Method to treat disease using nucleic acid vector encoding highly compact multi-input logic gate

Abstract

To provide contiguous DNA sequences encoding highly compact multi-input genetic logic gates for precise in vivo cell targeting, and methods of treating disease using a combination of in vivo delivery and such contiguous DNA sequences.SOLUTION: A contiguous polynucleic acid molecule encoding at least two cassettes is provided, where each cassette comprises a regulatory component and a response component.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field Disclosed herein are contiguous DNA sequences that encode highly compact multi-input genetic logic gates for precise in vivo cellular targeting, and methods of using a combination of in vivo delivery and such contiguous DNA sequences to treat disease. [Background technology]

[0002] background Gene therapy is emerging as a next-generation treatment option for genetic diseases and cancer. However, current gene therapy vectors suffer from low efficacy, high toxicity, and long development timelines to generate therapeutic leads. One reason for these shortcomings is the lack of strict control of therapeutic gene expression in gene therapy vectors, which leads to (i) gene expression in unintended cell types and tissues or (ii) either insufficient or too high dosages. In other words, precise control of gene expression, both in terms of gene product dosage (i.e., number of protein molecules per cell) and cell-type restricted expression, remains an unchallenged challenge in gene therapy. Summary of the Invention

[0003] Engineering contiguous DNA molecules that contain the multiple components required for multifunctional cellular probes and generate appropriate therapeutic effects is an extremely challenging task even when the original building blocks are partially known. This disclosure describes an approach to engineer contiguous DNA molecules that encode complex multi-input genetic logic circuits that can simultaneously probe multiple transcription factors and / or promoter activities, and optionally microRNA function. In light of this, the contiguous molecules are suitable for implementation in vectors with low packaging capacity but high therapeutic value (e.g., AAV, lentivirus, adenovirus), a wide variety of viral vectors including non-replicating and replicating viruses, and non-viral delivery vectors. The resulting viral and non-viral delivery vectors can be used to selectively target specific cell types or cell states both in vivo and in vitro and used as therapeutics.

[0004] In some aspects, the present disclosure relates to a contiguous polynucleic acid molecule encoding at least two cassettes, each cassette comprising a regulatory element and a response element.

[0005] In some embodiments, (i) at least one cassette comprises a 5' regulatory element comprising a transactivator response element, and a 3' response element comprising an output; and (ii) at least one cassette comprises a 5' regulatory element, and a 3' response element comprising a nucleic acid sequence encoding a transactivator protein; and wherein the transactivator of (ii) binds to and transactivates the transactivator response element of (i) when expressed as a protein. In some embodiments, the transactivators independently bind to and transactivate a transactivator response element.

[0006] In some embodiments, the 5' regulatory element of the cassette in (i) further comprises a transcription factor response element and / or a minimal promoter. In other embodiments, the transactivator binds to and transactivates the transactivator response element only in the presence of a transcription factor bound to the transcription factor response element.

[0007] In some embodiments, the 5' regulatory elements include, from 5' to 3', a transactivator response element, a transcription factor response element, and a minimal promoter. In some embodiments, the 5' regulatory elements include, from 5' to 3', a transcription factor response element, a transactivator response element, and a minimal promoter.

[0008] In some embodiments, the 5' regulatory element in (i) further comprises a promoter element. In some embodiments, the promoter element comprises a mammalian promoter or promoter fragment. In some embodiments, the 5' regulatory elements include, from 5' to 3', a transactivator response element and a promoter element, and optionally, a minimal promoter.

[0009] In some embodiments, the 5' regulatory component of the cassette in (ii) comprises a promoter element. In some embodiments, the promoter element comprises a transcription factor response element and a minimal promoter, and optionally, the transcription factor response element is unique. In some embodiments, the promoter element comprises a mammalian promoter or promoter fragment, and optionally, a minimal promoter.

[0010] In some embodiments, at least one cassette in (i) and at least one cassette in (ii) are in a convergent orientation. In some embodiments, at least one cassette in (i) and at least one cassette in (ii) are in a divergent orientation. In some embodiments, at least one cassette in (i) and at least one cassette in (ii) are in a head-to-tail orientation.

[0011] In some embodiments, the 3' response element of the cassette in (i) further comprises at least one microRNA target site. In some embodiments, the at least one microRNA target site is 3' to the output. In some embodiments, the at least one microRNA target site is 5' to the output or within the output.

[0012] In some embodiments, the cassette in (ii) comprises at least one microRNA target site. In some embodiments, the at least one microRNA target site is 3' to the DNA sequence encoding the transactivator protein. In some embodiments, the at least one microRNA target site is 5' to the DNA sequence encoding the transactivator protein or within the DNA sequence encoding the transactivator protein.

[0013] In some embodiments, the at least one microRNA target site of the cassette in (i) and the at least one microRNA target site of the cassette in (ii) are the same nucleic acid sequence or are different sequences that are regulated by the same microRNA.

[0014] In some embodiments, at least one cassette is adjacent to an insulator. In some embodiments, the transactivator of at least one cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA.

[0015] In some embodiments, the output is a protein or RNA molecule. In some embodiments, the output is a therapeutic agent. In some embodiments, the output is a fluorescent protein, a cytotoxin, an enzyme that catalyzes the activation of a prodrug, an immunomodulatory protein and / or RNA, a DNA modifier, a cell surface receptor, a gene expression regulator, a kinase, an epigenetic modifier, and / or a factor required for vector replication. In some embodiments, the immunomodulatory protein and / or RNA is a cytokine or a colony stimulating factor.

[0016] In some embodiments, the DNA modifying factor is a gene encoding a protein intended to correct genetic defects, a DNA modifying enzyme, and / or a component of a DNA modifying system. In some embodiments, the DNA modifying enzyme is a site-specific recombinase, a homing endonuclease, or a protein component of a CRISPR / Cas DNA modifying system. In some embodiments, the gene expression regulator is a protein capable of controlling gene expression, or a component of a multi-component system capable of controlling gene expression.

[0017] In some aspects, the disclosure relates to a contiguous polynucleic acid molecule encoding at least one cassette, wherein the cassette comprises: (i) a 5' regulatory element comprising a transactivator response element; and (ii) a 3' response element comprising an output, a transactivator, and an optional polycistronic expression element, wherein the output and the transactivator are optionally separated by the polycistronic expression element; wherein transcription of the response element produces a single mRNA; and wherein the transactivator of (ii), when expressed as a protein, binds to and transactivates the transactivator response element of (i).

[0018] In some embodiments, the transactivators independently bind to and transactivate a transactivator response element. In some embodiments, the 5' regulatory element in (i) further comprises a transcription factor response element and / or a minimal promoter. In some embodiments, the transactivator binds to and transactivates the transactivator response element only in the presence of a transcription factor bound to the transcription factor response element.

[0019] In some embodiments, the 5' regulatory elements include, from 5' to 3', a transactivator response element, a transcription factor response element, and a minimal promoter. In some embodiments, the 5' regulatory elements include, from 5' to 3', a transcription factor response element, a transactivator response element, and a minimal promoter.

[0020] In some embodiments, the 5' regulatory element in (i) further comprises a promoter element. In some embodiments, the promoter element comprises a mammalian promoter or promoter fragment. In some embodiments, the 5' regulatory element in (i) comprises, from 5' to 3', a transactivator response element and a promoter element.

[0021] In some embodiments, the 3' response element of (ii) further comprises at least one microRNA target site. In some embodiments, the at least one microRNA target site is 3' to the output and / or transactivator. In some embodiments, the at least one microRNA target site is 5' to the output and / or transactivator or is internal to the output and / or transactivator.

[0022] In some embodiments, at least one cassette is adjacent to an insulator. In some embodiments, the transactivator of at least one cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA.

[0023] In some embodiments, the output is a protein or an RNA molecule. In some embodiments, the output is a therapeutic protein or an RNA molecule. In some embodiments, the output is a fluorescent protein, a cytotoxin, an enzyme that catalyzes the activation of a prodrug, an immunomodulatory protein and / or an RNA, a DNA modifier, a cell surface receptor, a gene expression regulator, a kinase, an epigenetic modifier, and / or a factor required for vector replication. In some embodiments, the immunomodulatory protein and / or an RNA is a cytokine or a colony stimulating factor.

[0024] In some embodiments, the DNA modifying factor is a gene encoding a protein intended to correct genetic defects, a DNA modifying enzyme, and / or a component of a DNA modifying system. In some embodiments, the DNA modifying enzyme is a site-specific recombinase, a homing endonuclease, or a protein component of a CRISPR / Cas DNA modifying system. In some embodiments, the gene expression regulator is a protein capable of controlling gene expression, or a component of a multi-component system capable of controlling gene expression.

[0025] In some aspects, the present disclosure relates to a vector comprising a contiguous polynucleic acid molecule as described above.

[0026] In some aspects, the present disclosure relates to an engineered viral genome comprising a continuous polynucleic acid molecule as described above.In some embodiments, the viral genome is an adeno-associated virus (AAV) genome, a lentivirus genome, an adenovirus genome, a herpes simplex virus (HSV) genome, a vaccinia virus genome, a poxvirus genome, a Newcastle disease virus (NDV) genome, a coxsackievirus genome, a reovirus genome, a measles virus genome, a vesicular stomatitis virus (VSV) genome, a parvovirus genome, a Seneca Valley virus genome, a Maraba virus genome, or a common cold virus genome.

[0027] In some aspects, the present disclosure relates to a virion comprising an engineered viral genome as described above. In some aspects, the present disclosure relates to a method of stimulating a cell-specific event in a population of cells. In some embodiments, the method comprises contacting a population of cells with a sequential polynucleic acid molecule as described above, a vector as described above, an engineered viral genome as described above, or a virion as described above.

[0028] In some embodiments, the cell-specific event is controlled by an endogenous transcription factor that binds to and transactivates the regulatory element of at least one cassette; and / or the transcriptional activity of the promoter fragment; and / or at least one endogenous microRNA that is complementary to a microRNA target site of the response element of at least one cassette; or, alternatively, is controlled by an endogenous transcription factor that binds to and transactivates a transcription factor response element of the 5' regulatory element of at least one cassette; and / or the transcriptional activity of the promoter fragment; and / or at least one endogenous microRNA that is complementary to a microRNA target site of the 3' response element of at least one cassette.

[0029] In some embodiments, the population of cells comprises at least one target cell and at least one non-target cell. In some embodiments, target cells and non-target cells differ in (i) the protein level or activity of an endogenous transcription factor that binds to and transactivates the regulatory element of at least one cassette; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) the RNA level or activity of at least one endogenous microRNA that is complementary to the microRNA target site of the response element of at least one cassette; and wherein the different protein level or activity in (i), and / or transcriptional activity in (ii), and / or RNA level or activity in (iii) cause the target cells and non-target cells to differ in the expression level of the output of the response element of at least one cassette, thereby stimulating a cell-specific event.

[0030] In some embodiments, target cells and non-target cells differ in (i) the protein level or activity of an endogenous transcription factor that binds to and transactivates a transcription factor response element of a 5' regulatory element of at least one cassette; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) the RNA level of at least one endogenous microRNA that is complementary to a microRNA target site of a 3' response element of at least one cassette; and wherein the different protein levels in (i), and / or transcriptional activity of the promoter fragment in (ii), and / or RNA levels in (iii) cause target cells and non-target cells to differ in the expression levels of the output of the 3' response element of at least one cassette, thereby stimulating a cell-specific event.

