promoters

Abstract

The present invention features promoters providing for DNA transcription. The promoters comprise a nucleic acid sequence of any of (a) nucleotides 1-314 of SEQ ID NO: 1; (b) the nucleic acid sequence of SEQ ID NO: 2; (c) the nucleic acid sequence of SEQ ID NO: 3; or (d) the nucleic acid sequence of SEQ ID NO: 4. In some embodiments a promoter is operably linked to a heterologous sequence.

Description

PROMOTERSCROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 660,936 filed June 17, 2024, the disclosure of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (065830-30W01.xml; Size: 22,782 bytes; and Date of Creation: June 13, 2025) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Eukaryotic promoters comprise regulatory regions providing for RNA polymerase II recruitment and downstream DNA transcription into RNA. Regulatory elements affecting promoter activity can be located upstream or downstream of a core promoter region.

[0004] In mammals, the core promoter generally can be separated into two distinct classes: conserved TATA-box enriched promoters that initiate at a single transcription start site, and variable CpG-rich promoters containing multiple transcription start sites located upstream of a gene. (Barrett et al., Cell Mol Life Sci. 2012 Nov;69(21):3613-34.)

[0005] Regulatory elements that may impact promoter activity include those present in an untranslated region (UTR) encompassing the transcription start site. The UTR can also provide elements that modulate gene expression by regulating translation.

[0006] Based on a genome-wide functional scan of human genes, about 35% of human genes contain an intron in a UTR that is 5’ of the translation start site. Introns present in the 5’ UTR may impact promoter activity. (Barrett et al., Cell Mol Life Sci. 2012 Nov;69(21):3613 -34, and Rose Front. Genet., 2019 9:672.)BRIEF SUMMARY OF THE INVENTION

[0007] The present invention features promoters providing for DNA transcription. The promoters comprise a nucleic acid sequence of any of: (a) the nucleic acid sequence of SEQ ID NO: 13; (b) the nucleic acid sequence of SEQ ID NO: 2; (c) the nucleic acid sequence of SEQ ID NO: 3; or (d) the nucleic acid sequence of SEQ ID NO: 4. In some embodiments the promoter is operably linked to a heterologous sequence.

[0008] Reference to a “heterologous” sequence operably linked to a promoter, indicates the sequence is not natively associated with the promoter. In some embodiments the promoter is operatively linked to a transgene.

[0009] Reference to a “transgene” indicates a nucleic acid sequence that can be expressed to produce an encoded nucleic acid sequence, which in some embodiments is translated into a polypeptide. In some embodiments, the transgene is part of an expression cassette providing for one or more additional expression control elements facilitating transcription and / or translation.

[0010] Reference to “operably linked” indicates the promoter binds the necessary proteins to facilitate transcription of the heterologous sequence into RNA.

[0011] Different types of RNA sequences can be produced including mRNA coding for a protein and biologically active RNA. Biologically active RNA sequences include inhibitory nucleic acid such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, and an antisense RNA; stimulatory RNA such as small activating ribonucleic acid (saRNA); and nucleic acid providing for base editing such as CRISPR (clustered regularly interspaced short palindromic repeats).

[0012] Thus, a first aspect of the present invention describes a polynucleotide comprising a promoter, wherein said promoter comprises: a) the nucleic acid sequence of SEQ ID NO: 13; b) the nucleic acid sequence of SEQ ID NO: 2; c) the nucleic acid sequence of SEQ ID NO: 3; or d) the nucleic acid sequence of SEQ ID NO: 4.

[0013] In some embodiments, the polynucleotide comprises a transgene operably linked to the promoter. In some embodiments the polynucleotide comprising a transgene operatively linked to a promoter is part of an expression cassette comprising one or more additional expression control elements.

[0014] In certain aspects, the polynucleotide is a recombinant viral nucleic acid comprising (a) a transgene operably linked to a promoter and (b) 5’ and / or 3’ viral elements providing for viral packaging and replication.

[0015] Another aspect of the present invention is directed to a gene delivery vehicle comprising a polynucleotide described herein and a viral or non-viral vector. Gene delivery vehicles can be used for, example, for delivering a polynucleotide to a cell in vitro, ex vivo, or in vivo.

[0016] Another aspect of the present invention is directed to a method of producing a protein or biologically active RNA in a cell comprising the step of transfecting or transducing the cell with a gene delivery vehicle comprising a polynucleotide described herein.

[0017] Another aspect of the present invention is directed to a method of producing a protein or biologically active RNA in a subject comprising the step of administering a gene delivery vehicle comprising a transgene operably linked to a promoter, encoding the biologically active RNA or protein to the subject.

[0018] Additional aspects of the present invention include the polynucleotides described herein for use in medicine and in a preparation for producing a protein or biologically active RNA in a subject.

[0019] Other features and advantages of the present invention are apparent from additional descriptions provided herein, including different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. Such examples do not limit the claimed invention. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 A and FIG. IB are bar diagrams illustrating transgene mRNA expression from different promoter constructs. Transgene expression was measured by quantifying human neuropeptide Y2 receptor (huNPY2R) mRNA levels using qPCR. All constructs contain a promoter operably linked to huNPY2R cDNA, except for the construct designed CAG-eGFP, which comprises a CAG promoter operably linked to eGFP cDNA. FIG. 1 A illustrates transgene expression in HEK293T cells from promoters designated as CAG, EFla, PGK-EFla (PGK-EFla), CaMKII, Bexl (Bexlv2), and CMVe-Syn. FIG. IB shows transgene expression levels in SH-SY5Y cells (neuroblastoma cell line) from promoters designated CAG and CaMKII.

[0021] FIGs. 2A-2C are bar diagrams illustrating transgene expressed protein detection by flow cytometry. Transgene protein expression was driven by promoter constructs designated PGK- EFla and Bexl. Antibody staining was carried out using huNPY2R Alexa Fluor® 488- conjugated antibody. FIG. 2A illustrates results from Experiment #1 measuring relative fluorescence using 2 wells per condition, pooled for flow cytometry, with or without huNPY2R siRNAs #1 and #2 mixed together (20 nM). FIG. 2B and FIG. 2C illustrate results from Experiment #2 measuring relative fluorescence (FIG. 2B) or percent transgene protein-positive cells (FIG. 2C), 3 replicates per condition, 1 well per replicate, with or without huNPY2R siRNA #1 (50 nM).

[0022] FIG. 3A and FIG. 3B are bar diagrams illustrating transgene protein expression from promoter constructs designated PGK-EFla and Bexl. FIG. 3 A illustrates percent of transfected cells also expressing transgene protein, determined using a tdTomato reporter. FIG. 3B illustrates relative fluorescence of the transgene protein signal.

[0023] FIG. 4 is a bar diagram illustrating protein expression from EFla promoter-driven constructs. Transgene protein expression was detected by immunocytochemistry staining.

[0024] Fig. 5 is a bar diagram illustrating expression of a human transgene in mice, based on RNA transcripts from Bexlv2 promoter-driven constructs in different regions of mouse brain. Levels of transcript were measured by qRT-PCR, and abundance of the mRNA for the transgene is shown as normalized expression over the mouse ortholog (gray squares). Vehicle -treated animals (black ovals) showed no expression of the human transgene.DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention features promoters providing for DNA transcription. The promoters comprise a nucleic acid sequence of any of: (a) the nucleic acid sequence of SEQ ID NO: 13; (b) the nucleic acid sequence of SEQ ID NO: 2; (c) the nucleic acid sequence of SEQ ID NO: 3; or (d) the nucleic acid sequence of SEQ ID NO: 4. In some embodiments a promoter is operably linked to a heterologous sequence. The promoters provided herein can be used in general to drive transgene RNA transcription from expression cassettes in different types of polynucleotides, including plasmids and viral vectors.

[0026] Promoter regions affecting DNA transcription are generally located up to a few kilobases around and upstream of the transcription initiation site, and no further downstream than the translation start site. A promoter can comprise a UTR, where the promoter UTR refers to a region downstream of the promoter core region that is transcribed, but is generally not translated. The UTR corresponds to a gene 5 ‘UTR prior to the translation start site. The UTR can provide regulatory elements affecting transcription and / or translation. Additionally, in some contexts, a 5’ UTR region can be translated. In some embodiments, the promoter may encompass a promoter core region, a 5’ UTR, as well as an intron.

[0027] A promoter comprising the sequence of nucleotides 1-314 of SEQ ID NO: 1 provides a core Bexl promoter region (SEQ ID NO: 13). Additional sequences from SEQ ID NO: 1 may be present along with the core sequence (SEQ ID NO: 13) and / or additional sequences that are not present in SEQ ID NO: 1 may be associated with the core sequence (SEQ ID NO: 13). SEQ ID NO: 1, also referred to herein as Bexl or Bexlv2, was derived by starting with the BEX1 promoter, and contains a truncated intron and several engineered point mutations in non-coding exons. The BEX1 promoter provides for neuron expression. BEX1 is described, for example, in Alvarez et al., Gene, 357: 1, 2005, pages 18-28, hereby incorporated by reference herein in its entirety.

[0028] A promoter comprising the sequence of SEQ ID NO: 2, referred to herein as PGK-EFla or PGK-EFla, was derived starting with the human PGK promoter and the EFl a promoter / first intron. The PGK -EFl a promoter includes 511 bp of the PGK promoter and 943 bp of the EFla 5’ UTR plus intron (mostly intron), and was designed to be ubiquitous.

[0029] The promoter comprising the sequence of SEQ ID NO: 3, referred to herein as EFla, was derived starting with the human EFla promoter. Modifications to the human EFl a include a 4 bp deletion and a one base pair change. The promoter was designed to be ubiquitous.

[0030] The promoter comprising the sequence of SEQ ID NO: 4, referred to herein as CMVe- hSyn, was derived starting with the human CMV promoter / enhancer and the hSynl promoter. The 423 bp CMV enhancer was fused upstream of 448 bp human synapsin promoter. The promoter was designed to be neuron-specific. The hSynl promoter is described, for example, in Kugler et al., Gene Ther 10, 337-347 (2003).

[0031] The CAG promoter is a composite of the CMV enhancer, the chicken beta actin (CBA) promoter and the rabbit beta globin intron (see, e.g., Boshart et al., (1985) Cell, 41 :521-530).

[0032] Promoters described herein can be operatively linked to one or more transgenes, for example, mono-cistronic or multi-cistronic transgenes. Mono-cistronic genes provide for the production of a single gene product (e.g., protein or biologically active RNA) from a promoter. Multi-cistronic expression provides transcription of an RNA transcript from a promoter, where the RNA transcript encodes for more than one gene product. Translation of multiple proteins from a single transcript can be obtained, for example, through the use of an internal ribosome entry site and viral peptide 2A.

