Adenovirus helper plasmid and stable helper cell line for AAV production and uses thereof

Abstract

Provided are nucleic acids, vectors, and / or packaging systems providing helper functions for adeno-associated virus (AAV) preparation with minimal sets of adenoviral genes. The nucleic acids, vectors, and / or packaging systems reduce AAV production cost and reduce the presence of adenoviral genetic material during AAV production.

Description

[0001] ADENOVIRUS HELPER PLASMID AND STABLE HELPER CELL LINE FOR AAV PRODUCTION AND USES THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 625,849, filed January 26, 2024. entitled "NEXT GENERATION ADENO HELPER PLASMIDS AND STABLE HELPER CELL LINE FOR PRODUCTION OF AAV VECTORS” and U.S. Provisional Application No. 63 / 540,609, filed September 26, 2023, entitled “ADENOVIRAL GENE NECESSARY FOR RAAV PRODUCTION AND DEVELOPMENT OF AN IMPROVED, CONTROLLED SYSTEM OF RAAV PRODUCTION”, the entire contents of each of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (M105370051WO00-SEQ-KUW.xml; Size 373,444 bytes; and Date of Creation: September 24, 2024) is herein incorporated byreference in its entirety.

[0006] BACKGROUND

[0007] Adeno-associated viruses (AAV) are single stranded viruses of 4.7kb genome consisting of packaging replication (Rep) and functional capsid (Cap) genes flanked by inverted terminal repeats (ITRs). Adeno-associated viral (AAV) vectors have proven to be highly valuable for gene therapy due to several advantageous characteristics: they are nonpathogenic, provoke little immune response, allow targeting to specific tissues, and enable long-lasting gene expression. Additionally, AAVs are replication-deficient on their own and require co-infection with a helper virus - either adenovirus or herpes simplex virus - to replicate. The increasing number of AAV- based therapies receiving regulatory approval and currently in clinical testing demonstrates their usefulness. The demand for GMP grade AAV vectors is going to keep increasing as many more AAV-based therapies are expected to gain approval over the next decade.

[0008] Currently, AAV vectors are primarily produced by triple plasmid transient transfection in HEK293 cells. Briefly, in this method, three plasmids comprising of: i) packaging Rep and structural Cap genes, ii) recombinant AAV genome flanked by ITRs and iii) adenovirus helper genes are transfected in mammalian HEK293 cells and vectors harvested 72 hours post transfection. This method is easy to setup and gives good yields at smaller scales. However, high transfection costs, requirement of GMP grade plasmid DNA, limited scalability', and lack of reproducibility in yields at large scales are limitations stifling this method to meet the rising demand of AAV vectors. Any reduction in manufacturing related costs, for e.g. eliminating the requirement of plasmid DNA and using stable cell lines will have a direct impact on reducing the overall therapy cost. AAV production irrespective of the method also suffers from having a large fraction of empty' capsids.

[0009] In antibody manufacturing, high yield at large scales are made possible by use of stable cell lines. AAV Rep and helper adenovirus genes form the redundant set of genes for AAV production, independent of serotype and recombinant AAV genome. These genes are toxic to the cells and their integration into the genome causes cellular instability.

[0010] Previous research has identified El A / B, E2A, E4orf6 and VA I RNA genes to be sufficient for production of AAV vectors. E1A is a master regulator of expression of other adenovirus genes and of AAV genes. El B is an essential factor that enhances AAV second strand synthesis by forming a complex with E4orf6. E2A is a single stranded DNA binding protein (ssDBP) which stimulates viral DNA replication. It is also found to interact with Rep proteins and is also involved in Rep mRNA splicing. VA RNA I is mainly involved in inhibition of Protein kinase R (recognizes dsRNA and exerts antiviral effects by inhibiting translation and production of new viral particles). E4orf6 is involved in facilitating AAV mRNA export from the nucleus, enhancing second strand synthesis and degradation of Mrel l (part of MRN complex that inhibits AAV transduction and replication). Attempts to integrate helper genes have resulted in either drastic yield reduction and / or cellular instability. Tight and inducible control of these genes should result in truly stable cell line with maintained yields. For AAV production, adenovirus helper genes El A / B are stably integrated in HEK293 cells while E2A, E4orf6 and VA RNA I are supplied in the form of a helper plasmid. Currently used adenovirus helper plasmids (pDeltaF6 or pALD-X80) consist of many genes whose necessity in AAV production is not known. Knowledge of the importance of these additional adenovirus genes in the context of AAV production and identification of the minimal set of adenovirus genes for AAV production is needed.

[0011] SUMMARY

[0012] Provided are nucleic acids, vectors, packaging systems and methods of using the same for production of adeno-associated virus (AAV) vector particles using a minimal set of adenovirus helper genes. The nucleic acids, vectors, and / or packaging system comprise adenovirus helper genes that provide efficient packaging of AAV vector particles, while omitting additional adenovirus helper genes. Tn some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein.

[0013] In some aspects, a nucleic acid sequence encoding an adenovirus E2A protein. In some aspects, the polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus E4 protein. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding an adenovirus VA RNA I, an adenovirus VA RNA II, or a combination thereof. In some aspects, the polynucleotide, further comprising a nucleic acid sequence encoding an adenovirus 33k protein.

[0014] Also provided is a polynucleotide comprising a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein. In some aspects, the polynucleotide further comprising a nucleic acid sequence encoding an adenovirus E2A protein. In some aspects, the polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus E4 protein. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding an adenovirus VA RNA I, an adenovirus VA RNA II, or a combination thereof. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding an adenovirus 22k.

[0015] In some aspects, a polynucleotide comprises a further regulator)’ sequence. In some aspects, the further regulatory sequence is selected from a promoter, a polyadenylation / termination signal, and combinations thereof. In some aspects, the promoter is a constitutive promoter or an inducible promoter. In some aspects, the constitutive promoter is a CMV promoter or an EFla promoter. In some aspects, the constitutive promoter is operably linked to the nucleic acid encoding the E2A protein. In some aspects, the promoter is a pol III promoter. In some aspects, the pol III promoter is operably linked to the adenovirus VA RNA I, adenovirus VA RNA II, or combination thereof.

[0016] In some aspects, the inducible promoter is operably linked to the nucleic acid encoding the 22k protein, the nucleic acid encoding the 33k protein, and / or the nucleic acid encoding an E2A protein. In some aspects, wherein the transcription termination signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or a pol III termination signal.

[0017] Further provided is a polynucleotide comprising adenovirus helper genes for packaging adeno-associated virus (AAV), which polynucleotide comprises no more than 5244 nucleotides. In some aspects, the adenovirus helper genes for packaging AAV comprise an adenovirus 22k gene. In some aspects, the adenovirus helper genes for packaging AAV comprise an adenovirus 33k gene. In some aspects, the adenovirus helper genes for packaging AAV further comprise an adenovirus E2A gene. In some aspects, the adenovirus helper genes for packaging AAV further comprise an adenovirus VA RNA I, an adenovirus VA RNA II or a combination thereof.

[0018] In some aspects, the polynucleotide further comprises a regulatory sequence. In some aspects, the regulatory sequence is selected from a promoter, a polyadenylation / termination signal, and combinations thereof. In some aspects, the promoter is a constitutive promoter or an inducible promoter. In some aspects, the constitutive promoter is a CMV promoter or an EFla promoter.

[0019] In some aspects, the inducible promoter is operably linked to the adenovirus 22k gene, the adenovirus 33k gene, and / or the adenovirus E2A gene. In some aspects, the promoter is a pol III promoter. In some aspects, the pol III promoter is operably linked to the VA RNA 1, VA RNA 11, or combination thereof. In some aspects, the termination signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or a pol III termination signal

[0020] In some aspects, at least one nucleic acid sequence as described herein is present in a cellular genome and / or at least one of the adenovirus helper genes described herein is present in a cellular genome.