[0031] In some embodiments, the expression level of the output of the 3' response element differs between the target cell type and the non-target cell type by at least 2, at least 5, at least 10, at least 100, at least 1,000, or at least 10,000 fold. In some embodiments, the cells of the target cell population are tumor cells and the cell-specific event is cell death. In some embodiments, the tumor cell death is mediated by immune targeting through expression of an activating receptor ligand, a specific antigen, a stimulatory cytokine, or any combination thereof.

[0032] In some embodiments, the cells of the target cell population are senescent cells and the cell-specific event is cell death. In some embodiments, the method further comprises contacting the population of cells with a prodrug or with a non-toxic precursor compound that is metabolized by the output into a therapeutic agent or a toxic compound.

[0033] In some embodiments, cells of the target cell population differentially express a factor relative to a wild-type cell of the same type, and the cell-specific event is modulating the expression level of the factor. In some embodiments, output expression ensures survival of the target cell population, while non-target cells are eliminated due to lack of output expression and in the presence of irrelevant and non-specific cell death inducers.

[0034] In some embodiments, cells of the target cell population comprise a particular phenotype of interest, such that output expression is restricted to cells of this particular phenotype. In some embodiments, the cells of the target cell population are of a selected cell type, and the cell-specific event is encoding a novel function through expression of a gene that is not naturally present or is inactive in the selected cell type.

[0035] In some embodiments, the population of cells comprises a multicellular organism. In some embodiments, the multicellular organism is an animal. In some embodiments, the animal is a human. In some embodiments, the population of cells is contacted ex-vivo. In some embodiments, the population of cells is contacted in-vivo.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure that may be better understood by reference to one or more of these drawings in combination with specific embodiments presented herein. It should be understood that the data illustrated in the drawings are not intended to limit the scope of the present disclosure in any manner. [Brief description of the drawings]

[0037] [Figure 1]1A-1B. FIG. 1A. Overview representing various options of genetic interactions that can be implemented in the continuous DNA molecule described herein. Thin bars and thick annotated features represent various functional DNA-encoded building blocks. Only DNA, microRNA (miR) and protein components are explicitly shown. Blunt arrows represent the repression of gene expression by microRNAs, which occurs at the mRNA level. The mRNA is not explicitly shown, but it is also implied that the presence of microRNA targets such as TX and TY as part of the DNA sequence also leads to the presence of this same target in the transcribed mRNA, which is then targeted by the miRNA input. Open-ended arrows indicate activation of gene expression. Full-ended arrows display gene expression (shown as a direct transition from DNA to protein, no mRNA intermediates are shown). Abbreviations are as follows: TF-A: any transcription factor A; TF-B: any transcription factor B; miR-X: any microRNA X; miR-Y: any microRNA Y; TX: sequence targeted by miR-X; TY: sequence targeted by miR-Y; TF-A-RE: response recognized by any transcription factor A; TF-B-RE: response element B for any transcription factor; AA: auxiliary transactivator protein; AA-RE: response element recognized by auxiliary transactivator; Pmin: minimal promoter with low intrinsic leakage; output: coding gene for any protein or RNA; PR-E: any promoter or promoter fragment. Figure 1B. The presence of microRNA targets according to options 3a and 3b resulted in a very strong downregulation of output expression, i.e., more than 100-fold repression. Ctr miR stands for "control" miRNA, which is a miRNA sequence that does not elicit an effect on either miR-424 or miR-126 targets. The left bar in each grouping represents the output genes that contain the miR-424 target sequence, and the right bar in each grouping represents the output genes that contain the miR-126 target.

[0038] [Diagram 2] Figure 2. Sequential DNA structural variants. A genetic circuit similar to that in Figure 1A is depicted as implemented in a continuous DNA molecule. Each structural variant represents a different multi-input program; divergent and convergent configurations are shown. Abbreviations are as follows: viral sequence L: any sequence that is specific to the viral vector and must be in the vector independent of the genetic circuit payload, including inverted terminal repeats (ITRs), long terminal repeats (LTRs), Psi sequences, packaging signals, genes required for viral replication and packaging in the case of oncolytic vectors, etc.; viral sequence R: same as viral sequence L, but flanking the continuous DNA cassette from the right; polyA: polyadenylation sequence, and 3'-untranslated region (3'-UTR) of the gene; Rep gene: vector-specific gene(s) that trigger viral vector replication; TF-A: any transcription factor A; TF-B: any transcription factor B; miR-X: any microRNA X; miR-Y: any microRNA Y; TX: sequence targeted by miR-X; TY: sequence targeted by miR-Y; TF-A-RE: response element A for any transcription factor; TF-B-RE: response element B for any transcription factor; AA: auxiliary transactivator protein; AA-RE: response element that binds auxiliary transactivator; Pmin: minimal promoter with low intrinsic leakage; Output: coding gene for any protein or RNA; PR-E: any promoter or promoter fragment.

[0039] [Diagram 3]Figure 3A-3B. Figure 3A. Specific implementation compatible with adeno-associated viral vectors (AAV). Divergent and convergent variants were constructed either without a microRNA target or with the microRNA target indicated in the figure. Two different auxiliary transactivators (PIT-RelA fusion and PIT-VP16 fusion) were used. The continuous DNA construct is flanked on both sides by AAV2 ITRs (inverted terminal repeats) required for packaging in AAV virions. Figure 3B. Comparison between convergent and divergent variants in Huh-7 cells. mCherry fluorescence was measured when DNA molecules were transiently transfected into cells. The left two bars compare the convergent and divergent variants without a miRNA target. They showed comparable output expression levels. The right two bars compare the divergent and convergent variants including the miRNA target T-miR-424. The divergent variants clearly showed significantly improved gene expression over the convergent variants.

[0040] [Figure 4]4A-4B. Demonstration of an AAV virion that has implemented a genetic circuit according to the current disclosure. FIG. 4A. Overview of four different sequential DNA cassettes being constructed. Virions containing different DNA cassettes are shaded according to the miRNA target embedded in the cassette (no target; miR-126 target; miR-424 target; miR-122 target. Note that data for virion containing miR-122 target is only shown in FIGS. 6A-6B and 7A-7C). The DNA payload implements the logic mechanism "output" ~ Sox9 / 10 AND Hnf1A / B AND NOT (MiR-X), where X = 126 or 424 or 122; when no target is present, the DNA payload implements the mechanism "output" ~ Sox9 / 10 AND Hnf1A / B. There are two distinct outputs: the mCherry fluorescent reporter, and the enzyme HSV-TK (thymidine kinase) that converts the non-toxic prodrug ganciclovir into a toxic product that leads to cell death. The bar graphs show mCherry expression measured in different cell lines (left to right in each grouping: HepG2, Huh7, HCT-116, Hela) infected with virions carrying the respective DNA payload (circuit vector, left to right: HNF1A / B AND SOX9 / 10, HNF1A / B AND SOX9 / 10 AND NOT(mir-126), HNF1A / B AND SOX9 / 10 AND NOT(mir-424)). The cell lines HepG2 and Huh7 express high levels of Sox9 / 10 and HNF1A / B without expression of either miR-126 or miR-424, and were therefore predicted to result in high output expression. The cell lines HCT-116 and HeLa do not express either Sox9 / 10 or HNF1A / B and were therefore predicted to result in very low output expression. Indeed, in line with predictions, the data showed that virions caused very strong mCherry expression in the cell lines HepG2 and Huh-7, and nearly 1000-fold lower expression in the cell lines HeLa and HCT-116. Figure 4B.The overview on the right shows the logic program that controls the output and leads to cell death when both Sox9 / 10 and HNF1A / B are highly expressed and miRNAs are not expressed. The bar graphs on the left show robust cell death upon infection of HepG2 cells (left bars in each grouping) with the three types of virions, as expected, and negligible cell death in HeLa cells (right bars in each grouping). Circuit vectors, left to right: HNF1A / B AND SOX9 / 10, HNF1A / B AND SOX9 / 10 AND NOT(mir-126), HNF1A / B AND SOX9 / 10 AND NOT(mir-424). The two bars labeled "constitutive cherry" show that cells were not killed by AAV infection but via toxic output. The middle bar graph shows that both cell lines (left: HepG2; right: Hela) were killed by constitutively expressed HSV-TK, and thus the differential effect is attributable to the gene circuit payload.

[0041] [Figure 5A] Figures 5A-5C. Implementation of one embodiment in a lentiviral vector. Figure 5A. Overview of the sequential DNA cassette. Two different insulator pairs as well as an insulator-free construction were performed. [Figure 5B-5C]Figure 5B. Expression of fluorescent output in two cell lines predicted to result in high output expression (HuH-7 and HepG2) and one not predicted to lead to high expression, HCT-116. In general, the results were consistent with the predictions. The insulator pair A1 / A3 showed a good combination of high output expression in the intended cell line and low expression in the "negative" cell line. For each set of bars: left, none; middle, A1 / A3; right, F1 / C3. Figure 5C. Time course of output expression obtained from these integrated vectors in positive and negative cell lines. Good expression stability was observed over 60 days with constructs using the insulator pair A1 / A3. Top line: Huh7; middle line: HepG2; bottom line: HCT-116.

[0042] [Figure 6A] 6A-6B. Demonstration of specific cell targeting in vivo by virions carrying consecutive DNA cassettes. FIG. 6A. Schematic outlined structure of the consecutive DNA cassettes (see also FIG. 4A-4B). The program implemented by the gene circuit is HNF1A / B AND Sox9 / 10 AND NOT(miR-122). This was predicted to result in high expression in HepG2 tumor cells and low expression in mouse liver due to high expression of miR-122 in the liver. AAV-DJ virions were generated with these consecutive cassettes. In addition, consecutive cassettes were generated without miRNA-122 targets. Both consecutive cassette molecules generate mCherry output and serve to assess cell targeting specificity. The experimental workflow is illustrated below the DNA scheme.

[0043] [Figure 6B]Mice were injected with luciferase and YFP modified HepG2 cancer cells into the spleen. The cells spread to the liver and form multiple tumor foci resembling clinical presentations of liver cancer. After tumors were established, virions were injected systemically via the tail vein. After a few days, animals were euthanized and liver tissues as well as implanted tumors were examined for expression of mCherry output protein. Specific expression is achieved when the "yellow" signal, representing tumor cells, colocalizes with the "mCherry" signal, representing circuit output. Microscopic snapshots were taken from representative fresh liver slices. Images show, from top to bottom, phase contrast images of the slices; tumor location; and expression of vector output. From left to right, a vector with constitutive mCherry expression; a vector implementing the program HNF1A / B AND Sox9 / 10; and a vector implementing the program HNF1A / B AND Sox9 / 10 AND NOT(miR-122). The latter two vectors implement divergent orientations in their sequential DNA payloads.

[0044] [Figure 7A] Figures 7A-7C. Demonstration of anti-cancer efficacy of viral vectors carrying gene circuits. Figure 7A. The sequential DNA cassette shown in Figure 6A was modified to contain a gene encoding the HSV-TK enzyme as output. Figures 7A-7B. Tumors were established in mouse livers similar to that described in Figure 6B. AAV-DJ-type vectors were injected systemically twice into the tail vein; GCV administration was started 3 days after the first injection and once a day for the next 15 days.

