Multi-cistronic miRNA constructs for immune checkpoint suppression

Abstract

The present invention provides a ribonucleic acid comprising two non-natural pre-miRNA sequences, where each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein. The pre-miRNA sequence can target different genes or target different regions of the same gene. The invention provides a desoxyribonucleic acid, and the desoxyribonucleic acid is used for coding the ribonucleic acid. The deoxyribonucleic acids may further encode proteins, such as chimeric antigen receptors, cytokines, cell tags, and / or immune checkpoint inhibitors. The invention provides a carrier, and the carrier comprises the ribonucleic acid or the desoxyribonucleic acid. The invention provides a method for modifying the expression of a gene in a cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned desoxyribonucleic acid into the cell. The invention provides a method for producing a genetically engineered cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned desoxyribonucleic acid into the cell. In addition, the invention provides a genetically modified cell, and the genetically modified cell comprises the ribonucleic acid or the desoxyribonucleic acid. In addition, the present invention provides a composition comprising the aforementioned ribonucleic acid or the aforementioned desoxyribonucleic acid. The invention provides a kit, and the kit comprises the ribonucleic acid or the desoxyribonucleic acid. The present invention provides a method of treating a disease or condition in a subject, the method comprising administering the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the subject. The present invention provides a method of treating a disease or condition in a subject comprising administering the aforementioned cells to the subject. The present invention provides the use of the aforementioned ribonucleic acid or the aforementioned desoxyribonucleic acid in the manufacture of a medicament for modifying the expression of a gene. The present invention provides the use of the aforementioned ribonucleic acid or the aforementioned desoxyribonucleic acid in the manufacture of a medicament for the treatment of a disease or disorder in a subject.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on June 21, 2023, is named 391456_SL.xml, and is 501,736 bytes in size. Background Art

[0003] Immune checkpoint proteins are used to regulate the immune system. Positive immune checkpoint proteins are used to assist T cells in immune responses. At the same time, negative immune checkpoint proteins (such as PD-1, TIGIT, CD70 and CTLA-4) are used to downregulate immune responses, thereby preventing T cells from damaging or killing healthy cells. However, in individuals with cancer, this downregulation may also prevent T cells from killing cancerous cells, including T cells (CAR-T cells) modified to contain chimeric antigen receptors. As such, it is desirable to inhibit the activity of such checkpoint proteins.

[0004] Immune checkpoint inhibition has shown promise as an immunotherapy that prevents T-cell shutoff and promotes the activity of these cells. Examples of checkpoint inhibitor proteins that may be targeted by such therapy include, but are not limited to, PD1, PD-L1, CTLA-4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, and VISTA. One of the most studied checkpoint inhibition pathways is the PD-1 / programmed death ligand 1 (PD-L1) pathway, which plays a critical role in how tumor cells evade immune responses. Immunotherapy using PD-1 / PD-L1 blocking antibodies has been extensively evaluated in the clinic and has been shown to improve tumor regression in a variety of malignancies, particularly when administered in combination with CAR-T cells.

[0005] However, checkpoint inhibitor blocking antibodies do not perform consistently across cancer types. Furthermore, such antibodies may not adequately enter the tumor microenvironment, require repeated administration, and may lose effectiveness over time. Genome editing is an alternative approach and has the advantage of limiting checkpoint inhibitor blockade to only engineered cells. However, gene editing adds complexity to the manufacturing process, which increases the turnaround time and cost of cell therapies. Therefore, there is a continuing need in the art to obtain new checkpoint inhibitor therapies.

[0006] MicroRNAs (miRNAs) are small noncoding RNA molecules that can bind to mRNA molecules produced by targeted genes, thereby affecting their translation into proteins. Through this action, miRNAs silence genes.

[0007] In order to increase the silencing of the target gene, it is known that multiple miRNAs targeting different regions on the same gene are encoded in a polycistronic genetic construct, which can, for example, be delivered to cells via a vector. See Mueller et al., Molecular Therapy, 20: 590–600 (2012) (“Mueller”), see Figure 1. This method has been shown to achieve robust knockdown of target genes. Ibid. However, when a construct containing a repeating precursor miRNA (pre-miRNA) structure is used, the pre-miRNA stem-loop structure is at risk of alternate folding during transcription. This may produce alternatively processed miRNAs that may cause unexpected off-target gene silencing, thereby posing a safety risk. In addition, a construct containing a repeating pre-miRNA structure may allow recombination within the vector, resulting in an impure vector population with sequence variation.

[0008] The applicant solves these risks by designing polycistronic constructs that encode multiple miRNAs but each pre-miRNA is different and non-complementary to each other. In certain embodiments, at least about 10 nucleotides separate the pre-miRNA structure to help ensure the appropriate co-transcriptional folding of RNA. In addition, in certain embodiments, pre-miRNA is designed to maintain predicted stem-loop structure and internal loop based on endogenous human sequence, which is expected to reduce the risk of toxicity based on RNAi. The pre-miRNA in these constructs can target different genes or different regions of the same gene respectively.

[0009] The present invention relates to the use of polycistronic miRNA constructs to modify the expression of genes encoding immune checkpoint proteins. Summary of the invention

[0010] The present invention relates in part to a ribonucleic acid comprising two non-natural pre-miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein.

[0011] In certain embodiments, the non-natural pre-miRNA sequences have less than about 50% sequence identity with each other.

[0012] In certain embodiments, the nucleic acid sequence of at least one non-naturally occurring pre-miRNA sequence has at least about 90% sequence identity to the nucleic acid sequence of a naturally occurring pre-miRNA sequence.

[0013] In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 10 nucleotides.

[0014] In certain embodiments, each non-natural pre-miRNA sequence targets a different gene.

[0015] In certain embodiments, each non-natural pre-miRNA sequence targets a different region of the same gene.

[0016] In certain embodiments, each non-naturally occurring pre-miRNA comprises a backbone sequence that is identical to the corresponding backbone segment of a naturally occurring pre-miRNA.

[0017] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence from miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915.

[0018] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence from miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206.

[0019] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence from miR16, miR21, miR22, miR204, or miR206.

[0020] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence from: miR204 or miR206.

[0021] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to mRNA and thereby interfering with its translation and / or promoting its degradation.

[0022] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to mRNA under stringent hybridization conditions.

[0023] In certain embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1.

[0024] In certain embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1.

[0025] In certain embodiments, the immune checkpoint protein is CD70, PD-1, or TIGIT.

[0026] In certain embodiments, the immune checkpoint protein is PD-1.

[0027] The present invention also relates in part to a deoxyribonucleic acid encoding the ribonucleic acid of the present invention.

[0028] In certain embodiments, the DNA further encodes a protein.

[0029] In certain embodiments, the protein is a chimeric antigen receptor.

[0030] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain that binds an antigen that is overexpressed in cancer.

[0031] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain that binds CD19, CD33, MUC-16, or ROR-1.

[0032] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain that binds ROR-1.

[0033] In certain embodiments, the protein is a cytokine.

[0034] In certain embodiments, the protein comprises IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof.

[0035] In certain embodiments, the protein is a cellular tag.

[0036] In certain embodiments, the cell tag comprises HER1 domain III or a functional fragment or variant thereof and a truncated HER1 domain IV or a functional fragment or variant thereof.

[0037] In certain embodiments, the cell tag further comprises a CD28 transmembrane domain or a functional fragment or variant thereof.

[0038] In certain embodiments, the protein is an immune checkpoint inhibitor.

[0039] In certain embodiments, the deoxyribonucleic acid encodes: (a) a chimeric antigen receptor; (b) a protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof; and (c) a cell tag.

[0040] The present invention also relates in part to a vector comprising the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention.

[0041] In certain embodiments, the vector is a plasmid, a nanoplasmid, a viral vector, an episomal vector, or a non-viral vector.

[0042] In certain embodiments, the vector is a Sleeping Beauty transposon.

[0043] In certain embodiments, the vector is a viral vector.

[0044] In certain embodiments, the vector is an adenoviral vector.

[0045] The present invention also relates in part to a method for modifying the expression of a gene in a cell, wherein the method comprises introducing a ribonucleic acid of the present invention or a deoxyribonucleic acid of the present invention.

[0046] The present invention also relates in part to a method for modifying the expression of a gene in a cell, wherein the method comprises transfecting the cell with the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention.

[0047] In certain embodiments, the method comprises transfecting a cell with a vector of the invention.

[0048] In certain embodiments, the method further comprises transfecting the cell with a vector encoding a transposase.

[0049] The present invention also relates in part to a method for producing a genetically engineered cell, wherein the method comprises introducing into the cell a ribonucleic acid of the present invention or a deoxyribonucleic acid of the present invention.

[0050] The present invention also relates in part to a genetically modified cell comprising the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention.

[0051] The present invention also relates in part to a genetically modified cell produced by the method of the present invention.

[0052] The present invention also relates in part to a composition comprising the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention.

[0053] In certain embodiments, the compositions are for use in modifying the expression of a gene.

[0054] In certain embodiments, the compositions are for use in treating a disease or disorder in a subject.

[0055] The present invention also relates in part to a composition comprising a vector of the present invention or a cell of the present invention.

[0056] The present invention also relates in part to a kit comprising the ribonucleic acid of the present invention or the deoxynucleic acid of the present invention.

[0057] The invention also relates in part to a kit comprising the cells of the invention.

[0058] The present invention also relates in part to a method of treating a disease or condition in a subject, the method comprising administering to the subject a ribonucleic acid of the present invention or any of the deoxynucleic acids of the present invention.

[0059] The present invention also relates in part to a method of treating a disease or condition in a subject, the method comprising administering to the subject a cell of the present invention or any of the deoxynucleic acids of the present invention.

[0060] The present invention also relates in part to the use of the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention in the manufacture of a medicament for modifying the expression of a gene.

[0061] The present invention also relates in part to the use of a ribonucleic acid of the present invention or a deoxyribonucleic acid of the present invention in the manufacture of a medicament for treating a disease or condition in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A is an exemplary representation of a vector comprising a combination of one or more checkpoint inhibitor miRNAs and a chimeric receptor (SD=splice donor; SA=splice acceptor). Figure 1B is an exemplary illustration of the hairpin and loop designs for each pri-miRNA, including the target miRNA and complementary sequence at each 5' position or 3' position. Figure 1C are exemplary illustrations of the hairpin and loop designs of individual pri-miRNAs, including their positions within the transgene cassette.

[0063] Figure 2 is a graph showing the relative RNA expression of PD1 after transfection of various combinations of miRNA constructs in the presence or absence of MUC16-specific CAR. Constructs No. 1 to 8 shown on the X-axis are schematically presented as in Table 10.

[0064] Figure 3A , Figure 3B and Figure 3Cis a graph showing normalized absolute transcript counts obtained from genetic profiling of >700 genes using the Nanostring Human Genome Encoding Panel of CD33 CAR-T cells stimulated with CD3 / CD28 beads. Figure 3A In the figure, the Y-axis plots the transcript counts from CAR-T cells that contain introns encoding 2 checkpoint inhibitor miRNAs targeting PD-1 and TIGIT (CD33CAR-mbIL15-HER1t+miRNA(PD-1+TIGIT)), and the X-axis plots the transcripts from CAR-T cells alone (without any checkpoint inhibitory miRNAs). Circles represent genes of interest. Figure 3B In the figure, the Y axis plots the transcript counts from CAR-T cells containing PD-1 miRNA (CD33 CAR-mbIL15-HER1t+miRNA(PD-1+PD-1)), and the X axis plots the transcripts from CAR-T cells alone. Figure 3C A non-targeted miRNA control (CD33 CAR-mbIL15-HER1t+miRNA (scrambled*)) is plotted on the Y-axis, and CAR-T cells without miRNA introns are plotted on the X-axis. Circles represent genes of interest and are used to illustrate the on-target specificity of checkpoint inhibitor miRNA design. All three graphs are derived from one donor. *Scrambled controls are non-targeted miRNAs.

[0065] FIG. 4A to FIG. 4C is a graph showing normalized absolute transcript counts obtained from genetic analysis of >700 genes using the Nasri genome coding set of MUC16-specific CAR-T cells stimulated with CD3 / CD28 beads. Figure 4A In the figure, the Y-axis plots transcript counts from CAR-T cells that contain introns encoding miRNAs that target two different sequences within PD-1 and TIGIT (MUC16CAR-mbIL15-HER1t (collectively referred to as "MUC16CAR") + miRNA (PD-1 / PD-1 / TIGIT)), and the X-axis plots transcripts from CAR-T cells that do not contain miRNA introns (MUC16CAR-mbIL15-HER1t). Black circles represent genes of interest. Figure 4B In the figure, the X-axis is the same, and the Y-axis plots the transcript counts from CAR-T cells containing miRNA targeting dual PD-1 (MUC16CAR-mbIL15-HER1t+miRNA(PD-1 / PD-1)). Figure 4CA non-targeting miRNA control (MUC16CAR-mbIL15-HER1t+miRNA (scrambled)) is plotted on the Y-axis, and CAR-T cells without the miRNA intron are plotted on the X-axis. All three graphs are from one donor.

[0066] Figure 5A It is a graph showing the number of GFP+K562 cells expressing MUC16 changing over time. The line with black circle solid points at each time point represents the number of GFP+ target cells in the well without CAR-T cells. The line with square hollow points represents the target cell count in the well with MUC16 CAR-mbIL15-HER1t CAR-T cells ("with CAR-T cells") without miRNA introns. The line with gray circle solid points represents the target cell count in the well with CAR-T cells ("with CAR-T+miRNA cells"), which contain synthetic introns with miRNA targeting dual PD-1 (MUC16 CAR-mbIL15-HER1t+miRNA (PD-1 / PD-1)). Data are from one donor, and plotted are the mean + SD of triplicate wells. ***P<0.001 is based on a two-way ANOVA using Dunnett's Multiple Comparison post hoc test.

[0067] Figure 5B It is a graph showing the number of GFP+K562 / MUC16+ / PD-L1+ / CD155+ cells changing over time. The line with square solid points at each time point represents the number of GFP+ target cells only in the well. The line with hollow dots represents the target cell counts in the wells with MUC16-specific CAR-T cells (with CAR-T cells) without miRNA introns (MUC16 CAR-mbIL15-HER1t). The line with hollow circle solid points represents the target cell counts in the wells with CAR-T cells, which contain synthetic introns with miRNAs targeting dual PD-1 and TIGIT (MUC16CAR-mbIL15-HER1t+miRNA (PD-1 / PD-1 / TIGIT) (with CAR-T+miRNA cells)). The data are from one donor, and the mean value + SD of triplicate wells is plotted.

[0068] FIG. 6A to FIG. 6B Shown are cytokine expression levels of IFNγ and GM-CSF in MUC16 CAR-T cells with a combination of one or more checkpoint inhibitor miRNAs after co-culture with tumor target cells (K562 / MUC16t). FIG. 6C to FIG. 6DThe cytokine expression levels of IFNγ and GM-CSF in MUC16 CAR-T cells with a combination of one or more checkpoint inhibitor miRNAs in the absence of co-culture with tumor target cells are shown. Constructs No. 1 to 11 shown on the X-axis are schematically presented as in Table 11.

[0069] Figure 7 Shown are tumor burdens in mice treated with MUC16 CAR+mbIL-15+HER1t (shown as “MUC16 CAR”) in combination with various miRNAs.

[0070] Fig. 8A Shown are PD-1 levels in cell populations gated after hCD45 / CD3+ / HER1t+ expression in the blood of mice treated with MUC16CAR+mbIL15+HER1t (CAR only) and MUC16CAR+mbIL15+HER1t+miRNA(PD1 / PD-1) (CAR+miRNA(PD-1 / PD-1)). Figure 8B Shown are PD-1 levels measured by median fluorescence intensity (MFI) in CAR and CAR+miRNA (PD-1 / PD-1) treated mice.

[0071] Fig. 9A and Fig. 9B The PD-1 level and TIGIT MFI level in the cell population after gating hCD45 / CD3+ / HER1t+ expression in the blood of mice treated with various CARs and CAR+miRNAs are shown. Groups 1 to 9 shown on the X-axis are schematically presented as shown in Table 12.

[0072] Fig. 10A The PD1 silencing submodule produces guide miRNAs, and PD1 mRNA expression is correspondingly reduced in UltraCAR-T cells generated from five T cell donors. RT-qPCR results of guide miRNAs targeting PD1 are shown.

[0073] Fig. 10B The PD1 silencing submodule was shown to produce guide miRNAs and a corresponding reduction in PD1 mRNA expression in ultraCAR-T cells generated from five T cell donors. RT-qPCR results for PD1 mRNA are shown.

[0074] Fig.11 We show that the PD1 silencing module preferentially produces guide miRNAs targeting PD1 rather than non-targeting passenger miRNAs.

[0075] FIG. 12A to FIG. 12EWe show that guide miRNAs are the major small RNA species derived from the PD1 silencer module.

[0076] Fig.13 Quantification of mature miRNAs mapping to the PD1 silencing submodule as a percentage of total small RNAseq reads is shown.

[0077] Fig.14A and Fig. 14B Differential gene expression in ROR1+PD1 silencer cells compared to ROR1 UltraCAR-T control cells is shown.

[0078] FIG. 15A to FIG. 15D Comparison of predicted miRNA binding strength and transcript log fold change is shown.

[0079] Fig.16 Exemplary schemes of the genetic constructs of the present disclosure are provided.

[0080] Fig.17 Shown is TIGIT expression in TIGIT expressing cells after administration of anti-TIGIT miRNA relative to that in controls.

[0081] Fig.18 Shown is TIGIT expression in cells following administration of constructs containing a single anti-TIGIT pre-miRNA or two anti-TIGIT pre-miRNAs relative to TIGIT expression in controls.

