Engineered minimal core promoter and uses thereof

Abstract

The present disclosure provides for, among other things, methods and compositions comprising chimeric minimal promoters and engineered regulatory elements comprising the chimeric minimal promoters, that allow for highly selective and efficient transcriptional activity of an operably linked polynucleotide.

Description

Attorney Ref. No.: 33978 / 70016 / PC ENGINEERED MINIMAL CORE PROMOTER AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 535,541 filed on August 30, 2023, the contents of which are hereby incorporated in their entirety by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. The electronic copy, created on August 21, 2024, is named 70016_SeqListing.xml, and is 9,268 bytes in size. BACKGROUND

[0003] Core promoters can generate undesirable basal levels of gene expression when paired with upstream enhancer sequences, therefore, core promoters that minimize basal expression and maximize the intended transcriptional output are ideal for applications requiring tight and potent regulation of gene expression. Several synthetic and viral core promoters are capable of driving low levels of basal expression and moderate levels of transcription in the induced state, however, core promoters with low basal expression levels often produce rates of induced transcription that are significantly lower than those of core promoters exhibiting higher levels of basal activity such as minCMV or the SCP3 synthetic promoter. Additionally, core promoters with high transcriptional output often contain viral sequences that may be prone to silencing by eukaryotic cell machinery thereby compromising their ability to drive durable long-term expression. Accordingly, there is a need to develop core promoters composed of non-viral elements that produce high levels of transcription in the induced state while minimizing expression levels in the uninduced state. SUMMARY

[0004] In one embodiment, the present disclosure provides an engineered minimal promoter comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 – 5.

[0005] In another embodiment, the present disclosure provides an engineered minimal promoter comprising an upstream promoter element and at least one downstream promoter element, wherein the upstream promoter element comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atAttorney Ref. No.: 33978 / 70016 / PC least 99%, or 100% sequence identity to TCTAGAGGGTATATAATGGGGGCCATTCTCTGTGTAATTCAGTCCGGTAGTCCGGC (SEQ ID NO: 10).

[0006] In another embodiment, the present disclosure provides an engineered minimal promoter comprising a first polynucleotide sequence encoding a minimal promoter sequence derived from YB_TATA, a second polynucleotide sequence comprising an initiator sequence (Inr), a third polynucleotide sequence comprising at least one downstream promoter element, wherein the first polynucleotide sequence is operably linked to the second polynucleotide sequence and the second polynucleotide sequence is operably linked to the third polynucleotide sequence with a first linker sequence and second linker sequence, respectively, and wherein the first linker sequence and / or second linker sequence wherein the first linker sequence and / or the second linker sequence does not comprise a transcription factor binding site. In some embodiments, the at least one downstream promoter element comprises a first downstream promoter element and a second downstream promoter element. In some embodiments, the at least one downstream promoter element comprises a pause button motif. In some embodiments, the pause button motif comprises the consensus nucleotide sequence KCGRWCG. In some embodiments, the at least one downstream promoter element is MTE / DPE or DPR. In some embodiments, the initiator sequence comprises a nucleic acid sequence of TCAGTC. In some embodiments, the first polynucleotide sequence comprises SEQ ID NO: 6. In some embodiments, the downstream promoter element comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0007] In another embodiment, the present disclosure provides an engineered regulatory element comprising an enhancer region and the engineered minimal promoter of the present disclosure. In some embodiments, the enhancer region is upstream of the upstream promoter element. In some embodiments, the enhancer region is operably linked to the minimal promoter. In some embodiments, the enhancer region is heterologous to the engineered minimal promoter. In some embodiments, the engineered regulatory element comprises at least one spacer sequence between the engineered minimal promoter and the enhancer region.

[0008] In another embodiment, the present disclosure provides a heterologous construct comprising any one of the engineered minimal promoters or the engineered regulatory elements according to the present disclosure, wherein the engineered minimal promoter or the engineered regulatory element is operably linked to a polypeptide-encoding polynucleotide, wherein the polypeptide-encoding polynucleotide comprises a polynucleotide sequence encoding a polypeptide. In some embodiments, the polypeptide comprises at least one effector molecule. InAttorney Ref. No.: 33978 / 70016 / PC some embodiments, the polypeptide comprises a first effector molecule and a second effector molecule. In some embodiments, the polypeptide-encoding polynucleotide comprises a polynucleotide sequence encoding the first effector molecule, a linker polynucleotide sequence, and a polynucleotide sequence encoding the second effector. In some embodiments, the linker polynucleotide sequence encodes one or more 2A ribosome skipping elements. In some embodiments, the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A. In some embodiments, the at least one effector molecule belongs to a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, an antibody, a peptide, and an enzyme. In some embodiments, each of the first effector molecule and the second effector molecule is from a separate therapeutic class. In some embodiments, each of the at least one effector molecule is a human-derived effector molecule.

[0009] In another embodiment, the present disclosure provides a vector comprising the heterologous construct according to the present disclosure.

[0010] In another embodiment, the present disclosure provides a dual expression vector comprising the heterologous construct according to the present disclosure and a second construct comprising a second polypeptide-encoding polynucleotide sequence encoding a second effector protein.

[0011] In another embodiment, the present disclosure provides an engineered cell comprising the heterologous construct according to the present disclosure, the vector according to the present disclosure, or the dual expression vector according to the present disclosure. In some embodiments, the engineered cell expresses at least one effector molecule.

[0012] In another embodiment, the present disclosure provides a pharmaceutical composition comprising the engineered minimal promoter of the present disclosure, the engineered regulatory element according to the present disclosure, the heterologous construct according to the present disclosure, the vector of the present disclosure, the dual expression vector according to the present disclosure, or the engineered cell according to the present disclosure, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

[0013] In another embodiment, the present disclosure provides a method of increasing expression of a target gene, the method comprising use of the engineered minimal promoter of the present disclosure, the engineered regulatory element according to the present disclosure, the heterologous construct according to the present disclosure, the vector of the present disclosure,Attorney Ref. No.: 33978 / 70016 / PC the dual expression vector according to the present disclosure, or the engineered cell according to the present disclosure, to increase expression of the target gene.

[0014] In another embodiment, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering the engineered minimal promoter of the present disclosure, the engineered regulatory element according to the present disclosure, the heterologous construct according to the present disclosure, the vector of the present disclosure, the dual expression vector according to the present disclosure, or the engineered cell according to the present disclosure.

[0015] In another embodiment, the present disclosure provides a method of treating a subject having a disease or disorder, the method comprising administering the engineered minimal promoter of the present disclosure, the engineered regulatory element according to the present disclosure, the heterologous construct according to the present disclosure, the vector of the present disclosure, the dual expression vector according to the present disclosure, or the engineered cell according to the present disclosure, to the subject in need thereof.

[0016] In another embodiment, the present disclosure provides a kit for treating and / or preventing a disease or disorder, comprising the pharmaceutical composition according to the present disclosure. In some embodiments, the kit further comprises written instructions for using the pharmaceutical composition for treating and / or preventing a tumor in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG.1 depicts expression of GFP using various engineered minimal promoters combined with an enhancer in different cell lines (Astrocytes, IMR-32, and Huh7).

[0018] FIG.2A depicts expression of an activating CAR that is driven by various minimal promoters combined with Enhancer A, Enhancer B, or Enhancer C.

[0019] FIG.2B depicts expression of an inhibitory CAR that is driven by various minimal promoters combined with Enhancer A, Enhancer B, or Enhancer C. DETAILED DESCRIPTION I. Definitions

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosed subject- matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and theAttorney Ref. No.: 33978 / 70016 / PC following detailed description are exemplary and explanatory only and are not restrictive of any subject-matter claimed or otherwise provided herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject-matter described.

[0021] Singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0022] All numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90% to 100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

[0023] Agent: The term “agent” may refer to a compound, molecule, or entity of any chemical class including, for example, polypeptides, nucleic acids (e.g., engineered nucleic acids as described herein), saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man- made in that it is designed, engineered, modified, and / or produced through action of the hand of man and / or is not found in nature. In many aspects, the present disclosure provides for engineered nucleic acids comprising particular transcriptional regulatory elements as described herein. In some embodiments, an agent may be utilized in isolated or pure form (e.g., an isolated polynucleotide); in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present disclosure include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA / RNA hybrids, antisense oligonucleotides, and ribozymes), peptides, and peptide mimetics. In some embodiments, an agent as described herein is encoded on a coding nucleotide sequence that is operably linked to an engineered regulatory element as provided herein (e.g., an engineered regulatory element). In some embodiments, an agent is an effector molecule as described herein.

[0024] Approximately: The term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwiseAttorney Ref. No.: 33978 / 70016 / PC stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0025] Biologically active: The phrase “biologically active” refers to an agent or substance that has activity in a biological system (e.g., an isolated cell, a cell in a particular tissue, a cell in culture, or a cell in an organism, etc.). For instance, an agent or substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active, or have biological activity (e.g., a biologically active agent, bioactive agent, bioactive molecule, etc.). It will be appreciated by those skilled in the art that often only a portion or fragment of a biologically active substance is required (e.g., is necessary and sufficient) for an activity to be present; in such circumstances, that portion or fragment is considered to be a “biologically active” portion or fragment.

[0026] Expression cassette: An “expression cassette” is a polynucleotide construct, generated recombinantly or synthetically, comprising regulatory sequences operably linked to a selected polynucleotide (e.g., a coding sequence, such as a gene) to facilitate expression of said selected polynucleotide in a host cell, or in a cell-free environment. Heterologous constructs as described herein may include one or more expression cassettes.

