1, Rajkumar et al., 2016. In other preferred embodiments, the inducible promoter is YGP1pr from Figure 1, Rajkumar et al., 2016.

[0803] Osmotic pressure-induced promoters:

[0804] Inducible promoters can be osmotically induced. Zhang et al. described suitable osmotically induced promoters (Molecular Biology Reports, Vol. 39, pp. 7347-7353 (2012)), which is incorporated herein by reference.

[0805] Carbon source-induced promoters:

[0806] Inducible promoters can be induced by adding a specific carbon source (e.g., a non-sugar carbon source). Alternatively, inducible promoters can be induced by removing or lacking a carbon source. Suitable promoters induced by the presence or absence of multiple carbon sources are described in Weinhandl et al., 2014 (Weinhandl, K. et al. (2014) 'Carbon source dependent promoters in yeasts', Microbial Cell Factories, 13(1), pp. 1-17. doi: 10.1186 / 1475-2859-13-5), which is incorporated herein by reference.

[0807] Alcohol (e.g., ethanol) inducible promoters:

[0808] Inducible promoters can be induced by adding ethanol. Suitable promoters induced by ethanol are described by Matsuzawa et al. (Applied Microbiology and Biotechnology, Vol. 97, pp. 6835-6843 (2013)), which is incorporated herein by reference.

[0809] Amino acid inducible promoters:

[0810] Inducible promoters can be induced by adding one or more amino acids. Suitable amino acids can be aromatic. Suitable amino acids can be GABA (gamma-aminobutyric acid), which is also a neurotransmitter. Suitable promoters induced by aromatic amino acids and GABA are described by Kim et al. (Applied Microbiology and Biotechnology, Vol. 99, pp. 2705-2714 (2015)), which is incorporated herein by reference.

[0811] Hormone (e.g., ecdysone) inducible promoters:

[0812] Inducible promoters can be induced by steroid hormones. Appropriately, ecdysone can be used. A mammalian ecdysone inducible system was created by No, Yao, and Evans (No, D., Yao, TP and Evans, RM (1996) 'Ecdysone-inducible gene expression in mammalian cells and transgenic mice', Proceedings of the National Academy of Sciences of the United States of America, 93(8), pp. 3346-3351. doi: 10.1073 / pnas.93.8.3346), which is incorporated herein by reference. The expression of modified ecdysone receptors in mammalian cells allows for the induction of expression by ecdysone-responsive promoters upon the addition of ecdysone, such as those described by No, Yao, and Evans 1996. Figure 2 As shown. This system exhibits lower basal activity and higher inducibility than tetracycline-inducible systems, such as those of No, Yao, and Evans (1996). Figure 6 As shown. Suitable commercially available inducible systems are available from Agilent Technologies and are described in Agilent Technologies (2015) 'Complete Control Inducible Mammalian Expression System Instruction Manual', which is incorporated herein by reference.

[0813] Tetracycline-regulated promoters:

[0814] In some implementations, the promoter can be induced by the presence or absence of tetracycline or its derivatives.

[0815] The appropriate promoters induced in the absence of tetracycline or its derivatives are those in the tet-OFF system. In the tet-OFF system, the tetracycline-controlled transactivator (tTA) allows transcriptional activation of the tTA-dependent promoter in the absence of tetracycline or its derivatives. The tTA and tTA-dependent promoters were originally created by Gossen and Bujard, 1992 (Gossen, M. and Bujard, H. (1992) 'Tight control of gene expression in mammalian cells by tetracycline-responsive promoters', Proceedings of the National Academy of Sciences of the United States of America, 89(12), pp. 5547-5551. doi: 10.1073 / pnas.89.12.5547), which is incorporated herein by reference. tTA is created by fusing a tetracycline resistance operon (tet repressor) encoded in Tn10 of *Escherichia coli* with an activated cycline-controlled transactivator (tTA), while the tTA-dependent promoter is created by combining a tet operator sequence with a minimal promoter from the human cytomegalovirus promoter IE (hCMV-IE). Upon addition of tetracycline or a derivative thereof, tTA can no longer bind to its target sequence within the tTA-dependent promoter, and there is no expression from the tTA-dependent promoter. This is described in Jaisser, 2000. Figure 1AThe mechanism of conformational changes resulting from the binding of tetracyclines or their derivatives to tTA is described in Orth et al., 2000 (Orth, P. et al., (2000) 'Inducible gene expression and gene modification in transgenic mice', Journal of the American Society of Nephrology, 11(SUPPL.16), pp. 95-100), which is incorporated herein by reference. Tetracycline binding to TetR increases the dissociation of the attached DNA-binding domain, which eliminates TetR's affinity for manipulating DNA.

[0816] The appropriate promoter induced by the presence of tetracycline or its derivatives is the promoter in the tet-ON system. In the tet-ON system, the inverse tetracycline-controlled trans-activator (rtTA) allows transcriptional activation of the tTA-dependent promoter in the presence of tetracycline or its derivatives, as described by Gossen et al. (Science 23 Jun 1995: Vol. 268, Issue 5218, pp. 1766-1769 DOI: 10.1126 / science.7792603), which is incorporated herein by reference. In the absence of tetracycline or its derivatives, tTA can no longer bind to its target sequence within the tTA-dependent promoter, and there is no expression from the tTA-dependent promoter. This is described in Jaisser, 2000. Figure 1BThe above is shown in the table and explained on page 96 of Jaisser, 2000 (Jaisser, F. (2000) 'Inducible gene expression and gene modification in transgenic mice', Journal of the American Society of Nephrology, 11(SUPPL. 16), pp. 95-100), which is incorporated herein by reference.

[0817] Appropriately, an improved variant of the inverse tetracycline-controlled trans-activator (rtTA) is used.