[0021] Provided is an adeno-associated virus (AAV) packaging system comprising a polynucleotide described herein and a nucleic acid sequence comprising an inverted terminal repeat and encoding a gene of interest, a nucleic acid sequence encoding an AAV replication protein (Rep) and a nucleic acid sequence encoding an AAV capsid protein (Cap).

[0022] In some aspects, the AAV packaging system does not comprise any additional adenovirus helper gene encoding nucleic sequences.

[0023] Further provided is an adeno-associated virus (AAV) packaging system consisting essentially of a polynucleotide described herein, a nucleic acid sequence comprising an inverted terminal repeat and encoding a gene of interest, a nucleic acid sequence encoding an AAV replication protein (Rep) and a nucleic acid sequence encoding an AAV capsid protein (Cap).

[0024] Also provided is a vector comprising a polynucleotide described herein.

[0025] Further provided is a cell comprising a polynucleotide described herein, a packaging system described herein, or a vector described herein.

[0026] Provided is a method of preparing an adeno-associated virus (AAV), the method comprising introducing into a cell a polynucleotide described herein, a packaging system described herein, or a vector described herein; culturing the cell under conditions such that an AAV is generated in the cell; and isolating the AAV. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGs. 1A-1H show evaluation of adenovirus helper genes and identification of 22k / 33k genes for their impact on AAV production. FIG. 1A shows a schematic depicting the essential E2A, E4orf6 and VA RNA I adenovirus genes with their sizes (top) and the commonly used helper plasmid DF6 with its open reading frames (orfs) (bottom). Black arrows indicate the direction and length of protein coding sequence in the plasmid. Sliding waves indicate intronic sequence. FIG. IB shows a schematic of the production and quantification of AAV2 / 9 using equimolar amounts of Rep2 / Cap9, Luciferase-ITR and Helper plasmids (left). A bar chart (right) shows AAV9 titers in genome copies per mL (GC / mL) measured via qPCR when using DF6 versus a combination of E2A, E4orf6 and VA RNA I helper genes. FIG. 1C shows a schematic of new helper plasmids pJD108 (top) and pJD107 (bottom), both derived from DF6. FIG. ID shows bar charts showing titers in GC / mL (left) and luciferase intensity depicting transduction efficiency in relative light units (RLU) (right) for helper plasmids DF6, pJD108 and pJD107. FIG. IE shows a schematic of E4orf6-6 / 7 and E4 genes (top). E4orf6 / 7 and E4orf6 orfs are indicated as arrows. Sliding waves indicate intronic sequence. The bar charts (bottom) show titers in GC / mL (left) and transduction efficiency in RLU (right) for a combination of helper genes / plasmids, presence of which is indicated by + sign and absence is indicated by - sign. FIG. IF shows a schematic of 22k / 33k and 100k genes (top). 22k and 33k orfs are indicated as arrows. Sliding waves indicate intronic sequence. The bar charts (bottom) show titers in GC / mL (left) and luciferase intensity in relative light units (RLU) depicting transduction efficiency for a combination of helper genes / plasmids. FIG. 1G shows a bar chart of titers in GC / mL for combinations of helper genes. FIG. 1H shows a bar chart of titers in GC / mL for combinations of helper genes. The presence of helper genes is indicated by + sign and absence is indicated by - sign. Each bar in FIG. IB, FIG. D to FIG. 1H represents mean ± SD of biological triplicates. All samples had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) alongside the helper genes / plasmids in triple transfection. CMV, human cytomegalovirus promoter; Efl a, human elongation factor 1 alpha promoter; AAA, polyadenylation signal.

[0028] FIGs. 2A-2B show identification of minimal adenoviral helper gene set and their optimal amounts for production of AAV vectors. FIG. 2A shows a schematic depicting E2A, E4orf6, VA RNA I and 22k / 33k adenovirus genes with constitutive regulatory' sequences (top). The bar chart (bottom) displays titers in GC / mL for a combination of E2A, E4orf6, VA RNA I and 22k / 33k helper genes used in transfection for AAV production. Presence of a gene is indicated by + sign and absence is indicated by - sign. Titers in GC / mL with DeltaF6 (DF6) plasmid was used as a standard control value. FIG. 2B shows titration of VA RNA I (left), 22k / 33k (middle), E2A (right) helper genes. The bar charts represent titers in GC / mL measured via qPCR. The molar equivalent of the respective adenoviral helper gene(s) to pJD64 (Rep2-Cap9) plasmid is show n in the bottom of the charts. Each bar in FIG. 2A and FIG. 2B represents mean ± SD of biological duplicates or triplicates. All samples had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) alongside the helper genes / plasmids in triple transfection.

[0029] FIGs. 3A-3D show' next generation helper plasmids for AAV production. FIG. 3A shows a schematic depicting tw o helper plasmids pJD150 and pJD171 with their genes and regulatory' sequences. FIG. 3B show s a bar chart displaying titers in GC / mL for AAV9 vector produced using DF6, ALD-X80, pJD150 and pJD171 helpers in 24-well plate scale. FIG. 3C shows a bar chart displaying titers in GC / mL (left y axis) and number of viral particles in capsids / mL (right y axis) for AAV9 vector produced using DF6, pJD150 and pJD171 helper plasmids in three independent runs performed in 15cm dish scale. FIG. 3D shows transmission electron microscopy images of AAV9 vectors produced using DF6, pJD150 and pJD171 helper plasmids. Scale bar lOOnm. Magnification 980000x. Each bar in FIG. 3B represents mean ± SD of biological triplicates. Each bar in FIG. 3C represents mean ± SD at qPCR level of a single biological replicate. All samples in FIG. 3B and FIG. 3C had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) alongside the helper genes / plasmids in triple transfection. The helper plasmid used for production is denoted below the bars.

[0030] FIGs. 4A-4F show that AAV production using next generation helper plasmids is independent of serotypes, genomes and scale. FIG. 4A shows a bar chart displaying titers in GC / mL of AAV9 produced in 15 cm dishes using DF6, pJD150, or pJD171 helper plasmids. FIG. 4B shows a bar chart displaying titers in GC / mL of AAV2 / 6 and AAV2 / 8 produced using DF6, pJD150, or pJD171 helper plasmids. FIG. 4C shows a bar chart displaying titers in GC / mLfor AAV6, AAV8 and AAV1 1 vector produced using DF6, pJD150 and pJD171 helper plasmids in 24-well plate scale. pACM-delta AAV genome (CMV-ACMdelta) was used for AAV11 and pJD36 AAV genome (CMV-luciferase) was used for AAV6 and AAV8 production. FIG. 4D shows a schematic depicting the commonly used helper plasmid ALD-X80 with its open-reading frames (orfs). Black arrows indicate the direction and length of protein coding sequence in the plasmid. Sliding w aves indicate intronic sequence. FIG. 4E shows a bar chart displaying titers in GC / mL for AAV9 vector produced using ALD-X80, pJD150 and pJD171 helper plasmids in HYPERflask 500mL scale. FIG. 4F shows transduction efficiency measured through flow cytometry as absolute mScarlet fluorescence units for HEK293 cells transduced using AAV9 vectors produced with ALD-X80, pJD150 and pJD171 helper plasmids. pJD34 AAV genome (CMV-mScarlet) was used during production. Each bar in FIG. 4A, FIG. 4B, FIG. 4C. FIG, 4E. and FIG. 4F represents mean ± SD of biological duplicates or triplicates. Samples in FIG. 4A, FIG. 4B, FIG. 4C, FIG, 4E, and FIG. 4F used pJD64 (Rep2-Cap9) plasmid in triple transfection. The helper plasmid used for production is denoted below the bars.