[0027] Heterologous: The term “heterologous” with respect to a nucleotide sequence, amino acid sequence, or polypeptide, refers to a compound or agent which is either foreign (e.g., exogenous, such as it is not found in nature) to a given host cell, or which is naturally found in a given host cell (e.g., is endogenous), however, said compound or agent is in the context of a heterologous construct, e.g., employing heterologous nucleic acid, as described herein. A heterologous nucleotide sequence as found endogenously may also be produced in an unnatural, e.g., greater than expected or greater than naturally found, amount in a cell. A heterologous nucleotide sequence, or a nucleic acid comprising a heterologous nucleotide sequence, possibly differs in sequence from an endogenous nucleotide sequence but encodes the same protein as found endogenously. Specifically, heterologous nucleotide sequences are those not found in the same relationship to a host cell in nature. Any recombinant or artificial nucleotide sequence is understood to be heterologous. A non-limiting example of a heterologous polynucleotide, e.g., an engineered regulatory element as described herein, is a nucleotide sequence such as an enhancer, or enhancer region, operably linked to a promoter with which it is not natively associated. Heterologous polynucleotides comprising such engineered regulatory elements may further be used to control expression of coding sequences (e.g., genes) in place of the native, or wild-type, promoter of said coding sequence.

[0028] Identity: The term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules)Attorney Ref. No.: 33978 / 70016 / PC and / or between polypeptide molecules. Accordingly, reference to “identity” or “homology” in the context of a particular sequence (e.g., nucleotide sequence, amino acid sequence, etc.) have the same meaning unless the context indicates otherwise. Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Optimal alignment of sequences for comparison can also be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al.). Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0029] Inert Sequence: “Inert sequence,” refers to a polynucleotide sequence that does not include transcription factor binding sites within said polynucleotide sequence. In addition, anAttorney Ref. No.: 33978 / 70016 / PC inert sequence may be further modified such that 5’ and 3’ ends of the inert sequence also do not contain transcription factor binding sites when joined to an enhancer sequence of the of the present disclosure (e.g., via an ablation). The presence of an inert sequence in a regulatory element (e.g., an enhancer region) does not render the entire regulatory element inert.

[0030] Isolated: The term “isolated” refers to a substance and / or entity that has been: (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. A substance is “pure” if it is substantially free of other components. Calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0031] Minimal promoter: A “minimal promoter” or “minimal core promoter” refers to a minimal sequence of a promoter that maintains the ability to initiate, or induce, transcription of a target coding sequence, e.g., a gene. Accordingly, a minimal promoter can be a heterologous, engineered promoter.

[0032] Nucleic acid: The term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA.” “RNA.” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure. The terms “nucleotide sequence encoding an amino acid sequence” or “coding sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. NucleotideAttorney Ref. No.: 33978 / 70016 / PC sequences that encode proteins and / or RNA may include introns. A coding sequence may also refer to a nucleotide sequence that produces a bioactive nucleic acids (e.g., a mRNA, miRNA, siRNA, shRNA, etc.). Nucleic acids can be isolated or purified from natural sources, produced using recombinant expression systems and optionally isolated or purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. The term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, or more residues. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). In some embodiments, the present disclosure is directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.

[0033] Operably linked: The term “operably linked” refers to polynucleotide sequences or amino acid sequences placed into a functional relationship with one another. For instance, a regulatory sequence or regulatory element (e.g., a promoter and / or an enhancer region) is operably linked to a coding sequence if it regulates, or contributes to modulation of, the transcription of the coding sequence. Operably linked DNA sequences encoding regulatory sequences are typically contiguous to a coding sequence. However, enhancers (e.g., enhancer regions) can function when separated from a promoter by up to several kilobases or more.Attorney Ref. No.: 33978 / 70016 / PC Additionally, multi-cistronic constructs can include multiple coding sequences which use only one regulatory element by including a 2A self-cleaving peptide, an IRES element, etc. as described herein. Accordingly, some polynucleotide elements (e.g., engineered regulatory elements provided herein) may be operably linked to one or more coding sequences, but not contiguous with said one or more coding sequences.

[0034] Protein: The term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a biologically active portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0035] Specific: The term “specific”, when used with reference to an agent or entity having an activity, or inducing an activity (e.g., an engineered nucleic acid as provided herein), is understood by those skilled in the art to mean that the agent or entity discriminates between potential targets or states. In many aspects, provided agents or entities display specific activity in a particular cell type as compared to a different or alternative cell type. In many aspects of the present disclosure, an agent or entity is an engineered nucleic acid (e.g., an engineered regulatory element) that specifically induces transcription of one or more coding sequences in a particular cell type as compared to a different cell type; that is to say that induced transcription of the one or more coding sequence is higher in a particular cell type as compared to a different cell type. In some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of competing alternative targets. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target. In some embodiments, the agent or entity binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and / or increased stability to its target as compared with the competing alternative target(s).Attorney Ref. No.: 33978 / 70016 / PC

[0036] Substantially: The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0037] Subject: The term “subject” means a mammal (e.g., a human, in some embodiments including prenatal human forms, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. In some embodiments, a subject is an individual to whom therapy is administered, e.g., a recipient. In many aspects of the present disclosure, a subject has, or is susceptible to, a disease or disorder.

[0038] Therapeutic agent: The phrase “therapeutic agent” in general refers to any agent that elicits a desired pharmacological effect when administered to an organism (e.g., a subject). In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans.

[0039] Therapeutically effective amount: The term “therapeutically effective amount” refers to an amount of a therapeutic protein (e.g., an effector molecule) which confers a therapeutic effect on the treated subject, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e.,Attorney Ref. No.: 33978 / 70016 / PC subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0040] Treatment: The term “treatment” (also “treat” or “treating”) refers to any administration of a substance that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0041] Vector: “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. The terms “vector” and “plasmid” may be used interchangeably. In some embodiment, vectors are capable of extra-chromosomal replication and / or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and / or prokaryotic cell. Vectors capable of directing the expression of operatively linked coding sequences (e.g., genes) are referred to herein as “expression vectors.” An expression vector typically comprises an expression cassette. Vectors and plasmids include, but are not limited to,Attorney Ref. No.: 33978 / 70016 / PC replication vectors, probe generation vectors, sequencing vectors, integrating vectors, phagemids, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, viral vectors (e.g., animal virus vectors), cosmids, and artificial chromosomes. II. Engineered Minimal Promoters

[0042] The present disclosure provides methods and compositions comprising engineered nucleic acids that comprise engineered minimal promoters which allow for selective and efficient induction of transcription of an operably linked polynucleotide (e.g., a gene). In many embodiments, an engineered nucleic acid provided by the present disclosure is or comprises an engineered minimal promoter, as described herein. In some embodiments, an engineered minimal promoter is provided in an expression cassette, a heterologous construct, a vector, or other polynucleotide sequence.

[0043] In some embodiments, an engineered minimal promoter is operably linked to an enhancer region (e.g., native or engineered enhancer region), that exhibits remarkable selectivity for inducing transcriptional activity.

[0044] In some embodiments, the engineered minimal promoters comprise a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a nucleotide sequence selected from SEQ ID Nos: 1 – 5. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 1, or comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 1. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 2, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 2. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 3, or comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 3. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 4, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 4. In some embodiments, the engineered minimalAttorney Ref. No.: 33978 / 70016 / PC promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 5, or comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 5.

[0045] In some embodiments, the engineered minimal promoter comprises an upstream promoter element and at least one downstream promoter element. In some embodiments, the upstream promoter element comprises at least a portion of a minimal promoter. In some embodiments, the upstream promoter element comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to TCTAGAGGGTATATAATGGGGGCCATTCTCTGTGTAATTCAGTCCGGTAGTCCGGC (SEQ ID NO: 10).

[0046] In other embodiments, an engineered minimal promoter of the present disclosure comprises a first polynucleotide sequence encoding a minimal promoter or fragment thereof, a second polynucleotide sequence comprising an initiator consensus sequence, and a third polynucleotide sequence comprising a downstream promoter element. In some embodiments, the first polynucleotide sequence is operably linked to the second polynucleotide sequence and the second polynucleotide sequence is operably linked to the third polynucleotide sequence with a first linker sequence and second linker sequence, respectively. In some embodiments, the first linker sequence and / or the second linker sequence do not include transcription factor binding sites. In some embodiments, the first linker sequence and the second linker sequence are identical. In some embodiments, the first linker sequence and the second linker sequence comprise different sequences. In some embodiments, the initiator consensus sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of TCAGTC. In some embodiments, the first polynucleotide or the minimal promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6.

[0047] A “pause button” motif (e.g., a motif including the consensus sequence KCGRWCG) has been found to occur in a subset of promoters that have been stalled. In some instances, the pause button motif may be present in downstream promoter elements. In some embodiments of the disclosure, the downstream promoter element comprises at least one, at least two, or at least three pause button motifs. In some embodiments, the downstream promoter element comprises the consensus nucleotide sequence KCGRWCG, wherein “K” can be guanine or thymine,Attorney Ref. No.: 33978 / 70016 / PC wherein R can be a purine (e.g., guanine or adenine), and / or wherein “W” can be adenine or thymine.

[0048] Downstream promoter elements are thought to improve transcription by improving RNA Pol II binding. Inclusion of such downstream promoter elements may improve the transcriptional output of engineered promoters, potentially due to the presence of a pause button motif. These elements can include Motif Ten Element (MTE), which is a downstream promoter element found in Drosophila melanogaster and has been found to be a new core promoter element that improves transcription. In some cases, a downstream promoter element (DPE) can be combined with MTE forming a fusion of MTE and DPE (i.e., MTE / DPE).

[0049] Another downstream promoter element included in the present disclosure can be DPR (downstream core promoter region), the human ortholog of the MTE / DPE.