[0818] Suitable improved variants are described in Table 1 of Urlinger et al., 2000 (Urlinger, S. et al. (2000) 'Exploring the sequence space for tetracycline-dependent transcriptionalactivators: Novel mutations yield expanded range and sensitivity', Proceedings of the National Academy of Sciences of the United States of America, 97(14), pp. 7963-7968. doi: 10.1073 / pnas.130192197), which is incorporated herein by reference. Urlinger et al., 2000 Figure 3 The studies showed that variants rtTA-S2 and rtTA-M2 exhibited lower basal activity, indicating minimal background expression from the tTA-dependent promoter in the absence of tetracycline or its derivatives. Furthermore, as Urlinger et al. (2000) Figure 3 As shown, rtTA-M2 exhibits increased sensitivity to tetracyclines and their derivatives, and functions at concentrations up to 10 times lower than rtTA. In some preferred embodiments, an improved variant of rtTA is rtTA-M2 from Urlinger et al., 2000.

[0819] The alternative improved variants are described in Table 1 of Zhou et al., 2006 (Zhou, X. et al. (2006) 'Optimization of the Tet-On system for regulated gene expression through viral evolution', Gene Therapy, 13(19), pp. 1382-1390. doi: 10.1038 / sj.gt.3302780), which is incorporated herein by reference. (See Zhou et al., 2006...) Figure 3 As described above, most of these variants exhibit higher transcriptional activity and doxycycline sensitivity compared to rtTA. The highest-performing variants show 7-fold higher activity and 100-fold higher sensitivity to doxycycline. In some preferred embodiments, improved variants of rtTA are V14, V15, or V16 from Zhou et al., 2006.

[0820] A suitable commercially available tetracycline-inducible system is the T-Rex system from Life-Technologies (see, for example, Life-Technologies (2014) 'Inducible Protein Expression Using the T-REx™ System', 1, pp. 1-12, available at: www.lifetechnologies.com / de / de / home / references / protocols / proteins-expression-isolation-and-analysis / protein-expression-protocol / inducible-protein-expression-using-the-trex-system.reg.us.htmL / ).

[0821] Induction can be achieved, for example, in the absence of tetracycline and in the presence of estrogen:

[0822] Inducible promoters can be induced by the absence of a molecule and the presence of a different molecule. In some embodiments, inducible promoters can be induced by removing tetracycline and adding estrogen, as described by Iida et al., 1996 (Iida, A. et al. (1996) 'Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system.', Journal of virology, 70(9), pp. 6054-6059. Doi: 10.1128 / jvi.70.9.6054-6059.1996), which is incorporated herein by reference. This specific inducibility is achieved by adding the ligand-binding domain of the estrogen receptor to the carboxyl terminus of the tTA transactivator. As described by Iida et al., 1996 Figure 3 As shown, this modified transactivator induces high expression of the gene of interest in the absence of tetracycline and in the presence of estrogen.

[0823] Induction via small molecule enhancers:

[0824] Inducible promoters can be induced by small molecule enhancers. Suitable promoters induced by small molecule enhancers (such as aromatic carboxylic acids, isohydroxamic acids, and acetamides) are described by Allen et al. (Biotechnol. Bioeng. 2008;100:1193-1204), which are incorporated herein by reference.

[0825] Mifepristone (RU-486) ​​inducible promoter:

[0826] Inducible promoters can be induced by synthetic steroids. In some embodiments, inducible promoters can be induced by mifepristone (also known as RU-486). The heterozygous mifepristone-responsive transcription factor LexPR transactivator was created by Emelyanov and Parinov, 2008 (Emelyanov, A. and Parinov, S. (2008) 'Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish', Developmental Biology, 320(1), pp. 113-121. doi: 10.1016 / j.ydbio.2008.04.042, which is incorporated herein by reference) by fusing the DNA-binding domain of a bacterial LexA repressor, a truncated ligand-binding domain of a human progesterone receptor, and an activation domain of a human NF-κB / p65 protein. Following the addition of mifepristone, LexPR induces expression via promoter sequences containing LexA binding sites, as illustrated in Figure 1 of Emelyanov and Parinov's 2008 study. Figure 2 As shown in the figure. A suitable commercially available mifepristone inducible system is the GeneSwitch System (see, for example, Fisher, T. (1994) 'Inducible Protein Expression Using GeneSwitch™ Technology', pp. 1-25).

[0827] Cumate inducible promoter:

[0828] In some implementations, an inducible promoter can be induced by the presence or absence of cumate.

[0829] In the cumate switch system from Mullick et al., 2006 (Mullick, A. et al. (2006) 'The cumategene-switch: A system for regulated expression in mammalian cells', BMCBiotechnology, 6, pp. 1-18. doi: 10.1186 / 1472-6750-6-43, incorporated herein by reference), the repressor CymR blocks transcription from promoters containing a CuO sequence downstream of the promoter. Once cumate is added, the CymR repressor cannot bind to CuO, and transcription from promoters containing CuO can continue. This is from Mullick et al., 2006... Figure 1B and Figure 2 As shown in the image.

[0830] In the alternative cumate switch system, the chimeric transactivator (cTA) generated by the fusion of CymR with the activation domain of VP16 does not prevent transcription from a promoter containing a CuO sequence upstream of the promoter in the presence of cumate. In the absence of cumate, the chimeric transactivator (cTA) binds to the CuO sequence and prevents transcription. This is illustrated in Figure 1C from Mullick et al., 2006. Figure 3 As shown in the image.

[0831] In the third configuration, the inverse chimeric transactivator (rcTA) prevents transcription from a promoter containing a CuO sequence upstream of the promoter in the absence of cumate. In the presence of cumate, rcTA binds to the CuO sequence, and transcription from a promoter containing the CuO sequence can proceed. This is illustrated in Figure 1D from Mullick et al., 2006. Figure 7 As shown in the image.

[0832] Suitable commercially available cumate-inducible systems can be found at SBI Biosciences (see SBI (2020) 'Cumate-inducible Systems For the ultimate in gene expression control, use SBI's cumate-CUMATE-INDUCIBLE SYSTEMS', pp. 1-13, which are incorporated herein by reference).

[0833] 4-Hydroxytamoxifen (OHT) inducible promoter:

[0834] Inducible promoters can be induced by 4-hydroxytamoxifen (OHT). Suitable 4-hydroxytamoxifen inducible promoters were described by Feil et al. (Biochemical and Biophysical Research Communications, Vol. 237, No. 3, August 28, 1997, p. 752), which is incorporated herein by reference.