[0031] FIGs. 5A-5D show inducible stable helper cell lines for AAV production. FIG. 5A shows a schematic on the left displaying bi-directional tet inducible promoter (Trebi) driving expression of E2A and 22k / 33k genes. The inducible promoter is activated by tetracycline transactivator (tTA) provided as a separate plasmid. The bar chart on the left displays titers in GC / mL for AAV9 produced in 24-well plate scale using either DF6 or the inducible pJD172 helper plasmid. The bar chart on the right displays luciferase intensity, proxy of transduction efficiency, in relative light units (RLU). Presence of the helper plasmid is indicated by + sign and absence is indicated by - sign. For tTA, 0.5x or lx molar equivalent to pJD64 (Rep2-Cap9) plasmid was used for activation. Absence of inducer is indicated by - sign. FIG. 5B shows a schematic (top) showing the process of random integration of transfer vectors in the HEK293 genome using piggybac transposon followed by fluorescence-based sorting. Transfer vector with three or two helper genes is respectively shown on the left and right. mCitrine fluorescent protein expression is under control of an ETR promoter activated by an ET transactivator. ET, erythromycin-dependent transactivator. FIG. 5C shows a bar charts (top) displaying titers in GC / mL at different time points (Day 37. 55 and 66) for AAV9 produced in 24-well plate scale using stable helper cell lines containing three helper genes (top) and luciferase intensity in relative light units (RLU) for HEK293 cells transduced wi th AAV vectors made using stable helper cells containing three genes (bottom). FIG. 5D shows a bar chart (top) displaying titers in GC / mL at different time points (Day 37. 55 and 66) for AAV9 produced in 24-well plate scale using stable helper cell lines containing two helper genes and exogenously supplemented with VA RNA T. The bar chart (bottom) displays luciferase intensity in relative light units (RLU) for HEK293 cells transduced with AAV vectors made using stable helper cells containing two genes and exogenous VA RNA I. Presence of VA RNA I, Rep2-Cap9, AAV genome and tTA inducer plasmids during transfection is indicated by + sign and absence is indicated by - sign. Each bar in FIG. 5A, FIG. 5C, FIG. 5D represents mean ± SD of biological duplicates or triplicates.

[0032] FIGs. 6A-6D show evaluation of adenovirus helper genes and control samples in titers and transduction efficiency for their impact on AAV production. FIG. 6A is a bar chart showing luciferase intensity depicting transduction efficiency in relative light units (RLU) for HEK293 cells transduced with AAV9 vectors made using either DF6 vs a combination of E2A, E4orf6 and VA RNA I helper genes, presence of which is indicated by + sign and absence is indicated by - sign below the chart. FIG. 6B are bar charts showing titers in GC / mL (left) and luciferase intensity depicting transduction efficiency in relative light units (RLU) (right) for AAV9 vectors made using combinations of helper genes E2A, E4orf6, VA RNA T, 22k, 33k, and / or 23k, presence of which is indicated by + sign and absence is indicated by - sign below the chart. FIG. 6C are bar charts showing titers in GC / rnL (left) and luciferase intensity' depicting transduction efficiency in relative light units (RLU) (right) for AAV9 vectors made using a combination of helper genes E2A, E4orf6, VA RNA I, and / or VA RNA II, presence of which is indicated by + sign and absence is indicated by - sign below the chart. FIG. 6D shows a bar chart showing titers in GC / mL for AAV9 vectors made in control conditions. Titers in GC / mL using DF6 helper are shown for comparison. Each bar in FIG. 6A to FIG. 6D represents mean ± SD of biological triplicates. All samples had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) alongside helper gene plasmid(s) in triple transfection.

[0033] FIGs. 7A-7C show indispensability of 22k / 33k genes and dispensability' of E4orf6 gene for production of AAV vectors in terms of transduction efficiency and titers. FIG. 7A is a bar chart displaying fold change in titers relative to DF6. Refer to FIGs. 2A-2B for titer data. Presence of a gene is indicated by + sign and absence is indicated by - sign. FIG. 7B shows a schematic (left) of DF6 helper plasmid split in two plasmids and devoid of an E4 region. Black arrow(s) indicate the direction and length of protein coding sequence in the plasmid. Sliding waves indicate intronic sequence. The bar chart (right) represents titers in genome copies per mL (GC / mL) measured via qPCR for AAV9 vector produced using a combination of helper genes, the presence of yvhich is indicated by + sign and absence is indicated by - sign below the chart. DBP, DNA binding protein region of adenovirus gene containing multiple orfs. FIG. 7C is a bar chart displaying luciferase intensity, proxy for transduction efficiency, in relative light units (RLU) for HEK293 cells transduced using AAV9 vectors produced using single or combinations of E2A, E4orf6, VA RNA I and 22k / 33k helper genes. Presence of a gene is indicated by + sign and absence is indicated by - sign. RLU for DeltaF6 (DF6) plasmid yvas used for comparison. Each bar in FIG. 7A, FIG. 7B, and FIG. 7C represents mean ± SD of biological duplicates or triplicates. All samples had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) alongside helper gene(s) plasmid(s) in transfection for AAV production.

[0034] FIGs. 8A-8B shoyv transduction efficiency of AAV9 vector preparations made using different helper plasmids. The bar charts display luciferase intensity, proxy for transduction efficiency, in relative light units (RLU) for HEK293 cells transduced using AAV9 vectors produced using helper plasmids DF6 or ALD-X80 or JD150 or JD171 at 24well plate scale (FIG. 8A) and 15 cm dish scale (FIG. SB). Each bar in FIG. 8A represents mean ± SD of biological triplicates. Each bar in FIG. 8B represents mean ± SD obtained during measurement of luciferase intensity of a biological replicate. All samples had pJD64 (Rep2-Cap9) and pJD36 (AAV genome: CMV-luciferase) in transfection for AAV production.

[0035] FIGs. 9A-9E show viability and flow cytometry plots of sorting of stable helper cell lines. FIG. 9A shows a graph displaying viability of inducible stable helper cell lines containing either two or three helper genes. FIG. 9B shows flow cytometry plots depicting all events and the region used for scatter plots as part of the gating strategy for sorting mCitrine positive cells, proxy for positive integrants containing three (middle) or two (bottom) helper genes. Top, gating strategy in HEK293 cells with no integration used as control. FIG. 9C shows flow cytometry plots depicting scatter plots and the region used for forward scatter (fsc) singlets as part of the gating strategy for sorting mCitrine positive cells, proxy for positive integrants containing three (middle) or two (bottom) helper genes. Top, gating strategy in HEK293 cells with no integration used as control. FIG. 9D shows flow cytometry' forward scatter singlets plots and the region used for side scatter (ssc) singlets used in the gating strategy for sorting mCitrine positive cells, proxy for positive integrants containing three (middle) or two (bottom) helper genes. Top, gating strategy in HEK293 cells with no integration used as control. FIG. 9E shows flow cytometry' side scatter singlet plots showing mCitrine positive cells, proxy for positive integrants containing three (middle) or two (bottom) helper genes. Top, gating strategy in HEK293 cells with no integration used as control. Refer to Example 1 for more details on generation of stable helper cell lines and sorting.

[0036] DETAILED DESCRIPTION

[0037] Provided are nucleic acids, vectors, packaging systems, and methods of using the same for production of adeno-associated virus (AAV) vector particles. The nucleic acids and vectors comprise adenovirus helper genes for efficient packaging of AAV vector particles, while omitting adenovirus helper genes that are identified herein as having no impact on AAV vector particle packaging. The nucleic acids, vectors, and packaging system described herein provide advantages regarding clinical safety based on the use of the fewest adenovirus helper genes possible for clinical AAV vector particle production with lower potential for adenoviral proteins or nucleic acid being present in clinical AAV product. Further, nucleic acids, vectors, and packaging system described herein allow a reduction of vector particle production cost due to smaller size of nucleic acids and vectors used for AAV production.