[0082] Fig.19 Shown is CD70 expression in cells relative to CD70 expression in control after administration of constructs containing: a single anti-CD70 pre-miRNA in the 5' intron; two anti-PD1 pre-miRNAs and one anti-CD70 pre-miRNA in the 5' intron; two anti-PD1 pre-miRNAs in the 5' intron and two anti-CD70 pre-miRNAs in the 3'UTR; and two anti-PD1 pre-miRNAs in the 5' intron and one anti-CD70 pre-miRNA in the 3'UTR. DETAILED DESCRIPTION

[0083] The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can be varied accordingly. Those skilled in the art will recognize that the scope of the present disclosure encompasses variations and modifications of the present disclosure.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0085] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0086] Although various features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosure may be described in the context of a single embodiment for clarity, the disclosure may also be implemented in a single embodiment.

[0087] I. Definitions

[0088] The following definitions supplement those in the art and are specific to the present application and are not attributed to any related or unrelated circumstances, such as any commonly owned patents or applications.The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0089] In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0090] In this application, unless otherwise stated, the use of "or" means "and / or". As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C. The term "or" may be used in conjunction or disjunction unless the context specifically refers to disjunction.

[0091] Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0092] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments of the present disclosure.

[0093] As used in this specification and one or more claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude otherwise unlisted elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0094] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" associated with a reference value may also include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the value. Alternatively, particularly for biological systems or processes, the term "about" may mean within an order of magnitude, preferably within 5 times of a value, and more preferably within 2 times. When particular values ​​are described in the present application and claims, unless otherwise indicated, the term "about" should be assumed to mean that the particular value is within an acceptable error range.

[0095] "Therapeutically effective amount" or "therapeutically effective dose" refers to an effective amount or dose that achieves the desired therapeutic effect over a necessary period of time. The amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the nucleic acid sequences of the invention to elicit a desired response in the individual.

[0096] "Polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides or nucleic acids of any length, i.e., ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term encompasses double-stranded deoxyribonucleic acid (DNA) and single-stranded DNA, triplex DNA, as well as double-stranded ribonucleic acid and single-stranded RNA. The term also encompasses modified, e.g., by methylation and / or by end-capping, as well as unmodified forms of polynucleotides. The term is also meant to encompass molecules that contain non-naturally occurring or synthetic nucleotides and nucleotide analogs.

[0097] Unless otherwise indicated, nucleic acid sequences in the text of the present specification are given in the 5' to 3' direction when reading from left to right.

[0098] As used herein, the terms "transfection", "transformation", "nucleofection" or "transduction" refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by the use of physical, chemical and / or electrical methods. The nucleic acid sequences and vectors disclosed herein can be introduced into cells or organisms by any such method, including, for example, electroporation, calcium phosphate coprecipitation, strontium phosphate DNA coprecipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polycation-mediated transfection, tungsten particle-promoted microparticle bombardment, viral and / or non-viral mediated transfection. In some cases, the method of introducing nucleic acids into cells or organisms involves the use of viral vectors, retroviral vectors, lentiviral vectors, or transposon vectors or transposable element-mediated (e.g., Sleeping Beauty) vectors.

[0099] As used herein, "polypeptide", "peptide" and their grammatical equivalents refer to a polymer of amino acid residues. The polypeptide may optionally contain glycosylation or other typical modifications of a given protein in a given cellular environment. The polypeptides disclosed herein (including functional fragments and functional variants thereof) may contain synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. The present disclosure further contemplates that the expression of the polypeptide or protein described herein in the engineered cell can be associated with post-translational modification of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, disulfide bond formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, proteolipidation, and iodination.

[0100] The term "conservative amino acid substitution" or "conservative mutation" refers to an amino acid replaced by another amino acid with common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, amino acid groups can be defined, wherein the amino acids within the group are preferentially exchanged with each other, and therefore are most similar to each other in terms of their impact on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include amino acid substitutions of amino acids within the subgroups below, for example, lysine replaces arginine, and vice versa, so that a positive charge can be maintained; glutamic acid replaces aspartic acid, and vice versa, so that a negative charge can be maintained; serine replaces threonine, so that free-OH can be maintained; and glutamine replaces asparagine, so that free-NH 2 Exemplary conservative amino acid substitutions are shown in the following table:

[0101] Types of Amino Acids Substitutable amino acids Hydrophilicity Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Thiol Cys Fatty Val, Ile, Leu, Met Alkaline Lys, Arg, His Aromatic Phe, Tyr, Trp

[0102] Amino acid sequences that differ from a reference amino acid sequence only by conservative amino acid substitutions will be referred to herein as "conservatively substituted variants" of the reference sequence.

[0103] In some embodiments, the functional variant may comprise an amino acid sequence of a reference protein having at least one non-conservative amino acid substitution. The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, lysine replaces tryptophan, or phenylalanine replaces serine, etc. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is enhanced compared to the homologous parent protein. Amino acid substitutability is discussed in more detail, for example, LYYampolsky and A.Stoltzfus, "The Exchangeability of Amino acids in Proteins," Genetics, August 2005; 170 (4): 1459-1472.

[0104] In the context of two nucleic acid sequences or amino acid sequences of polypeptides, the term "identical" and its grammatical equivalents or "sequence identity" as used herein refers to the residues in the two sequences that are identical when aligned over a specified comparison window to achieve maximum correspondence. As used herein, a "comparison window" refers to a segment of at least about 20, typically about 50 to about 200, more typically about 100 to about 150 consecutive positions, wherein the sequence can be compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci USA, 85:2444 (1988); computerized implementations of these algorithms (including but not limited to CLUSTAL in the PC / Gene program of Intelligentics, Mountain View, California, the Genetics Computer Group (GCG), 575 Science Avenue, Madison, Wisconsin, USA) Dr., Madison, Wis., USA); the CLUSTAL program is described in detail in the following: Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also usually performed by inspection and manual alignment. In one class of embodiments, a polypeptide herein is at least 80%, 85%, 90%, 98%, 99% or 100% identical to a reference polypeptide (i.e., its full length) or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL or any other available alignment software) using default parameters.Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., a nucleic acid can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid (i.e., its full length) or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL or any other available alignment software) using default parameters. When a molecule has a certain percentage of sequence identity with a larger molecule, this means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a matching residue in the larger molecule according to the order in which the two molecules are optimally aligned.

[0105] For the purposes of this specification and claims, it is understood that the phrase "having at least 50% sequence identity to a reference sequence," or any range cited therein (e.g., "having at least 80% sequence identity to a reference sequence") encompasses the reference sequence itself. Thus, for example, a claim reciting "a nucleic acid having at least 80% sequence identity to SEQ ID NO:0" encompasses SEQ ID NO:0 itself.

[0106] The term "substantially identical" and its grammatical equivalents as applied to nucleic acid or amino acid sequences means that the nucleic acid or amino acid sequences comprise sequences having at least 95% sequence identity compared to a reference sequence using programs such as BLAST described above using standard parameters.

[0107] Homology is usually inferred from the sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of identity between sequences that can be used to establish homology varies with the nucleic acid and protein in question, but usually only 25% sequence identity is used to establish homology. A higher level of sequence identity, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. The method for determining sequence identity percentage (for example, BLASTP and BLASTN using default parameters) is described herein and is usually available. When nucleic acid and / or nucleotide sequence are derived from common ancestral nucleic acid or nucleotide sequence naturally or artificially, these nucleic acid and / or nucleotide sequence are "homologous". When the coding DNA of protein and / or protein sequence is derived from common ancestral nucleic acid or nucleotide sequence naturally or artificially, these protein and / or protein sequence are "homologous". Homologous molecules can be referred to as homologues. For example, any naturally occurring protein can be modified by any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

[0108] Nucleic acid molecules that hybridize to the disclosed sequences are also contemplated and encompassed herein. Hybridization conditions may be mild, moderate or stringent, as appropriate.

[0109] Appropriate stringent conditions that promote DNA hybridization, such as 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by washing with 2×SSC at 50° C., are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. “Stringent hybridization conditions” are conditions comprising a salt concentration of 1.0 M NaCl in 50% formamide at 37° C. for 4 to 12 hours, followed by washing in 0.1×SSC at 60° C. to 65° C.

[0110] Skilled practitioners will appreciate that minor changes in the nucleic acid sequence will not necessarily change the amino acid sequence of the encoded polypeptide. The present disclosure relates to the degeneracy of codon usage, as understood by those of ordinary skill in the art. For example, as is known in the art, different codons will encode the same amino acid, as illustrated in the following chart.

[0111] Amino Acids Codon Ala / A GCT, GCC, GCA, GCG Arg / R CGT, CGC, CGA, CGG, AGA, AGG Asn / N AAT、AAC Asp / D GAT、GAC Cys / C TGT, UGC Gln / Q CAA, CAG Glu / E GAA, GAG Gly / G GGT, GGC, GGA, GGG His / H CAT、CAC Ile / I ATT, ATC, ATA Leu / L TTA, TTG, CTT, CTC, CTA, CTG Lys / K AAA、AAG Met / M ATG Phe / F TTT, TTC Pro / P CCT, CCC, CCA, CCG Ser / S TCT, TCC, TCA, TCG, AGT, AGC Thr / T ACT, ACC, ACA, ACG Trp / W TGG Tyr / Y TAT、TAC Val / V GTT, GTC, GTA, GTG Starting ATG termination TAG, TGA, TAA

[0112] As used herein, the phrase "codon degenerate variant" when used in reference to a nucleic acid sequence means a nucleic acid sequence that differs from a reference sequence but encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference sequence.

[0113] Additionally, it will be appreciated by those skilled in the art that partial sequences are usually as effective as full-length versions. The method for changing or shortening nucleotide sequences is well known to those skilled in the art, as is the method for the suitability or effectiveness of the gene that tests changes. In certain embodiments, the suitability and / or effectiveness of the gene that changes can be easily tested by, for example, conventional gas chromatography. Therefore, all such variations of gene are included as a part of this disclosure.

[0114] As used herein, the term "isolated" and its grammatical equivalents refer to removal of a nucleic acid from its natural environment. However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and still be isolated for practical purposes.

[0115] As used herein, the term "purified" and its grammatical equivalents refer to molecules or compositions of increased purity, whether taken from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, wherein "purity" is a relative term, not "absolute purity". For example, when used for introduction into cells, nucleic acids are usually mixed with an acceptable carrier or diluent. As used herein, the term "substantially purified" and its grammatical equivalents refer to nucleic acid sequences, polypeptides, proteins or other compounds, which are substantially free, that is, more than about 50% free, more than about 70% free, more than about 90% free of polynucleotides, proteins, polypeptides and other molecules naturally associated with nucleic acids, polypeptides, proteins or other compounds.

[0116] As used herein, "T cell" or "T lymphocyte" is a lymphocyte that plays a central role in cell-mediated immunity. T cells or T lymphocytes can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of T cell receptors (TCRs) on the cell surface.

[0117] "Transposon", "transposable element" or "TE" refers to a DNA sequence that can change its position in the genome, sometimes generating or reversing mutations and changing the genome size of the cell. Transposition usually results in the replication of the transposon. Class I transposons replicate in two stages: first, these class I transposons are transcribed from DNA into RNA, and the resulting RNA is reverse transcribed into DNA. The replicated DNA is then inserted into the genome at the new location. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the transposon itself. The properties of retrotransposons are similar to those of retroviruses (such as HIV). The cut-and-paste transposition mechanism of class II transposons does not involve an RNA intermediate. Transposition is catalyzed by several transposases. Some transposases bind non-specifically to any target site in the DNA, while other transposases bind to specific DNA sequence targets. The transposases perform staggered cuts at the target site, producing single-stranded 5' or 3' DNA overhangs (sticky ends). This step cuts off the DNA transposon, which is then connected to the new target site; this process involves the activity of the DNA polymerase that fills the gap and the DNA ligase that closes the sugar-phosphate backbone. This will result in the duplication of the target site. The insertion site of the DNA transposon can be identified by the following repeat sequence: a short direct repeat sequence produced by the staggered cutting carried out in the target DNA and the filling carried out by the DNA polymerase, followed by a series of inverted repeat sequences that are important for the transposon excision of the transposase. If the transposition of the cut-and-paste transposon occurs during the S phase of the cell cycle in which the donor site has been replicated but the target site has not been replicated, then these transposons can be replicated. In both class I transposons and class II transposons, transposition can be classified as "autonomous" or "non-autonomous". Autonomous transposons can move on their own, while non-autonomous transposons require the presence of another transposon to move. This is usually because non-autonomous transposons lack transposases (for class II) or reverse transcriptases (for class I).

[0118] "Transposase" refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut-and-paste mechanism or a replicative transposition mechanism. In some embodiments, the catalytic activity of a transposase can be used to move a gene from a vector to a genome.

[0119] "Expression vector" or "vector" is any genetic element, e.g., a plasmid, a minicircle, a nanoplasmid, a chromosome, a virus, a transposon, which behaves as an autonomous unit of intracellular polynucleotide replication. (i.e., capable of replication under its own control) or is endowed with replication capability by insertion into a host cell chromosome to which another polynucleotide segment is attached, thereby causing replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. The vector may contain polynucleotide sequences that are necessary to achieve vector connection or insertion into a desired host cell and to achieve expression of the attached segment. Such sequences vary from host organism to host organism; these sequences include promoter sequences that achieve transcription, enhancer sequences that increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, an expression vector is capable of directly expressing the nucleic acid sequence product encoded therein without connecting or integrating the vector into a host cell DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome" that is capable of replicating in a host cell and persisting as an extrachromosomal segment of DNA in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids utilizing Epstein Barr Nuclear Antigen 1 (EBNA1) and Epstein Barr Virus (EBV) replication origin (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and the vector pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of episomal vectors that use T antigen and SV40 replication origins instead of EBNA1 and oriP. The vector may also include a selectable marker gene. In certain embodiments utilizing nanoplasmids, strains utilizing antisense RNA selection markers (eg, sucrose tolerance), such as R6K, may be used.

[0120] The term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or excluded in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described in, for example, International Patent Application Publications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981); Mulligan and Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981); Sa Interre et al., Gene, 30:147 (1984); Kent et al., Science, 237:901-903 (1987); Wigler et al., Cell, 11:223 (1977); Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962); Lowy et al., Cell, 22:817 (1980); and in U.S. Pat. Nos. 5,122,464 and 5,770,359.

[0121] The term "coding sequence" refers to a segment of a polynucleotide that encodes a protein or polypeptide. This region or sequence is bounded by a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame.

[0122] The term "operably connected" refers to the physical and / or functional connection of a DNA segment to another DNA segment, and the mode of its connection allows these segments to play a role in its expected manner. When the DNA sequence encoding a gene product is connected to a regulatory sequence (e.g., a promoter, an enhancer and / or a silencer) in a manner that allows the transcription of the DNA sequence to be directly or indirectly regulated, the DNA sequence is operably connected to the regulatory sequence. For example, when the DNA sequence is connected to the promoter downstream of the transcription initiation site of the promoter and in the correct reading frame relative to the transcription initiation site, the sequence is operably connected to the promoter, thereby allowing transcription extension to be carried out by the DNA sequence. When an enhancer or silencer is connected to a DNA sequence in a manner that increases or decreases the transcription of a DNA sequence, the enhancer or silencer is operably connected to the DNA sequence encoding the gene product. Enhancers and silencers can be located upstream or downstream of the coding region of a DNA sequence or embedded in the coding region. If the signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA for the signal sequence is operably connected to the DNA encoding the polypeptide. Linking of the DNA sequence to the regulatory sequences is generally accomplished by ligation at appropriate restriction sites or by inserting adapters or linkers into the sequences using restriction endonucleases known to those skilled in the art.

[0123] As used herein, the terms "induce," "induction," and their grammatical equivalents refer to an increase in transcription, promoter activity, and / or expression of a nucleic acid sequence caused by a transcriptional regulator relative to some basal transcription level.

[0124] The term "transcriptional regulator" refers to a biochemical element that acts to prevent or inhibit transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or a biochemical element that allows or stimulates transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or enhancer).

[0125] As used herein, the term "enhancer" refers to a DNA sequence that increases, for example, the transcription of a nucleic acid sequence to which it is operably connected. The enhancer can be positioned many kilobases away from the nucleic acid sequence coding region, and can mediate the binding of regulatory factors, changes in DNA methylation patterns or DNA structure. A large number of enhancers from a variety of different sources are well known in the art and can be used as cloned polynucleotides or obtained in cloned polynucleotides (from, for example, depository institutions, such as ATCC and other commercial or individual sources). Many polynucleotides comprising promoters (such as the commonly used CMV promoter) also include enhancer sequences. The enhancer can be located upstream or downstream of the coding sequence, or in the coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (μ) 5' enhancer, the light chain (κ) 5' enhancer, the κ intronic enhancer, and the μ intronic enhancer, as well as the 3' enhancer (see generally Paul WE (ed.), Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Pat. No. 5,885,827).

[0126] The term "promoter" refers to a polynucleotide region that initiates transcription of a coding sequence. The promoter is located near the transcription start site of a gene, on the same chain, and upstream of the DNA (towards the 5' region of the sense strand). Some promoters are constitutive because they are active in all cases in the cell, while other promoters (e.g., inducible promoters) are regulated and become active in response to a specific stimulus. As used herein, the term "promoter activity" and its grammatical equivalents refer to the expression level of the nucleotide sequence of the promoter that is operably connected to the activity being measured. Promoter activity can be measured directly by determining the amount of the RNA transcript produced (e.g., by Northern blot analysis), or indirectly by determining the amount of the product encoded by the connected nucleic acid sequence (such as a reporter nucleic acid sequence connected to the promoter).

[0127] "Inducible promoter" refers to a promoter that is induced to be active in the presence or absence of a transcriptional regulator (e.g., a biological or abiotic factor). Inducible promoters are useful because the expression of genes operably linked to them can be turned on or off at certain stages of organism development or in specific tissues. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. Inducible promoters can be part of a gene switch or a genetic switch.

[0128] As used herein, "T cell" or "T lymphocyte" is a lymphocyte that plays a central role in cell-mediated immunity. T cells or T lymphocytes can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of T cell receptors (TCRs) on the cell surface.

[0129] As used herein, the phrase "functional fragment" when used to refer to a polypeptide refers to a fragment of such a polypeptide that has the primary function of the referenced polypeptide. For example, a functional fragment of a polypeptide that acts as a transmembrane domain is a fragment of this polypeptide that also acts as a transmembrane domain. In certain embodiments, the functional fragment of a polypeptide is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N-terminus and / or C-terminus than the referenced polypeptide. When used to refer to a nucleic acid, the phrase "functional fragment" refers to a fragment of the referenced nucleic acid that encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid.

