Polycistronic miRNA constructs for immune checkpoint inhibition

Abstract

The ribonucleic acid contains two non-natural pre-miRNA sequences, and each pre-miRNA sequence contains a guide miRNA that inhibits the expression of immune checkpoint proteins. The pre-miRNA sequences may target different genes or different regions of the same gene. The deoxyribonucleic acid encodes the above ribonucleic acid. The deoxyribonucleic acid may further encode proteins such as chimeric antigen receptors, cytokines, cell tags, and / or immune checkpoint inhibitors. The vector contains the above ribonucleic acid or the above deoxyribonucleic acid. The method for modifying gene expression in a cell includes introducing the above ribonucleic acid or the above deoxyribonucleic acid into the cell. The method for generating genetically modified cells includes introducing the above ribonucleic acid or the above deoxyribonucleic acid into the cell. The genetically modified cells contain the above ribonucleic acid or the above deoxyribonucleic acid. The composition contains the above ribonucleic acid or the above deoxyribonucleic acid. The kit contains the above ribonucleic acid or the above deoxyribonucleic acid. The method for treating a disease or disorder in a subject includes administering the above ribonucleic acid or the above deoxyribonucleic acid to the subject. The method for treating a disease or disorder in a subject includes administering the above cells to the subject. Use of the above ribonucleic acid or the above deoxyribonucleic acid in the manufacture of a medicament for modifying gene expression. Use of the above ribonucleic acid or the above deoxyribonucleic acid in the manufacture of a medicament for treating a disease or disorder in a subject.

Description

Background Art

[0001] Reference to Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy was created on June 21, 2023, named 391456_SL.xml, and is 501,736 bytes in size.

[0002] Immune checkpoint proteins play a role in regulating the immune system. Positive immune checkpoint proteins help T cells carry out immune responses. On the other hand, negative immune checkpoint proteins such as PD-1, TIGIT, CD70, and CTLA-4 play a role in downregulating immune responses, thereby preventing T cells from damaging or killing healthy cells. However, in cancer patients, such downregulation may also prevent T cells, including T cells modified to contain chimeric antigen receptors (CAR-T cells), from killing cancer cells. Therefore, it is desirable to inhibit the activity of such checkpoint proteins.

[0003] Immune checkpoint inhibition that can prevent the switch-off of T cells and promote the activity of these cells is expected as an immunotherapy. Examples of checkpoint inhibitor proteins that can be targeted by such treatments include 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 Cell Death Ligand 1 (PD-L1) pathway, which plays an important role in how tumor cells evade immune responses. Immunotherapy using PD-1 / PD-L1 inhibitory antibodies has been widely evaluated clinically and has been shown to improve tumor regression across multiple types of malignancies, particularly when used in combination with CAR-T cells.

[0004] However, antibodies that block checkpoint inhibitors have not functioned consistently across all cancer types. Furthermore, such antibodies have limited access to the tumor microenvironment, need to be administered repeatedly, and may lose effectiveness over time. Genome editing is an alternative approach that has the advantage of restricting the blockade of checkpoint inhibitors to genetically engineered cells only. However, gene editing complicates the manufacturing process and increases the time and cost of cell therapy. Therefore, there is a continuing need in the art to obtain novel checkpoint inhibitor therapies.

[0005] MicroRNA (miRNA) is a small non-coding RNA molecule that binds to mRNA molecules produced from target genes and affects their translation into proteins. By such action, miRNA silences genes. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] To enhance the silencing of a target gene, it is known to encode multiple miRNAs targeting different regions on the same gene in a single polycistronic gene construct that may be delivered to cells, for example, via a vector. See Mueller et al., Molecular Therapy, 20:590-600 (2012) (refer to FIG. 1 of “Mueller”). Such an approach has been shown to achieve a strong knockdown of the target gene. Id. However, using constructs containing repetitive precursor miRNA (pre-miRNA) structures risks the alternating folding of pre-miRNA stem-loop structures during transcription. This can result in alternatively processed miRNAs, which may cause unintended off-target gene silencing and pose a safety risk. Additionally, constructs containing repetitive pre-miRNA structures enable recombination within the vector and may lead to the generation of an impure vector population with sequence variations.

[0007] Applicants address these risks by designing polycistronic constructs that encode multiple miRNAs, where each pre-miRNA is different and not complementary to each other. In certain embodiments, at least about 10 nucleotides separate the pre-miRNA structures to assist in ensuring proper co-transcriptional folding of the RNA. Further, in certain embodiments, the pre-miRNAs are designed to maintain the stem-loop structures and internal loops predicted based on endogenous human sequences, thereby hopefully reducing the risk of RNAi-based toxicity. The pre-miRNAs within these constructs can each target different genes or different regions of the same gene.

[0008] The present invention relates to the use of a polycistronic miRNA construct for modifying the expression of a gene encoding an immune checkpoint protein.

Means for Solving the Problems

[0009] The present invention relates, in part, to a ribonucleic acid comprising two non-natural pre-miRNA sequences, each pre-miRNA sequence comprising a guide miRNA that inhibits the expression of an immune checkpoint protein.

[0010] In certain embodiments, the non-natural pre-miRNA sequences have less than about 50% sequence identity to each other.

[0011] In certain embodiments, the nucleic acid sequence of at least one non-natural pre-miRNA sequence has at least about 90% sequence identity to the nucleic acid sequence of a native pre-miRNA sequence.

[0012] In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 10 nucleotides.

[0013] In certain embodiments, each non-natural pre-miRNA sequence targets a different gene.

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

[0015] In certain embodiments, each non-natural pre-miRNA comprises the same backbone sequence as the corresponding backbone segment of a native pre-miRNA.

[0016] In certain embodiments, each non-natural pre-miRNA comprises miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915.

[0017] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence from miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206.

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

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

[0020] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that can bind to the mRNA, thereby interfering with its translation and / or promoting its degradation.

[0021] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that can bind to the mRNA under stringent hybridization conditions.

[0022] In certain embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1.

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

[0024] In certain embodiments, the immune checkpoint protein is CD70, PD-1, or TIGIT.

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

[0026] In addition, the present invention also relates, in part, to deoxyribonucleic acid encoding the ribonucleic acid of the present invention.

[0027] In certain embodiments, the deoxyribonucleic acid further encodes a protein.

[0028] In certain embodiments, the protein is a chimeric antigen receptor.

[0029] In certain embodiments, the chimeric antigen receptor comprises an antigen-binding domain that binds to an antigen overexpressed in cancer.

[0030] In certain embodiments, the chimeric antigen receptor comprises an antigen-binding domain that binds to CD19, CD33, MUC-16, or ROR-1.

[0031] In certain embodiments, the chimeric antigen receptor comprises an antigen-binding domain that binds to ROR-1.

[0032] In certain embodiments, the protein is a cytokine.

[0033] 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.

[0034] In certain embodiments, the protein is a cell tag.

[0035] In certain embodiments, the cell tag comprises domain III of HER1, or a functional fragment or variant thereof, and truncated domain IV of HER1, or a functional fragment or variant thereof.

[0036] In certain embodiments, the cell tag further comprises the transmembrane domain of CD28 or a functional fragment or variant thereof.

[0037] In certain embodiments, the protein is an immune checkpoint inhibitor.

[0038] 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.

[0039] The invention also relates, in part, to a vector comprising the ribonucleic acid or deoxyribonucleic acid of the invention.

[0040] In certain embodiments, the vector is a plasmid, nanoplasmid, viral vector, episomal vector, or non-viral vector.

[0041] In certain embodiments, the vector is a Sleeping Beauty transposon.

[0042] In certain embodiments, the vector is a viral vector.

[0043] In certain embodiments, the vector is an adenoviral vector.

[0044] The invention also relates, in part, to a method for modifying gene expression in a cell, the method comprising introducing the ribonucleic acid or deoxyribonucleic acid of the invention.

[0045] The invention also relates, in part, to a method for modifying gene expression in a cell, the method comprising transfecting the cell with the ribonucleic acid or deoxyribonucleic acid of the invention.

[0046] In certain embodiments, the method comprises transfecting the cell with the vector of the invention.

[0047] In certain embodiments, the method further comprises transfecting the cell with a vector encoding a transposase.

[0048] The invention also relates, in part, to a method for generating genetically engineered cells, the method comprising introducing into the cell a ribonucleic acid or a deoxyribonucleic acid of the invention.

[0049] The invention also relates, in part, to genetically modified cells comprising a ribonucleic acid or a deoxyribonucleic acid of the invention.

[0050] The invention also relates, in part, to genetically modified cells generated by the method of the invention.

[0051] The invention also relates, in part, to a composition comprising a ribonucleic acid or a deoxyribonucleic acid of the invention.

[0052] In certain embodiments, the composition is for use in modifying the expression of a gene.

[0053] In certain embodiments, the composition is for use in the treatment of a disease or disorder in a subject.

[0054] The invention also relates, in part, to a composition comprising a vector or a cell of the invention.

[0055] The invention also relates, in part, to a kit comprising a ribonucleic acid or a deoxyribonucleic acid of the invention.

[0056] The invention also relates, in part, to a kit comprising a cell of the invention.

[0057] The invention also relates, in part, to a method for treating a disease or disorder in a subject, the method comprising administering to the subject a ribonucleic acid or any deoxyribonucleic acid of the invention.

[0058] The present invention also relates, in part, to a method of treating a subject disease or disorder, the method comprising administering to the subject a cell of the present invention or any one deoxyribonucleic acid of the present invention.

[0059] 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 gene expression.

[0060] 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 disorder in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0061]

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Mode for Carrying Out the Invention

[0062] The following description and examples illustrate in detail embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can be modified. Those skilled in the art will recognize that there are variations and modifications of the present disclosure, and they will be included within the scope of the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0064] The section headings used herein are for the purpose of organization only and should not be construed as limiting the subject matter described.

[0065] Although the various features of the present disclosure can be described in relation to a single embodiment, these features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described in relation to separate embodiments for clarity herein, the present disclosure can also be implemented in a single embodiment.

[0066] I. Definitions The following definitions supplement definitions in the art and are specific to this application and do not pertain to any related or unrelated cases, such as those attributable to co-owned patents or co-filed applications. The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0067] In this application, unless otherwise specified, the use of the singular form includes the plural. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0068] In this application, unless otherwise specified, the use of "or" means "and / or". The terms "and / or" and "any combination thereof", as well as their grammatically equivalent forms used herein, may be used interchangeably. These terms can convey that any combination is specifically contemplated. Merely for illustration purposes, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A individually; B individually; C individually; A and B; B and C; A and C; as well as A, B, and C". The term "or (or)" can be used conjunctively or disjunctively unless the context specifically indicates a disjunctive use.

[0069] Furthermore, the term "including", as well as other forms such as "include", "includes", and "included", is not limiting.

[0070] References herein 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 of the present disclosure, but not necessarily in all embodiments.

[0071] As used in this specification and the claims, the terms "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 additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or configuration of the present disclosure, and vice versa. Further, the methods of the present disclosure can be achieved using the compositions of the present disclosure.

[0072] 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., on the limitations of the measuring system. For example, "about" can mean within one or more standard deviations, depending on the convention used in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, an amount "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" with respect to a reference numerical value can include ranges of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Alternatively, especially with respect to biological systems or processes, the term "about" can mean within an order of magnitude of the value, preferably within fivefold, more preferably within twofold. Where a particular value is recited in this application and the claims, the term "about" meaning within an acceptable error range for the particular value should be assumed unless otherwise stated.

[0073] "Therapeutically effective amount" or "therapeutically effective dose" refers to an amount or dose that is effective over a period necessary to achieve a desired therapeutic result. The amount can vary depending on factors such as the individual's disease state, age, gender, weight, and the ability of the nucleic acid sequence of the present invention to induce a desired response in the individual.

[0074] "Polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides or nucleic acids of any length, i.e., ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded deoxyribonucleic acid (DNA), triple-stranded DNA, as well as double-stranded and single-stranded ribonucleic acid (RNA). Also included are, for example, modifications by methylation and / or capping, as well as unmodified forms of polynucleotides. This term also means that it includes molecules containing non-natural or synthetic nucleotides, as well as nucleotide analogs.

[0075] Unless otherwise indicated, nucleic acid sequences in the text of this specification are shown in the 5' to 3' direction when read from left to right.

[0076] 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 using physical, chemical, and / or electrical methods. The nucleic acid sequences and vectors disclosed herein can be introduced into cells or organisms by any method, such as electroporation, calcium phosphate coprecipitation, strontium phosphate DNA coprecipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polycation-mediated transfection, tungsten particle-facilitated microparticle bombardment, virus and / or non-virus-mediated transfection, etc. In some cases, the method of introducing nucleic acid into a cell or organism involves the use of a virus, retrovirus, lentivirus, transposon, or transposable element-mediated (e.g., Sleeping Beauty) vector.

[0077] As used herein, "polypeptide", "peptide", and their grammatical equivalents refer to polymers of amino acid residues. A polypeptide can optionally include glycosylation and other modifications typical of a given protein in a given cellular environment. The polypeptides and proteins (including functional fragments and functional variants thereof) disclosed herein can include synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are known in the art and include, by way of example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline 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-hydroxy-lysine, 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 polypeptides or proteins described herein in engineered cells can be associated with post-translational modification of one or more amino acids of the polypeptide or protein.Examples of post-translational modifications include, but are not limited to, phosphorylation, acetylation and acylation including formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylization, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glycolipidation, lipoylation and iodination.

[0078] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid having common characteristics. A functional way to define the common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, multiple amino acids within a group are preferentially exchanged with each other, thereby defining groups of amino acids that are most similar to each other in terms of the impact on the overall protein structure (Schulz, G.E. and Schirmer, R.H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the following subgroups, for example, substitution from lysine to arginine or vice versa so as to maintain a positive charge; substitution from glutamic acid to aspartic acid or vice versa so as to maintain a negative charge; substitution from threonine to serine so as to maintain a free -OH; and substitution from glutamine to asparagine so as to maintain a free -NH2. Examples of conservative amino acid substitutions are shown in the following chart.

Number

[0079] An amino acid sequence that differs from a reference amino acid sequence only by conservative amino acid substitutions is herein referred to as a "conservative substitution variant" of the reference sequence.

[0080] In some embodiments, a functional variant can comprise an amino acid sequence of a reference protein having at least one non-conservative amino acid substitution. The term "non-conservative mutation" includes amino acid substitutions between different groups, such as an amino acid substitution from lysine to tryptophan, or from phenylalanine to serine. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with the functional variant or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the homologous parent protein. The substitutability of amino acids is described in detail, for example, in L.Y. Yampolsky and A. Stoltzfus, "The Exchangeability of Amino acids in Proteins", Genetics August 2005; 170(4):1459-1472.