[0050] In some embodiments, the downstream promoter element includes MTE / DPE or DPR. In some embodiments, the downstream promoter element comprises MTE / DPE or DPR, or any combination thereof. In some embodiments, the downstream promoter element comprises a nucleotide sequence having at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9, or comprises 1, 2, 3, 4, or 5 nucleotide substitutions compared to SEQ ID NO: 8 or SEQ ID NO: 9. Table 1: Polynucleotide Sequences of Engineered Minimal Promoters and ComponentsAttorney Ref. No.: 33978 / 70016 / PCIII. Engineered Regulatory Elements Comprising an Engineered Minimal Promoter

[0051] The present disclosure also provides methods and compositions comprising engineered regulatory elements comprising the engineered minimal promoter of the present disclosure and an enhancer region.

[0052] In some embodiments, an engineered regulatory element comprises an engineered enhancer region. In some embodiments, an engineered enhancer region is engineered in that it includes a portion where at least one nucleotide motif therein is modified. In some embodiments, an engineered enhancer region allows for high selectivity for inducing transcriptional activity of a particular coding sequence.

[0053] In some embodiments, an engineered regulatory element comprises an enhancer region that is or comprises a wild-type enhancer or engineered enhancer (e.g., an synthetic enhancer comprising one or more engineered regulatory components). In some embodiments, an enhancer region is or comprises one or more enhancer segments. In some embodiments, one or more enhancer segments of an enhancer region are contiguous. In some embodiments, one or more enhancer segments of an enhancer region are non-contiguous. In some embodiments, one or more enhancer segments of an enhancer region are contiguous, and one or more enhancer regions of the same enhancer region are non-contiguous. In some embodiments, a nucleotide sequence of about 1, about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides is located between the first enhancer segment and the second enhancer segment. In some embodiments, a nucleotide sequence of about 1 to about 200, about 1 to about 100, about 1 to about 50, about 1 to about 25, about 10 to about 200, about 10 to about 100, about 10 to about 50, about 10 to about 25, about 25 to about 200, about 25 to about 100, about 25 to about 50, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 50 to about 75, about 100 to about 200, about 100 to about 150, about 25 to about 100, about 30 to about 100, about 40 to about 100, about 25 to about 75, or about 25 to about 50 nucleotides is located between the first enhancer segment and the second enhancer segment.

[0054] In some embodiments, an engineered regulatory element induces expression of an operably linked coding sequence (e.g., a gene) in a cell at a level of specificity of at least aboutAttorney Ref. No.: 33978 / 70016 / PC 5, at least about 10, at least 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 150, at least about 200, at least about 350, at least about 400, at least about 450, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1250, at least about 1500, at least about 2000, at least about 2500, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 25,000, at least about 30,000 times, or more than induced expression.

[0055] In some embodiments, an engineered regulatory element induces expression of an operably linked coding sequence (e.g., a gene) at a comparable level, or strength, to an alternative regulatory element, e.g., as measured by percentage of transcriptional activity as compared to said alternative promoter such as a CAG promoter, which is not intended to be a limiting example. In some embodiments, an engineered minimal promoter and an enhancer region induces expression of an operably linked coding sequence at a strength of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more as compared to an alternative promoter such as a CAG promoter, which is not intended to be a limiting example.

[0056] In some embodiments, an engineered regulatory element comprises an enhancer region, such as a native enhancer region or an engineered enhancer region that is about 50 base pairs, about 60 base pairs, about 70 base pairs, about 80 base pairs, about 90 base pairs, about 100 base pairs, about 110 base pairs, about 120 base pairs, about 130 base pairs, about 140 base pairs, about 150 base pairs, about 160 base pairs, about 170 base pairs, about 180 base pairs, about 190 base pairs, about 200 base pairs, about 220 base pairs, about 240 base pairs, about 260 base pairs, about 280 base pairs, about 300 base pairs, about 320 base pairs, about 340 base pairs, about 360 base pairs, about 380 base pairs, about 400 base pairs, about 420 base pairs, about 440 base pairs, about 460 base pairs, about 480 base pairs, about 500 base pairs, about 520 base pairs, about 540 base pairs, about 560 base pairs, about 580 base pairs, about 600 base pairs, about 620 base pairs, about 640 base pairs, about 660 base pairs, about 680 base pairs, about 700 base pairs, about 720 base pairs, about 740 base pairs, about 760 base pairs, about 780 base pairs, about 800 base pairs, about 820 base pairs, about 840 base pairs, about 860 base pairs, about 880 base pairs, about 900 base pairs, about 920 base pairs, about 940 base pairs, about 960 base pairs, about 980 base pairs, about 1000 base pairs, about 1020 base pairs, about 1040 base pairs, about 1060 base pairs, about 1080 base pairs, or about 1100 base pairs in length.Attorney Ref. No.: 33978 / 70016 / PC

[0057] In some embodiments, an engineered regulatory element comprises an enhancer region, such as a native enhancer region or an engineered enhancer region (e.g., an ablated enhancer region) that is at least about 50 base pairs, at least about 60 base pairs, at least about 70 base pairs, at least about 80 base pairs, at least about 90 base pairs, at least about 100 base pairs, at least about 110 base pairs, at least about 120 base pairs, at least about 130 base pairs, at least about 140 base pairs, at least about 150 base pairs, at least about 160 base pairs, at least about 170 base pairs, at least about 180 base pairs, at least about 190 base pairs, at least about 200 base pairs, at least about 220 base pairs, at least about 240 base pairs, at least about 260 base pairs, at least about 280 base pairs, at least about 300 base pairs, at least about 320 base pairs, at least about 340 base pairs, at least about 360 base pairs, at least about 380 base pairs, at least about 400 base pairs, at least about 420 base pairs, at least about 440 base pairs, at least about 460 base pairs, at least about 480 base pairs, at least about 500 base pairs, at least about 520 base pairs, at least about 540 base pairs, about at least 560 base pairs, at least about 580 base pairs, at least about 600 base pairs, at least about 620 base pairs, at least about 640 base pairs, at least about 660 base pairs, at least about 680 base pairs, at least about 700 base pairs, at least about 720 base pairs, at least about 740 base pairs, at least about 760 base pairs, at least about 780 base pairs, at least about 800 base pairs, at least about 820 base pairs, at least about 840 base pairs, at least about 860 base pairs, about at least 880 base pairs, at least about 900 base pairs, at least about 920 base pairs, at least about 940 base pairs, at least about 960 base pairs, at least about 980 base pairs, at least about 1000 base pairs, about at least 1020 base pairs, at least about 1040 base pairs, at least about 1060 base pairs, at least about 1080 base pairs, or at least about 1100 base pairs in length.

[0058] In some embodiments, an engineered regulatory element comprises an enhancer region, such as a native enhancer region or an engineered enhancer region (e.g., an ablated enhancer region) that is up to about 50 base pairs, up to about 60 base pairs, up to about 70 base pairs, up to about 80 base pairs, up to about 90 base pairs, up to about 100 base pairs, up to about 110 base pairs, up to about 120 base pairs, up to about 130 base pairs, up to about 140 base pairs, up to about 150 base pairs, up to about 160 base pairs, up to about 170 base pairs, up to about 180 base pairs, up to about 190 base pairs, up to about 200 base pairs, up to about 220 base pairs, up to about 240 base pairs, about up to 260 base pairs, up to about 280 base pairs, about up to 300 base pairs, up to about 320 base pairs, up to about 340 base pairs, up to about 360 base pairs, up to about 380 base pairs, up to about 400 base pairs, up to about 420 base pairs, up to about 440 base pairs, up to about 460 base pairs, up to about 480 base pairs, up to about 500 base pairs, up to about 520 base pairs, up to about 540 base pairs, up to about 560 base pairs, up to about 580 base pairs, up to about 600 base pairs, up to about 620 base pairs, up to about 640 base pairs, upAttorney Ref. No.: 33978 / 70016 / PC to about 660 base pairs, up to about 680 base pairs, about up to 700 base pairs, up to about 720 base pairs, up to about 740 base pairs, up to about 760 base pairs, up to about 780 base pairs, about up to 800 base pairs, up to about 820 base pairs, up to about 840 base pairs, up to about 860 base pairs, up to about 880 base pairs, up to about 900 base pairs, up to about 920 base pairs, up to about 940 base pairs, up to about 960 base pairs, up to about 980 base pairs, up to about 1000 base pairs, up to about 1020 base pairs, up to about 1040 base pairs, up to about 1060 base pairs, up to about 1080 base pairs, or up to about 1100 base pairs in length.

[0059] In some embodiments, an engineered regulatory element as described herein comprises at least one spacer sequence. In some embodiments, an engineered regulatory element comprises at least one spacer located between an enhancer region (e.g., a native enhancer region, or engineered enhancer region) and a promoter (e.g., a minimal promoter). In some embodiments, the at least one spacer sequence does not include transcription factor binding sites.

[0060] In some embodiments, an engineered regulatory element comprises a post-transcriptional regulatory element (PRE). PREs can enhance gene expression via enabling tertiary RNA structure stability and 3’ end formation. Non-limiting examples of PREs include the Hepatitis B virus PRE (HPRE) and the Woodchuck Hepatitis Virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises the alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE comprises the alpha component of the WPRE element.

[0061] In some embodiments, engineered nucleic acids (e.g., heterologous constructs, as described herein) are configured to produce multiple agents (e.g., one or more effector molecules) that can be encoded in one or more coding sequences that are operably linked to an engineered an engineered regulatory element, as provided herein. For example, engineered nucleic acids may be configured to produce 2-20 different agents. In some embodiments, engineered nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2- 13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3- 13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5- 11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8- 18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-Attorney Ref. No.: 33978 / 70016 / PC 16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17- 19, 17-18, 18-20, 18-19, or 19-20 agents. In some embodiments, nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 agents. Provided agents are, in many cases, therapeutic agents, such as those described herein.