[0130] As used herein, the phrase "functional variant" when used to refer to a polypeptide refers to a polypeptide that is different from the referenced polypeptide but has the main function of the referenced polypeptide. For example, a functional variant of a polypeptide that acts as a transmembrane domain is a fragment of this polypeptide that also acts as a transmembrane domain. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the referenced amino acid sequence and / or is a conservative substitution variant of the referenced sequence. When used to refer to a nucleic acid, the phrase "functional variant" refers to a nucleic acid that is different from the referenced nucleic acid but encodes a polypeptide that has the same main function as the polypeptide encoded by the referenced nucleic acid. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the referenced nucleic acid sequence, hybridizes with the complementary sequence of the referenced nucleic acid sequence under stringent hybridization conditions, or is a codon degenerate variant of the nucleic acid sequence.

[0131] The term "antibody", also known as immunoglobulin (Ig), as used herein, may refer to a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and bound to different epitopes of the same antigen. Antibodies can be from any animal source. The antibody can be IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. In some embodiments, the antibody can be a single-chain whole antibody. Antibodies are generally composed of four polypeptides: two identical copies of heavy (H) chain polypeptides and two identical copies of light (L) chain polypeptides. Each heavy chain in the heavy chain contains an N-terminal variable (V H ) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains an N-terminal variable (V L ) region and a C-terminal constant (C L The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. H Area and V L The regions have a similar overall structure, each of which contains four framework regions whose sequences are relatively conservative. The framework regions are connected by three complementary determining regions (CDRs). These three CDRs, referred to as CDR1, CDR2 and CDR3, form the "hypervariable region" of the antibody responsible for antigen binding. These specific regions have been described by the following documents: Kabat et al., J.Biol.Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J.Mol.Biol.196:901-917 (1987); and MacCallum et al., J.Mol.Biol.262:732-745 (1996), wherein the definition includes overlaps or subsets of amino acid residues when compared to each other. Preferably, the term "CDR" is a CDR defined by Kabat based on sequence comparison. CDRH1, CDRH2 and CDRH3 represent heavy chain CDRs, while CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

[0132] The terms "antibody fragment", "antibody fragment", "antibody fragment", "antigen binding portion" and their grammatical equivalents are used interchangeably herein to refer to one or more antibody fragments or portions that retain the ability to specifically bind to an antigen (see generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Antibody fragments desirably contain, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Non-limiting examples of antibody fragments include (1) Fab fragments, which are monovalent fragments consisting of V and B regions. L Domain, V H Domain, C L (2) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the stem region; (3) Fv fragment, which consists of the V domain of a single arm of the antibody L Domain and V H (4) single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., V L and V H ) that enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)); and (5) diabodies, which are dimers of polypeptide chains, each of which comprises a V domain connected to a V domain by a peptide linker. L V H , the peptide linker is too short to allow V on the same polypeptide chain H and V L Pairing between them to drive different V H -V L Pairing between complementary domains on the polypeptide chains produces a dimeric molecule with two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, for example, US Patent No. 8,603,950.

[0133] The terms "antigen recognition portion", "antigen recognition domain", "antigen binding domain" and "antigen binding region" refer to a molecule or a portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition portion is an antibody, antibody-like molecule or a fragment thereof.

[0134] The term "proliferative disease" refers to the unifying concept that excessive proliferation of cells and / or turnover of cell matrix contribute significantly to the pathogenesis of diseases, including cancer. In some embodiments, the proliferative disease is cancer.

[0135] A "patient" or "subject" refers to a mammalian subject diagnosed with or suspected of having or suffering from a proliferative disorder, such as cancer. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of suffering from a proliferative disorder, such as cancer. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. A "patient in need thereof" or "subject in need thereof" refers to a patient diagnosed with or suspected of having a disease or disorder, such as, but not limited to, cancer.

[0136] "Administering" herein refers to providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration (e.g., injection) of the composition can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be performed, for example, by bolus injection or gradual infusion over time. Alternatively or concurrently, administration can be performed by an oral route. Additionally, administration can also be performed by surgical deposition of cell clusters or pellets, or placement of a medical device.

[0137] As used herein, the terms "treatment," "treating," and their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms attributable to the disease. In some embodiments, the term "treating" may include "preventing" a disease or condition.

[0138] As used herein, a "treatment interval" refers to a treatment cycle, eg, a course of administration of a therapeutic agent that can be repeated, eg, on a fixed schedule. In some embodiments, during a treatment interval, a dosage regimen can have one or more periods during which the therapeutic agent is not administered.

[0139] As used herein, "co-administration", "co-administration", "co-administering" and "co-providing" mean delivering two (or more) different treatments to a subject during the course of the subject's suffering from a disease, for example, delivering the two or more treatments after the subject has been diagnosed with the disease and before the disease has been cured or eliminated or treatment has been discontinued for other reasons. In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, so that there is an overlap in administration. This is sometimes referred to as "simultaneous" or "concurrent delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective, for example, an equivalent effect is seen with less of the second treatment, or the second treatment alleviates the symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen in the case of the first treatment. In some embodiments, delivery results in a reduction in symptoms or a reduction in other parameters associated with the disease that is greater than the reduction or reduction that would be observed in a treatment delivered in the absence of another treatment. The effects of the two treatments may be partially cumulative, fully cumulative, or greater than cumulative. Delivery may be such that the effects of the first treatment delivered are still detectable when the second treatment is delivered.

[0140] In some embodiments, the first treatment and the second treatment can be administered simultaneously (simultaneously) (e.g., at the same time), with the same or different compositions, or sequentially. Sequential administration refers to administering a treatment before administering another treatment, for example, a secondary treatment (e.g., immediately before, less than 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes; 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours or more hours; 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or more days; 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more weeks). The order of administration of the first treatment and the secondary treatment can also be reversed.

[0141] The terms "therapeutically effective amount", "therapeutic amount", "immunologically effective amount", "anti-tumor effective amount", "tumor suppressive effective amount" and their grammatical equivalents refer to an effective amount to achieve the desired therapeutic result at the necessary dosage and time period. The therapeutically effective amount can vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the compositions described herein to elicit the desired response in one or more subjects. The exact amount of the composition of the present disclosure to be administered can be determined by the physician taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.

[0142] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject can be "prophylactic", i.e., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" refers to an effective amount at the dosage and time period necessary to achieve the desired prophylactic effect (e.g., preventing the onset of a disease).

[0143] As used herein, the term "immune checkpoint protein" refers to a molecule that transmits an inhibitory signal or has an immunosuppressive function. Examples of such immune checkpoint proteins include, but are not limited to, CTLA-4, PD-1, PD-L1 (programmed cell death-ligand 1), PD-L2 (programmed cell death-ligand 2), LAG-3 (lymphocyte activation gene 3), TIM3 (T cell immunoglobulin mucin-3), BTLA (B and T lymphocyte attenuator), B7H3, B7H4, CD160, CD39, CD70, CD73, A2aR (adenosine A2a receptor), KIR (killer inhibitory receptor), VISTA (V domain Ig-containing inhibitor of T cell activation), IDO1 (indoleamine 2,3-dioxygenase), arginase I, TIGIT (T cell immunoglobulin and ITIM domain) CD70, CD115, etc. (see, Nature Reviews Cancer, 12, pp. 252-264, 2012 and Cancer Cell, 27, pp. 450-461, 2015).

[0144] As used herein, terms used to identify biological parts may or may not include a dash "—" in the term. The presence or absence of a dash does not change the intended meaning or identification of the biological part. By way of illustration only, and not limited to these biological parts, each of the following pairs of terms (shown with / without dashes) indicate and identify the same biological entity: CCR-4 / CCR4, CD-3 / CD3, CD-4 / CD4, CD-33 / CD33, EGFR-2 / EGFR2, FLT-1 / FLT1, HER-1 / HER1, HER-1t / HER1t, IL-12 / IL12, IL-15 / IL15, IL-15Rα / IL15Rα, MUC-1 / MUC1, MUC-16 / MUC16, ROR-1 / ROR1, ROR-1R / ROR1R, TGF-β / TGFβ, VEGF-1 / VEGF1, VEGF-R2 / VEGFR2. "

[0145] II. miRNA

[0146] As used herein, the terms "miR," "mir," and "miRNA" are used to refer to microRNA, a class of small non-coding RNA molecules that are capable of affecting the expression of genes ("target genes") by modulating the translation of the messenger RNA transcribed therefrom (increasing or decreasing the expression of the gene) and / or destabilizing such messenger RNA.

[0147] The term "primary miRNA", abbreviated as "pri-miRNA", refers to a miRNA containing at least one RNA hairpin. The RNA hairpin is cut from the pri-miRNA in the nucleus to form one or more precursor miRNAs ("pre-miRNA"). The pre-miRNA is exported to the cytoplasm, where the stem-loop structure is cut to produce a double-stranded miRNA containing a miRNA-5p strand from the front 5' arm of the hairpin loop and a miRNA-3p strand from the front 3' arm of the hairpin loop. Arguin then binds to the double-stranded miRNA and releases one of the strands (miRNA-5p sequence or miRNA-3p sequence). The remaining bound strand becomes the "guide strand", and the released strand is called the "passenger strand" and is preferably degraded. The guide strand then continues to interact with the messenger RNA derived from the target gene, thereby affecting the translation of the messenger RNA.

[0148] The miRNA-5p chain sequence and the miRNA-3p chain sequence will be referred to as "mature miRNA" sequence in this article. The remainder of pri-miRNA or pre-miRNA (part 5' of its miRNA-5p sequence, part 3' of its miRNA-3p sequence, and the stem-loop sequence between the miRNA-5p sequence and the miRNA-3p sequence) will be collectively referred to as the miRNA backbone sequence. The term "5' backbone sequence" will be used herein to refer to the backbone sequence of the 5' of the miRNA-5p sequence in pri-miRNA or pre-miRNA. The term "3' backbone sequence" will be used herein to refer to the backbone sequence of the 3' of the miRNA-3p sequence in pri-miRNA or pre-miRNA. The term "loop sequence" refers to the backbone sequence between the miRNA-5p sequence and the miRNA-3p sequence in pri-miRNA or pre-miRNA.

[0149] Unless otherwise indicated, the term "miRNA" generally refers to mature, primary and precursor forms of a specific microRNA and its functional fragments and variants.

[0150] The miRNA may be non-naturally occurring. The terms "non-naturally occurring," "non-natural," "synthetic," and "artificial" as used herein to describe miRNA are used interchangeably and refer to a miRNA having a sequence that does not occur in nature.

[0151] The present invention relates in part to a ribonucleic acid comprising two non-natural pre-miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein. In certain embodiments, the RNA comprises more than two such non-natural pre-miRNA sequences, for example, three, four, five, six, seven, eight, nine, ten or more such sequences. It should be understood that each guide miRNA can target the same gene or different genes. In embodiments where two or more guide miRNAs target the same gene, such guide miRNAs can target the same region or different regions of such a gene.

[0152] In certain embodiments, each non-natural pre-miRNA sequence in the ribonucleic acid forms a stem-loop secondary structure that is different and non-complementary to the stem-loop secondary structure formed by a different non-natural pre-miRNA sequence in the ribonucleic acid. In certain embodiments, the non-natural pre-miRNA sequences have less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 55%, or less than about 50% sequence identity to each other.

[0153] In certain embodiments, the secondary structure of each non-natural pre-miRNA is sufficiently similar to the secondary structure of a naturally occurring pre-miRNA sequence to reduce or prevent RNAi-based cellular anti-pathogen toxicity. In certain such embodiments, the nucleic acid sequence of the non-natural pre-miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence of the naturally occurring pre-miRNA and / or can hybridize to the naturally occurring pre-miRNA under stringent hybridization conditions.

[0154] In certain embodiments, the secondary structure of each pri-miRNA containing a non-natural pre-miRNA (hereinafter "non-natural pri-miRNA") is sufficiently similar to the secondary structure of a naturally occurring pri-miRNA sequence to reduce or prevent RNAi-based cellular anti-pathogen toxicity. In certain such embodiments, the nucleic acid sequence of the non-natural pri-miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence of the naturally occurring pri-miRNA and / or can hybridize with the naturally occurring pri-miRNA under stringent hybridization conditions.

[0155] The non-natural pre-miRNAs of the present invention can be generated from naturally occurring pre-miRNAs by removing native mature miRNA sequences and replacing these sequences with non-native mature miRNA sequences, one of which is capable of acting as a guide miRNA targeting a gene of interest.

[0156] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence derived from a naturally occurring pre-miRNA, such as a pre-miRNA present in a mouse, rat, or human. In certain embodiments, the backbone sequence (3' backbone sequence, 5' backbone sequence, and loop sequence) of the non-natural pre-miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the corresponding backbone sequence of the naturally occurring pre-miRNA and / or can hybridize with such corresponding backbone segments under stringent hybridization conditions. In certain embodiments, the backbone segments of the non-natural pre-miRNA sequences are identical to the corresponding backbone segments of the naturally occurring pre-miRNA. In certain embodiments, the naturally occurring pre-miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In certain embodiments, the naturally occurring pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNA is miR204 or miR206.

[0157] In certain embodiments, each non-natural pri-miRNA comprises a backbone sequence derived from a naturally occurring pri-miRNA, such as a pri-miRNA present in a mouse, a rat or a human. In certain embodiments, the backbone sequence (3' backbone sequence, 5' backbone sequence and loop sequence) of the non-natural pri-miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the corresponding backbone sequence of the naturally occurring pri-miRNA and / or can hybridize with such corresponding backbone segments under stringent hybridization conditions. In certain embodiments, the backbone segment of the non-natural pri-miRNA sequence is identical to the corresponding backbone segment of the naturally occurring pri-miRNA. In certain embodiments, the naturally occurring pri-miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In certain embodiments, the naturally occurring pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNA is miR204 or miR206.

[0158] Although miRNA-5p and miRNA-3p sequences hybridize to each other, they are not necessarily completely complementary. When designing non-naturally occurring miRNAs, compensatory mutations may be made in the miRNA-5p sequence and / or the miRNA-3p sequence to maintain the RNA folding and free energy of the native miRNA. In certain embodiments, the sequence encoding the miRNA-3p sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the complementary sequence of the sequence encoding the miRNA-5p sequence or is capable of hybridizing with the sequence encoding the miRNA-5p sequence under stringent hybridization conditions.

[0159] In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides. nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, or at least about 250 nucleotides. In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by about 5 to 250 nucleotides, about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 10 to 40 nucleotides, about 10 to 30 nucleotides, about 10 to 20 nucleotides, about 16 to 250 nucleotides, about 16 to 200 nucleotides, about 16 to 150 nucleotides, about 16 to 100 nucleotides, about 16 to 50 nucleotides, about 16 to 40 nucleotides, about 16 to 30 nucleotides, about 16 to 20 nucleotides, about 20 to 200 nucleotides, about 20 to 150 nucleotides, about 20 to 100 nucleotides, about 20 to 50 nucleotides, about 20 to 45 nucleotides, about 20 to 40 nucleotides, about 20 to 35 nucleotides, about 20 to 30 nucleotides, about 20 to 25 nucleotides, about 30 to 200 nucleotides, about 30 to 150 nucleotides, about 30 to 100 nucleotides, about 30 to 50 nucleotides or about 30 to 40 nucleotides.In certain embodiments, two non-natural pre-miRNA sequences are separated from each other by at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 1 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides.

[0160] In certain embodiments, two non-natural pri-miRNA sequences are adjacent to each other, wherein the 3' nucleotide of one pri-miRNA is directly bonded to the 5' nucleotide of the other pri-miRNA. In such embodiments, the nucleotides separating the respective non-natural pre-miRNAs contained in each pri-miRNA form part of the pri-miRNA sequence.

[0161] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to mRNA and thereby interfering with its translation and / or promoting its degradation. The mRNA may be produced by expression of a target gene.

[0162] In certain embodiments, the target gene encodes an immune checkpoint protein. Therefore, the pre-miRNA sequence inhibits the expression of the immune checkpoint protein by targeting the gene expressing the immune checkpoint protein. In certain such embodiments, the immune checkpoint protein is PD-1, PD-L1, CTLA4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM3 or VISTA. In certain such embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR or PIK3IP1. In certain embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1. In certain embodiments, the immune checkpoint protein is CD70, PD-1, or TIGIT. In certain embodiments, the immune checkpoint protein is PD-1.

[0163] In certain embodiments, each non-natural pre-miRNA targets a different gene or a different region of the same gene.

[0164] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD-1; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD-1.

[0165] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD-1; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD-1.

[0166] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting PD-1; and (b) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting PD-1.

[0167] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting PD-1; and (b) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting PD-1.

[0168] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0169] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0170] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0171] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0172] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT.

[0173] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT.

[0174] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0175] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0176] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT.

[0177] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT.

[0178] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0179] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0180] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0181] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0182] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0183] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0184] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0185] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0186] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0187] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0188] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0189] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting TIGIT.

[0190] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0191] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting TIGIT.

[0192] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0193] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting TIGIT.

[0194] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting CD70.

[0195] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting CD70.

[0196] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0197] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0198] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting CD70.

[0199] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting CD70.

[0200] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0201] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0202] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70.

[0203] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting CD70.

[0204] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0205] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0206] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1.

[0207] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting TIGIT; and (b) a pie-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1.

[0208] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pre-miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting TIGIT.

[0209] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pri-miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting TIGIT.

[0210] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1.

[0211] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting TIGIT; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1.

[0212] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pre-miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting TIGIT.

[0213] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pri-miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting TIGIT.

[0214] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pre-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0215] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pri-miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting CD70.

[0216] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0217] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0218] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pre-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pre-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0219] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD1; (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD1; and (c) a pri-miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting CD70.

[0220] The present invention also relates in part to a deoxyribonucleic acid encoding any one of the aforementioned ribonucleic acids.