[0033] Viral 2A peptides are generally around 18 to 22 amino acids in length and cause ribosome skipping during translation, resulting in the translation of different proteins. Examples of viral 2A peptides include E2A (equine rhinitis A virus), F2A (foot-and-mouth disease virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A). (Chng et al., MAbs. 2015;7(2):403-12; and Liu. Sci Rep. 2017 May 19;7(1):219.) Adding a cleavage recognition site, such as a furin recognition site upstream of the 2A cleavage site, can be useful for removing 2A residues from the upstream protein. (Chng et al., MAbs. 2015;7(2):403-12.)

[0034] Internal ribosome entry sites allow the translational machinery to start protein synthesis by internal initiation. (Renaud-Gabardos et al., World J Exp Med. 2015 Feb 20;5(l): 11 -20.)

[0035] In some embodiments the polynucleotide comprising the promoter is less than 5.5 kb. In some embodiments the polynucleotide is less than 5.2 kb, less than 5.1 kb, less than 5.0 kb, less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb; between 4 kb to 5.2 kb, 3.0 kb to 5.5 kb, 4.0 kb to 5.0 kb, 4.3 kb to 4.8 kb; or about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, or about 5.0 kb.

[0036] Recombinant AAV comprising rAAV nucleic acid and an AAV capsid can be used to facilitate introduction of the rAAV nucleic acid into a cell. In some embodiments, a rAAV vector is used for transgene delivery.

[0037] The use of a rAAV vector for gene delivery can be limited by the AAV capsid packagingcapacity of approximately 5.0 kb. Recombinant AAV expression cassettes comprising a transgene operatively linked to a promoter, may also contain additional regulatory elements involved in DNA transcription and protein translation. Reducing the promoter size can facilitate the ability of rAAV vector to deliver larger transgenes and / or multiple transgenes.

[0038] In some embodiments, the rAAV comprises a transgene coding region that is about 3.8 kb or greater, 3.9 kb or greater, 4.0 kb or greater, 4.1 kb or greater, 4.2 kb or greater, or 4.3 kb or greater; wherein in some embodiments the size of the transgene coding region is up to 4.4 kb.

[0039] In some embodiments, the promoter constructs are used to deliver multiple transgenes to a cell or subject. Transgene delivery to a subject can be useful, for example, in a subject to treat a disease or disorder, and in a non-human subject or cell culture for protein production in general.

[0040] Reference to “subject” indicates a mammal, such as a human; non-human primate such as ape, gibbon, gorilla, chimpanzee, orangutan, and macaque; domestic animal such as a dog and cat; farm animal such as poultry, duck, horse, cow, goat, sheep and pig; and experimental animal such as mice, rat, rabbit, sheep, and guinea pig. A preferred subject is a human.

[0041] Reference to an indicated percent identity to one or more reference sequences, and similar language throughout the specification providing for an indicated percent identity to one or more reference sequences, provides the indicated percent identity or percent identity range independently to each of the referenced sequences. Reference to a percent “identical”, “identity” and similar terminology are with respect to two sequences having maximal alignment in a particular area. The provided area is with respect to the indicated reference sequence. For example, sequence “identical” or “identity” to a reference polypeptide or region can be calculated by determining the number of identical amino acids in aligned sequences, where the amino acid sequence of the polypeptide or region being compared to the reference polypeptide is aligned for maximal alignment, dividing by the total number of amino acids in the reference polypeptide or region, and multiplying by 100. Percent “identical” or “identity” for nucleic acid sequences can be determined in an analogous manner where sequences are aligned to achieve maximal alignment taking into account nucleotide differences and gaps, determining the number of identical nucleotides, dividing by the total number of nucleotides in the reference sequence and multiplying by 100.

[0042] Throughout the application, unless clearly indicated otherwise by the context, when different variable components are provided, any variable of one component may be used with any variable of another component.

[0043] In discussing nucleic acids, a sequence or structure of a particular polynucleotide can be described herein according to the convention providing the sequence in the 5’ to 3’ direction.

[0044] In some embodiments, nucleic acids include genomic DNA, RNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (poly -and oligo-nucleotide), chromosomal DNA, spliced or unspliced mRNA, rRNA, tRNA, inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans -splicing RNA, or antisense RNA), locked nucleic acid analogue (LNA), oligonucleotide DNA (ODN) single and double stranded, immunostimulating sequence (ISS), riboswitches and ribozymes.

[0045] In some embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single, double, or triplex, linear or circular, and can be of different lengths.

[0046] According to some embodiments, the polynucleotide is a single-stranded (ssDNA) or a double-stranded DNA (dsDNA) molecule. According to some embodiments, the dsDNA molecule is a minicircle, a nanoplasmid, open linear duplex DNA or a closed-ended linear duplex DNA (CELiD / ceDNA / doggybone DNA). According to some embodiments, the ssDNA molecule is a closed circular or an open linear DNA.

[0047] Some embodiments are directed to “CpG reduced” or “CpG depleted” nucleic acid.“CpG reduced” or “CpG depleted” refer to (i) a nucleotide sequence wherein one or more of the CpG dinucleotides (or motifs) are removed from a reference nucleic acid sequence; and / or (ii) the percentage of CpGs in a referred to polynucleotide is 0% to 15%. In some embodiments, the CpG percentage is 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.

[0048] CpG motifs can be suitably reduced or eliminated in a nucleotide sequence encoding a protein and in other sequences that are present in particular constructs (e.g., expression cassettes and viral vectors). Other sequences that may be present include non-coding sequences such as vector 5’ and 3’ untranslated regions, stuffer sequences, promoter, enhancer, polyadenylation signal, ITRs, and introns.

[0049] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0050] The conjunctive term “and / or” between multiple recited elements encompasses both individual and combined options. For instance, where two elements are conjoined by “and / or”, a first option refers to the applicability of the first option without the second, a second option refers to the applicability of the second option without the first, and a third option refers to theapplicability of the first and second options together. Any one of the options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or”. Concurrent applicability of more than one of the options is also understood to fall within the meaning of the term “and / or.”

[0051] Unless clearly indicated otherwise by the context employed the terms “or” and “and” have the same meaning as “and / or”.

[0052] Reference to terms such as “including”, “for example”, “e.g.,” “such as” followed by different members or examples, are open-ended descriptions where the listed members or examples are illustrative and other member or examples can be provided or used.

[0053] The terms “polypeptide,” “protein” and “peptide” can be used interchangeably to refer to an amino acid sequence without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids. Amino acids include naturally occurring amino acids and amino acids provided by cellular modification.

[0054] Reference to “comprise”, and variations such as “comprises” and “comprising”, used with respect to an element or group of elements is open-ended and does not exclude additional unrecited elements or method steps. Terms such as “including”, “containing” and “characterized by” are synonymous with comprising. In the different aspects and embodiments described herein reference to an open-ended term such as “comprising” can be replaced by “consisting” or “consisting essentially of’.

[0055] Reference to “consisting of’ excludes any element, step, or ingredient not specified in the listed claim elements, where such element, step or ingredient is related to the claimed invention.

[0056] Reference to “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0057] The term “about” refers to a value within 10% of the underlying parameter (z.e., plus or minus 10%). For example, “about 1 : 10” includes 1.1 :10.1 or 0.9:9.9, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a string of values modifies each of the values by 10%. In some embodiments the term “about” provides for a value within 5% of the underlying parameter.

[0058] All numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5% and so forth and reference to a numerical range, such as “1 -4” includes 1, 2, 3, 4as well as 1.1, 1.2, 1.3, 1.4 and so forth. As a further illustration, “1 to 4 weeks” includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0059] Further, reference to a numerical range, such as “0.01 to 10” includes 0.011, 0.012, 0.013 etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9 and so forth. For example, a dosage of about “0.01 mg / kg to about 10 mg / kg” body weight of a subject includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg and so forth.

[0060] Reference to an integer with more (greater) or less than includes numbers greater or less than the reference number, respectively. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and administration “two or more” times includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.

[0061] Various references including articles and patent publications are cited or described in the background and throughout the specification. Each of these references is herein incorporated by reference in their entirety. None of the references are admitted to be prior art with respect to any inventions disclosed or claimed. In some cases, particular references are indicated to be incorporated by reference herein to highlight the incorporation.

[0062] The definitions provided herein, including those in the present section and other sections of the application, apply throughout the present application.

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

[0064] The description has been separated into various sections and paragraphs, and provides examples of various embodiments. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiment. The provided descriptions have broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest the scope of the disclosure, including the claims (unless otherwise provided in the claims), is limited to these examples.

[0065] Cross-reference to a particular section of the present application, includes all the provided subsections.

[0066] The instant invention is generally disclosed herein using affirmative language to describe the numerous embodiments of the instant invention. The instant invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, insome embodiments of the instant invention, materials and / or method steps are excluded. Thus, even though the instant invention is generally not expressed herein in terms of what the instant invention does not include, embodiments that are not expressly excluded in the instant invention are nevertheless disclosed herein.I. Expression Cassettes

[0067] Polynucleotide expression cassettes comprise a promoter operatively linked to a transgene and generally contain one or more additional expression control elements. Expression control can be affected, for example, at the level of transcription, translation, splicing, and message stability. Expression control elements are typically located 5’ (“upstream”) or 3’ (“downstream”) of a transcribed nucleic acid. Expression control elements can also be located within the transcript (e.g., in an intron), adjacent to or at a distance away from the transcribed sequence. One or more expression control elements of the same or different type may be present.

[0068] Expression control elements that may be present include an intron, an enhancer, a polyadenylation signal sequence, a Kozak sequence, a post translational regulatory element, 5’ and 3’ inverted repeats (ITRs), regulatable elements, cell-specific regulators (e.g., micoRNA binding elements), internal ribosome entry sites or other elements that affect expression or stability of the encoded sequence, or protein processing.

[0069] Polyadenylation signal sequences provide for the formation of a polyA tail, which facilitates nuclear export, translation and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signal sequences include SV40 late polyadenylation signal, bovine growth hormone polyA (bGHpA) signal sequence, synthetic polyA, mouse 0-globin pA, rabbit 0-globin pA, and H4-based pA. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0070] Enhancers are DNA regions that increase promoter transcription. Enhancers can be adjacent or inside a promoter or can be distal. Typically, enhancers are located upstream of a promoter, but can be located downstream or within a promoter sequence.