[0081] As used herein, the term "identical" and its grammatical equivalents, or "sequence identity" in the context of two nucleic acid or amino acid sequences of a polypeptide, refers to residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. As used herein, a "comparison window" refers to at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 segments that can be compared with a reference sequence of the same number of contiguous positions in a given sequence. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted 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. U.S.A., 85:2444 (1988); computer implementations of these algorithms (e.g., CLUSTAL in the PC / Gene programs by Intelligenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package of Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA), but are not limited to these.The CLUSTAL programs are described in detail by 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). Also, the alignments are often performed by inspection and manual alignment. In some classes of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99% or 100% identical to a reference polypeptide (i.e., its full length) or a fragment thereof 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, for example, they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof as measured by BLASTN (or CLUSTAL or any other available alignment software) using default parameters. When one molecule is said to have a given percentage sequence identity with a larger molecule, this means that when the two molecules are optimally aligned, the percentage of residues of the smaller molecule that are identical to the residues of the larger molecule, following the order in which the two molecules are optimally positioned.

[0082] For the purposes of this specification and the claims, the reference sequence and the expression "having at least 50% sequence identity", or any range therein (e.g., "having at least 80% sequence identity"), are understood to include the reference sequence itself. Thus, for example, a claim that recites "a nucleic acid having at least 80% sequence identity to SEQ ID NO: 0" includes SEQ ID NO: 0 itself.

[0083] When applied to a nucleic acid or amino acid sequence, the terms "substantially identical" and its grammatically equivalent terms mean that the nucleic acid or amino acid sequence, when compared to a reference sequence using the above programs, e.g., BLAST, using standard parameters, includes a sequence having at least 95% sequence identity.

[0084] "Homology" is generally inferred from sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of identity between sequences useful for establishing homology varies depending on the nucleic acids and proteins in question, but a sequence identity of only 25% is commonly used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining the percentage of sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and / or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" if their encoding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules can be referred to as "homologs". For example, a native protein can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide homologous to the protein encoded by the original nucleic acid.

[0085] Also intended and included herein are nucleic acid molecules that hybridize to the disclosed sequences. Hybridization conditions may, of course, be mild, moderate, or stringent.

[0086] Appropriate stringent conditions for promoting DNA hybridization, for example, washing at about 45°C in 6× sodium chloride / sodium citrate (SSC) followed by washing at 50°C in 2× SSC, are known and can also be found in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 - 6.3.6. "Stringent hybridization conditions" include conditions such as a salt concentration of 1.0 M NaCl in 50% formamide, a temperature of 37°C for 4 - 12 hours, followed by washing at 60 - 65°C in 0.1× SSC.

[0087] As will be understood by those skilled in the art, minor changes in nucleic acid sequences do not necessarily change the amino acid sequence of the encoded polypeptide. The present disclosure includes, as will be understood by those skilled in the art, the degeneracy of codon usage. For example, as is known in the art, different codons will encode the same amino acid as shown in the following chart.

Number

[0088] As used herein, the phrase "codon degeneracy variant" with respect 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 amino acid sequence encoded by the reference sequence.

[0089] Furthermore, those skilled in the art will understand that sub - sequences often function as effectively as the full - length version. Methods for altering or shortening nucleotide sequences are well - known to those skilled in the art, as are methods for testing the fitness or effectiveness of the modified genes. In certain embodiments, the fitness and / or effectiveness of the modified genes can be readily tested, for example, by conventional gas chromatography. Thus, all such variations of the gene are included as part of the present disclosure.

[0090] As used herein, the term "isolated" and its grammatical equivalents refer to the removal of nucleic acids from their natural environment. However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and further isolated for practical purposes.

[0091] As used herein, the term "purified" and its grammatical equivalents refer to a molecule or composition of enhanced purity, whether taken from nature (including genomic DNA and mRNA), synthesized (including cDNA), and / or amplified under laboratory conditions, where "purity" is a relative term, not "absolute purity". For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells. As used herein, the term "substantially purified" and its grammatical equivalents refer to nucleic acid sequences, polypeptides, proteins, and other compounds that essentially do not contain other molecules naturally associated with the polynucleotide, protein, polypeptide, and such nucleic acid, polypeptide, protein, and other compounds, that is, contain less than about 50%, less than about 70%, less than about 90% of them.

[0092] As used herein, "T cell" or "T lymphocyte" refers to a type of lymphocyte that plays a central role in cellular immunity. These can be distinguished from other lymphocytes, such as B cells and natural killer (NK) cells, by the presence of a T - cell receptor (TCR) on the cell surface.

[0093] A "transposon", "transposable element", or "TE" is a DNA sequence that can change its position within a genome, sometimes causing or reversing mutations and changing the size of the cell's genome. Transposons are often duplicated by translocation. Class I transposons are copied in two steps: first, they are transcribed from DNA to RNA, and then the resulting RNA is reverse transcribed into DNA. This copied DNA is then inserted into a new location in the genome. The reverse transcription step is catalyzed by a reverse transcriptase enzyme that can be encoded by the transposon itself. Retrotransposons are characterized by being similar to retroviruses such as HIV. The transfer mechanism by cleavage and attachment of Class II transposons does not involve an RNA intermediate. The transfer is catalyzed by several transposase enzymes. Some transposases bind non-specifically to any target site of DNA, while other transposases bind to specific DNA sequence targets. Transposases make staggered cuts at the target site, resulting in single-stranded 5' or 3' DNA overhangs (sticky ends). This step excises the DNA transposon, which is then ligated to a new target site. This process involves the activity of a DNA polymerase that fills in the gaps and the activity of a DNA ligase that closes the sugar-phosphate backbone. This results in duplication of the target site. The insertion site of a DNA transposon can be identified by short direct repeats that can be generated by staggered cutting of the target DNA and filled in by DNA polymerase, followed by a series of inverted repeats that are important for excision of the transposon by the transposase. When translocation occurs during the S phase of the cell cycle when the donor site has already been replicated but the target site has not yet been replicated, the transposon that has been cleaved and attached can be replicated. Transfers can be classified as either "autonomous" or "non-autonomous" for both Class I and Class II transposons. Autonomous transposons can move on their own, while non-autonomous transposons require the presence of another transposon to move.This is because, in many cases, non-autonomous transposons lack transposase (in the case of Class II) or reverse transcriptase (in the case of Class I).

[0094] "Transposase" refers to an enzyme that binds to the ends 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 transposase can be utilized to move a gene(s) from a vector to the genome.

[0095] An "expression vector" or "vector" is any genetic element that functions as an autonomous unit of polynucleotide replication within a cell (i.e., can replicate under its own control), or enables replication by insertion into the host cell chromosome to cause replication and / or expression of an added polynucleotide segment, such as a plasmid, minicircle, nanoplasmid, chromosome, virus, transposon. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. The vector can contain polynucleotide sequences necessary to achieve ligation or insertion of the vector into the desired host cell and to achieve expression of the added segment. Such sequences vary depending on the host organism. These include promoter sequences that affect transcription, enhancer sequences that increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, an expression vector may be able to directly express an internally encoded nucleic acid sequence product without ligation or integration of the vector into the host cell DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome" that can replicate within the host cell and persists as an extrachromosomal segment of DNA within the host cell in the presence of an appropriate selection 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 that utilize Epstein-Barr virus-derived nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). pREP4, pCEP4, pREP7, and pcDNA3.1, vectors manufactured by Invitrogen (Carlsbad, Calif.), and pBK-CMV, manufactured by Stratagene (La Jolla, Calif.), represent non-limiting examples of episomal vectors that use the T antigen and the origin of replication of SV40 instead of EBNA1 and oriP. Also, the vector can contain a selectable marker gene.In certain embodiments where nanoplasmids are utilized, strains such as R6K that utilize an antisense RNA selectable marker (e.g., sucrose tolerance) can be used.

[0096] The term "selectable marker gene" refers to a nucleic acid sequence that enables cells expressing the nucleic acid sequence to be specifically selected or not in the presence of the corresponding selective agent. Suitable selectable marker genes are known in the art and are described, for example, in WO 92 / 08796 and WO 94 / 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); Santerre 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 U.S. Pat. Nos. 5,122,464 and 5,770,359.

[0097] The term "coding sequence" refers to a segment of a polynucleotide that encodes a protein or polypeptide. The region or sequence is bounded at the near 5'-end by a start codon and at the near 3'-end by a stop codon. The coding sequence is also referred to as an open reading frame.

[0098] As used herein, the term "operably linked" refers to the physical and / or functional linkage of a DNA segment to another DNA segment such that the segment functions as intended. A DNA sequence encoding a gene product is operably linked to regulatory sequences such as, for example, promoters, enhancers, and / or silencers, when the regulatory sequences directly or indirectly enable the regulation of transcription of the DNA sequence. For example, a DNA sequence is operably linked to a promoter when the DNA sequence is linked to the promoter downstream of the transcription start site of the promoter and within the correct reading frame relative to the transcription start site such that transcription elongation proceeds through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when linked to the DNA sequence to increase or decrease transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream or downstream of, or embedded within, the coding region of the DNA sequence. DNA of a signal sequence is operably linked to DNA encoding a polypeptide when the signal sequence is expressed as a preprotein involved in the secretion of the polypeptide. The joining of a DNA sequence to a regulatory sequence is typically accomplished using restriction endonucleases known to those of skill in the art by ligation at appropriate restriction sites, or via adapters or linkers inserted into the sequences.

[0099] As used herein, the terms "induce", "inducing" and their grammatical equivalents refer to an increase in the transcription, promoter activity and / or expression of a nucleic acid sequence effected by a transcriptional regulatory factor as compared to the basal level of transcription.

[0100] As used herein, the term "transcription regulatory factor" refers to a biochemical element (e.g., a repressor or nuclear inhibitory protein) that acts to prevent or inhibit transcription of a promoter-driven DNA sequence under a given environmental condition, or a biochemical element (e.g., an inducer or enhancer) that acts to permit or stimulate transcription of a promoter-driven DNA sequence under a given environmental condition.

[0101] As used herein, the term "enhancer" refers to, for example, a DNA sequence that increases transcription of a nucleic acid sequence to which it is operably linked. An enhancer can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A number of enhancers from a variety of different sources are well known in the art and are available as cloned polynucleotides or within them (e.g., from depository institutions such as ATCC and other commercial or individual sources). Some polynucleotides, including promoters (such as the commonly used CMV promoter), also contain enhancer sequences. An enhancer can be located upstream or downstream of, or within, 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 and μ intron enhancers, and the 3' enhancer (see generally Paul W.E. (ed.), Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Patent No. 5,885,827)).

[0102] As used herein, the term "promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. A promoter is located near the transcription start site of a gene and upstream (towards the 5' region of the sense strand) on the same strand of DNA. Some promoters are constitutive as they are active in all situations within a cell, while other promoters are regulated to become active in response to specific stimuli, such as inducible promoters. As used herein, the terms "promoter activity" and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operably linked to the promoter whose activity is being measured. Promoter activity can be measured directly, for example, by determining the amount of RNA transcripts produced by Northern blot analysis, or indirectly by determining the amount of a product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0103] An "inducible promoter" refers to a promoter whose activity is induced by the presence or absence of a transcriptional regulator (e.g., a biological or abiotic factor). Inducible promoters are useful because they can turn the expression of genes operably linked to them on or off at a given stage of an organism's development or within a specific tissue. Examples of inducible promoters include, but are not limited to, alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenicity-regulated promoters, temperature-regulated promoters, and light-regulated promoters. Inducible promoters can be part of a gene switch or a genetic switch.

[0104] As used herein, "T cell" or "T lymphocyte" refers to a type of lymphocyte that plays a central role in cellular immunity. These can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their cell surface.

[0105] As used herein, the term "functional fragment", when used with respect to a polypeptide, refers to a fragment of the polypeptide that has the major function of the polypeptide being referred to. For example, a functional fragment of a polypeptide that functions as a transmembrane domain is a fragment of that polypeptide that also functions as a transmembrane domain. In certain embodiments, a functional fragment of a polypeptide is at the N-terminus and / or C-terminus, up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than the polypeptide being referred to. When used with respect to a nucleic acid, the phrase "functional fragment" refers to a fragment of the nucleic acid being referred to that encodes a polypeptide having the same primary function as the polypeptide encoded by the nucleic acid being referred to.

[0106] As used herein, the phrase "functional variant", when used with respect to a polypeptide, refers to a polypeptide that is different from the polypeptide being referred to but has the major function of the polypeptide being referred to. For example, a functional variant of a polypeptide that functions as a transmembrane domain is a fragment of that polypeptide that also functions as a transmembrane domain. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence being referred to and / or is a conservative substitution variant of the sequence being referred to. When used with respect to a nucleic acid, the phrase "functional variant" refers to a nucleic acid that is different from the nucleic acid being referred to but encodes a polypeptide having the same primary function as the polypeptide encoded by the nucleic acid being referred to. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence being referred to, hybridizes with the complement of the nucleic acid sequence under stringent hybridization conditions, or is a codon degeneracy variant of the nucleic acid sequence.

[0107] As used herein, the term "antibody" is also known as an immunoglobulin (Ig) and can refer to a monoclonal antibody or a polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells that binds to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells that bind to different epitopes of the same antigen. The antibody can be of any animal origin. The antibody can be IgG (including IgG1, 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. An antibody typically consists of four polypeptides, namely two identical copies of the heavy (H) chain polypeptide and two identical copies of the light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (V H ) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each of the light chains contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The V H region and the V LThe regions have a similar general structure, each region contains four framework regions, and their arrangement is relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody responsible for antigen binding. These specific regions are described in 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), where these definitions include overlaps or subsets of amino acid residues when compared to each other. Preferably, the term "CDR" is the CDR as defined by Kabat based on sequence comparison. CDRH1, CDRH2, and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light chain CDRs.

[0108] The terms "antibody fragment", "antibody fragment", "fragment of an antibody", "antigen - binding portion" and their grammatically equivalent terms are used interchangeably herein to mean one or more fragments or portions of an antibody 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 include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Examples of antibody fragments include (1) a Fab fragment which is a monovalent fragment consisting of a V L 、V H 、C L 、and CH1 domains; (2) an F(ab’)2 fragment which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the stalk region; (3) the V L domain of a single arm of the antibody and V HAn Fv fragment consisting of domains; (4) A single-chain Fv (scFv) which is a monovalent molecule consisting of two domains of the Fv fragment (i.e., V L and V H ) linked by a linker that enables synthesis as a single polypeptide chain (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) A diabody which is a dimer of polypeptide chains, each polypeptide chain containing V H and V L connected to V L by a peptide linker that is too short to allow pairing between V H thereby promoting pairing between complementary domains on different V H -V L polypeptide chains to generate a dimer molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in detail, for example, in U.S. Patent No. 8,603,950.

[0109] The terms "antigen recognition portion", "antigen recognition domain", "antigen binding domain", and "antigen binding region" refer to a molecule or a part of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition portion is an antibody, an antibody-like molecule, or a fragment thereof.

[0110] The term "proliferative disease" refers to a unified concept that excessive cell proliferation and / or turnover of the cell matrix significantly contribute to the development of diseases including cancer. In some embodiments, the proliferative disease is cancer.

