DNA promoters

Abstract

The present invention features promoters providing for DNA transcription. The promoters comprise: (1) a promoter core derived from the human peptidyl-prolyl isomerase G (PPIG) promoter, ATP citrate lyase (ACLY) promoter, actin gamma (ACTG1) promoter, actin related protein 2 (ACTR2) promoter, actin related protein 2 / 3 complex subunit 2 (ARPC2) promoter, or the vinculin (VCL) promoter; and / or (2) ocular specific transcription factor binding motifs from CRX, OTX2, and / or NRL.

Description

Attorney Docket Number: 065830.11507 / 32WO1 DNA PROMOTERS CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 661,003 filed on 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 (065830-32WO1.xml; Size: 211,959 bytes; and Date of Creation: June 3, 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 a 5’ UTR that encompass the transcription start site. The 5’ UTR can also 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 the 5’ UTR. 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: (1) a promoter core derived from the human peptidyl-prolyl isomerase G (PPIG) promoter, ATP citrate lyase (ACLY) promoter, actin gamma (ACTG1) promoter, actin related protein 2 (ACTR2) promoter, actin related protein 2 / 3 complex subunit 2 (ARPC2) promoter, or the vinculin (VCL) promoter; and / or (2) ocular specific transcription factor binding motifs from Cone-Rod Homeobox; (CRX), Orthodenticle Homeobox 2 (OTX2) and / or Neural Retina Leucine Zipper (NRL).

[0008] A promoter “core” derived from the PPIG promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 15. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 15.

[0009] A promoter “core” derived from the ACLY promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 10. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 10.

[0010] A promoter “core” derived from the ACTG1 promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 11. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 11.

[0011] A promoter “core” derived from the ACTR2 promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 12. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 12.

[0012] A promoter “core” derived from the ARPC2 promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 13. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 13.

[0013] A promoter “core” derived from the VCL promoter indicates a nucleic acid with a sequence identity of at least 95% to the nucleic acid sequence of SEQ ID NO: 14. In certain embodiments the promoter core has a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 14.

[0014] The promoter “UTR” refers to a region downstream of the promoter core region that is transcribed, but is 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. In certain embodiments, the UTR may be operatively linked to an intron.

[0015] Thus, a first aspect of the present invention describes a polynucleotide comprising a protomer wherein said protomer comprises either: a) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 15, wherein said promoter does not comprise the nucleic acid sequence of the wild-type PPIG promoter, in a further embodiment the promoter does not comprise the nucleic acid sequence SEQ ID NO: 8;b) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 10, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 3; c) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 11, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 4; d) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 12, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 5; e) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 13,wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 6; orf) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 14, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 7.

[0016] Another aspect of the present invention describes a polynucleotide comprising a promoter, wherein the promoter comprises a promoter core nucleic acid sequence with a sequence identity of at least 90% to SEQ ID NO: 15, and the polynucleotide does not comprise a sequence of SEQ ID NO: 77, immediately upstream of the promoter core nucleic acid sequence.

[0017] Another aspect of the present invention is directed to a polynucleotide comprising a promoter, wherein the promoter comprises a core-UTR-Intron, wherein a) the core has a nucleic acid sequence with a sequence identity of at least 95% with SEQ ID NO: 15; b) the UTR has a sequence identity of at least 90% to a sequence selected from any of SEQ ID NOs: 17-19; and c) the Intron has a sequence identity of at least 90% to a sequence selected from any of SEQ ID NOs: 20-29.

[0018] The Intron from the UTR-Intron comprises splice donor and acceptor sites.

[0019] Another aspect of the invention is directed to a polynucleotide comprising a promoter, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 95% to any of SEQ ID NOs: 30-75 and 82.

[0020] Another aspect of the invention is directed to a polynucleotide comprising a promoter operatively linked to a heterologous transgene sequence, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 95% to any of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9.

[0021] A heterologous sequence in relation to a promoter, is a sequence that is not natively operatively linked to the promoter. For example, a PPIG derived promoter drives transcription of PPIG, and a heterologous sequence with respect to a PPIG derived sequence would be a sequence other than one encoding for PPIG. In certain embodiments the heterologous sequence is coding. In certain embodiments the heterologous sequence is non-coding.

[0022] Another aspect of the present invention is directed to a promoter comprising the sequence of any one of SEQ ID NOs: 84-93, 95-104, 106-115, 117-126, 128-137, 139-148, and 150-159.

[0023] Another aspect of the present invention is directed to a recombinant viral nucleic acid comprising (a) a transgene operatively linked to a promoter described herein and (b) 5’ and / or 3’ viral elements providing for viral packaging and replication.

[0024] Another aspect of the present invention is directed to a gene delivery vehicle comprising a polynucleotide that comprises a promoter described herein operatively linked to a transgene; and a viral or non-viral vector.

[0025] Another aspect of the present invention is directed to a method of producing a protein or biologically active nucleic acid in a cell comprising the step of transducing or transfecting the cell with a gene delivery vehicle comprising a promoter described herein operatively linked to a transgene. In certain embodiments, the gene delivery is a viral vector comprising the promoter operatively linked to a transgene encoding for a protein.

[0026] Another aspect of the present invention is directed to a method of producing a protein or biologically active nucleic acid in a subject comprising the step of administering a gene delivery vehicle comprising a promoter described herein operatively linked to a transgene, to the subject. In certain embodiments, the gene delivery vehicle is a viral vector comprising the promoter operatively linked to a transgene sequence encoding for a protein.

[0027] Another aspect of the present invention is directed to the use of a promoter described herein operatively linked to a transgene encoding for a protein or biologically active nucleic acid, for producing the protein or biologically active nucleic acid in a subject.

[0028] 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

[0029] FIGs.1A-1E illustrate the activity of different promoters in different cell lines. FIG. 1A illustrates activity in ARPE19 (epithelial) cells. FIG.1B illustrates activity in Y79 (photoreceptor-like) cells. FIG. 1C illustrates activity in Neuro2A (neuroblastoma) cells. FIG. 1D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.1E illustrates activity in HEK293 (embryonic kidney) cells. The dotted line indicates EF1alpha promoter activity. “CAG” refer tothe cytomegalovirus immediate‑early enhancer / chicken β‑actin promoter; “EF1A” refers to the elongated factor 1 alpha promoter; ACLY (SEQ ID NO: 3), ACTG1 (SEQ ID NO: 4), ACTR2 (SEQ ID NO: 5), ARPC2 (SEQ ID NO: 6), VCL (SEQ ID NO: 7), and PPIG-Core-Full-Intron (SEQ ID NO: 9).

[0030] FIGs.2A-2E illustrate the activity of different promoters plotted as a percentage of their parental sequence designs. FIG. 2A illustrates activity in ARPE19 (epithelial) cells. FIG. 2B illustrates activity in Y79 (photoreceptor-like) cells. FIG. 2C illustrates activity in Neuro2A (neuroblastoma) cells. FIG.2D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.2E illustrates activity in HEK293 (embryonic kidney) cells. ACLY-core (SEQ ID NO: 10), ACTG1-core (SEQ ID NO: 11), ACTR2-core (SEQ ID NO: 12), ARPC2-core (SEQ ID NO: 13), VCL-core (SEQ ID NO: 14), and PPIG-core (SEQ ID NO: 16, providing a PPIG-core+sequence).

[0031] FIGs.3A-3E illustrate the activity of different promoter core regions plotted as a percentage of the PPIG (SEQ ID NO: 8) promoter, containing the PPIG promoter distal region. FIG.3A illustrates activity in ARPE19 (epithelial) cells. FIG. 3B illustrates activity in Y79 (photoreceptor-like) cells. FIG. 3C illustrates activity in Neuro2A (neuroblastoma) cells. FIG. 3D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.3E illustrates activity in HEK293 (embryonic kidney) cells. The dotted line notes PPIG promoter activity. PPIG (SEQ ID NO: 8); PPIG-core-Full-intron (SEQ ID NO: 9), PPIG-core (SEQ ID NO: 16), IgH Intron (promoter with IgH intron, SEQ ID NO: 30), VCL intron (promoter with VCL intron, SEQ ID NO: 31), Promega intron (promoter with Promega intron, SEQ ID NO: 32).

[0032] FIG.4 show promoter activity in HEK293 cells transiently transfected with an ablation series of promoters produced by tiled deletions across the PPIG (SEQ ID NO: 8). FIX expression was plotted as a percent of control (dotted line). The deletions are indicated in the schematic of the promoter structure shown below.