[0071] Expression control elements also can impact expression in a manner that is regulatable by a signal or stimuli increasing or decreasing expression. A regulatable element increasing expression of transcribed nucleic acid in response to a signal or stimuli is also referred to as an “inducible element” (i.e., is induced by a signal). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present. Particular examples include zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone- inducible mouse mammary tumor virus (MMTV) promoter; the tetracycline -repressible system (Gossen, etal., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-induciblesystem (Gossen et al., Science 268: 1766-1769 (1995); see also Harvey etal., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang etal., Gene Ther. 4:432-441 (1997); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997); and Rivera etal., Nat. Medicine. 2: 1028- 1032 (1996)). Other examples of regulatable control elements include those regulated by a specific physiological state such as temperature, acute phase, or development.

[0072] In some embodiments the expression cassette further comprises one or more introns independent of an intron present in the promoter UTR. A variety of different introns can be used to enhance gene expression. Examples of introns that may be used include the rabbit P-globin intron with splice donor / splice acceptor, SV40 intron with splice donor / splice acceptor, human P-globin introns, intron 2 of the human hemoglobin beta gene, hFIX inti (intron 1 of the human coagulation factor IX gene), CBA-rHHB (synthetic intron derived from the fusion of the intron 1 of the chicken beta actin gene and intron 2 of the rabbit hemoglobin beta), CBA (intron 1 of the chicken beta actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (synthetic intron derived from different portions of the human coagulation factor IX gene and present in the pLIVE vector, Mirus Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and optimized HBB2; and chimeric introns such as introns made up of the 5'- splice donor of the first human P-globin intron and the branch and 3 '-acceptor site from the intron that is between the leader and the body of the immunoglobulin gene heavy chain variable region. (Buck etal., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti etal., Mol. Ther. Methods Clin Dev. (2016) Jul 20;3: 16049; and the HBB -I GG intron provided by the pCMVNT™ vector.)

[0073] In some embodiments the expression cassette comprises a post-transcriptional regulatory element. Post-translational regulatory elements such as Woodchuck post-transcriptional regulatory element (WPRE) and Hepatitis B regulatory element can increase gene expression. (Buck etal., Int. J. Mol. Sci. (2020), 21, 4197.)

[0074] In some embodiments, the expression cassette comprises a Kozak consensus sequence (also referred to herein as a Kozak sequence) or a variation thereof. Kozak consensus sequences play a role in translation initiation. The Kozak consensus sequence and variations are provided in, for example, McClements et al., (2021 ) Molecular Vision, 27, 233-242, hereby incorporated by reference herein,

[0075] In some embodiments the expression cassette comprises from 5’ to 3’ operatively linked to a protein encoding sequence: a promoter or promoter / enhancer, an intron, a Kozak sequence, the protein encoding sequence and a polyaden lation signal.

[0076] In some embodiments the expression cassette comprises a miRN'A target sequence, which in some embodiments is incorporated into the 3’ UTR of the expression cassete. AmiRNA target sequence is recognized by miRNA present in particular cells or tissues leading to degradation of mRN transcripts. Based on the presence of certain miRNA in particular cells, incorporating miRNA target sequence(s) can be used to reduce expression in certain cells or tissue types. Multiple tandem repeats of miRNA target sequences can be used to increase degradation. (Geisle etal., (2016) World Journal of Experimental Medicine 6(2): 37-54.)

[0077] In some embodiments, the expression cassette nucleotide sequence contains 0-5, 0-10, 0- 15, 0-50, or 0-100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.I. A, Therapeutic Proteins

[0078] A transgene can encode a variety of nucleic acid sequences and can encode for different proteins, including therapeutic protein and base editing protein. Examples of transgenes encoding therapeutic protein include those providing a healthy copy of gene in a subject where the gene is defective; a modified gene that can help treat a disease or disorder; or a new gene encoding for protein providing a beneficial effect.

[0079] In some embodiments, a transgene encodes GAA (acid alpha-glucosidase) for treatment of Pompe disease; TPP1 (tripeptidyl peptidase-1) for treatment of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2); ATP7B (copper transporting ATPase2) for treatment of Wilson’s disease; alpha galactosidase for treatment of Fabry disease; ASS1 (arginosuccinate synthase) for treatment of Citrullinemia Type 1 ; beta-glucocerebrosidase for treatment of Gaucher disease Type 1; beta-hexosaminidase A for treatment of Tay-Sachs disease; SERPING1 (Cl protease inhibitor or Cl esterase inhibitor) for treatment of hereditary angioedema (HAE), also known as Cl inhibitor deficiency type I and type II); or glucose-6-phosphatase for treatment of glycogen storage disease type I (GSDI).

[0080] In some embodiments, the transgene encodes insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growthfactors I or II (IGF-I or IGF-II), TGF0, activins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 or NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 or netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog or tyrosine hydroxylase.

[0081] In some embodiments, the transgene encodes thrombopoietin (TPO), an interleukin (IL-1 through IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocytemacrophage colony stimulating factor, Fas ligand, tumor necrosis factors a or 0, interferons a, 0, or y, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD or IgE, chimeric immunoglobulins, an antibody, humanized antibody, single chain antibody, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I or class II MHC molecules. Antibodies and immunoglobulins can, for example, be provided targeting cancer cells or other disease or disorder causing cells.

[0082] In some embodiments, the transgene encodes CFTR (cystic fibrosis transmembrane regulator protein), a blood coagulation (clotting) factor (Factor XIII, Factor IX (FIX), Factor VIII (FVIII), Factor X, Factor VII, Factor Vila, or protein C) a gain of function blood coagulation factor, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, 0-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, 0-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a and 0, a cytokine, a-interferon, 0-interferon, interferon-y, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxy cytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRl, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (choroideremia), LCA 5 (LCA-lebercilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), X-linked retinitis pigmentosa GTPase (XLRP), MER proto-oncogene tyrosine kinase (MERTK) (autosomal recessive (AR) forms of retinitis pigmentosa (RP)), ABCA4 (Stargardt), ACHM 2, 3and 4 (achromatopsia), an anti-vascular endothelial growth factor (VEGF) agent polypeptide (e.g., bevacizumab, brolucizumab, ranibizumab, aflibercept), DFNB1 (connexin 26 deafness), USH1C (Usher’s syndrome 1C), PKD-1 or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase-1), a sulfatase, N-acetylglucosamine-1 -phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activator protein, or one or more donor sequences used as repair templates for genome editing.

[0083] In some embodiments, the transgene encodes erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for treatment of various immune disorders, viral infections and cancer; an interleukin (IL), including any one of IL-1 through IL-36, and corresponding receptors, for treatment of various inflammatory diseases or immuno-deficiencies; a chemokine, including chemokine (C-X-C motif) ligand 5 (CXCL5) for treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for treatment of immune disorders such as Crohn’s disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for treatment of various human inflammatory diseases; macrophage colony stimulating factor (M-CSF) for treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for treatment of epithelial tissue damage; chemokines such as monocyte chemoattractant protein-1 (MCP-1) for treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for treatment of various immune disorders; alphal -antitrypsin for treatment of emphysema or chronic obstructive pulmonary disease (COPD); alpha-L-iduronidase for treatment of mucopolysaccharidosis I (MPS I); ornithine transcarbamoylase (OTC) for treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for treatment of phenylketonuria (PKU); lipoprotein lipase for treatment of lipoprotein lipase deficiency; apolipoproteins for treatment of apolipoprotein (Apo) A-I deficiency; low-density lipoprotein receptor (LDL-R) for treatment of familial hypercholesterolemia (FH); albumin for treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for treatment of homocystinuria; branched chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl CoA carboxylase; methylmalonyl-CoA mutase; glutaryl CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; or dystrophin.

[0084] In some embodiments the transgene encodes a protein for treating a disease or disorder selected from the group consisting of: hereditary angioedema, Pompe disease, hemophilia A,hemophilia B, Fabry, wet macular degeneration, Leber hereditary optic neuropathy, and Stargardt disease.LB, Inhibitory Nucleic Acid

[0085] DNA vectors can provide a variety of different transgenes encoding for a variety of different inhibitory nucleic acid such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, and an antisense RNA. In some embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a disease or disorder selected from huntingtin (HTT) gene, a gene associated with dentatorubropallidoluysian atrophy (atrophin 1, ATN1), androgen receptor on the X chromosome in spinobulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-dependent calcium channel (CACNA1 A), TATA-binding protein, Ataxin 8 opposite strand (ATXN8OS), serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMRI (fragile X mental retardation 1) in fragile X syndrome, FMRI (fragile X mental retardation 1) in fragile X- associated tremor / ataxia syndrome, FMRI (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; myotonin -protein kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich’s ataxia; a mutant of superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene involved in pathogenesis of Parkinson’s disease and / or Alzheimer’s disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function kidney transplant or kidney injury acute renal failure; protein kinase N3 (PKN3) in advance recurrent or metastatic solid malignancies; LMP2, LMP2 also known as proteasome subunit beta-type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-type 8 (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit betatype 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis suppressor B -cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors;Furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Reddl also known as DNA damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascularendothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischaemic optic neuropathy; keratin 6A N17K mutant protein in pachyonychia congenital; influenza A virus genome / gene sequences in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequences in SARS infection; respiratory syncytial virus genome / gene sequences in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola infection; hepatitis B and C virus genome / gene sequences in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequences in HSV infection; coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) like torsin A (TORI A) in primary dystonia, pan-class I and HLA-allele specific in transplant; and mutant rhodopsin gene (RHO) in autosomal dominantly inherited retinitis pigmentosa (adRP).I.C Gene Editing

[0086] The DNA vector can provide a variety of different transgenes encoding for a variety of different base editing nucleic acid / protein such as CRISPR / Cas-based RNA-guided DNA endonucleases, and gene editing proteins such as zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Gene editing nucleases can be used, for example, to modify proteins described herein to correct a defect, enhance activity, and / or decrease expression. In some embodiments the gene editing nucleic acid edits a subject’s DNA to provide a therapeutic protein as provided in Section I. A. supra., or disrupt a gene as provided in Section I.B. supra.II. Recombinant Viral Vector Nucleic Acid

[0087] Recombinant viral vector nucleic acid can be used for transgene delivery and expression. Recombinant viral vector nucleic acid contain 5’ and / or 3’ viral elements providing for viral packaging and may provide for additional activities such as self -priming, DNA replication, promoter activity, genome integration, or episomal concatemerization. The 5’ and 3’ elements are generally located at or near the 5’ and 3’ terminal end of the recombinant viral vector nucleic acid and can be naturally occurring or modified versions of naturally occurring sequences. Examples of 5’ and 3’ elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequence; and retrovirus 5’ and 3’ long terminal repeats (LTRs) and packaging sequences. (Naso etal., (2017) BioDrugs, 31 (4), 317-334; Bulcha et al, (2021 ) Sig. Transduct. Target Ther. 6:53 (2021); and Liu and Seol (2020) BMB Reports; 53(11): 565 -575.)