[0221] Examples of deoxyribonucleic acid sequences encoding backbone sequences that can be used in the practice of the present invention include, but are not limited to, the deoxyribonucleic acid sequences listed in Table 1 below. The symbols "X" and "Y" in Table 1 indicate nucleic acid sequences encoding guide miRNA (which may be miRNA-5p or miRNA-3p) and passenger miRNA (which may be miRNA-5p or miRNA-3p), respectively, and the symbol "n" indicates the number of nucleotides in such sequences, for example, 16 to 30, preferably 18 to 25. In some embodiments, n may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. In certain embodiments, the deoxyribonucleic acid encoding the backbone sequence is a sequence that hybridizes to the complementary sequence of any one of the sequences listed in Table 1 under stringent hybridization conditions.

[0222] Table 1: DNA sequences encoding miRNA backbone sequences

[0223]

[0224] In any of the foregoing embodiments, the sequence encoding the pre-miRNA comprises:

[0225] SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively;

[0226] SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively;

[0227] SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively;

[0228] SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0229] SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0230] SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively;

[0231] SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;

[0232] SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 respectively;

[0233] SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively;

[0234] SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 respectively;

[0235] SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33 respectively;

[0236] SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36 respectively;

[0237] SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, respectively;

[0238] SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively;

[0239] SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively;

[0240] SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively;

[0241] SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively;

[0242] SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, respectively;

[0243] SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, respectively;

[0244] SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, respectively;

[0245] SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63 respectively;

[0246] SEQ ID NO:338, SEQ ID NO:339 and SEQ ID NO:340, respectively;

[0247] SEQ ID NO:341, SEQ ID NO:342 and SEQ ID NO:343, respectively; or

[0248] SEQ ID NO:344, SEQ ID NO:345 and SEQ ID NO:346, respectively;

[0249] Or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the aforementioned sequences or capable of hybridizing to the complement of such sequences under stringent hybridization conditions.

[0250] Table 2 lists non-limiting examples of nucleic acid sequences encoding guide miRNAs for targeted genes, which encode such checkpoint inhibitors. Table 2 also lists sequences encoding passenger chains. As previously mentioned, guide chains and passenger chains are not necessarily complementary. It is expected that passenger chains can also be used to target messenger RNAs associated with target genes. It is also expected that sequences that hybridize to the complementary sequences of the sequences listed in Table 2 under stringent hybridization conditions can also be used. The mature miRNA sequence used can be combined with a specific pri-miRNA backbone. Table 2 also lists the backbones that can be combined with the mature guide miRNAs and passenger miRNAs listed therein.

[0251] Table 2: DNA sequences encoding mature miRNA sequences

[0252]

[0253]

[0254]

[0255] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 64-83, 85, 87-171, 293-322, and 704-713 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 64-83, 85, 87-171, 293-322, and 704-713 under stringent hybridization conditions. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 64-83, 85, 87-171, 293-322, and 704-713. NO:64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156 , 158, 160, 162, 164, 166, 168, 170, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709 and 710 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity or can be hybridized under stringent conditions with SEQ ID NO:64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 1 10, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 1 A nucleic acid sequence that hybridizes to the complement of any one of 46, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709 and 710.

[0256] In certain embodiments, the sequence encoding the guide miRNA sequence is the same as SEQ ID NO: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160 , 162, 164, 166, 168, 170, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709 and 710 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity; or can hybridize under stringent hybridization conditions to the complement of any of such sequences.

[0257] In certain embodiments, the sequence encoding the passenger miRNA sequence is the same as SEQ ID NO: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 706-708 and 711-713 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity; or can hybridize under stringent hybridization conditions to the complement of any of such sequences.

[0258] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting CTLA. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 65-71 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 65-71 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 64, 66, 68 and 70 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 64, 66, 68 and 70 under stringent hybridization conditions.

[0259] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 72-83, 85, 87 and 704-713 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 72-83, 85, 87 and 704-713 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:72, 74, 76, 78, 80, 82, 704, 705, 709 and 710 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs:72, 74, 76, 78, 80, 82, 704, 705, 709 and 710 under stringent hybridization conditions.

[0260] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting TIGIT. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 88-145 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 88-145 under stringent hybridization conditions. In certain embodiments, the invention relates to a deoxyribonucleic acid comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 and 138 or capable of hybridizing under stringent conditions with SEQ ID NO: 64, 66, 68 and 70 and SEQ ID NO: 73. A nucleic acid sequence that hybridizes to the complementary sequence of any one of NO:88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 and 138.

[0261] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting TIM3. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 146-157 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 146-157 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 146, 148, 150, 152, 154 and 156 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 146, 148, 150, 152, 154 and 156 under stringent hybridization conditions.

[0262] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting LAG3. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 158-161 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 158-161 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 158 and 160 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 158 and 160 under stringent hybridization conditions.

[0263] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting GITR. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 162-165, or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 162-165 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 162 and 164, or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 162 and 164 under stringent hybridization conditions.

[0264] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PIK3IP1. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 166-171 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 166-171 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 166, 168 and 170 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 166, 168 and 170 under stringent hybridization conditions.

[0265] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting CD70. In certain such embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 293-322 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 293-322 under stringent hybridization conditions. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319 and 321 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319 and 321 under stringent hybridization conditions.

[0266] In certain embodiments, the present invention relates to a deoxyribonucleic acid, wherein each sequence encoding a pre-miRNA comprises:

[0267] a) a sequence encoding the 5' miRNA backbone sequence;

[0268] b) a sequence encoding a guide miRNA sequence;

[0269] c) a sequence encoding a stem-loop sequence;

[0270] d) a sequence encoding a passenger miRNA sequence; and

[0271] e) Sequence encoding the 3' backbone sequence.

[0272] In certain embodiments, the sequence encoding the pre-miRNA comprises:

[0273] a) a guide miRNA sequence that is identical to SEQ ID NO: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 704,

[0277] Any of 705, 709 and 710 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%

[0278] or 99% sequence identity or capable of hybridizing under stringent hybridization conditions to the complement of any of such sequences; and

[0279] b) a passenger sequence that is identical to SEQ ID NO: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,

[0284] Any of 706-708 and 711-713 has at least 80%, 85%, 90%, 95%, 96%, 97%,

[0285] 98% or 99% sequence identity or the ability to hybridize under stringent hybridization conditions to the complement of any of such sequences.

[0286] Table 3 describes nucleic acids encoding exemplary non-natural pre-miRNA sequences that target specific checkpoint inhibitors. In certain embodiments, the deoxyribonucleic acid may comprise a sequence that is capable of hybridizing to the complement of any of the sequences listed in Table 3 under stringent hybridization conditions.

[0287] Table 3: DNA sequences encoding non-natural miRNA sequences

[0288]

[0289]

[0290]

[0291]

[0292] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 347-447 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 347-447 under stringent hybridization conditions.

[0293] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 178-263 and 323-337 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 178-263 and 323-337 under stringent hybridization conditions.

[0294] In certain embodiments, the miRNA targets CTLA4. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 347, 419, 420 and 421, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 347, 419, 420 and 421. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 178 and 250-252, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 178 and 250-252.

[0295] In certain embodiments, the pre-miRNA targets PD-1. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 348, 349 and 410-418, or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 348, 349 and 410-418. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 179, 180 and 241-249, or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 179, 180 and 241-249. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 348 or 349, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NO: 348 or 349. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 179 or 180, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NO: 179 or 180.

[0296] In certain embodiments, the pre-miRNA targets TIGIT. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 350-377 and 404-409 or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 350-377 and 404-409. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 181-208 and 235-240 or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 181-208 and 235-240.

[0297] In certain embodiments, the pre-miRNA targets TIM3. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 378-389 or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 378-389. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 209-220 or capable of hybridizing under stringent hybridization conditions with the complementary sequence of any one of SEQ ID NOs: 209-220.

[0298] In certain embodiments, the pre-miRNA targets LAG3. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 390-396, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 390-396. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 221-227, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 221-227.

[0299] In certain embodiments, the pre-miRNA targets GITR. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 397-403, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 397-403. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 228-234, or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 228-234.

[0300] In certain embodiments, the pre-miRNA targets PIK3IP1. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 422-424 or capable of hybridizing under stringent hybridization conditions with a complementary sequence of any one of SEQ ID NOs: 422-424. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 253-255 or capable of hybridizing under stringent hybridization conditions with a complementary sequence of any one of SEQ ID NOs: 253-255.

[0301] In certain embodiments, the pre-miRNA targets CD70. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 433-447 or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 433-447. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 323-337 or capable of hybridizing under stringent hybridization conditions to the complementary sequence of any one of SEQ ID NOs: 323-337.

[0302] In an embodiment of the present invention, two or more pre-miRNAs encoded by deoxyribonucleic acid may each contain a guide miRNA sequence targeting the same target gene, or each guide miRNA may target different genes. In addition, each pre-miRNA design of the pri-miRNA containing these pre-miRNAs may be based on a different native miRNA backbone to reduce the possibility of misfolding of one miRNA with another miRNA. Table 4 provides examples of deoxyribonucleic acid sequences encoding two or more pri-miRNAs.

[0303] Table 4: DNA sequences containing two or more pri-miRNAs

[0304]

[0305]

[0306]

[0307] In certain such embodiments, the invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 267-290 and 448-460 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 267-290 and 448-460 under stringent hybridization conditions.

[0308] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 267 and 282 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 267 and 282 under stringent hybridization conditions.

[0309] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting TIGIT. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 269-274, 287, 288 and 290 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 269-274, 287, 288 and 290 under stringent hybridization conditions.

[0310] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1 and a pre-miRNA targeting TIGIT. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 275-280 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 275-280 under stringent hybridization conditions.

[0311] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting CTLA4. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 281, 283 and 284 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 281, 283 and 284 under stringent hybridization conditions.

[0312] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting TIGIT and a pre-miRNA targeting CTLA4. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 285, 286 and 289 or capable of hybridizing to the complementary sequence of any one of SEQ ID NOs: 285, 286 and 289 under stringent hybridization conditions.

[0313] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD1 and a pre-miRNA targeting CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 448 or 451 or capable of hybridizing to the complementary sequence of SEQ ID NO: 448 or 451 under stringent hybridization conditions.

[0314] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD1 and two pre-miRNAs targeting CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 449 or capable of hybridizing to the complementary sequence of SEQ ID NO: 449 under stringent hybridization conditions.

[0315] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD1 and a pre-miRNA targeting CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 450 or capable of hybridizing to the complementary sequence of SEQ ID NO: 450 under stringent hybridization conditions.

[0316] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs, each targeting CD70. In certain embodiments, the invention relates to a deoxyribonucleic acid comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 452-460 or capable of hybridizing to the complement of any one of SEQ ID NOs: 452-460 under stringent hybridization conditions.

[0317] III. Protein of Interest

[0318] In certain embodiments, the deoxyribonucleic acid encoding the pre-miRNA is contained in the same genetic construct as a genetic construct containing one or more genes encoding a protein of interest (e.g., a chimeric antigen receptor, cytokine, cellular tag, or checkpoint inhibitor).

[0319] A. Chimeric receptors

[0320] In any of the foregoing embodiments, the deoxyribonucleic acid of the present disclosure may further encode a chimeric receptor, such as a chimeric antigen receptor (CAR) or a chimeric T cell receptor (TCR). Therefore, the deoxyribonucleic acid of the present disclosure may encode miRNA and a chimeric receptor, such as a CAR or a TCR.

[0321] In certain embodiments, the deoxyribonucleic acid of the invention encodes CAR and is introduced into T cells to generate chimeric antigen receptor T cells (CAR-T cells).

[0322] CAR is an engineered receptor that is transplanted to an exogenous specificity on an immune effector cell. In some cases, CAR includes an extracellular domain (extracellular domain), which includes an antigen binding domain, a transmembrane domain, and an intracellular (intracellular domain) domain. The intracellular domain includes an intracellular signaling domain. In certain embodiments, the extracellular domain further includes an amino acid region (i.e., spacer) between the antigen binding domain and the transmembrane domain.

[0323] The antigen binding domain may comprise the complementary determining regions of a monoclonal antibody and / or its antigen binding fragment. Complementarity determining regions (CDRs) are short amino acid sequences present in the variable domains of antigen receptor (e.g., immunoglobulins and T cell receptor) proteins that bind to antigens and thus provide the receptor with its specificity for this particular antigen. Each polypeptide chain of an antigen receptor may contain three CDRs (CDR1, CDR2, and CDR3).

[0324] In certain embodiments, the antigen binding domain comprises an antibody or a functional fragment or variant thereof that binds to a target antigen. The functional fragment or variant may comprise a heavy chain (V H ) and / or the light chain (V L ) or a functional fragment or variant thereof. In certain embodiments, the antigen binding domain comprises an Fv fragment, a Fab fragment, a Fab 2 fragment, Fab' fragment, F(ab') 2 Fragment or F(ab') 3 In certain embodiments, the antigen binding domain comprises scFv, sc(Fv) 2 , dsFv, diabody, minibody, nanobody or their binding fragments. In certain embodiments, the antigen binding domain further comprises an Fc fragment of an antibody, for example, the antigen binding domain may comprise a scFv connected to an Fc fragment.

[0325] In some embodiments, CAR targets antigens overexpressed in cancer cells, in autoimmune cells, or in cells infected by viruses, bacteria, or parasites. Pathogens that can be targeted include, but are not limited to, Plasmodium, Trypanosoma, Aspergillus, Candida, Hepatitis A, Hepatitis B, Hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola virus (Ebola) pathogens. Autoimmune diseases may include graft-versus-host disease, rheumatoid arthritis, lupus, celiac disease, Crohn's disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, and autoimmune uveitis.

[0326] The pathogen identified by CAR can be basically any kind of pathogen, but in some embodiments, the pathogen is a fungus, a bacterium or a virus. Exemplary viral pathogens include adenoviridae, Epstein-Barr virus (Epstein-Barr virus, EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, HPV, HSV, HHV virus family, hepatitis virus family, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, Ebola virus and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes and Salmonella. In some embodiments, the pathogen receptor Dectin-1 can be used to generate CARs that recognize carbohydrate structures on the cell wall of fungi (such as Aspergillus). In another embodiment, CARs can be made based on antibodies that recognize viral determinants (e.g., glycoproteins from CMV and Ebola viruses) to interfere with viral infection and pathology.

[0327] In certain embodiments, the CAR comprises an antigen binding domain that binds an antigen that is overexpressed in the cancer.

[0328] In some embodiments, the CAR comprises an antigen binding domain that binds to an epitope on B7H4, BCMA, BTLA, CAIX, CA125, CCR4, CD3, CD4, CD5, CD7, CD16, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / v8, CD47, CD52, CD56, CD70, CD79b, CD80, CD81, CD86, CD123, CD133, CD137, CD138, CD151, CD17 1. CD174, CD276, CEA, CEACAM6, CLL-1, c-MET, CS1, CSPG4, CTLA-4, DLL3, EDB-F, EGFR, EGFR2, EGFRvIII, EGP-2, EGP-40, EphA2, FAP, FLT1, FLT4, folate binding protein, folate receptor, folate receptor α, α-folate receptor, frizzled protein, GD2, GD3, GHR, GHRHR, GITR, GPC3, Gp100, gp130, HBV antigen, HER1, HER2, HER3, HER4, HER1 / HER3, h5T4, HP V antigen, HVEM, IGF1R, Igκ, IL-1-RAP, IL-2R, IL6R, IL-11Rα, IL-13R-a2, KDR, KRASG12V, LewisA, LewisY, L1-CAM, LIFRP, LRP5, LTPR, MAGE-A, MAGE-A1, MAGE-A10, MAGE-A3, MAGEA3 / A6, MAGE-A4, MAGE-A6, MART-1, MCAM, mesothelin, PSCA, mucins such as MUC1, MUC-4 or MUC16, NGFR, NKG2D, Notch-1-4, N Y-ESO-1, O-acetyl GD2, O-acetyl GD3, OX40, P53, PD1, PDE10A, PD-L1, PD-L2, PMSA, PRAME, PSCA, PSMA, PTCH1, RANK, Robol, ROR1, ROR1R, ROR2, TACI, TAG-72, TCRa, TCRp, TGF, TGFβ, TGFβ-II, TGFBR1, TGFBR2, titin, TLR7, TLR9, TNFR1, TNFR2, TNFRSF4, TRBC1, TWEAK-R, VEGF, VEGF-R2, or WT-1.

[0329] In some embodiments, the CAR described herein comprises an antigen binding domain that binds to an epitope on CD19, CD33, MUC1, MUC16, ROR1, HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1 and / or type II collagen.

[0330] In some embodiments, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD19, CD33, MUC1, MUC16, and / or ROR1.

[0331] In some embodiments, CAR comprises an antigen binding domain that binds to an epitope on CD 19. Examples of CARs that bind to an epitope on CD 19 are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2016 / 033570; WO 2015 / 123642; and WO 2015 / 187528.

[0332] In some embodiments, the CAR comprises an antigen binding domain that binds to an epitope on CD33. Examples of CARs that bind to an epitope on CD33 are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2017 / 214333.

[0333] In some embodiments, the CAR comprises an antigen binding domain that binds to an epitope on MUCl.Examples of CARs that bind to an epitope on MUCl are known to those of skill in the art and have been described.

[0334] In some embodiments, the CAR comprises an antigen binding domain that binds to an epitope on MUC16. Examples of CARs that bind to an epitope on MUC16 are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2019 / 236577.

[0335] In some embodiments, the CAR comprises an antigen binding domain that binds to an epitope on ROR1. Examples of CARs that bind to an epitope on ROR1 are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2020 / 014366.

[0336] Antigen binding can be evaluated by flow cytometry or cell-based analysis or any other equivalent assay. Cell-based assays can utilize cell types that express the antigen of interest on the surface to evaluate antigen binding. Antigens or fragments thereof expressed as soluble proteins can be used to evaluate antigen binding using flow cytometry or similar assays. The improvement of antigen binding can be indirectly evaluated by functional measurements of antigen binding domains or chimeric receptors. For example, as described herein, the improvement of antigen binding of chimeric receptors or CARs can be measured by an increase in specific cytotoxicity against target cells expressing the antigen.