[0033] FIGs.5A-5E illustrate the activity of different promoters. FIG. 5A illustrates activity in ARPE19 cells. FIG.5B illustrates activity in Y79 cells. FIG. 5C illustrates activity in Neuro2A cells. FIG.5D illustrates activity in Huh7 cells. FIG. 5E illustrates activity in HEK293 cells. Abl-combi 7plus 9 (SEQ ID NO: 68); Abl-combi 1to3 (SEQ ID NO: 67), Composite-Abl-1 (SEQ ID NO: 69), Composite-Abl-2 (SEQ ID NO: 70), Composite-Abl-3 (SEQ ID NO: 71), Composite-Abl-4 (SEQ ID NO: 72), Composite-Abl-5 (SEQ ID NO: 73), Composite-Abl-6 (SEQ ID NO: 74), Composite-Abl-7 (SEQ ID NO: 75) and PPIG (SEQ ID NO: 8).

[0034] FIG.6 illustrates expression of ACLY variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by different promoters. Mean intensity was assessed relative to the CAGpromoter. The different promoters were designated: Var1 (SEQ ID NO: 84); Var2 (SEQ ID NO: 85); Var3 (SEQ ID NO: 86); Var4 (SEQ ID NO: 87); Var5 (SEQ ID NO: 88); Var6 (SEQ ID NO: 89); Var7 (SEQ ID NO: 90); Var8 (SEQ ID NO: 91); Var9 (SEQ ID NO: 92); and wild-type (SEQ ID NO: 3).

[0035] FIG.7 illustrates expression of ACTG variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 95); Var2 (SEQ ID NO: 96); Var6 (SEQ ID NO: 100); Var9 (SEQ ID NO: 103); and wild-type (SEQ ID NO: 4).

[0036] FIG.8 illustrates expression of ACTR2 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 106); Var2 (SEQ ID NO: 107); Var3 (SEQ ID NO: 108); Var5 (SEQ ID NO: 110); Var6 (SEQ ID NO: 111); Var8 (SEQ ID NO: 113); and wild-type (SEQ ID NO: 5).

[0037] FIG.9 illustrates expression of ARPC2 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 117); Var2 (SEQ ID NO: 118); Var3 (SEQ ID NO: 119); Var4 (SEQ ID NO: 120); Var5 (SEQ ID NO: 121); Var6 (SEQ ID NO: 122); Var7 (SEQ ID NO: 123); Var9 (SEQ ID NO: 125); Var10 (SEQ ID NO: 126) and wild-type (SEQ ID NO: 6).

[0038] FIG.10 illustrates expression of PPIG-Core-UTR-Full-Intron variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 128); Var4 (SEQ ID NO: 131); Var6 (SEQ ID NO: 133); Var7 (SEQ ID NO: 134); Var8 (SEQ ID NO: 135); Var9 (SEQ ID NO: 136); and wild-type (SEQ ID NO: 138).

[0039] FIG.11 illustrates expression of PPIG-Composite 6 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoter were designated: Var2 (SEQ ID NO: 140); Var3 (SEQ ID NO: 141); Var4 (SEQ ID NO: 142); Var5 (SEQ ID NO: 143); Var8 (SEQ ID NO: 146); Var9 (SEQ ID NO: 147); Var10 (SEQ ID NO: 148); and wild-type (SEQ ID NO: 149).

[0040] FIG.12 illustrate expression of PPIG-Composite 7 variants comprising transcriptionfactor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by the different promoters. Mean intensity was assessed relative to the CAG promoter. The different promoter were designated: Var1 (SEQ ID NO: 150); Var2 (SEQ ID NO: 151); Var3 (SEQ ID NO: 152); Var4 (SEQ ID NO: 153); Var6 (SEQ ID NO: 155); Var7 (SEQ ID NO: 156); Var8 (SEQ ID NO: 157); Var9 (SEQ ID NO: 158); Var10 (SEQ ID NO: 159); and wild-type (SEQ ID NO: 160). DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention features promoters providing for DNA transcription. The promoters comprise: (1) a promoter core derived from the human peptidyl-prolyl isomerase G (PPIG) promoter, ATP citrate lyase (ACLY) promoter, actin gamma (ACTG1) promoter, actin related protein 2 (ACTR2) promoter, actin related protein 2 / 3 complex subunit 2 (ARPC2) promoter, or the vinculin (VCL) promoter; and / or (2) ocular specific transcription factor binding motifs from CRX, OTX2, or NRL. In certain embodiments, a promoter comprises all or a portion of a 5’ UTR downstream from core region. The examples provided below illustrate the ability to reduce promoter size, for example, by removing the distal regions and / or modifying the 5’ UTR; and to use ocular specific transcription factor binding motifs from CRX, OTX2, or NRL to enhance ocular transgene expression.

[0042] The promoters provided herein can be used in general to drive transgene transcription in different setting, such plasmids and viral vectors. Smaller promoter size offers advantages for use in vectors having a limited size.

[0043] In certain embodiments, the promoter provides for expression in multiple cell types.

[0044] In certain embodiments, promoters comprising ocular specific transcription factor binding motifs from CRX, OTX2, or NRL provide for increased ocular expression and / or increased selectivity for ocular expression. Reference to “ocular” expression indicates expression in one or more ocular cell. Reference to ocular expression does not exclude expression in non-ocular cells.

[0045] A “transgene” provides a sequence that is transcribed into RNA. Transcription can be achieved by operatively linking the transgene to a promoter which drives transcription to RNA.

[0046] Reference to “recombinant” provides a different combination than found in nature. For example, the transgene can be present in a vector and / or can be a heterologous sequence with respect to other components such as the transgene.

[0047] In different embodiments, the promoter provides for at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 100% activity compared to the corresponding full-length native promoter in one or more cell types.

[0048] Transgenes can be mono-cistronic or multi-cistronic. 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 mRNA transcript from a promoter, where the mRNA 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.

[0049] 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-12A), 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; both of which are hereby incorporated by reference herein in their entirety.) 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, hereby incorporated by reference herein in its entirety.)

[0050] 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(1):11-20, hereby incorporated by reference herein in its entirety.)

[0051] In certain embodiments the polynucleotide comprising the promoter is less than 5.5 kb. In further 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.

[0052] In certain embodiments, the polynucleotide is a recombinant adeno-associated virus (rAAV) viral nucleic acid. Recombinant AAV nucleic acid can be produced using AAV 3’ and 5’ ITRs elements providing for viral replication and packaging. The 3’ and 5’ ITRs can be added to an expression cassette, comprising a promotor operatively linked to a nucleic acid region, to facilitate transcription of the region. In certain embodiments the nucleic acid sequence encodes a protein.

[0053] 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 certain embodiments, a rAAV vector is used for transgene delivery.

[0054] The use of a rAAV vector for gene delivery can be limited by the AAV capsid packaging capacity of approximately 5.0 kb. Recombinant AAV expression cassettes comprising a transgene, may also contain additional regulatory elements involved in DNA transcription andprotein translation. Reducing the promoter size can facilitate the ability of rAAV vector to deliver larger transgenes and / or multiple transgenes.

[0055] In certain embodiments, the rAAV nucleic acid 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 a further embodiment the size of the transgene coding region is up to 4.4 kb.

[0056] In certain embodiments, the promoter constructs are used to deliver multiple transgenes to a cell or subject.

[0057] Transgene delivery to a subject can be used, for example, to express an encoded protein or biologically active nucleic acid in the subject. 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.

[0058] 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.

[0059] 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. For example, in the case where a core protomer comprises a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: X is combined with a UTR comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y, differentembodiments are explicitly provided for each of the combinations and ranges, for example: (1) a promoter core comprising a sequence identity of at least 95% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; a promoter core comprising a sequence identity of at least 96% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; a promoter core comprising a sequence identity of at least 97% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; a promoter core comprising a sequence identity of at least 98% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; a promoter core comprising a sequence identity of at least 99% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; a promoter core comprising a sequence identity of 100% to SEQ ID NO: X, is combined with a nucleic acid sequence comprising a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y; (2) specific combinations of each of the individual values are provided, for example a core promoter comprising a sequence identity of at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: Y, specifically includes the combinations of at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 95% to SEQ ID NO: Y, at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 96% to SEQ ID NO: Y, at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 97% to SEQ ID NO: Y, at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 98% to SEQ ID NO: Y, at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of at least 99% to SEQ ID NO: Y, and at least 95% to SEQ ID NO: X in combination with a nucleic acid sequence of 100% to SEQ ID NO: Y; and (3) further combinations with a third nucleic acid sequence provides embodiments were each of the third nucleic acid sequence embodiment is independently combined with (1) and (2).

[0060] 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.