[0088] The term “recombinant,” as a modifier of nucleic acid or a vector indicates a combination of elements that does not occur in nature. For example, recombinant viral vectornucleic acid provides 5’ and / or 3’ viral elements along with an expression cassette or transgene containing one or more elements not naturally associated with the 5’ and / or 3’ elements. Similarly, a viral vector, such as an rAAV vector may contain a naturally occurring or modified capsid, encapsidating recombinant viral vector nucleic acid.

[0089] In some embodiments, the viral vector comprises a polyA signal operatively linked to the 3’ ITR, where the polyA signal antagonizes potential transcription initiating from the 3’ -ITR. The operatively linked polyA signal is upstream of the 3 ’-ITR.

[0090] In some embodiments, the viral vector nucleic acid contains 0-5, 0-10, 0-15, 0-50, 0-100, or O to 150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 44, 46, 47, 48, 49 or 50 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14% or up to about 15% CpGs.III. Viral Vectors

[0091] Viral vectors comprise a protein capsid encapsidating recombinant viral vector nucleic acid. The viral vector can deliver the viral vector nucleic acid to cells or tissues. Depending on the particular vector, the viral vector may further comprise a viral envelope. Examples of viral vectors that can be used for gene delivery include adenovirus vectors, rAAV, retrovirus vectors and herpes simplex vectors.

[0092] Different serotypes exist within different types of viruses. The different serotypes can provide for different activities, such as cell or tissue tropism and likelihood of generating a host immune response. The term “serotype” broadly refers to both serologically distinct viruses as well as viruses not serologically distinct that can be within a subgroup or a variant of a given serotype. Serologic distinctiveness can be determined based on the lack of cross -reactivity between antibodies to one capsid as compared to another capsid. Such cross -reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes).

[0093] As more naturally occurring virus isolates are discovered or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where a new virus has no serological difference, this new virus would be a subgroup or variant of the corresponding serotype.III, A. Adenovirus Vectors

[0094] Adenoviruses are non-enveloped double-stranded DNA viruses. Recombinant adenovirus vectors comprise recombinant adenovirus nucleic acid lacking one or more protein involved in viral replication, and further comprise an adenoviral capsid. Recombinant adenovirus vectors can be produced containing different amounts of adenoviral DNA. The Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs) varying in length from 30-371 bp at its termini. The ITRs serve as self-priming structures that promote primase-independent DNA replication. A packaging signal located at the left arm of the genome is required for viral genome packaging. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha etal., (2021) Sig. Transduct. Target Ther. 6:53.)

[0095] In some embodiments, the recombinant adenovirus vector is a third-generation vector, which is also referred to as “gutless” or “helper-dependent”. Gutless vectors can be produced from recombinant adenovirus nucleic acid where all, or substantially all viral sequences, except for the ITRs and the packaging signal, are not present. Gutless adenovirus vectors are high capacity vectors able to accommodate up to about 36 kb of DNA insert. Preferred recombinant adenovirus nucleic acid is about 27 kb to about 37 kb. Stuff er sequences can be added to recombinant adenovirus nucleic acid to increase nucleic acid size and capsid incorporation. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports; 53(11):565-575; Bulcha etal., (2021) Sig. Transduct. Target Ther. 6:53; and Sandig etal, PNAS (2000) 97(3): 1002-1007, each of which are hereby incorporated by reference herein in their entirety.)

[0096] In some embodiments, recombinant adenovirus vectors can be produced based on rare human serotypes or chimpanzee serotypes. The use of chimpanzee and rare human serotypes may be helpful in reducing host immune response against recombinant adenovirus vectors due to preexisting immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7):1679- 1685 and Bulcha et al., (2021 ) Sig. Transduct. Target Ther. 6:53.)

[0097] Adenovirus vectors can be produced by supplying viral proteins needed for vector production in trans using for example, appropriate helper viruses or plasmids and cell lines. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha el al., (2021) Sig. Transduct. Target Ther. 6:53.)III.B, Recombinant AAV Vectors

[0098] Recombinant adeno-associated viral (also referred to herein as “rAAV”) vectors are based on the adeno-associated virus (AAV). AAV is a single-strand DNA virus containing a 4.7- kb genome flanked by 145 -nt ITRs on both ends of the genome. ITR activity is important forself-priming and packaging, and may also provide additional activity such as promoter activity. AAV 5’ and 3’ ITRs can vary in size and the 5’ and 3’ ITRs need not be exact inverted repeats.

[0099] A rAAV vector contains AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In some embodiments, the rAAV vector contains an AAV 5’ and / or 3’ ITR along with a DNA insert. In some embodiments, rAAV nucleic acid comprise a 5’ ITR and / or 3’ ITR independently selected from 5’ and 3’ ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 and AAV3B ITRs. In some embodiments 5’ and 3’ ITRs are present, and both ITRs are from the same serotype genome.

[0100] In some embodiments, the rAAV nucleic acid includes a stuffer sequence and the total length including the stuffer sequence is 4-5.2 kb, 3.0-5.5 kb, 4.0-5.0 kb, 4.3-4.8 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. If needed, stuffer sequences can be used to increase rAAV nucleic acid size and packaging efficiency. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences.

[0101] In some embodiments, the rAAV is a self-complementary adeno -associated virus vector (scAAV) or short hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide for a double-stranded rAAV nucleic acid that can be incorporated into an AAV capsid. scAAV and shAAV comprise inverted dimeric repeats providing intramolecular double -stranded DNA. scAAV can be produced by mutating an ITR terminal resolution site so that rep fails to nick the terminal resolution site. shAAV can utilize a short hairpin to produce double-stranded AAV nucleic acid. scAAV and shAAV being double-stranded DNA, provide an advantage in circumventing the DNA synthesis step required for single-stranded rAAV nucleic acid upon entry into a cell. A potential disadvantage of scAAV and shAAV is the size of DNA inserts that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid. (U.S. Patent No. 10,457,940; Xie et al., Mol Ther. (2017) 25(6): 1363-1374; and McCarty Mol. Ther. (2008) 16(10): 1648-1656; each of which are hereby incorporated by reference herein in their entirety.)

[0102] Naturally occurring AAV capsids contain viral proteins VP1, VP2 and VP3 in a ratio of about 1 :1 :10. Recombinant AAV vectors can be produced where all three viral proteins are based upon a particular serotype or where one, two or all three viral proteins are based on different serotypes.

[0103] Recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other. In some embodiments, a rAAV nucleic acid has the same serotype genome (e.g., ITRs) as the encapsidating capsid protein.

[0104] In some embodiments, the rAAV capsid comprises a protein having a sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9% identical to, or comprising the sequence of a VP1, VP2 or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-218, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAVl / rh.10; or VPl of SEQ ID NO: 6.

[0105] Recombinant AAV capsids comprising VP1 of SEQ ID NO: 6 is described, for example, in U.S. Patent No. 9840719; hereby incorporated herein by reference.

[0106] In some embodiments, AAV capsids comprises VP1, VP2 and VP3 each independently having a sequence identity of at least 80%, at least 90%, at least 95% or 100% to a VP1, VP2 or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-218, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAVl / rh.10, SEQ ID NO: 6; as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof. (See, for example, U.S. Patent Nos. 9,909,142 and 9,840,719 disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6; the disclosures of which are herein incorporated in their entirety.)

[0107] In some embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 6; VP2 having the sequence of SEQ ID NO: 7; and VP3 having the sequence of SEQ ID NO: 8.

[0108] In some embodiments, the AAV capsid can cross the blood brain barrier and provide for central nervous system (CNS) expression. Examples of such AAV capsids and the design of AAV capsids able to provide for CNS expression are provided in Chen et al., (2021) J. Control. Release 333, 129-138 e.g., AAV9, AAV-PHP-B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAVl / rh.10), U.S. Patent No. 9,585,971, and Goertsen et al., (2022) Nat. Neurosci. 25, 106-115 (2022), each of which are incorporated by reference herein in its entirety.

[0109] The AAV genome contains two main genes: rep and cap. Transcription from the rep gene is initiated from two different promoters resulting in the production of nonstructural proteins designated Rep78, Rep68, Rep52, and Rep40. The rep proteins function in genome replication and / or encapsidation. The cap gene encodes for structural proteins making up the capsid (VP1, VP2 and VP3); a non-structural assembly -activating protein (APP), which performs functions related to capsid assembly; and the membrane -associated accessory protein, which may be associated with production phases of the replication cycle. (Maurer and Weitzman(2020) Hum. Gene Ther. 31(9-10):499-511, hereby incorporated by reference herein in its entirety.)

[0110] AAV requires helper virus functions to complete its replication cycle. Helper virus functions can be supplied by different viruses in permissive cell lines. Permissive cell lines are cell lines able to support viral replication. Examples of helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HBoVl which can be used in conjunction with, for example, permissive primate cells; and baculovirus which can be used in conjunction with, for example, permissive insect cells such as sf9. (Maurer and Weitzman Hum. Gene Ther. (2020) 31(9- 10):499-511 and Meier et al., (2020) Viruses 19;12(6):662, both of which are herein incorporated by reference herein in their entirety.)

[0111] Recombinant AAV can be produced by supplying viral proteins needed for vector production in trans using for example, appropriate helper viruses or plasmids and cell lines. In some embodiments, rAAV is produced using a rAAV vector genome (vg) plasmid. The plasmid comprises that portion of the rAAV nucleic acid ultimately packaged or encapsidated to form a viral (e.g., rAAV) vector. The “plasmid backbone,” contains elements important for propagation and recombinant virus production. Except for possible 3’ ITR and / or 5’ ITR cloning remnants the plasmid backbone is not itself packaged or encapsidated into virus particles.

[0112] The vector genome plasmid may contain regions such as an origin of replication and a selectable marker. Additional sites that may be present include cloning sites.

[0113] Recombinant AAV can be produced from different types of cell lines including HeLa, A549, BHK, Vero, and HEK293, or derivatives thereof. In some embodiments, HEK293 cells are used (American Type Culture Collection Accession Number ATCC CRL1573). Other host cell lines appropriate for rAAV vector production are described in, for example, Robert etal., (2017) Biotechnol. J. (2017) 12(3), 1600193; and International Application No.PCT / US2017 / 024951, the disclosures of which are herein incorporated in its entirety.

[0114] Recombinant AAV can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV manufacturing include Clement and Grieger (2016) Mol. Ther. Methods Clin. Dev. 16;3 : 16002; Robert etal., (2017) Biotechnol. J. 12(3), 1600193; and Adeno-Associated Virus Vectors (2019), Ed. Castle., 1stEdition, Springer Protocols, New York, NY.; each of which are hereby incorporated by reference herein in their entirety.)