[0062] In some embodiments, engineered nucleic acids (e.g., heterologous constructs, as described herein) may comprise multicistronic regions, i.e., more than one separate polypeptide (e.g., therapeutic agents, effector molecules, and the like) can be produced from a single mRNA transcript transcribed from said multicistronic region. Multicistronic regions may be created through the use of various linkers, e.g., a first coding sequence can be linked to a second coding sequence with a linker, for example creating a construct comprising, from 5’ to 3’, a first coding sequence, a linker, and a second coding sequence. A linker polynucleotide sequence can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A.2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence, such as such a flexible linker (e.g., a Gly-Ser-Gly sequence), that further promotes cleavage. In some embodiments, a multicistronic region comprises up to two, about to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, up to fifteen, up to twenty, or more coding sequences each linked by a linker (e.g., a first linker, a second linker, a third linker, a fourth linker, and so on).

[0063] The present disclosure provides for, among other things, engineered regulatory elements that comprise an engineered minimal promoter. In some embodiments, the engineered minimal promoter is operably linked to an enhancer region. In many embodiments, provided engineered regulatory elements comprise an enhancer region operably linked to an engineered minimal promoter exhibit remarkable selectivity for activity in a target cell type as compared to a non- target cell type.

[0064] In some embodiments, an engineered minimal promoter comprises an engineered minimal promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NOs: 1 - 5. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%Attorney Ref. No.: 33978 / 70016 / PC sequence identity to SEQ ID NO: 1, or comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 1. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 2, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 2. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 3, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 3. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 4, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 4. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 5, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 5. In some embodiments, the engineered minimal promoters comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 5, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions compared to SEQ ID NO: 10.

[0065] In some embodiments, an engineered regulatory element comprises an enhancer region that is engineered in that it includes a portion where at least one nucleotide motif therein is modified. In some embodiments, an engineered enhancer region is engineered in that it includes one or more components from different regulatory elements. In some embodiments, an engineered enhancer region allows for high selectivity for inducing transcriptional activity of a particular coding sequence. In some embodiments, an engineered regulatory element comprises an engineered enhancer region. In some embodiments, an engineered regulatory element comprises an engineered enhancer region that is operably linked to a promoter (e.g., a minimal promoter). III. Heterologous Constructs

[0066] Certain aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) comprising one or more engineered regulatory elements as described herein toAttorney Ref. No.: 33978 / 70016 / PC produce a heterologous construct. In some embodiments, a provided heterologous construct further comprises one or more expression cassettes.

[0067] In some embodiments, a heterologous construct comprises an engineered regulatory element that is operably linked to a coding polynucleotide sequence (e.g., a gene, or coding sequence for a bioactive molecule). In some embodiments, a heterologous construct comprises an engineered regulatory element that is operably linked to a polynucleotide sequence encoding at least one effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth). In some embodiments, an effector molecule comprises a bioactive molecule. In some embodiments, an effector molecule comprises a polypeptide. In some embodiments, an effector molecule comprises a polynucleotide (e.g., a mRNA, miRNA, siRNA, shRNA, etc.). In some embodiments, an effector molecule is a human-derived effector molecule.

[0068] In some embodiments, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules under the transcriptional control of an engineered regulatory element of the present disclosure. In some embodiments, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules each under the transcriptional control of separate engineered regulatory elements.

[0069] In some embodiments, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules that are in the same reading frame and are expressed as a single polypeptide chain. In some embodiments, two or more effector molecules that are expressed as a single polypeptide chain may comprise one or more peptide cleavage sites (e.g., auto-cleavage sites, or cleavage sites for an intracellular protease) which when cleaved separate the two or more effector molecules. Suitable peptide cleavage sites may include, without limitation, a T2A peptide cleavage site, a P2A peptide cleavage site, an E2A peptide cleavage sire, and an F2A peptide cleavage site.

[0070] In some embodiments, two or more effector molecules that are expressed as a single polypeptide chain comprise a T2A peptide cleavage site. In some embodiments, two or more effector molecules that are expressed as a single polypeptide chain comprise an E2A peptide cleavage site. In some embodiments, two or more effector molecules that are expressed as a single polypeptide chain comprise a T2A and an E2A peptide cleavage site.

[0071] In some embodiments, a polynucleotide sequence encoding a first effector molecule is linked to a polynucleotide sequence encoding a second effector molecule by a linker polynucleotide sequence. In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding at least one 2A ribosome skipping element. In some embodiments, a 2A ribosome skipping element is a T2A, a P2A, a E2A, or a F2A element.Attorney Ref. No.: 33978 / 70016 / PC

[0072] Any suitable effector molecule known in the art can be encoded in or expressed by a polynucleotide within a provided heterologous construct. In some embodiments, an effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth) is a therapeutic molecule. Suitable effector molecules can be grouped into therapeutic classes based on structure similarity, sequence similarity, or function. Effector molecule therapeutic classes include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, receptors, ligands, antibodies, polynucleotides, peptides, shRNAs, miRNAs, and enzymes. Accordingly, in some embodiments, an effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth) belongs to a therapeutic class selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a receptor, a ligand, an antibody, a peptide, an RNA molecule (e.g., mRNA, miRNA, siRNA, shRNA, etc.), and an enzyme.

[0073] In some embodiments, an effector molecule is a chemokine. Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC, all of which exert biological effects by binding selectively to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by polynucleotides in a heterologous construct of the present disclosure include: CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCl25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5 CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or combinations thereof.

[0074] In some embodiments, an effector molecule is a cytokine. Non-limiting examples of cytokines that may be encoded by polynucleotides in a heterologous construct of the present disclosure include: IL-1-alpha, IL-1-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17A, IL-17B / C / D, IL-17E (IL-25), IL-17F, 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-34, IL-35, IL-36-alpha, IL-36-beta, IL-36-gamma, IL-37, IL-38, IFN-alpha, IFN-beta, IFN- gamma, TGF-beta, GM-CSF, CSF-1 / M-CSF, G-CSF, SCF, TNF-alpha, TNF-beta, growth hormone (GH), prolactin (PRL), erythropoietin (EPO), leptin, FLT3 ligand, or combinations thereof.

[0075] In some embodiments, an effector molecule is a tumor microenvironment modifier. Suitable tumor microenvironment modifiers for use as an effector molecule include, but are notAttorney Ref. No.: 33978 / 70016 / PC limited to, adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof.

[0076] In some embodiments, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one TGF-beta inhibitor. Suitable TGF-beta inhibitors for use as an effector molecule include, but are not limited to, an anti-TGF-beta peptide, an anti-TGF-beta antibody, a TGF-beta-TRAP, or combinations thereof.

[0077] In some embodiments, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one immune checkpoint inhibitor. Suitable immune checkpoint inhibitors for use as an effector molecule include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti- BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNF-alpha antibodies, anti-TREM1 antibodies, and anti- TREM2 antibodies, or combinations and / or functional fragments thereof.

[0078] Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK- 3475 / Keytruda® - Merck), nivolumamb (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 – Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio® - Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq® - Roche / Genentech), BMS- 936559 (anti-PD-L1 - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma / AstraZeneca).

[0079] In some embodiments, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one VEGF inhibitor. Suitable VEGF inhibitors for use as an effector molecule include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitors comprise anti- VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0080] In some embodiments, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one therapy that treats an CNS / neuronal disease or disorder. In some embodiments, a CNS / neuronal disease or disorder includes neuronal disorders, including stroke, ischemia, epilepsy, head and spinal cord trauma, Parkinson's diseases, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and neurogenetic disorders.Attorney Ref. No.: 33978 / 70016 / PC

[0081] In some embodiments, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one gene replacement therapy. A gene therapy is a therapy that replaces a non-functional, partially functional, or missing disease-related gene with an engineered version of the same disease-related gene, thereby providing a therapeutically relevant amount of the disease-related gene product. In some embodiments, a gene replacement therapy is a therapy that treats a disease or disorder.

[0082] In some embodiments, an effector molecule as described may comprise a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the effector molecule’s N- terminus. Without wishing to be bound by theory, a secretion signal peptide or signal sequence is understood to direct newly synthesized proteins destined for secretion or membrane insertion to the proper protein processing pathways. In embodiments with two or more effector molecules, each effector molecule can comprise a secretion signal.

[0083] In some embodiments, a secretion signal peptide operably associated with a effector molecule can be a native secretion signal peptide (e.g., a secretion signal peptide that is natively associated with the given effector molecule). In some embodiments, a secretion signal peptide operably associated with an effector molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments of interest, e.g., those related to diseases or disorders. IV. Vectors / Plasmids

[0084] Another aspect of the disclosure relates to a vector comprising a nucleotide sequence encoding an engineered regulatory element (e.g., a heterologous construct), as described herein. In some embodiments, a vector is an expression vector. Such an expression vector comprises a nucleotide sequence encoding any engineered regulatory element disclosed herein operably linked a coding nucleotide sequence (e.g., for an effector molecule, e.g., as described herein) to allow for expression of said coding nucleotide sequence in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a nucleic acid molecule encoding engineered regulatory elements of the present disclosure including, without limitation, viral expression vectors, prokaryotic expression vectors, eukaryotic expression vectors (e.g., yeast expression vectors, insect expression vectors, mammalian expression vectors, etc.), and cell-free extract expression vectors.