[0061] In certain 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 shorthairpin (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.

[0062] In certain embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. In certain embodiments, nucleic acids can be single, double, or triplex, linear or circular, and can be of different lengths.

[0063] According to certain embodiments, the polynucleotide is a single-stranded (ssDNA) or a double-stranded DNA (dsDNA) molecule. According to certain 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 certain embodiments, the ssDNA molecule is a closed circular or an open linear DNA.

[0064] In certain embodiments, the nucleic acid is “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 different 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.

[0065] 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 (UTRs), stuffer sequences, promoter, enhancer, polyadenylation signal, ITRs, and introns.

[0066] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0067] 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 the applicability 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”. Concurrentapplicability of more than one of the options is also understood to fall within the meaning of the term “and / or.”

[0068] 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.

[0069] 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.

[0070] 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”.

[0071] 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.

[0072] 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.

[0073] The term “about” refers to a value within 10% of the underlying parameter (i.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 certain embodiments the term “about” provides for a value within 5% of the underlying parameter.

[0074] 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, 4 as 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] The definitions provided herein, including those in the present section and other sections of the application, apply throughout the present application.

[0079] 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.

[0080] 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.

[0081] Cross-reference to a particular sections of the present application, includes all the provided subsections.

[0082] 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, in certain 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. Derived Promoters

[0083] Reference to a derived promoter indicates the promoter comprises one or more elements of the reference promoter. In certain embodiments the derived promoter comprises a core region and / or ocular specific transcription factor binding motifs from CRX, OTX2, or NRL. In further embodiments, the promoter is a mini promoter comprising up to an indicated number of contiguous nucleotides from a reference sequence.

[0084] In certain embodiments, promoters described herein comprising ocular specific transcription factor binding motifs from CRX, OTX2, or NRL provide for increased ocular expression and / or ocular cell selectivity compared to the corresponding wild-type PPIG, ACLY, ACTG, or ARPC2 promoter.

[0085] In certain embodiments, the promoter comprises a sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NOs: 3, 4, 5, 6, 7, 8, or 9. In a further embodiment, the is promoter operatively coupled with a heterologous transgene I.A. PPIG Derived Promoter

[0086] PPIG derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 15, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0087] Aspect 1A-1 is directed to a polynucleotide comprising a promoter, wherein the promoter comprises a sequence at least 95% identical to the sequence of SEQ ID NO: 15. In different embodiments the promoter does not comprise the wild-type PPIG sequence or the nucleic acid sequence of SEQ ID NO: 8.

[0088] Embodiment E1 further describes aspect A1-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprises, the nucleic acid sequence of SEQ ID NO: 15.

[0089] Embodiment E2 further describes aspect A1-1 and E1, wherein the promoter does not comprise SEQ ID NO: 77 immediately upstream of the promoter core nucleic acid sequence.

[0090] Embodiment E3 further describes aspect A1-1 and E1, wherein the promoter does not comprise a sequence having a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 77 immediately upstream of the promoter core nucleic acid sequence.

[0091] A fourth embodiment E4 further describes aspect A1-1, E1, E2 and E3, wherein the promoter does not comprise a sequence having a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 76 immediately upstream of the promoter core nucleic acid sequence; and / or does not comprise a sequence having a sequence identity ofat least 95%, at least 96%, at least 97%, at least 98% or 100%, to the 3’ region of at least 30 nucleotides, least 35 nucleotide, at least 40 nucleotides, or at least 50 nucleotides of SEQ ID NO: 76, immediately upstream of the promoter core nucleic acid sequence.

[0092] Embodiment E5 further describes aspect A1-1, E1, E2, E3, and E4, wherein the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 8. In further embodiments, the promoter comprises up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, or up to 100 contiguous nucleotides provided in SEQ ID NO: 8.

[0093] Reference to comprises “up to” a certain number contiguous sequence of a reference sequence excludes longer length contiguous sequences from the reference sequence, but does not exclude other sequences.

[0094] Embodiment E6 further describes aspect A1-1, E1, E2, E3, E4, or E5, wherein the promoter further a 5’-UTR-Intron, wherein the UTR comprises a sequence at least 90%, identical to a sequence selected from any of SEQ ID NOs: 17-19; and the Intron comprises a sequence at least 90% identical to a sequence selected from any of SEQ ID NOs: 20-29.

[0095] Reference to “5’-UTR-Intron” indicates the UTR-Intron is at the 5’ end of the referenced promoter sequence.

[0096] Embodiment E7 further describes E6, wherein the UTR comprises a sequence at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprises, a sequence selected from any of SEQ ID NOs: 17-19

[0097] Embodiment E8 further describes E6 and E7, wherein the Intron comprises a sequence at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprises, a sequence selected from any of SEQ ID NOs: 20-29.

[0098] Embodiment E9 further describes the aspect A1-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 95% to any of SEQ ID NOs: 30-75, 82 or 83. In further embodiments the promotor comprises a sequence with a sequence identity (1) of at least 96%, at least 97%, at least 98%, at least 99% or 100% to the nucleic acid sequence of any of SEQ ID NOs: 30-75, 82 and 83; and (2) the provided sequence identity is with respect to any of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 74, 75 or 82.

[0099] Aspect 1A-2 is directed to a polynucleotide comprising a promoter operatively linked to a downstream heterologous transgene, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 90% to SEQ ID NO: 15. The heterologous sequence withrespect to a promoter derived from the PPIG promoter is a nucleic acid sequence that does not encode native PPIG.

[0100] Aspect 1A-3 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of SEQ ID NOs: 8 or 9, wherein the promoter does not comprise the wild-type PPIG sequence.

[0101] Aspect 1A-4 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 128-137, 139- 148, and 150-159. I.B. ACLY Derived Promoter

[0102] ACLY derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 10, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0103] Aspect 1B-1 is directed to a polynucleotide comprising a promoter nucleic acid sequence at least 95% identical to the sequence of SEQ ID NOs: 10; and in a further embodiment the promoter does not comprise the sequence of SEQ ID NO: 3.

[0104] Embodiment E10 further describes aspect 1B-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% to, or comprises, the nucleic acid sequence of SEQ ID NO: 10.

[0105] Embodiment E11 further describes aspect 1B-1 and E10, wherein the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 3. In further embodiments, the promoter comprises up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, or up to 300, contiguous nucleotides provided in SEQ ID NO: 3.

[0106] Aspect 1B-2 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 3, wherein the promoter does not comprise the wild-type ACLY sequence.

[0107] Aspect 1B-3 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 84-93. I.C. ACTG Derived Promoter

[0108] ACTG derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 11, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0109] Aspect 1C-1 is directed to a promoter comprising a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 11; and in further embodiments the promoter does not comprise the sequence of SEQ ID NO: 4.

[0110] Embodiment E12 further describes aspect 1C-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% to, or comprising, the nucleic acid sequence of SEQ ID NO: 11.

[0111] Embodiment E13 further describes aspect 1C-1 and E12, wherein the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 4. In further embodiments, the promoter comprises up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, or up to 150 contiguous nucleotides provided in SEQ ID NO: 4.

[0112] Aspect 1C-2 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 4, wherein the promoter does not comprise the wild-type ACLY sequence.

[0113] Aspect 1C-3 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 95-104. I.D. ACTR2 Derived Promoter

[0114] ACTR2 derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 12, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0115] Aspect 1D-1 is directed to a promoter comprising a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 12, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 5.

[0116] Embodiment E14 further describes aspect 1D-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% to, or comprising, the nucleic acid sequence of SEQ ID NO: 12.

[0117] Embodiment E15 further describes aspect 1D-1 and E14, wherein, the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 5. In further embodiments, the promoter comprises up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, or up to 150 contiguous nucleotides provided in SEQ ID NO: 5.

[0118] Aspect 1D-2 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 106-115. I.E. ARPC2 Derived Promoter

[0119] ARPC2 derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 13, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0120] Aspect 1E-1 is directed to a promoter comprising a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 13, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 6.

[0121] Embodiment E16 further describes aspect 1E-1, wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% or comprising the nucleic acid sequence of SEQ ID NO: 13.

[0122] Embodiment E17 further describes aspect 1E-1 and E16, wherein the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 6. In further embodiments, the promoter comprises up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, or up to 150 contiguous nucleotides provided in SEQ ID NO: 6.

[0123] Aspect 1E-2 is directed to a polynucleotide comprising a promoter sequence, wherein the promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 117-126. I.F. VCL Derived Promoter

[0124] VCL derived promoters comprise a nucleic acid sequence having a sequence identity of least 95% to the core sequence of SEQ ID NO: 14, wherein the promoter does not comprise the full-length wild-type sequence; and / or comprises ocular specific transcription factor binding motifs from CRX, OTX2, or NRL.