[0337] Can, for example, use the determination based on flow cytometry to assess the cell surface expression level of polypeptide of the present disclosure.The increase of expression of antigen-binding polypeptide can be measured in the form of the average density of the percentage of the analyzed cells expressing the antigen-binding polypeptide or alternatively the surface of the cell of the antigen-binding polypeptide.Other suitable methods that can be used to assess the cell surface expression of antigen-binding polypeptide described herein include Western blotting or any other equivalent assay method.

[0338] B. Cytokines

[0339] In any of the foregoing embodiments, the deoxyribonucleic acid of the present disclosure may further encode a cytokine. Thus, the deoxyribonucleic acid of the present disclosure may encode a miRNA and a cytokine.

[0340] In certain embodiments, the deoxyribonucleic acid also encodes CAR and introduces it into T cells, thereby producing CAR-T cells. In certain other embodiments, the deoxyribonucleic acid of the present invention encoding miRNA and cytokines is introduced into CAR-T cells.

[0341] In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor or a variant or combination thereof. In certain embodiments, the cytokine is a ligand of interferon, GM-CSF, G-CSF, M-CSF, LT-β, ​​TNF-α, growth factor, hGH and / or human Toll-like receptor (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IFN-α, IFN-β or IFN-γ).

[0342] In certain embodiments, the cytokine is an interleukin. In some cases, the interleukin is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 or a functional variant or fragment thereof.

[0343] In certain embodiments, the cytokine may be IL-12 or a functional fragment or variant thereof. In certain embodiments, IL-12 is single-chain IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, embedded IL-12. In some cases, IL-12 variants are described in International Application Publication Nos. WO2015 / 095249, WO2016 / 048903, and WO2017 / 062953.

[0344] In certain embodiments, the cytokine may be IL-15 or a functional fragment or variant thereof. In certain embodiments, IL-15 or a functional fragment or variant thereof is membrane-bound. This may occur when IL-15 or a functional fragment or variant thereof binds to a membrane-bound IL-15Rα or a functional fragment or variant thereof. Therefore, certain embodiments of the present invention may relate to a fusion protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof. In certain embodiments, IL-15 or a functional fragment or variant thereof is connected to IL-15Rα or a functional fragment thereof via a linker. Examples of fusion proteins comprising IL-15 or a functional fragment or variant thereof bound to a membrane-bound IL-15Rα or a functional fragment or variant thereof are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2014 / 186469.

[0345] In certain embodiments, cytokines are connected to signal peptides. Any signal for use in eukaryotic cells (including the signal used with CAR described above) can be connected to cytokines. In certain embodiments, cytokines are connected to IgE signal peptides.

[0346] C. Cell Labeling

[0347] In any of the foregoing embodiments, the DNA of the present disclosure may further encode a cell tag. Thus, the DNA of the present disclosure may encode a miRNA and a cell tag.

[0348] In certain embodiments, deoxyribonucleic acid also encodes a CAR and introduces it into T cells, thereby generating CAR-T cells. In certain other embodiments, the deoxyribonucleic acid of the present invention encoding miRNA and a cell tag is introduced into CAR-T cells. In certain such embodiments, the deoxyribonucleic acid also encodes a cytokine. In certain embodiments, the deoxyribonucleic acid of the present invention encodes: (a) a CAR; (b) a protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof; and (c) a cell tag.

[0349] In certain embodiments, the cell tag is used as a kill switch, a selection marker, a biomarker, or a combination thereof.

[0350] In certain embodiments, the cell tag is capable of being bound by a predetermined binding partner. In certain such embodiments where the deoxyribonucleic acid encoding the cell tag is introduced into a cell and the cell is introduced into a subject, administration of the predetermined binding partner to the subject permits depletion of the cells. For example, administration of cetuximab or any antibody that recognizes HER1 permits elimination of cells expressing a cell tag comprising a truncated non-immunogenic HER1.

[0351] In certain such embodiments, the cell tag is non-immunogenic. This can be achieved, for example, where the cell tag comprises a polypeptide that is truncated such that the cell tag is non-immunogenic. For example, truncation of the HER1 sequence eliminates the possibility of EGF ligand binding, homodimerization and heterodimerization of EGFR, and / or EGFR-mediated signal transduction, while leaving cetuximab binding ability intact (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Vol. 37, pp. 353-373).

[0352] In certain embodiments, the cell tag comprises at least one of a truncated non-immunogenic HER1 polypeptide, a truncated non-immunogenic LNGFR polypeptide, a truncated non-immunogenic CD20 polypeptide, or a truncated non-immunogenic CD52 polypeptide, or a functional fragment or variant thereof.

[0353] In certain embodiments, the cell tag comprises HER1 domain III or a functional fragment or variant thereof and a truncated HER1 domain IV or a functional fragment or variant thereof. Examples of such cell tags are known to those skilled in the art and are described in, for example, International Application Publication No. WO 2014 / 186469.

[0354] In certain embodiments, the cell tag comprises a truncated non-immunogenic CD20 or CD20t-1 or a functional fragment or variant thereof.

[0355] In certain embodiments, the cell tag further comprises a transmembrane domain. The transmembrane domain may be derived from a natural or synthetic source. In the case where the source is natural, the domain may, for example, be derived from any membrane-bound protein or transmembrane protein. Suitable transmembrane domains may include transmembrane domains of α, β or ζ chains of T cell receptors; or transmembrane domains from CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 or their functional fragments or variants. In certain embodiments, the cell tag further comprises a CD28 transmembrane domain or its functional fragment or variant. Alternatively, the transmembrane domain may be synthetic and may include hydrophobic residues, such as leucine and valine. In certain embodiments, a triplet of phenylalanine, tryptophan and valine is present at one or both ends of the synthetic transmembrane domain.

[0356] In certain embodiments, the cell tag comprises a truncated HER1 or a functional fragment or variant thereof and a transmembrane domain or a functional fragment or variant thereof.

[0357] In certain embodiments, the cell tag is connected to a signal peptide. The signal peptide can be any signal peptide suitable for use in eukaryotic cells, including the signal peptide described for CAR herein. In certain embodiments, the signal peptide is an Igκ signal peptide or a functional fragment or variant thereof.

[0358] D. Immune checkpoint inhibitors

[0359] In any of the foregoing embodiments, the DNA of the present disclosure may further encode an immune checkpoint inhibitor. Therefore, the DNA of the present disclosure may encode miRNA and an immune checkpoint inhibitor. Therefore, the use of such DNA provides two modes of action for reducing the activity of immune checkpoints.

[0360] In certain embodiments, the immune checkpoint inhibitor inhibits the activity of an immune checkpoint protein such as PD1, PD-L1, CTLA-4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, or VISTA.

[0361] In certain embodiments, the immune checkpoint inhibitor is an antibody or a functional fragment or variant thereof.

[0362] In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody, such as cemiplimab, pembrolizumab, nivolumab, torpalimab, sintilimab, LY3434172, JTX-4014, 609A, Sym021, LZM009, budigalimab, IB, SCT-I10A, SG001, AMP-224, AMG 404, AK112, CS1003, MEDI0680, RO7121661, F520, sasanlimab, BI 754091, cetrelimab, HerinCAR-PD-1, HX008, zimberelimab, retifanlimab, balstilimab, pidilizumab, teripalimab, CBT-501, BAT1306, tislelizumab, AK105, spartalizumab, prolgolimab, serplulimab, dostarlimab, camrelizumab, IBI319, KY1043, STI-1110, CA05100948, Nb97, ENUM 388D4, hAb-10D3, ANB030, MCLA-134, and hAb21; anti-CTLA-4 antibodies, such as ipilimumab (YERVOY) and tremelimumab; anti-PD-L1 antibodies, such as BMS935559 (MDX-1105), atezolizumab, avelumab, or durvalumab; anti-CD28 antibodies; anti-TIGIT antibodies; anti-LAG3 antibodies, such as BMS-986016 and LAG525; anti-TIM3 antibodies; anti-GITR antibodies; anti-4-1BB antibodies, such as PF-05082566; or anti-OX-40 antibodies, such as MEDI6469, MEDI0562, and MOXR0916.

[0363] IV. Genetic Constructs

[0364] As previously discussed, in certain embodiments, the deoxyribonucleic acid encoding the pre-miRNA is contained in the same genetic construct as a genetic construct containing one or more genes encoding a protein of interest (eg, a chimeric antigen receptor, cytokine, or cell marker).

[0365] In some such embodiments, this genetic construct comprises a nucleic acid sequence encoding a 5' untranslated region (5'UTR), which is directly located upstream of the gene encoding the protein of interest, and the pre-miRNA sequence is contained in the 5'UTR. In some embodiments, this genetic construct comprises a nucleic acid sequence encoding a 3' untranslated region (3'UTR), which is directly located downstream of the gene encoding the protein of interest, and the pre-miRNA sequence is contained in the 3'UTR. In some embodiments, this genetic construct comprises a nucleic acid sequence encoding a 5'UTR and a 3'UTR, wherein each such region contains at least one pre-miRNA sequence (e.g., each UTR may comprise a pre-miRNA, the 5'UTR may comprise a pre-miRNA and the 3'UTR may comprise 2 pre-miRNAs, the 5'UTR may comprise two pre-miRNAs and the 3'UTR may comprise a pre-miRNA, both UTRs may comprise two pre-miRNAs, etc.).

[0366] In embodiments where the sequence encoding the pri-miRNA is included in a sequence corresponding to the 5'UTR, the transcribed RNA may include additional sequences, such as splicing donors, branch points and / or acceptor site sequences. The inclusion of splicing donors, branch points and acceptor sites is important for splicing miRNA from transcribed RNA. In the absence of splicing, highly structured miRNA sequences may hinder ribosome scanning to the translation initiation sequence associated with the gene of interest. Examples of sequences encoding such splicing donor / acceptor sites include SEQ ID NOs: 291 and 292, sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with such sequences and sequences capable of hybridizing with the complementary sequence of such sequences under stringent hybridization conditions.

[0367] Therefore, in certain embodiments, the deoxyribonucleic acid of the present invention further comprises: a) a nucleic acid sequence that has at least 80% sequence identity with SEQ ID NO: 291 or is capable of hybridizing with the complementary sequence of SEQ ID NO: 291 under stringent hybridization conditions; and b) a nucleic acid sequence that has at least 80% sequence identity with SEQ ID NO: 292 or is capable of hybridizing with the complementary sequence of SEQ ID NO: 292 under stringent hybridization conditions.

[0368] In some embodiments, the portion of the deoxyribonucleic acid encoding the pre-miRNA is contained within a segment of an intron within a gene encoding a protein of interest. In some such embodiments, at least one pre-miRNA is contained within an intron located within the 5'UTR (hereinafter referred to as a "5'UTR intron").

[0369] Fig.16 Exemplary polynucleotides that can be used as templates for expressing various genes and other regulatory elements in cells are shown. It should be understood that various elements can be included or omitted from the polynucleotide, and different options are shown for various exemplary sites within the polynucleotide.

[0370] As Fig.16 shown, the polynucleotide can contain an integration signal for integrating the polynucleotide attP / attB phage into the bacterial genome. The polynucleotide can further contain a 5' homologous arm or 5' terminal repeat sequence and a 3' homologous arm or 3' terminal repeat sequence. The polynucleotide can further contain insulators, boundary elements, and S / MARs located adjacent to the 3' of the 5' homologous arm or 5' terminal repeat sequence and adjacent to the 5' of the 3' homologous arm or 3' terminal repeat sequence. Between the insulators, boundary elements, or S / MARs, the polynucleotide can contain, from 5' to 3', a promoter, which can contain silencers, enhancers, TF binding modules, and a core promoter; a 5' untranslated region, which can contain stability modules, translation control elements, and intron-embedded elements, such as miRNA coding sequences; one or more genes, which can contain signal peptides, extracellular domains, transmembrane domains, signaling domains, antibody domains, peptide linkers, inteins, and epitope tags; and a 3' untranslated region, which can contain stability modules, translation control, 3' end processing signals, and transcription terminators.

[0371] As previously discussed, miRNAs can be encoded in the same genetic construct as additional proteins of interest (e.g., CARs, cytokines, and / or cell tags). The advantage of expressing two or more of such components using a single genetic construct is the stoichiometric expression of such components.

[0372] As will be understood by those skilled in the art, genes encoding polypeptides of interest can be linked by linkers. Any suitable linker known for linking genes can be used in the practice of the present invention. Examples of such linkers include linkers encoding internal ribosome entry sites (IRESs), cleavable peptides, and ribosome skipping peptides. Examples of cleavable peptides encoded by such linkers include Furinlink linkers, fmdv linkers, and 2A linkers (e.g., P2A, GSG-P2A, FP2A, T2A, and Furin-T2A) or functional fragments or variants thereof.

[0373] The polynucleotides of the present invention can be present in the construct in an operably linked manner to a promoter. Appropriate promoters can be selected based on the host cell and effect sought. Suitable promoters include constitutive promoters and inducible promoters. Promoters can be tissue-specific, and such promoters are well known in the art.

[0374] Examples of constitutive promoters for use in the present invention include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter; human elongated growth factor 1α1 (hEF1A1); the simian virus 40 (SV40) early promoter; the mouse mammary tumor virus (MMTV); the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter; the MoMuLV promoter; the avian leukosis virus promoter; the Epstein-Barr virus immediate early promoter; the Rous sarcoma virus promoter; and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter; and functional fragments and variants thereof.

[0375] Compared to constitutive promoters, the use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when such expression is desired, or can turn off such expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible di-polypeptide ecdysone receptor-based gene switch, such as Gene switch.

[0376] The present invention relates in part to a deoxyribonucleic acid comprising a genetic construct as described above.

[0377] V. Vectors and Delivery Systems

[0378] The RNA and / or DNA of the present invention can be delivered to cells on long oligonucleotides, which are then inserted into specific genomic locations. In certain embodiments, the RNA and / or DNA of the present invention can be integrated into the genome of the cell by using a gene editing system using CRISPR, TALEN or zinc finger nucleases.

[0379] The polynucleotides of the present invention can be delivered to target cells by any suitable delivery system, including non-viral delivery systems and viral delivery systems. Therefore, the present invention also relates in part to a vector comprising the ribonucleic acid or deoxyribonucleic acid of the present invention.

[0380] Any vector known in the art for use in delivering RNA or DNA can be used in the practice of the present invention. In certain embodiments, the vector is a plasmid, a mini-ring DNA, a nanoplasmid, a viral vector, an additional vector or a non-viral vector. Examples of viral vectors for use in the present invention include lentiviral vectors and retroviral vectors. Examples of non-viral vectors for use in the present invention include the Sleeping Beauty transposon. In certain embodiments, the vector may include a sequence for serine recombinase-mediated integration (e.g., for aatP sites or attB sites). In the case where the vector is a plasmid, a mini-ring DNA or a nanoplasmid, the plasmid, the mini-ring DNA or the nanoplasmid may further include a bacterial replication origin, for example, a replication origin from a ColE1 plasmid.

[0381] The example of the non-viral vector used in delivering the deoxyribonucleic acid or ribonucleic acid of the present invention is a lipid formulation. Any lipid formulation known in the art for delivering such nucleic acids can be used in the practice of the present invention. In certain embodiments, nucleic acids can be associated with lipids. For example, nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, attached to liposomes via a connecting molecule associated with liposomes and oligonucleotides, captured in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, containing micelles or compounded with micelles, or otherwise associated with lipids.

[0382] Another example of a non-viral vector is a transposon. Any transposon known in the art for delivering deoxyribonucleic acid or ribonucleic acid can be used in the practice of the present invention. When a transposon is used to deliver nucleic acid, a transposase or a nucleic acid encoding it is also typically delivered to the cell. A transposase is an enzyme that binds to a transposon and catalyzes its integration into the genome of a cell. In certain embodiments, the vector is a Sleeping Beauty transposon. When used, a Sleeping Beauty transposase or a functional fragment or variant thereof, or a nucleic acid encoding it is also delivered to the cell. Examples of such transposases include, but are not limited to, SB10 transposase, SB11 transposase, SB100x transposase, and SB110 transposase. The Sleeping Beauty transposon system is known in the art and is described in, for example, U.S. Patent Nos. 6,489,458 and 8,227,432.

[0383] Any viral vector known in the art for delivering deoxyribonucleic acid or ribonucleic acid can be used in the practice of the present invention. Examples of such vectors include, but are not limited to, adenoviral vectors (e.g., adenoviral-based Per.C6 systems available from Crucell, Inc. (Leiden, The Netherlands)), adeno-associated virus-based vectors, lentiviral-based vectors (e.g., lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)), retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH), and herpes virus-based vectors.

[0384] In one embodiment, the viral vector is an adenoviral vector.

[0385] In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Slow viral vectors have additional advantages than vectors derived from tumor retroviruses (such as murine leukemia viruses) because they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0386] In order to evaluate the expression of one or more miRNA and CAR described herein or part thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and select expression cells from a cell population seeking to be transfected or infected by a viral vector or a non-viral vector. In other aspects, selectable markers may be carried on a separate fragment of DNA and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Available selectable markers include, for example, antibiotic resistance genes, such as neomycin (neomycin) resistance gene (neo) and ampicillin (ampicillin) resistance gene, etc. In certain embodiments, truncated epidermal growth factor receptor (HER1t or HER1t-1) labels may be used as selectable marker genes.

[0387] Reporter gene can be used to identify cells of potential transfection and for evaluating the functionality of regulatory sequences. Generally, reporter gene is such a gene, which is not present in a receptor organism or tissue or is not expressed by the receptor organism or the tissue and encodes a polypeptide whose expression is shown by some easily detectable characteristics (e.g., enzymatic activity). The expression of reporter gene is measured at the appropriate time after DNA has been introduced into the receptor cell. Suitable reporter gene includes genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479:79-82 (2000)). Suitable expression systems are well-known and can be prepared or commercially available using known techniques. Generally, the construct with the minimum 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter region can be connected to a reporter gene and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0388] In certain embodiments, the vector comprises: (a) a nucleic acid sequence encoding a ribonucleic acid comprising at least one pri-miRNA; (b) a nucleic acid sequence encoding a CAR; (c) a nucleic acid encoding a cytokine; and (d) a nucleic acid encoding a cell tag. The miRNA, CAR, cytokine, and cell tag encoded in a single construct allow for manufacturing consistency.