[0085] It is further understood that expression vectors useful to practice aspects of methods described herein may include additional promoters (e.g., inducible, constitutive, cell-specific), enhancer elements, or both. Expression vectors may include polynucleotides encoding proteinAttorney Ref. No.: 33978 / 70016 / PC tags, or epitope tags, to aid in isolation, purification or selection (e.g., poly-His tags, FLAG-tags, hemagglutinin tags, fluorescent protein tags, bioluminescent tags, and nuclear localization tags). As described herein, coding sequences for such protein tags, or epitope tags, can be fused to a coding sequence or can be included in a separate expression cassette. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences- Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.

[0086] In some embodiments, a vector comprises a transposon / transposase system to incorporate one or more nucleotides of the present disclosure into a host cell genome. In some embodiments, a transposon system used in accordance with the present disclosure is a Sleeping Beauty transposon / transposase or the piggyBac transposon / transposase.

[0087] In some embodiments, an expression vector of the present disclosure may be provided to a cell in the form of a viral vector. Suitable viral vector systems are well known in the art. For example, viral vectors may be derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, a vector of the present disclosure is a lentiviral vector. Lentiviral vectors are suitable for long-term gene transfer as such vectors allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors are also advantageous over vectors derived from onco-retroviruses (e.g., murine leukemia viruses) in that lentiviral vectors can transduce non-proliferating cells. In some embodiments, a vector of the present disclosure is an adenoviral vector (A5 / 35).

[0088] In some embodiments, a vector of the present disclosure contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and US 6,326,193). A number of viral based systems have been developed for gene transfer into mammalian cells. A selected gene (e.g., alone, or within an expression cassette, or heterologous construct) can be inserted into a vector and packaged in retroviral particles using techniques known in the art. A recombinant virus can then be isolated and delivered to mammalian cells either in vivo or ex vivo. A number of retroviral systems are known in the art.

[0089] In some embodiments, vectors of the present disclosure comprise regulatory elements such as enhancers that regulate the frequency of transcriptional initiation. Enhancers are typically located in a region that is 30 bp to 110 bp upstream of a transcription start site,Attorney Ref. No.: 33978 / 70016 / PC although a number of promoters have been shown to contain functional elements downstream of the start site as well. In general, the spacing between regulatory elements may be flexible, so that transcriptional function is preserved when regulatory elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements may function either cooperatively or independently to activate transcription. Exemplary promoters may include, without limitation, a SFFV gene promoter, an EFS gene promoter, a CMV IE gene promoter, an EF1a promoter, a ubiquitin C promoter, a phosphoglycerokinase (PGK) promoter, or functional fragments or combinations thereof.

[0090] In some embodiments, a vector of the present disclosure may further comprise a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator, an element allowing episomal replication, and / or elements allowing for selection.

[0091] In some embodiments, a vector of the present disclosure can further comprise a selectable marker gene and / or a reporter gene to facilitate identification and selection of certain cells (e.g., those comprising an engineered regulatory element as described herein) from a population of cells that have been transduced with said vector. In some embodiments, a selectable marker may be encoded by a polynucleotide that is separate from a vector and used in a co-transfection procedure. A selectable marker or a reporter gene may be flanked with appropriate regulatory sequences to allow expression in a host cell. In some embodiments, a selectable marker comprises an antibiotic resistance gene. Examples of antibiotic resistance genes include, without limitation, kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, and combinations thereof.

[0092] In some embodiments, a reporter gene may be used for identifying transduced cells and for evaluating the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide that has an easily detectable property, such as enzymatic activity or fluorescence. Expression of a reporter gene can be assayed at a suitable time after a polynucleotide comprising said reporter gene has been introduced into a recipient or host cell that is capable of expressing said reporter gene. Examples of reporter genes include, without limitation, genes encoding for luciferase (or variants or fragments of luciferase, e.g., nanoluciferase), genes encoding for beta- galactosidase, genes encoding for chloramphenicol acetyl transferase, genes encoding for secreted alkaline phosphatase, and genes encoding for green fluorescent protein (GFP) (or common variants or fragments of GFP, e.g., RFP, YFP, etc.). Suitable expression systems are well known in the art and may be prepared using known techniques or obtained commercially.Attorney Ref. No.: 33978 / 70016 / PC V. Engineering Cells

[0093] The present disclosure further provides for methods and compositions for preparing and using engineered cells comprising a heterologous construct comprising an engineered regulatory element as provided herein.

[0094] In some embodiments, engineered cells comprise an engineered enhancer region or an engineered regulatory element provided herein.

[0095] Also provided herein are compositions and methods for engineering cells that are capable of producing one or more effectors molecules (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth). Effector molecules of the present disclosure may be encoded in one or more heterologous constructs or expression cassettes as described herein or otherwise known in the art.

[0096] In some embodiments, cells of the present disclosure are engineered to produce effector molecules through introduction (i.e., delivery) of one or more polynucleotides that encode for one or more effector molecules (e.g., those provided herein). For example, polynucleotide heterologous constructs or expression cassettes encoding one or more effector molecules can be any of the engineered nucleic acids described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means (e.g., including all methods described herein). One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered. A. Viral-Mediated Delivery

[0097] Viral vector-based delivery platforms can be engineered to deliver certain nucleic acids of interest into a host cell in order to create an engineered cell as described herein. In general, a viral vector-based delivery platform can be used to create an engineered cell by introducing (i.e., delivering, or transducing) a heterologous nucleic acid, e.g., a transgene, expression cassette, or a heterologous construct as described herein, into a particular host cell. In many embodiments of the present disclosure, a viral vector-based delivery platform delivers a nucleic acid comprising an engineered neuron -specific regulatory element as described herein, or a heterologous construct as described herein. In some embodiments, a transduced nucleic acid is integrated into a host cell genome. Viruses created for use in viral vector-based delivery platforms can be referred to as recombinant viruses, or engineered viruses. It is understood that a recombinant virus may encode one or more viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), in someAttorney Ref. No.: 33978 / 70016 / PC cases referred to as cis-acting elements or cis-acting genes, in addition to nucleic acids to be delivered to a host cell.

[0098] In some embodiments, one or more recombinant viruses may be used to deliver nucleic acids encoding one or more genes (e.g., transgenes), expression cassettes, or heterologous constructs. In many embodiments, delivered nucleic acids comprise one or more nucleotide sequences comprising engineered neuron -specific element as described herein. In some embodiments, delivered nucleic acids (e.g., heterologous constructs) are configured to express one or more effector molecules.

[0099] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding genes (e.g., transgenes), expression cassettes, or heterologous constructs described herein, and referred to as trans-acting elements or trans-acting genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and / or viral production on one or more additional separate vectors in addition to a vector encoding a transgene expression cassette, or heterologous construct. The number of viral vectors used can depend on the packaging capacity of the above-mentioned viral vector-based platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0100] In general, any of viral vector-based system can be used for the in vitro expression of engineered nucleic acids (e.g., heterologous constructs), e.g., for the production of molecules, such as effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids comprising nucleotide sequences encoding one or more effector molecules under transcriptional control of an engineered neuron -specific regulatory element. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.

[0101] Viral vector-based delivery platforms can utilize RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10,Attorney Ref. No.: 33978 / 70016 / PC 616—629), or lentivirus, including but not limited to second, third or hybrid second / third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873- 9880).

[0102] Provided engineered nucleic acid sequences may be preceded with one or more nucleic acid sequences that by themselves, or via their encoded polypeptide sequence, target a subcellular compartment. Upon introduction (i.e., delivery) of engineered nucleic acids into a host cell, infected cells (i.e., engineered cells) can express polypeptides encoded by said engineered nucleic acids (e.g., one or more effector molecules), and in some case secrete, said polypeptides. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No.4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

[0103] The viral vector-based delivery platforms described herein can utilize a virus that targets a tumor cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus comprising one more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs, etc.).Attorney Ref. No.: 33978 / 70016 / PC

[0104] In some embodiments, a recombinant virus is created from a virus selected from: a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0105] A viral vector-based delivery platform as used in accordance with the present disclosure can be retrovirus-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of said vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs, etc.) into a target cell to provide permanent integration and / or expression of said engineered nucleic acids. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al, J. Virol.66:1635-1640 (1992); Sommnerfelt et al., Virol.176:58-59 (1990); Wilson et al, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol.65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retrovirus system.

[0106] A viral vector-based delivery platform as used in accordance with the present disclosure can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). Lentiviral-based delivery platforms can be SIV, or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; 6,955,919, each herein incorporated by reference for all purposes.

[0107] A viral vector-based delivery platform as used in accordance with the present disclosure can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail inAttorney Ref. No.: 33978 / 70016 / PC U.S. Pat. Nos.5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO 96 / 13597, each herein incorporated by reference for all purposes.

[0108] A viral vector-based delivery platform as used in accordance with the present disclosure can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of an engineered nucleic acids, e.g., those encoding one or more effector molecules (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos.4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; US patent publications US 2003 / 0138772, US 2007 / 0036760, and US 2009 / 0197338; Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.94:1351 (1994), each of which are herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No, 5,173,414; Tratschin et al., Mol. Cell. Biol.5:3251- 3260 (1985); Tratschin et al., Mol. Cell, Biol.4:2072-2081 (1984); Muzyczka, PNAS 81:64666470 (1984); and Samuiski et al., J. Virol.63:03822-3828 (1989), each of which are herein incorporated by reference for all purposes. In general, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11 and variants thereof.