[0045] [Figure 7B-7C]FIG. 7B. Plot showing tumor burden in whole liver assessed by whole organ bioluminescence at the end of the study. The three groups include mice injected with viral vector alone, those treated with GCV alone, and those treated with a combination of viral injection and GCV. Only in the latter group was tumor size significantly reduced compared to the control. The images on the right substantiate this claim. FIG. 7C. The column "Total Liver Bioluminescence" shows the luminescence signal from the whole liver after mortem. The second column shows phase images of representative liver slices. The third column shows a representative slice of fresh liver with signals indicating tumor foci. As expected, only mice treated with viral vector and GCV exhibited significantly reduced tumor burden. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Detailed Description Classifier gene circuits are artificial genetic networks or circuits (engineered sets of interacting genes and genetic elements) that can transduce specific combinations of cytoplasmic molecular features into specific cellular responses according to programmable rules, e.g., activating genes only in cells where certain molecules are absent or present (Xie Z. et al., Science. 2011 Sep 2; 333(6047): 1307-11; Benenson Y., Nat. Rev. Genet. 2012 Jun 12; 13(7): 455-468). The ability to precisely control cellular behavior holds great promise for research, biotechnology and biomedical applications.

[0047] However, the potential of this technology for therapeutic applications is hampered by the size and complexity of the genetic circuitry required to perform therapeutically relevant actions, and by the fact that such circuits are inherently incompatible with therapeutically accepted modalities such as viral vectors and engineered cells. Instead, most, if not all, reports of classifier circuits disclose sets of plasmids delivered to cultured cells using transient transfection rather than by viral carriers with potential for medical practical applications.

[0048] Indeed, the medical translation of these developments requires efficient DNA delivery to somatic cells in vivo, which is currently performed with the aid of specialized viral vectors, and increasingly non-viral delivery vectors, selected based on the route of administration, the tissue to be targeted, and other specific requirements. Combining existing classifier circuit technologies with these vectors remains a significant challenge. As the circuit complexity (number of input features and number of circuit genes) required for specific cell targeting increases, packaging the circuit into a viral carrier becomes increasingly difficult for at least two reasons.

[0049] First, any viral delivery vehicle has a limited cargo capacity, making it difficult to accommodate all the genetic components required for circuit functionality and therapeutic output of interest. Similarly, the performance of non-viral vectors may deteriorate as DNA size increases due to the increasing size of the particle complex formed between DNA and packaging material.

[0050] Second, the risk of context effects (e.g., transcriptional read-through, promoter context, junction composition) increases with the number of independent transcription units on the same vector. Context effects are difficult to predict and may affect circuit performance or even collectively alter predicted behavior.

[0051] Disclosed herein are sequential polynucleic acid molecules encoding classifier genetic circuits that are compatible with commonly used gene therapy viral and non-viral vectors (Figure 1). Also disclosed herein are methods for implementing complex multi-input control over the expression of genes of interest in populations of cells.

[0052] Composition of a continuous polynucleic acid molecule In some aspects, the present disclosure relates to a continuous polynucleic acid molecule comprising at least one expression cassette. As used herein, the term "continuous polynucleic acid molecule" refers to a single continuous nucleic acid molecule (i.e., each expression cassette is encoded on a single polynucleic acid molecule) or two complementary continuous nucleic acid molecules (i.e., each expression cassette is encoded on a double-stranded polynucleic acid molecule comprising two complementary strands). In some embodiments, the continuous polynucleic acid is RNA (e.g., single-stranded or double-stranded). In some embodiments, the continuous polynucleic acid is DNA (e.g., single-stranded or double-stranded). In some embodiments, the continuous polynucleic acid is a DNA:RNA hybrid.

[0053] In some embodiments, the continuous polynucleic acid molecule comprises at least two cassettes. In some embodiments, the two cassettes are in divergent orientation. The term "divergent orientation" as used herein refers to the following configuration: (i) the transcription of the first cassette and the second cassette proceeds on different strands of the continuous polynucleic acid molecule, and (ii) the transcription of the first cassette is directed away from the second cassette, and the transcription of the second cassette is directed away from the first cassette. Figure 2 provides examples of various divergent configurations.

[0054] In some embodiments, the two cassettes are in convergent orientation. As used herein, the term "convergent orientation" refers to the following configuration: (i) the transcription of the first cassette and the second cassette proceeds on different strands of a continuous polynucleic acid molecule, and (ii) the transcription of the first cassette is directed toward the second cassette, and the transcription of the second cassette is directed toward the first cassette. In some embodiments, the two convergent cassettes have a polyadenylation sequence in common. Figure 2 provides examples of various convergent configurations.

[0055] In some embodiments, at least two cassettes are in a head-to-tail orientation. As used herein, the term "head-to-tail" refers to the following configuration: (i) transcription or translation of the first cassette and the second cassette proceed on the same strand of a continuous polynucleic acid molecule, and (ii) transcription or translation of the first cassette is directed toward the second cassette and transcription or translation of the second cassette is directed away from the first cassette (5'...→...→...3').

[0056] As used herein, the terms "expression cassette" or "cassette" are used interchangeably and refer to a polyacetate containing at least one control element and at least one response element, where the control element regulates transcription of the response element, RNA levels of the response element, and / or protein production from the response element.

[0057] In some embodiments, at least one cassette of the consecutive polynucleic acid molecule is flanked by an insulator.An insulator is a nucleic acid sequence that, when bound by an insulator-binding protein, protects a control or response element from the effect of other nearby control elements.For example, flanking consecutive polynucleic acid molecules can protect each cassette from the effect of the control elements of other cassettes.Examples of insulators are known to those skilled in the art.

[0058] Control components The cassette of the continuous polynucleic acid molecule comprises at least one control element. The control element may comprise one or more of a transactivator response element, a transcription factor response element, a promoter element, or a minimal promoter. Those skilled in the art will understand that these elements may be oriented in various configurations. For example, the transactivator response element may be 5' or 3' to the promoter element and / or the transcription factor response element; the transcription factor response element may be 5' or 3' to the promoter element and / or the transactivator response element; the promoter element may be 5' or 3' to the transcription factor response element and / or the transactivator response element.

[0059] The term "transactivator" or "transactivator protein" as used herein refers to a protein encoded on a contiguous polynucleic acid molecule that transactivates expression of an output (i.e., a gene of interest) and binds to a transactivator response element operably linked to a nucleic acid sequence encoding the output (i.e., a gene of interest). A transactivator response element is "operably linked" to a nucleic acid encoding an output when it is in the correct functional position and orientation relative to the nucleic acid sequence that it controls to control ("drive") transcription initiation and / or expression of that sequence. In some embodiments, a transactivator binds to and transactivates a transactivator response element independently (i.e., in the absence of any additional factors). In other embodiments, a transactivator binds to and transactivates a transactivator response element only in the presence of a transcription factor bound to the transcription factor response element.

[0060] In some embodiments, the transactivator protein comprises a DNA binding domain.In some embodiments, the DNA binding domain is engineered to bind to a DNA sequence that is different from naturally occurring sequence (i.e., not naturally occurring).Examples of DNA binding domains are known to those skilled in the art, and include, but are not limited to, the DNA binding domain that is derived from using zinc finger technology or TALEN technology or from the mutant response regulator of two-component signaling pathway from bacteria.

[0061] In some embodiments, the DNA binding domain is derived from a mammalian protein. In other embodiments, the DNA binding domain is derived from a non-mammalian protein. For example, in some embodiments, the DNA binding domain is derived from a protein originating from bacteria, yeast, or plants. In some embodiments, the DNA binding domain requires additional components (e.g., protein or RNA) to target transactivator response elements. For example, in some embodiments, the DNA binding domain is that of a CRISPR / Cas protein (e.g., Cas1, Cas2, Cas3, Cas5, Cas4, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, Cpf1, C2c1, C2c2, C2c3) that requires additional components of guide RNA to target transactivator response elements.

[0062] In some embodiments, the transactivator protein is derived from a naturally occurring transcription factor, where the DNA binding domain of the naturally occurring transcription factor is mutated, resulting in altered DNA binding specificity relative to the wild-type transcription factor. In some embodiments, the transactivator is a naturally occurring transcription factor.

[0063] In some embodiments, the transactivator protein further comprises a transactivation domain (i.e., a fusion protein comprising a DNA binding domain and a transactivation domain). As used herein, the term "transactivation domain" refers to a protein domain that functions to recruit transcription machinery to a minimal promoter. In some embodiments, the transactivation domain does not independently trigger gene activation. In some embodiments, the transactivation domain is naturally-occurring. In other embodiments, the transactivation domain is engineered. Examples of transactivation domains are known to those skilled in the art and include, but are not limited to, the RelA transactivation domain, VP16, VP48, and VP64. In some embodiments, the transactivator of at least one cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA. See, e.g., Angelici B. et al., Cell Rep. 2016 Aug 30; 16(9): 2525-2537.

[0064] In some embodiments, the regulatory component comprises a transactivator response element. A "transactivator response element" can comprise a minimal DNA sequence that is bound to and recognized by a transactivator protein. In some embodiments, a transactivator response element comprises more than one copy (i.e., repeats) of a minimal DNA sequence that is bound to and recognized by a transactivator protein. In some embodiments, a transactivator response element comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 repeats of a minimal DNA sequence that is bound to and recognized by a transactivator protein.

[0065] In some embodiments, the repeats are tandem repeats. In some embodiments, the transactivator response element comprises a combination of minimal DNA sequences. In some embodiments, the minimal DNA sequences are interspersed with spacer sequences. In some embodiments, the spacer sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 nucleotides in length.

[0066] In some embodiments, the regulatory component comprises a transcription factor response element. The term "transcription factor response element" refers to a DNA sequence that is bound to and recognized by a transcription factor. As used herein, the term "transcription factor" refers to a protein not encoded on a contiguous polynucleic acid that regulates gene transcription. In some embodiments, the transcription factor is a transcription activator (i.e., increases transcription). In other embodiments, the transcription factor is a transcription inhibitor (i.e., inhibits transcription). In some embodiments, the transcription factor is an endogenous transcription factor of the cell.

[0067] A "transcription factor response element" can comprise a minimal DNA sequence that is bound and recognized by a transcription factor. In some embodiments, a transcription factor response element comprises more than one copy (i.e., repeats) of a minimal DNA sequence that is bound and recognized by a transcription factor. In some embodiments, a transcription factor response element comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 repeats of a minimal DNA sequence that is bound and recognized by a transcription factor. In some embodiments, the repeats are tandem repeats.

[0068] In some embodiments, the transcription factor response element comprises a combination of minimal DNA sequences. In some embodiments, the minimal DNA sequences are interspersed with spacer sequences. In some embodiments, the spacer sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 nucleotides in length. In some embodiments, the transcription factor response element is unique (i.e., the continuous polynucleic acid contains only one copy of the transcription factor response element). In other embodiments, the transcription factor response element is not unique.

[0069] In some embodiments, the regulatory component comprises a promoter element. In some embodiments, the promoter element comprises a transcription factor response element and a minimal promoter. In some embodiments, the promoter element comprises a mammalian promoter or promoter fragment. In some embodiments, the mammalian promoter or promoter fragment is unique (i.e., the contiguous polynucleic acid contains only one copy of the mammalian promoter or promoter fragment). In other embodiments, the mammalian promoter or promoter fragment is not unique.