[0115] In some embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. A host cell having AAV helper functions can be referred to as a “helper cell” or “packaging helper cell.” AAV helper constructs are thussometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be, for example, in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as plasmids pAAV / Ad and pIM29+45 which encode both rep and cap expression products. A number of other vectors are known which encode rep and / or cap expression products. Recombinant AAV can be produced, for example, as described in U.S. Patent No. 9,408,904; and International Application Nos. PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are herein incorporated in their entirety.

[0116] In some embodiments, a rAAV vector is produced by a rAAV production cell comprising rAAV helper virus activity. The genome of the rAAV production cell comprises rAAV nucleic acid, the rep gene and the cap gene.

[0117] In some embodiments, a rAAV vector is produced by culturing a rAAV permissive cell comprising an AAV genome plasmid, where the rAAV permissive cell further comprises rep and cap genes provided either as part of the cell genome and / or by one or more separate plasmids; and helper virus activity either as part of the cell genome and / or provided by one or more separate plasmids. In some embodiments, (a) the rAAV permissive cell line is a packaging cell, wherein the genome of the packaging cell comprises the cap gene and the rep gene; (b) the rep gene, cap gene, and helper activity are provided from the same plasmid; or (c) the rep gene and cap gene are provided by a rep / cap plasmid and helper activity is provided by a helper plasmid.

[0118] In some embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding for at least UL5, UL8, UL52, and ICP8.

[0119] In some embodiments involving the use of adenovirus helper functions, the helper functions are provided by genes encoding for at least El A, E1B19K, E1B55K, E2A, E4orf6 and VA RNA. In some embodiments El , E2A and VR RNA functions are provided by a helper plasmid, where additional helper functions are provided by a host strain.

[0120] In some embodiments, rAAV vector is obtained by producing rAAV using methods described herein and purifying the rAAV. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based, and combined methods. (See, e.g., Ayuso etal., (2010), Curr Gene Ther. (2010) 10(6):423-36, hereby incorporated by reference herein in its entirety.) 1III.C. Retrovirus Vectors

[0121] Retroviruses are enveloped, single-stranded RNA viruses comprising 5’ and 3’ LTRs, and a signal packaging sequence located just outside of the LTR. Different types of retrovirus vectors can contain different amounts of viral genome. In some embodiments, the retrovirus vector is a lentiviral vector based on HIV, retaining all cis-acting sequences needed for viral RNA packaging, reverse transcription and proviral DNA integration, while removing all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to about 9 kb. If needed, stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced by supplying viral proteins needed for vector production in trans using appropriate plasmids and cell lines. (Bulcha etal., (2021 ) Sig. Transduct. Target Ther. 6:53.)

[0122] In some embodiments, the promoter sequence operably linked to a transgene is encoded for by RNA, such as provided by a retrovirus vector, which produces DNA. The produced DNA comprises the promoter operably linked to the transgene, which can be used to facilitate transcription.IV. Non- Viral Vectors

[0123] In some embodiments, the promoter operatively linked to a transgene, or expression cassette, is delivered to a cell with a non-viral vector. Non-viral vectors include nanoparticles and naked nucleic acid. Non-viral vectors can be used to deliver different types of nucleic acid and nucleic acid vectors into a cell.

[0124] In some embodiments, the non-viral vector is a nanoparticle. A variety of different nanoparticles can be employed including lipid nanoparticles (LNP), polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, e.g., Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; Neshat et al., (2020) Current Opin. Biotechnol. 66:1 -10; Ouranidis et al., (2022) Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21 ;7(1): 166, each of which are hereby incorporated by reference herein in their entirety.)

[0125] If desired, a nanoparticle can target a cell type using, for example, targeting ligands recognizing a target cell receptor. Examples of targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and protein / peptides (e.g., RGD, epidermal growth factor, and lowdensity lipoprotein) and peptides. (For example, Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41.)

[0126] Nanoparticles can be used to deliver transgenes to a cell. In some embodiments, nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds is provided in different nanoparticles. Reference to compound includes small molecules and large molecules (e.g., therapeutic proteins and antibodies).

[0127] The production of different nanoparticles and incorporation of nucleic acid and other compounds is well known in the art. Examples of publications illustrating incorporation of nucleic acid in a particular nanoparticle such as an LPNP and a LNP include Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41 ; Bochicchio etal., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira etal., (2017) Prog. Lipid Res. Oct;68:l-11 (each of which are hereby incorporated by reference herein in their entirety). Factors that may impact small molecule incorporation into a nanoparticle include hydrophobicity and the presence of an ionizable moiety. (See, e.g., Nii and Ishii, Int. J. Pharm. (2005) 298: 198-205; and Chen etal., J. Control. Release (2018) 286:46-54.)

[0128] International Publication No. W02023 / 004437 (hereby incorporated by reference herein in its entirety), describes a variety of different non-viral vector that can used to deliver transgenes to a cell or subject, including lipid-based delivery systems, polymer-based nanoparticles, lipid polymer nanoparticles, protein and peptide-based nanoparticles, peptide cage nanoparticles and exosomes.V, Pharmaceutical Compositions

[0129] Pharmaceutical compositions comprise a pharmaceutical acceptable carrier facilitating administration and / or storage of polynucleotides comprising a transgene, viral vectors or non- viral vectors. Reference to “pharmaceutically acceptable” indicates the components do not cause substantial undesirable biological effects at the amount utilized. Pharmaceutically acceptable carriers can contain different components such as one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include salt, sugar, buffer, solvent, preservative, protein and surfactant. A particular excipient can have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used with viral vectors are provided in, for example, International Patent Publication No.WO2021 / 071835.

[0130] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery. Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions, which preparations are typicallysterile and can be isotonic with the blood of the intended recipient. Illustrative examples include water, buffered saline, Hanks’ solution, Ringer’s solution, dextrose, fructose, ethanol, animal vegetable and synthetic oils. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0131] In an embodiment, the pharmaceutical composition contains a formulation capable of injection into a subject. Examples of injectable formulation components include isotonic, sterile, saline solutions, salts (e.g., monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and mixtures of such salts), buffered saline, sugars (e.g., dextrose), and water for injection. Pharmaceutical compositions include dry, for example, freeze-dried compositions which upon addition of sterilized water or physiological saline, permit the constitution of solutions suitable for administration.

[0132] Additionally, suspensions can be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilizers or agents which increase compound solubility facilitating the preparation of concentrated solutions.

[0133] An “effective amount” or “sufficient amount” refers to an amount providing an indicated or desired effect. The effective amount can be administered, in single or multiple doses, alone or in combination, with one or more other compositions (e.g., additional therapeutic or immunosuppressive agents), treatments, protocols, or therapeutic regimens; and provide for a long- or short-term response.

[0134] Pharmaceutical compositions comprising transgenes encoding therapeutic protein can be delivered to a subject, so as to allow production of the encoded protein. Delivery can be in vivo or ex vivo. In some embodiments, pharmaceutical compositions comprise sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a protein in the subject.

[0135] A “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired or indicated biological or medicinal response in a subject. A therapeutically effective amount can be determined, for example, based on observed symptoms and / or through the use of biomarkers associated with a particular disease or disorder. Selection of a particular effective dose can be optimized taking into account different factors, including the disease or disorder to be treated or prevented, the symptoms involved, the targeted disease or disorder, safety and effectiveness in animal models, the patient’s body mass, and the patient’s immune status. The optimal dose to be employed in the formulation will also depend on theroute of administration, and the severity of the disease or disorder, and can be evaluated depending upon patient’s circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0136] In some embodiments, a pharmaceutical composition comprising a rAAV vector comprises empty AAV capsids. In some embodiments, in a pharmaceutical composition comprising rAAV vectors and empty AAV capsids, the ratio of empty AAV capsids to rAAV vector is within or between about 100:1 -50: 1, from about 50: 1-25: 1, from about 25: 1-10:1, from about 10: 1-1 : 1, from about 1 : 1-1 : 10, from about 1 : 10-1 :25, from about 1 :25-1 :50, or from about 1 : 50- 1 : 100. In some embodiments, the ratio of the empty AAV capsids to the rAAV vector is about 2:1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10:1.

[0137] Additional guidance and examples of pharmaceutical compositions and delivery systems are provided in, for example, Remington: The Science and Practice of Pharmacy (2020) 23th ed., University of the Science in Philadelphia, published by Elsevier; The Merck Index (2013) 15th ed., Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD.VI. Administration and Treatment

[0138] Polynucleotides comprising a transgene encoding therapeutic protein, viral vectors and non-viral vectors can be administered to a subject, preferably a human subject, to provide for prophylactic treatment reducing the likelihood or severity of a disease or disorder and / or treating a diagnosed disease or disorder. In some embodiments, the particular therapeutic agent, route of administration, and / or pharmaceutical composition is selected taking into account the particular disease or disorder being treated.

[0139] Optimal doses can vary depending upon different factors such as a particular therapeutic, desired endpoint and administration route. The dose amount, number, frequency or duration can be proportionally increased or reduced, taking into account adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject.

[0140] A “unit dosage form” refers to a physically discrete unit containing a predetermined effective amount of active ingredient in combination with a pharmaceutically acceptable carrier. Unit dosage forms can be provided within, for example, ampules and vials, which can include a pharmaceutically acceptable carrier, or a composition in a freeze-dried or lyophilized state. In the case of a freeze-dried or lyophilized state, a sterile liquid carrier can be added prior to administration. Individual unit dosage forms can be included in multi-dose kits or containers.

[0141] An “effective amount” achieves the desired or indicated effect. For example, an effective amount for treatment decreases one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces disease or disorder progression. Preferred effective amounts for treatment are effective to decrease multiple or all adverse symptoms.

[0142] In some embodiments a suitable dosage is from about 0.01 mg / kg to about 10 mg / kg of vector per kg body weight of a subject, about 0.01 mg / kg to about 0.1 mg / kg of vector per kg body weight of a subject, about 0.1 mg / kg to about 1.0 mg / kg of vector per kg body weight of a subject, or about 1.0 mg / kg to about 10 mg / kg of vector per body weight of a subject.