[0109] A viral vector-based delivery platform as used in accordance with the present disclosure can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. Production of VLPs are described in more detail in Seow et al. (Mol Ther.2009 May; 17(5): 767–777), herein incorporated by reference for all purposes.Attorney Ref. No.: 33978 / 70016 / PC

[0110] A viral vector-based delivery platform as used in accordance with the present disclosure can be engineered to target (i.e., infect or transduce) a range of cells, target a narrow subset of cells, or target a specific cell. In general, an envelope protein chosen for a viral vector-based delivery platform will determine viral tropism. A virus used in a viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. A viral vector-based delivery platform can be pantropic and infect a range of cells. For example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. A viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting a desired cell type. B. Lipid Structure Delivery Systems

[0111] Engineered nucleic acids of the present disclosure (e.g., any of engineered nucleic acid as described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0112] A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. A “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid, such as any engineered nucleic acid as described herein, within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Attorney Ref. No.: 33978 / 70016 / PC Biophys. Bioeng.9; 467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each of which are herein incorporated by reference for all purposes

[0113] A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing self-rearrangement. A desired cargo (e.g., a polypeptide, a nucleic acid, a small molecule drug, a engineered nucleic acid, such as any engineered nucleic acid as described herein, a viral vector, a viral-based delivery system, etc.) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with a lipid bilayer.

[0114] A liposome used in accordance with the present disclosure can be made by different methods, as would be known to one of ordinary skill in the art. Preparations of liposomes are described in further detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each of which are hereby incorporated by reference for all purposes.

[0115] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No.5,962,016; 5,030,453; 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications WO 03 / 015757A1, WO 04029213A2, and WO 02 / 100435A1, each of which are herein incorporated by reference in their entirety

[0116] Lipid-mediated gene delivery methods are described, for instance, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No.5,279,833; WO91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each of which are herein incorporated by reference for all purposes.

[0117] The term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any engineered nucleic acid as described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated fromAttorney Ref. No.: 33978 / 70016 / PC the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is hereby incorporated by reference in its entirety. Exosomes useful for the delivery of nucleic acids are known to those skilled in the art, e.g., the exosomes described in more detail in U.S. Pat. No.9,889,210, herein incorporated by reference for all purposes.

[0118] The term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The cargo can comprise nucleic acids (e.g., any engineered nucleic acid as described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0119] The term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any engineered nucleic acid as described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) orAttorney Ref. No.: 33978 / 70016 / PC other molecules. A nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

[0120] Lipid nanoparticles (LNPs), in general, are engineered lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo / payloads, such as any engineered nucleic acid or viral system described herein, by absorbing into a membrane of a target cell and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be engineered or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability. In an example, the lipid composition comprises dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity, which will be appreciated by those skilled in the art.

[0121] Micelles, in general, are spherical engineered lipid structures that are formed using single-chain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther.2017 Jul 5; 25(7): 1501–1513), herein incorporated by reference for all purposes.

[0122] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of a nucleic acid by serum nucleases or off-target stimulation of an immune system by free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and / or neutralization of a viral delivery system. Therefore, encapsulation of an engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off- target affects. In certain examples, an engineered nucleic acid and / or viral delivery system isAttorney Ref. No.: 33978 / 70016 / PC fully encapsulated within a delivery vehicle, such as within an aqueous interior of an LNP, or other vesicle or lipid system as described herein. Encapsulation of an engineered nucleic acid and / or viral delivery system within an LNP or other lipid system can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, a desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to a droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that a delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, delivery vehicles encapsulating a cargo / payload (e.g., an engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration. C. Nanoparticle Delivery

[0123] Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, can be made of non-immunogenic materials and generally avoid eliciting immunity to a delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery—A Review. Nanomaterials 2017, 7(5), 94), each of which are herein incorporated by reference for all purposes. D. Genomic Editing Systems

[0124] A genomic editing system can be used to engineer a host genome to encode a engineered nucleic acid, such as any engineered nucleic acid described herein. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vector-based delivery platform (e.g., those described herein).

[0125] A transposon system can be used to integrate an engineered nucleic acid, e.g., an engineered nucleic acid as described herein, into a host genome. Transposons generallyAttorney Ref. No.: 33978 / 70016 / PC comprise terminal inverted repeats (TIR) that flank a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the TIR- flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo / payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tc1 / mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol.2017 Aug;52(4):355-380), and U.S. Patent Nos.6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos.6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes

[0126] A nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. Without wishing to be bound by theory, in general, nuclease-mediated gene editing systems used to introduce an exogenous gene, or nucleic acid, take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into a cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with a lesion that is included between the 5’ and 3’ complimentary ends within a HRT (e.g., a gene or a portion of a gene, or engineered nucleic acid as described herein) can be incorporated (i.e., “integrated”) into a given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a second region of an endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any engineered nucleic acid as described herein, e.g., those encoding one or more effector molecules).

[0127] In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.Attorney Ref. No.: 33978 / 70016 / PC

[0128] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of a HR template, with respect to an exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of an endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of an endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

[0129] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.

[0130] A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator- like effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0131] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid as described herein, e.g., those encoding one or more effector molecules described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by reference for all purposes. In general, a CRISPR- mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 systems comprised of a Cas9 nuclease and a RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with andAttorney Ref. No.: 33978 / 70016 / PC thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpf1 system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double-stranded break typically produced by Cas9 enzymes.

[0132] In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPR systems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced (i.e., “delivered”) into a cell’s cytosol and / or nucleus, e.g., a T cell’s cytosol and / or nucleus. The in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro (i.e., synthesized and purified) using a variety of protein production techniques known to those skilled in the art. CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro (i.e., synthesized and purified) using a variety of RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0133] An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can be also be used at different amounts in a CRISPR-mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

[0134] In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see description of exemplaryAttorney Ref. No.: 33978 / 70016 / PC multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

[0135] Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

[0136] Engineered cells described herein can be engineered using non-viral methods, e.g., nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of other viral-based delivery methods described herein.

[0137] In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

[0138] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an engineered site-specific nuclease, which is composed of the DNA-binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a double- stranded break. TALEN-based systems are described in more detail in U.S. Ser. No.12 / 965,590; U.S. Pat. No.8,450,471; U.S. Pat. No.8,440,431; U.S. Pat. No.8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No.13 / 738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;Attorney Ref. No.: 33978 / 70016 / PC 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos.2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties for all purposes. E. Other Engineering Delivery Systems

[0139] Various additional means to introduce engineered nucleic acids (e.g., any engineered nucleic acid as described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0140] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo / payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of a target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of engineered nucleic acid as described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, type of cell or other recipient entity, cargo to be delivered, efficiency of internalization desired, and viability desired. Optimization of such criteria are within the scope of those skilled in the art. A variety of devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

[0141] Other means for introducing engineered nucleic acids (e.g., any of engineered nucleic acid as described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.

[0142] Compositions and methods for delivering engineered mRNAs in vivo, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther.2019 Apr 10; 27(4): 710–728) and Kaczmarek et al. (Genome Med.2017; 9: 60.), each of which are herein incorporated by reference for all purposes.Attorney Ref. No.: 33978 / 70016 / PC VI. Methods of Use

[0143] Methods and compositions for treating a subject with a disease or disorder are encompassed by the present disclosure. Provided methods include administering to a subject with a disease or disorder a therapeutically effective amount of any engineered nucleic acid (e.g., a heterologous construct) or engineered cell (e.g., an isolated engineered cell) described herein.

[0144] In some embodiments, methods and compositions provided herein may be used to treat a disease or disorder. In this regard, for example, the disclosure provides use of, e.g., a heterologous construct described herein or engineered cell described herein for the manufacture of a medicament for the treatment of a disease or disorder. In some embodiments, use of a heterologous construct described herein or an engineered cell disclosed for the treatment of a disease or disorder is provided.

[0145] The disclosure also provides a method of increasing expression of a target gene using any of the engineered minimal promoters described herein, any of the engineered regulatory elements described herein, any of the heterologous constructs described herein, any vector described herein, any dual expression vector described herein, or any engineered cell described herein. In some embodiments, any of the engineered nucleic acids (e.g., any heterologous construct) or engineered cell (e.g., isolated engineered cell) may be used for the manufacture of a medicament for increasing expression of a target gene. The disclosure also contemplates use of any of the engineered minimal promoters described herein, any of the engineered regulatory elements described herein, any of the heterologous constructs described herein, any vector described herein, any dual expression vector described herein, or any engineered cell described herein to increase expression of a target gene.

[0146] In various aspects of the method or use, the engineered minimal promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 – 5. VII. Pharmaceutical Compositions

[0147] Provided engineered nucleic acids or engineered cells can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the engineered nucleic acids or engineered cells, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. TheAttorney Ref. No.: 33978 / 70016 / PC precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0148] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or engineered oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0149] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.

[0150] Composition provided herein can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. VIII. Kits

[0151] Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of disease or disorder. In certain embodiments, a disease or disorder is a cancer (e.g., solid tumors). In certain embodiments, a kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more engineered nucleic acids (e.g., isolate engineered nucleic acids) of the present disclosure, vectors of the present disclosure, and / or engineered cells of the present disclosure. In some embodiments, a kit comprises a sterile container. In some embodiments, such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. A container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments

[0152] In some embodiments, a therapeutic or prophylactic composition is provided together with instructions for administering said therapeutic or prophylactic composition to a subject having or at risk of developing a particular disease or disorder. In some embodiments, the instructions may include information about use of a composition for the treatment and / or prevention of a disease or disorder. In some embodiments, the instructions include, without limitation, a description of a therapeutic or prophylactic composition, a dosage schedule, an administration schedule for treatment or prevention of a disease or disorder or a symptom thereof, precautions, warnings, indications, counter-indications, over-dosage information,Attorney Ref. No.: 33978 / 70016 / PC adverse reactions, animal pharmacology, clinical studies, and / or references. In some embodiments, the instructions can be printed directly on a container (when present), or as a label applied to a container, or as a separate sheet, pamphlet, card, or folder supplied in or with a container.