[0038] The results presented herein identified novel 22k / 33k adenovirus genes (also identified by PCT Publication WO2024 / 1079851) to be indispensable for AAV production. Furthermore, a minimal adenovirus helper gene set for production of AAV vectors was identified, which minimal gene set comprises E2A, VA RNA I and 22k / 33k adenovirus genes and / or E2A. VA RNA I and 22k, and / or E2A, VA RNA I and 33k. It was demonstrated that E4orf6 is dispensable for AAV production. After identifying the minimal gene set, new helper plasmids, JD150 and JD171, were constructed that were comparable in yields and transduction efficiency to the commonly used pDeltaF6 and pALD-X80 helper plasmids. Furthermore, inducible stable helper cells were produced by integrating adenoviral helper genes and such cells successfully produced AAV vectors upon activation of inducible promoters that control expression of the integrated genes. However, the stable helper cells had 6-10 fold lower yields than the transient transfection approach. Overall, the disclosures provided herein deconstruct adenovirus helper gene plasmids, identify the minimal adenovirus gene set for AAV production and establish a proof-of-concept of stably integrating adenoviral helper genes for production of AAV vectors.

[0039] I. Polynucleotides

[0040] Provided are polynucleotides comprising adenoviral helper genes for packaging of adeno-associated virus (AAV) particles. In some aspects, the polynucleotides provided comprise minimal sets of adenoviral helper genes for packaging AAV particles when used together with a recombinant AAV genome, and genes encoding AAV replication (Rep) and AAV capsid (Cap) proteins.

[0041] Adenovirus Nucleic Acid Sequences

[0042] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein. The term “adenovirus 22k protein,’' as used herein, refers to a protein encoded by a L4-22k mRNA transcribed from a late transcription unit 4 (L4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus E2A protein. The term “adenovirus E2A protein,” as used herein, refers to a protein encoded by a mRNA transcribed from an early transcription unit 2 (E2) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein. The term “adenovirus 33k protein,” as used herein, refers to a protein encoded by a L4- 33k mRNA transcribed from a late transcription unit 4 (L4) gene of an adenovirus. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus 23k protein. The term “adenovirus 23k protein,” as used herein, refers to a protein encoded by a L4- 23k mRNA transcribed from a late transcription unit 4 (L4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding VA RNA 1. The term “VA RNA I,” as used herein refers to a major species of adenovirus virus-associated non-coding RNA. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding a fragment of VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding VA RNA II. The term “VA RNA II,” as used herein refers to a minor species of adenovirus virus-associated non-coding RNA. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding a fragment of VA RNA II. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding VA RNA II. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus 23k protein. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus 100k protein. The term “adenovirus 100k protein,” as used herein, refers to a protein encoded by a L4-100k mRNA transcribed from a late transcription unit 4 (L4) gene of an adenovirus. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding VA RNA II. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus E4 protein. The term “adenovirus E4 protein,” as used herein, refers to a protein encoded by a mRNA transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus pTP protein or fragment thereof. The term “pTP protein,” as used herein refers to a protein encoded by a mRNA transcribed from an E2B gene of an adenovirus.

[0043] In some aspects, a polynucleotide comprises adenovirus helper genes for packaging adeno-associated virus (AAV), e.g., adenovirus helper genes sufficient for packaging AAV vector particles. In some aspects, the adenovirus helper genes for packaging AAV vector particles comprise an adenovirus 22k gene. In some aspects, the adenovirus helper genes for packaging AAV vector particles comprise an adenovirus 33k gene. In some aspects, the adenovirus helper genes for packaging AAV vector particles further comprise an adenovirus E2A gene. In some aspects, the adenovirus helper genes for packaging AAV vector particles further comprise an adenovirus VA RNA I, an adenovirus VA RNA II or a combination thereof. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 138. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 138.

[0044] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein comprising an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 138. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein comprising an amino acid sequence that has 60%. 65%. 70%. 75%. 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. 99.5%. or 100% identity to SEQ ID NO: 138.

[0045] In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least

[0046] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0047] 94%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 139. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 139.

[0048] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein comprising an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 139. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein comprising an amino acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 139.

[0049] In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least

[0050] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0051] 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 140. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 140.

[0052] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus E2A protein comprising an amino acid sequence that has at least 60%, at least 65%, at least 70%. at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 140. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus E2A protein comprising an amino acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 140.

[0053] In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 141. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 141.

[0054] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 23k protein comprising an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO:

[0055] 141. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 23k protein comprising an amino acid sequence that has 60%. 65%. 70%. 75%. 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 141.

[0056] In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 142. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 142.

[0057] In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 132. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity' to SEQ ID NO: 132. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus E2A protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 33k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 33k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus V A RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus VA RNA II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 23k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0058] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. The term ■‘adenovirus E4orfl protein,’’ as used herein, refers to a protein encoded by a first open reading frame transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. The term “adenovirus E4orf2 protein.” as used herein, refers to a protein encoded by a second open reading frame transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. The term "‘adenovirus E4orf3 protein,” as used herein, refers to a protein encoded by a third open reading frame transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. The term “adenovirus E4orf4 protein,” as used herein, refers to a protein encoded by a fourth open reading frame transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. The term “adenovirus E4orf6 protein.” as used herein, refers to a protein encoded by a sixth open reading frame transcribed from an early transcription unit 4 (E4) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein. The term “adenovirus E4orf6 / 7 protein.” as used herein, refers to a protein encoded by a fusion of open reading frame E4orf 6 and E4orf7 generated by RNA splicing. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus precursor terminal protein (pTP) or a fragment thereof.

[0059] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. The term “adenovirus fiber protein,” as used herein, refers to a major adenovirus capsid protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid comprising an adenovirus U exon. The term “adenovirus U exon,” as used herein, refers to a nucleic acid encoding a late adenoviral U exon protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. The term “adenovirus pVIII protein,” as used herein, refers to a minor adenovirus capsid protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise an adenovirus E3 12.5k encoding sequence or fragment thereof. The term “adenovirus E3 12.5k encoding sequence or a fragment thereof,” as used herein, refers to a nucleic acid sequence, or a fragment thereof, of an early transcription unit 3 (E3) gene of an adenovirus. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise an adenovirus pllla protein encoding sequence or fragment thereof. The term “adenovirus pllla protein encoding sequence or fragment thereof,” as used herein, refers to a nucleic acid sequence, or fragment thereof, of a pllla minor adenovirus capsid protein gene.

[0060] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0061] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 33k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 33k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4 protein.

[0062] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0063] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid comprising an adenovirus U exon.

[0064] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus 23k protein.

[0065] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein.

[0066] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence.

[0067] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding or an adenovirus pTP protein or fragment thereof.

[0068] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orf l protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II. an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0069] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus 23k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0070] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0071] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding a hexon associated precursor protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein, and does not comprise a nucleic acid encoding an adenovirus precursor terminal protein (pTP) or a fragment thereof.

[0072] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus hexon associated precursor protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus precursor terminal protein (pTP) or a fragment thereof.

[0073] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus E2A protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0074] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0075] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, an adenovirus VA RNA II, an adenovirus 23k protein, an adenovirus 100k protein, or an adenovirus pTP protein or fragment thereof.

[0076] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, and a nucleic acid sequence encoding adenovirus E2A. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid sequence encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0077] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus pVIII protein.

[0078] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise an incomplete adenovirus E3 12.5k coding sequence.

[0079] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0080] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II. an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0081] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid sequence encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0082] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA 1 and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0083] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0084] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus 23k protein, an adenovirus VA RNA II, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0085] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a nucleic acid sequence encoding an adenovirus VA RNA II or a combination of VA RNA I and II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0086] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0087] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus VA RNA II. or a combination of VA RNA I and II and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0088] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA 1. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid sequence encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I. and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not compnse a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0089] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0090] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid sequence encoding an adenovirus VA RNA I and does not compnse a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II. an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus 23k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0091] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA 1 and II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein.