[0143] Generally, rAAV doses range from at least IxlO8vector genomes per kilogram (vg / kg) of the weight of the subject, or more, for example, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13or IxlO14, or more, vector genomes per kilogram (vg / kg) of the weight of the subject, to achieve a therapeutic effect. In some embodiments the rAAV dose is about 5xl0nrAAV vg / kg or greater than about 5xl0nrAAV vg / kg; about IxlO12rAAV vg / kg or greater than about IxlO12rAAV vg / kg; about 2x1012rAAV vg / kg or greater than about 2x1012rAAV vg / kg; about 3x1012rAAV vg / kg or greater than about 3xl012rAAV vg / kg; about 4xlO12rAAV vg / kg or greater than about 4xl012rAAV vg / kg; about 5xl012rAAV vg / kg or greater than about 5xl012rAAV vg / kg; about IxlO13rAAV vg / kg or greater than about IxlO13rAAV vg / kg; about 2xlO13rAAV vg / kg or greater than about 2x1013rAAV vg / kg; about 3xl013rAAV vg / kg or greater than about 3x1013rAAV vg / kg; about 4xl013rAAV vg / kg or greater than about 4xl013rAAV vg / kg; about 5xl013rAAV vg / kg or greater than about 5xl013rAAV vg / kg; about 6xl013rAAV vg / kg or greater than about 6x1013rAAV vg / kg.

[0144] Examples of dose ranges of rAAV vg / kg include a dose range from about 5xl0nto about 6x1013rAAV vg / kg; a dose range from about 5xl0nto about 5.5xl0nrAAV vg / kg; a dose range from about 5.5xl0nto about 6xlOnrAAV vg / kg; a dose range from about 6xlOnto about 6.5xlOnrAAV vg / kg; a dose range from about 6.5xlOnto about 7xlOnrAAV vg / kg; a dose range from about 7xlOnto about 7.5xlOnrAAV vg / kg; a dose range from about 7.5xlOnto about 8xl0nrAAV vg / kg; a dose range from about 8xl0nto about 8.5xl0nrAAV vg / kg; a dose range from about 8.5xl0nto about 9xlOnrAAV vg / kg; a dose range from about 9xlOnto about 9.5xlOnrAAV vg / kg; a dose range from about 9.5xlOnto about IxlO12rAAV vg / kg; a dose range from about IxlO12to about 1.5xl012rAAV vg / kg; a dose range from about 1.5xl012to about 2xl012rAAV vg / kg; a dose range from about 2xl012to about 2.5xl012rAAV vg / kg; a dose range from about 2.5xl012to about 3xl012rAAV vg / kg; a dose range from about 3xl012to about 3.5xl012rAAV vg / kg; a dose range from about 3.5xl012to about 4xl012rAAV vg / kg; a dose range from about 4xl012to about 4.5xl012rAAV vg / kg; a dose range from about 4.5xl012to about 5xl012rAAV vg / kg; a dose range from about 5xl012to about 5.5xl012rAAV vg / kg; a dose range from about 5.5xl012to about 6xl012rAAV vg / kg; a dose range from about 6xl012to about 6.5xl012rAAV vg / kg; a dose range from about 6.5xl012to about 7xl012rAAV vg / kg; a dose range from about 7xl012to about 7.5xl012rAAV vg / kg; a dose range from about 7.5xl012to about 8xl012rAAV vg / kg; a dose range from about 8xl012to about 8.5xl012rAAV vg / kg; a dose range from about 8.5xl012to about 9xl012rAAV vg / kg; a dose range from about 9xl012to about 9.5xl012rAAV vg / kg; a dose range from about 9.5xl012to about IxlO13rAAV vg / kg; a dose range from about IxlO13to about 1.5xl013rAAV vg / kg; a dose range from about 1.5xl013to about 2xl013rAAV vg / kg; a dose range from about 2xl013to about 2.5xl013rAAV vg / kg; a dose range from about 2.5xl013to about 3xl013rAAV vg / kg; a dose range from about 3xl013to about 3.5xl013rAAV vg / kg; a dose range from about 3.5xl013to about 4xl013rAAV vg / kg; a dose range from about 4xl013to about 4.5xl013rAAV vg / kg; a dose range from about 4.5xl013to about 5xl013rAAV vg / kg; a dose range from about 5xl013to about 5.5xl013rAAV vg / kg; a dose range from about 5.5x1013to about 6x1013rAAV vg / kg; or a dose range from about 6xl013to about IxlO14rAAV vg / kg.

[0145] In some embodiments, rAAV vg / kg are administered at a dose of about 5xl0nvg / kg, about 6xlOnvg / kg, about 7xlOnvg / kg, about 8xl0nvg / kg, about 9xlOnvg / kg, about IxlO12vg / kg, about 2xl012vg / kg, about 3xl012vg / kg, about 4xl012vg / kg, about 5xl012vg / kg, about 6xl012vg / kg, about 7xl012vg / kg, about 8xl012vg / kg, about 9xl012vg / kg, about IxlO13vg / kg, about 2xl013vg / kg, about 3xl013vg / kg, about 4xl013vg / kg, about 5xl013vg / kg, or about 6x1013vg / kg.

[0146] In some embodiments doses and dose ranges for other viral vectors are as provided herein with respect to rAAV. For example, in some embodiments the dose and dose range for recombinant adenovirus vectors, recombinant retrovirus vectors (e.g., lentivirus), and recombinant herpes simplex virus vectors is the same as illustrated above with respect to rAAV.

[0147] In some embodiments, polynucleotide constructs, viral vectors and non-viral vectors described herein are administered in combination with additional compounds or treatments for a particular disease of disorder; and / or in combination with a compound decreasing an immune response generated against the provided or produced polypeptide, polynucleotide, and / or delivery vehicle. Additional compounds or treatments can be provided in different modalities such as administered separately; and administered or performed prior to, substantially contemporaneously with or following administration of the polynucleotide constructs, viral vectors and non-viral vectors described herein.

[0148] In some embodiments, administration of polynucleotides comprising a transgene encoding a therapeutic protein, viral vectors and non-viral vectors described herein is incombination with an immunosuppressive agent or regimen. Such agents and regimens can be utilized, as needed, to achieve immune tolerance or mitigate the immune response to the produced therapeutic protein, the provided polynucleotides, or the provided delivery vehicles. Examples of immunosuppressive agents and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)- rapamycin (rapamycin encapsulated in a biodegradable nanoparticle)), ImmTOR-IL™ (ImmTOR with Treg-selective IL-2 agonist), B-cell depletion, immunoadsorption, and plasmapheresis.

[0149] In some embodiments, the viral vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents, where one or more immunosuppressive agent is administered prior to, substantially at the same time as, or after, administering the vector or non- viral vector. In some embodiments, the one or more immunosuppressive agent is administered concomitantly with a vector or non-viral vector. In some embodiments, the one or more immunosuppressive agents is administered 1-12, 12-24 or 24-48 hours; or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days prior to viral or non-viral vector administration. In some embodiments, the one or more immunosuppressive agent is administered 1-12, 12-24 or 24-48 hours; or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30- 50 days, or more than 50 days, following viral or non-viral vector administration.Administration of immunosuppressive agents after a period of time following administering vector or non-viral vector can be done, for example, if there is a decrease in the encoded protein after the initial expression levels for a period of time, e.g., 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 or more than 200 days following vector or non-viral vector administration.

[0150] In some embodiments, the immunosuppressive agent is an anti-inflammatory agent. In some embodiments, the immunosuppressive agent is a steroid, e.g., a corticosteroid. In some embodiments, the immunosuppressive agent is prednisone, prednisolone, calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g. CellCept®, Myfortic®), CD52 inhibitor (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, belatacept), anti-CD3 mAb, anti-LFA- 1 mAb (e.g., efalizumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epratuzumab), anti-CD20 mAb (e.g., rituximab, orelizumab, ofatumumab, veltuzumab), proteasome inhibitor (e.g., bortezomib), TACI-Ig (e.g., atacicept), anti-C5 mAb (e.g., eculizumab), mycophenolate, azathioprine, sirolimus everolimus, TNFR-Ig, anti-TNF mAb, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 mAb (e.g., secukinumab), anti -IL-6 mAb (e.g., anti-IL-6 antibody sirukumab, anti-IL-6 receptor antibody tocilizumab (Actemra®), IL-10 inhibitor, TGF-beta inhibitor, a B cell targeting antibody (e.g., rituximab), a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin), synthetic vaccine particle (SVP™)-rapamycin(rapamycin encapsulated in a biodegradable nanoparticle), intravenous gamma globulin (IVIG), omalizumab, methotrexate, a tyrosine kinase inhibitor (e.g., ibrutinib), cyclophosphamide, fingolimod, an inhibitor of B-cell activating factor (BAFF) (e.g., anti-BAFF mAb, e.g., belimumab), an inhibitor of a proliferation-inducing ligand (APRIL), anti-IL-lb mAb (e.g., canakinumab (Haris®)), a C3a inhibitor, a Tregitope (see, e.g., U.S. Patent No. 10,213,496), or a combination and / or derivative thereof.

[0151] Immune-suppression protocols, including the use of rapamycin, alone or in combination with IL- 10, can be used to decrease, reduce, inhibit, prevent or block humoral and cellular immune responses to the therapeutic protein. Hepatic gene transfer with viral vector (e.g., r AAV) and non-viral vector can be used to induce immune tolerance to the therapeutic protein through induction of regulatory T cells (Tregs).

[0152] Strategies to reduce (overcome) or avoid humoral immunity to viral vectors, such as rAAV in systemic gene transfer include, administering high vector doses; use of AAV empty capsids as decoys to adsorb anti-AAV antibodies; administration of immunosuppressive drugs to decrease, reduce, inhibit, prevent or eradicate the humoral immune response to rAAV; changing the rAAV capsid serotype or engineering the rAAV capsid to be less susceptible to neutralizing antibodies; use of plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titer; and use of delivery techniques such as balloon catheters followed by saline flushing. Such strategies are described in Mingozzi etal., (2013) Blood, 122:23-36. Additional strategies include using AAV-specific plasmapheresis columns to selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma, as described in Bertin etal., 2020, Set. Rep. 10:864. Similar techniques and strategies can be used for other types of viral vectors.

[0153] Empty capsids used as decoy probes can be provided in different ratios to viral vectors. Amounts of empty capsids administered can be calibrated based upon the amount (titer) of antibodies produced in a particular subject. In some embodiments, the ratio of the empty AAV capsids to the rAAV vector is within or between about 100: 1 -50:1, from about 50:1 to 25:1, from about 25:1 to 10: 1, from about 10: 1 to 1 :1, from about 1 :1 to 1 :10, from about 1 : 10 to 1 :25, from about 1 :25 to 1 :50, or from about 1 :50 to 1 : 100. In particular aspects, the ratio of the administered empty AAV capsids to rAAV vector is about 2:l, 3: 1, 4:1, 5:1, 6: 1, 7:1, 8: 1, 9: 1, or 10: 1. Preferably, the serotype of the empty capsids is the same as the rAAV serotype.