[0070] In some embodiments, the control element comprises a minimal promoter. As used herein, the term "minimal promoter" refers to a nucleic acid sequence that is necessary but not sufficient to initiate the expression of an output. In some embodiments, the minimal promoter is naturally occurring. In other embodiments, the minimal promoter is engineered, such as by modifying and / or shortening a naturally occurring sequence, combining a naturally occurring sequence, or combining a naturally occurring sequence with a non-naturally occurring sequence; in each case, the engineered minimal promoter is a non-naturally occurring sequence. In some embodiments, the minimal promoter is engineered from a viral or non-viral source. Examples of minimal promoters are known to those skilled in the art.

[0071] In some embodiments, the control elements include a transactivator response element, a transcription factor response element, and a minimal promoter. In some embodiments, the control elements of the cassette include, 5' to 3', a transactivator response element, a transcription factor response element, and a minimal promoter. In some embodiments, the control elements include, 5' to 3', a transcription factor response element, a transactivator response element, and a minimal promoter.

[0072] In some embodiments, the control components of the cassette comprise a transactivator response element and a promoter element. In some embodiments, the control components of the cassette comprise, from 5' to 3', a transactivator response element and a promoter element. In some embodiments, the control components of the cassette comprise a transactivator response element, a promoter element, and a minimal promoter. In some embodiments, the control components of the cassette comprise, from 5' to 3', a transactivator response element, a promoter element, and a minimal promoter.

[0073] In some embodiments, the control components of the cassette comprise, from 5' to 3', a promoter element and a transactivator response element. In some embodiments, the control components of the cassette comprise, from 5' to 3', a promoter element, a transactivator response element and a minimal promoter. In some embodiments, the promoter element is a mammalian promoter. In some embodiments, the promoter element is a promoter fragment.

[0074] In some embodiments, a regulatory element (e.g., a transactivator response element, and / or a transcription factor response element, and / or a promoter element, and / or a minimal promoter) is operably linked to a nucleic acid encoding a transactivator protein and / or output. A regulatory element is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence it controls to control ("drive") transcription initiation and / or expression of that sequence. A regulatory element may be bound to a transcription factor and / or transactivator protein that increases or decreases expression of the transactivator protein and / or output.

[0075] Response Component The cassette of sequential polynucleic acid molecules comprises at least one response element. In some embodiments, the response element comprises a nucleic acid sequence that encodes an output or a gene of interest. In some embodiments, the output is an RNA molecule. In some embodiments, the RNA molecule is an mRNA that encodes a protein. In some embodiments, the output is a non-coding RNA molecule. Examples of non-coding RNA molecules are known to those skilled in the art and include, but are not limited to, transfer RNA (tRNA), ribosomal RNA, (rRNA), microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, and long ncRNA.

[0076] In some embodiments, the output is a therapeutic molecule (i.e., associated with the treatment of a disease), such as a therapeutic protein or RNA molecule. Examples of therapeutic molecules include, but are not limited to, antibodies (e.g., monoclonal or polyclonal; chimeric; humanized; including antibody fragments and antibody derivatives (bispecific, trispecific, scFv, and Fab)), enzymes, hormones, inflammatory molecules, anti-inflammatory molecules, immunomodulatory molecules, anti-cancer molecules, short hairpin RNA, small interfering RNA, and microRNA. Specific examples of the types of therapeutic molecules mentioned above are known in the art, any of which may be used according to the present disclosure.

[0077] In some embodiments, the output is a detectable protein, such as a fluorescent protein. In some embodiments, the output is a cytotoxin. As used herein, the term "cytotoxin" refers to a substance that is toxic to cells. For example, in some embodiments, the output is a cytotoxic protein. Examples of cytotoxic proteins are known to those skilled in the art and include, but are not limited to, granulysin, perforin / granzyme B, Fas / Fas ligand, and various cytokines / chemokines (e.g., IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, CCR3, CXCR3, CXCR4, and CCR10).

[0078] In some embodiments, the output is the enzyme that catalyzes the activation of prodrugs. Examples of enzymes that catalyze the activation of prodrugs are known to those skilled in the art and include, but are not limited to, carboxylesterase, acetylcholinesterase, butylcholinesterase, paraxonase, matrix metalloproteinase, alkaline phosphatase, β-glucuronidase, valacyclovirase, prostate specific antigen, purine-nucleoside phosphorylase, carboxypeptidase, amidase, β-lactamase, β-galactosidase, and cytosine deaminase. For example, see Yang Y. et al., Enzyme-mediated hydrolytic activation of prodrugs. Acta. Pharmaceutica. Sinica B. 2011 Oct; 1(3): 143-159.

[0079] Likewise, various prodrugs are known to those skilled in the art and include, for example, acyclovir, allopurinaol, azidothymidine, bambuterol, becampicillin, capecitabine, captopril, carbamazepine, carisoprodol, cyclophosphamide, diethylstilbestrol diphosphate, dipivefrin, enalapril, famciclovir, fludarabine triphosphate, fluorouracil, fosamprenavir, fosphenytoin, fursultiamine, gabapentin enacarbil, encarbil), ganciclovir, gemcitabine, hydrazide MAO inhibitors, leflunomide, levodopa, methanamine, mercaptopurine, mitomycin, molsidomine, nabumetone, olsalazine, omeprazole, paliperidone, phenacetin, pivampicillin, primidone, proguanil, psilocybin, ramipril, S-methyldopa, simvastatin, sulfasalazine, sulindac, tegafur, terfenadine, valacyclovir, valganciclovir, and zidovudine.

[0080] In some embodiments, the output is an immunomodulatory protein and / or RNA. As used herein, the term "immunomodulatory protein" (or immunomodulatory RNA) refers to a protein (or RNA) that modulates (stimulates (i.e., immunostimulatory protein or RNA) or inhibits (i.e., immunoinhibitory protein or RNA)) the immune system by inducing activation and / or increasing activity of components of the immune system. Various immunomodulatory proteins are known to those skilled in the art. See, for example, Shahbazi S. and Bolhassani A. Immunostimulants: Types and Functions. J. Med. Microbiol. Infec. Dis. 2016; 4(3-4): 45-51. In some embodiments, the immunomodulatory protein is a cytokine or a colony-stimulating factor.

[0081] In some embodiments, the output is a DNA modification factor. The term "DNA modification factor" as used herein refers to a factor that modifies the structure of DNA and / or modifies the sequence of DNA (for example, by inducing genetic recombination or introducing mutations). In some embodiments, the DNA modification factor is a gene that encodes a protein intended to correct a genetic defect, a DNA modification enzyme, and / or a component of a DNA modification system. In some embodiments, the DNA modification enzyme is a site-specific recombinase, a homing endonuclease, or a protein component of a CRISPR / Cas DNA modification system.

[0082] In some embodiments, the output is a cell surface receptor. In some embodiments, the output is a kinase. In some embodiments, the output is a gene expression regulator. The term "gene expression regulator" as used herein refers to any factor that increases or decreases the transcription of at least one gene when present. In some embodiments, the gene expression regulator is a protein. In some embodiments, the gene expression regulator is an RNA. In some embodiments, the gene expression regulator is a component of a multi-component system that can control gene expression.

[0083] In some embodiments, the output is an epigenetic modifier.The term "epigenetic modifier" as used herein refers to the factor (e.g., protein or RNA) that increases, decreases or modifies epigenetic modification.Examples of epigenetic modification are known to those skilled in the art and include, but are not limited to, DNA methylation and histone modification. In some embodiments, the output is a factor required for vector replication. Examples of factors required for vector replication are known to those skilled in the art.

[0084] In some embodiments, the response element comprises a nucleic acid sequence encoding a transactivator. In some embodiments, the response element comprises a polycistronic expression element. The term "polycistronic response element" as used herein refers to a nucleic acid sequence that facilitates the production of two or more proteins from a single mRNA. The polycistronic response element may comprise an internal recognition sequence (IRES) or a polynucleic acid that encodes a 2A peptide. For example, see Liu et al., Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep. 2017 May 19; 7(1): 2193.

[0085] In some embodiments, the response element comprises a nucleic acid sequence encoding an output, a transactivator, and a polycistronic expression element, where transcription of the response element produces a single mRNA. In some embodiments, the output and the transactivator are separated by a polycistronic expression element. In some embodiments, the response element comprises at least one polyadenylation sequence. In some embodiments, the polyadenylation sequence is suitable for transcription termination and polyadenylation in a mammalian cell.

[0086] In some embodiments, the response element comprises at least one microRNA target site. In some embodiments, the response element comprises at least one, at least two, at least three, at least four, at least five, or at least six microRNA target sites. MicroRNAs are a class of small non-coding RNAs, typically 21-25 nucleotides in length, that downregulate the levels of RNA to which they bind in a variety of ways, including translational repression, mRNA cleavage, and deadenylation.

[0087] The term "microRNA target site" as used herein refers to the sequence that is complementary to and controlled by the microRNA sequence.The microRNA target site can have at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the microRNA that binds and controls the microRNA target site. In some embodiments, at least one microRNA target site is 3' to the output. In some embodiments, at least one microRNA target site is 5' to the output.

[0088] In some embodiments, the response element comprises, 5' to 3', an output and at least one microRNA target site. In some embodiments, the response element comprises, 5' to 3', a nucleic acid sequence encoding a transactivator protein and at least one microRNA target site. In some embodiments, the response element comprises, 5' to 3', a nucleic acid sequence encoding a transactivator protein, a nucleic acid sequence encoding an output, and at least one microRNA target site.

[0089] In some embodiments, the multiple cassettes of the contiguous polynucleic acid molecule comprise at least one microRNA target site. In some embodiments, each microRNA target site of the contiguous polynucleic acid is unique (i.e., the contiguous polynucleic acid contains only one copy of the microRNA target). In some embodiments, the contiguous polynucleic acid molecule comprises at least two cassettes each comprising at least one microRNA target site that is the same nucleic acid sequence. In some embodiments, the contiguous polynucleic acid molecule comprises at least two cassettes each comprising at least one microRNA target site, where the at least one microRNA target site of each cassette comprises different nucleic acid sequences controlled by the same microRNA. For example, a first cassette may comprise microRNA target site X, and a second cassette may comprise microRNA target site Y, and microRNA Z controls target site X and target site Y.

[0090] In some embodiments, the continuous polynucleic acid molecule comprises at least one cassette, wherein the cassette comprises: (i) a 5' regulatory element comprising a transactivator response element, a transcription factor response element, and a minimal promoter; and (ii) a 3' response element comprising an output, a transactivator, and an optional polycistronic expression element, wherein the output and the transactivator are optionally separated by the polycistronic expression element; wherein transcription of the response element produces a single mRNA; and wherein the transactivator of (ii) when expressed as a protein binds to and transactivates the transactivator response element of (i).

[0091] In some embodiments, the continuous polynucleic acid molecule encodes at least two cassettes, where at least one cassette comprises (i) a 5' regulatory element comprising a transactivator response element, a transcription factor response element, and a minimal promoter, and a 3' response element comprising an output; and at least one cassette comprises (ii) a promoter element operably linked to a nucleic acid encoding a transactivator protein; and where the transactivator of (ii) when expressed as a protein binds to and transactivates the transactivator response element of (i).