[0125] Aspect 1F-1 is directed to a promoter comprising a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 14, wherein the promoter does not comprise an nucleic acid sequence of SEQ ID NO: 7.

[0126] Embodiment E18 further describes aspect 1F-1 wherein the promoter comprises a nucleic acid sequence with a sequence identity of at least 96%, at least 97%, at least 98%, at least 99% to, or comprising, the nucleic acid sequence of SEQ ID NO: 14.

[0127] Embodiment (E19) further describes aspect 1F-1 and E18, wherein the promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 7. In further embodiments, the promoter comprises up to 750 or up to 700 contiguous nucleotides provided in SEQ ID NO: 7. II. Transgene

[0128] Promoter constructs described throughout Section I supra. (including the provided subsections), can be used to drive transcription of a variety of different operatively linkedtransgene sequences. In certain embodiments, the transgene is a heterologous sequence, encodes a protein and / or encodes a sequence that is not translated (non-coding sequence).

[0129] In different embodiments, the transgene encodes a protein and / or biologically active RNA such as an inhibitory nucleic acid or a stimulatory nucleic acid. Examples of inhibitory nucleic acid include short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, and an antisense RNA. An example of stimulatory nucleic acid is saRNA.

[0130] In certain embodiments, the transgene encodes for a therapeutic protein.

[0131] In certain embodiments, the transgene encodes a Rep and Cap protein.

[0132] In certain embodiments, the transgene encodes for inhibitory nucleic acid.

[0133] In certain embodiments, the transgene encodes for a miRNA.

[0134] Transgenes can be used, for example, in gene therapy to deliver a desired protein or biologically active nucleic acid. In certain embodiments, the transgenes encodes a healthy copy of protein in a subject where the subject protein is defective; the transgene provides a modified gene encoding a protein that can help treat a disease or disorder; or the transgene encodes a new protein providing a beneficial effect.

[0135] In different 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 (C1 protease inhibitor or C1 esterase inhibitor) for treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency type I and type II); or glucose-6- phosphatase for treatment of glycogen storage disease type I (GSDI).

[0136] In different 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 α (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I or II (IGF-I or IGF-II), TGFβ, 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.

[0137] In different embodiments, the transgene encodes thrombopoietin (TPO), an interleukin (IL-1 through IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α or β, interferons α, β, or γ, 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.

[0138] In different 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 VIIa, or protein C) a gain of function blood coagulation factor, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-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 α and β, a cytokine, α-interferon, β-interferon, interferon-γ, 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, deoxycytidine 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 hCDR1, 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, 3 and 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.

[0139] In different embodiments, the transgene encodes erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, or 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; alpha1-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.

[0140] In further 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.III. Expression Cassettes

[0141] Polynucleotide expression cassettes comprise a transgene operatively linked to a promoter 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. Examples of expression control elements include a promoter, enhancer, an intron, polyadenylation signal, a Kozak sequence, post-transcriptional regulator elements and a termination sequence.

[0142] Enhancers are DNA regions that increase promoter transcription. Enhancers can be adjacent, 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.

[0143] 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, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science 268: 1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol.2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech.15:239-243 (1997) and Wang et al., Gene Ther.4:432-441 (1997); and the rapamycin-inducible system (Magari et al., J. Clin. Invest.100:2865-2872 (1997); and Rivera et al., 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.

[0144] In certain 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 β-globin intron with splice donor / splice acceptor, SV40 intron with splice donor / splice acceptor, human β-globin introns, intron 2 of the human hemoglobin beta gene, hFIX int1 (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 thechicken 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 β-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 et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al., Mol. Ther. Methods Clin Dev. (2016) Jul 20;3:16049; and the HBB-IGG intron provided by the pCMVNT™ vector.)

[0145] In certain 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 et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0146] 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 β-globin pA, rabbit β-globin pA, and H4-based pA. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0147] In certain embodiments, the expression cassette comprises a Kozak consensus 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.

[0148] In certain 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 polyadenylation signal.

[0149] In certain embodiments the expression cassettes further comprises a miRNA target sequences, which in further embodiments is incorporated into the 3’ UTR of the expression cassette. A miRNA target sequence is recognized by miRNA present in particular cells or tissues leading to degradation of mRNA 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 et al., (2016) World Journal of Experimental Medicine 6(2): 37– 54.)

[0150] In certain 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. IV. Recombinant Viral Vector Nucleic Acid

[0151] Recombinant viral vector can be used for transgene delivery. 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 et al., (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.)

[0152] 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 vector nucleic acid provide 5’ and / or 3’ viral elements along with an expression cassette 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.

[0153] In certain 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.

[0154] In certain embodiments, the viral vector nucleic acid contains 0-5, 0-10, 0-15, 0-50, 0- 100, or 0 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 about8%, 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. V. Viral Vectors

[0155] 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.

[0156] 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).

[0157] 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. V.A. Adenovirus Vectors

[0158] 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 et al., (2021) Sig. Transduct. Target Ther.6:53.)

[0159] In certain 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 highcapacity 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. Stuffer 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 et al., (2021) Sig. Transduct. Target Ther. 6:53; and Sandig et al., PNAS (2000) 97(3):1002-1007, each of which are hereby incorporated by reference herein in their entirety.)

[0160] In certain 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.)

[0161] 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 et al., (2021) Sig. Transduct. Target Ther.6:53.) V.B. Recombinant AAV Vectors

[0162] Recombinant adeno-associated viral (also referred to herein as “rAAV”) vector are based on the adeno-associated virus. The adeno-associated virus 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 for self-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.

[0163] 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 certain embodiments, the rAAV vector contains an AAV 5’ and / or 3’ ITR along with a DNA insert. In certain 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 further embodiments 5’ and 3’ ITRs are present, and both ITRs are from the same serotype genome.

[0164] Recombinant adeno-associated viral vectors typically accept inserts of DNA having a size range generally about 4 kb to about 5.2 kb. If needed, stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency. In different embodiments, the rAAV nucleic acid including stuffer is 4-5.2 kb, 3.0-5.5 kb, 4.0-5.0 kb, 4.3-4.8 kb, about 4.2 kb, about4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences.

[0165] In certain 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 caspid. 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.)

[0166] 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 serotypes or where one, two or all three viral protein are based on different serotypes.

[0167] 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 certain embodiments, a rAAV nucleic acid has the same serotype genome (e.g., ITRs) as the encapsidating capsid protein.

[0168] In different 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% or 100% identical 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-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 78.

[0169] Recombinant AAV capsids comprising VP1 of SEQ ID NO: 78 is described, for example, in U.S. Patent No.9,840,719, hereby incorporated herein by reference.

[0170] In certain 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 orVP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, or SEQ ID NO: 78; 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.)

[0171] In certain embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 78; VP2 having the sequence of SEQ ID NO: 79; and VP3 having the sequence of SEQ ID NO: 80.

[0172] In certain embodiments, the AAV capsid can cross the blood brain barrier and provide for 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 AAV1 / rh.10), U.S. Patent No. 9,585,971, and Goertsen et al., Nat. Neurosci.25, 106– 115 (2022), each of which are incorporated by reference herein in its entirety.

[0173] 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.)

[0174] 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 HBoV1 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.)

[0175] 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. Incertain embodiments, rAAV is produced using a rAAV vector genome 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.

[0176] The vector genome plasmid may contain regions such an origin of replication and a selectable marker. Additional sites that may be present include cloning sites.

[0177] Recombinant AAV can be produced from different types of cell lines including HeLa, A549, BHK, Vero, and HEK293, or derivatives thereof. In certain 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 et al., (2017) Biotechnol. J.12(3), 1600193; and International Application No. PCT / US2017 / 024951, the disclosures of which are herein incorporated in its entirety.

[0178] Recombinant AAV can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV manufacturing include Clément and Grieger (2016) Mol. Ther. Methods Clin. Dev.16;3:16002; Robert et al., (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.)

[0179] In certain 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 thus sometimes 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.

[0180] In certain 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.

[0181] In certain 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 further 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.

[0182] In certain embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding for at least UL5, UL8, UL52, and ICP8.

[0183] In certain embodiments involving the use of adenovirus helper functions, the helper functions are provided by genes encoding for at least E1A, E1B19K, E1B55K, E2A, E4orf6 and VA RNA. In certain embodiments E1, E2A and VR RNA functions are provided by a helper plasmid, where additional helper functions are provided by a host strain.