[0154] Strategies to reduce humoral immunity to rAAV (which can be applied to other viral vectors) include methods to remove, deplete, capture, and / or inactivate AAV antibodies, commonly referred to as apheresis and more particularly, plasmapheresis where blood products are involved. Apheresis or plasmapheresis, is a process where a human subject’s plasma iscirculated ex vivo (extracorporal) through a device that modifies the plasma through addition, removal and / or replacement of components before its return to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from a blood product (e.g., plasma). This procedure can be employed to deplete, capture, inactivate, reduce or remove immunoglobulins (antibodies) that bind AAV thereby reducing the titer of AAV antibodies in the treated subject that can contribute to rAAV neutralization. An example is using a device composed of an AAV capsid affinity matrix column, and passing blood product (e.g., plasma) through an AAV capsid affinity matrix resulting in binding of AAV antibodies of different isotypes. (See, e.g., Bertin et al., (2020) Sci. Rep. 10, 864, hereby incorporated by reference herein in its entirety.)

[0155] In some embodiments the polypeptide constructs, encoding polynucleotide constructs, viral vectors and non-viral vectors can be used in combination with an agent that blocks, inhibits, or reduces the interaction of IgG with the neonatal Fc receptor (FcRn), such as an anti- FcRn antibody, to reduce IgG recycling and enhance IgG clearance in vivo: and / or an agent that decreases the circulating antibodies that bind to a therapeutic an encoded therapeutic protein, encoding nucleic acid, or delivery vehicle. In some embodiments, antibody binding is reduced or inhibited by an agent that reduces interaction of IgG with FcRn, a protease or a glycosidase. Such treatment can, for example, be carried out as part of the vector administration to reduce or clear vector neutralizing antibodies.

[0156] In some embodiments, the polypeptide constructs, polynucleotide constructs, viral vectors and non-viral vectors described herein are used in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or a modified variant thereof, or an endoglycosidase (e.g., S. pyogenes EndoS) or a modified variant thereof. Such treatment can, for example, be carried out to reduce or clear neutralizing antibodies and enable treatment of patients previously viewed as not eligible for treatment. Such strategies are described in, for example, Leborgne et al., (2020) Nat. Med., 26:1096-1101 ; and U.S. Patent Application No. 2023142731.VII. Kits

[0157] The present invention includes kits with packaging material and one or more components. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo. A kit can contain a collection components, for example, a polynucleotide comprises a transgene, a viral or a non-viral vector, and optionally a second active, such as another compound, agent, drug or composition.

[0158] A kit refers to a physical structure housing one or more components. Packaging material can maintain the components sterilely, and can be made of material suitable for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampules, vials, and tubes.

[0159] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information on a disease for which a kit component can be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and information for practicing any of the methods, uses, treatment protocols including prophylactic or therapeutic regimes described herein.

[0160] Labels or inserts can include information on one or more benefits a component can provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that can be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

[0161] Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD-or DVD-ROM / RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH media or memory type cards.VIII. Additional Aspects and Embodiments

[0162] Additional aspects, embodiments, and examples of combinations thereof include:

[0163] A first aspect of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises nucleotides 1-169 of SEQ ID NO: 1, also provided for by SEQ ID NO: 11.

[0164] Aspect 1 A of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises nucleotides 1 -314 of SEQ ID NO: 1, also provided for by SEQ ID NO: 13

[0165] A second aspect of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises a sequence of SEQ ID NO: 2.

[0166] A third aspect of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises a sequence of SEQ ID NO: 3.

[0167] A fourth aspect of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises a sequence of SEQ ID NO: 4.

[0168] Embodiment El a further describes the first aspect, wherein the promoter comprises, consists, or consists essentially of nucleotides 1-169, nucleotides 1-200, nucleotides 1-225, nucleotides 1-250, nucleotides 1-275, nucleotides 1-300, nucleotides 1 -325, nucleotides 1-350, nucleotides 1-375, nucleotides 1-400, nucleotides 1-450, nucleotides 1-500, nucleotides 1-550, nucleotides 1-600, or nucleotides 1-650 of SEQ ID NO: 1, or the nucleic acid sequence of SEQ ID NO: 1.

[0169] Embodiment Elb further describes the first aspect, wherein the promoter (a) comprises nucleotides 1-169; and (b) comprises up to 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, or 650 contiguous nucleotides of SEQ ID NO: 1. Reference to “comprises” used with “up to”, an indicated number of contiguous sequences excludes the presence of more than the indicated number of contiguous sequence from SEQ ID NO: 1 in the promoter, but allows for the presence of other sequence regions.

[0170] Embodiment El c further describes Aspect 1A, wherein the promoter comprises, consists, or consists essentially of nucleotides 1 -314, nucleotides 1-350, nucleotides 1-375, nucleotides 1-400, nucleotides 1-450, nucleotides 1-500, nucleotides 1-550, nucleotides 1 -600, or nucleotides 1-650 of SEQ ID NO: 1, or the nucleic acid sequence of SEQ ID NO: 1.

[0171] Embodiment El d further describes Aspect 1A, wherein the promoter (a) comprises nucleotides 1-314; and (b) comprises up to 325, 350, 375, 400, 450, 500, 550, 600, or 650 contiguous nucleotides of SEQ ID NO: 1. Reference to “comprises” used with “up to”, an indicated number of contiguous sequences excludes the presence of more than the indicated number of contiguous sequence from SEQ ID NO: 1 in the promoter, but allows for the presence of other sequence regions.

[0172] Embodiment E2 further describing the first aspect, Aspect Al, the second aspect, the third aspect, the fourth aspect, and El a, Elb, Elc and Eld, wherein the promoter is operably linked to a heterologous sequence.

[0173] Embodiment E3 further describing E2, wherein the heterologous sequence encodes a biologically active RNA or a protein.

[0174] Embodiment E4 further describing E3, wherein the polynucleotide further comprises one more expression control elements affecting RNA transcription, processing, or translation of encoded heterologous RNA. In some embodiments, expression control elements comprise a Kozak sequence, promoter enhancer sequence and / or polyadenylation sequence.

[0175] Embodiment E5 further describing the first aspect, Aspect Al, the second aspect, the third aspect, the fourth aspect, and each of embodiments El a, Elb, Elc, Eld, E2, E3 and E4, wherein the polynucleotide is a plasmid further comprising an origin of replication. In some embodiments the plasmid comprises a selectable marker conferring an ability to grow under selective pressure such in the presence of an antibiotic and / or certain growth media.

[0176] Embodiment E6 further describing the first aspect, Aspect Al, the second aspect, the third aspect, the fourth aspect, and each of embodiments El a, Elb, Elc, Eld, E2, E3, E4, and E5, wherein the polynucleotide is a recombinant viral vector nucleic acid and comprises 5’ and / or 3’ viral elements providing for viral packaging and replication. In some embodiments, 5’ and 3 ’ viral elements are present.

[0177] Embodiment E7 further describes E6, wherein the polynucleotide is an adeno-associated virus (AAV) vector nucleic acid comprising an inverted terminal repeat (ITR) flanking the 5 ’ terminus of said polynucleotide and an AAV ITR flanking the 3’ terminus of said polynucleotide recombinant. In some embodiments, the AAV vector nucleic acid is single stranded DNA or double-stranded DNA (e.g., self-complementary adeno-associated virus vector (scAAV) or short hairpin adeno-associated virus vector nucleic acid).

[0178] Embodiment E8 further describing E7, wherein the 5’ and 3’ ITR are either AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 or AAV3B, 5’ and 3’ ITRs.

[0179] Embodiment E9 further describing E7 and E8, wherein the polynucleotide is about 3 kb to about 5.2 kb in length. In some embodiments the polynucleotide is less than 5.2 kb, less than 5.1 kb, less than 5.0 kb, less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb; between 4 kb to 5.2 kb, 3.0 kb to 5.5 kb, 4.0 kb to 5.0 kb, 4.3 kb to 4.8 kb; or about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, or about 5.0 kb.

[0180] A fifth aspect provides a gene delivery vehicle comprising the polynucleotide provided for in the first aspect, Aspect Al, the second aspect, the third aspect, the fourth aspect, and each of embodiments El a, Elb, Elc, Eld, E2, E3, E4, E5, E6, E7, E8 and E9 wherein the gene delivery is a viral or non-viral vector.

[0181] Embodiment El 0 further describing the fifth aspect, wherein the gene delivery vehicle is a rAAV vector. In some embodiments: (1) the rAAV vector comprises a capsid comprising a VP1, VP2 or VP3 protein at least 90% identical, at least 95% identical, or comprising the VP1, VP2 and / or VP3 protein sequence of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-218, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAVl / rh.10, or SEQ ID NO: 6; or (2) comprises VP1 of SEQ ID NO: 6, VP2 of SEQ ID NO: 7, and VP3 of SEQ ID NO: 8.

[0182] A sixth aspect is directed to a method of producing a protein or biologically active RNA in a cell or subject comprising the step of transducing or transfecting the cell or subject with the gene delivery vehicle of any one of the fifth aspect or E10, wherein the protein or biologically active RNA is expressed in said cell or subject.IX, Sequences

[0183] Table 1 provides different sequences. In some embodiments, a polynucleotide comprises a nucleic acid sequence having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% to any of the nucleic acid sequences provided in Table 1. In some embodiments, a polypeptide comprises an amino acid sequence having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% to any of the amino acid sequences provided in Table 1.Table 1EXAMPLES

[0184] Examples are provided below further illustrating different features of the present invention and methodology for practicing the invention. The provided examples do not limit the claimed invention.

[0185] Example 1 : Promoter Driven mRNA Expression

[0186] The ability of different promoter constructs to drive expression was evaluated by measuring mRNA levels in transfected cells. The different promoter constructs comprise a promoter operably linked to DNA encoding human neuropeptide Y receptor (huNPY2R). The encoded huNPY2R has the amino acid sequence of SEQ ID NO: 9 and the encoding sequence is provided by SEQ ID NO: 10. The tested promoters and the corresponding sequences are provided in Table 2. CAG-eGFP was used a control.