[0389] In some embodiments, the ribonucleic acid comprises two pri-miRNAs. In certain embodiments, both miRNAs target PD-1. In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD-1; and (b) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD-1.

[0390] In some embodiments, CAR comprises an antigen binding domain as described elsewhere herein, for example, an antigen binding domain that binds to an epitope on the following items: CD19, CD33, MUC1, MUC16, ROR1, HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1 and / or type II collagen. In certain embodiments, CAR comprises an antigen binding domain that binds to an epitope on the following items: CD19, CD33, MUC1, MUC16 and / or ROR1. In certain such embodiments, CAR comprises an antigen binding domain that binds to an epitope on ROR1.

[0391] In some embodiments, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 or their functional variants or fragments. In certain embodiments, the cytokine is IL-15 or its functional fragment or variant. In certain embodiments, IL-15 or its functional fragment or variant is membrane-bound. In certain embodiments, the vector encodes a fusion protein comprising IL-15 or its functional fragment or variant and IL-15Rα or its functional fragment or variant.

[0392] In some embodiments, the cell tag comprises HER1 domain III or a functional fragment or variant thereof and a truncated HER1 domain IV or a functional fragment or variant thereof. In certain embodiments, the cell tag further comprises a CD28 transmembrane domain or a functional fragment or variant thereof.

[0393] In certain embodiments, the nucleic acid encoding CAR and the nucleic acid encoding cytokine are connected by the nucleic acid encoding joint (e.g., furin-T2A joint).In certain embodiments, the nucleic acid encoding cytokine and the nucleic acid encoding cell label are connected by the nucleic acid encoding (e.g., T2A joint).

[0394] In some embodiments, the vector further encodes a splice donor site and a splice acceptor site. In certain embodiments, the splice donor side comprises SEQ ID NO: 291, and the splice acceptor site comprises SEQ ID NO: 292.

[0395] In some embodiments, the vector comprises: (a) a nucleic acid sequence encoding the following: (i) a pri-miRNA comprising a main chain sequence from miR204 and a guide miRNA targeting PD-1; and (ii) a pri-miRNA comprising a main chain sequence from miR206 and a guide miRNA targeting PD-1; (b) a nucleic acid sequence encoding a CAR, the CAR comprising an antigen binding domain that binds to an epitope on a ROR; (c) a nucleic acid encoding a fusion protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof; and (d) a nucleic acid encoding a cell tag, the cell tag comprising: (i) HER1 domain III or a functional fragment or variant thereof; (ii) a truncated HER1 domain IV or a functional fragment or variant thereof; and (iii) a CD28 transmembrane domain or a functional fragment or variant thereof. In certain such embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the cytokine are connected by a nucleic acid encoding a furin-T2A linker, and the nucleic acid encoding the cytokine and the nucleic acid encoding the cell tag are connected by a nucleic acid encoding a T2A linker. In certain such embodiments, the vector is in the form of a splice donor Sleeping Beauty transposon. In some embodiments, the vector further encodes a splice donor side comprising SEQ ID NO: 291 and a splice acceptor site comprising SEQ ID NO: 292.

[0396] VI. Methods for Introducing miRNA into Cells

[0397] The present invention relates in part to a method for modifying the expression of a gene in a cell, wherein the method comprises introducing into the cell a ribonucleic acid of the present invention or a deoxyribonucleic acid of the present invention. The present invention also relates in part to the use of a ribonucleic acid of the present invention or a deoxyribonucleic acid of the present invention in the manufacture of a medicament for modifying the expression of a gene.

[0398] The present invention also relates in part to a method for producing a genetically engineered cell, wherein the method comprises introducing the ribonucleic acid of the present invention or the deoxyribonucleic acid of the present invention into the cell.

[0399] In certain embodiments of the above methods, the method comprises transfecting cells with the RNA of the invention or the DNA of the invention. In certain embodiments, transfection involves electroporation.

[0400] In certain embodiments, the deoxyribonucleic acid may comprise a transposon, for example, a Sleeping Beauty transposon. In embodiments using a Sleeping Beauty transposon, a Sleeping Beauty transposase or a functional fragment or variant thereof or a nucleic acid encoding the same may be introduced into the cell. In certain embodiments of transfecting cells with a transposon, the method further comprises transfecting the cell with a vector encoding the transposase.

[0401] In certain embodiments of the above methods, cells are transduced with the RNA of the invention or the DNA of the invention. Cells can be transfected with a viral vector comprising such RNA or DNA.

[0402] Methods for introducing genes into cells and expressing genes are known in the art. In the context of expression vectors, the vector can be easily introduced into a host cell (e.g., a mammal, a bacterium, a yeast, or an insect cell) by any method known in the art. For example, the vector can be transferred into a cell by physical, chemical, or biological means.

[0403] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In some embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection. In some embodiments, the method for introducing polynucleotides into host cells is electroporation.

[0404] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0405] As a biological method, the ribonucleic acid or deoxyribonucleic acid of the present invention can be introduced into cells, for example, using a virus-based delivery system. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), adeno-associated virus-based vectors, lentivirus-based vectors (e.g., lentivirus-based pLPI from Life Technologies (Carlsbad, Calif.), retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH), and herpes virus-based vectors. In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncorretroviruses (such as murine leukemia viruses) because they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. Typically and in embodiments, suitable vectors contain a replication origin that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0406] Regardless of the method used to introduce exogenous nucleic acid into the host cell, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR, and "biochemical" assays, for example, detecting the presence or absence of specific peptides by immunological methods (ELISA and protein blots).

[0407] VII. Genetically Modified Cells

[0408] The present invention relates in part to a genetically modified cell comprising a ribonucleic acid or deoxyribonucleic acid of the present invention. The cell can be produced by any method known in the art for introducing such ribonucleic acid or deoxyribonucleic acid into a cell. In certain embodiments, the cell is produced using the methods described herein.

[0409] In certain embodiments, the cell is a modified immune effector cell. In certain embodiments, the modified immune effector cell is a modified T cell, natural killer (NK) cell or macrophage. In certain embodiments, the modified T cell is a modified cytotoxic T cell, for example, a T cell that destroys virus-infected cells and / or tumor cells.

[0410] In certain embodiments, immune effector cells are obtained from a subject, for example, by separation from cord blood, peripheral blood, human embryonic stem cells, iPSCs, bone marrow, lymph node tissue, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Such cells can then be modified, for example, by the methods described herein to contain ribonucleic acids or deoxyribonucleic acids of the invention. Thus, the present invention contemplates that in methods for producing genetically engineered cells, such as the methods described herein, there may be an initial step of obtaining cells from a subject.

[0411] For example, after being modified by transfection or transduction, cells can be immediately infused into a subject or can be cryopreserved. In certain embodiments, cells are incubated less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day or less than 12 hours after transfection or transduction, and then (for example, by infusion into a subject) delivered to the subject. In certain embodiments, cells are manufactured to allow delivery to a subject in 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day or less than 12 hours after transfection or transduction. In certain embodiments, before delivery (for example, by infusion) to a subject, cells do not undergo breeding, activation, incubation or cultivation.

[0412] In some aspects, after transfection or transduction, cells can be stored in a cytokine bath that can include IL-2 and / or IL-21 until ready for infusion. In some aspects, after modification, cells can be propagated in vitro for several days, weeks or months as a body colony in about 1 day, 2 days, 3 days, 4 days, 5 days or longer after gene transfer in cells. In another aspect, after modification, modified cells are cloned, and clones show the presence of a single integrated or episomal expression cassette or plasmid, and in vitro amplification of miRNA and / or the expression of the protein of interest.

[0413] Recombinant T cells can be expanded by stimulation with IL-2 or other cytokines that bind to the common γ chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). Recombinant T cells can also be expanded by stimulation with artificial antigen presenting cells (aAPCs) or antibodies (such as OKT3) that cross-link CD3 on the surface of T cells.

[0414] VIII. Kits and Compositions

[0415] The present invention relates in part to a kit or composition comprising a ribonucleic acid or deoxyribonucleic acid of the present invention. In certain embodiments, the kit or composition comprises a vector of the present invention. In certain embodiments, the kit or composition comprises a genetically modified cell of the present invention.

[0416] The present invention also relates in part to a kit or composition as described above for use in modifying the expression of a gene. The present invention also relates in part to a kit or composition as described above for use in treating a disease or condition in a subject or for use in producing a medicament for treating a disease or condition in a subject.

[0417] In certain embodiments, a kit or composition comprises a transposase.

[0418] In certain embodiments, a kit or composition comprises a gene switch component such as Components of gene switch components.

[0419] In certain embodiments, the composition further comprises a carrier, a diluent and / or an excipient. Any carrier, diluent or excipient known in the art for use with nucleic acids, vectors or cells is contemplated for use in the practice of the present invention. For example, the composition of the present invention may comprise: a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose, dextran or mannitol; a protein; a polypeptide or amino acid example such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0420] In certain embodiments, the kit comprises a carrier, a package, a label or a container. Suitable containers include, for example, bottles, vials, syringes and test tubes.

[0421] IX. Treatment Methods

[0422] The present invention also relates to a method for treating a disease or condition in a subject, the method comprising administering to the subject an RNA, DNA, vector, cell or composition of the present invention. Administration can be performed in a therapeutically effective amount. In certain embodiments, the subject is a mammal, e.g., a human.

[0423] The present invention also relates to the use of the ribonucleic acid, deoxyribonucleic acid, vector, cell or composition of the present invention in the manufacture of a medicament for treating a disease or disorder in a subject.

[0424] In certain embodiments, the disease or condition is one in which reduction or silencing of the expression of an immune checkpoint protein would provide a benefit. In certain such embodiments, the disease or condition is cancer, an autoimmune disorder, or is caused by a viral, bacterial, or parasitic infection.

[0425] In some embodiments, the disease is cancer. The cancer can be a blood tumor or a solid tumor. In some cases, the cancer is metastatic. Examples of cancers that can be treated include, but are not limited to, human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, breast adenocarcinoma, e.g., triple negative breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell carcinoma, leukemias, e.g., acute myeloid leukemia, acute lymphocytic leukemia, mantle cell lymphoma, acute lymphocytic leukemia, and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroblastic leukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); diffuse large B-cell lymphoma; and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

[0426] In some embodiments, the disease is an autoimmune disease. Examples of such autoimmune diseases include, but are not limited to, graft-versus-host disease, rheumatoid arthritis, lupus, celiac disease, Crohn's disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, and autoimmune uveitis.

[0427] In some embodiments, the disease is a disease caused by a viral, bacterial or parasitic infection. Examples of such diseases include, but are not limited to, diseases caused by Plasmodium, Trypanosoma, Aspergillus, Candida, Hepatitis A, Hepatitis B, Hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus and Ebola virus pathogens.

[0428] In some embodiments, the disease or disorder is associated with overexpression of an antigen. In certain embodiments, the antigen is CD19, CD33, ROR1, MUC1 or MUC16.

[0429] In some embodiments, the disease is associated with overexpression of MUC16. In certain such embodiments, the disease is ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, or lung cancer.

[0430] In some embodiments, the disease is associated with overexpression of CD33. In certain such embodiments, the disease is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

[0431] In some embodiments, the disease is associated with overexpression of ROR1. In certain such embodiments, the disease is related to a blood tumor, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In certain such embodiments, the disease is related to a solid tumor, for example, a breast adenocarcinoma, including triple-negative breast cancer (TNBC), pancreatic cancer, ovarian cancer and lung adenocarcinoma.

[0432] In certain embodiments, the method involves administering a nucleic acid, vector, cell or composition as described herein. Such methods can be performed in any manner known in the art, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The nucleic acid, vector, cell or composition described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection or intraperitoneally.

[0433] In certain embodiments, the method involves administering genetically modified cells to a subject. The cells may be cells of the invention as described herein. Such methods may include obtaining a cell sample from a subject, modifying cells of the sample with the RNA or DNA of the invention, and administering the modified cells to the subject, for example, by infusion.

[0434] In certain embodiments, the modified immune cells are directly applied to a specific part of the body, for example, targeted cancer via direct regional delivery to tumor tissue. For example, in ovarian cancer, modified immune effector cells can be delivered to the abdomen or peritoneal cavity intraperitoneally (IP). This IP delivery can be carried out via a port placed for delivering chemotherapy drugs or a pre-existing port. Other methods of regional delivery of modified cells may include catheter infusion into the resection cavity, ultrasound-guided intratumoral injection, hepatic artery infusion, or intrathoracic delivery.

[0435] In some embodiments, before the step of applying the modified cells to the subject, the subject is subjected to lymphocyte removal. As used herein, "lymphocyte removal" relates to a method for reducing the number of lymphocytes in the subject by administering a lymphocyte removal agent, for example. Examples of lymphocyte removal include non-myeloablative lymphocyte removal chemotherapy, myeloablative lymphocyte removal chemotherapy. Lymphocyte removal can also be achieved by local or systemic fractionated radiotherapy. Lymphocyte removal agents can be compounds or compositions that can reduce the number of functional lymphocytes of mammals when applied to mammals. Such agents and dosages are known and can be selected by treating physicians according to the subject to be treated. Examples of lymphocyte removal agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or a combination thereof. In some embodiments, before the step of applying the modified immune effector cells to the subject, the subject is not subjected to lymphocyte removal.

[0436] In some embodiments, the patient or subject does not undergo lymphodepletion prior to drawing blood to produce autologous modified immune effector cells.

[0437] In some embodiments, the modified immune effector cells are autologous to the subject. In some embodiments, the modified immune effector cells are allogeneic to the subject.

[0438] The dosage of the above treatment to be administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment. The proportional scaling of the dosage administered to a human can be performed according to practices accepted in the art. For adult patients or pediatric patients, the appropriate dosage can be adjusted accordingly.

[0439] In some cases, the effective amount of modified cells for administration comprises about 10 4 to about 10 9 modified cells / kg, about 10 4 to about 10 5modified cells / kg, about 10 5 to about 10 6 modified cells / kg, about 10 6 to about 10 7 Modified effector cells / kg, >10 4 But ≤10 5 modified cells / kg, >10 5 But ≤10 6 Modified effector cells / kg or >10 6 But ≤10 7 Modified effector cells / kg.

[0440] Alternatively, a typical amount of modified cells administered to a mammal (e.g., a human) can be, for example, in the range of one hundred, one thousand, ten thousand, one million to 100 billion cells; however, amounts below or above this exemplary range are within the scope of the invention. For example, a dose of such cells can be about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 100 million cells, about 150 million cells, about 160 million cells, about 170 million cells, about 180 million cells, about 190 million cells, about 200 million cells, about 210 million cells, about 220 million cells, about 230 million cells, about 240 million cells, about 250 million cells, about 260 million cells, about 270 million cells, about 280 million cells, about 290 million cells, about 300 million cells, about 310 million cells, about 310 million cells, about 320 million cells, about 330 million cells, about 340 million cells, about 360 million cells, about 370 million cells, about 380 million cells, about 390 million cells, about 400 million cells, about 410 million cells, about 420 million cells, about 430 million cells, about 440 million cells, about 450 million cells, about 460 million cells, about 470 million cells, about 480 million cells, about cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the foregoing values).

[0441] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0442] The therapeutic or preventive efficacy can be monitored by regular assessment of the treated patient. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired disease symptom suppression occurs. However, other dosage regimens are also available and within the scope of the present invention. The desired dose can be delivered by the following methods: a single bolus administration of the composition, multiple bolus administration of the composition, or continuous infusion administration of the composition.

[0443] In some embodiments, an amount of the modified cells is administered to a subject in need thereof, and the amount is determined based on the potency and potential to induce cytokine-related toxicity.

[0444] In some embodiments, the compositions described herein can be administered as a combination therapy with an additional therapeutic agent.Examples of such agents include biologics and small molecules.

[0445] Examples

[0446] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. The following table includes abbreviations and special terms that apply only to the examples. These abbreviations and special terms are not otherwise limiting and do not replace or narrow the broader definitions above, which should continue to apply to the claims.

[0447] Table 5: Abbreviations and special terms used in the examples

[0448]

[0449]

[0450] Example 1. PD1 module design

[0451] The PD1 silencing module of the miRNA-expressing ROR1 UltraCAR-T cells encodes two artificial miRNAs that are designed to specifically reduce the expression of PD-1 mRNA within UltraCAR-T cells while avoiding off-target silencing of other endogenous transcripts. The two artificial miRNAs of the miRNA-expressing ROR1 UltraCAR-T cells are encoded in dual primary miRNA (pri-miRNA) sequences placed within the 5'UTR splicing unit of the UltraCAR-T transgene cassette ( Figure 1BThe dual pri-miRNAs form stem-loop structures that are recognized and processed by cellular complexes to produce two unique 21-24 nucleotide mature guide miRNAs that are homologous to specific sequences in PD1 target transcripts. The interaction of guide miRNAs with PD1 target sequences is expected to trigger silencing of PD1 by inducing RNA degradation or translational inhibition (Guo et al., 2010).