[0111] As used herein, the term "patient" or "subject" refers to a mammalian subject that has been diagnosed with or is suspected of having a proliferative disease such as cancer, or has developed such a proliferative disease. In some embodiments, the term "patient" refers to a mammalian subject that has a higher than average likelihood of developing a proliferative disease 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 treatments disclosed herein. Exemplary human patients can be male and / or female. The term "patient in need thereof" or "subject in need thereof" means, for example, a patient who has been diagnosed with or is suspected of having a disease or disorder (but not limited to cancer).

[0112] As used herein, "administering" refers to providing one or more of the compositions described herein to a patient or subject. By way of example and not limitation, administration of a composition, such as an injection, can be carried out by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be used. Parenteral administration can be effected, for example, by bolus injection or by infusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be effected by surgical deposition of a bolus or pellet of cells, or by positioning of a medical device.

[0113] As used herein, the terms "treatment", "treating", and their grammatically equivalent terms 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 the adverse symptoms caused by the disease. In some embodiments, the term "treating" can include "preventing" a disease or condition.

[0114] As used herein, "treatment interval" refers to a treatment cycle, e.g., the administration process of a therapeutic agent that can be repeated on a regular schedule. In some embodiments, the dosing schedule can have one or more periods during which no therapeutic agent is administered during the treatment interval.

[0115] As used herein, the terms "co-administered", "simultaneously administered", "administered simultaneously", and "provided simultaneously" mean that two (or more) different therapeutic agents are delivered to a subject while the subject is suffering from a disease, e.g., the two or more therapeutic agents are delivered after the subject has been diagnosed with the disease and before the disease has been cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, delivery of the first therapeutic agent is still occurring when delivery of the second therapeutic agent begins, such that there is overlap with respect to administration. This may be referred to herein as "simultaneous" or "co-delivery". In other embodiments, delivery of one therapeutic agent ends before delivery of the other therapeutic agent begins. In some embodiments of either case, the treatment is more effective for combination administration. For example, the second therapeutic agent is more effective, e.g., when fewer of the second therapeutic agent are administered, or when the symptoms are reduced more significantly than when the second therapeutic agent is administered without the first therapeutic agent, or when a similar situation as with the first therapeutic agent is seen, an equivalent effect is observed. In some embodiments, the delivery is such that reduction of other parameters related to the symptom or disorder is greater than when one therapeutic agent is delivered in the absence of the other. The effects of the two therapeutic agents may be partially additive, fully additive, or supra-additive. The delivery can be such that the effect of the first therapeutic agent delivered is still detectable when the second therapeutic agent is delivered.

[0116] In some embodiments, the first therapeutic agent and the second therapeutic agent can be administered simultaneously (e.g., at the same time), in the same composition or separate compositions, or sequentially. Sequential administration refers to administering the first therapeutic agent prior to administration of an additional therapeutic agent, e.g., the second therapeutic agent (e.g., immediately prior, less than 5, 10, 15, 30, 45, 60 minutes; more than 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, or 96 hours; more than 4, 5, 6, 7, 8, 9 days; more than 1, 2, 3, 4, 5, 6, 7, 8 weeks before). The order of administration of the first therapeutic agent and the second therapeutic agent can also be reversed.

[0117] The terms “therapeutically effective amount,” “therapeutic amount,” “immunologically effective amount,” “anti-tumor effective amount,” “tumor-inhibiting effective amount,” and their grammatically equivalent terms refer to an amount effective over a dosage and period necessary to achieve a desired therapeutic result. The therapeutically effective amount can vary depending on factors such as the individual's disease state, age, gender, weight, and the ability of the compositions described herein to elicit a desired response in one or more subjects. The exact dosage of the compositions of the present disclosure can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0118] Alternatively, the pharmacological and / or physiological effects of administration of one or more of the compositions described herein to a patient or subject can be “preventive,” i.e., an effect that completely or partially prevents a disease or its symptoms. A “preventively effective amount” refers to an amount effective over a dosage and period necessary to achieve a desired preventive result (e.g., prevention of the onset of a disease).

[0119] As used herein, the term "immune checkpoint protein" refers to a molecule that transmits an inhibitory signal or a molecule that 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 and 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 T cell activation suppressor), IDO1 (indoleamine 2,3-dioxygenase), arginase I, TIGIT (T cell immunoglobulin and ITIM domain), CD70, CD115, etc. (see Nature Reviews Cancer, 12, p. 252-264, 2012 and Cancer Cell, 27, p. 450-461, 2015).

[0120] As used herein, a term used to identify a biological moiety may or may not contain a dash "-" within the term. The presence or absence of the dash does not change the intended meaning or identification of the biological moiety. For purposes of illustration only and not limited to these biological moieties, each of the following pairs of terms (shown with / without a dash) denote 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-beta / TGF beta, VEGF-1 / VEGF1, VEGF-R2 / VEGFR2.

[0121] II. miRNA As used herein, the terms "miR", "mir", and "miRNA" are used to refer to microRNA, a class of small non-coding RNA molecules that can affect the expression of a gene ("target gene") by regulating the translation of a transcribed messenger RNA (increasing or decreasing gene expression) and / or destabilizing such messenger RNA.

[0122] The term "primary miRNA", abbreviated as "pri-miRNA", refers to a miRNA that contains at least one RNA hairpin. The RNA hairpin is cleaved from the pri-miRNA in the cell nucleus to form one or more precursor miRNAs ("pre-miRNA"). This pre-miRNA is transported to the cytoplasm where the stem-loop structure is cleaved to generate a double-stranded miRNA, which comprises a miRNA-5p strand from the former 5' arm of the hairpin loop and a miRNA-3p strand from the former 3' arm of the hairpin loop. Next, an argonaute protein binds to the double-stranded miRNA and one of the strands (either the miRNA-5p sequence or the miRNA-3p sequence) is released. The remaining bound strand becomes the "guide strand", while the released strand is known as the "passenger strand" and is preferably degraded. Thereafter, the guide strand continues to interact with messenger RNA derived from the target gene, thus affecting translation.

[0123] Both the miRNA-5p strand sequence and the miRNA-3p strand sequence are referred to herein as the "mature miRNA" sequence. The remaining portions of the pri-miRNA or pre-miRNA (the 5' portion relative to the miRNA-5p sequence, the 3' portion relative to the miRNA-3p sequence, and the stem-loop sequence between the miRNA-5p and miRNA-3p sequences) are collectively referred to as the miRNA backbone sequence. As used herein, the term "5' backbone sequence" is used to refer to the backbone sequence that is 5' to the miRNA-5p sequence in the pri-miRNA or pre-miRNA. As used herein, the term "3' backbone sequence" is used to refer to the backbone sequence that is 3' to the miRNA-3p sequence in the pri-miRNA or pre-miRNA. The term "loop sequence" refers to the backbone sequence that is between the miRNA-5p sequence and the miRNA-3p sequence in the pri-miRNA or pre-miRNA.

[0124] Unless otherwise indicated, the term "miRNA" collectively refers to the mature form, primary form, and precursor form of a particular microRNA, as well as its functional fragments and variants.

[0125] The miRNA may not be naturally occurring. The terms "non-natural", "unnatural", "synthetic", and "artificial", as used herein to describe the miRNA, are used interchangeably and refer to miRNAs having sequences that do not occur naturally.

[0126] The present invention also relates, in part, to a ribonucleic acid comprising two non-natural pre-miRNA sequences, each pre-miRNA sequence comprising 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, such as 3, 4, 5, 6, 7, 8, 9, 10, or more such sequences. It is 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 or different regions of such gene.

[0127] In certain embodiments, each non-natural pre-miRNA sequence in the ribonucleic acid forms a non-complementary stem-loop secondary structure, which is different from the 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.

[0128] In certain embodiments, the secondary structure of each non-natural pre-miRNA is sufficiently similar to the secondary structure of a native pre-miRNA sequence so as to reduce or prevent RNAi-based anti-pathogen toxicity in a cell. 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 native pre-miRNA and / or can hybridize to the native pre-miRNA under stringent hybridization conditions.

[0129] 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 the native pri-miRNA sequence so as to reduce or prevent anti-pathogen toxicity based on RNAi in cells. In such certain 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 native pri-miRNA and / or can hybridize with the native pri-miRNA under stringent hybridization conditions.

[0130] The non-natural pre-miRNAs of the present invention can be generated from native pre-miRNAs by removing the native mature miRNA sequences and replacing them with non-natural mature miRNA sequences, where one of the sequences can function as a guide miRNA targeting a gene of interest.

[0131] In certain embodiments, each non-natural pre-miRNA comprises a backbone sequence derived from a native pre-miRNA, such as a native pre-miRNA present in mouse, rat, or human. In certain embodiments, the backbone sequences (3' backbone sequence, 5' backbone sequence, and loop sequence) of the non-natural pre-miRNA have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the corresponding backbone sequences of the native pre-miRNA and / or are capable of hybridizing to the corresponding backbone segments under stringent hybridization. In certain embodiments, the backbone segments of the non-natural pre-miRNA sequence are identical to the corresponding backbone segments of the native pre-miRNA. In certain embodiments, the native 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 native pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain specific embodiments, the native pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain specific embodiments, the native pre-miRNA is miR204 or miR206.

[0132] In certain embodiments, each non-natural pri-miRNA comprises a backbone sequence derived from a native pri-miRNA, such as a pri-miRNA present in a mouse, rat, or human. In certain embodiments, the backbone sequences (3' backbone sequence, 5' backbone sequence, and loop sequence) of the non-natural pri-miRNA have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the corresponding backbone sequences of the native pri-miRNA and / or can hybridize with the corresponding backbone segments under stringent hybridization conditions. In certain embodiments, the backbone segments of the non-natural pri-miRNA sequence are identical to the corresponding backbone segments of the native pri-miRNA. In certain embodiments, the native 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 native pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the native pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain embodiments, the native pre-miRNA is miR204 or miR206.

[0133] The miRNA-5p sequence and the miRNA-3p sequence hybridize to each other but are not necessarily exactly complementary. In the design of non-natural miRNAs, compensatory mutations can be introduced into the miRNA-5p and / or miRNA-3p sequences to maintain the RNA folding and free energy of the natural 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 to a sequence complementary to the sequence encoding the miRNA-5p sequence, or can hybridize to the sequence encoding the miRNA-5p sequence under stringent hybridization conditions.

[0134] In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, or at least about 250 nucleotides. In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by about 5 - 250 nucleotides, about 10 - 250 nucleotides, about 10 - 200 nucleotides, about 10 - 150 nucleotides, about 10 - 100 nucleotides, about 10 - 50 nucleotides, about 10 - 40 nucleotides, about 10 - 30 nucleotides, about 10 - 20 nucleotides, about 16 - 250 nucleotides, about 16 - 200 nucleotides, about 16 - 150 nucleotides, about 16 - 100 nucleotides, about 16 - 50 nucleotides, about 16 - 40 nucleotides, about 16 - 30 nucleotides, about 16 - 20 nucleotides, about 20 - 200 nucleotides, about 20 - 150 nucleotides, about 20 - 100 nucleotides, about 20 - 50 nucleotides, about 20 - 45 nucleotides, about 20 - 40 nucleotides, about 20 - 35 nucleotides, about 20 - 30 nucleotides, about 20 - 25 nucleotides, about 30 - 200 nucleotides, about 30 - 150 nucleotides, about 30 - 100 nucleotides, about 30 - 50 nucleotides, or about 30 - 40 nucleotides.In certain embodiments, the 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, 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, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides.

[0135] In certain embodiments, two non-natural pri-miRNA sequences are adjacent to each other with the 3’ nucleotide of one pri-miRNA directly linked 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.

[0136] In certain embodiments, the non-natural pre-miRNA contains a mature miRNA sequence that can bind to mRNA, thereby interfering with its translation and / or promoting its degradation. The mRNA can be generated from the expression of a target gene.

[0137] In certain embodiments, the target gene encodes an immune checkpoint protein. Thus, the pre-miRNA sequence inhibits the expression of the immune checkpoint protein by targeting the gene that expresses the immune checkpoint protein. In 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 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.

[0138] In certain embodiments, each non-natural pre-miRNA targets a different gene. In certain embodiments, they are in specific different regions of the same gene.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] The present invention also relates, in part, to deoxyribonucleic acids encoding any of the above ribonucleic acids.

[0196] 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, those listed in Table 1 below. The symbols "X" and "Y" in Table 1 indicate nucleic acid sequences encoding a guide miRNA (which may be either miRNA-5p or miRNA-3p) and a passenger miRNA (which may be either miRNA-5p or miRNA-3p), respectively, while the symbol "n" indicates the number of nucleotides in the sequence, e.g., 16-30, preferably 18-25. In some embodiments, n can 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 hybridizes under stringent hybridization conditions to a complement of any one of the sequences listed in Table 1.

Table 1

[0197] In any of the above embodiments, the sequence encoding the pre-miRNA comprises: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, respectively; SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively; SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively; SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO: 340, respectively; SEQ ID NO: 341, SEQ ID NO: 342, and SEQ ID NO: 343, respectively; or SEQ ID NO: 344, SEQ ID NO: 345, and SEQ ID NO: 346, respectively; Or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the above sequences, or a sequence capable of hybridizing with the complement of the sequence under stringent hybridization conditions.

[0198] Non-limiting examples of nucleic acid sequences encoding guide miRNA target genes encoding such checkpoint inhibitors are listed in Table 2. Table 2 also lists sequences encoding passenger strands. As described above, the guide strand and the passenger strand are not necessarily complementary. It is also conceivable that the passenger strand can act to target messenger RNA associated with the target gene. It is also conceivable that sequences that hybridize with the complement of the sequences listed in Table 2 under stringent hybridization conditions can also be used. The mature miRNA sequences used may bind to a specific pri-miRNA backbone. Table 2 also lists backbones that can be combined with the listed mature guide and passenger miRNAs.

Table 2

[0199] In certain embodiments, the invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 64-83, 85, 87-171, 293-322, and 704-713, or a deoxyribonucleic acid capable of hybridizing under stringent hybridization conditions with a complement of any one of SEQ ID NOs: 64-83, 85, 87-171, 293-322, and 704-713. In such a particular embodiment, 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 with any one of SEQ ID NOs: 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, or a deoxyribonucleic acid capable of hybridizing under stringent hybridization conditions with a complement of any one of SEQ ID NOs: 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.

[0200] In certain embodiments, the sequence encoding the guide miRNA sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 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, or can hybridize with a complement of any one of the sequences under stringent hybridization conditions.

[0201] In certain embodiments, the sequence encoding the passenger miRNA sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 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, or can hybridize with a complement of any one of the sequences under stringent hybridization conditions.

[0202] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets CTLA. In such certain 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: 65 - 71, or a polynucleotide capable of hybridizing under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 65 - 71. In certain 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: 64, 66, 68, and 70, or a deoxyribonucleic acid capable of hybridizing under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 64, 66, 68, and 70.