[0835] Gas-induced promoters:

[0836] Inducible promoters can be gas-inducible promoters, such as acetaldehyde-inducible promoters. Suitable gas-inducible promoters are described in Weber et al., 2004 (Weber, W. et al. (2004) 'Gas-inducible transgene expression in mammalian cells and mice', Nature Biotechnology, 22(11), pp. 1440-1444. doi:10.1038 / nbt1021), which is incorporated herein by reference. The natural acetaldehyde-inducible AlcR-PalcA system from Asperigillus nidulans has been developed by introducing AlcR-specific manipulation modules into the human minimal promoter (collectively referred to as P). AIR (And suitable for use in mammals, such as) Figure 1A As shown in Figure 1C, when AlcR is constitutively expressed in cells of interest, the introduction of acetaldehyde causes it to bind to AlcR, thereby inducing the expression of the gene of interest under the control of the PAIR promoter. Figure 2 and Figure 3 As shown, in the absence of acetaldehyde, the genes of interest are not expressed.

[0837] Riboswitch, ribozyme, and aptamer inducible promoters:

[0838] Inducible promoters can be induced with or without a ribozyme. Ribozymes can then be induced by ligands.

[0839] Inducible promoters can be induced in the absence of metabolites. In some embodiments, the metabolite may be glucosamine-6-phosphate responsive. Suitable ribozymes that act as repressors of glucosamine-6-phosphate responsive genes are described by Winkler et al., 2004 (Winkler, WC et al. (2004) 'Control of gene expression by a natural metabolite-responsive ribozyme', Nature, 428(6980), pp. 281-286. doi: 10.1038 / nature02362), which is incorporated herein by reference. Figure 2 As shown in C, the ribozyme is activated in a concentration-dependent manner by glucosamine-6-phosphate and cleaves the messenger RNA of the glmS gene. With modification, this natural system can be applied to control genes of interest other than the glmS gene.

[0840] Ligand-inducible aptamers can also downregulate protein expression. Protein expression can be downregulated by aptamers that induce mRNA degradation through small molecule-induced ribozyme self-cleavage, see Zhong et al., 2016 (Zhong, G. et al. (2016) 'Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells', eLife, 5(NOVEMBER2016). doi: 10.7554 / eLife.18858), which is incorporated herein by reference. Suitable aptamers in (Zhong et al., 2016) Figure 4 As shown in A. These aptamers reduce the relative expression of genes of interest, such as (Zhong et al., 2016). Figure 4 As shown.

[0841] Protein expression can also be upregulated by small molecule-dependent ribozymes. These ribozymes can be tetracycline-dependent. Beilstein et al. described suitable tetracycline-dependent ribozymes that can enable protein expression by preventing ribozyme cleavage, which otherwise cleaves mRNA in the absence of a ligand (ACS Synth. Biol. 2015, 4, 5, 526-534), which is incorporated herein by reference.

[0842] Protein expression can also be regulated by guanine-dependent aptamerases, as described by Nomura et al. (Chem. Commun., 2012, 48, 7215-7217), which is incorporated herein by reference.

[0843] In addition, Kumar et al. described an RNA structure that combines a drug-induced allosteric ribozyme with a microRNA precursor analog, which allows for the chemical induction of RNAi in mammalian cells (J. Am. Chem. Soc. 2009, 131, 39, 13906-13907), which is incorporated herein by reference.

[0844] Metallothionein inducible promoters:

[0845] Metallothionein inducible promoters have been described in the literature. See, for example, Shinichiro Takahashi, “Positive and negative regulators of the metallothionein gene,” Molecular Medicine Reports, March 9, 2015, pp. 795-799, which is incorporated herein by reference.

[0846] Rapamycin-inducible promoter

[0847] Inducible promoters can be induced by small molecule drugs such as rapamycin. Humanized systems using rapamycin for pharmacological control of gene expression are described in Rivera et al., 1996 (Rivera et al., Nature Medicine, Vol. 2, pp. 1028-1032 (1996)), which is incorporated herein by reference. The native ability of rapamycin to bind to FKBP12 and, conversely, the ability of this complex to bind to FRAP, was used by Rivera et al., 1996 to induce rapamycin-specific expression of genes of interest. This was achieved by fusing one of the FKBP12 / FRAP proteins to the DNA-binding domain and the other to the activation domain. If FKBP is fused to the DNA-binding domain and FRAP is fused to the activation domain, there will be no transcription of the gene of interest in the absence of rapamycin because FKBP and FRAP do not interact, as shown in Figure 1b. In the presence of rapamycin, FKBP and FRAP interact, bringing the DNA-binding domain and activation domain into close contact, resulting in transcription of the gene of interest, such as... Figure 2 and Figure 3 As shown.

[0848] Chemically induced neighbor-inducible promoters

[0849] Inducible promoters can be controlled by chemically induced proximity. A suitable small molecule-based system for controlling protein abundance or activity is described by Liang et al. (Sci Signal. 2011 Mar 15;4(164): rs2. doi:10.1126 / scisignal.2001449), which is incorporated herein by reference.

[0850] Gene expression can be induced by chemically induced proximity, which occurs by the association of two proteins to molecules on their binding surfaces, as illustrated by Belshaw et al., 1996 (Belshaw, PJ et al., (1996) 'Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins', Proceedings of the National Academy of Sciences of the United States of America, 93(10), pp.4604-4607), which is incorporated herein by reference. Figure 3 The transcriptional activation of a gene of interest is demonstrated by chemically induced proximity, which is achieved by combining molecules on two protein-binding surfaces.

[0851] Rheoswitch® inducible promoter:

[0852] Inducible promoters can be induced by small synthetic molecules. In some embodiments, these small synthetic molecules can be dihydrazide ligands. Suitable systems for inducible upregulation and downregulation of gene expression are described in Cress et al. (Vol. 66, Supplement 8, pp. 27) or Barrett et al. (Cancer Gene Therapy, Vol. 25, pp. 106-116 (2018)), which are incorporated herein by reference. The RheoSwitch® system consists of two chimeric proteins derived from the ecdysone receptor (EcR) and RXR, which are fused to the DNA-binding domain and the acidic transcription activation domain, respectively. Upon binding to the small synthetic ligand, the nuclear receptor can heterodimerize to create a functional transcription factor and activate transcription by a responsive promoter linked to the gene of interest.