[0153] Throughout the description, where agents, compounds, entities, and so forth (e.g., provided engineered nucleic acids) are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are agents, compounds, entities, and so forth of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0154] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0155] Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way. Enumerated Embodiments: Embodiment 1 An engineered minimal promoter comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 – 5. Embodiment 2 An engineered minimal promoter comprising: a. an upstream promoter element; and b. at least one downstream promoter element, and c. wherein the upstream promoter element comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to TCTAGAGGGTATATAATGGGGGCCATTCTCTGTGTAATTCAGTCCGGT AGTCCGGC (SEQ ID NO: 10). Embodiment 3 An engineered minimal promoter comprising:Attorney Ref. No.: 33978 / 70016 / PC a. a first polynucleotide sequence encoding a minimal promoter sequence derived from YB_TATA; b. a second polynucleotide sequence comprising an initiator sequence (Inr); and c. a third polynucleotide sequence comprising at least one downstream promoter element, and d. wherein the first polynucleotide sequence is operably linked to the second polynucleotide sequence and the second polynucleotide sequence is operably linked to the third polynucleotide sequence with a first linker sequence and second linker sequence, respectively, and wherein the first linker sequence and / or the second linker sequence does not comprise a transcription factor binding site. Embodiment 4 The engineered minimal promoter of embodiment 2 or 1.b, wherein the at least one downstream promoter element comprises a first downstream promoter element and a second downstream promoter element. Embodiment 5 The engineered minimal promoter of any one of embodiments 2 - 2, wherein the at least one downstream promoter element comprises a pause button motif. Embodiment 6 The engineered minimal promoter of embodiment 3, wherein the pause button motif comprises the consensus nucleotide sequence KCGRWCG. Embodiment 7 The engineered minimal promoter of any one of embodiments 2 - 3, wherein the at least one downstream promoter element is MTE / DPE or DPR. Embodiment 8 The engineered minimal promoter of any one of embodiments 1.b – 2, wherein the initiator sequence comprises a nucleic acid sequence of TCAGTC. Embodiment 9 The engineered minimal promoter of any one of embodiments 1.b – Error! Reference source not found., wherein the first polynucleotide sequence comprises SEQ ID NO: 6. Embodiment 10 The engineered minimal promoter of any one of embodiments 1-5, wherein the downstream promoter element comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9.Attorney Ref. No.: 33978 / 70016 / PC Embodiment 11 An engineered regulatory element comprising: a. an enhancer region; and b. the engineered minimal promoter of any one of embodiments 1-3. Embodiment 12 The engineered regulatory element of embodiment 5, wherein the enhancer region is upstream of the upstream promoter element. Embodiment 13 The engineered regulatory element of embodiment 5 or 6, wherein the enhancer region is operably linked to the minimal promoter. Embodiment 14 The engineered regulatory element of any one of embodiments 5-6, wherein the enhancer region is heterologous to the engineered minimal promoter. Embodiment 15 The engineered regulatory element of any one of embodiments 5-6, wherein the engineered regulatory element comprises at least one spacer sequence between the engineered minimal promoter and the enhancer region. Embodiment 16 A heterologous construct comprising the engineered minimal promoter of any one of embodiments 1-3 or the engineered regulatory element according to any one of embodiments 5-6, wherein the engineered minimal promoter or the engineered regulatory element is operably linked to a polypeptide-encoding polynucleotide, wherein the polypeptide-encoding polynucleotide comprises a polynucleotide sequence encoding a polypeptide. Embodiment 17 The heterologous construct of embodiment 7, wherein the polypeptide comprises at least one effector molecule. Embodiment 18 The heterologous construct of embodiment 7 or embodiment 7, wherein the polypeptide comprises a first effector molecule and a second effector molecule. Embodiment 19 The heterologous construct of any one of embodiments 7-7, wherein the polypeptide-encoding polynucleotide comprises a polynucleotide sequence encoding the first effector molecule, a linker polynucleotide sequence, and a polynucleotide sequence encoding the second effector. Embodiment 20 The heterologous construct of embodiment 8, wherein the linker polynucleotide sequence encodes one or more 2A ribosome skipping elements.Attorney Ref. No.: 33978 / 70016 / PC Embodiment 21 The heterologous construct of embodiment 8, wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A. Embodiment 22 The heterologous construct of any one of embodiments 7-8, wherein the at least one effector molecule belongs to a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, an antibody, a peptide, and an enzyme. Embodiment 23 The heterologous construct of embodiment 9, wherein each of the first effector molecule and the second effector molecule is from a separate therapeutic class. Embodiment 24 The heterologous construct of any one of embodiments 7-9, wherein each of the at least one effector molecule is a human-derived effector molecule. Embodiment 25 A vector comprising the heterologous construct according to any one of embodiments 7-9. Embodiment 26 A dual expression vector comprising the heterologous construct according to any one of embodiments 7-9 and a second construct comprising a second polypeptide- encoding polynucleotide sequence encoding a second effector protein. Embodiment 27 An engineered cell comprising the heterologous construct according to any one of embodiments 7-9, the vector according to embodiment 10, or the dual expression vector according to embodiment 10. Embodiment 28 The engineered cell of embodiment 11, wherein the engineered cell expresses at least one effector molecule. Embodiment 29 A pharmaceutical composition comprising the engineered minimal promoter of any one of embodiments 1-3, the engineered regulatory element according to any one of embodiments 5-6, the heterologous construct according to any one of embodiments 7-9, the vector of embodiment 10, the dual expression vector according to embodiment 10, or the engineered cell according to embodiment 11 or 11, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereofAttorney Ref. No.: 33978 / 70016 / PC Embodiment 30 A method of increasing expression of a target gene, the method comprising use of the engineered minimal promoter of any one of embodiments 1-3, the engineered regulatory element according to any one of embodiments 5-6, the heterologous construct according to any one of embodiments 7-9, the vector of embodiment 10, the dual expression vector according to embodiment 10, or the engineered cell according to embodiment 11 or 11, to increase expression of the target gene. Embodiment 31 A method of treating a subject in need thereof, the method comprising administering the engineered minimal promoter of any one of embodiments 1-3, the engineered regulatory element according to any one of embodiments 5-6, the heterologous construct according to any one of embodiments 7-9, the vector of embodiment 10, the dual expression vector according to embodiment 10, or the engineered cell according to embodiment 11 or 11. Embodiment 32 A method of treating a subject having a disease or disorder, the method comprising administering the engineered minimal promoter of any one of embodiments 1-3, the engineered regulatory element according to any one of embodiments 5-6, the heterologous construct according to any one of embodiments 7-9, the vector of embodiment 10, the dual expression vector according to embodiment 10, or the engineered cell according to embodiment 11 or 11, to the subject in need thereof. Embodiment 33 A kit for treating and / or preventing a disease or disorder, comprising the pharmaceutical composition according to embodiment 12. Embodiment 34 The kit according to embodiment 15, wherein the kit further comprises written instructions for using the pharmaceutical composition for treating and / or preventing a tumor in a subject. Embodiment 35 Use of a heterologous construct of Embodiment 7 for the manufacture of a medicament for treatment of increasing expression of a target gene. Embodiment 36 Use of the engineered cell of Embodiment 11 for the manufacture of a medicament for the treatment of increasing expression of a target gene.Attorney Ref. No.: 33978 / 70016 / PC EXAMPLES

[0156] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way. Example 1: Development and Characterization of Engineered Minimal Promoters

[0157] In this study, engineered minimal promoters were developed and evaluated for their ability to drive expression of a payload when used in combination with enhancer regions (i.e., engineered regulatory elements). The engineered minimal promoters were designed such that they contain a portion of the synthetic minimal promoter, YB_TATA, an initiator (Inr) sequence, and a downstream promoter element such as MTE / DPE or DPR. The engineered minimal promoter was further modified to remove transcription factor binding sites, such as those associated with viruses, in regions not within the synthetic minimal promoter, the Inr, or the downstream promoter element. Engineered minimal promoters were evaluated by flow cytometry by measuring expression of GFP or mCherry as a readout of the activity of the engineered regulatory element having the chimeric minimal promoter. Cell lines

[0158] Surrogate cell lines used in the study are: IMR-32, Huh-7, and primary human astrocytes. Primary human astrocyte served as ON-target cell, and Huh-7, a hepatoma cell line, and IMR-32 neuroblastoma cell line, serve as OFF-target cell lines. Transfection

[0159] Cell lines were co-transfected with two DNA vectors: one vector coding for mCherry driven by CAG promoter serving as an activity control, a second vector coding for GFP driven by screened enhancer and / or promoter sequences. Transfections were performed using commonly used transfection reagents, according to the manufacturer’s guidelines, with specific detailed changes found to be beneficial.

[0160] ON Core promoters were designed and inserted downstream of an enhancer sequence expected to drive ON-target astrocyte-specific expression. DNA synthesis was performed by GenScript. Primary human astrocyte cells cultured on poly-L-lysine-coated flasks were seeded for transfection at a density of 20,0000 cells per well in a poly-L-lysine-coated 96-well plate. Cells were cotransfected one day post-seeding with 100ng of total DNA using Lipofectamine LTX with plus reagents. Transfected cells were then incubated for 48 hours at 37 degrees Celsius post-transfection. After 48 hours, cells were trypsinized for 5 minutes in 75 uL of 1xAttorney Ref. No.: 33978 / 70016 / PC TrypLE Express, centrifuged at 400xg for 4 minutes and supernatant removed, resuspended in PBS + 2.5mM of EDTA with 1:1000 Sytox Blue, and taken for flow cytometry. Fluorescent protein expression was analyzed on a CytoFlex LX at a flow rate of 100 uL per minute.