[0184] In certain 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 et al., (2010), Curr Gene Ther.10(6):423-36, hereby incorporated by reference herein in its entirety.) V.C. Retrovirus Vectors

[0185] 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 certain 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 et al., (2021) Sig. Transduct. Target Ther. 6:53.)VI. Non-Viral Vectors

[0186] In certain embodiments, the transgene is used 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.

[0187] Preferred non-viral vectors are nanoparticles. 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.)

[0188] 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 low density lipoprotein) and peptides. (For example, Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41.)

[0189] Nanoparticles can be used to deliver transgenes to a cell. In different 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).

[0190] 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 et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct;68:1-11 (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 et al., J. Control. Release (2018) 286:46-54.)

[0191] International Publication No. WO2023 / 004437 (hereby incorporated by reference herein in its entirety), describes a variety of different non-viral vector that can used to delivertransgenes 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. VII. Pharmaceutical Compositions

[0192] 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 Nos. WO2021 / 071835 and WO2024 / 138129.

[0193] 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 typically sterile 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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 certain embodiments, pharmaceutical compositions comprise sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a protein in the subject.

[0198] 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 the route 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.

[0199] In certain embodiments, a pharmaceutical composition comprising a rAAV vector comprises empty AAV capsids. In certain 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 certain 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.

[0200] 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.VIII. Administration and Treatment

[0201] 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 certain embodiments, the particular therapeutic agent, route of administration, and / or pharmaceutical composition is selected taking into account the particular disease or disorder being treated.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] In different 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.

[0206] Generally, rAAV doses range from at least 1x108vector genomes per kilogram (vg / kg) of the weight of the subject, or more, for example, 1x109, 1x1010, 1x1011, 1x1012, 1x1013or 1x1014, or more, vector genomes per kilogram (vg / kg) of the weight of the subject, to achieve a therapeutic effect. In different embodiments the rAAV dose is about 5x1011rAAV vg / kg or greater than about 5x1011rAAV vg / kg; about 1x1012rAAV vg / kg or greater than about 1x1012rAAV vg / kg; about 2x1012rAAV vg / kg or greater than about 2x1012rAAV vg / kg; about 3x1012rAAV vg / kg or greater than about 3x1012rAAV vg / kg; about 4x1012rAAV vg / kg or greater than about 4x1012rAAV vg / kg; about 5x1012rAAV vg / kg or greater than about 5x1012rAAV vg / kg; about 1x1013rAAV vg / kg or greater than about 1x1013rAAV vg / kg; about 2x1013rAAV vg / kgor greater than about 2x1013rAAV vg / kg; about 3x1013rAAV vg / kg or greater than about 3x1013rAAV vg / kg; about 4x1013rAAV vg / kg or greater than about 4x1013rAAV vg / kg; about 5x1013rAAV vg / kg or greater than about 5x1013rAAV vg / kg; about 6x1013rAAV vg / kg or greater than about 6x1013rAAV vg / kg.

[0207] Examples of dose ranges of rAAV vg / kg include a dose range from about 5x1011to about 6x1013rAAV vg / kg; a dose range from about 5x1011to about 5.5x1011rAAV vg / kg; a dose range from about 5.5x1011to about 6x1011rAAV vg / kg; a dose range from about 6x1011to about 6.5x1011rAAV vg / kg; a dose range from about 6.5x1011to about 7x1011rAAV vg / kg; a dose range from about 7x1011to about 7.5x1011rAAV vg / kg; a dose range from about 7.5x1011to about 8x1011rAAV vg / kg; a dose range from about 8x1011to about 8.5x1011rAAV vg / kg; a dose range from about 8.5x1011to about 9x1011rAAV vg / kg; a dose range from about 9x1011to about 9.5x1011rAAV vg / kg; a dose range from about 9.5x1011to about 1x1012rAAV vg / kg; a dose range from about 1x1012to about 1.5x1012rAAV vg / kg; a dose range from about 1.5x1012to about 2x1012rAAV vg / kg; a dose range from about 2x1012to about 2.5x1012rAAV vg / kg; a dose range from about 2.5x1012to about 3x1012rAAV vg / kg; a dose range from about 3x1012to about 3.5x1012rAAV vg / kg; a dose range from about 3.5x1012to about 4x1012rAAV vg / kg; a dose range from about 4x1012to about 4.5x1012rAAV vg / kg; a dose range from about 4.5x1012to about 5x1012rAAV vg / kg; a dose range from about 5x1012to about 5.5x1012rAAV vg / kg; a dose range from about 5.5x1012to about 6x1012rAAV vg / kg; a dose range from about 6x1012to about 6.5x1012rAAV vg / kg; a dose range from about 6.5x1012to about 7x1012rAAV vg / kg; a dose range from about 7x1012to about 7.5x1012rAAV vg / kg; a dose range from about 7.5x1012to about 8x1012rAAV vg / kg; a dose range from about 8x1012to about 8.5x1012rAAV vg / kg; a dose range from about 8.5x1012to about 9x1012rAAV vg / kg; a dose range from about 9x1012to about 9.5x1012rAAV vg / kg; a dose range from about 9.5x1012to about 1x1013rAAV vg / kg; a dose range from about 1x1013to about 1.5x1013rAAV vg / kg; a dose range from about 1.5x1013to about 2x1013rAAV vg / kg; a dose range from about 2x1013to about 2.5x1013rAAV vg / kg; a dose range from about 2.5x1013to about 3x1013rAAV vg / kg; a dose range from about 3x1013to about 3.5x1013rAAV vg / kg; a dose range from about 3.5x1013to about 4x1013rAAV vg / kg; a dose range from about 4x1013to about 4.5x1013rAAV vg / kg; a dose range from about 4.5x1013to about 5x1013rAAV vg / kg; a dose range from about 5x1013to about 5.5x1013rAAV vg / kg; a dose range from about 5.5x1013to about 6x1013rAAV vg / kg; or a dose range from about 6x1013to about 1x1014rAAV vg / kg.

[0208] In certain embodiments, rAAV vg / kg are administered at a dose of about 5x1011vg / kg, about 6x1011vg / kg, about 7x1011vg / kg, about 8x1011vg / kg, about 9x1011vg / kg, about 1x1012vg / kg, about 2x1012vg / kg, about 3x1012vg / kg, about 4x1012vg / kg, about 5x1012vg / kg, about6x1012vg / kg, about 7x1012vg / kg, about 8x1012vg / kg, about 9x1012vg / kg, about 1x1013vg / kg, about 2x1013vg / kg, about 3x1013vg / kg, about 4x1013vg / kg, about 5x1013vg / kg, or about 6x1013vg / kg.

[0209] In certain embodiments doses and dose ranges for other viral vectors is as provided herein with respect to rAAV. For example, in certain 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.

[0210] In certain 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.

[0211] In certain embodiments, administration of polynucleotides comprising a transgene encoding a therapeutic protein, viral vectors and non-viral vectors described herein is in combination 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-ILTM(ImmTOR with Treg-selective IL-2 agonist), B-cell depletion, immunoadsorption, and plasmapheresis.

[0212] In certain 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 certain embodiments, the one or more immunosuppressive agent is administered concomitantly with a vector or non-viral vector. In certain 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 certain 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 vectoradministration. 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.

[0213] In certain embodiments, the immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, the immunosuppressive agent is a steroid, e.g., a corticosteroid. In certain 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-1b 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.

[0214] 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., rAAV) and non-viral vector can be used to induce immune tolerance to the therapeutic protein through induction of regulatory T cells (Tregs).

[0215] 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, therebyreducing anti-AAV antibody titer; and use of delivery techniques such as balloon catheters followed by saline flushing. Such strategies are described in Mingozzi et al., (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 et al., 2020, Sci. Rep. 10:864. Similar techniques and strategies can be used for other types of viral vectors.

[0216] 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 certain 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 2:1, 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.

[0217] 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 is circulated 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.)

[0218] In certain 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 certain 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.

[0219] In certain 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. IX. Ocular Administration and Treatment

[0220] In certain embodiment the promoters described herein are used for ocular transgene expression and / or treatment of an ocular disease or disorder. In further embodiments, the transgene encodes a polypeptide or biological active nucleic acid useful in treating or preventing (e.g., decreasing the likelihood or severity) or an ocular disease or disorder.

[0221] In further embodiments, the transgene encodes a polypeptide for treating an ocular disease or disorder, such as guanylate cyclase 2D (LCA-GUCY2D); retinal pigment epithelium- specific 65 kDa protein (RPE65); rab escort protein-1 (REP-1); LCA 5 (LCA-lebercilin); ornithine ketoacid aminotransferase retinoschisin; X-linked retinitis pigmentosa GTPase; MER proto-oncogene tyrosine kinase (MERTK); ABCA4; ACHM 2, 3 and 4; or a vascular endothelial growth factor (VEGF) antagonist.