[0853] CRISPR inducible promoters:

[0854] Gene expression can be induced by CRISPR-based transcriptional regulators. Nuclease-deficient Cas9 can be directed to sequences of interest by designing its associated single guide RNA (sgRNA), and it can modulate gene expression through tethering of effector domains on the sgRNA-Cas9 complex, as illustrated in Figure 1 of Ferry, Lyutova, and Fulga, 2017 (Ferry, QRV, Lyutova, R. and Fulga, TA (2017) 'Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs', Nature Communications. Nature Publishing Group, 8, pp. 1-10. doi:10.1038 / ncomms14633), which is incorporated herein by reference. A suitable, multifunctional, inducible CRISPR-TR platform based on minimally engineered sgRNA is described in Ferry, Lyutova, and Fulga, 2017.

[0855] CRISPR-based transcriptional regulation can be drug-induced. A suitable drug-inducible CRISPR-based transcriptional regulation system is described in Zhang et al., 2019 (Zhang, J. et al., (2019) 'Drug Inducible CRISPR / CasSystems', Computational and Structural Biotechnology Journal. Elsevier BV, 17, pp. 1171-1177. doi: 10.1016 / j.csbj.2019.07.015), which is incorporated herein by reference.

[0856] In one embodiment, contacting cells with an inducer or applying suitable inducing conditions to cells induces the expression of at least one toxic protein.

[0857] The inducible promoters described herein can further control the expression of inducers or repressors of the inducible promoter (e.g., inducers or repressors of a second different promoter, or inducers or repressors of the promoter itself). In one embodiment, the cell includes a first inducible promoter operatively linked to a repressive element that can prevent protein expression.

[0858] In one implementation, the first inducible promoter further encodes a protein that represses the expression of the first inducible promoter.

[0859] In one embodiment, the cell contains a first inducible promoter that further encodes a protein that induces the expression of a second inducible promoter.

[0860] Toxic genes

[0861] The various aspects of the invention described herein relate to the use of any inducible promoters presented herein to control the expression of toxic proteins in cells (e.g., viral vector-produced cells). As used herein, a "toxic protein" refers to a gene product that, when expressed in a cell, has a toxic effect on the cell, negatively impacting cellular physiological function and potentially causing reduced cell proliferation, apoptosis, and ultimately cell death. Toxic proteins are not initially highly toxic, but their expression can, for example, kill cells over time.

[0862] Certain viral proteins required for viral vector replication are toxic when expressed in cells, thus requiring strict control over the expression of these genes during viral vector production. For example, the replication (rep) and capsid (cap) proteins required for AAV viral vector replication are toxic when expressed in AAV-producing cells. rep and cap each express related protein families from different open reading frames and are generated by alternative mRNA splicing and different transcription and translation initiation sites. Rep proteins (Rep78, Rep68, Rep52, and Rep40) are involved in the replication, rescue, and integration of the AAV genome. Rep78 and Rep68 have the same N-terminal sequence and share the same natural promoter p5, but Rep78 contains an exon that is alternatively spliced ​​out of rep68. Similarly, Rep52 and Rep40 have the same N-terminal sequence and share the natural p19 promoter, which is downstream of the p5 promoter, but rep52 contains an exon that is alternatively spliced ​​out of rep68. The Cap gene encodes three capsid proteins (VP1, VP2, and VP3) and an assembly activation protein (AAP) that promotes capsid formation, resulting in the viral particle capsid. Cap gene transcription is driven by the p40 promoter. Helper viral genes used in AAV vector production can also produce toxic products. Commonly used helper genes in AAV production include E1 (E1A and E1B), E2A, E4, and VA RNA. Furthermore, polymerases (pol), histocyte antigens (gag), and envelope proteins required for the proliferation of viral vectors, such as lentiviral or adenoviral vectors, become toxic over time when expressed in viral vector-producing cells. The env protein is essential for viral envelope formation. Gag is an abbreviation for histocyte antigen (ag). Histocyte antigens form the viral core structure, are RNA genome-binding proteins, and are the main proteins containing the nucleoprotein core particle. Pol (reverse transcriptase) is an enzyme essential for reverse transcription, which converts the RNA genome into a pre-integrated double-stranded DNA form. The pol gene further encodes integrase activity and RNase H activity that function during genome reverse transcription. When expressed over time, tat and rev proteins can also become toxic to cells.

[0863] As described above, the expression of the Rep protein is controlled by a second regulated promoter or a regulated transcription factor. In one embodiment, the second regulated promoter is an inducible promoter or a binding site containing a regulated transcription activator.

[0864] In one embodiment, the nucleic acid or individual nucleic acids encoding the Rep protein are operatively linked to an inducible promoter. Suitable inducible promoters are described elsewhere herein.

[0865] In one embodiment, the nucleic acid encoding the Rep protein, or the individual nucleic acids thereon, is operatively linked to a promoter containing a target site for binding a regulated transcriptional activator. Suitable regulated transcriptional activators are described elsewhere herein, such as zinc finger transcriptional activators (ZF-TA).

[0866] In one embodiment, the first Rep protein and the second Rep protein are encoded by nucleic acid sequences, optionally by one or more nucleic acid sequences. In one embodiment, the first Rep protein is Rep78 and the second Rep protein is Rep52. In one embodiment, the nucleic acids or individual nucleic acids encoding the first and second Rep proteins are under the control of a second regulated promoter or a regulated transcription activator. In one embodiment, the individual nucleic acids encoding the first and second Rep proteins are under the control of a regulated transcription activator. In one embodiment, the individual nucleic acids encoding the first and second Rep proteins are under the control of the same regulated transcription activator. In one embodiment, the transcription activator is a zinc finger transcription activator (ZF-TA).

[0867] In one embodiment, the nucleic acid encoding the Rep protein includes a modified p19 promoter. In one embodiment, the nucleic acid encoding the Rep78 protein includes a modified p19 promoter. In one embodiment, the modified p19 promoter is modified to attenuate expression. In one embodiment, the modified p19 promoter includes one or more mutations.