[0092] Other Compositions In another aspect, the present disclosure relates to a composition of a vector. In some embodiments, the vector comprises a contiguous polynucleic acid molecule as described above. In other aspects, the present disclosure relates to the composition of engineered viral genome.In some embodiments, the viral genome comprises the continuous polynucleic acid molecule described above.In some embodiments, the viral genome is adeno-associated virus (AAV) genome, lentivirus genome, adenovirus genome, herpes simplex virus (HSV) genome, vaccinia virus genome, poxvirus genome, Newcastle disease virus (NDV) genome, coxsackievirus genome, reovirus genome, adenovirus genome, herpes simplex virus (HSV) genome, vaccinia virus genome, poxvirus genome, Newcastle disease virus (NDV) genome, coxsackievirus genome, reovirus genome, measles virus genome, vesicular stomatitis virus (VSV) genome, parvovirus genome, Seneca Valley virus genome, Maraba virus genome, or common cold virus genome.

[0093] In other aspects, the present disclosure relates to compositions of virions.As used herein, the term "virion" refers to the infectious form of a virus outside a host cell (including, for example, a DNA / RNA genome and a protein capsid).In some embodiments, the virion comprises the engineered viral genome described above.

[0094] Methods for stimulating cell-specific events In another aspect, the present disclosure relates to a method for stimulating cell-specific events in a population of cells.In some embodiments, the method comprises contacting a population of cells with the sequential polynucleic acid molecule described above, the vector described above, the engineered viral genome described above, or the virion described above, wherein the cell-specific events are controlled by at least one endogenous transcription factor and / or at least one endogenous microRNA.

[0095] In some embodiments, the contact between the host cell and the above-described continuous polynucleic acid molecule or the above-described vector occurs via non-viral delivery methods.Examples include, but are not limited to, transfection (e.g., DEAE-dextran-mediated transfection, CaPO4-mediated transfection, lipid-mediated uptake, PEI-mediated uptake, and laser transfection), transformation (e.g., calcium chloride, electroporation, and heat shock), gene transfer, and particle bombardment. In some embodiments, the population of cells is contacted ex-vivo (i.e., the population of cells is isolated from an organism and the population of cells is contacted outside of the organism). In some embodiments, the population of cells is contacted in-vivo.

[0096] As used herein, the term "endogenous" refers to a factor (e.g., protein or RNA) found in a cell in a natural state in the context of a cell. In some embodiments, an endogenous transcription factor can bind and activate a promoter element (e.g., a transcription factor response element) of a control element of at least one cassette. In some embodiments, an endogenous microRNA can be complementary to a microRNA target site of a control element or response element of at least one cassette.

[0097] In some embodiments, the "transactivator" and corresponding "transactivator response element" are selected such that the transactivator specifically binds to the "transactivator response element" but binds as little as possible to response elements naturally present in the cell. In some embodiments, the DNA binding domain of the transactivator protein does not bind efficiently to native regulatory sequences present in the cell and therefore does not trigger excessive side effects.

[0098] In some embodiments, the population of cells comprises at least one target cell and at least one non-target cell. The target cell and the non-target cell type differ in the level of at least one endogenous transcription factor and / or the expression intensity of at least one endogenous promoter or fragment thereof and / or at least one endogenous microRNA. In some embodiments, the target cell and the non-target cell are different cell types. For example, in some embodiments, the target cell is a cancerous cell and the non-target cell is a non-cancerous cell. Similarly, in some embodiments, the target cell is a hepatocyte and the non-target cell is a non-hepatocyte (e.g., muscle cell). In other embodiments, the target cell and the non-target cell are the same cell type (e.g., both are hepatocyte), but still differ in the level of at least one transcription factor and / or at least one endogenous microRNA. For example, the target cell can be a senescent muscle cell and the non-target cell can be a non-senescent muscle cell.

[0099] In some embodiments, the expression level of the response element output differs between target and non-target cells by at least 2, at least 5, at least 10, at least 100, at least 1,000, or at least 10,000 fold. In some embodiments, the cells of the target cell population are tumor cells and the cell-specific event is cell death. In some embodiments, the cells of the target cell population are senescent cells and the cell-specific event is cell death. In some embodiments, the cell death is mediated by immune targeting through expression of an activating receptor ligand, a specific antigen, a stimulatory cytokine, or any combination thereof. In some embodiments, the method further comprises contacting the population of cells with a prodrug or a non-toxic precursor compound that is metabolized into a therapeutic agent or a toxic compound by output.

[0100] In some embodiments, cells of the target cell population differentially express a factor relative to a wild-type cell of the same type, and the cell-specific event is modulating the expression level of the factor. In some embodiments, output expression ensures survival of the target cell population, while non-target cells are eliminated due to lack of output expression and in the presence of a cell death inducer, In other embodiments, output expression ensures survival of the non-target cell population, while target cells are eliminated due to lack of output expression and in the presence of an unrelated and non-specific cell death inducer.

[0101] In some embodiments, cells of the target cell population comprise a particular phenotype of interest, such that output expression is restricted to cells of this particular phenotype. In some embodiments, the cells of the target cell population are of a selected cell type, and the cell-specific event is encoding a novel function through expression of a gene that is not naturally present or is inactive in the selected cell type. In some embodiments, the population of cells comprises a multicellular organism. In some embodiments, the multicellular organism is an animal. In some embodiments, the animal is a human.

[0102] Advantages over prior art The compositions and methods disclosed herein exhibit advantages over the prior art in a variety of ways, examples of which are provided below. First, the approaches and methods described herein exhibit increased precision: the ability to sense multiple highly informative molecules and combine their information allows expression to be restricted more precisely than using previously described natural tissue-specific promoters (which are usually expressed to different degrees in multiple tissues / cell types) or detargeting.

[0103] Secondly, the circuit architecture described herein shows excellent dynamic range with strict Off and full expression comparable to that of strong constitutive promoter.This approach beats classical transcription targeting, which often suffers from weak and leaky expression.Strong absolute expression is the key to many gene therapies, and high dynamic range is especially important in targeting toxic genes (for example, cancer suicide therapy).

[0104] Third, the structure of the circuits described herein is modular in nature (i.e., individual sensing sequences can be swapped to change circuit specificity), and single signals are combined in predictable ways. As a result, single sensors can be combined according to a growing number of TFs and miRNA expression data sets to rationally design vectors with different specificities.

[0105] Moreover, these approaches can be used to develop new gene and cell therapies (GCTs) with improved specificity and higher efficacy. The logic circuits can be programmed to sense specific conditions and respond with genes, therapeutic proteins, modified miRNAs, or combinations of multiple discrete outputs. The programmable inputs, compact size, and resulting ability to fit comfortably with many different viral vectors open up a wide range of applications.

[0106] The circuit can be packaged into the most appropriate AAV serotype and programmed to drive functional genes only in tissues of interest for precise somatic gene therapy. In a variant of this application, suicide gene therapy, the killing gene is specifically expressed in cancer cells but not in other healthy tissues in the body, as described herein for specific targeting of hepatocellular carcinoma. This represents an ideal benchmark for the technology, as it requires high expression levels in cancer cells coupled with strict control in other tissues, where leaky expression may lead to toxic effects.

[0107] The circuit is small enough to be packaged into many oncolytic viruses (e.g., adenovirus, HSV) without compromising the replication machinery, and can therefore be easily used to precisely target viral replication in cancer cells. The circuit design described herein can also be used in conjunction with lentiviral vectors for ex-vivo engineering of cells for use as stem cells or in immunotherapy. In this case, the circuit is designed to execute a specific genetic program only when certain in vivo conditions are met.

[0108] As an example, stem cells can be transduced with the circuit as described herein, differentiated and selected in vitro; and, in the differentiated cells reinfused into the patient, the circuit can continuously monitor the status of key pluripotency markers and kill the cells as they appear to avoid teratoma formation. The demonstration that the circuit described herein can be inserted between insulators and packaged into lentiviral particles without significantly affecting circuit performance represents the first proof of concept in this direction.

[0109] The compositions and methods disclosed herein represent specific advances in the following areas: Targeted expression of genes of interest in gene therapy: Restricting expression of a gene of interest (GOI) to a tissue / cell type of interest remains a challenging problem in virotherapy. The problem is particularly acute, for example, in (i) cancer virotherapy due to the high similarity between healthy and cancer tissues, and (ii) in indications where the GOI must be very tightly controlled due to deleterious side effects in unintended tissues.

[0110] The main approach to control the specificity of a gene of interest embedded in a viral vector in the prior art is "transcriptional targeting" (Dorer DE and Nettelbeck DM, Adv. Drug Deliv. Rev. 2009. 61(7-8): 554-571; Robson T. and Hirst DG, J. Biomed. Biotechnol. 2003; 2003(2): 110-137; Navarro SA et al., Expert Opin. Ther. Pat. 2016 Sep; 26(9): 1095-1104). In this approach, the GOI is placed under the control of a cancer-specific or tissue-specific promoter. The disadvantages of this method are numerous. For example, this approach has limited specificity. Promoters defined as "specific" are in fact often expressed in multiple tissues. This is especially true for so-called "cancer-specific promoters" since they are usually linked to the overexpression of genes that are physiological in other tissues. In addition, this approach is also characterized by extremely low expression differences between targeted tissues or cell types and those that should not be targeted.

[0111] Most of the tissue-specific or cancer-specific promoters are both weak and somewhat leaky (for example, the AFP promoter, a commonly used tissue-specific promoter, is 500-fold less active than CAG) (Kanegae Y. et al., Nucleic Acids Res. 2011 Jan; 39(2): e7). This low expression differential limits the ability to restrict the expression of highly cytotoxic genes and proteins required at high doses to only those cells that require it. Proposed solutions to increase the power of selective promoters include two-stage transcriptional amplification (TSTA) (Iyer M. et al., Proc. Natl. Acad. Sci. USA 2001 Dec 4; 98(25): 14595-14600; US7527942B2; US2009 / 0192101A1) or mixing specific promoters with sequence elements from strong constitutive promoters (Sakaguchi M. et al., Oncol. Rep. 2017 Aug; 38(2): 1108-1114). Both solutions are partially successful and partially unsuccessful, since they increase promoter strength at the expense of leakiness and often result in reduced specificity.

[0112] A more recent strategy, often referred to as "miRNA detargeting", combines a constitutive promoter with miRNA targets that are abundant in tissues that are excluded from the expression profile (WO2007 / 000668A2). This approach allows high transgene dosage to be reached due to the use of constitutive promoters. While useful for refining targeting accuracy, it is difficult to reach high selectivity by detargeting alone in applications that rely on systemic delivery, as it requires one-to-one exclusion of a large number of tissues through a large set of miRNA targets. As the number of targets increases, cassette design becomes more difficult due to compositional effects and potential genetic instability (Ruiz AJ et al., J. Virol. 2016 Mar 28; 90(80): 4078-4092). Moreover, strict Off levels require highly abundant miRNAs, potentially limiting the number of molecular targets that can be used.

[0113] A version of miRNA targeting using plasmid-encoded genetic circuits previously proposed by Rinaudo et al. (Rinaudo K. et al., Nature Biotechnol. 2007 Jul; 25(7): 795-801) was attempted in lentiviral vectors (Amendola M. et al., Mol. Ther. 2013 May; 21(5): 934-946). Interestingly, the study implemented two distinct lentiviral vector approaches and thus illustrated that packaging complex genetic circuits into continuous DNA molecules is a difficult task.