[0203] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PD-1. In such certain 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: 72 - 83, 85, 87, and 704 - 713, or a polynucleotide capable of hybridizing under stringent hybridization conditions to a complementary sequence of any one of SEQ ID NOs: 72 - 83, 85, 87, and 704 - 713. In certain 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: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710, or a deoxyribonucleic acid capable of hybridizing under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710.

[0204] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets TIGIT. In such certain 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: 88 to 145, or a polynucleotide capable of hybridizing to a complement of any one of SEQ ID NOs: 88 to 145 under stringent hybridization conditions. In certain 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: 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 a deoxyribonucleic acid capable of hybridizing to a complement of any one of SEQ ID NOs: 64, 66, 68, and 70, SEQ ID NOs: 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 under stringent hybridization conditions.

[0205] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets TIM3. In such certain 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 a polynucleotide capable of hybridizing to a complement 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 a deoxyribonucleic acid capable of hybridizing to a complement of any one of SEQ ID NOs: 146, 148, 150, 152, 154, and 156 under stringent hybridization conditions.

[0206] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets LAG3. In such certain 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 a polynucleotide capable of hybridizing to a complement 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 a deoxyribonucleic acid capable of hybridizing to a complement of any one of SEQ ID NOs: 158 and 160 under stringent hybridization conditions.

[0207] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets GITR. In such certain 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 a polynucleotide capable of hybridizing to a complement 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 a deoxyribonucleic acid capable of hybridizing to a complement of any one of SEQ ID NOs: 162 and 164 under stringent hybridization conditions.

[0208] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PIK3IP1. In such certain 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 a polynucleotide capable of hybridizing to a complement 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 a deoxyribonucleic acid capable of hybridizing to a complement of any one of SEQ ID NOs: 166, 168, and 170 under stringent hybridization conditions.

[0209] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets CD70. In such certain 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 with any one of SEQ ID NOs: 293 to 322, or a polynucleotide capable of hybridizing with a complement of any one of SEQ ID NOs: 293 to 322 under stringent hybridization conditions. In certain 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 with any one of SEQ ID NOs: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, and 321, or a deoxyribonucleic acid capable of hybridizing with a complement 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.

[0210] In certain embodiments, the invention relates to a deoxyribonucleic acid in which each sequence encoding a pre-miRNA comprises the following: a) a sequence encoding a 5'miRNA backbone sequence; b) a sequence encoding a guide miRNA sequence; c) a sequence encoding a stem-loop sequence; d) a sequence encoding a passenger miRNA sequence; and e) a sequence encoding a 3' backbone sequence.

[0211] In certain embodiments, the sequence encoding a pre-miRNA comprises the following: a) A guide miRNA sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 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, 705, 709, and 710, or one that can hybridize with a complement of any one of these sequences under stringent hybridization conditions; and b) A passenger sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 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, or one that can hybridize with a complement of any one of these sequences under stringent hybridization conditions.

[0212] Table 3 lists deoxyribonucleic acids encoding exemplary unnatural pre-miRNA sequences that target specific checkpoint inhibitors. In certain embodiments, the deoxyribonucleic acid can include a sequence that can hybridize with a complement of any one of the sequences listed in Table 3 under stringent hybridization conditions.

Table 3

[0213] In certain embodiments, the invention relates to deoxyribonucleic acids 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 under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 347-447.

[0214] In certain embodiments, the invention relates to deoxyribonucleic acids 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 under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 178-263 and 323-337.

[0215] In certain embodiments, the miRNA targets CTLA4. In such certain embodiments, the invention relates to deoxyribonucleic acids 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 a complement of any one of SEQ ID NOs: 347, 419, 420, and 421. In such certain embodiments, the invention relates to deoxyribonucleic acids 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 a complement of any one of SEQ ID NOs: 178 and 250-252.

[0216] In certain embodiments, the pre-miRNA targets PD-1. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 348, 349, and 410-418 under stringent hybridization conditions. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 179, 180, and 241-249 under stringent hybridization conditions. In certain embodiments, the present invention relates to deoxyribonucleic acids 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 or 349, or those capable of hybridizing to a complement of any one of SEQ ID NOs: 348 or 349 under stringent hybridization conditions. In certain embodiments, the present invention relates to deoxyribonucleic acids 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 or 180, or those capable of hybridizing to a complement of any one of SEQ ID NOs: 179 or 180 under stringent hybridization conditions.

[0217] In certain embodiments, the pre-miRNA targets TIGIT. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 350-377 and 404-409. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 181-208 and 235-240.

[0218] In certain embodiments, the pre-miRNA targets TIM3. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 378-389. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 209-220.

[0219] In certain embodiments, the pre-miRNA targets LAG3. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 390-396 under stringent hybridization conditions. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 221-227 under stringent hybridization conditions.

[0220] In certain embodiments, the pre-miRNA targets GITR. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 397-403 under stringent hybridization conditions. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 those capable of hybridizing to a complement of any one of SEQ ID NOs: 228-234 under stringent hybridization conditions.

[0221] In certain embodiments, the pre-miRNA targets PIK3IP1. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 422-424 under stringent hybridization conditions. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 253-255 under stringent hybridization conditions.

[0222] In certain embodiments, the pre-miRNA targets CD70. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 433-447 under stringent hybridization conditions. In such certain embodiments, the present invention relates to deoxyribonucleic acids 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 to a complement of any one of SEQ ID NOs: 323-337 under stringent hybridization conditions.

[0223] In embodiments 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 various guide miRNAs may target different genes. Further, the design of each pre-miRNA, or the pri-miRNA containing them, may be based on different natural miRNA backbones in order to reduce the possibility of misfolding between one miRNA and the other. Table 4 provides examples of deoxyribonucleic acid sequences encoding two or more pri-miRNAs.

Table 4

[0224] In such specific embodiments, the present invention relates to deoxyribonucleic acid containing a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 267 to 290 and 448 to 460, or capable of hybridizing with a complement of any one of SEQ ID NOs: 267 to 290 and 448 to 460 under stringent hybridization conditions.

[0225] In such specific embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1. In a specific embodiment, the present invention relates to deoxyribonucleic acid containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 267 and 282, or capable of hybridizing with a complement of any one of SEQ ID NOs: 267 and 282 under stringent hybridization conditions.

[0226] In such a specific embodiment, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting TIGIT. In a specific embodiment, 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 with any one of SEQ ID NOs: 269-274, 287, 288, and 290, or capable of hybridizing with a complement of any one of SEQ ID NOs: 269-274, 287, 288, and 290 under stringent hybridization conditions.

[0227] In such a specific embodiment, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1 and one pre-miRNA targeting TIGIT. In a specific embodiment, 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 with any one of SEQ ID NOs: 275-280, or capable of hybridizing with a complement of any one of SEQ ID NOs: 275-280 under stringent hybridization conditions.

[0228] In such a specific embodiment, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting CTLA4. In a specific embodiment, 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 with any one of SEQ ID NOs: 281, 283, and 284, or capable of hybridizing with a complement of any one of SEQ ID NOs: 281, 283, and 284 under stringent hybridization conditions.

[0229] In such a specific embodiment, the deoxyribonucleic acid encodes a pre-miRNA targeting TIGIT and a pre-miRNA targeting CTLA4. 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: 285, 286, and 289, or capable of hybridizing under stringent hybridization conditions to a complement of any one of SEQ ID NOs: 285, 286, and 289.

[0230] In such a specific embodiment, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD1 and one pre-miRNA 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 SEQ ID NO: 448 or 451, or capable of hybridizing under stringent hybridization conditions to a complement of SEQ ID NO: 448 or 451.

[0231] In such a specific embodiment, the deoxyribonucleic acid encodes a pre-miRNA targeting PD1 and two pre-miRNAs 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 SEQ ID NO: 449, or capable of hybridizing under stringent hybridization conditions to a complement of SEQ ID NO: 449.

[0232] In such a specific embodiment, 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 with SEQ ID NO: 450, or capable of hybridizing with a complement of SEQ ID NO: 450 under stringent hybridization conditions.

[0233] In such a specific embodiment, the deoxyribonucleic acid encodes two pre-miRNAs each of which constitutes 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 with any one of SEQ ID NOs: 452 - 460, or capable of hybridizing with a complement of any one of SEQ ID NOs: 452 - 460 under stringent hybridization conditions.

[0234] III. Protein of Interest In certain embodiments, the deoxyribonucleic acid encoding the pre-miRNA is included in the same gene construct as one or more genes encoding a protein of interest (e.g., a chimeric antigen receptor, a cytokine, a cell tag, or a checkpoint inhibitor).

[0235] A. Chimeric Receptor In any of the above embodiments, the deoxyribonucleic acid of the present disclosure can further encode a chimeric receptor such as a chimeric antigen receptor (CAR) or a chimeric T cell receptor (TCR). Thus, the deoxyribonucleic acid of the present disclosure can encode an miRNA and a chimeric receptor such as a CAR or a TCR.

[0236] In certain embodiments, the deoxyribonucleic acid of the present invention encodes a CAR, which is introduced into T cells, thereby generating chimeric antigen receptor T cells (CAR-T cells).

[0237] A CAR is an engineered receptor that confers exogenous specificity to immune effector cells. In some examples, a CAR comprises an extracellular domain (ectodomain) that includes an antigen-binding domain, a transmembrane domain, and an intracellular (endodomain) domain. The intracellular domain includes an intracellular signaling domain. In certain embodiments, the extracellular domain further includes a region of amino acids (i.e., a spacer) between the antigen-binding domain and the transmembrane domain.

[0238] The antigen-binding domain can include the complementarity-determining regions of a monoclonal antibody and / or its antigen-binding fragment. Complementarity-determining regions (CDRs) are short amino acid sequences found in the variable domains of antigen receptor (such as immunoglobulins and T cell receptors) proteins that bind to an antigen, thus conferring specificity for that particular antigen to the receptor. Each polypeptide chain of an antigen receptor can include three CDRs (CDR1, CDR2, and CDR3).

[0239] In certain embodiments, the antigen-binding domain includes an antibody that binds to a target antigen, or a functional fragment or variant thereof. The functional fragment or variant can include the variable domain of the heavy chain (VH) of the antibody and / or the variable domain of the light chain (VL) of the antibody, or a functional fragment or variant thereof. In certain embodiments, the antigen-binding domain includes an Fv, Fab, Fab2, Fab’, F(ab’)2, or F(ab’)3 fragment of an antibody. In certain embodiments, the antigen-binding domain includes a scFv, sc(Fv)2, dsFv, diabody, minibody, nanobody, or a binding fragment thereof. In certain embodiments, the antigen-binding domain further includes an Fc fragment of an antibody, for example, this can include an scFv linked to the Fc fragment.

[0240] In some embodiments, the CAR targets an antigen that is overexpressed in cancer cells, autoimmune cells, or cells infected with a virus, bacterium, or parasite. 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 the Ebola hemorrhagic fever pathogen. 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, autoimmune uveitis, and the like.

[0241] The pathogen recognized by the CAR can be essentially any type of pathogen, but in some embodiments, the pathogen is a fungus, bacterium, or virus. Exemplary viral pathogens include those of the Adenoviridae family, Epstein - Barr virus (EBV), Cytomegalovirus (CMV), Respiratory syncytial virus (RSV), JC virus, BK virus, HPV, HSV, viruses of the HHV family, viruses of the hepatitis family, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, Ebola hemorrhagic fever, rubella, etc. Exemplary pathogenic fungi include the genera Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include the genera Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochaeta, and Salmonella. In some embodiments, a CAR can be generated using the pathogen receptor dectin - 1 to recognize carbohydrate structures on the cell wall of fungi such as Aspergillus. In another embodiment, the CAR can be generated based on an antibody that recognizes viral determinants (e.g., glycoproteins from CMV and Ebola) to block viral infection and disease symptoms.

[0242] In certain embodiments, the CAR comprises an antigen - binding domain that binds to an antigen overexpressed in cancer.

[0243] 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, CD171, 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 alpha, alpha-folate receptor, Frizzled receptor, GD2, GD3, GHR, GHRHR, GITR, GPC3, Gp100, gp130, HBV antigen, HER1, HER2, HER3, HER4, HER1 / HER3, h5T4, HPV antigen, HVEM, IGF1R, Ig kappa, IL-1-RAP, IL-2R, IL6R, IL-11R alpha, IL-13R-a2, KDR, KRASG12V, Lewis A, Lewis Y, L1-CAM, LIFRP, LRP5, LTPR, MAGE-A, MAGE-A1, MAGE-A10, MAGE-A3, MAGEA3 / A6, MAGE-A4, MAGE-A6, MART-1, MCAM, mesothelin, PSCA, MUC1, mucin such as MUC-4 or MUC16, NGFR, NKG2D, Notch-1-4, NY-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, TGF beta, TGF beta-II, TGFBR1, TGFBR2, tyrosin, TLR7, TLR9, TNFR1, TNFR2, TNFRSF4, TRBC1, TWEAK-R, VEGF, VEGF-R2, or WT-1.

[0244] In some embodiments, the CARs described herein include antigen-binding domains that bind to epitopes on CD19, CD33, MUC1, MUC16, ROR1, HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, and / or type II collagen.

[0245] In some embodiments, the CARs described herein include antigen-binding domains that bind to epitopes on CD19, CD33, MUC1, MUC16, and / or ROR1.

[0246] In some embodiments, the CAR includes an antigen-binding domain that binds to an epitope on CD19. Examples of CARs that bind to epitopes on CD19 are known to those skilled in the art and are described, for example, in International Publication No. WO 2016 / 033570; International Publication No. WO 2015 / 123642; and International Publication No. WO 2015 / 187528.

[0247] In some embodiments, the CAR includes an antigen-binding domain that binds to an epitope on CD33. Examples of CARs that bind to epitopes on CD33 are known to those skilled in the art and are described, for example, in International Application Publication No. WO 2017 / 214333.

[0248] In some embodiments, the CAR includes an antigen-binding domain that binds to an epitope on MUC1. Examples of CARs that bind to epitopes on MUC1 are known to those skilled in the art and are described.

[0249] In some embodiments, the CAR includes an antigen-binding domain that binds to an epitope on MUC16. Examples of CARs that bind to epitopes on MUC16 are known to those skilled in the art and are described, for example, in International Application Publication No. WO 2019 / 236577.

[0250] In some embodiments, the CAR comprises an antigen-binding domain that binds to an epitope on ROR1. Examples of CARs that bind to epitopes on ROR1 are known to those skilled in the art and are described, for example, in International Application Publication No. WO 2020 / 014366.

[0251] Antigen binding can be evaluated by flow cytometry, cell-based assays, or other equivalent assays. In cell-based assays, cell types expressing the antigen of interest on their surface can be utilized to evaluate antigen binding. Antigen binding can be evaluated using flow cytometry or similar assays with an antigen expressed as a soluble protein or a fragment thereof. Improvement in antigen binding can be indirectly evaluated by measuring the functional activity of the antigen-binding domain or chimeric receptor. For example, improvement in antigen binding of the chimeric receptors or CARs described herein can be measured by an increase in specific cytotoxicity against target cells expressing the antigen.