[0092] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0093] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA 11 or a combination of VA RNA I and II, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pllla protein.

[0094] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid sequence encoding an adenovirus VA RNA II, or a combination of VA RNA I and II and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or a fragment thereof.

[0095] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I. wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I. and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0096] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0097] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA T, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0098] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus E2A protein, a nucleic acid sequence encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0099] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0100] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA 11 or a combination of VA RNA I and II, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding a hexon associated precursor protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0101] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus E2A protein, a nucleic acid sequence encoding an adenovirus VA RNA II, or a combination of VA RNA I and II and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0102] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid sequence encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA 1, and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0103] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA T, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0104] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid sequence encoding an adenovirus E2A protein, a nucleic acid sequence encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0105] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA 1 and VA RNA II. wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0106] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A, and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA II or a combination of VA RNA I and VA RNA II, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA 11 or a combination of VA RNA 1 and VA RNA II. and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid encoding an adenovirus 33k protein, and a nucleic acid encoding adenovirus E2A. and a nucleic acid encoding a VA RNA IT or a combination of VA RNA I and VA RNA IT, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0107] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid sequence encoding an adenovirus E2A protein, a nucleic acid sequence encoding an adenovirus VA RNA IT, or a combination of VA RNA I and VA RNA IT and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0108] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus E2A protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA II. Tn some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus 23k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0109] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 1 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0110] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0111] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf? protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus pllla protein or fragment thereof or an adenovirus pTP protein or fragment thereof.

[0112] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4 protein.

[0113] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0114] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0115] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus 23k protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orI3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0116] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus E2A protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0117] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus VA RNA II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus 23k protein, an adenovirus pTP protein or fragment thereof, an adenovirus VA RNA II, an adenovirus 100k protein, or an adenovirus pllla protein or fragment thereof.

[0118] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0119] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA T and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0120] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus 23k. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0121] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus VA RNA II, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0122] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA 1 and II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a nucleic acid encoding an adenovirus VA RNA II, or a combination of VA RNA I and II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0123] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0124] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus VA RNA II, or a combination of VA RNA I and II and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0125] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0126] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus VA RNA II.

[0127] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA I, and does not comprise an adenovirus pTP protein or fragment thereof. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, a nucleic acid encoding an adenovirus VA RNA I and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II, an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0128] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II and does not comprise a nucleic acid encoding an adenovirus E4orfl protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf 2 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf3 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf4 protein.

[0129] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus E4orf6 / 7 protein.

[0130] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus fiber protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid comprising an adenovirus U exon. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pVIII protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 23k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus 100k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise an incomplete adenovirus E3 12.5k coding sequence. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pllla protein. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, and a nucleic acid encoding adenovirus VA RNA II or a combination of VA RNA I and II, and does not comprise a nucleic acid encoding an adenovirus pTP protein or fragment thereof.

[0131] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid encoding an adenovirus E2A protein, a nucleic acid encoding an adenovirus VA RNA II. or a combination of VA RNA I and II and does not comprise a nucleic acid encoding any one of an adenovirus E4 protein, an adenovirus E4orfl protein, an adenovirus E4orf2 protein, an adenovirus E4orf3 protein, an adenovirus E4orf4 protein, an adenovirus E4orf6 protein, an adenovirus E4orf6 / 7 protein, an adenovirus 23k protein, an adenovirus 100k protein, an adenovirus VA RNA II. an adenovirus fiber protein, an adenovirus U exon protein, an adenovirus pVIII protein, an adenovirus E3 12.5k protein or fragment thereof, an adenovirus pllla protein or fragment thereof, or an adenovirus pTP protein or fragment thereof.

[0132] Regulatory Elements

[0133] The term '‘regulatory element,” as used herein, refers to a nucleotide sequence or structural component of a nucleic acid described herein which regulates expression of components of a nucleic acid (e.g.. a 22k gene). Regulatory elements include, but are not limited to, promoters as described herein, enhancers, silencers, insulators, response elements, initiation sites, termination signals, and ribosome binding sites. In some aspects, regulatory elements can be one or more naturally occurring and / or engineered regulator}' elements. In some aspects, provided is a polynucleotide that comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein. In some aspects, provided is a polynucleotide that comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus E2A protein. Tn some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus VA RNA I. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0134] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0135] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus E2A protein, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus VA RNA I. wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein, a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein, a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus E2A protein, and a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus VA RNA I, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0136] In some aspects, a promoter is a constitutive promoter. Examples of constitutive promoters include, without limitation, a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer, an EFla promoter, a pol III promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer, a SV 40 promoter, the dihydrofolate reductase promoter, an actin promoter, and a phosphoglycerol kinase (PGK.) promoter. The term “pol III promoter,” as used herein, refers to an RNA polymerase III promoter. The term “EFla promoter,” as used herein, refers to a human elongation factor-1 alpha promoter.

[0137] In some aspects, a promoter is an inducible promoter. In some aspects, an inducible promoter is regulated by an exogenously supplied compound, an environmental factor such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied compounds, include, a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary' tumor virus (MMTV) promoter, ta T7 polymerase promoter system, an ecdysone insect promoter, a tetracycline-repressible system (see, e.g.. Gossen et al. Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992), a tetracyclineinducible system (see, e g., Gossen et al, Science, 268: 1766-1769, 1995), see also Han ey et al, Curr. Opin. Chem. Biol., 2:512-518, 1998), a RU486-inducible system (see, e.g., Wang et al, Nat. Biotech., 15:239-243, 1997. and Wang et al, Gene Then, 4:432-441, 1997) and a rapamycin-inducible system (see, e.g.. Magari et al, J. Clin. Invest.. 100:2865-2872, 1997). Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0138] In some aspects, a promoter is a native promoter. A native promoter may be preferred when it is desired that expression of the gene mimics the native expression. A native promoter may be used when expression of the gene must be regulated temporally7or developmentally, or in a cell specific manner, or in response to specific transcriptional stimuli. In some aspects, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0139] In some aspects, a polynucleotide comprises an EFla promoter operably linked to a nucleic acid encoding a 22K protein. The term “operably linked” as used herein refers to a nucleic acid sequence joined to another nucleic acid sequence as part of the same nucleic acid molecule, wherein, e.g., a promoter is positioned and oriented for transcription of a gene of interest, e.g., a 22k gene described herein. In some aspects, a polynucleotide comprises an EFla promoter operably linked to a nucleic acid encoding a 33K protein. In some aspects, a polynucleotide comprises an EF 1 a promoter operably linked to a nucleic acid encoding a 22K and 33k protein. In some aspects, a polynucleotide comprises a CMV promoter operably linked to a nucleic acid encoding a E2A protein. In some aspects, a polynucleotide comprises a CMV promoter operably linked to a nucleic acid encoding an E4orf 6 protein.

[0140] In some aspects, a polynucleotide comprises a pol III promoter operably linked to a VA RNA I. In some aspects, a polynucleotide comprises a pol III promoter operably linked to a VA RNA II. In some aspects, a polynucleotide comprises a pol III promoter operably linked to a combination of VA RNA I and VA RNA II.

[0141] In some aspects, a polynucleotide described herein comprises a termination signal. In some aspects, the termination signal comprises a poly adenylation signal (poly A). In some aspects, a poly adenylation signal comprises a bovine growth hormone (bGH) polyA. some aspects, a polyadenylation signal comprises a simian virus 40 (SV40) polyA. In some aspects, a termination signal comprises a pol III termination signal. The term “pol III termination signal,” as used herein, refers to a nucleotide sequence that terminates transcription by an RNA polymerase III.

[0142] In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k protein and a termination signal selected from a bGH poly and a SV40 polyA. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 33k protein and a termination signal selected from a bGH poly and a SV40 polyA. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus 22k and a 33k protein and a termination signal selected from a bGH poly and a SV40 polyA. In some aspects, a polynucleotide comprises a nucleic acid sequence encoding an adenovirus E2A protein and a termination signal selected from a bGH poly and a SV40 polyA. In some aspects, a polynucleotide compnses a nucleic acid sequence encoding an adenovirus 23k protein and a termination signal selected from a bGH poly and a SV40 polyA. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 111.