[0868] In one implementation, the modified p19 promoter comprises the following nucleic acid sequence:

[0869]

[0870] In one embodiment, the nucleic acid sequence encoding a Rep protein includes a modified start codon. In one embodiment, the nucleic acid sequence encoding a large Rep protein (e.g., Rep78 protein) includes a modified start codon. In one embodiment, the nucleic acid sequence encoding a large Rep protein (e.g., Rep78 protein) includes a modified start codon selected from ACC, AUC, CUG, and AGG. In such embodiments, the nucleic acid sequence encoding a small Rep protein (e.g., Rep52) includes a typical start codon. In such embodiments, the nucleic acid sequence encoding a small Rep protein (e.g., Rep52 protein) includes the ATG start codon.

[0871] In one implementation, the nucleic acid sequence encoding a large Rep protein (e.g., Rep78 protein) contains the CUG start codon.

[0872] In one embodiment, the nucleic acid encoding the modified Rep protein containing the modified start codon comprises the following sequence, wherein the modified start codon is shown in bold:

[0873]

[0874] In one embodiment, the Rep protein is a modified Rep protein. The modified Rep protein is Rep78. In one embodiment, the modified Rep protein has a lysine-to-arginine mutation at amino acid position 84. Those skilled in the art can generate the modified Rep protein using a assay based on standard site-directed mutagenesis PCR. DNA sequencing can be used to detect Rep proteins with, for example, amino acid substitutions.

[0875] In one embodiment, the modified Rep protein is a modified Rep78 and contains the following sequence, wherein the lysine-to-arginine mutation is shown in bold:

[0876]

[0877] In one embodiment, the nucleic acid encoding the Rep protein further includes a nucleic acid encoding a ribozyme protein located at its 3' end.

[0878] In any embodiment of any aspect, the Rep protein or individual Rep proteins may include any combination of the above features to optimize expression.

[0879] In one embodiment, the Rep protein comprises a modified p19 promoter and an amino acid substitution mutation. In any embodiment, the Rep protein comprises a modified p19 promoter and a nucleic acid encoding a ribozyme located at its 3' end. In one embodiment, the Rep protein comprises a modified start codon and a modified p19 promoter. In one embodiment, the Rep protein comprises a modified start codon and a nucleic acid encoding a ribozyme located at its 3' end. In one embodiment, the Rep protein comprises a modified start codon and an amino acid substitution. In one embodiment, the Rep protein comprises an amino acid substitution and a nucleic acid encoding a ribozyme located at its 3' end.

[0880] In any embodiment of any aspect, the Rep protein comprises an amino acid substitution, is a modified Rep protein, and further comprises nucleic acid encoding a ribozyme at its 3' end. In any embodiment of any aspect, the Rep protein comprises a modified start codon, an amino acid substitution, and further comprises nucleic acid encoding a ribozyme at its 3' end. In any embodiment of any aspect, the Rep protein comprises an amino acid substitution, comprises a modified p19 promoter, and comprises a modified start codon. In any embodiment of any aspect, the Rep protein comprises a modified p19 promoter, a modified start codon, and nucleic acid encoding a ribozyme at its 3' end.

[0881] In one embodiment, the nucleic acid encoding the Rep protein, which includes a modified start codon, amino acid substitutions, and a modified p19 promoter, comprises the following sequences, wherein these features are shown consecutively in bold:

[0882]

[0883]

[0884]

[0885] In one embodiment, the Rep protein, which includes a modified start codon, amino acid substitutions, and a modified p19 promoter, comprises the following sequences:

[0886]

[0887] In any embodiment of any aspect, the Rep protein comprises a modified start codon, a modified p19 promoter, is a modified Rep protein, and further comprises a nucleic acid encoding a ribozyme located at its 3' end.

[0888] In any embodiment of any aspect, the Rep protein comprises a CUG start codon, a modified p19 promoter, a lysine-to-arginine mutation at amino acid position 84, and further comprises a nucleic acid encoding a ribozyme located at its 3' end.

[0889] In one implementation, large Rep (e.g., Rep78) and small Rep (e.g., Rep52) are controlled by individual or different tunable elements, such that the expression of each Rep protein is independent of each other. In this manner, the expression of small Rep can be easily controlled.

[0890] In one embodiment, the toxic protein is any viral protein known in the art (e.g., any component or product of a virus). Viral proteins include, but are not limited to, any structural, non-structural, regulatory, and accessory proteins of any virus. In one embodiment, the toxic protein is a capsid protein, envelope protein, membrane fusion protein, non-structural protein, or viral accessory protein. In one embodiment, the toxic protein is the Rep protein as explained above.

[0891] Viral membrane fusion proteins are classified into four distinct classes, each identified by a characteristic structural conformation: (Class I) fusion conformations possess a distinctive central coiled-coil structure, consisting of a characteristic trimer of α-helical hairpins (e.g., HIV glycoprotein, gp41); (Class II) lack the central coiled-coil structure and contain a characteristic elongated β-sheet extracellular domain that refolds to form a hairpin trimer (e.g., dengue virus E protein and West Nile virus E protein); (Class III) structural conformations are a combination of features from Class I and Class II viral membrane fusion proteins (e.g., rabies virus glycoprotein G); and (Class IV) viral fusion proteins are fusion-associated small transmembrane (FAST) proteins and do not themselves form hairpin trimers or hairpin structures. FAST proteins are encoded by members of the non-enveloped reoviridae family of viruses.

[0892] Viral nonstructural proteins are proteins encoded by the viral genome and expressed in infected cells. However, these proteins do not assemble within the viral particle; rather, they perform important functions influencing replication and assembly processes. Some viral nonstructural proteins function as transactivation of genes encoding viral structural proteins, replicon formation, and immune regulation. For example, in hepatitis C virus, viral nonstructural proteins interact with the cell vesicle membrane transport protein hVAP-33 to assemble replicons. The viral nonstructural 4b (NS4B) protein alters the host cell membrane and initiates the formation of the replication complex. Other viral nonstructural proteins (such as NS5A, NS5B, and NS3) are also recruited into the complex, with which NS4B interacts and binds to viral RNA. Exemplary immunomodulatory proteins include the viral nonstructural protein NS1 in West Nile virus, which prevents complement activation by binding to the complement control protein factor H.