[0252] The cell surface expression level of the polypeptides of the present disclosure can be evaluated, for example, using an assay based on flow cytometry. Improvement in the expression of the antigen-binding polypeptide can be measured as the percentage of cells expressing the antigen-binding polypeptide among the cells analyzed, or alternatively, as the average density of the antigen-binding polypeptide on the cell surface. Additional suitable methods that can be used to evaluate the cell surface expression of the antigen-binding polypeptides described herein include Western blotting and other equivalent assays.

[0253] B. Cytokine In any of the above embodiments, the deoxyribonucleic acid of the present disclosure can further encode a cytokine. Thus, the deoxyribonucleic acid of the present disclosure can encode miRNA and a cytokine.

[0254] In certain embodiments, the deoxyribonucleic acid also encodes a CAR and is introduced into T cells, thereby generating CAR-T cells. In other specific embodiments, the deoxyribonucleic acid of the present invention encoding miRNA and cytokine is introduced into CAR-T cells.

[0255] In some cases, the cytokine includes at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variants or combinations thereof. In certain embodiments, the cytokine is interferon, GM-CSF, G-CSF, M-CSF, LT-β, TNF-α, growth factor, hGH, and / or a ligand of human toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IFN-α, IFN-β, or IFN-γ.

[0256] In certain embodiments, the cytokine is 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.

[0257] In certain embodiments, the cytokine may be IL-12, or a functional fragment or variant thereof. In some embodiments, IL-12 is single-chain IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or inserted IL-12. In some examples, the IL-12 variant is as described in International Application Publication No. WO 2015 / 095249, International Application Publication No. WO 2016 / 048903, and International Application Publication No. WO 2017 / 062953.

[0258] 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 can occur when IL-15, or a functional fragment or variant thereof, binds to membrane-bound IL-15Rα, or a functional fragment or variant thereof. Accordingly, certain embodiments of the present invention can include a fusion protein comprising IL-15 or a functional fragment or variant thereof and IL-15Rα or a functional fragment or functional variant thereof. In certain embodiments, IL-15, or a functional fragment or variant thereof, is linked via a linker to IL-15Rα, or a functional fragment thereof. Examples of fusion proteins comprising IL-15, or a functional fragment or variant thereof, bound to membrane-bound IL-15Rα, or a functional fragment or variant thereof, are known to those of skill in the art and are described, for example, in WO 2014 / 186469.

[0259] In certain embodiments, the cytokine is bound to a signal peptide. Any signal for use in eukaryotic cells, including the signals described above for use in CARs, can be attached to the cytokine. In certain embodiments, the cytokine is bound to an IgE signal peptide.

[0260] C. Cell Tags In any of the above embodiments, the deoxyribonucleic acid of the present disclosure can further encode a cell tag. Accordingly, the deoxyribonucleic acid of the present disclosure can encode a miRNA and a cell tag.

[0261] In certain embodiments, the deoxyribonucleic acid also encodes a CAR and is introduced into T cells, thereby generating CAR-T cells. In certain other embodiments, the deoxyribonucleic acid of the present invention encoding miRNA and cell tags is introduced into CAR-T cells. In certain 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.

[0262] In certain embodiments, the cell tag is used as a kill switch, a selection marker, a biomarker, or a combination thereof.

[0263] In certain embodiments, the cell tag is bindable by a predetermined binding partner. In certain embodiments where the deoxyribonucleic acid encoding the cell tag is introduced into a cell and the cell is introduced into a subject, depletion of the cell can be achieved by administering the predetermined binding partner to the subject. For example, administration of cetuximab or any antibody that recognizes HER1 can eliminate cells expressing a cell tag comprising a truncated non-immunogenic HER1.

[0264] In such certain embodiments, the cell tag is non-immunogenic. This can be achieved, for example, when the cell tag comprises a polypeptide that is truncated to be non-immunogenic. For example, truncation of the HER1 sequence eliminates the possibility of EGF ligand binding, EGFR homo- and hetero-dimerization, and / or EGFR-mediated signal transduction while maintaining cetuximab binding ability (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Vol. 37, pp. 353-373).

[0265] 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.

[0266] 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, for example, in International Application Publication No. 2014 / 186469.

[0267] In certain embodiments, the cell tag comprises a truncated non-immunogenic CD20, or CD20t-1, or a functional fragment or variant thereof.

[0268] In certain embodiments, the cell tag further comprises a transmembrane domain. The transmembrane domain can be derived from either a natural or synthetic source. When the source is natural, the domain can be derived from, for example, any membrane-bound or transmembrane protein. Suitable transmembrane domains include the transmembrane domains of the α, β, or ζ chains of the T cell receptor; or transmembrane domains derived from CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, or functional fragments or variants thereof. In certain embodiments, the cell tag further comprises the CD28 transmembrane domain or a functional fragment or variant thereof. Alternatively, the transmembrane domain can be synthetic and can contain hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at one or both ends of the synthetic transmembrane domain.

[0269] 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.

[0270] In certain embodiments, the cell tag is linked to a signal peptide. The signal peptide can be any signal peptide suitable for use in eukaryotic cells, including those described herein with respect to the CAR. In certain embodiments, the signal peptide is an Igκ signal peptide, or a functional fragment or variant thereof.

[0271] D. Immune checkpoint inhibitors In any of the above embodiments, the deoxyribonucleic acid of the present disclosure can further encode an immune checkpoint inhibitor. Thus, the deoxyribonucleic acid of the present disclosure can encode an miRNA and an immune checkpoint inhibitor. Therefore, the use of such deoxyribonucleic acids provides two mechanisms of action for reducing the activity of immune checkpoints.

[0272] 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.

[0273] In certain embodiments, the immune checkpoint inhibitor is an antibody or a functional fragment or variant thereof.

[0274] In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody such as semiprimab, pembrolizumab, nivolumab, toripalimab, sintilimab, LY3434172, JTX-4014, 609A, Sym021, LZM009, budigalimab, IB, SCT-I10A, SG001, AMP-224, AMG404, AK112, CS1003, MEDI0680, RO7121661, F520, sasanalimab, BI754091, cetrelimab, helin CAR-PD-1, HX008, ginberelimab, retifanlimab, balstilimab, pidilizumab, teriparlimab, CBT-501, BAT1306, tislelizumab, AK105, spartalizumab, prorgolimab, serplulimab, dostarlimab, camrelizumab, IBI319, KY1043, STI-1110, CA05100948, Nb97, ENUM388D4, hAb-10D3, ANB030, MCLA-134, and hAb21; an anti-CTLA-4 antibody such as ipilimumab (YERVOY) and tremelimumab; an anti-PD-L1 antibody such as BMS935559 (MDX-1105), atezolizumab, avelumab, or durvalumab; an anti-CD28 antibody; an anti-TIGIT antibody; an anti-LAG3 antibody such as BMS-986016 and LAG525; an anti-TIM3 antibody; an anti-GITR antibody; an anti-4-1BB antibody such as PF-05082566; or an anti-OX-40 antibody such as MEDI6469, MEDI0562, MOXR0916.

[0275] IV. Gene construct As described above, in certain embodiments, the deoxyribonucleic acid encoding the pre-miRNA is contained in the same gene construct as one or more genes encoding a protein of interest (e.g., a chimeric antigen receptor, a cytokine, or a cell tag).

[0276] In such specific embodiments, the gene construct comprises a nucleic acid sequence encoding a 5' untranslated region (5'UTR) directly upstream of the gene encoding the protein of interest, and the pre-miRNA sequence is contained in the 5'UTR. In certain embodiments, the gene construct comprises a nucleic acid sequence encoding a 3' untranslated region (3'UTR) directly downstream of the gene encoding the protein of interest, and the pre-miRNA sequence is contained in the 3'UTR. In certain embodiments, the gene construct comprises nucleic acid sequences encoding both the 5'UTR and the 3'UTR, and each such region contains at least one pre-miRNA sequence (e.g., each UTR can contain one pre-miRNA sequence, the 5'UTR can contain one pre-miRNA, the 3'UTR can contain two pre-miRNAs, the 5'UTR can contain two pre-miRNAs, the 3'UTR can contain one pre-miRNA, both UTRs can contain two pre-miRNAs, etc.).

[0277] In embodiments where the sequence encoding pri-miRNA is contained in the sequence corresponding to the 5'UTR, the transcribed RNA can contain additional sequences such as splice donor sequences, branch point sequences, and / or acceptor site sequences. Inclusion of splice donors, branch points, and acceptor sites is important for splicing miRNA from the transcribed RNA. Without splicing, the highly structured miRNA sequence is likely to interfere with ribosome scanning to the translation initiation sequence associated with the gene of interest. Examples of sequences encoding such splice 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 to such sequences, and sequences capable of hybridizing to the complement of such sequences under stringent hybridization conditions.

[0278] Thus, in certain embodiments, the deoxyribonucleic acid of the present invention further comprises: a) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 291, or a nucleic acid sequence capable of hybridizing with the complement of SEQ ID NO: 291 under stringent hybridization conditions; and b) a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 292, or a nucleic acid sequence capable of hybridizing with the complement of SEQ ID NO: 292 under stringent hybridization conditions.

[0279] In certain embodiments, the portion of the deoxyribonucleic acid encoding pre-miRNA is included in a section corresponding to an intron within a gene encoding a protein of interest. In such certain embodiments, at least one pre-miRNA is included in an intron located within the 5’UTR (hereinafter referred to as “5’UTR intron”).

[0280] Exemplary polynucleotides that can be used as templates for the expression of various genes and other regulatory elements in cells are shown in FIG. 16. It should be understood that various elements can be included in or omitted from the polynucleotide, and different options are shown for various exemplary sites within the polynucleotide.

[0281] As shown in FIG. 16, the polynucleotide can include an integration signal for attP / attB phage integration of the polynucleotide into the bacterial genome. The polynucleotide can further include a 5' homology arm or 5' terminal repeat, and a 3' homology arm or 3' terminal repeat. The polynucleotide can further include insulators, boundary elements, and S / MARs located on the 3' side of the 5' homology arm or 5' terminal repeat, and on the 5' side of the 3' homology arm or 3' terminal repeat. Between the insulator, boundary element, or S / MAR, the polynucleotide can include, from 5' to 3', a promoter that can include a silencer, enhancer, TF binding module, and a core promoter; a 5' untranslated region that can include intron-embedded elements such as a stability module, translation control element, miRNA coding sequence, etc.; one or more genes that can include a signal peptide, extracellular domain, transmembrane domain, signaling domain, antibody domain, peptide linker, intein, and epitope tag; and a 3' untranslated region that can include a stability module, translation control, 3' end processing signal, and transcription terminator.

[0282] As described above, the miRNA may be encoded within the same gene construct along with additional proteins of interest (e.g., CAR, cytokine, and / or cell tag). The advantage of expressing two or more of the components using one gene construct is the stoichiometric expression of the components.

[0283] As will be understood by those skilled in the art, genes encoding the polypeptide of interest can be linked via a linker. Any suitable linker known for linking genes can be used in the practice of the present invention, and examples of such linkers include internal ribosome entry sites (IRES), cleavable peptides, and linkers encoding ribosome skipping peptides. Examples of cleavable peptides encoded by such linkers include furin linkers, fmdv, and 2A linkers (e.g., P2A, GSG-P2A, FP2A, T2A, and furin-T2A), or functional fragments or variants thereof.

[0284] The polynucleotides of the present invention can be present in a construct operably linked to a promoter. Based on the host cell and the desired effect, an appropriate promoter can be selected. Suitable promoters include constitutive promoters and inducible promoters. The promoter can be tissue-specific, and such promoters are well known in the art.

[0285] Examples of constitutive promoters for use in the present invention include, but are not limited to: the immediate early cytomegalovirus (CMV) promoter; human elongation growth factor 1α1 (hEF1A1); simian virus 40 (SV40) early promoter; mouse mammary tumor virus (MMTV); human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter; MoMuLV promoter; avian leukosis virus promoter; Epstein-Barr virus early promoter; Rous sarcoma virus promoter; and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, etc. (however, not limited thereto). As well as functional fragments and variants thereof.

[0286] In contrast to constitutive promoters, the use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked when expression is desired and turn off the expression when 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 two-polypeptide ecdysone receptor-based gene switch such as the RHEOSWITCH® gene switch.

[0287] The present invention also relates, in part, to deoxyribonucleic acid comprising the above-described gene construct.

[0288] V. Vectors and Delivery Systems The ribonucleic acid and / or deoxyribonucleic acid of the present invention can be delivered to cells on long oligonucleotides and then inserted into specific genomic locations. In certain embodiments, the ribonucleic acid and / or deoxyribonucleic acid of the present invention can be integrated into the genome of a cell through a gene editing system that utilizes CRISPR, TALEN, or zinc finger nucleases.

[0289] 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. Accordingly, the present invention also relates, in part, to vectors comprising the ribonucleic acid or deoxyribonucleic acid of the present invention.

[0290] Vectors known in the art for use in delivering ribonucleic acid or deoxyribonucleic acid can be used in the practice of the present invention. In certain embodiments, the vector is a plasmid, minicircle DNA, nanoplasmid, viral vector, episomal vector, or non-viral vector. Examples of viral vectors used in the present invention include lentiviral vectors, retroviral vectors, and the like. Examples of non-viral vectors used in the present invention include the Sleeping Beauty transposon. In certain embodiments, the vector can include sequences for serine recombinase-mediated integration (e.g., those for the aatP or attB sites). When the vector is a plasmid, minicircle DNA, or nanoplasmid, the plasmid, minicircle DNA, or nanoplasmid can further include a bacterial origin of replication, such as an origin of replication derived from the ColE1 plasmid.

[0291] An example of a non-viral vector used for delivering 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, the nucleic acid can associate with the lipid. For example, the nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule that associates with both the liposome and an oligonucleotide, trapped within a liposome, form a complex with a liposome, be dispersed in a solution containing the lipid, be mixed with the lipid, be bound to the lipid, be included as a suspension in the lipid, be included with or form a complex with a micelle, or otherwise associate with the lipid.

[0292] 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, typically, the transposase or the nucleic acid encoding it is also delivered to the cell. A transposase is an enzyme that binds to the transposon and catalyzes its integration into the genome of the cell. In certain embodiments, the vector is a Sleeping Beauty transposon. When used, the Sleeping Beauty transposase, or a functional fragment or variant thereof, or the nucleic acid encoding it is also delivered to the cell. Examples of such transposases include, but are not limited to, SB10, SB11, SB100x, and SB110 transposases. The Sleeping Beauty transposon system is known in the art and is described, for example, in U.S. Pat. Nos. 6,489,458 and 8,227,432.