[0452] The guide miRNA encoded within the miRNA-expressing ROR1 UltraCAR-T cells was designed to be highly specific for the PD1 target transcript NM_005018.3 by implementing an in-house designed computational workflow based on three validated rule-based siRNA prediction algorithms using a combination of twenty-one ranking parameters (Amarzguioui and Prydz, 2004; Reynolds et al., 2004; Ui-Tei et al., 2004). Multi-level specificity profiling was performed against the human reference exome (RefSeq) and the activated T cell transcriptome (Zhao et al., 2014) to ensure that the mature miRNA was highly specific for the PD1 target gene. To further reduce the risk of off-target silencing of the non-targeting PD1 passenger strand miRNA, a pri-miRNA scaffold was selected that produced a high ratio of guide miRNA:passenger miRNA (Miniarikova et al., 2016). The guide miRNA PD1_1843 targeting PD1 was incorporated into a pri-miRNA scaffold based on human miRNA hsa-miR-204 (accession number MI0000284), while the PD1_2061 guide miRNA was incorporated into the hsa-miR-206 (accession number MI0000490) scaffold. Mutations were generated on the passenger strand side of each pri-miRNA to ensure that the specific miRNA structure was maintained and that the thermodynamic stability was not substantially altered. The RNA structure was predicted using the CLC Main Workbench software. The PD1 silencing module contains the miR204 PD1_1843pri-miRNA, which is directly upstream of the miR206 PD1_2061pri-miRNA within the synthetic splicing unit in the 5'UTR of the CAR-T transgenic expression cassette. The splicing unit is ordered from IDT as gBlock and can be cloned into the Sleeping Beauty CAR vector and can be cut with ClaI / NheI for cloning into the 5'UTR of any other Sleeping Beauty CAR vector.

[0453] Example 2. miRNA reduces the expression of PD1 transcripts

[0454] Primary human T cells were transfected with the constructs listed in Table 6 and expanded in vitro using AaPC ​​cells expressing the cognate antigen, grown in bulk and then aliquoted for freezing. 6 The resulting CAR-T cells were further activated with 1:1 beads: T cell ratio per T cell / mL for 48 hours, and then the cells were collected for RNA isolation. RNA isolation was performed according to the manufacturer's recommended protocol (Qiagen), and RT-qPCR analysis was performed using SuperScript VILO premix (Invitrogen) with ezDNA enzyme and TaqMan FAST advanced premix for qPCR to evaluate PD-1 expression levels using specific primers / probes (human PD1: Hs00169472_m1; human TIGIT: Hs00545087_m1; and human ACTb: Hs99999903_m1). All samples were also normalized to β-actin expression levels. Relative expression values ​​were derived based on the ΔΔCT method by normalization relative to construct No. 1 (MUC16 CAR-T cells only). The results are shown in Figure 2 Middle. Shown are the mean ± SD from 3 donors.

[0455] Table 6: Figure 2 Description of constructs 1-8 used in (the sequences of miRNAs are described in Table 4)

[0456]

[0457] The data demonstrate the specificity of the PD-1 checkpoint inhibitor miRNA targeting the expected sequence. CAR constructs expressing scrambled miRNA (constructs 2 and 3) and CAR constructs that do not express mbIL-15 (construct 8) did not show any reduction in PD-1 expression, while CAR constructs containing PD-1 miRNA (construct 4) or a combination of PD-1 and TIGIT miRNA (constructs 6 and 7) did show a reduction in PD-1 expression. On the other hand, the CAR construct containing only the TIGIT miRNA sequence (construct 5) did not show any reduction in PD-1 mRNA expression levels, further demonstrating specificity in targeting.

[0458] Example 3. Downregulation of targeted mRNA

[0459] Primary human T cells were transfected with a vector encoding a CD33-specific CAR or a vector encoding a MUC16-specific CAR. The vector contains a synthetic intron or a non-targeted scrambled control miRNA, which contains a miRNA sequence targeting PD-1, PD-1 and / or TIGIT. Antigen presenting cells with homologous tumor antigens were used and T cell cultures were amplified in vitro at a ratio of 1: 1 (AaPC cells: T cells). These cells were K562 cells modified to express CD33 or MUC16 plus other co-stimulatory molecules (based on "clone 1"). The resulting CAR+ cells were then stimulated with anti-CD3 / anti-CD28 beads (1: 1 beads: T cell ratio) for 48 hours in the absence of cytokines. According to the manufacturer's protocol, RNA was isolated using a Qiagen kit (AllPrep Universal DNA / RNA / miRNA Kit No. 80224) and used for a human pan-cancer immune gene panel kit from Nasri. Briefly, RNA was hybridized to capture probe sets and reporter probe sets according to the manufacturer's protocol, samples were processed, and then hybridized to slides using the nCounter preparation station, and transcript counts were generated by the nCounter digital analyzer. Data validation, QC, and normalization were performed by nSolver software (Nanostring Technologies).

[0460] If the samples were identical, the distribution of the transcript count data on the graph would be a line with a slope of 1 (from lower left to upper right). Data points that fall on this line represent changes in transcript counts between the two samples. Counts found below the main line represent decreased expression, while counts above the main line represent increased expression in the compared samples. Transcripts targeted by the miRNA, whether PD-1 or TIGIT expressed in CAR-T cells, specifically showed lower expression of transcripts for their respective targeted genes compared to CAR-T cells without the miRNA. See FIG. 3A to FIG. 3B In addition, to further demonstrate the specificity of targeting, CAR-T cells with scrambled control miRNA showed no change in transcript levels for PD-1 or TIGIT, as the data distribution in this graph was still close to the diagonal line, indicating little change in transcript levels. Figure 3C Similar results were observed in T cells transfected with a MUC16 CAR vector containing a synthetic intron containing miRNA sequences targeting PD-1, PD-1, and / or TIGIT (see FIG. 4A to FIG. 4C ).

[0461] Example 4. Enhancement of tumor cytotoxicity of miRNA MUC16 CAR-T cells

[0462] Primary human T cells were transfected with vectors encoding MUC16-specific CAR and miRNA sequences that target two sequences within the PD-1 transcript, or with vectors encoding MUC16-specific CAR but not encoding miRNA. The CAR-T cells used in this assay were amplified in vitro, CAR expression was normalized, and then these CAR-T cells were inoculated in triplicate in 96-well plates with GFP+K562 cells ("tumor cells") expressing MUC16 at a ratio of 3:1E:T. The plate was loaded into the IncuCyteS3 instrument, and 4 images were taken per well every 2 hours for 7 days. The data was analyzed using IncuCyte software and GFP+ cell counts / images were normalized relative to the 0 hour time point.

[0463] The growth assay determines the growth rate of target cells in the presence or absence of CAR-T cells over the course of 7 days in culture. Lower target cell counts in cultures containing CAR-T cells indicate CAR-T cell lytic activity. Figure 5A The difference between tumor target cells only (black circles, solid) and cells expressing only MUC16-specific CAR (hollow squares) is shown, demonstrating the killing ability of MUC16 CAR-T cells. Compared with cells expressing only MUC16-specific CAR (hollow squares), cells further expressing miRNA targeting PD-1 (grey solid circles) further demonstrated an increase in cytolytic activity, based on the low GFP+ counts that persisted in the time course evaluation. The data demonstrate that the cytolytic activity of CAR-T cells containing miRNA targeting PD-1 is enhanced.

[0464] Similar experiments were performed using GFP+ K562 cells expressing MUC16, PD-L1, and CD155. Figure 5B As shown, the difference between tumor target cells only (square, solid) and cells expressing only CAR (circle, hollow) demonstrates the killing ability of MUC16 CAR-T cells.Compared with MUC16 CAR-T cells only (circle, solid), CAR-T cells (circle, solid) incorporating miRNAs targeting both PD-1 and TIGIT further demonstrated the improvement of cytolytic activity, which is based on the continuous low GFP+ counts in the time course evaluation.The data demonstrate that the cytolytic activity of CAR-T cells containing 3 targeting miRNAs (2 for PD-1 and 1 for TIGIT) in a single construct is enhanced.

[0465] Example 5. Enhancement of cytokine expression

[0466] Primary human T cells were transfected with a single miRNA targeting sequence or a combined vector encoding a MUC16-specific CAR but not a miRNA targeting sequence and a vector encoding a MUC16-specific CAR and a miRNA targeting sequence of PD-1 and TIGIT (see Table 7). CAR-T cells amplified in vitro were normalized for CAR expression, and then these CAR-T cells were inoculated in triplicate in 96-well plates with a ratio of effector to target 1:1, together with K562 tumor target cells (9MUC16t) expressing truncated MUC16, or these CAR-T cells were cultured only in culture medium. Culture supernatants were collected after 3 days of co-cultivation. According to the manufacturer's protocol, culture supernatants were collected, and interferon-γ (IFNγ) and granulocyte / macrophage colony stimulating factor (GM-CSF) were evaluated by multiple cytokine analysis (Luminex). Figure 6 shows the mean ± SD from duplicate wells.

[0467] For CAR-T cell constructs cultured in culture medium, only basal levels of IFNγ and GM-CSF were detected. No cytokines were observed using target cells or culture medium alone. The supernatant after co-culturing only MUC16 CAR-T cells (vector 1) with tumor cells provided baseline values ​​for the expression levels of IFNγ and GM-CSF. By including checkpoint inhibitor miRNAs in the CAR constructs, increased cytokine expression can be observed, particularly inhibition of the PD-1 pathway. In constructs containing a combination of dual PD-1 (construct 3) or single PD-1 and TIGIT miRNA (construct 6) or dual PD-1 and TIGIT miRNA (constructs 10 and 11), higher levels of cytokine expression are provided.

[0468] Table 7: FIG. 6A to FIG. 6D Description of constructs 1 to 11 in (miRNA sequences are described in Table 4)

[0469] Construct miRNA Effector gene 1 not applicable MUC16 CAR-mbIL15-HER1t 2 Random comparison 1 MUC16 CAR-mbIL15-HER1t 3 miR204 PD1+miR206 PD1 MUC16 CAR-mbIL15-HER1t 4 miR17 TIGIT MUC16 CAR-mbIL15-HER1t 5 miR17 TIGIT+miR206 PD1 MUC16 CAR-mbIL15-HER1t 6 miR150 TIGIT+miR206 PD1 MUC16 CAR-mbIL15-HER1t 7 miR17 TIGIT+miR204 PD1+miR206 PD1 MUC16 CAR-mbIL15-HER1t 8 miR17 TIGIT+miR204 PD1+miR206 PD1 extension v1 MUC16 CAR-mbIL15-HER1t 9 miR17 TIGIT+miR204 PD1+miR206 PD1 extension v2 MUC16 CAR-mbIL15-HER1t 10 miR204 PD1+miR206 PD1+miR17 TIGIT MUC16 CAR-mbIL15-HER1t 11 miR204 PD1+miR206 PD1+miR17 TIGIT extension v1 MUC16 CAR-mbIL15-HER1t

[0470] Example 6. Tumor Burden in Treated Mice

[0471] On day 0, fLUC-GFP+SK-OV-3 tumor cells expressing MUCt were implanted intraperitoneally into non-obese diabetic / severe combined immunodeficiency (NOD / SCID) γ mice (NSG). Throughout the study, the tumor burden of these mice was monitored using an in vivo imaging system (IVIS) by luminescence from an IVIS spectral instrument (Perkin Elmer). IVIS data were analyzed based on the defined area of ​​interest using Live Image Software (version 4.1) to obtain total flux values ​​(photons / second). Before administering CAR-T cells, mice were randomly assigned to different groups based on tumor burden and body weight, and then the test articles were administered on day 6. All tested CARs expressed MUC16-specific CARs as well as mbIL15 and HER1t and were referred to as MUC16 CARs. All test articles were compared to 0.5×10 6 CAR-T cells / mouse were normalized and administered intraperitoneally. IVIS imaging was performed twice a week to monitor the overall tumor burden of the mice. The data shown are means ± SEM from n = 4 to 8 mice / group.

[0472] like Figure 7 As shown, tumors in mice given only saline (grey solid circles) continued to grow, as evidenced by the increase in total flux levels observed throughout the study. Ultimately, these mice succumbed to tumor burden and were euthanized. Mice given only MUC16 CAR (black solid squares) were able to control tumor burden. Based on the reduction in tumor flux values ​​to background levels, it was found that CAR-T cells expressing checkpoint inhibitor miRNAs for PD-1 and TIGIT within the construct (open squares and circles) maintained anti-tumor activity. In addition, CAR-T cells expressing checkpoint inhibitor miRNAs for PD-1 and TIGIT showed a faster time frame and rate of tumor burden reduction compared to MUC16 CAR constructs alone.

[0473] Example 7. In vivo phenotyping experiments

[0474] On day 6 of the study, CAR-T cells (expressing MUC16-specific CAR, mbIL15 and HER1t) with CAR alone or CAR with PD-1 / PD-1 miRNA were administered to SKOV-3 / MUC16 tumor-bearing mice. On day 31 of the study, whole blood was collected from mice for phenotypic evaluation of the administered CAR-T cells by flow cytometry. Briefly, whole blood samples were stained with a mixture of fluorescently conjugated antibodies, and then these whole blood samples were fixed in parallel using a one-step fixation / lysis buffer, and red blood cells were lysed at the same time. The fixed samples were read on a flow cytometer (BD LSRFortessaX-20). CAR-T cells were identified based on gating of hCD45 / CD3+ / HER1t+ expression. Fig. 8A In the CAR sample alone (dashed line) showed high PD-1 expression, while the CAR with PD-1 / PD-1 miRNA (solid line) showed a significant decrease in the detected PD-1 expression level. To further quantify the decrease in PD-1 expression detected on the CAR+miRNA (PD-1 / PD-1) group, the median fluorescence intensity (MFI) ( Figure 8B ). The average MFI of PD1 expression in mice given only CAR-T cells (striped columns) was about 709, while the average MFI of CAR-T cells with PD-1 / PD-1 miRNA (solid columns) was reduced to about 236. Means ± SEM from 5 to 8 mice are shown.

[0475] Example 8. Specific PD-1 and TIGIT downregulation

[0476] On study day 6, CAR-T cells (expressing MUC16-specific CAR ("MUC16 CAR"), mbIL15, and HER1t) were administered to SKOV-3 tumor-bearing mice with either CAR alone or CAR with different checkpoint miRNA inhibitors (PD-1 and TIGIT). See Table 8.

[0477] Table 8: FIG. 9A to FIG. 9B Description of groups 1 to 9 in

[0478] Group CAR-T cells 1 Saline control 2 MUC16 CAR / mbIL15 / HER1t 3 MUC16 CAR / mbIL15 / HER1t+anti-PD1 4 MUC16 CAR / mbIL15 / HER1t+scrambled miRNA 2 5 MUC16 CAR / mbIL15 / HER1t+miR206 PD1 6 MUC16 CAR / mbIL15 / HER1t+miR17 TIGIT 7 MUC16 CAR / mbIL15 / HER1t+miR204 PD1 / miR206 PD1 8 MUC16 CAR / mbIL15 / HER1t+miR150 TIGIT / miR206 PD-1 9 MUC16 CAR / mbIL15 / HER1t+miR204 PD1 / miR206 PD1 / miR150 TIGIT

[0479] On the 45th day (D45) of the study, whole blood of mice was collected to evaluate the phenotype of the administered CAR-T cells by flow cytometry. In brief, whole blood samples were stained with a mixture of fluorescent conjugated antibodies (including specific antibodies for human PD-1 and human TIGIT), and then fixed with a one-step fixation / lysis buffer. The fixed samples were read on a flow cytometer (BD LSRFortessaX-20). CAR-T cells were identified in mice based on gating of hCD45 / CD3+ / HER1t+ expression. In order to further evaluate the specificity of the miRNA used in the CAR vector to the checkpoint inhibitors, the median fluorescence intensity (MFI) ( Fig.9A and Fig. 9B ). The MFI for PD-1 is shown on the left, and the MFI for TIGIT is shown on the right to quantitatively evaluate the expression level of the same set of vectors. Fig.9A As shown, compared with only CAR vector, for the groups indicated by downward arrows (solid lines for PD-1), reduced expression of PD-1 was seen on CAR-T cells, which were constructs with PD-1miRNA (single PD-1miRNA, double PD-1miRNA, and PD-1miRNA in combination with another miRNA checkpoint inhibitor). On the right (downward dashed arrows), cell populations with reduced TIGIT expression seen on CAR-T cells are highlighted. The down-regulated expression of TIGIT corresponds to samples containing miRNA for TIGIT (as single miRNA or miRNA in combination with other checkpoint miRNA inhibitors). Mean ± SEM from 5 to 8 mice are shown.

[0480] Example 9. Expression of miRNA targeting PD1 in ROR1-targeted CAR-T cells.

[0481] Briefly, miRNA-expressing ROR1 UltraCAR-T cells or control ROR1 UltraCAR-T cells were generated from T cells from five donors. PanT cells from five healthy donors were transfected with the indicated transposon vectors (VVN-5355 or VVN-5351) plus the SB11 transposase vector, and these cells were expanded by weekly stimulation with ROR1 antigen-presenting cells for 4 weeks (approximately 35 days before adding beads). After a 7-8 day rest period, the cells were transfected with CD3 / CD28 magnetic beads (1:1 beads:T cell ratio, 1 × 10 T cells) and then cultured for 4 weeks. 6UltraCAR-T cells were activated for 48 hours 7 to 8 days after the last AaPC ​​stimulation, and RNA was then harvested. The expression of PD1-targeted guide miRNAs and the effect on PD1 mRNA expression were verified by RT-qPCR. Small RNAseq, an established method for identifying predicted miRNA sequences and alternative miRNA sequences that may be produced by pri-miRNAs (Borel et al., 2018; Miniarikova et al., 2016) was performed to compare the expression levels of guide miRNAs targeting PD1 with other small RNA species (such as passenger miRNAs that may be produced by the PD1 silencer module). Small RNAseq is also used to assess potential changes in global endogenous miRNA expression (Mueller et al., 2012). RNAseq analysis was performed to evaluate global transcript expression and identify changes in molecular pathway signaling or off-target gene silencing attributed to PD1 silencers. In silico miRNA target prediction was performed using the miRanda algorithm to identify the most likely targets of miRNAs produced by the PD1 silencer module. The expression of predicted target genes was evaluated by RNAseq. Details of each method are included in the sections below.

[0482] To characterize the expression of miRNAs encoded by the PD1 silencer and the effects on PD1 transcript levels, RT-qPCR and small RNAseq were performed. To characterize changes in specific genes or cellular pathways, RNAseq was performed.

[0483] Nucleic acids were purified from cell pellets using Qiagen's AllPrep DNA / RNA / miRNA Universal Kit (Cat. No. 80224) following the manufacturer's protocol. Total RNA was eluted with 50 uL of nuclease-free water and the concentration was measured on a Nanodrop™ 2000 spectrophotometer.