[0143] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 33k protein, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 112.

[0144] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22 k protein and an adenovirus 33k protein, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 108.

[0145] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus E2A protein, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 105. In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA I, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 106.

[0146] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA II, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least

[0147] 60%. at least 65%. at least 70%. at least 75%, at least 80%, at least 85%, at least 86%, at least

[0148] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0149] 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 115.

[0150] . In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA I + II, and a termination signal, wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least

[0151] 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least

[0152] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0153] 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 107.

[0154] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus E2A protein, and a termination signal; wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0155] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA I, and a termination signal; wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0156] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 33k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus E2A protein, and a termination signal; wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0157] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 33k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA I, and a termination signal; wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0158] In some aspects, a polynucleotide comprises a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 22k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus 33k protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus E2A protein, and a termination signal; and a nucleic acid sequence comprising a promoter, a nucleic acid sequence encoding an adenovirus VA RNA I. and a termination signal; wherein the polynucleotide comprises between about 5000 nucleotides and about 5500 nucleotides.

[0159] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 130. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 130. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 130.

[0160] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 131. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity' to SEQ ID NO: 131. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 131.

[0161] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 129. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 129. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 129.

[0162] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 126. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 126. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 126.

[0163] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 135. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 135. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 135.

[0164] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 127. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 127. Tn some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 127.

[0165] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 132. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 132. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 132.

[0166] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 128. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 128. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 128.

[0167] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 110. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 110. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 110.

[0168] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 109. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 109. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 109.

[0169] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 114. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 114. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 114.

[0170] In some aspects, a polynucleotide comprises a nucleic acid sequence that is about 60% to about 100% identical to SEQ ID NO: 113. In some aspects, a polynucleotide comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99.5% identity to SEQ ID NO: 113. In some aspects, a polynucleotide comprises a nucleic acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 113.

[0171] II. Vectors, Cells

[0172] Provided herein are vectors comprising a polynucleotide as described herein. In some aspects, a vector is a plasmid. In some aspects, a vector comprises additional nucleotide sequence, e.g., for replication of the vector and / or selection of a cell comprising the vector. Sequence for polynucleotide replication in a cell can be chosen by a person of ordinary skill in the art based on the type of cell the vector is to be replicated in. For example, a vector to be replicated in a bacterial cell comprises sequences for replication in the bacterial cell. In some aspects, a vector to be replicated in a mammalian cell comprise sequences for replication in the mammalian cell.

[0173] Also provided are cells comprising a polynucleotide as described herein. In some aspects, the cells comprise a polynucleotide as described herein, which polynucleotide is expressed transiently in the cell. In some aspects, the cells comprise a polynucleotide as described herein, which polynucleotide is integrated in the cell’s genome and is expressed in the cell either transiently (e.g., when the polynucleotide comprises an inducible promoter) or constitutively (e.g., when the polynucleotide comprises a constitutive promoter). A cell comprising a polynucleotide as described herein can comprise adenoviral sequences encoding an E1A and / or E1B protein. In some aspects, the adenoviral sequences encoding an El A and / or E1B protein are present non-integrated in the cell’s genome. In some aspects, the adenoviral sequences encoding an El A and / or E1B protein are integrated in the cell’s genome.

[0174] Cells suitable for AAV vector particle preparation are known in the art and can readily be used by a person of ordinary skill in the art using the disclosures provided herein.

[0175] III. Packaging System

[0176] Provided are packaging systems comprising adenoviral genes that supplement helper functions for packaging of AAV vector particles. In some aspects, a packaging system comprises adenoviral helper genes that result in successful packaging of AAV vectors. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises no more than about 10000 nucleotides. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises no more than about 8000 nucleotides. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises no more than about 7900 nucleotides, about 7800 nucleotides, about 7700 nucleotides, about 7600 nucleotides, about 7500 nucleotides, about 7400 nucleotides, about 7300 nucleotides, about 7200 nucleotides, about 7100 nucleotides, about 7000 nucleotides, about 6900 nucleotides, about 6800 nucleotides, about 6700 nucleotides, about 6600 nucleotides, about 6500 nucleotides, about 6400 nucleotides, about 6300 nucleotides, about 6200 nucleotides, about 6100 nucleotides, about 6000 nucleotides, about 5900 nucleotides, about 5800 nucleotides, about 5700 nucleotides, about 5600 nucleotides, about 5500 nucleotides, about 5400 nucleotides, about 5300 nucleotides, about 5200 nucleotides, about 5100 nucleotides, about 5000 nucleotides, about 4900 nucleotides or about 4800 nucleotides. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises between about 8000 nucleotides and about 4800 nucleotides, or about 4801 nucleotides to about 4900 nucleotides, or about 4901 nucleotides to about 5000 nucleotides, or about 5001 nucleotides to about 5100 nucleotides, about 5101 to about 5200 nucleotides, about 5201 nucleotides to about 5300 nucleotides, about 5301 nucleotides to about 5400 nucleotides, about 5401 to about 5500 nucleotides, about 5501 nucleotides to about 5600 nucleotides, about 5601 nucleotides to about 5700 nucleotides, about 5701 to about 5800 nucleotides, about 5801 nucleotides to about 5900 nucleotides, about 5901 nucleotides to about 6000 nucleotides, about 6001 to about 6100 nucleotides, about 6101 nucleotides to about 6200 nucleotides, about 6201 nucleotides to about 6300 nucleotides, about 6301 to about 6400 nucleotides, about 6401 nucleotides to about 6500 nucleotides, about 6501 nucleotides to about 6600 nucleotides, about 6601 to about 6700 nucleotides, about 6701 nucleotides to about 6800 nucleotides, about 6801 nucleotides to about 6900 nucleotides, about 6901 to about 7000 nucleotides, about 7001 nucleotides to about 7100 nucleotides, about 7101 nucleotides to about 7200 nucleotides, about 7201 to about 7300 nucleotides, about 7301 nucleotides to about 7400 nucleotides, about 7401 nucleotides to about 7500 nucleotides, about 7501 to about 7600 nucleotides, about 7601 nucleotides to about 7700 nucleotides, about 7701 nucleotides to about 7800 nucleotides, about 7801 to about 7900 nucleotides, or about 7901 nucleotides to about 8000 nucleotides. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises between about 4800 nucleotides and about 5000 nucleotides, about 4850 nucleotides and about 5950 nucleotides, about 4900 nucleotides and about 5900 nucleotides, about 4950 nucleotides and about 5850 nucleotides, about 5000 nucleotides and about 5800 nucleotides, about 5050 nucleotides and about 5750 nucleotides, about 5100 nucleotides and about 5700 nucleotides, about 5150 nucleotides and about 5650 nucleotides, or about 5200 nucleotides and about 5600 nucleotides.

[0177] In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises about 4800 nucleotides, about 4850 nucleotides, about 4900 nucleotides, about 4950 nucleotides, about 5000 nucleotides, about 5050 nucleotides, about 5100 nucleotides, about 5150 nucleotides, about 5200 nucleotides, about 5250 nucleotides, about 5300 nucleotides, about 5350 nucleotides, about 5400 nucleotides, about 5450 nucleotides, about 5500 nucleotides, about 5550 nucleotides, about 5600 nucleotides, about 5650 nucleotides, about 5700 nucleotides, about 5750 nucleotides, about 5800 nucleotides, about 5850 nucleotides, about 5900 nucleotides, about 5950 nucleotides, about 6000 nucleotides, about 6050 nucleotides, about 6100 nucleotides, about 6150 nucleotides, about 6200 nucleotides, about 6250 nucleotides, about 6300 nucleotides, about 6350 nucleotides, about 6400 nucleotides, about 6450 nucleotides, about 6500 nucleotides, about 6550 nucleotides, about 6600 nucleotides, about 6650 nucleotides, about 6700 nucleotides, about 6750 nucleotides, about 6800 nucleotides, about 6850 nucleotides, about 6900 nucleotides, about 6950 nucleotides, about 7000 nucleotides, or any number of nucleotides therein between. In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises about 5244 nucleotides.