[0293] 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., the adenovirus-based Per.C6 system available from Crucell (Leiden, The Netherlands)), adeno-associated virus-based vectors, lentiviral vectors (e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)), retroviral vectors (e.g., pFB-ERV and pCFB-EGSH), and herpesvirus-based vectors.

[0294] In one embodiment, the viral vector is an adenoviral vector.

[0295] 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 enable long-term and stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. These have the additional advantage of low immunogenicity.

[0296] To assess the expression of one or more of the miRNAs and CARs or portions thereof described herein, the expression vector introduced into the cells can also contain a selectable marker gene and / or a reporter gene, or both, to facilitate the identification and selection of the expressing cells from the cell population to be transfected or infected via a viral or non-viral vector. In other embodiments, the selectable marker is carried on a separate DNA fragment and can be used in a co-transfection procedure. Both the selectable marker and the reporter gene can be adjacent to appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the ampicillin resistance gene. In some embodiments, the subtype epidermal growth factor receptor (HER1t or HER1t-1) tag can be used as a selectable marker gene.

[0297] Reporter genes can be used to identify cells that may have been transfected or to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that does not exist or is not expressed in the recipient organism or tissue and encodes a polypeptide whose expression is revealed by some readily detectable property, such as enzyme activity. At a suitable time after the DNA has been introduced into the recipient cells, the expression of the reporter gene is assayed. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, a construct having a minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to regulate promoter-driven transcription.

[0298] 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; (d) a nucleic acid encoding a cell tag. Encoding miRNA, CAR, cytokine, and cell tags in a single construct allows for manufacturing consistency.

[0299] 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.

[0300] In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen-binding domain described elsewhere herein, such as an epitope on CD19, CD33, MUC1, MUC16, ROR1, HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, and / or type II collagen. In certain embodiments, the CAR comprises an antigen-binding domain that binds to an epitope on CD19, CD33, MUC1, MUC16, and / or ROR1. In such certain embodiments, the CAR comprises an antigen-binding domain that binds to an epitope on ROR1.

[0301] 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 a functional variant or fragment thereof. In certain embodiments, the cytokine is IL-15, or a functional fragment or variant thereof. In certain embodiments, IL-15, or a functional fragment or variant thereof, is membrane-bound. In certain embodiments, the vector encodes a fusion protein comprising IL-15, or a functional fragment or variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof.

[0302] In some embodiments, the cell tag comprises domain III of HER1, or a functional fragment or variant thereof, and truncated domain IV of HER1, or a functional fragment or functional variant thereof. In certain embodiments, the cell tag further comprises the CD28 transmembrane domain or a functional fragment or functional variant thereof.

[0303] In certain embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the cytokine are linked by a nucleic acid encoding a linker, such as a Furin-T2A linker. In certain embodiments, the nucleic acid encoding the nucleic acid encoding the cytokine and the nucleic acid encoding the cell tag are linked by a nucleic acid encoding a linker, such as a T2A linker.

[0304] In some embodiments, the vector further encodes a splice donor site and a splice acceptor site. In certain embodiments, the splice donor side contains SEQ ID NO: 291 and the splice acceptor site contains SEQ ID NO: 292.

[0305] In some embodiments, the vector comprises: (a) a nucleic acid sequence encoding: (i) a pri-miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting PD-1; and (ii) a pri-miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting PD-1; (b) a nucleic acid sequence encoding a CAR comprising an antigen-binding domain that binds to an epitope on 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 functional variant thereof; and (d) a nucleic acid encoding a 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 such specific embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the cytokine are linked 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 linked by a nucleic acid encoding a T2A linker. In such specific 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.

[0306] VI. Method for Introducing miRNA into Cells The present invention also relates, in part, to a method for modifying gene expression in a cell, the method comprising 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 an agent for modifying gene expression.

[0307] The present invention also relates, in part, to a method for generating genetically engineered cells, the method comprising introducing into a cell the ribonucleic acid or deoxyribonucleic acid of the present invention.

[0308] In certain embodiments of the above method, the method comprises transfecting a cell with the ribonucleic acid or deoxyribonucleic acid of the present invention. In certain embodiments, transfection comprises electroporation.

[0309] In certain embodiments, the deoxyribonucleic acid can comprise a transposon, such as a Sleeping Beauty transposon. In embodiments where a Sleeping Beauty transposon is used, a Sleeping Beauty transposase, or a functional fragment or variant thereof, or a nucleic acid encoding them can be introduced into the cell. In certain embodiments of transfecting a cell with a transposon, the method further comprises transfecting the cell with a vector encoding the transposase.

[0310] In certain embodiments of the above method, a cell is transduced with the ribonucleic acid or deoxyribonucleic acid of the present invention. A viral vector containing such ribonucleic acid or deoxyribonucleic acid may be transduced into the cell.

[0311] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, the vector can be introduced into the cell by physical, chemical, or biological means.

[0312] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating 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 a polynucleotide into a host cell is calcium phosphate transfection or polyethyleneimine (PEI) transfection. In some embodiments, the method for introducing a polynucleotide into a host cell is electroporation.

[0313] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0314] As a biological method, the ribonucleic acid or deoxyribonucleic acid of the present invention can be introduced into cells using, for example, a virus-based delivery system. Representative virus expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell (Leiden, the Netherlands)), adeno-associated virus-based vectors, lentivirus-based vectors (e.g., the lentivirus-based pLPI from Life Technologies (Carlsbad, CA)), retroviral vectors (e.g., pFB-ERV and pCFB-EGSH), and herpesvirus-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 for long-term and stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity. Generally, and in embodiments, suitable vectors include an origin of replication, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers that function in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0315] Regardless of the method used to introduce the exogenous nucleic acid into the host cell, various 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 blotting and Northern blotting, RT-PCR and PCR, and "biochemical" assays such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot).

[0316] VII. Genetically Modified Cells The present invention also relates, in part, to genetically modified cells comprising the ribonucleic acid or deoxyribonucleic acid of the present invention. Such cells can be generated by any method known in the art for introducing the ribonucleic acid or deoxyribonucleic acid into the cell. In certain embodiments, the cells are generated using the methods described herein.

[0317] In certain embodiments, the cells are modified immune effector cells. In certain embodiments, the modified immune effector cells are modified T cells, natural killer (NK) cells, or macrophages. In certain embodiments, the modified T cells are modified cytotoxic T cells, e.g., T cells that destroy virus-infected cells and / or tumor cells.

[0318] In certain embodiments, the immune effector cells are obtained, for example, from umbilical cord blood, peripheral blood, human embryonic stem cells, iPSCs, bone marrow, lymph node tissue, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Such cells can then be modified to contain the ribonucleic acid or deoxyribonucleic acid of the present invention, for example, by the methods described herein. Accordingly, the present invention contemplates that there can be an initial step of obtaining cells from a subject in a method such as the methods described herein for producing genetically engineered cells.

[0319] For example, after modification by transfection or transduction, the cells can be immediately injected into a subject or cryopreserved. In certain embodiments, the cells are incubated for 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 before being delivered (e.g., by injection) to the subject. In certain embodiments, the cells are manufactured to be deliverable to the subject within 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, the cells do not undergo proliferation, activation, incubation, or culturing prior to delivery (e.g., by injection) to the subject.

[0320] In certain aspects, after transfection or transduction, the cells can be stored in a cytokine bath that can contain IL-2 and / or IL-21 until ready for injection. In certain aspects, after modification, the cells can be expanded ex vivo for several days, weeks, or months as a bulk population within about 1, 2, 3, 4, 5 or more days after gene transfer into the cells. In a further aspect, after modification, the modified cells are cloned and the clones are developed ex vivo for the presence of a single integrated or episomally maintained expression cassette or plasmid and for the expression of the miRNA and / or protein of interest.

[0321] Recombinant T cells can be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (such as IL-7, IL-12, IL-15, IL-21, etc.). Recombinant T cells can also be expanded by stimulation with an antibody such as OKT3 that crosslinks CD3 on the surface of artificial antigen presenting cells (aAPCs) or T cells.

[0322] VIII. Kits and Compositions The present invention also relates, in part, to a kit or composition comprising the 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.

[0323] The present invention also relates, in part, to the above kit or composition for use in modifying gene expression. Further, the present invention also relates, in part, to the above kit or composition for use in treating a disease or disorder in a subject or for use in the manufacture of a medicament for treating a disease or disorder in a subject.

[0324] In certain embodiments, the kit or composition comprises a transposase.

[0325] In certain embodiments, the kit or composition comprises a gene switch component, such as a component of the RHEOSWITCH® gene switch construct.

[0326] In certain embodiments, the composition further comprises a carrier, diluent, and / or 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 compositions of the present invention can include neutral buffered saline, buffers such as phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0327] In certain embodiments, the kit comprises a carrier, package, label, or container. Suitable containers include, for example, bottles, vials, syringes, and test tubes.

[0328] IX. Method of Treatment The present invention also relates to a method of treating a disease or disorder in a subject, which comprises administering to the subject the ribonucleic acid, deoxyribonucleic acid, vector, cell, or composition of the present invention. The administration may be in a therapeutically effective amount. In certain embodiments, the subject is a mammal, such as a human.

[0329] 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.

[0330] In certain embodiments, the disease or disorder is a disease or disorder in which reduction or silencing of the expression of an immune checkpoint protein would be beneficial. In such certain embodiments, the disease or disorder is cancer, an autoimmune disease, or is caused by an infection by a virus, bacterium or parasite.

[0331] In some embodiments, the disease is cancer. The cancer can be a hematological malignancy or a solid tumor. In some cases, the cancer is metastatic. Examples of treatable cancers include human sarcomas and carcinomas, such as 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, such as 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, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute myeloid leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, acute lymphocytic leukemia, and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); diffuse large B-cell lymphoma; and polycythemia vera, lymphoma (Hodgkin's and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease, but are not limited thereto.

[0332] In some embodiments, the disease is an autoimmune disease. Examples of such autoimmune diseases 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, but are not limited thereto.

[0333] In some embodiments, the disease is a disease caused by an infection by a virus, bacterium, or parasite. Examples of such diseases include, but are not limited to, those caused by Plasmodium, Trypanosoma, Aspergillus, Candida, hepatitis A virus, hepatitis B virus, hepatitis C virus, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, and Ebola pathogen.

[0334] 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.

[0335] In some embodiments, the disease is associated with overexpression of MUC16. In certain embodiments, the disease is ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, or lung cancer.

[0336] In some embodiments, the disease is associated with overexpression of CD33. In certain embodiments, the disease is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

[0337] In some embodiments, the disease is associated with overexpression of ROR1. In certain embodiments, the disease includes hematological tumors such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL). In certain embodiments, the disease includes solid tumors such as triple-negative breast cancer (TNBC), pancreatic cancer, ovarian cancer, and breast cancer including lung adenocarcinoma.

[0338] In certain embodiments, the method includes administration of the nucleic acids, vectors, cells, or compositions described herein. Such methods can be carried out by any method known in the art, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The nucleic acids, vectors, cells, or compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intra-articular, intramedullary, intramuscular, intravenous (i.v.) injection, or intraperitoneal administration.

[0339] In certain embodiments, the method includes administering genetically modified cells to a subject. The cells may be the cells of the invention described herein. Such methods can include obtaining a sample of cells from the subject, modifying the sample of cells with the ribonucleic acid or deoxyribonucleic acid of the invention, and administering the modified cells to the subject, for example by injection.

[0340] In certain embodiments, the modified immune cells are administered directly to a particular site in the body, for example, by targeting cancer and delivering them directly locally to the tumor tissue. For example, in ovarian cancer, the modified immune effector cells can be delivered intraperitoneally (IP) to the abdomen or peritoneal cavity. Such IP delivery can be carried out via a port placed for delivery of chemotherapeutic agents or an existing port. Other methods of local delivery of modified cells include catheter injection into the resection cavity, ultrasound-guided intratumoral injection, hepatic artery injection, or intrapleural delivery.

[0341] In some embodiments, prior to the step of administering the modified cells to a subject, the subject undergoes lymphodepletion. As used herein, "lymphodepletion" includes methods of reducing the number of lymphocytes in a subject, for example, by administration of a lymphodepleting agent. Examples of lymphodepletion include non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy. Lymphodepletion can also be achieved by fractionated radiation therapy of a part or the whole body. A lymphodepleting agent can be a compound or composition that, when administered to a mammal, can reduce the number of functional lymphocytes in the mammal. Such agents and dosages are known and can be selected by the treating physician depending on the subject being treated. Examples of lymphodepleting agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof. In some embodiments, prior to the step of administering the modified immune effector cells to a subject, the subject does not undergo lymphodepletion.

[0342] In some embodiments, the patient or subject has not been lymphodepleted prior to being drawn for the production of autologous modified immune effector cells.

[0343] 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.

[0344] The dosages of the above-described treatments administered to a patient vary depending on the exact nature of the condition being treated and the recipient of the treatment. Adjustment of dosages for administration to humans can be done according to practices recognized in the art. Appropriate dosages can be adjusted according to whether the patient is an adult or a pediatric patient.

[0345] In some cases, the effective amount of modified cells for administration is from about 10 4 to about 10 9 modified cells / kg, from about 10 4 to about 10 5 modified cells / kg, from about 10 5 to about 106 modified cells / kg, about 10 6 ~ about 10 7 modified cells / kg, > 10 4 provided that ≦ 10 5 modified cells / kg, > 10 5 provided that ≦ 10 6 modified effector cells / kg, or > 10 6 provided that ≦ 10 7 includes modified effector cells / kg.

[0346] Alternatively, a typical amount of modified cells administered to a mammal (e.g., a human) may be, for example, in the range of 100, 1000, 10000, 1 million to 100 billion cells. However, amounts below or above this exemplary range are also within the scope of the present invention. For example, such a cell dosage 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 above 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 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 above values), or about 100 million to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million, about 450 million, about 650 million, about 800 million, about 900 million, about 3 billion, about 30 billion, about 45 billion, or a range defined by any two of the above values).

[0347] Note that the dosage may vary depending on the type and severity of the symptoms to be alleviated. Further, for any given subject, the specific dosing regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be understood that the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition.

[0348] The therapeutic or prophylactic effect can be monitored by regularly evaluating the treated patients. When administering repeatedly over several days or more, depending on the condition, the treatment is repeated until the desired suppression of the disease symptoms occurs. However, other dosing regimens are also useful and are within the scope of the present invention. The desired dosage can be delivered by single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion administration of the composition.

[0349] 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 efficacy and the potential to induce cytokine-related toxicity.

[0350] In some embodiments, the compositions described herein can be administered as combination therapy with additional therapeutic agents. Examples of such agents include biological agents and small molecules.

Examples

[0351] These examples are provided for illustrative purposes only and do not limit 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 limiting in other respects, nor do they supersede or narrow the broader definitions above, and shall continue to apply to the claims.