[0222] Reference to a VEGF “antagonist” refers to a polypeptide inhibiting VEGF activity. VEGF activity can be inhibited, for example, by polypeptides interacting with VEGF or inhibiting a VEGF receptor.

[0223] Examples of ocular diseases and disorders include glaucoma, retinitis pigmentosa, X- linked retinitis pigmentosa, autosomal dominant retinitis pigmentosa, recessive retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, Stargardt disease, choroideremia, choroidal neovascularization, early dry AMD, intermediate dry AMD, geographic atrophy, gyrate atrophy, retinoschisis, X-linked retinoschisis, diabetic macular edema (DME), diabetic retinopathy associated with DME, non-proliferative diabetic retinopathy, neovasular diabetic retinopathy, wet age-related macular degeneration (wAMD or wet AMD), macular edema following retinal vein occlusion, non-arteritic ischaemic optic neuropathy, achromatopsia, and Dominant Optic Atrophy (DOA), X-linked recessive Optic Atrophy, and Multiple sclerosis.

[0224] Examples of particular ocular diseases or disorders and therapeutic protein include MER proto-oncogene, tyrosine kinase (MERTK), phosphodiesterase 6B (PDE6B), and / or retinaldehyde binding protein 1 (RLBP1) for treating retinitis pigmentosa; NADH dehydrogenase subunit 1 (ND1) and / or NADH dehydrogenase for treating Leber hereditary optic neuropathy; retinoschisin 1 (RS1) for treating X-linked juvenile retinoschisis; CNGB3 and CNGA3 for treating achromatosis; REP1 for treating choroideremia; RPRG for treating X- linked retinitis pigmentosa; CD59 for treating non-neovascular age related macular degeneration; anti-VEGF polypeptides for treating wet AMD; or anti-VEGF polypeptides for treating diabetic retinopathy and / or diabetic macular edema. (He et al., Progress and Prospects. Research 2023;6:Article 0291, hereby incorporated by reference herein in its entirety). In certain embodiments, the VEGF antagonist is an antibody or antibody fragment targeting VEGF.

[0225] Antibody fragments are polypeptides comprising an antibody variable region. Examples of antibody fragments include a diabody, a Fab, a Fab′, a F(ab′)2, scFab, an Fv fragment, a single-chain antibody molecule (scFv), a single domain antibody (sdAb), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a nanobody, a domain antibody and a bivalent domain antibody.

[0226] In certain embodiments, the transgene encodes OPA1, C3 inhibitor, C5 inhibitor, CD59, CFH, GDNF, OPA1, OPA2, OPA3, OPA4, OPA5, OPA6, OPA8, MT-ND1, MT-ND4, MT- ND4L, MT-ND6, Catalase, SOD, Anti-IL6, or Anti-TNF-alpha protein.

[0227] In certain embodiments the transgene encodes a biologically active nucleic acid. In certain embodiments, the biologically active nucleic acid is an inhibitory RNA, a stimulatory RNA (e.g., saRNA), and / or an editing RNA. Examples of biologically active nucleic acid include short hair pin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, or small activating RNA (saRNA).

[0228] In certain embodiments, the biologically active nucleic acid inhibits RTP801 / Redd1 (DNA damage-inducible transcript 4 protein) expression.

[0229] In certain embodiments, the biologically active nucleic acid inhibits vascular endothelial growth factor receptor 1 (VEGFR1) and / or VEGF expression.

[0230] In certain embodiments the nucleic acid inhibit Sterile Alpha and TIR Motif Containing 1 (SARM1) expression.X. Kits

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.XI. Additional Aspects and Embodiments

[0236] Additional aspects, embodiments, and examples of combinations thereof include the following: 1. A polynucleotide comprising a protomer wherein said protomer comprises either: a) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 15, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 8; b) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 10, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 3; c) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 11, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 4; d) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to, or comprising, the sequence of SEQ ID NO: 12, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 5; e) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 13, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 6; or f) a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 14, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 7. 2. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 15, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 8. 3. The polynucleotide of 2, wherein said promoter comprises the sequence of SEQ ID NO: 15. 4. The polynucleotide of 3, wherein said promoter does not comprise the sequence of SEQ ID NO: 77. 5. The polynucleotide of any one of 2-4, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 8. 6. The polynucleotide of any one of 2-4, wherein said promoter comprises up to 100 contiguous nucleotides of SEQ ID NO: 8.7. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 10, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 3. 8. The polynucleotide or 7, wherein said promoter comprises the sequence of SEQ ID NO: 10. 9. The polynucleotide of 7 or 8, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 3. 10. The polynucleotide of any one of 7-9, wherein said promoter comprises up to 300 contiguous nucleotides of SEQ ID NO: 3. 11. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 11, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 4. 12. The polynucleotide of 11, wherein said promoter comprises the sequence of SEQ ID NO: 4. 13. The polynucleotide of 11 or 12, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 4. 14. The polynucleotide of any one of 11-13, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO: 4. 15. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 12 and the promoter does not comprise the nucleic acid sequence of SEQ ID NO: 5. 16. The polynucleotide of 15, wherein said promoter comprises the sequence of SEQ ID NO: 5. 17. The polynucleotide of 15 or 16, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 5. 18. The polynucleotide of any one of 15-17, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO: 5. 19. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 13, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 6.20. The polynucleotide of 19, wherein said promoter comprises the sequence of SEQ ID NO: 5. 21. The polynucleotide of 19 or 20, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 6. 22. The polynucleotide of any one of 19-21, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO: 6. 23. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, the sequence of SEQ ID NO: 14, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 7. 24. The polynucleotide of 23, wherein said promoter comprises the sequence of SEQ ID NO: 7. 25. The polynucleotide 23 or 24, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 7. 26. The polynucleotide of any one of 23-25, wherein said promoter comprises up to 700 contiguous nucleotides of SEQ ID NO: 7. 27. The polynucleotide of any one of 1-4, wherein said promoter further comprises a 5’- UTR-Intron, wherein said UTR comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, a sequence selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, a sequence selected from any of SEQ ID NOs: 20-29. 28. The polynucleotide of 5 or 6, wherein said promoter further comprises a 5’-UTR- Intron, wherein said UTR comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, a sequence selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or comprising, a sequence selected from any of SEQ ID NOs: 20-29. 29. The polynucleotide of 27 or 28, wherein said UTR comprises a selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence selected from any of SEQ ID NOs: 20-29. 30. The polynucleotide of 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of any of SEQ ID NOs: 30-75 or 82. 31. The polynucleotide of 30, wherein said promoter comprises a nucleic acid sequence selected from any of SEQ ID NOs: 67-75 or 82.32. The polynucleotide of 1, wherein said promoter comprises the sequence of any one of SEQ ID NOs: 84-93, 95-104, 106-115, 117-126, 128-137, 139-148, and 150-159. 33. The polynucleotide of any of 1-32, wherein the polynucleotide comprises an expression cassette comprising said promoter operatively linked to a transgene. 34. The polynucleotide of 33, wherein said expression cassette further comprises a polyadenylation signal downstream of said transgene. 35. The polynucleotide of 33 or 34, wherein said transgene encodes a protein and said expression cassette further comprises a Kozak sequence operatively linked to said transgene. 36. A recombinant viral vector nucleic acid comprising the polynucleotide of any one of 1-35, wherein said recombinant viral nucleic acid comprises 5’ and / or 3’ viral elements providing for viral packaging and replication. 37. The recombinant viral vector nucleic acid of 36, wherein said recombinant viral vector nucleic acid is DNA and 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 said polynucleotide. 38. The recombinant viral vector nucleic acid of 37, 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. 39. The recombinant viral vector nucleic acid of 37 or 38, wherein said recombinant viral vector nucleic acid is 3 kb to 5.2 kb in length. 40. The recombinant viral vector nucleic acid 39, wherein said recombinant viral vector nucleic acid is 4 kb to 5.1 kb in length. 41. A gene delivery vehicle comprising the polynucleotide of any one of 1-35 or the recombinant viral vector nucleic acid of any one of 36-40 and a viral or non-viral vector. 42. The gene delivery vehicle of 41, wherein said gene delivery vehicle is a viral vector comprising the recombinant viral vector nucleic acid of any one of claims 36-41. 43. The gene delivery vehicle of 42, 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%, 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 comprises, 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-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10 or SEQ ID NO: 78.44. The gene delivery vehicle of 43, wherein said capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or said capsid comprises VP1 of SEQ ID NO: 78. 45. The gene delivery vehicle of 43, wherein said capsid comprises VP1 of SEQ ID NO: 78, VP2 of SEQ ID NO: 79, and VP3 of SEQ ID NO: 80. 46. A method of producing a protein in a cell comprising the step of transducing or transfecting said cell with the gene delivery vehicle of any one of 41-45, wherein said viral vector nucleic acid comprises said transgene encoding said protein. 47. A method of producing a protein in a subject comprising the step of administering the gene delivery vehicle of any one of 41-45 to said subject, wherein said viral vector nucleic acid comprises said transgene encoding said protein. XII. Sequences

[0237] Table 1 provides different sequences. In certain 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% to, or comprises, any of the nucleic acid sequences provided in Table 1. Table 1EXAMPLES

[0238] 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 ed invention.