[0187] Table 2

[0188] HEK293T cells were transfected with plasmids comprising the different promoter constructs using lipid-based transfection (HEK293T cells) or Magnetofection™ (SH-SY5Y cells), and mRNA expression was measured using the following procedure:Seed HEK293T cells at 30,000 cells / well, or SH-SY5Y cells at 60,000 cells / well in a 96- well plate 24-hours before transfectionTransfect HEK293T with jetOPTIMUS at a 3: 1 reagent to DNA ratio (0.13 pg DNA) Transfect SH-SY5Y with Magnetofection™ reagent (NeuroMag™) at a 4: 1 reagent to DNA ratio (0.2 pg DNA)Harvest cell pellets 48-hours after transfection for downstream qPCR of RNA Gently remove media and rinse cells with sterile, IX PBSAdd 50 pL of 0.03% trypsin / EDTA solution and incubate at 37 °C for 3 minutes Promptly add 100 pL of cell culture media containing at least 10% FBS to inactivate the trypsinPipette thoroughly to form a single cell suspension. Transfer suspension to new tubes Centrifuge cells at 200g for 5 minutes at 4 °C, remove media, rinse once with cold PBS, and centrifuge againIsolate RNA from cell pellets (pool 3 wells of same condition) using AllPrep DNA / RNA / miRNA Universal Kit (Qiagen; #80224), nanodrop, and dilute each RNA sample to 2.5 ng / pL in Rnase-free waterPerform RT-qPCR for detection of NPY2R transcripts (and Gapdh internal control) using a primer / probe sets targeting NPY2R transcripts and Luna Probe One-Step RT-qPCR 4X MasterMix w / UDG (NEB; #M3019)

[0189] The level of mRNA expression from the different promoter constructs are illustrated in FIG. 1A and FIG. IB. FIG. 1A illustrates transgene expression in HEK293 T cells from promoters designated CAG, EFla, PGK-EFla, CaMKII, Bexl, and CMVe-hSyn. FIG. IB illustrates transgene expression in SH-SY5Y cells from promoters designated CAG andCaMKII.

[0190] Example 2: PGK-EFla and Bexlv2 Promoter Driven Expression

[0191] Transgene protein expression from promoter constructs designated PGK-EFla (SEQ ID NO: 2) and Bexlv2 (SEQ ID NO: 1) were detected by flow cytometry. The different promoter constructs comprise a promoter operably linked to DNA encoding human neuropeptide Y Receptor (huNPY2R). HEK293T cells were transfected with DNA plasmids using the protocol provided in Example 1. siRNA, when provided, was added at time of transfection to demonstrate signal specificity.

[0192] Blocking: After transfection, PBS was removed from cell pellets and cells were resuspended in 100 pL Fc blocking buffer (BD Pharmingen Human BD Fc Block (#564219)) at 2.5 pg / lM cells. Cells were incubated for 10 minutes in a dark at room temperature, then spun down at 200g for 5 minutes at 4 °C and the supernatant was removed.

[0193] Ab Staining: Primary Conjugated Ab staining was performed using huNPY2R Alexa Fluor® 488-conjugated antibody (R&D Systems FAB10211G). The conjugated primary Ab provides a direct correlation between the number of Y2R receptors and Y2R signal intensity. One fluorophore per receptor. The primary antibody was diluted in suspension buffer (FACS / Suspension / Staining buffer: 1% BSA in IX PBS), to 0.2 pg / pL (-1 :400 dilution (0.25 pg / lM cells)): 0.25 pL Ab in 100 pL buffer per sample).

[0194] Antibody staining was performed after blocking. Fc Block solution was removed, cells suspended in primary Ab dilution, and incubated in the dark for 30 minutes at 4 °C. Cells were spun down (200 g / 5 min / 4 °C), supernatant removed, and washed twice with suspension buffer. Samples were resuspended in -200 pL suspension buffer, followed by flow cytometry detection.

[0195] Flow cytometry results are shown in FIGs. 2A-2C. FIG. 2A illustrates results from Experiment #1 measuring relative fluorescence using 2 wells per condition, pooled for flow cytometry, huNPY2R siRNAs #1 and #2 mixed together (20 nM). FIG. 2B and FIG. 2C illustrates results from Experiment #2 measuring relative fluorescence (FIG. 2B) or percent huNPY2R-positive cells (FIG. 2C), 3 replicates per condition, 1 well per replicate, huNPY2R siRNA #1 (50 nM).

[0196] FIG. 3A and FIG. 3B are bar diagrams illustrating transgene protein expression from promoter constructs designated PGK-EFla and Bexl, using different antibody staining conditions. The results are from one experiment, 2 wells / condition (average of 9 fields of view per well, each at 6 depths of field), tdTomato reporter in the plasmid backbone was used to identify transfected cells. FIG. 3A illustrates percent of transfected cells also expressing transgene protein, determined using the tdTomato reporter. FIG. 3B illustrates relative fluorescence of the huNPY2R protein signal.

[0197] Example 3: Effect of siRNA on PGK-EF la Promoter Driven Expression

[0198] The effect of siRNA on transgene expression from the PGK-EFla (SEQ ID NO: 2) promoter was measured in HEK293T cells. Promoters were operatively linked to DNA encoding human neuropeptide Y Receptor (huNPY2R). HEK293T cells were transfected with plasmid using the same protocol as in Example 1 ; and siRNA, when provided, was added at time of transfection. Flow cytometry was carried out as described in Example 2.

[0199] FIG. 4 is a bar diagram illustrating protein expression from EFl a promoter-driven constructs. The results are from one experiment, 3 wells / condition (average of 9 fields of view per well, each at 6 depths of field) huNPY2R siRNA (50 nM), control (Ctrl) siRNA (50 nM).

[0200] Example 4: Bexlv2 Promoter Driven Expression in the Striatum and Cortex of Mouse Brains

[0201] 3el0 AAV-encapsidated vector genomes of a single-stranded AAV construct, comprising the Bexlv2 promoter operably linked to a human gene, were delivered by stereotactic single bilateral intrastriatal injection (3 gl total volume per side) into 10-week-old C57BL / 6J mice. At 3 months post-injection, mice were sacrificed, and fresh brain was dissected into left and right striatum, frontal cortex, and cerebellum. Total RNA was extracted from each tissue and levels of gene expression were measured by qRT-PCR using specific TaqMan® assays and an absolute quantification method. Gene expression levels (mRNA abundance) of the human gene are reported as normalized expression over the mouse ortholog (gray squares) (FIG. 5). Vehicle-treated animals (black ovals) showed no detectable expression of the human gene.

[0202] C57BL / 6J mice received bilateral intrastriatal injections of 3el0 AAV-encapsidated vector genomes of a single-stranded AAV construct encoding a reporter gene under the control of the Bexlv2 promoter, using an AAV capsid that broadly transduces a variety of cell types in the CNS. Formalin-fixed paraffin-embedded (FFPE) serial coronal brain sections (8 gm) were obtained at 3 months post-injection and co-stained with DAPI, a fluorescent-labeled antibody (Alexa-488) against NeuN (a neuronal specific marker), and a fluorescently-labeled RNAscope™ probe (Bio-Techne) against the reporter gene mRNA. Extensive co-localization of the RNAscope™ signal and the NeuN marker was observed, indicating neuronally -restricted activity of the Bexlv2 promoter.

[0203] While the invention has been described and illustrated with reference to some embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention.

Claims

CLAIMSI / we claim:

1. A polynucleotide comprising a promoter, wherein said promoter comprises a) the nucleic acid sequence of SEQ ID NO: 13; b) the nucleic acid sequence of SEQ ID NO: 2; c) the nucleic acid sequence of SEQ ID NO: 3; or d) the nucleic acid sequence of SEQ ID NO: 4.

2. The polynucleotide of claim 1 , wherein said promoter comprises the nucleic acid sequence of SEQ ID NO: 13.

3. The polynucleotide of claim 2, wherein said promoter comprises the sequence of SEQ ID NO: 1.

4. The polynucleotide of claim 1, wherein said promoter comprises the sequence of SEQ ID NO: 2.

5. The polynucleotide of claim 1, wherein said promoter comprises the sequence of SEQ ID NO: 3.

6. The polynucleotide of claim 1, wherein said promoter comprises the sequence of SEQ ID NO: 4.

7. The polynucleotide of any one of claims 1 -6, wherein said promoter is operably linked to a heterologous sequence.

8. The polynucleotide of claim 7, wherein said heterologous sequence encodes a biologically active RNA.

9. The polynucleotide of claim 7, wherein said heterologous sequence encodes a protein.

10. The polynucleotide of claim 8 and 9, wherein said polynucleotide further comprises a polyadenylation signal downstream of said heterologous sequence.

11. The polynucleotide of claim 9, wherein said polynucleotide further comprises a polyadenylation signal downstream of said heterologous sequence, and Kozak sequence.

12. The polynucleotide of any one of claims 1-11, where said polynucleotide is a plasmid further comprising an origin of replication.

13. The polynucleotide of any one of claims 1 -11, wherein said polynucleotide is a recombinant viral vector nucleic acid and comprises 5’ and / or 3’ viral elements providing for viral packaging and replication.

14. The polynucleotide of claim 13, wherein said recombinant viral vector nucleic acid comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR) flanking the 5’ terminus of said polynucleotide and an AAV ITR flanking the 3’ terminus of saidpolynucleotide.

15. The polynucleotide of claim 15, wherein said recombinant viral vector nucleic acid comprises the 5’ ITR and the 3’ ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 or AAV3B.

16. The polynucleotide of any one of claims 13-15, wherein said recombinant viral vector nucleic acid further comprises one or more stuffer sequences.

17. The polynucleotide of any one of claims 13-16, wherein said polynucleotide is 3 kb to 5.2 kb in length.

18. The polynucleotide of claim 17, wherein said polynucleotide is 4 kb to 5.1 kb in length.

19. A gene delivery vehicle comprising the polynucleotide of any one of claims 1 -18, wherein said gene delivery vehicle is a viral or non-viral vector.

20. The gene delivery vehicle of claim 19, wherein said gene delivery vehicle is a viral vector.

21. The gene delivery vehicle of claim 20, wherein said gene delivery vehicle is a recombinant adeno-associated virus (rAAV) vector, and said rAAV vector comprises a capsid comprising a VP1, VP2 or VP3 protein at least 90% identical to a VP1, VP2 or VP3 protein sequence of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-218, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAVl / rh.10, or SEQ ID NO: 6.

22. The gene delivery vehicle of claim 21, wherein said capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-218, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAVl / rh.10 capsid; or said capsid comprises VP1 of SEQ ID NO: 6.

23. The gene delivery vehicle of claim 22, wherein said capsid comprises VP1 of SEQ ID NO: 6, VP2 of SEQ ID NO: 7, and VP3 of SEQ ID NO: 8.

24. A method of producing a protein or biologically active RNA in a cell comprising the step of transducing or transfecting said cell with the gene delivery vehicle of any one of claims 19-23, wherein said protein or biologically active RNA is expressed in said cell.

25. The method of claim 24, wherein said polypeptide encodes for a protein and said method further comprising purifying said protein.

26. A method of producing a protein or biologically active RNA in a subject comprising the step of administering the gene delivery vehicle of any one of claims 19-23, to said subject, wherein said protein or biologically active RNA is expressed in said cell.

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