Table 5

[0352] Example 1. Design of the PD1 module The PD1 silencing module of ROR1 UltraCAR-T cells expressing miRNA encodes two artificial miRNAs designed to specifically reduce the expression of PD-1 mRNA in UltraCAR-T cells while avoiding off-target silencing of other endogenous transcripts. The two artificial miRNAs of miRNA-expressing ROR1 UltraCAR-T cells are encoded within a duplex primary miRNA (pri-miRNA) sequence located within the 5'UTR splice unit of the UltraCAR-T transgene cassette (Figure 1B). The duplex pri-miRNA forms a stem-loop structure and is recognized and processed by cellular complexes to generate two unique mature guide miRNAs of 21-24 nucleotides that are homologous to specific sequences within the PD1 target transcript. Interaction between the guide miRNA and the PD1 target sequence is expected to cause silencing of PD1 by induction of RNA degradation or translational inhibition (Guo et al, 2010).

[0353] The guide miRNAs encoded within ROR1 UltraCAR-T cells that express miRNA were designed to be highly specific for the PD1 target transcript NM_005018.3 by implementing an in-house designed computational workflow using 21 combinations of ranking parameters based on an siRNA prediction algorithm based on three validation rules (Amarzguioui and Prydz, 2004; Reynolds et al, 2004; Ui-Tei et al, 2004). Multilevel 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 by non-PD1 target passenger strand miRNAs, a pri-miRNA scaffold that generates a high ratio of guide:passenger miRNA was selected (Miniarikova et al, 2016). The PD1 target guide miRNA PD1_1843 was incorporated into the pri-miRNA scaffold based on the human miRNA hsa-miR-204 (accession number MI0000284), and the PD1_2061 guide miRNA was incorporated into the hsa-miR-206 (accession number MI0000490) scaffold. Mutations were introduced on the passenger strand side of each pri-miRNA to ensure that the specific miRNA structure was maintained and the thermodynamic stability was substantially unchanged. The RNA structure was predicted using CLC Main Workbench software. The PD1 silencing module contains the miR204PD1_1843 pri-miRNA placed directly upstream of the miR206PD1_2061 pri-miRNA within the synthetic splice unit of the 5’UTR of the CAR-T transgene expression cassette. The splice unit was ordered as a gBlock from IDT, can be cloned into the Sleeping Beauty CAR vector, and can be cleaved using ClaI / NheI for cloning into the 5’UTR of other Sleeping Beauty CAR vectors.

[0354] Example 2. Reduction of PD1 transcript expression by miRNA Primary human T cells were transfected with the constructs listed in Table 6, grown in large batches, and then grown in vitro using AaPC cells expressing the allogeneic antigen that had been frozen in aliquots. Next, the generated CAR-T cells were further activated for 48 hours using anti-CD3 / anti-CD28 beads at a bead:T cell ratio of 1:1 and 1×10 6 T cells / mL before harvesting the cells for RNA isolation. RNA isolation was performed according to the manufacturer's recommended protocol (Qiagen), and then RT-qPCR analysis was performed using specific primers / probes (human PD1: Hs00169472_m1; human TIGIT: Hs00545087_m1; and human ACTb: Hs99999903_m1) with SuperScript VILO Master Mix containing ezDNase (Invitrogen) and TaqMan FAST Advanced Master Mix for qPCR to evaluate PD-1 expression levels. Also, all samples were normalized against the β-actin expression level. The relative expression values were based on the ΔΔCT method by normalizing against construct #1 (MUC16 CAR-T cells only). The results are shown in Figure 2. Mean ± SD from 3 donors are shown.

Table 6

[0355] The data demonstrate the specificity of the PD-1 checkpoint inhibitor miRNA targeting the sequence of interest. CAR constructs expressing scrambled miRNA (constructs 2 and 3), along with the CAR construct that did not express mbIL-15 (construct #8), did not show a decrease in PD-1 expression, whereas the CAR construct containing PD-1 miRNA (construct 4), or the combination of PD-1 and TIGIT miRNA (constructs 6 and 7), showed a decrease in PD-1 expression. On the other hand, the CAR construct containing only the TIGIT miRNA sequence (construct 5) did not show a decrease in PD-1 mRNA expression level, further demonstrating the specificity of the difference in targeting.

[0356] Example 3. Downregulation of target mRNA Primary human T cells were transfected with a vector encoding a CD33-specific CAR or a vector encoding a MUC16-specific CAR. The vectors contained a synthetic intron containing miRNA sequences targeting PD-1, PD-1, and / or TIGIT, or a non-targeting scrambled control miRNA. T cell cultures were grown in vitro at a ratio of 1:1 (AaPC: T cells) using antigen-presenting cells together with an allogeneic tumor antigen, which was K562 cells modified to express other costimulatory molecules (based on "Clone 1") in addition to either CD33 or MUC16. The generated CAR+ cells were then stimulated with anti-CD3 / anti-CD28 beads (bead: T cell ratio 1:1) for 48 hours in the absence of cytokines. RNA was isolated using the Qiagen kit (AllPrep Universal DNA / RNA / miRNA kit #80224) according to the manufacturer's protocol and utilized in the Nanostring Human PanCancer Immune gene set panel kit. Briefly, RNA was hybridized with capture and reporter probe sets, the samples were processed, then hybridized to slides using the nCounter Prep Station, and transcript counts were generated by the nCounter Digital Analyzer according to the manufacturer's protocol. Data validation, QC, and normalization were performed by nSolver software (Nanostring Technologies).

[0357] If the samples are identical, the distribution of transcript number data on the graph will be a line with a slope of 1 (from the lower left corner to the upper right corner). Data points that deviate from this line represent the variation in transcript numbers between the two samples. Counts found below the main line represent a decrease in expression, whereas counts above the main line represent an increase in expression relative to the sample being compared. miRNA targeting either PD-1 or TIGIT expressed in CAR-T cells showed that the transcripts of their respective target genes had particularly low expression compared to CAR-T cells without miRNA. See FIGS. 13A - B. Further, to further demonstrate the specificity of targeting, in CAR-T cells containing scrambled control miRNA, the data distribution of this graph remained close to the diagonal as there was little variation in the transcription levels, indicating that there was little variation in the transcription levels as neither PD-1 nor TIGIT transcription levels changed. See FIG. 3C. Similar results were observed in T cells transfected with an MUC16CAR vector containing a synthetic intron with miRNA sequences targeting PD-1, PD-1 and / or TIGIT (see FIGS. 4A - C).

[0358] Example 4. Enhancement of the tumor cell cytotoxic effect of miRNA MUC16 CAR-T cells Primary human T cells were transfected with a vector encoding a MUC16-specific CAR and a miRNA sequence targeting two sequences within the PD-1 transcript, or a vector encoding a MUC16-specific CAR but not encoding miRNA. The CAR-T cells used in this assay were grown in vitro and normalized for CAR expression, after which GFP+ K562 cells expressing MUC16 (“tumor cells”) were seeded in triplicate in 96-well plates at an E:T ratio of 3:1. The plates were loaded into an IncuCyte S3 device, and four images per well were taken every 2 hours for 7 days. Data were analyzed using IncuCyte software, and the number of GFP+ cells / image was normalized to the 0-hour time point.

[0359] In the proliferation assay, the proliferation rate of target cells is determined over a 7-day culture period in the presence or absence of CAR-T cells. A low number of target cells in the culture containing CAR-T cells indicates the lytic activity of CAR-T cells. Figure 5A shows the difference between tumor target cells only (filled black circles) and cells expressing only the MUC16-specific CAR (white squares), demonstrating the killing ability of MUC16 CAR-T cells. Cells further expressing a miRNA targeting PD-1 (gray-filled circles) further demonstrate an improvement in cell lysis activity based on the sustained low GFP+ counts over time-course evaluation compared to cells expressing only the MUC16-specific CAR (white squares). This data demonstrates that the cell lysis activity of CAR-T cells containing a PD-1-targeting miRNA was enhanced.

[0360] A similar experiment was performed using GFP+ K562 cells expressing MUC16, PD-L1, and CD155. As shown in Figure 5B, the difference between tumor target cells only (filled squares) and cells expressing only the CAR (white circles) demonstrates the killing ability of MUC16 CAR-T cells. CAR-T cells incorporating miRNAs targeting both PD-1 and TIGIT (filled circles) further demonstrate an improvement in cell lysis activity based on the sustained low GFP+ numbers over time-course evaluation compared to MUC16 CAR-T cells only (filled circles). This data demonstrates that the cell lysis activity of CAR-T cells containing three target miRNAs (two for PD-1 and one for TIGIT) in a single construct is enhanced.

[0361] Example 5. Improvement of cytokine expression Primary human T cells were transfected with a vector encoding a MUC16-specific CAR but not encoding miRNA target sequences, and a vector encoding a MUC16-specific CAR and a single or combination of miRNA target sequences of PD-1 and TIGIT (see Table 7). CAR-T cells expanded in vitro were expanded in vitro and normalized for CAR expression, and then K562 tumor target cells expressing truncated MUC16 (9MUC16t) were seeded in triplicate in 96-well plates at an effector-to-target ratio of 1:1, or CAR-T cells were cultured in medium alone. After co-culture for 3 days, the culture supernatant was collected. The culture supernatant was collected and interferon γ (IFNγ) and granulocyte / macrophage colony-stimulating factor (GM-CSF) were evaluated by multiplex cytokine analysis (Luminex) according to the manufacturer's protocol. Shown in Figure 6 are the mean ± SD from two wells.

[0362] For CAR-T cell constructs cultured in medium, only basal levels of IFNγ and GM-CSF were detected. No cytokines were observed when using target cells or medium alone. The supernatant after co-culturing MUC16 CAR-T cells alone (vector 1) with tumor cells became the baseline value of the expression levels of IFNγ and GM-CSF. Incorporating checkpoint inhibitor miRNAs into the CAR construct can observe an improvement in cytokine expression, especially through inhibition by the PD-1 pathway. Higher levels of cytokine expression were obtained with dual PD-1 (construct #3), or a combination of single PD-1 and TIGIT miRNA (construct #6), or constructs containing dual PD-1 and TIGIT miRNA (constructs #10 and 11).

Table 7

[0363] Example 6. Tumor volume in treated mice Non-obese diabetic / severe combined immunodeficiency (NOD / SCID) gamma mouse (NSG) mice were intraperitoneally transplanted with fLUC-GFP+SK-OV-3 tumor cells expressing MUC1 on day 0. The tumor burden in these mice was monitored throughout the study using an in vivo imaging system (IVIS) by luminescence with an IVIS Spectrum device (Perkin Elmer). IVIS data were analyzed using Living Image software (version 4.1) based on defined regions of interest to obtain total flux values (photons / sec). Prior to the administration of CAR-T cells, the mice were randomly divided into different groups based on tumor burden and body weight, and then the test articles were administered on day 6. All CARs tested expressed MUC16-specific CAR together with mbIL15 and HER1t. This is referred to as MUC16 CAR. All test articles were normalized to 0.5×106 CAR-T cells / mouse and administered intraperitoneally. IVIS imaging was performed twice a week to monitor the overall tumor burden in the mice. The data shown are mean ± SEM from n = 4 - 8 mice / group.

[0364] As shown in Figure 7, mice given only saline (filled gray circles) showed continuous tumor growth, as evidenced by the increase in total flux levels observed throughout the study. Eventually, these mice died due to tumor burden and were euthanized. Mice administered only MUC16 CAR (filled black squares) were able to control tumor burden. CAR-T cells expressing checkpoint inhibitor miRNAs against PD-1 and TIGIT within the construct (white squares and white circles) were found to maintain anti-tumor activity based on the decrease in tumor flux values to background levels. Furthermore, CAR-T cells expressing checkpoint inhibitor miRNAs against PD-1 and TIGIT showed a faster time frame and rate of decrease in tumor burden compared to the construct of MUC16 CAR only.

[0365] Example 7. In vivo phenotypic analysis experiment SKOV-3 / MUC16 tumor-bearing mice were administered on day 6 of the study with either CAR-only or CAR-T cells (expressing MUC16-specific CAR, mbIL15, and HER1t) containing either PD-1 / PD-1 miRNA. Whole blood was collected from the mice on day 31 of the study, and phenotypic evaluation of the administered CAR-T cells was performed by flow cytometry. Briefly, whole blood samples were stained using a cocktail of fluorescent-conjugated antibodies and then fixed with one-step Fix / Lyse buffer for simultaneous erythrocyte lysis. The fixed samples were read on a flow cytometer (BD LSRFortessa X-20) instrument. CAR-T cells were identified based on gating of hCD45 / CD3+ / HER1t+ expression. In Figure 8A, the CAR-only sample (dotted line) shows high PD-1 expression, whereas the CAR containing PD-1 / PD-1 miRNA (solid line) shows a significant decrease in the detected PD-1 expression level. To further quantify the decrease in PD-1 expression detected in the CAR+miRNA (PD-1 / PD-1) group, the mean fluorescence intensity (MFI) was examined (Figure 8B). The mean MFI of PD1 expression in mice given CAR-T cells only (striped bars) was approximately 709, whereas the mean MFI of CAR-T cells containing PD-1 / PD-1 miRNA (solid bars) decreased to approximately 236. Mean ± SEM of 5 - 8 mice are shown.

[0366] Example 8. Specific PD-1 and TIGIT Downregulation Mice bearing SKOV-3 tumors were administered on day 6 of the study with either CAR-only or CAR-T cells (expressing MUC16-specific CAR (「MUC16 CAR」), mbIL15, and HER1t) having various checkpoint miRNA inhibitors (PD-1 and TIGIT). See Table 8.

Table 8

[0367] On day 45 (D45) of the study, whole blood was collected from the mice, and phenotypic evaluation of the administered CAR-T cells was performed by flow cytometry. Briefly, whole blood samples were stained using a cocktail of fluorescently conjugated antibodies including specific antibodies against human PD-1 and human TIGIT, and then fixed using one-step Fix / Lyse buffer. The fixed samples were read on a flow cytometer (BD LSRFortessa X-20) instrument. CAR-T cells were identified in the mice based on gating of hCD45 / CD3+ / HER1t+ expression. To further evaluate the specificity of the miRNA for checkpoint inhibitors used in the CAR vector, the median fluorescence intensity (MFI) for the expression of PD-1 and TIGIT was analyzed (Figures 9A and B). For the quantitative evaluation of the expression levels of the same vector set, the MFI of PD-1 is shown on the left side and the MFI of TIGIT is shown on the right side. As shown in Figure 9A, a decrease in the expression of PD-1 was observed in the CAR-T cells of the group indicated by the downward arrow (dotted line for PD-1), which is a construct having PD-1 miRNA (alone, double, and combinations with other miRNA checkpoint inhibitors), when compared to the CAR vector alone. The right side (downward dashed arrow) highlights the cell population with a decreased expression of TIGIT expression seen in the CAR-T cells. The downregulated expression of TIGIT corresponded to samples containing miRNA of TIGIT (alone or in combination with other checkpoint miRNA inhibitors). Mean ± SEM of 5 - 8 mice are shown.