[0239] Example 1: Promoter Activity in Different Cells lines

[0240] The activity of different promoter constructs were evaluated in different cells. Promoter expression was evaluated using a tdTomato reporter or Factor IX (FIX) expression. The tdTomato assay involves inserting the promoter upstream of nucleic acid encoding tdTomato red fluorescent protein; and measuring activity by flow cytometer and data analysis via FlowJo. FIX expression was determined via an ELISA utilizing FIX-EIA antibodies from Affinity Biologicals read on microplate reader and quantified in SoftMax. Promoter expression in ARPE19, Y79, Neuro2A cells were measured using the TdTomato promoter. Promoter expression in Huh7 and HEK293 cells were measured by measuring FIX expression.

[0241] The activity of different promoters were tested: the CAG promoter (SEQ ID NO: 1), the EF1A promoter (SEQ ID NO: 2), along with promoter constructs ACLY (SEQ ID NO: 3), ACTG1 (SEQ ID NO: 4), ACTR2 (SEQ ID NO: 5), ARPC2 (SEQ ID NO: 6), VCL 5 (SEQ ID NO: 7), and PPIG-Core-Full-Intron (SEQ ID NO: 9). The test constructs are further described in Table 2. Table 2

[0242] Promoter constructs were transfected via a lipid-based methods in up to five cell lines, which were used as surrogates for different types of tissues. FIG. 1A illustrates activity in ARPE19 (retinal pigment epithelial-like) cells. FIG. 1B illustrates activity in Y79 (photoreceptor-like) cells. FIG. 1C illustrates activity in Neuro2A (neuroblastoma) cells. FIG. 1D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.1E illustrates activity in HEK293 (embryonic kidney) cells. The dotted line indicates EF1alpha promoter activity.

[0243] Example 2: Promoter core Activity

[0244] Constructs ACLY-core (SEQ ID NO: 10), ACTG1-core (SEQ ID NO: 11), ACTR2-core (SEQ ID NO: 12), ARPC2-core (SEQ ID NO: 13), VCL-core (SEQ ID NO: 14), and PPIG-core (SEQ ID NO: 16) were assayed for promoter function and plotted as a percentage of their parental sequence designs. Parental sequence designs are provided in Table 2. Promoter expression was measured using a tdTomato reporter or measuring FIX expression as described in Example 1.

[0245] FIGs.2A-2E illustrate the activity of the different promoter core regions plotted as a percentage of their parental sequence designs. FIG.2A illustrates activity in ARPE19 (epithelial) cells. FIG. 2B illustrates activity in Y79 (photoreceptor-like) cells. FIG.2C illustrates activity in Neuro2A (neuroblastoma) cells. FIG. 2D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.2E illustrates activity in HEK293 (embryonic kidney) cells. The dotted line indicates EF1alpha promoter activity.

[0246] Example 3: Promoter Modifications

[0247] The PPIG promoter was modified by intron swapping, distal promoter region removal, and providing a partial 5’ UTR. The partial 5’ UTR modification removed a terminal 3’ region of 22 nucleotides. The different test constructs are summarized in Table 3. Table 3

[0248] Promoter expression was measured as described in Example 1. FIGs.3A-3E show the percent gene expression relative to PPIG (SEQ ID NO: 8). FIG. 3A illustrates activity in ARPE19 (epithelial) cells. FIG. 3B illustrates activity in Y79 (photoreceptor-like) cells. FIG. 3C illustrates activity in Neuro2A (neuroblastoma) cells. FIG. 3D illustrates activity in Huh7 (hepatocyte-like) cells. FIG.3E illustrates activity in HEK293 (embryonic kidney) cells. The dotted line notes PPIG promoter activity.

[0249] Example 4: Tile Ablations

[0250] The impact of deleting PPIG promoter regions was determined by tiled ablation in which 30 base pair windows of sequence were deleted. Promoter activity was measured in HEK293 cells as described in Example 1. The results are shown in FIG.4.

[0251] Example 5: Promoter Miniaturization

[0252] Different promoters were produced incorporating various deletions to reduce promoter size. Miniaturized variants of the PPIG (SEQ ID NO: 8) promoter spanning ~300-700bp were produced and tested using the reporter genes tdTomato (ARPE19, Y79, Neuro2A) or FIX (Huh7 and HEK293). Table 4 summarizes constructs Abl-combi 7 plus 9, Abl-combi 1to3, Composite-Abl- 1, Composite-Abl-2, Composite-Abl-3, Composite-Abl-4, Composite-Abl-5, Composite-Abl-6, Composite-Abl-7. Table 4 reference to UTR sequence and intron sequence provides the location of the different regions. Table 4

[0253] FIGs 5A-5E illustrate the impact of different promoter modifications in different cell types. FIG.5A illustrates activity in ARPE19 cells. FIG. 5B illustrates activity in Y79 cells. FIG.5C illustrates activity in Neuro2A cells. FIG. 5D illustrates activity in Huh7 cells. FIG. 5E illustrates activity in HEK293 cells. Abl-combi 7 plus 9 (SEQ ID NO: 68); Abl-combi 1to3 (SEQ ID NO: 67), Composite-Abl-1 (SEQ ID NO: 69), Composite-Abl-2 (SEQ ID NO: 70), Composite-Abl-3 (SEQ ID NO: 71), Composite-Abl-4 (SEQ ID NO: 72), Composite-Abl-5 (SEQ ID NO: 73), Composite-Abl-6 (SEQ ID NO: 74), Composite-Abl-7 (SEQ ID NO: 75) and PPIG (SEQ ID NO: 8).

[0254] Promoter strength showed variable dependence on UTRs and / or native intron sequences in different cell lines. Miniaturization of PPIG produced variants of ~1.1kb to ~700bp in length with minimal loss in potency in different cell types, and variants as small as ~300bp retained strong gene expression in ARPE19 and Y79 cells.

[0255] Example 6: Ocular Transcription factor binding motifs

[0256] Transcription factor binding motifs for the ubiquitous promoters ACLY, ACTG, ACTR2, ARPC2, and PPIG were mapped and annotated. In each instance, the promoters were modified by picking two types of annotated motifs and replacing those endogenous sequences with ocular specific transcription factor binding motifs (CRX, OTX2, or NRL). Up to 10 variants containing motif replacements were generated per ubiquitous promoter candidate. Because the CRX, OTX2, and NRL motifs permit some degenerate bases at specific nucleotide positions (that is, a unique base is not specified at every single position), the replacement motifs were sampled from a broad range of possibilities that match the position weight matrix (PWM). Variant promoters were cloned into a pAAV backbone plasmid containing tdTomato as the reporter gene. The variants have a unique 5’ barcode for the creation of a pooled library.

[0257] Plasmids containing the promoters were individually transiently transfected into Y79 cells and FACS was performed to assess tdTomato expression driven by different promoter variants. The data was analyzed via FlowJo and reported as a percentage of CAG promoter and graphed in GraphPad. The results are shown in FIGs.6-12.

[0258] FIG.6 illustrates expression of ACLY variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 84); Var2 (SEQ ID NO: 85); Var3 (SEQ ID NO: 86); Var4 (SEQ ID NO: 87); Var5 (SEQ ID NO: 88); Var6 (SEQ ID NO:89 );Var7 (SEQ ID NO: 90); Var8 (SEQ ID NO: 91); Var9 (SEQ ID NO: 92); and wild-type (SEQ ID NO: 3).

[0259] FIG.7 illustrates expression of ACTG variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 95); Var2 (SEQ ID NO: 96); Var6 (SEQ ID NO: 100); Var9 (SEQ ID NO: 103); and wild-type (SEQ ID NO: 4).