[0484] To quantify the expression of PD1 guide miRNA and passenger miRNA, total RNA was used as input for cDNA synthesis using Qiagen's miRCURY LNA RT kit (No. 339340). According to the manufacturer's protocol, cDNA was diluted 1:60 with nuclease-free water, and 3 μL of the diluted cDNA was used as a positive control for qPCR using the miRCURY LNA SYBR Green PCR Kit (Qiagen No. 339345), which has custom miRCURYLNA primers specific for two PD1 guide miRNAs. Endogenous miRNA hsa-let-7a-5p was quantified as a reference small RNA to allow positive control normalization (Qiagen product No. 339306 with custom number YP00205727). Samples were run in a 384-well format on a QuantStudio 6Flex instrument. Relative quantification (dCT) calculations were performed in Microsoft Excel, and data charts were drawn in GraphPad Prism 9. dCT calculations were performed for each technical repeat as follows:

[0485] dCT=CT(guide miRNA)–CT(hsa-let-7a)

[0486] ddCT = dCT (miRNACART replicates) – dCT (average of VVN-5355 technical replicates)

[0487] Fold change = 2^-ddCT

[0488] The VVN-5355ROR1 UltraCAR-T control sample was used as a reference control sample for comparison with miRNA-expressing ROR1 UltraCAR-T cells within each donor set. The mean fold change and standard deviation of the technical replicates were calculated and presented in Fig. 10A Report in.

[0489] To quantify and compare the production of guide and passenger miRNAs derived from the PD1 silencing module, RT-qPCR was performed as described above, except that this second experiment included a primer assay to detect the passenger miRNA as well as an additional endogenous reference small RNA (RNU1A1). Expression calculations were performed to compare the expression of each guide strand mature miRNA or passenger strand mature miRNA relative to the mean of the endogenous control small RNA as follows:

[0490] dCT = CT (mature miRNA) – CT (average of hsa-let-7a and RNU1A1)

[0491] Fold change = 2^-dCT

[0492] The average fold change was calculated based on three technical replicates. These values ​​are plotted on Fig.11 , where the means and standard deviations are shown for the tested donor sample sets.

[0493] In order to quantify the expression of endogenous PD1 mRNA, cDNA synthesis was performed using Invitrogen's SuperScript IV VILO premix and ezDNA enzyme kit (No. 11766050) using a final RNA concentration of 5ng / μL. Multiple Taqman qPCR was performed using Invitrogen's TaqMan Mast advanced premix (No. 4444963) and 1 microliter of cDNA and Invitrogen (ThermoFisher Scientific) Taqman assay. Human PD1 Taqman assay (Invitrogen No. Hs00169472_m1) was FAM labeled, and internal housekeeping gene ACTb (Invitrogen No. Hs99999903_m1) was VIC labeled. Samples were run in 384-well format on a QuantStudio 6Flex instrument. As described above, relative quantification (dCT) calculations were performed in Microsoft Excel, and data charts were drawn in GraphPad Prism9.

[0494] Table 9: Test and control articles

[0495]

[0496] The PD1 silencer module was designed to produce two mature guide miRNAs that bind to PD1 transcripts to silence PD1 expression. The expression of two guide miRNAs targeting PD1 (termed PD1_1843 and PD1_2061) was confirmed in miRNA expressing ROR1 UltraCAR-T cells generated from multiple donors ( Fig. 10A A corresponding reduction in PD1 mRNA expression was validated in miRNA-expressing ROR1 UltraCAR-T cells from all tested donors ( Fig. 10B ). This result indicates that the PD1 silencer module can produce the expected guide miRNA and function as designed.

[0497] To reduce the risk of silencing genes other than PD1, PD1 silencer modules were designed using pri-miRNA scaffolds that preferentially produced guide miRNAs targeting PD1 over non-targeting passenger miRNAs. Guide mature miRNAs and passenger mature miRNAs were quantified by RT-qPCR from miRNA-expressing ROR1 UltraCAR-T cells, which verified that guide miRNAs targeting PD1 were the predominant species compared to non-targeting passenger strand miRNAs (Figure 4). Small RNAseq confirmed a strong processing preference for guide miRNAs targeting PD1, with 99.7% of reads mapping to the PD1 silencer module matching the expected guide miRNA targeting PD1 ( FIG. 12A to FIG. 12E In addition, the start and end positions of the miRNAs were as expected, with miRNAs of 21 to 23 nucleotides detected, with identical 5' ends and variable lengths at the 3' ends ( FIG. 12A to FIG. 12E The extremely low incidence of passenger strand miRNAs and the lack of unexpected small RNAs generated by aberrant RNA processing greatly reduce the risk of off-target gene silencing.

[0498] To ensure that expression of the PD1 silencer module did not have an extreme effect on the internal cellular RNAi machinery, endogenous miRNA counts of ROR1 UltraCAR-T cells expressing miRNAs were compared to control ROR1 UltraCAR-T cells. Examination of the top twenty expressed endogenous miRNAs showed no statistically significant changes in expression between samples (Table 11). In addition, mature miRNAs produced by the PD1 silencer module accounted for approximately 4% of all quantified small RNAs ( Fig.13 ). The data suggest that miRNA expression from the PD1 silencing submodule does not saturate the cellular RNAi machinery and has no detectable effect on global endogenous miRNA expression.

[0499] Table 10: Small RNAseq comparison of guide miRNA and passenger miRNA counts

[0500]

[0501] Table 11: Top 20 expressed endogenous mature miRNAs detected by small RNAseq

[0502]

[0503]

[0504] Example 10: The PD1 silencing submodule specifically reduces the expression of PD-1.

[0505] Using the in silico miRNA target prediction algorithm, miRanda (Betel et al., 2008; Betel et al., 2010), the most likely target transcripts for the guide miRNA and passenger miRNA produced by the PD1 silencer module were predicted. This algorithm assigns a score to each potential miRNA target gene, with higher scores indicating a higher likelihood of silencing by the positive control miRNA sequence. Table 12 lists the summary of the top ten hits for each mature miRNA produced by the PD1 silencing module. PD1 is the only gene with perfect homology to either the guide miRNA or the passenger miRNA and has the highest predicted miRanda score among any potential target genes. The expression of each predicted target gene was characterized from the RNAseq differential expression dataset. PD1 was the most downregulated among all predicted target genes, with a log2 fold change (LFC) of -2.63 (the PD1 in ROR1 UltraCAR-T cells expressing miRNA was reduced by approximately 84% compared to control ROR1 CAR-T cells), and the adjusted p-value was highly significant. The expression of other predicted target genes did not change; the LFCs of those genes with adjusted p-values below 0.05 were in the range of -0.33 to 0.22, i.e., the expression was reduced or increased by <20%. An exception was the weakly predicted target gene HDAC9 of the PD1_2061 guide miRNA, with an LFC of -1.51. HDAC9 is a transcriptional repressor, and mechanistically, it is associated with PD1 expression via BCL6 (Xie et al., 2017; Gil et al., 2016). It is possible that HDAC9 is not directly targeted by the PD1_2061 guide miRNA, and the reduction of HDAC9 is an indirect effect of the decreased PD1 expression.

[0506] Table 12: In silico predicted miRNA target genes

[0507]

[0508]

[0509] Changes in PD1 expression were expected to affect the expression of other genes in downstream pathways. As expected, analysis of the RNAseq data confirmed the differential expression of several genes in ROR1 UltraCAR-T cells expressing miRNA compared to control ROR1 UltraCAR-T cells ( Fig.14A and Fig. 14B, Table 13). To elucidate direct versus indirect changes in gene expression, the differential expression (LFC) of miRNA-expressing ROR1UltraCAR-T cells compared to control ROR1 CAR-T cells was plotted relative to the predicted binding potential (predicted free energy) of PD1 miRNAs for genes that were predicted in silico as potential PD1 miRNA targets ( FIG. 15A to FIG. 15D ). Genes with highly negative free energies and statistically significant expression reductions are likely directly targeted by miRNAs, while downregulated genes with weak free energies are likely indirectly affected by miRNAs. PD1 is clearly distinguished from all other genes in the figure, with strongly reduced expression and high miRNA binding potential, suggesting that PD1 silencer miRNAs strongly and preferentially target PD1 directly, rather than other genes.

[0510] Table 13: Top 10 down-regulated and up-regulated genes in ROR1+PD1 silencer cells relative to control ROR1 UltraCAR-T cells

[0511]

[0512]

[0513] Example 11: Dual miRNA design for reducing the expression of TIGIT.

[0514] Individual non-natural pri-miRNAs containing guide miRNAs targeting TIGIT were screened.

[0515] Jurkat cells overexpressing TIGIT were transfected with the following vectors: an expression vector encoding CAR, membrane-bound IL15 and truncated HER1, wherein the nucleic acid encoding the TIGIT-targeting pri-miRNA was located in the segment corresponding to the 5'UTR intron; or a control vector expressing CAR, membrane-bound IL15 and truncated HER1 but not expressing pri-miRNA.

[0516] Two days after transfection, flow cytometry was performed to quantify TIGIT expression (geometric mean fluorescence). The results are shown in Table 14 and Fig.17 middle.

[0517] Table 14

[0518]

[0519]

[0520] After the initial screening described above, additional screening was performed using the same protocol, except that the expression vectors tested here contained either one pri-miRNA targeting TIGIT or two pri-miRNAs targeting TIGIT. As in the previous screening, each test expression vector encoded CAR, membrane-bound IL15, and truncated HER1, wherein the nucleic acid encoding the pri-miRNA was located in a segment corresponding to the 5'UTR intron. The control vector expressed CAR, membrane-bound IL15, and truncated HER1, but did not express the pri-miRNA. The results are shown in Table 15 and Fig.18 middle.

[0521] Table 15

[0522]

[0523]

[0524] Example 12: Design of miRNA for reducing the expression of CD70.

[0525] Individual constructs encoding non-natural pri-miRNAs containing guide miRNAs targeting CD70 were screened.

[0526] Jurkat JRFTCR cells were transfected with the following vectors: expression vectors encoding anti-MUC16 (4A5) CAR, membrane-bound IL-15, and truncated HER1 (HER1t). The expression vector further encodes: (i) a single pri-miRNA targeting CD70 located in the 5'UTR intron; (ii) two pri-miRNAs targeting PD1 and a single pri-miRNA targeting CD70 located in the 5'UTR intron; (iii) two pri-miRNAs targeting PD1 located in the 5'UTR intron and two pri-miRNAs targeting CD70 located in the 3'UTR intron; or (iv) two pri-miRNAs targeting PD1 located in the 5'UTR intron and a single pri-miRNA targeting CD70 located in the 3'UTR intron. In addition, some cells were transfected with control vectors expressing: (i) anti-MUC16 (4A5) CAR, membrane-bound IL-15, and truncated HER1, but no pri-miRNA; or (ii) anti-MUC16 (4A5) CAR, membrane-bound IL-15, and truncated HER1, and two PD1-targeting pre-miRNAs located in the 5'UTR intron.

[0527] Cells were activated with PMA / ion to stimulate CD70 expression 1 day after transfection, and flow cytometry was performed 2 days after transfection to quantify CD70 expression (percentage of CD70 positive cells in the activated transfected population and geometric mean fluorescence). The results are shown in Table 16 and Fig.19 middle.

[0528] Table 16

[0529]

[0530]

[0531]

[0532] The sequence of the dual pri-miRNA targeting PD-1 used in all the above constructs is SEQ ID NO:267.

[0533] Example 13

[0534] As shown in the above examples, the dual miR204+miR206 combination has been shown to provide robust gene silencing of all target human genes tested to date (PD1, TIGIT, CD70). In addition, small RNAseq analysis has confirmed that about 99.9% of the small RNAs generated by the dual miR204+miR206 combination map to the predicted guide mature miRNA sequence or the passenger mature miRNA sequence (about 93% guide, 7% passenger), thus confirming the proper folding and processing of the dual pri-miRNA design.

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

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[0543]

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[0550]

[0551]

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[0569]

[0570]

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[0573]

[0574]

[0575] Splice donor and splice acceptor site sequences

[0576]

[0577] Exemplary Control Sequences

[0578]

Claims

1. A ribonucleic acid comprising two non-natural pre-miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein.

2. The ribonucleic acid of claim 1, wherein the non-natural pre-miRNA sequences have less than about 50% sequence identity with each other.

3. The ribonucleic acid of claim 1, wherein the nucleic acid sequence of at least one non-natural pre-miRNA sequence has at least about 90% sequence identity with the nucleic acid sequence of a naturally occurring pre-miRNA sequence.

4. The ribonucleic acid of claim 1, wherein the two non-natural pre-miRNA sequences are separated from each other by at least 10 nucleotides.

5. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA sequence targets a different gene.

6. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA sequence targets a different region of the same gene.

7. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA comprises a backbone sequence identical to a corresponding backbone segment of a naturally occurring pre-miRNA.

8. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA comprises a backbone sequence from miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915.

9. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA comprises a backbone sequence from miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206.

10. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA comprises a backbone sequence from miR16, miR21, miR22, miR204, or miR206.

11. The ribonucleic acid of claim 1, wherein each non-natural pre-miRNA comprises a backbone sequence from: miR204 or miR206.

12. The ribonucleic acid of claim 1, wherein the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to mRNA and thereby interfering with its translation and / or promoting its degradation.

13. The ribonucleic acid of claim 1, wherein the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to mRNA under stringent hybridization conditions.

14. The ribonucleic acid of claim 1, wherein the immune checkpoint protein is CTLA4, CD70, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR or PIK3IP1.

15. The ribonucleic acid of claim 1, wherein the immune checkpoint protein is CTLA4, CD70, PD-1, TIGIT, TIM3, LAG3, GITR or PIK3IP1.

16. The RNA according to claim 1, wherein the immune checkpoint protein is CD70, PD-1 or TIGIT.

17. The RNA of claim 1, wherein the immune checkpoint protein is PD-1.

18. A deoxyribonucleic acid encoding the ribonucleic acid according to any one of claims 1 to 17.

19. The deoxyribonucleic acid according to claim 18, further encoding a protein.

20. The DNA of claim 19, wherein the protein is a chimeric antigen receptor.

21. The deoxyribonucleic acid of claim 20, wherein the chimeric antigen receptor comprises an antigen binding domain that binds an antigen overexpressed in cancer.

22. The deoxyribonucleic acid of claim 21, wherein the chimeric antigen receptor comprises an antigen binding domain that binds CD19, CD33, MUC-16, or ROR-1.

23. The deoxyribonucleic acid of claim 22, wherein the chimeric antigen receptor comprises an antigen binding domain that binds ROR-1.

24. The deoxyribonucleic acid of claim 19, wherein the protein is a cytokine.

25. The deoxyribonucleic acid according to claim 24, wherein the protein comprises IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof.

26. The deoxyribonucleic acid of claim 19, wherein the protein is a cell tag.

27. The deoxyribonucleic acid according to claim 26, wherein the cell tag comprises HER1 domain III or a functional fragment or variant thereof and a truncated HER1 domain IV or a functional fragment or variant thereof.

28. The deoxyribonucleic acid according to claim 27, wherein the cell tag further comprises a CD28 transmembrane domain or a functional fragment or variant thereof.

29. The deoxyribonucleic acid according to claim 19, further encoding: (a) a chimeric antigen receptor; (b) a protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or variant thereof; and (c) a cell tag.

30. The deoxyribonucleic acid of claim 19, wherein the protein is an immune checkpoint inhibitor.

31. A vector comprising the ribonucleic acid according to any one of claims 1 to 17 or the deoxyribonucleic acid according to any one of claims 18 to 30.

32. The vector of claim 31, wherein the vector is a plasmid, a nanoplasmid, a viral vector, an episomal vector or a non-viral vector.

33. The vector of claim 32, wherein the vector is a Sleeping Beauty transposon.

34. The vector of claim 32, wherein the vector is a viral vector.

35. The vector of claim 34, wherein the vector is an adenoviral vector.

36. A method for modifying the expression of a gene in a cell, wherein the method comprises introducing into the cell the ribonucleic acid according to any one of claims 1 to 17 or the deoxyribonucleic acid according to any one of claims 18 to 30.

37. A method for modifying the expression of a gene in a cell, wherein the method comprises transfecting the cell with the ribonucleic acid according to any one of claims 1 to 17 or the deoxyribonucleic acid according to any one of claims 18 to 30.

38. A method for modifying the expression of a gene in a cell, wherein the method comprises transfecting the cell with the vector according to claim 31.

39. The method of claim 38, further comprising transfecting the cell with a vector encoding a transposase.

40. A method for producing a genetically engineered cell, wherein the method comprises introducing into the cell a ribonucleic acid according to any one of claims 1 to 17 or a deoxyribonucleic acid according to any one of claims 18 to 30.

41. A genetically modified cell comprising the ribonucleic acid according to any one of claims 1 to 17 or the deoxyribonucleic acid according to any one of claims 18 to 30.

42. A genetically modified cell produced by the method of claim 40.

43. A composition comprising the ribonucleic acid according to any one of claims 1 to 17 or the deoxyribonucleic acid according to any one of claims 18 to 30.

44. A composition comprising the vector of claim 31.

45. A composition comprising the cell of claim 41.

46. ​​The composition of claim 43, for use in modifying the expression of a gene.

47. The composition of claim 43, for use in treating a disease or disorder in a subject.

48. A kit comprising the ribonucleic acid according to any one of claims 1 to 17 or the deoxynucleic acid according to any one of claims 18 to 30.

49. A kit comprising the cell of claim 41.

50. A method of treating a disease or condition in a subject, the method comprising administering to the subject a ribonucleic acid according to any one of claims 1 to 17 or a deoxynucleic acid according to any one of claims 18 to 30.

51. A method of treating a disease or condition in a subject, the method comprising administering to the subject the cell of claim 41.

52. Use of a ribonucleic acid according to any one of claims 1 to 17 or a deoxyribonucleic acid according to any one of claims 18 to 30 in the manufacture of a medicament for modifying the expression of a gene.

53. Use of a ribonucleic acid according to any one of claims 1 to 17 or a deoxyribonucleic acid according to any one of claims 18 to 30 in the manufacture of a medicament for treating a disease or condition in a subject.

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