[0114] Synthetic biology: A variety of circuits that exhibit complex logic behaviors have been described. The implementation of all possible one-input and two-input logic gates in mammalian cells has been shown before using transcriptional and translational regulators (Auslander S. et al., Nature. 2012 Jul 5; 487(7405): 123-27). More recently, several groups have demonstrated and described the implementation of molecular logic and memory based on recombinases (WO2014 / 093852A1; WO2015 / 188191A1). These efforts have demonstrated remarkable progress in cell control and logic implementation using biomolecules. However, all these systems suffer from two shortcomings when it comes to their use for in vivo cell targeting: (i) interface matching and (ii) circuit size.

[0115] Previously described circuits have been tested in cell cultures using well-characterized and externally regulated ectopic input molecules, and there is no evidence of their performance when they come into contact with endogenous cellular molecules. While there has been significant progress in developing logic integration, the creation of logic circuit-cell environment interfaces still lags behind. An example of endogenous miRNA as an effective logic input has been shown before using a set of recombinant plasmids delivered to cultured cell lines (WO2012 / 012739A2). Logic gene circuits capable of processing endogenous transcription factor inputs have been shown using two full-length tissue-specific promoters, each controlling a split protein fragment (Nissim L. and Bar-Ziv RH, Mol. Syst. Biol. 2010 Dec 21; 6:444) or Cas9 and gRNA (Liu YC et al., Nat. Commun. 2014 Nov. 6; 5: 5393).

[0116] Systems in which single endogenous transcription factors can be robustly attached to synthetic circuits, as well as pairs of unrelated transcription factors, have also been demonstrated (Angelici B. et al., Cell Rep. 2016 Aug 30; 16(9): 2525-2537). However, in all of the above cases, experimental implementations are limited to mixtures of recombinant plasmids transfected into cultured cells, and especially in Angelici B. et al. (Cell Rep. 2016 Aug 30; 16(9): 2525-2537). In no case has it been shown that logic circuits can be implemented in continuous nucleic acid molecules or in medically relevant vector systems, or that they have been shown to treat disease in animal models.

[0117] In addition, most of the designs mentioned above aim for maximum flexibility of circuit function, and this is usually achieved by constructing additional recombinant plasmids. Indeed, many complex circuits require 10 separate plasmids to operate in a transient transfection scenario. As an example, the two-input AND gate described by Auslander et al. (Nature. 2012 Jul 5; 487(7405): 123-27) requires five different plasmids, and the recombinase system described by W. Wong (WO2015 / 188191A1) relies on three plasmids (two recombinases plus the output expression cassettes they operate on), and neither has been tested in conjunction with endogenous cellular inputs.

[0118] Moreover, previously described systems designed to implement AND logic required three plasmids (Liu YC et al., Nat. Commun. 2014 Nov. 6; 5: 5393; Angelici B. et al., Cell Rep. 2016 Aug 30; 16(9): 2525-2537). These approaches also require the use of a complete mammalian promoter as an interface with the cellular background, and in one case, the use of a large protein (CAS9) for circuit integration results in extremely large circuit sizes.

[0119] Gene circuits for therapeutic applications: The implementation of multi-component gene circuits in therapeutically relevant viral and non-viral vectors remains extremely rare, due in no small part to the fact mentioned above that many state-of-the-art circuits require increasingly complex and large DNA footprints. The additional challenges of integrating multiple genes on a contiguous DNA molecule discussed above (e.g., read-through, intergenic regulatory interference, etc.) have hindered medical translation of many of the fundamental advances described above.

[0120] Existing examples of logic circuits for targeting are limited to lentiviral vectors due to their higher cargo capacity (Morel M. et al., Proc. Natl. Acad. Sci. USA 2016 Jul 19; 113(29): 8133-8138; Nissim L. et al., Cell. 2017 Nov 16; 171(5): 1138-1150). While lentiviral backbones are useful for ex-vivo applications (especially immune cell engineering), they are far from ideal for in vivo therapy due to their ability to integrate into the host genome, causing mutations and / or unwanted gene activation. The genetic circuit shown in Nissim L. et al. (Cell. 2017 Nov 16; 171(5): 1138-1150) is implemented using a pair of lentiviral constructs rather than a single vector, which underlies the difficulty of encoding complex genetic circuits in a continuous DNA molecule as shown herein.

[0121] As shown in the examples herein and in Figures 3A-3B, 5A-5C, 6A-6B, and 7A-7B, non-integrated single-component AAV vectors represent a safe and proven alternative, with a wide selection of serotypes optimized for efficient delivery in different cell types and a high ability to penetrate tumors, but limited in terms of cargo capacity. Data in Morel et al. (Morel M. et al., Proc. Natl. Acad. Sci. USA 2016 Jul 19; 113(29): 8133-8138) also show how the poor On:Off properties of natural specific promoters affect circuit performance, imposing a severe trade-off between sensitivity and specificity. Despite the shortcomings of these tools, the specific methods used in Morel M. et al. (Proc. Natl. Acad. Sci. USA 2016 Jul 19; 113(29): 8133-8138) and Nissim L. et al. (Cell. 2017 Nov 16; 171(5): 1138-1150) are not overlapping with the present disclosure.

[0122] example Example 1. Functionality implemented in a continuous DNA molecule . Disclosed herein are sequential DNA constructs capable of implementing multi-input logic, for example between two transcription factors (TFs), between an endogenous promoter (or promoter fragment) and an optional transcription factor, and optionally one or more microRNAs (miRNAs). The inputs are typically chosen such that their combination is specific to one or more physiological cell states that the circuit is designed to target. A general circuit architecture overview is shown in FIG. 1A. An optional transcription factor A (TF-A) binds to a response element (TF-A-RE) in the promoter region of the output gene next to the minimal promoter (Pmin). Elsewhere in this promoter region are binding sites for proteins named "auxiliary transactivators" (AA).

[0123] AA is a transcriptional transactivator that can activate gene expression when bound to its response element (AA-RE) and in the presence of a minimal promoter. The promoter region containing response elements for transcription factor A and auxiliary transactivator AA has synergistic behavior, that is, the expression of the output driven by either transcription factor A alone or auxiliary transactivator AA is less than the expression of the output in the presence of both the former and the latter (Angelici B. et al., Cell Rep. 2016 Aug 30; 16(9): 2525-2537).

[0124] In "Option 1a" of the circuit in Figure 1A, AA protein expression is driven by an optional transcription factor B that binds to a response element (TF-B-RE) in the promoter region of the AA-encoding gene next to a minimal promoter. In this option, the output is strongly expressed when both TF-A and TF-B are strongly active, which implements the AND-like circuit behavior between TF-A and TF-B "TF-A AND TF-B" (Table 1). Alternatively, AA expression can be driven by the promoter of the endogenous gene (PR-E) or one or more fragments of such a promoter ("Option 1b" of the circuit in Figure 1A). In this case, the output is strongly expressed only when the endogenous promoter is active at the same time as the highly active TF-A, which implements the logic "PR-E AND TF-A" (Table 1).

[0125] [Table 1]

[0126] In "option 2" of the circuit in Figure 1A, AA protein expression is coupled to output expression, e.g., via a T2A linker. In this option, output is proportional to the power of TF-A (or alternatively and / or in addition to PR-E) amplified by AA action (Angelici B. et al., Cell Rep. 2016 Aug 30; 16(9): 2525-2537).

[0127] [Table 2]

[0128] In "Option 3" of the circuit in FIG. 1A, the output is further controlled by an optional microRNA (miR-X) via a target site in the mRNA encoding the output. In "Option 3a", only the output gene is controlled by the miRNA, while in "Option 3b" (only compatible with option 1 above), the gene encoding AA is controlled by the same miRNA. "Option 3a" and "Option 3b" can be used together or separately.

[0129] "Option 1a" in combination with "Option 3a", "Option 3b", or both, results in a logical behavior of "TF-A AND TF-B AND NOT(miR-X)", i.e., the output is highly expressed in the simultaneous presence of TF-A and TF-B and in the absence of miR-X (Table 2). Similarly, "Option 1b" can be combined with "Option 3a", "Option 3b", or both to implement a logical behavior of "PR-E AND TF-A AND NOT(miR-X)" type (Table 2).

[0130] [Table 3]

[0131] "Option 2" in combination with "Option 3a" results in the logical behavior of "TF-B AND NOT (miR-X)", i.e., the output is highly expressed in the presence of TF-B and in the absence of miR-X.

[0132] [Table 4]

[0133] In "Option 4" of the circuit in FIG. 1A, the output is further controlled by an optional microRNA (miR-Y) via a target site in the mRNA encoding the output. In "Option 4a", only the output gene is controlled by the miRNA, while in "Option 4b" (only compatible with "Option 1" above), the gene encoding AA is controlled by the same miRNA. "Option 4a" and "Option 4b" can be used together or separately.

[0134] "Option 1a" in combination with "Option 3a", "Option 3b", or both, and in combination with "Option 4a", "Option 4b", or both, results in a logical behavior of "TF-A AND TF-B AND NOT(miR-X) AND NOT(miR-Y)", i.e., the output is highly expressed in the simultaneous presence of TF-A and TF-B and in the absence of miR-X and miR-Y. The utility of this configuration is to prevent output expression in tissues where miR-X or miR-Y or both are strongly expressed, thus improving safety characteristics while retaining strong expression in cells that express both TF-A and TF-B but neither of miR-X or miR-Y. Similarly, "Option 1b" can be combined with "Option 3a", "Option 3b", or both, and "Option 4a", "Option 4b", or both to implement the logical behavior "PR-E AND TF-B AND NOT(miR-X) AND NOT(miR-Y)".

[0135] "Option 2" in combination with "Option 3a" and "Option 4a" results in the logical behavior of "TF-A AND NOT(miR-X) AND NOT(miR-Y)", i.e., the output is highly expressed in the presence of TF-B and in the absence of miR-Y. Additional miRNA inputs and their binding sites can be added, and the circuit structure and logic behavior can be extended in a similar manner (e.g., miR-Z as another input feature, and structural "option 5a" and "option 5b", etc.). Specific genetic implementations of these regulatory programs are illustrated by the structures in FIG. 2.

[0136] Example 2. Implementation of consecutive DNA cassettes and integration into viral vectors . As described herein, a representative example of the circuit described in Example 1 was implemented in a continuous DNA molecule and further integrated into a viral vector genome, whereupon viral particles were produced and tested for their ability to selectively target cells in vitro and in vivo and to halt tumor growth in vivo. Thus, among other things, specific examples of the therapeutic practical application of this approach are disclosed. Genes are integrated in the continuous DNA construct in either a convergent or divergent orientation. In the latter, the response element for the transcription factor input is located in the center of the DNA molecule (between the two coding sequences) and the miRNA target and polyA are immediately adjacent to the chosen viral backbone (Figure 2).

[0137] Genes can also be integrated in a head-to-tail (collinear) fashion; however, this has not been experimentally tried, as similar systems have had problems in the past, but it is possible that a head-to-tail orientation is also functional. Furthermore, insulators can be placed between the individual genetic modules to improve their isolation from each other and from the viral vector context. Finally, the approach can lead to conditionally replicating ("oncolytic") vectors, whose output gene is the gene(s) natively responsible for viral vector replication (Figure 2).