[0178] In some aspects, a packaging system comprises a polynucleotide comprising adenoviral helper genes for packaging AAV vector particles, which polynucleotide comprises no more than about 6000 nucleotides, about 5950 nucleotides, about 5900 nucleotides, about 5850 nucleotides, about 5800 nucleotides, about 5750 nucleotides, about 5700 nucleotides, about 5650 nucleotides, about 5600 nucleotides, about 5550 nucleotides, about 5500 nucleotides, about 5450 nucleotides, about 5400 nucleotides, about 5350 nucleotides, about 5300 nucleotides, about 5250 nucleotides, about 5200 nucleotides, about 5150 nucleotides, about 5100 nucleotides, about 5000 nucleotides, about 4900 nucleotides, or about 4800 nucleotides.

[0179] In some aspects, a packaging system comprises a polynucleotide described herein and does not comprise any additional adenovirus helper gene encoding nucleic acid sequences. In some aspects, a packaging system comprises a polynucleotide comprising, e.g.. an adenoviral 22k gene, an adenoviral 33k gene, and adenoviral E2A gene, and an adenoviral VA RNA I and does not comprise any additional adenovirus helper genes.

[0180] In some aspects, a packaging system further comprises a polynucleotide described herein and a polynucleotide comprising an AAV Rep gene. In some aspects, a packaging system further comprises a polynucleotide comprising an AAV Cap gene. In some aspects, a packaging system comprises a polynucleotide described herein and a polynucleotide comprising an AAV Rep gene and an AAV Cap gene. In some aspects, a packaging system comprises a polynucleotide as described herein, a polynucleotide comprising an AAV Rep gene, a polynucleotide comprising an AAV Cap gene, or a polynucleotide comprising both an AAV Rep and an AAV Cap gene, and a polynucleotide comprising at least one AAV inverted terminal repeat (ITR) and a gene of interest. In some aspects, a packaging system comprises a polynucleotide comprising an ITR- gene of interest-ITR nucleic acid, e.g., a recombinant AAV genome. The term “gene of interest,'’ as used herein refers to any gene that can be transferred to a cell by means of an AAV vector particle. In some aspects, a gene of interest is a therapeutic gene. The term “therapeutic gene,” as used herein refers to any gene that when transferred to a cell exercises a therapeutic effect on the cell, tissue which contains the cell, or subject which contains the cell and / or tissue. The therapeutic gene used will depend on the underlying disease or condition. Therapeutic genes that can be transferred to a cell by means of an AAV vector particle are known in the art and can readily be prepared by a person of ordinary skill in the art. In some aspects, a gene of interest is a marker gene. Marker genes that can be transferred to a cell by means of an AAV vector particle are known in the art and can readily be prepared by a person of ordinary skill in the art.

[0181] In some aspects, a packaging system consists essentially of a polynucleotide as described herein, a polynucleotide comprising an AAV Rep gene, a polynucleotide comprising an AAV Cap gene, or a polynucleotide comprising both an AAV Rep and an AAV Cap gene, and a polynucleotide comprising AAV inverted terminal repeat(s) flanking a gene of interest. In some aspects, a packaging system consists essentially of a polynucleotide comprising an adenoviral 22k gene, an adenoviral 33k gene, and adenoviral E2A gene, and an adenoviral VA RNA I, a polynucleotide comprising an AAV Rep gene, a polynucleotide comprising an AAV Cap gene, or a polynucleotide comprising both an AAV Rep and an AAV Cap gene, and a polynucleotide comprising AAV inverted terminal repeat(s) flanking a gene of interest. In some aspects, a packaging system does not comprise any of the following nucleic acid sequences or fragments thereof: a nucleic acid sequence encoding an adenoviral 23k protein or a fragment thereof; a nucleic acid sequence encoding an adenoviral fiber protein, or a fragment thereof; a nucleic acid sequence comprising an adenoviral U exon or a fragment thereof; a nucleic acid sequence encoding an adenoviral VA RNA II or a fragment thereof; a nucleic acid sequence encoding an adenoviral pVIII protein or a fragment thereof; a nucleic acid sequence encoding an adenoviral 100k or a fragment thereof; a nucleic acid sequence encoding an adenoviral E4orfl or a fragment thereof; a nucleic acid sequence encoding an adenoviral E4orf2 or a fragment thereof; a nucleic acid sequence encoding an adenoviral E4orf3 or a fragment thereof; a nucleic acid sequence encoding an adenoviral E4orf4 or a fragment thereof; a nucleic acid sequence encoding an adenoviral E4orf6 / 7 or a fragment thereof; a nucleic acid sequence encoding an adenoviral E3 12.5k protein or a fragment thereof; or a nucleic acid sequence encoding an adenoviral pllla protein or a fragment thereof or pTP protein or a fragment thereof

[0182] In some aspects, AAV Rep and Cap sequences are independently obtained from an AAV source and may be introduced into a polynucleotide described herein in any manner known to one of ordinary skill in the art. In some aspects, a recombinant AAV genome described herein may be encapsidated within a wild-type AAV capsid of various AAV serotypes as described herein, or any one of the AAV capsids variants known in the art (e.g.. comprising a capsid protein comprising one or more amino acid substitutions). Any AAV genome can be used as source for AAV ITR, Rep and Cap sequences, e.g., as disclosed in Patent Application Publication US2024 / 01 17378. which is incorporated herein by reference in its entirety. Nonlimiting examples of AAVs include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1, AAV 12, AAV 13, AAVrh.2, AAVrh.8, AAVrh.10, AAVrh.13, AAVhu. 14, AAV3a / 3b, AAVrh32.33, AAVrh.39, AAVrh.43, AAvrh.46, AAVrh.64. AAV- HSC15, AAV-HSC17. AAVhu. 13, AAVhu.26, AAvhu.29, AAVhu.37. AAVhu.53, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV218, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV2.5T, AAV- HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2.15, AAV2.4, AAVM41, Anc80, AAV2.7m8, AAVr3.45, and pseudotyped AAV capsids.

[0183] In some aspects, Rep genes used in the packaging system described herein can be from different AAV sources. For example, a packaging system can comprises Rep78 / 68 sequences from AAV2, whereas the Rep52 / 40 sequences may be from AAV8. In some aspects, Rep and Cap genes can be from different AAV sources to generate, e.g., pseudoty ped recombinant AAV vector particles.

[0184] IV. Methods

[0185] Provided are methods for preparing AAV vector particles using the polynucleotides, cells, and / or packaging system described herein. In some aspects, a method for preparing an AAV vector particle comprises introducing into a cell a polynucleotide as described herein, a vector described herein., or a packaging system described herein; culturing the cell under conditions such that an AAV is generated in the cell; and isolating the AAV.

[0186] Methods for preparing AAV vector particles are known in the art and a person of ordinary skill in the art can practice a method for preparing AAV vector particles using the disclosures provided herein combined with the knowledge in the art.