[0368] Example 9. Expression of PD1-targeted miRNA in ROR1-targeted CAR-T cells. Briefly, miRNA-expressing ROR1 UltraCAR-T cells or control ROR1 UltraCAR-T cells were generated from T cells from 5 donors. Pan T cells from 5 healthy donors were transfected with the indicated transposon vector (VVN-5355 or VVN-5351) + SB11 transposase vector and expanded by weekly stimulation with ROR1 antigen-presenting cells for 4 weeks (about 35 days before bead addition). After a resting period of 7-8 days, UltraCAR-T cells were activated with CD3 / CD28 Dynabeads (bead:T cell ratio 1:1, T cells 1×10 6 cells / mL) for 48 hours before RNA collection 7-8 days after the last AaPC stimulation. The effects on PD1 target-guided miRNA expression and PD1 mRNA expression were verified by RT-qPCR. Small RNAseq (Borel et al, 2018; Miniarikova et al, 2016), an established method for identifying predicted miRNA sequences and alternative miRNA sequences that may arise from pri-miRNA, was performed to compare the expression levels of PD1 target-guided miRNAs and additional small RNA species (such as passenger miRNAs) that can be generated from the PD1 silencer module. Small RNAseq was also used to evaluate potential changes in overall endogenous miRNA expression (Mueller et al, 2012). RNAseq analysis was performed to evaluate overall transcriptional expression and identify changes in molecular pathway signaling or off-target gene silencing due to the PD1 silencer. In silico miRNA target prediction was performed using the miRanda algorithm to identify the most likely targets of miRNAs generated from the PD1 silencer module. The expression of the predicted target genes was evaluated by RNAseq. Details of each method are described in the following section.

[0369] To characterize the effects of the miRNA encoded by the PD1 silencer on the expression of miRNA and the PD1 transcription level, RT-qPCR and Small RNAseq were performed. RNAseq was performed to characterize changes in specific genes or cell pathways.

[0370] The AllPrep DNA / RNA / miRNA Universal Kit (catalog number 80224) from Qiagen was used to purify nucleic acids from cell pellets according to the manufacturer's protocol. Total RNA was eluted with 50 μL of nuclease-free water, and the concentration was measured with a Nanodrop™ 2000 spectrophotometer.

[0371] To quantify the expression of PD1 guide miRNA and passenger miRNA, total RNA was used as the input for cDNA synthesis using the miRCURY LNA RT Kit (#339340) from Qiagen. According to the manufacturer's protocol, the cDNA was diluted 1:60 with nuclease-free water, and 3 μL of the diluted cDNA was used as the input for qPCR using the miRCURY LNA SYBR Green PCR Kit (Qiagen #339345) with custom miRCURY LNA primers specific for two PD1 guide miRNAs. The endogenous miRNA hsa-let-7a-5p was quantified as a reference small RNA enabling input normalization (Qiagen product #339306, custom #YP00205727). Samples were run in 384-well format on a QuantStudio 6 Flex instrument. Relative quantification (dCT) calculations were performed in Microsoft Excel, and the data was graphed in GraphPad Prism9. The dCT calculations were performed for each technical replicate as follows. dCT = CT(guide miRNA) - CT(hsa-let-7a) ddCT = dCT(miRNACART replicate) - dCT(average of VVN-5355 technical replicates) Fold change = 2^-ddCT

[0372] The VVN-5355 ROR1 Ultra CAR-T control sample functions as a reference control sample for comparison to ROR1 Ultra CAR-T cells expressing miRNA within each donor set. The mean fold change and standard deviation for technical replicates were calculated and reported in Figure 10A.

[0373] 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 primer assays for detecting passenger miRNA and the additional endogenous reference small molecule RNA, RNU1A1. Expression calculations were performed to compare the expression of mature miRNA for each guide or passenger strand to the mean of the endogenous control small molecule RNAs as follows: dCT = CT(mature miRNA) - CT(mean of hsa-let-7a and RNU1A1) Fold change = 2^-dCT

[0374] The mean fold change was calculated from three technical replicates. These values were plotted in Figure 11 along with the mean and standard deviation shown for the donor sample sets tested.

[0375] To quantify the expression of endogenous PD1 mRNA, cDNA synthesis was performed at a final RNA concentration of 5 ng / μL using Invitrogen SuperScript IV VILO Master Mix together with the ezDNase enzyme kit (#11766050). Multiplex Taqman qPCR was performed using Invitrogen TaqMan MastAdvanced Master Mix (#4444963) containing 1 microliter of cDNA and Taqman assays from Invitrogen (ThermoFisher Scientific). The Taqman assay for human PD1 (Invitrogen #Hs00169472_m1) was labeled with FAM, and the internal normalization gene ACTb (Invitrogen #Hs99999903_m1) was labeled with VIC. Samples were run in 384-well format on a QuantStudio 6 Flex instrument. Relative quantification (dCT) calculations were performed in Microsoft Excel as described above, and the data were graphed in GraphPad Prism9.

Table 9

[0376] The PD1 silencer module is designed to generate two mature guide miRNAs that bind to the PD1 transcript and suppress PD1 expression. The expression of two PD1 target guide miRNAs, called PD1_1843 and PD1_2061, was confirmed in the miRNAs expressed by ROR1UltraCAR-T cells generated from multiple donors (Figure 10A). A corresponding decrease in PD1 mRNA expression was verified in miRNA-expressing ROR1UltraCAR-T cells from all donors tested (Figure 10B). This result demonstrated that the PD1 silencer module generates the guide miRNAs of interest and functions as designed.

[0377] To reduce the risk of silencing genes other than PD1, the PD1 silencer module was designed using a pri-miRNA scaffold that preferentially generates PD1-targeting guide miRNAs over non-target passenger miRNAs. When both guide mature miRNA and passenger mature miRNA were quantified by RT-qPCR from the miRNAs expressed in ROR1 UltraCAR-T cells, it was confirmed that the PD1-targeting guide miRNA was the dominant species compared to the non-target passenger strand miRNA (Figure 4). Strong processing preference for the PD1-targeting guide miRNA was confirmed by small RNAseq, and 99.7% of the reads were mapped to the PD1 silencer module that matched the intended PD1-targeting guide miRNA (Figures 12A-E). Furthermore, the start and stop positions of the miRNAs were as expected, and miRNAs of 21-23 nucleotides with the same 5'- and 3'-end variable lengths were detected (Figures 12A-E). Since the incidence of the passenger strand miRNA was extremely low and there were no unexpected small RNAs generated by abnormal RNA processing, the risk of off-target gene silencing was significantly reduced.

[0378] To ensure that the expression of the PD1 silencer module does not overwhelm the intracellular RNAi machinery, the number of endogenous miRNAs was compared between ROR1 UltraCAR-T cells expressing miRNAs and control ROR1 UltraCAR-T cells. When the top 20 expressed endogenous miRNAs were examined, no statistically significant change in expression was seen across the samples (Table 11). Furthermore, the mature miRNAs generated from the PD1 silencer module accounted for approximately 4% of all the small RNAs quantified (Figure 13). These data showed that the expression of miRNAs from the PD1 silencer module did not saturate the cellular RNAi machinery and had no detectable effect on overall endogenous miRNA expression.

Table 10

Table 11

[0379] Example 10: The PD1 silencer module specifically reduces the expression of PD-1. Using miRanda (Betel et al, 2008; Betel et al, 2010), an in silico miRNA target prediction algorithm, the most likely target transcripts for the guide miRNA and passenger miRNA generated from the PD1 silencer module were predicted. This algorithm assigns a score to each potential miRNA target gene, and the higher the score, the more likely it is that silencing is caused by the input miRNA sequence. The summary of the top 10 hits for each mature miRNA generated from the PD1 silencing module is shown in Table 12. 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 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 (an approximately 84% decrease in PD1 in ROR1 UltraCAR-T cells expressing miRNA compared to control ROR1 CAR-T cells), and it was a highly significant adjusted p-value. The expression of other predicted target genes did not change. Genes with an adjusted p-value less than 0.05 had an LFC in the range of -0.33 to 0.22, which means that the decrease or increase in expression was less than 20%. One exception was HDAC9, a weakly predicted target gene of the PD1_2061 guide miRNA, with an LFC of -1.51. HDAC9 is a transcriptional repressor that is mechanistically related to PD1 expression via BCL6 (Xie et al, 2017; Gil et al, 2016). HDAC9 is not directly targeted by the PD1_2061 guide miRNA, and the decrease in HDAC9 may be an indirect effect of the decrease in PD1 expression.

Table 12

[0380] Changes in PD1 expression are expected to affect the expression of other genes in the downstream pathway. As expected, analysis of the RNAseq data confirmed differential expression of several genes in miRNA-expressing ROR1UltraCAR-T cells compared to control ROR1UltraCAR-T cells (Figs. 14A and B, Table 13). To elucidate the direct and indirect changes in gene expression, the differential expression (LFC) in miRNA-expressing ROR1UltraCAR-T cells compared to control ROR1CAR-T cells was plotted against the predicted binding potential (predicted free energy) of PD1 miRNA to the gene, and these were predicted in silico as potential PD1 miRNA targets (Figs. 15A–D). Genes with a very negative free energy and a statistically significantly decreased expression are likely to be directly targeted by the miRNA, while genes with a weak downregulation of free energy are likely to be indirectly affected by the miRNA. PD1 is clearly separated from all other genes in the plot, with a significantly decreased expression and a high miRNA binding ability, suggesting that the PD1 silencer miRNA strongly and preferentially directly targets PD1 rather than other genes.

Table 13

[0381] Example 11: Dual miRNA design for reducing the expression of TIGIT. Screening of various unnatural pri-miRNAs containing guide miRNAs targeting TIGIT was performed.

[0382] Jurkat cells overexpressing TIGIT were transfected with an expression vector encoding CAR, membrane-bound IL15, and truncated HER1 together with a nucleic acid encoding a pri-miRNA targeting TIGIT located in the section corresponding to the 5’UTR intron; or a control vector expressing CAR, membrane-bound IL15, and truncated HER1 but not expressing the pri-miRNA.

[0383] Two days after transfection, flow cytometry was performed to quantify TIGIT expression (geometric mean fluorescence). The results are shown in Table 14 and Figure 17.

Table 14

[0384] After the above initial screening, additional screening was performed using the same protocol, except that the expression vectors tested here contained one or two TIGIT-targeting pri-miRNAs. Similar to the previous screening, each test expression vector encoded CAR, membrane-bound IL15, and truncated HER1 together with a nucleic acid encoding a pri-miRNA located in the section 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 Figure 18.

Table 15

[0385] Example 12: miRNA design for reducing the expression of CD70. Screening of various constructs encoding unnatural pri-miRNAs containing guide miRNAs targeting CD70 was performed.

[0386] Jurkat JRFTCR cells were transfected with expression vectors encoding anti-MUC16 (4A5) CAR, membrane-bound IL-15, and truncated HER1 (HER1t). The expression vectors further encoded: (i) a single CD70-targeting pri-miRNA located in the 5'UTR intron; (ii) two PD1-targeting pri-miRNAs and one CD70-targeting pri-miRNA located in the 5'UTR intron; (iii) two PD1-targeting pri-miRNAs located in the 5'UTR intron and two CD70-targeting pri-miRNAs located in the 3'UTR; or (iv) two PD1-targeting pri-miRNAs located in the 5'UTR intron and one CD70-targeting pri-miRNA located in the 3'UTR. Additionally, certain cells were transfected with: (i) a control vector that expresses anti-MUC16 (4A5) CAR, membrane-bound IL-15, and truncated HER1 but does not express pri-miRNA; or (ii) a control vector that expresses anti-MUC16 (4A5) CAR, membrane-bound IL-15, truncated HER1, and two PD1-targeting pre-miRNAs located in the 5'UTR intron.

[0387] One day after transfection, the cells were activated with PMA / ion to stimulate CD70 expression, and flow cytometry was performed two days after transfection to quantify CD70 expression (the percentage of CD70-positive cells and the geometric mean fluorescence in the activated transfected population). The results are shown in Table 16 and Figure 19.

Table 16

[0388] The sequence of the dual pri-miRNA targeting PD-1 for all of the above constructs was SEQ ID NO: 267.

[0389] Example 13 As shown in the above example, the dual combination of miR204 + miR206 has been demonstrated to provide potent gene silencing of all target human genes (PD1, TIGIT, CD70) tested to date. Furthermore, small RNAseq analysis confirmed that approximately 99.9% of the small RNAs generated from the dual miR204 + miR206 combination mapped to the predicted guide or passenger mature miRNA sequences (approximately 93% guide, 7% passenger), confirming proper folding and processing of the dual pri-miRNA design.

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Claims

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

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

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

4. The ribonucleic acid according to claim 1, wherein each non-natural pre-miRNA comprises a skeletal sequence from miR204 or miR206.

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

6. The ribonucleic acid according to claim 1, wherein the immune checkpoint protein is PD-1.

7. The ribonucleic acid according to claim 1, wherein the immune checkpoint protein is CD70.

8. The ribonucleic acid according to claim 1, comprising (a) a pre-miRNA containing a skeletal sequence from miR204 and a guide miRNA targeting PD-1; and (b) a pre-miRNA containing a skeletal sequence from miR206 and a guide miRNA targeting PD-1.

9. The ribonucleic acid according to claim 1, comprising (a) a pre-miRNA containing a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA containing a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA containing a skeletal sequence from miR17 and a guide miRNA targeting TIGIT.

10. The ribonucleic acid according to claim 1, comprising (a) a pre-miRNA containing a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA containing a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA containing a skeletal sequence from miR22 and a guide miRNA targeting CD70.

11. Deoxyribonucleic acid encoding ribonucleic acid as described in claim 1.

12. The deoxyribonucleic acid according to claim 11, further encoding a protein.

13. The deoxyribonucleic acid according to claim 12, wherein the protein is a chimeric antigen receptor.

14. The deoxyribonucleic acid according to claim 13, wherein the chimeric antigen receptor comprises an antigen-binding domain that binds to CD19, CD33, MUC-16, or ROR-1.

15. The deoxyribonucleic acid according to claim 11, further encoding (a) a chimeric antigen receptor, (b) a protein comprising IL-15 or a functional fragment or functional variant thereof, and IL-15Rα or a functional fragment or functional variant thereof, and (c) a cell tag.

16. A vector comprising ribonucleic acid according to any one of claims 1 to 10, or deoxyribonucleic acid according to any one of claims 11 to 15.

17. The vector according to claim 16, wherein the vector is a Sleeping Beauty transposon.

18. A method for modifying gene expression in cells in vitro, comprising introducing ribonucleic acid according to any one of claims 1 to 10 or deoxyribonucleic acid according to any one of claims 11 to 15 into the cells.

19. Genetically modified cells comprising ribonucleic acid according to any one of claims 1 to 10, or deoxyribonucleic acid according to any one of claims 11 to 15.

20. A composition for use in the treatment of a disease or disorder in a subject, comprising ribonucleic acid according to any one of claims 1 to 10, or deoxyribonucleic acid according to any one of claims 11 to 15.

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