[0893] Viral accessory proteins (also known as helper proteins) are encoded by the genome of retroviruses. Most viral accessory proteins perform their functions only in specific types of cells and do not have a significant impact on viral replication. However, in some cases, the function of viral accessory proteins is required to maintain viral replication.

[0894] In one implementation, the toxic gene is a Cas protein. Exemplary Cas proteins include, but are not limited to: Cas9 (also known as Csn1 and Csx12), And Cas13c.

[0895] In one implementation, the Cas protein is Cas9 or a variant of Cas9 (e.g., SpCas9 isolated from the bacterium *Streptococcus pyogenes*). A CRISPR-associated nuclease binds to a guide RNA (gRNA), which directs the nuclease to a desired target sequence (e.g., a preprotospacer adjacent motif (PAM) sequence located downstream of the target sequence) for its cleavage. Once Cas9 recognizes the PAM sequence (5'-NGG-3 in the case of SpCas9, where N is any nucleotide), it creates a double-strand break (DSB) at the target locus. Cas9 activity is a collaborative effort of two parts of the protein: the recognition leaf that senses the complementary sequence of the gRNA and the nuclease leaf that cleaves the DNA.

[0896] In one implementation, the Cas protein is an enhanced-specific spCas9 (eSpCas9) variant. The eSpCas9 variant is further described in Slaymaker et al., Science. 2016; 351(6268): 84-88, which is incorporated herein by reference in its entirety.

[0897] In one implementation, the Cas protein is a natural Cas variant. Cas9 variants include, for example, Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), and Campylobacter jejuni (CjCas9), which are used in CRISPR experiments (to name only). The nuclease can be determined based on a preferred PAM sequence or size; for example, the SaCas9 nuclease is about 1 kb smaller than SpCas9, making it easier to package into viral vectors. CasX and CasY (Burstein, David, et al., New CRISPR-Cas systems from uncultivated microbes. Nature 542.7640 (2017): 237, incorporated herein by reference in its entirety) are two of the closest naturally occurring CRISPR variants.

[0898] Cas9 sequences from various species are known in the art. For example, Staphylococcus aureus Cas9 (saCas9) has the sequence SEQ ID NO: 154.

[0899] SEQ ID NO: 154 is the amino acid sequence encoding Staphylococcus aureus Cas9.

[0900]

[0901] In one embodiment, the Cas protein is Cas9 derived from Campylobacter jejuni. This Campylobacter jejuni Cas9 (CjCas9) is further described, for example, in International Patent Application WO2016021973A1, which is incorporated herein by reference in its entirety.

[0902] SEQ ID NO: 155 is the amino acid sequence encoding CjCas9.

[0903]

[0904]

[0905] In one implementation, the Cas protein is Cas12a (also known as Cpf1). Because Cas9 requires a guanine-rich PAM sequence from NGG, it is less suitable for targeting AT-rich sequences. Zetsche et al. characterized a nuclease (see, for example, the sequence and variants in US Patent Application US 2016 / 0208243, which is incorporated herein by reference in its entirety) that, when targeting AT-rich DNA sequences, CRISPR (Cpf1; now classified as Cas12a) from *Prevotella* and *Francisella* 1 can be used. Cpf1 creates staggered double-strand cuts in the target DNA, unlike the blunt-end cuts produced by SpCas9, and is useful for experiments dependent on HDR repair outcomes. Furthermore, Cpf1 is smaller than SpCas9 and does not require a tracer RNA. Therefore, the guide RNA required for Cpf1 is shorter, making its production more economical.

[0906] The Cpf1 sequence of many species is known in the art. For example, the Cpf1 of *Acidaminococcus* sp. has the sequence SEQ ID NO: 156.

[0907] SEQ ID NO: 156 is the amino acid sequence encoding the amino acid Cpf1 of the genus C.

[0908]

[0909] In one implementation, the Cas protein is an engineered variant of Cas9 (e.g., Cas9 cleavage enzyme, or dead Cas9 used in CRISPRi or CRISPRa systems). For example, variants that create nicks in a single DNA strand instead of producing double-strand breaks (see, for example, Cong, Le, et al., Multiplex genome engineering using CRISPR / Cassystems. Science (2013): 1231143; Mali, Prashant, et al., CAS9 transcriptionalactivators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology 31.9 (2013): 833; Ran, F. Ann, et al., Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154.6 (2013): 1380-1389; Cho, Seung Woo, et al., Analysis of off-target effects of CRISPR / Cas-derived RNA-guided endonucleases and nickases. Genome research 24.1 (2014): 132-141; each incorporated in its entirety by reference). In some implementations, two guide RNAs are used in conjunction with nCAS9. Alternatively, eSpCas9, which utilizes a single gRNA, can be used. Although nickases exhibit high specificity, they rely on two guide RNAs to reach the target site, thus reducing the number of potential target sites in the genome. An alternative has been created with an engineered version of Cas9 that uses a single guide RNA to improve fidelity (see, for example, Qi, Lei S et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152.5 (2013): 1173-1183, which is incorporated herein by reference in its entirety).

[0910] In one implementation, the Cas protein is SpCas9-HF1 or HypaCas9Kleinstiver (see, for example, Benjamin P. et al., High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529.7587 (2016): 490; Chen, Janice S. et al., Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550.7676 (2017): 407, both of which are incorporated herein by reference in their entirety).

[0911] In one implementation, the Cas protein is the xCas9 nuclease, which recognizes a wide range of PAM sequences, increasing the number of target sites in the genome to one-quarter (see, for example, Hu, Johnny H, et al., Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature (2018), which is incorporated herein by reference in its entirety).