[0161] OFF-target expression was evaluated in Huh7 cells. Cells were seeded at a density of 20,000 cells in a 96-well plate. Cells were cotransfected one day post-seeding with 100ng of total DNA using the FuGENE HD reagent. Transfected cells were then incubated for 48 hours at 37 degrees Celsius post-transfection. After 48 hours, cells were trypsinized for 5-10 minutes in 75 uL of 1x TrypLE Express, centrifuged at 400xg for 4 minutes and supernatant removed, resuspended in PBS + 2.5mM of EDTA with 1:1000 Sytox Blue, and taken for flow cytometry. Fluorescent protein expression was analyzed on a CytoFlex LX at a flow rate of 100 uL per minute.

[0162] OFF-target expression was evaluated in differentiated IMR-32 cells. Basal IMR-32 cells were maintained in 5 uM Brdu for 13 days for cell differentiation. Differentiated IMR-32 cells were harvested, resuspended in supplemented Lonza P3 buffer, and seeded at a density of 200,000 cells per well in a 96-well Lonza nucleofection plate.1 ug of total DNA was added to each sample. Cells were then transfected using the Lonza Nucleofector 4D with the DR-114 pulse code, immediately rescued with 80 uL of low-calcium media, and incubated for 15 minutes.50,000 cells from each sample were then seeded in a 96-well plate in triplicate and maintained in complete EMEM. After 24 hours, cells were trypsinized for 5 minutes in 75 uL of 1x TrypLE Express, centrifuged at 90xg for 10 minutes and supernatant removed, resuspended in PBS + 2.5mM of EDTA with 1:1000 Sytox Blue, and taken for flow cytometry. Fluorescent protein expression was analyzed on a CytoFlex LX at a flow rate of 100 uL per minute. Analysis

[0163] Reporter gene expression data acquired from flow cytometry was used to calculate promoter strength in ON target cell types and specificity against OFF target cell types. Briefly, data for each promoter in each cell type was collapsed to a representative expression curve using a binned-average method; the area under the curve (AUC) for each promoter expression curve was compared to that of a benchmark promoter, CAG, to calculate strength; the AUC for each promoter in the ON target cell type was compared with the counterpart AUC in each of the OFF target cell types to calculate specificity.

[0164] Table 2 and FIG.1 shows the various exemplary constructs consisting of the engineered regulatory elements (Enhancer and engineered minimal promoters) as previously described and their promoter strength (as a percentage of CAG promoter activity) and cell-type specificity. The engineered minimal promoters (Minimal Promoters 1-4) demonstrate increasedAttorney Ref. No.: 33978 / 70016 / PC potency as compared to Enhancer A linked to the YB_TATA synthetic minimal promoter alone. While the SCP3 promoter exhibited increased potency and selectivity, it contains sequence derived from CMV, which may be prone to transcriptional silencing.

[0165] Table 2: Engineered Minimal Promoter PerformanceExample 2: Engineered Minimal Core Promoter Expression of Chimeric Antigen Receptors

[0166] In this study, the engineered minimal promoters of the present disclosure were used to evaluate the expression of a CAR (aCAR (FIG.2A) and iCAR (FIG.2B)). The strength of minimal promoters minCMV, SCP3, Min. Promoter 1 (SEQ ID NO: 1), and Min. Promoter 5 (SEQ ID NO: 5) were evaluated in the context of three different enhancers (Enh. A, Enh. B, and Enh. C). These minimal promoters were placed downstream of the enhancers and upstream of our multi-component payload comprising an aCAR and an iCAR with peptide tags linked together by 2A peptides. These promoter-payload constructs were packaged into self- inactivating (SIN) γ-retroviruses through co-transfection with envelope plasmids into GP2-293 cells, collected, concentrated, and transduced into primary NK cells. Post-transduction, NK cells were allowed to rest for six days in culture, and surface expression of aCAR and iCAR proteins were detected by flow cytometry of fluorescent conjugated antibodies that recognize specific CAR-associated peptide tags. Promoter strength was quantified as mean fluorescent intensity (MFI) of aCAR and iCAR of live CAR+ NK cell populations.

[0167] The engineered minimal promoters (Min. Promoter 1 and Min, Promoter 5) paired with different enhancers (Enh. A, Enh. B, and Enh. C) demonstrated varying levels of intrinsicAttorney Ref. No.: 33978 / 70016 / PC activity in the expression of an aCAR (FIG.2A) and an iCAR (FIG.2B). In NK cells, promoters containing the engineered minimal promoters generally outperformed their counterparts containing the minCMV base promoter, and to a level that is similar or above even positive control promoters containing the SCP3 base promoter.Attorney Ref. No.:33978 / 70016 / PC INCORPORATION BY REFERENCE

[0168] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0169] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. 54 IPTS / 124230058.5

Claims

Attorney Ref. No.: 33978 / 70016 / PC CLAIMS What is claimed is:

1. An engineered minimal promoter comprising: a. a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 – 5; or b. an upstream promoter element and at least one downstream promoter element, wherein the upstream promoter element comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to TCTAGAGGGTATATAATGGGGGCCATTCTCTGTGTAATTCAGTCCGGT AGTCCGGC (SEQ ID NO: 10); or c. a first polynucleotide sequence encoding a minimal promoter sequence derived from YB_TATA, a second polynucleotide sequence comprising an initiator sequence (Inr), and a third polynucleotide sequence comprising at least one downstream promoter element, wherein the first polynucleotide sequence is operably linked to the second polynucleotide sequence and the second polynucleotide sequence is operably linked to the third polynucleotide sequence with a first linker sequence and second linker sequence, respectively, and wherein the first linker sequence and / or the second linker sequence does not comprise a transcription factor binding site, optionally wherein the initiator sequence comprises a nucleic acid sequence of TCAGTC, optionally wherein the first polynucleotide sequence comprises SEQ ID NO: 6.Attorney Ref. No.: 33978 / 70016 / PC 2. The engineered minimal promoter of claim 1, wherein the at least one downstream promoter element comprises a first downstream promoter element and a second downstream promoter element, optionally wherein the at least one downstream promoter element is MTE / DPE or DPR.

3. The engineered minimal promoter of claim 2, wherein the at least one downstream promoter element comprises a pause button motif, optionally wherein the pause button motif comprises the consensus sequence KCGRWCG.

4. The engineered minimal promoter of any one of claims 1-5, wherein the downstream promoter element comprises a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

9.

5. An engineered regulatory element comprising: a. an enhancer region; and b. the engineered minimal promoter of any one of claims 1-3.

6. The engineered regulatory element of claim 5, wherein the enhancer region is upstream of the upstream promoter element, optionally wherein the enhancer region is operably linked to the minimal promoter, optionally wherein the enhancer region is heterologous to the engineered minimal promoter, optionally wherein the engineered regulatory element comprises at least one spacer sequence between the engineered minimal promoter and the enhancer region.

7. A heterologous construct comprising the engineered minimal promoter of any one of claims 1-3 or the engineered regulatory element according to any one of claims 5-6, wherein the engineered minimal promoter or the engineered regulatory element is operably linked to a polypeptide-encoding polynucleotide, wherein the polypeptide- encoding polynucleotide comprises a polynucleotide sequence encoding a polypeptide,Attorney Ref. No.: 33978 / 70016 / PC optionally wherein the polypeptide comprises at least one effector molecule, optionally wherein the polypeptide comprises a first effector molecule and a second effector molecule.

8. The heterologous construct of claim 7, wherein the polypeptide-encoding polynucleotide comprises a polynucleotide sequence encoding the first effector molecule, a linker polynucleotide sequence, and a polynucleotide sequence encoding the second effector, optionally wherein the linker polynucleotide sequence encodes one or more 2A ribosome skipping elements, optionally wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A.

9. The heterologous construct of any one of claims 7-8, wherein the at least one effector molecule belongs to a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a co- activation molecule, a tumor microenvironment modifier, a receptor, a ligand, an antibody, a peptide, and an enzyme, optionally wherein each of the first effector molecule and the second effector molecule is from a separate therapeutic class, optionally wherein each of the at least one effector molecule is a human-derived effector molecule.

10. A vector comprising the heterologous construct according to any one of claims 7-9, optionally wherein the vector further comprises a second construct comprising a second polypeptide-encoding polynucleotide sequence encoding a second effector protein.

11. An engineered cell comprising the heterologous construct according to any one of claims 7-9, the vector according to claim 10, or the dual expression vector according to claim 10, optionally wherein the engineered cell expresses at least one effector molecule.

12. A pharmaceutical composition comprising the engineered minimal promoter of any one of claims 1-3, the engineered regulatory element according to any one of claims 5-6, the heterologous construct according to any one of claims 7-9, the vector of claim 10, the dual expression vector according to claim 10, or the engineered cell according to claimAttorney Ref. No.: 33978 / 70016 / PC 11, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof 13. A method of increasing expression of a target gene, the method comprising use of the engineered minimal promoter of any one of claims 1-3, the engineered regulatory element according to any one of claims 5-6, the heterologous construct according to any one of claims 7-9, the vector of claim 10, the dual expression vector according to claim 10, or the engineered cell according to claim 11, to increase expression of the target gene.

14. A method of treating a disease or disorder in subject in need thereof, the method comprising administering the engineered minimal promoter of any one of claims 1-3, the engineered regulatory element according to any one of claims 5-6, the heterologous construct according to any one of claims 7-9, the vector of claim 10, the dual expression vector according to claim 10, or the engineered cell according to claim 11.

15. A kit for treating and / or preventing a disease or disorder, comprising the pharmaceutical composition according to claim 12, optionally wherein the kit further comprises written instructions for using the pharmaceutical composition for treating and / or preventing a tumor in a subject.

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