[0138] Example 3. Preferred embodiments and functional demonstration . One preferred embodiment is a divergent head-to-tail configuration. In this configuration, the physical organization of the components maximizes functionality and minimizes unpredictable context effects, resulting in a robust modular system. Divergent genes avoid the risk of transcriptional run-through. Transcriptional control is directed to a response element located at the center of the construct, surrounded by genetic components that remain relatively constant (a minimal promoter usually remains unchanged, while transactivators and output genes are selected from a list of well-characterized compounds).

[0139] Thus, the intended transcriptional regulation is shielded from cryptic regulators or TF binding sites present on the viral backbone. miRNA targets act through post-transcriptional regulation, and therefore they are not affected by spurious binding of transcription factors, for example, to neighboring viral sequences. The superior performance of the embodiment with divergent configurations is shown in Figures 3A-3B, where there is a clear gain in gene expression when "option 1" is combined with "option 3a" or "option 3b". This experiment was performed using naked DNA prior to viral packaging; thus, in subsequent viral vector embodiments, only divergent configurations were employed.

[0140] The functionality of the sequential DNA cassettes was demonstrated in adeno-associated viral vectors (AAV) and in lentiviral vectors. In Figure 4A-4B, the in vitro functionality of several AAV vectors implementing divergent configurations, as well as the circuit architecture combining "option 1" with "option 3a" and "option 3b" are shown (Figure 1A). DNA was packaged into AAV-DJ type virions (Grimm D. et al., J. Virol., 2008 Jun; 82(12): 5887-5911). Each vector was generated in two variants: (i) fluorescent output mCherry to test target specificity and (ii) cytotoxic output HSV-TK to test selective antitumor activity. In Figure 4A, the different vectors with fluorescent output were tested in a panel of cell lines. The circuit was programmed to detect the combination of Sox9 / 10 and HNF1A / B expression, which is typical of liver cancer (Zhou D. et al., Tumour Biol. 2014 Oct; 35(10):9935-40; Guo X. et al., Diagn. Pathol. 2012 Apr 19; 7:44.).

[0141] In addition, miRNA control elements were added to create a safety switch in healthy liver, based on the fact that these miRNAs are highly expressed in mouse liver but not in hepatoma cells (internal profiling data). Hepatoma cell lines HepG2 and Huh7 were used as positive controls, while the other two non-hepatoma cell lines HeLa and HCT-116 were used as negative controls. As the bar graph in Figure 4A shows, the fluorescent output was expressed at high levels in the two hepatoma cell lines, but not in the negative control cell line.

[0142] In Figure 4B, the cytotoxic activity is shown when the fluorescent output is replaced with the HSV-TK gene and combined with the prodrug ganciclovir (GCV). HSV-TK (thymidine kinase) converts GCV into a toxic product that leads to cell death. Here, as expected, the hepatoma cell line HepG2 is targeted by the vector, resulting in greatly reduced viability. The control cell line HeLa remains viable. The chart on the right shows that both cell lines are susceptible to HSV-TK+GCV action when HSV-TK is driven by a constitutive promoter and expressed at similar levels in both cell lines.

[0143] An additional divergent cassette implementing the circuit "option 1" (Figure 5A) was embedded into a lentiviral vector and tested for selective output expression in vitro using fluorescent output. Here, insulators were used to flank the cassette, and two different insulator pairs were employed next to a construct without an insulator pair. All constructs showed comparable output expression in the positive control cell lines Huh7 and HepG2, and very low expression in the negative control cell line HCT-116 (Figure 5B). These integrated vectors were also tracked over time for up to two months with only minor loss in gene expression. In parallel, the negative control cell line showed consistent low expression (Figure 5C).

[0144] The AAV viral vectors carrying the divergent DNA cassettes and circuits according to "Option 1" and "Option 3a" implementing the cell targeting program "Sox9 / 10 AND HNF1A / B AND NOT(miR-122)" were further tested in vivo in an orthotopic mouse model of disseminated liver cancer. Nod-SCID-Gamma (NSG) immune-deficient mice underwent surgery in which HepG2 cancer cells were injected into the spleen, disseminated to the liver via portal circulation, and formed multiple tumor foci (Figure 6A). To prevent primary tumor formation in the spleen, the spleen was surgically removed. The cells were pre-augmented with YFP fluorescent reporter and luciferase genes to allow in vivo tracking of tumor burden and postmortem examination of tumor foci.

[0145] Following tumor cell injection and tumor establishment, AAV-DJ virions were injected systemically into the tail vein. In the first experiment (Figure 6B), the fluorescent reporter protein mCherry was used to measure tumor-specific expression of output. A variant of the circuit without T-miR-122 features (only "option 1") was also tested as a reference, as well as a control vector expressing mCherry under a constitutive promoter. As the data in Figure 6B show, virions encoding circuits that process all three inputs can target output gene expression to tumors, while circuits that only implement "option 1" result in third-party output expression in healthy liver.

[0146] We constructed a vector that implements a three-input cell targeting program with the cytotoxic output HSV-TK leading to cell death in the presence of the small molecule prodrug ganciclovir (GCV) (Figure 7A). The data show that treated animals (viral vector injected twice into the tail vein followed by daily administration of ganciclovir) had a much lower tumor burden compared to the control group (Figure 7B-7C). We thus demonstrated the antitumor potential for virions that package sequential DNA cassettes implementing a multi-input classifier circuit.

[0147] References [Table 5-1] [Table 5-2] [Table 5-3]

[0148] Other Aspects All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various modifications and improvements to the present disclosure to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0149] Equivalent While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art can readily envision a variety of other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and such variations and / or improvements are recognized to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) in which the teaching(s) of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein.

[0150] It is therefore to be understood that the foregoing embodiments have been presented by way of example only, and that, within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is encompassed within the inventive scope of the present disclosure, unless such features, systems, articles, materials, kits, and / or methods are mutually inconsistent.

[0151] All definitions as defined and used herein must be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the terms defined. All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter cited therefor, and in some cases may cover the entirety of the document. The indefinite articles "a" and "an," as used herein in the specification and claims, unless expressly stated to the contrary, should be understood to mean "at least one."

[0152] The term "and / or," as used herein in the specification and claims, should be understood to mean "one or both" of the elements so coordinated, i.e., coordinately in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so coordinated. Other elements, whether related or unrelated to those specifically identified elements, other than the elements specifically identified by the "and / or" clause may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); and so forth.

[0153] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when dividing items into lists, "or" or "and / or" shall be interpreted as inclusive, i.e., the inclusion of at least one, but also more than one of a number or list of elements, and optionally also including additional unlisted items. Only terms expressly stated to the contrary, such as "only one of," or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or," as used herein, shall be interpreted as representing exclusive alternatives (i.e., "one or the other but not both") only when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." As used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0154] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily inclusive of at least one of each and every element specifically listed in the list of elements, and not to exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the specifically identified elements in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A (and optionally including elements other than B) in the absence of B; in another embodiment, to at least one, optionally including more than one, B (and optionally including elements other than A) in the absence of A; in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); and so forth.

[0155] It should also be understood that, in any method including more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described, unless expressly stated to the contrary.

[0156] In the claims and in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are understood to be open-ended, i.e., meaning including but not limited to. As provided for in the United States Patent Office Guidelines for Patent Examination, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described in this document using open-ended transitional phrases (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features recited by the open-ended transitional phrases. For example, if the disclosure recites "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. A contiguous polynucleic acid molecule encoding at least two cassettes, each cassette comprising a control element and a response element.

2. (i) at least one cassette comprises a 5' regulatory element comprising a transactivator response element, and a 3' response element comprising an output; and (ii) at least one cassette comprises a 5' regulatory element and a 3' response element comprising a nucleic acid sequence encoding a transactivator protein; and 2. The continuous polynucleic acid molecule of claim 1, wherein the transactivator of (ii) binds to and transactivates the transactivator response element of (i) when expressed as a protein.

3. 3. A contiguous polynucleic acid molecule according to claim 2, wherein the transactivators independently bind to and transactivate transactivator response elements.

4. 4. The continuous polynucleic acid molecule of claim 2 or 3, wherein the 5' regulatory element of the cassette in (i) further comprises a transcription factor response element and / or a minimal promoter.

5. 5. A continuous polynucleic acid molecule according to claim 4, wherein the transactivator binds to and transactivates a transactivator response element only in the presence of a transcription factor bound to the transcription factor response element.

6. 5. The contiguous polynucleic acid molecule of claim 4, wherein the 5' regulatory element comprises, from 5' to 3', a transactivator response element, a transcription factor response element, and a minimal promoter.

7. 5. The contiguous polynucleic acid molecule of claim 4, wherein the 5' regulatory element comprises, from 5' to 3', a transcription factor response element, a transactivator response element, and a minimal promoter.

8. The continuous polynucleic acid molecule according to any one of claims 2 to 4, wherein the 5' regulatory element in (i) further comprises a promoter element.

9. 9. The contiguous polynucleic acid molecule of claim 8, wherein the promoter element comprises a mammalian promoter or a promoter fragment.

10. 10. A continuous polynucleic acid molecule according to claim 8 or 9, wherein the 5' regulatory element in (i) comprises, from 5' to 3', a transactivator response element and a promoter element, and optionally a minimal promoter.

11. A contiguous polynucleic acid molecule according to any one of claims 2 to 10, wherein the 5' regulatory element of the cassette in (ii) comprises a promoter element.

12. 12. The contiguous polynucleic acid molecule of claim 11, wherein the promoter element comprises a transcription factor response element and a minimal promoter, and optionally, the transcription factor response element is unique.

13. 12. The contiguous polynucleic acid molecule of claim 11, wherein the promoter element comprises a mammalian promoter or promoter fragment, and optionally a minimal promoter.

14. 14. A contiguous polynucleic acid molecule according to any one of claims 2 to 13, wherein at least one cassette of (i) and at least one cassette of (ii) are in a convergent orientation.

15. 14. A contiguous polynucleic acid molecule according to any one of claims 2 to 13, wherein at least one cassette of (i) and at least one cassette of (ii) are in a divergent orientation.

16. 15. A contiguous polynucleic acid molecule according to any one of claims 2 to 14, wherein at least one cassette of (i) and at least one cassette of (ii) are in a head-to-tail orientation.

17. The continuous polynucleic acid molecule according to any one of claims 2 to 16, wherein the 3' response element of the cassette in (i) further comprises at least one microRNA target site.

18. 20. The contiguous polynucleic acid molecule of claim 17, wherein at least one microRNA target site is 3' to the output.

19. 19. The continuous polynucleic acid molecule of claim 17 or 18, wherein at least one microRNA target site is 5' to the output or within the output.

20. The continuous polynucleic acid molecule according to any one of claims 2 to 13, wherein the cassette in (ii) further comprises at least one microRNA target site.

21. 21. The contiguous polynucleic acid molecule of claim 20, wherein at least one microRNA target site is 3' to a DNA sequence encoding a transactivator protein.

22. 22. The continuous polynucleic acid molecule of claim 20 or 21, wherein at least one microRNA target site is 5' to or within a DNA sequence encoding a transactivator protein.

23. 23. The continuous polynucleic acid molecule of any one of claims 20 to 22, wherein at least one microRNA target site of the cassette in (i) and at least one microRNA target site of the cassette in (ii) are the same nucleic acid sequence or are different sequences controlled by the same microRNA.

24. 24. The continuous polynucleic acid molecule according to any one of claims 2 to 23, wherein at least one cassette is flanked by insulators.