[0912] In one implementation, the Cas protein is a split Cas9 that can be fused with a fluorescent protein such as GFP. This would allow imaging of genomic loci (see “Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System” Chen B et al., Cell, 2013), but in an inducible manner. Therefore, in some implementations, one or more of the Cas9 moieties can be associated (particularly fused) with a fluorescent protein such as GFP. Generally, any use that can be made from Cas9 (whether wt, nickase, or dead-Cas9 (with or without associated functional domains)) can be performed using the split Cas9 method.

[0913] In one implementation, the Cas protein is a dimerized CRISPR RNA-guided Fokl nuclease (see, for example, Tsai SG et al., Nat Biotechnol. 2014. 32(6): 569-576, which is incorporated herein by reference in its entirety).

[0914] In one implementation, the Cas protein is inactive Cas9, dead Cas9 (also known as dCas9). The dead Cas9 (dCas9) CRISPR variant is obtained by simply inactivating the catalytic nuclease domain while retaining the recognition domain, which allows guide RNA-mediated targeting of specific DNA sequences (Komor, Alexis C, et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533.7603 (2016): 420, incorporated herein by reference in its entirety). dCas9 is known to silence gene expression by physically blocking transcription. dCas9 is also fused with other proteins and used in a variety of applications. For example, gene activators or inhibitors can be fused with dCas9 to activate or repress gene expression (CRISPRa and CRISPRi). Furthermore, labeling dCas9 with fluorescent dyes allows for the visualization of specific DNA fragments in the genome (Gaudelli, Nicole M, et al., Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage, Nature 551.7681 (2017): 464, which is incorporated herein by reference in its entirety). In one implementation, dCas9 fused with FokI is used (Abudayyeh, Omar O., et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 353.6299 (2016): aaf557314, which is incorporated herein by reference in its entirety).

[0915] In one implementation, the inactivated Cas protein is a functional gene-editing nuclease used as a base editor. The base-editing enzyme consists of a dead Cas9 domain fused to the catalytic enzyme cytidine aminoase (which converts GC to AT), or, for example, a tRNA adenosine deaminase fused to Cas9 to convert AT to GC, thereby allowing full-range nucleotide exchange across the genome: see, for example, Komor, Alexis C. et al., Programmable editing of a target basein genomic DNA without double-stranded DNA cleavage Nature 533.7603 (2016):420; Gaudelli, Nicole M. et al., Programmable base editing of A•T to G•C ingenomic DNA without DNA cleavage. Nature 551.7681 (2017):464; both are incorporated herein by reference in their entirety.

[0916] Gene regulatory system

[0917] The various aspects of the invention described herein relate to controlling gene expression using any regulated promoters (e.g., inducible or repressive promoters) presented herein or any regulated transcriptional activators described herein. Regulated repressors are also envisioned for use in controlling gene expression.

[0918] In any embodiment of the invention, the cell may further comprise a synthetic gene regulation system, wherein the synthetic gene regulation system comprises a target DNA-binding protein or a nucleic acid sequence encoding a target DNA-binding protein, operatively linked to a promoter; and a nucleic acid sequence encoding a gene of interest, operatively linked to a target promoter, wherein the target DNA-binding protein is capable of binding the target sequence, wherein the target sequence is located within the target promoter and / or the nucleic acid sequence encoding the gene of interest, thereby regulating or preventing the expression of the gene of interest.

[0919] In one embodiment, a gene regulation system is provided on one or more vectors. In one embodiment, the vector is contained within a cell. Suitable vectors are described elsewhere herein.

[0920] The nucleic acid sequences of synthetic gene regulatory systems can be parts of the same nucleic acid molecule or different nucleic acid molecules. Therefore, correspondingly, one or more nucleic acid sequences of synthetic gene regulatory systems can be contained in the same vector or different vectors.

[0921] Synthetic gene regulation systems can contain different components based on desired targets and outcomes. For example, DNA-binding proteins may be provided as proteins or encoded in nucleic acids, and genes of interest may be provided that are encoded in nucleic acids or already present in the cell. Target sequences may be located within the nucleic acid sequence of a target promoter or encoding a gene of interest.

[0922] In one implementation, the gene of interest is a gene that is desired to be repressed. Therefore, it is desirable to prevent the gene from being expressed in the production cell line. In one implementation, the gene of interest may be a therapeutic gene as described elsewhere herein. Thus, in such an implementation, the gene of interest encoded in nucleic acids can be provided, i.e., it is exogenous to the cell.

[0923] Alternatively, the gene of interest may be an unwanted gene, whose expression product is not needed or desired when expressing the nucleic acids encoding viral proteins / RNA and Rep proteins of the present invention. In one embodiment, as described elsewhere herein, the unwanted gene may be toxic to the cell or the organism containing the cell upon expression. In one embodiment, the unwanted gene may impose a metabolic burden on the cell or the organism containing the cell upon expression. Therefore, in such an embodiment, the unwanted gene may already be present in the cell, i.e., it is endogenous to the cell.

[0924] In one implementation, the nucleic acid sequence of the synthetic gene regulatory system is provided in an AAV vector, and a DNA-binding protein is targeted to regulate or prevent the expression of a gene of interest by the AAV vector. Therefore, the DNA-binding protein downregulates the expression of the gene of interest by the AAV vector. Thus, the gene of interest may be referred to as a "payload gene." Therefore, the DNA-binding protein is targeted to regulate or prevent the expression of the payload gene by the AAV vector. Therefore, the DNA-binding protein downregulates the expression of the payload gene by the AAV vector. Therefore, the "payload gene" may be a therapeutic gene.

[0925] In one embodiment, a DNA-binding protein can prevent or silence the expression of a gene of interest (transcriptional gene silencing) by blocking transcription of the gene. In such embodiments, the DNA-binding protein may be a regulated transcriptional repressor. Alternatively, a DNA-binding protein can prevent or silence the expression of a gene of interest by preventing the elongation of mRNA transcripts to produce incomplete mRNA transcripts and incomplete, truncated, or nonfunctional expression products. In such embodiments, the DNA-binding protein may be a regulated translational repressor.