[0260] FIG.8 illustrates expression of ACTR2 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. The different promoters were designated: Var1 (SEQ ID NO: 106); Var2 (SEQ ID NO: 107); Var3 (SEQ ID NO: 108); Var5 (SEQ ID NO: 110); Var6 (SEQ ID NO: 111); Var8 (SEQ ID NO: 113); and wild-type (SEQ ID NO: 5).

[0261] FIG.9 illustrates expression of ARPC2 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var1 (SEQ ID NO: 117); Var2 (SEQ ID NO: 118); Var3 (SEQ ID NO: 119); Var4 (SEQ ID NO: 120); Var5 (SEQ ID NO: 121); Var6 (SEQ ID NO: 122); Var7 (SEQ ID NO: 123); Var9 (SEQ ID NO: 125); Var10 (SEQ ID NO: 126) and wild- type (SEQ ID NO: 6).

[0262] FIG.10 illustrates expression of PPIG-Core-UTR-Full-Intron variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. The different promoters were designated: Var1 (SEQ ID NO: 128); Var4 (SEQ ID NO: 131); Var6 (SEQ ID NO: 133); Var7 (SEQ ID NO: 134); Var8 (SEQ ID NO: 135); Var9 (SEQ ID NO: 136); and wild-type (SEQ ID NO: 138).

[0263] FIG.11 illustrates expression of PPIG-Composite 6 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. Mean intensity was assessed relative to the CAG promoter. The different promoters were designated: Var2 (SEQ ID NO: 140); Var3 (SEQ ID NO: 141); Var4 (SEQ ID NO: 142); Var5 (SEQ ID NO: 143); Var8 (SEQ ID NO: 146); Var9 (SEQ ID NO: 147); Var10 (SEQ ID NO: 148); and wild-type (SEQ ID NO: 149).

[0264] FIG.12 illustrates expression of PPIG-Composite 7 variants comprising transcription factor binding motifs from CRX, OTX2, or NRL in Y79 cells. FACS was performed to assess tdTomato expression driven by different promoter. The different promoters were designated: Var1 (SEQ ID NO: 150); Var2 (SEQ ID NO: 151); Var3 (SEQ ID NO: 152); Var4 (SEQ ID NO: 153); Var6 (SEQ ID NO: 155); Var7 (SEQ ID NO: 156); Var8 (SEQ ID NO: 157); Var9 (SEQ ID NO: 158); Var10 (SEQ ID NO: 159); and wild-type (SEQ ID NO: 160).

[0265] While the invention has been described and illustrated with reference to certain particular 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

CLAIMS I / we claim:

1. A polynucleotide comprising a protomer wherein said protomer comprises either: a) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 15, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 8; b) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 10, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 3; c) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 11, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 4; d) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 12, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 5; e) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 13, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO: 6; or f) a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 14, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO:

7.

2. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 15, wherein the promoter does not comprise the nucleic acid sequence of SEQ ID NO:

8.

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

15.

4. The polynucleotide of claim 3, wherein said promoter does not comprise the sequence of SEQ ID NO:

77.

5. The polynucleotide of any one of claims 2-4, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO:

8.

6. The polynucleotide of any one of claims 2-4, wherein said promoter comprises up to 100 contiguous nucleotides of SEQ ID NO:

8.

7. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 10, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO:

3.

8. The polynucleotide of claim 7, wherein said promoter comprises the sequence of SEQ ID NO:

10.

9. The polynucleotide of claims 7 or 8, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO:

3.

10. The polynucleotide of any one of claims 7-9, wherein said promoter comprises up to 300 contiguous nucleotides of SEQ ID NO: 3.

11. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 11, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO:

4.

12. The polynucleotide of claim 11, wherein said promoter comprises the sequence of SEQ ID NO:

4.

13. The polynucleotide of claims 11 or 12, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO:

4.

14. The polynucleotide of any one of claims 11-13, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO:

4.

15. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 12 and the promoter does not comprise the nucleic acid sequence of SEQ ID NO:

5.

16. The polynucleotide of claim 15, wherein said promoter comprises the sequence of SEQ ID NO:

5.

17. The polynucleotide of claims 15 or 16, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO:

5.

18. The polynucleotide of any one of claims 15-17, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO:

5.

19. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 13, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO:

6.

20. The polynucleotide or claim 19, wherein said promoter comprises the sequence of SEQ ID NO:

5.

21. The polynucleotide of claims 19 or 20, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO:

6.

22. The polynucleotide of any one of claims 19-21, wherein said promoter comprises up to 150 contiguous nucleotides of SEQ ID NO:

6.

23. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of SEQ ID NO: 14, wherein said promoter does not comprise the nucleic acid sequence of SEQ ID NO:

7.

24. The polynucleotide of claim 23, wherein said promoter comprises the sequence of SEQ ID NO:

7.

25. The polynucleotide claims 23 or 24, wherein said promoter comprises up to 800 contiguous nucleotides of SEQ ID NO: 7.

26. The polynucleotide of any one of claims 23-25, wherein said promoter comprises up to 700 contiguous nucleotides of SEQ ID NO:

7.

27. The polynucleotide of any one of claims 1-4, wherein said promoter further comprises a 5’-UTR-Intron, wherein said UTR comprises a sequence at least 90% identical to a sequence selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence at least 90% identical to a sequence selected from any of SEQ ID NOs: 20-29.

28. The polynucleotide of claims 5 or 6, wherein said promoter further comprises a 5’- UTR-Intron, wherein said UTR comprises a sequence at least 90% identical to a sequence selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence at least 90% identical to a sequence selected from any of SEQ ID NOs: 20-29.

29. The polynucleotide of claims 27 or 28, wherein said UTR comprises a selected from any of SEQ ID NOs: 17-19; and said Intron comprises a sequence selected from any of SEQ ID NOs: 20-29.

30. The polynucleotide of claim 1, wherein said promoter comprises a nucleic acid sequence at least 95% identical to the sequence of any of SEQ ID NOs: 30-75 or 82.

31. The polynucleotide of claim 30, wherein said promoter comprises a nucleic acid sequence selected from any of SEQ ID NOs: 67-75 or 82.

32. The polynucleotide of claim 1, wherein said promoter comprises the sequence of any one of SEQ ID NOs: 84-93, 95-104, 106-115, 117-126, 128-137, 139-148, and 150-159.

33. The polynucleotide of any one of claims 1-32, wherein the polynucleotide comprises an expression cassette comprising said promoter operatively linked to a transgene.

34. The polynucleotide of claim 33, wherein said expression cassette further comprises a polyadenylation signal downstream of said transgene.

35. The polynucleotide of claims 33 or 34, wherein said transgene encodes a protein and said expression cassette further comprises a Kozak sequence operatively linked to said transgene.

36. A recombinant viral vector nucleic acid comprising the polynucleotide of any one of claims 1-35, wherein said recombinant viral nucleic acid comprises 5’ and / or 3’ viral elements providing for viral packaging and replication.

37. The recombinant viral vector nucleic acid of claim 36, wherein said recombinant viral vector nucleic acid is DNA and comprises an adeno-associated virus (AAV) invertedterminal repeat (ITR) flanking the 5’ terminus of said polynucleotide and an AAV ITR flanking the 3’ terminus of said polynucleotide.

38. The recombinant viral vector nucleic acid of claim 37, 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.

39. The recombinant viral vector nucleic acid of claims 37 or 38, wherein said recombinant viral vector nucleic acid is 3 kb to 5.2 kb in length.

40. The recombinant viral vector nucleic acid claim 39, wherein said recombinant viral vector nucleic acid is 4 kb to 5.1 kb in length.

41. A gene delivery vehicle comprising the polynucleotide of any one of claims 1-35 or the recombinant viral vector nucleic acid of any one of claims 35-40 and a viral or non-viral vector.

42. The gene delivery vehicle of claim 41, wherein said gene delivery vehicle is a viral vector comprising the recombinant viral vector nucleic acid of any one of claims 36-41.

43. The gene delivery vehicle of claim 42, 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-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO:

78.

44. The gene delivery vehicle of claim 43, wherein said capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or said capsid comprises VP1 of SEQ ID NO:

78.

45. The gene delivery vehicle of claim 43, wherein said capsid comprises VP1 of SEQ ID NO: 78, VP2 of SEQ ID NO: 79, and VP3 of SEQ ID NO:

80.

46. A method of producing a protein in a cell comprising the step of transducing or transfecting said cell with the gene delivery vehicle of any one of claims 41-45, wherein said viral vector nucleic acid comprises a transgene encoding a protein.

47. A method of producing a protein in a subject comprising the step of administering the gene delivery vehicle of any one of claims 41-45 to said subject, wherein said viral vector nucleic acid comprises said transgene encoding said protein.

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