25. 25. The continuous polynucleic acid molecule according to any one of claims 1 to 24, wherein the transactivator of at least one cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA.

26. The continuous polynucleic acid molecule according to any one of claims 1 to 25, wherein the output is a protein or an RNA molecule.

27. The continuous polynucleic acid molecule of any one of claims 1 to 26, wherein the output is a therapeutic agent.

28. 28. The continuous polynucleic acid molecule of claim 26 or 27, wherein the output is a fluorescent protein, a cytotoxin, an enzyme catalyzing the activation of a prodrug, an immunomodulatory protein and / or an RNA, a DNA modifier, a cell surface receptor, a gene expression regulator, a kinase, an epigenetic modifier, and / or a factor required for vector replication.

29. 29. The contiguous polynucleic acid molecule of claim 28, wherein the immunomodulatory protein and / or RNA is a cytokine or a colony stimulating factor.

30. 29. The contiguous polynucleic acid molecule of claim 28, wherein the DNA modifier is a gene encoding a protein intended to correct a genetic defect, a DNA modifying enzyme, and / or a component of a DNA modifying system.

31. 31. The contiguous polynucleic acid molecule of claim 30, wherein the DNA modifying enzyme is a site-specific recombinase, a homing endonuclease, or a protein component of a CRISPR / Cas DNA modifying system.

32. 29. A continuous polynucleic acid molecule according to claim 28, wherein the gene expression control element is a protein capable of controlling gene expression or a component of a multi-component system capable of controlling gene expression.

33. A vector comprising a contiguous polynucleic acid molecule according to any one of claims 1 to 32.

34. An engineered viral genome comprising a contiguous polynucleic acid molecule according to any one of claims 1 to 32.

35. 35. The engineered viral genome of claim 34, wherein the viral genome is an adeno-associated virus (AAV) genome, a lentivirus genome, an adenovirus genome, a herpes simplex virus (HSV) genome, a vaccinia virus genome, a poxvirus genome, a Newcastle disease virus (NDV) genome, a coxsackievirus genome, a reovirus genome, a measles virus genome, a vesicular stomatitis virus (VSV) genome, a parvovirus genome, a Seneca Valley virus genome, a Maraba virus genome, or a common cold virus genome.

36. A virion comprising the engineered viral genome of claim 34 or 35.

37. A contiguous polynucleic acid molecule encoding at least one cassette, wherein the cassette comprises: (i) a 5' regulatory element comprising a transactivator response element; and (ii) a 3' response element comprising an output, a transactivator, and optionally a polycistronic expression element, where the output and the transactivator are optionally separated by a polycistronic expression element; wherein transcription of the response element produces a single mRNA; and wherein the transactivator of (ii) binds to and transactivates the transactivator response element of (i) when expressed as a protein, said contiguous polynucleic acid molecule.

38. 38. A contiguous polynucleic acid molecule according to claim 37, wherein the transactivators independently bind to and transactivate transactivator response elements.

39. 39. The continuous polynucleic acid molecule of claim 37 or 38, wherein the 5' regulatory element in (i) further comprises a transcription factor response element and / or a minimal promoter.

40. 40. The continuous polynucleic acid molecule of claim 39, wherein the transactivator binds to and transactivates a transactivator response element only in the presence of a transcription factor bound to the transcription factor response element.

41. 40. The contiguous polynucleic acid molecule of claim 39, wherein the 5' regulatory element comprises, from 5' to 3', a transactivator response element, a transcription factor response element, and a minimal promoter.

42. 40. The contiguous polynucleic acid molecule of claim 39, wherein the 5' regulatory element comprises, from 5' to 3', a transcription factor response element, a transactivator response element, and a minimal promoter.

43. 40. The continuous polynucleic acid molecule according to any one of claims 37 to 39, wherein the 5' regulatory element in (i) further comprises a promoter element.

44. 44. The contiguous polynucleic acid molecule of claim 43, wherein the promoter element comprises a mammalian promoter or a promoter fragment.

45. 45. A contiguous polynucleic acid molecule according to claim 43 or 44, wherein the 5' regulatory element in (i) comprises, from 5' to 3', a transactivator response element and a promoter element.

46. The continuous polynucleic acid molecule of any one of claims 37 to 45, wherein the 3' response element of (ii) further comprises at least one microRNA target site.

47. 47. The contiguous polynucleic acid molecule of claim 46, wherein at least one microRNA target site is 3' to the output and / or transactivator.

48. 48. The continuous polynucleic acid molecule of claim 46 or 47, wherein at least one microRNA target site is 5' to the output and / or transactivator or internal to the output and / or transactivator.

49. 49. A continuous polynucleic acid molecule according to any one of claims 37 to 48, wherein at least one cassette is flanked by insulators.

50. 50. The continuous polynucleic acid molecule of any one of claims 37 to 49, wherein the transactivator of at least one cassette is tTA, rtTA, PIT-RelA, PIT-VP16, ET-VP16, ET-RelA, NarLc-VP16, or NarLc-RelA.

51. The continuous polynucleic acid molecule according to any one of claims 37 to 50, wherein the output is a protein or an RNA molecule.

52. The continuous polynucleic acid molecule according to any one of claims 37 to 51, wherein the output is a therapeutic protein or RNA molecule.

53. 53. The continuous polynucleic acid molecule of claim 51 or 52, wherein the output is a fluorescent protein, a cytotoxin, an enzyme catalyzing the activation of a prodrug, an immunomodulatory protein and / or an RNA, a DNA modifier, a cell surface receptor, a gene expression regulator, a kinase, an epigenetic modifier, and / or a factor required for vector replication.

54. 54. The contiguous polynucleic acid molecule of claim 53, wherein the immunomodulatory protein and / or RNA is a cytokine or a colony stimulating factor.

55. 54. The contiguous polynucleic acid molecule of claim 53, wherein the DNA modifier is a gene encoding a protein intended to correct a genetic defect, a DNA modifying enzyme, and / or a component of a DNA modifying system.

56. 56. The sequential polynucleic acid molecule of claim 55, wherein the DNA modifying enzyme is a site-specific recombinase, a homing endonuclease, or a protein component of a CRISPR / Cas system.

57. 54. A continuous polynucleic acid molecule according to claim 53, wherein the gene expression control element is a protein capable of controlling gene expression or a component of a multi-component system capable of controlling gene expression.

58. A vector comprising a contiguous polynucleic acid molecule according to any one of claims 37 to 57.

59. 58. An engineered viral genome comprising a contiguous polynucleic acid molecule according to any one of claims 37 to 57.

60. 60. The engineered viral genome of claim 59, wherein the viral genome is an adeno-associated virus (AAV) genome, a lentivirus genome, an adenovirus genome, a herpes simplex virus (HSV) genome, a vaccinia virus genome, a poxvirus genome, a Newcastle disease virus (NDV) genome, a coxsackievirus genome, a reovirus genome, a measles virus genome, a vesicular stomatitis virus (VSV) genome, a parvovirus genome, a Seneca Valley virus genome, a Maraba virus genome, or a common cold virus genome.

61. 61. A virion comprising the engineered viral genome of claim 59 or 60.

62. 35. A method of stimulating a cell specific event in a population of cells comprising contacting the population of cells with a continuous polynucleic acid molecule according to any one of claims 2 to 32, a vector according to claim 33, an engineered viral genome according to claim 34 or 35, or a virion according to claim 36, wherein the cell specific event comprises: (i) an endogenous transcription factor that binds to and transactivates the regulatory elements of at least one of the cassettes; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) controlled by at least one endogenous microRNA that is complementary to a microRNA target site of the response element of at least one cassette.

63. 10. A method of stimulating a cell specific response in a population of cells comprising contacting the population of cells with a continuous polynucleic acid molecule according to any one of claims 37 to 57, a vector according to claim 58, an engineered viral genome according to claim 59 or 60, or a virion according to claim 61, wherein the cell specific event is (i) an endogenous transcription factor that binds to and transactivates a transcription factor response element of the 5′ regulatory element of at least one cassette; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) at least one endogenous microRNA that is complementary to a microRNA target site of the 3' response element of at least one cassette.

64. The population of cells comprises at least one target cell and at least one non-target cell, wherein the target cell and the non-target cell are (i) the protein levels or activity of endogenous transcription factors that bind to and transactivate the regulatory elements of at least one cassette; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) the RNA level or activity of at least one endogenous microRNA that is complementary to a microRNA target site of the response element of at least one cassette; and The method of claim 62 or 63, wherein the different protein levels or activity in (i), and / or transcriptional activity of the promoter fragment in (ii), and / or RNA levels or activity in (iii) cause target cells and non-target cells to differ in expression levels of the output of at least one response element of the cassette, thereby stimulating a cell-specific event.

65. The population of cells comprises at least one target cell and at least one non-target cell, wherein the target cell and the non-target cell are (i) the protein level or activity of an endogenous transcription factor that binds to and transactivates a transcription factor response element of the 5′ regulatory element of at least one cassette; and / or (ii) the transcriptional activity of the promoter fragment; and / or (iii) the RNA level or activity of at least one endogenous microRNA that is complementary to a microRNA target site of the 3′ response element of at least one cassette; and The method of claim 64, wherein the different protein levels or activity in (i), and / or transcriptional activity of the promoter fragment in (ii), and / or RNA levels or activity in (iii) cause target cells and non-target cells to differ in expression levels of the output of the 3' response element of at least one cassette, thereby stimulating a cell-specific event.

66. 66. The method of claim 64 or 65, wherein the expression level of the 3' response element output differs between the target cell type and the non-target cell type by at least 2, at least 5, at least 10, at least 100, at least 1,000, or at least 10,000 fold.

67. The method of any one of claims 64 to 66, wherein the cells of the target cell population are tumor cells and the cell-specific event is cell death.

68. 68. The method of claim 67, wherein tumor cell death is mediated by immune targeting through expression of an activating receptor ligand, a specific antigen, a stimulatory cytokine, or any combination thereof.

69. 67. The method of any one of claims 64 to 66, wherein the cells of the target cell population are senescent cells and the cell-specific event is cell death.

70. 70. The method of any one of claims 67-69, further comprising contacting the population of cells with a prodrug or a non-toxic precursor compound that is metabolized to a therapeutic or toxic compound by the output.

71. 67. The method of any one of claims 64 to 66, wherein cells of the target cell population differentially express the factor relative to wild-type cells of the same type, and the cell-specific event is modulating the expression level of the factor.

72. 67. The method of any one of claims 64 to 66, wherein output expression ensures survival of the target cell population, while non-target cells are eliminated due to lack of output expression and in the presence of an irrelevant and non-specific cell death inducer.

73. 67. The method of any one of claims 64 to 66, wherein cells of the target cell population comprise a particular phenotype of interest, such that output expression is restricted to cells of this particular phenotype.

74. 67. The method of any one of claims 64 to 66, wherein the cells of the target cell population are of a selected cell type and the cell-specific event is encoding a novel function through expression of a gene that is not naturally present or is inactive in the selected cell type.

75. 75. The method of any one of claims 62 to 74, wherein the population of cells comprises a multicellular organism.

76. 76. The method of claim 75, wherein the multicellular organism is an animal.

77. 77. The method of claim 76, wherein the animal is a human.

78. 78. The method of any one of claims 62 to 77, wherein the population of cells is contacted ex vivo.

79. 78. The method of any one of claims 62 to 77, wherein the population of cells is contacted in vivo.

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