[0926] In one embodiment, the targeted DNA-binding protein includes a ligand-binding domain. Therefore, the targeted DNA-binding protein may include multiple DNA-binding domains. These multiple DNA-binding domains may optionally be linked by a linker. In one embodiment, the targeted DNA-binding protein may consist of multiple DNA-binding domains. In one embodiment, the targeted DNA-binding protein may consist of multiple linked DNA-binding domains. In one embodiment, the targeted DNA-binding protein may include one or more DNA-binding domains that do not have other functional domains. Therefore, in one embodiment, the targeted DNA-binding protein may not have any other functional domains besides one or more DNA-binding domains. In one embodiment, the targeted DNA-binding protein may consist only of one or more DNA-binding domains, which may optionally be linked by a non-functional linker. In one embodiment, the targeted DNA-binding protein may include one or more DNA-binding domains that do not have other functionalities.

[0927] The DNA-binding domain recognizes and binds to the target sequence. In one embodiment, the DNA-binding domain may comprise at least one of the following: bacterial helix-turn-helix protein, homology domain, wing-helix-turn-helix, ETS domain, helix-turn-helix, basic region-leucine zipper, basic region helix-loop-helix, zinc finger, zinc-binding domain, β-sheet recognition, TATA-binding protein, and Rel homology domain (Garvie & Wolberger, 2001). Alternatively, the DNA-binding domain may comprise a TAL effector central repeat domain (DNA-binding domain), a DNA-binding domain from a restriction enzyme, and a PAM interaction domain from cas9 loaded with guide RNA.

[0928] In one embodiment, the DNA-binding domain comprises at least one zinc finger (ZF) domain. In one embodiment, the DNA-binding domain may comprise at least six ZF domains. In one embodiment, the ZF domains are linked by a linker. In one embodiment, the ZF domain may be a Cys2-His2 domain. Alternatively, the DNA-binding domain may comprise a mixture of domains selected from the group consisting of: bacterial helix-turn-helix proteins, homologous domains, wing-helix-turn-helix, ETS domains, helix-turn-helix, basic region-leucine zipper, basic region helix-loop-helix, zinc finger, zinc-binding domain, β-sheet recognition, TATA-binding protein, Rel homologous domain, TAL effector center repeat domain (DNA-binding domain), DNA-binding domain from restriction enzymes, DNA-binding domain from transcription factors, and PAM interaction domain from cas9 loaded with guide RNA.

[0929] In one embodiment, the targeting DNA-binding domain consists of six ZF domains linked by a adapter. In another embodiment, the targeting DNA-binding domain consists of nine ZF domains linked by a adapter. In yet another embodiment, the targeting DNA-binding domain consists of ten ZF domains linked by a adapter. In yet another embodiment, the targeting DNA-binding domain consists of thirteen ZF domains linked by a adapter. In yet another embodiment, the targeting DNA-binding domain consists of fourteen ZF domains linked by a adapter.

[0930] In one implementation, a targeting DNA-binding protein binds to a target sequence within a target promoter, the target promoter being operatively linked to a nucleic acid sequence encoding a gene of interest and / or a nucleic acid sequence within the nucleic acid sequence encoding a gene of interest.

[0931] In one implementation, the targeting DNA-binding protein may be adapted to recognize a target sequence and, after binding to the target sequence, prevent the expression of the gene of interest.

[0932] In one implementation, the target sequence is located within the target promoter. It is not desirable to be theoretically bound by the expectation that targeting the DNA-binding protein to bind to the target sequence within the target promoter will prevent transcription of the gene of interest, because targeting the DNA-binding protein will prevent the binding and function of DNA polymerases, transcription factors, and / or transcription mechanisms (i.e., transcription will not be initiated).

[0933] In one implementation, the target sequence may be located within the nucleic acid sequence encoding the gene of interest. Unintentionally, it is anticipated that the target DNA-binding protein binding to the target sequence within the gene of interest will prevent the elongation of the mRNA of the gene of interest (preventing the elongation phase of transcription), resulting in incomplete, non-functional mRNA of the gene of interest. Regardless of whether the target sequence is located within the target promoter of the gene of interest and / or within the nucleic acid sequence encoding the gene of interest itself, it is anticipated that the target DNA-binding protein binding to the target sequence will cause downregulation of the expression of the gene of interest.

[0934] Furthermore, the target sequence may be located within an untranslated region (e.g., the 5' UTR). The target sequence may span the 3' end of the target promoter and the 5' end of the nucleic acid sequence encoding the gene of interest. In one embodiment, the target sequence may be partially located within the target promoter and partially within an untranslated region or partially within the nucleic acid sequence encoding the gene of interest.

[0935] In one implementation, a targeted DNA-binding protein can modulate the expression of a gene of interest by downregulating its expression. This can be achieved by blocking the expression of some (but not all) of the genes of interest.

[0936] In one implementation, the targeting DNA-binding protein downregulates the expression of the gene of interest by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more compared to a suitable control. As described herein, a suitable control refers to the expression of the gene of interest in cells that have not been exposed to the targeting DNA-binding protein.

[0937] Alternatively, targeting DNA-binding proteins can silence the expression of genes of interest. This can be achieved, for example, by preventing any expression of the gene of interest.

[0938] A promoter operably linked to a nucleic acid sequence encoding a target DNA-binding protein may be different from the target promoter, or a promoter operably linked to a nucleic acid sequence encoding a target DNA-binding protein may be the target promoter. When a promoter operably linked to a nucleic acid sequence encoding a target DNA-binding protein is different from the target promoter, it is referred to herein as an "expression promoter".

[0939] In one embodiment, the expression promoter may be a constitutive promoter. Constitutive expression promoters may be selected from: CMV-IE, EF1a, SV40, PGK1, CAG, ...

Claims

1. A nucleic acid construct, said nucleic acid construct comprising: A nucleic acid sequence comprising at least one of the following: a nucleic acid sequence encoding an E4 protein, a nucleic acid sequence encoding an E2A protein, and a nucleic acid sequence encoding VA RNA, wherein, Each nucleic acid sequence encoding any one of the E4 protein, E2A protein, and VA RNA is operatively linked to a first regulated promoter; as well as The nucleic acid sequence encoding the Rep protein, wherein the nucleic acid encoding the Rep protein is under the control of a second regulated promoter or a heterologous transcription activator. The first adjustable promoter and the second adjustable promoter are different.

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