[0187] In some aspects, a method of preparing an AAV vector particle comprises introducing one or more polynucleotides as described herein into a cell without introducing any additional polynucleotides into the cell. In some aspects, a method of preparing an AAV vector particle comprises introducing, e.g., one or more polynucleotides comprising an adenoviral 22k gene, an adenoviral 33k gene, an adenoviral E2A, and an adenoviral VA RNA I into a cell without introducing any additional polynucleotides comprising adenoviral helper genes into the cell. In some aspects, a method of preparing an AAV vector particle comprises providing a cell that comprises one or more polynucleotides, e.g., one or more polynucleotides comprising an adenoviral 22k gene, an adenoviral 33k gene, an adenoviral E2A. and an adenoviral VA RNA I but does not comprise any additional exogenously^ provided adenoviral helper genes. In some aspects, a method of preparing an AAV vector particle comprises providing a cell that comprises one or more polynucleotides, e.g., one or more polynucleotides comprising an adenoviral 22k gene, an adenoviral 33k gene, an adenoviral E2A, and an adenoviral VA RNA I as nonintegrated polynucleotides and does not comprise any additional exogenously provided adenoviral helper genes. In some aspects, a method of preparing an AAV vector particle comprises providing a cell that comprises one or more polynucleotides as described herein, e.g., one or more polynucleotides comprising an adenoviral 22k gene, an adenoviral 33k gene, an adenoviral E2A, and an adenoviral VA RNA I integrated into the cell’s genome and the cell does not comprise any additional exogenously provided adenoviral helper genes, either non-integrated or integrated into the cell’s genome.

[0188] V. Kits

[0189] Also provided are kits comprising a nucleic acid, vector and / or packaging system as described herein and components for preparing AAV vector particles using the nucleic acid, vector and / or packaging system. In some aspects, a kit comprises buffers and / or components in addition to the a nucleic acid, vector and / or packaging system to perform the method of preparing AAV vector particles as described herein.

[0190] EXAMPLES

[0191] Example 1. Methods

[0192] Cloning

[0193] For different kits used, manufacturer’s instructions were followed unless indicated otherwise. Plasmids and cloning procedures are listed in Table 2. DNA amplification was performed using Phusion High Fidelity DNA Polymerase (NEB, Cat#M0530). De-salted primers / oligonucleotides (Table 3) were ordered from Azenta or IDT. De-salted gene fragments / synthetic DNA sequences / gBlocks (Table 4) were ordered from IDT. Digestion fragments were purified using MinElute PCR purification kit (QIAGEN, Cat#28004). Gel extraction and purification was performed using Zymo Gel DNA recovery kit (Zymo, Cat#D4001). Restriction digestion was performed at temperatures recommended by the manufacturer. Ligation reaction was performed at room temperature using T4 DNA ligase (NEB, Cat#M0202). Mix and Go DH5 alpha E.coli transformation kit (Zymo, Cat#T3001) or NEB Stable cells (NEB, Cat#C3040H) or NEB DH5alpha cells (NEB, Cat#C2987H) were used for transformation. Screening of positive clones was performed using colony PCR with Quick-Load Taq 2x Master Mix (NEB, Cat#M0271L). Plasmid isolation from positive clones was performed using Zymo PURE Plasmid mini-prep kit (Zymo, Cat#D4210). All the plasmids were verified using Sanger sequencing service provided by Azenta or whole plasmid sequencing service provided by MGH CCIB DNA Core. Transformed bacteria were cultured in LB broth (RPI, Cat#L24040-2000) supplemented with appropriate antibiotics (Carbenicillin disodium salt 100 mg / rnL (Boston bioproduct, Cat#P760) and Kanamycin 50 mg / mL (Boston Bioproducts, Cat#P- 810) or Chloramphenicol (RPI, Cat#C61000-25)). lx NEB HiFi DNA assembly mix (NEB, Cat#E2621L) was used to perform Gibson assembly at 50 °C for 1 hour by mixing vector (50 ng) and insert(s) (3-5 molar equivalent) in a final volume of 10 pL. Oligo cloning required phosphorylation and annealing of oligonucleotides prior to ligation with the backbone fragment. Phosphorylation of oligonucleotides was performed by adding together 1.5 pL of oligonucleotide (100 pM), 2.5 pL lOxPNK buffer, 2.5 pL ATP (10 mM), 0.5 pL of T4 PNK (10 U / mL) (NEB, Cat#M0201S) and 16.5 pL ddFLO followed by incubation at 37 °C for 30 minutes. Annealing was performed by mixing 25 pL each of the phosphorylated oligonucleotides and then incubating in a thermocycler at 95 °C for 2 minutes followed by a decrease of 0.5 °C every minute for the next 44 minutes. 1 pL of 1 : 10 or 1 :20 diluted (with ddFLO) annealed oligonucleotides was used for ligation reaction. Golden gate assembly was performed using Bsal or BbsI enzy me with publicly available protocols.

[0194] Cell culture

[0195] All the experiments were performed in HEK293 cells (Invitrogen, Cat# 11631-017), cultured at 37 °C, 5% CO2, in 0.2 pm (Coming, Cat#431097) filtered DMEM (Coming, Cat#10- 013-CV) supplemented with 10% FBS (R&D Systems, Cat#S12450) and 1 % Penicillin / Streptomycin solution (Coming, Cat#30-002CI). Splitting was performed every 3-4 days using 0.25 %Trypsin-EDTA (Coming, Cat#25-053-CI). Cell cultures were propagated for at most two months before being replaced by fresh cell stock.

[0196] AAV production in 24-well plate

[0197] 24-well plates (Coming, Cat#3524) were pre-coated with poly-l-lysine (Sigma, Cat#P4707) by following manufacturer’s instmctions. HEK293 cells were counted using TC20 automated cell counter (BioRad, Cat# 1450102) and seeded on pre-coated plates at a density of 1.65*105cells per well in 500 pL complete DMEM (supplemented with 10% FBS and 1% Penicillin / Streptomycin) for achieving a confluency of 85-95% at the time of transfection. The seeded plates were incubated at 37 °C, 5 % CO2 for ~24 hours. A ratio of 1 : 1.375 was used of DNA (pg) to PEImax (pg) (Polysciences, Cat#24765-1) for transfection. Appropriate volume of PEImax was taken and serum-free DMEM was used to make final volume of 50 pL of PEImax- DMEM mix. Appropriate plasmid DNA amounts were taken and mixed with serum-free DMEM to make final volume of 50 pL of DNA-DMEM mix. The DMEM-DNA mix was incubated for 5 minutes before mixing with an appropriate amount of PEImax-DMEM mix. The resulting DNA- PEImax mix was then incubated at RT for 10-20 minutes. Media was gently removed from the wells of 24-well plate and supplied with 400 pL serum-free DMEM (supplemented with 1% Penicillin / Streptomycin). 100 pL of DNA-PEImax mix was added dropwise to the respective wells.

[0198] The plasmids used to generate the data represented in FIGs. 1A-1H, FIGs. 2A-2B, FIGs. 6A-6D, and FIGs. 7A-7C were combined in equal molar ratios prior to being transfected. For reference, 258 ng each of Rep2-Cap9 (pJD64), CMV -luciferase AAV genome (pJD36) plasmids and 520 ng of pDF6 helper plasmid pertaining to -3.3* 1010copies were used for transfection of a 24-well plate. Refer to Table 1 for plasmid sizes for plasmids utilized in each sample of each figure. New helper plasmids (pJD150 and pJD171) were utilized in molar ratio of 1 : 0.8 : 1 corresponding to Rep2-Cap9 : helper : AAV genome in FIGs. 3A-3D, FIGs. 4A-4F and FIGs. 8A-8B. The plasmids pJD64 (encoding Rep2-Cap9), pJD36 (containing the AAV genome), pJD33 (providing Efla-tTA) and pJD80 (supplying VA RNA I) were utilized in transfections as required for generating the samples represented in FIGs. 5A-5D and FIGs. 9A-9E. No helper. No transfection and No cap gene control conditions were also tested (FIG. 6D). Three cycles of freezing (-80°C for 30 minutes) and thawing (37°C for 30 minutes) of plate / samples was performed 72 hours post transfection. Then, the plates / samples were centrifuged at >3000g for 20 minutes and supernatant was used for downstream analysis (qPCR and transduction assays).

[0199] AAV production in 15 cm dish

[0200] 15cm dishes (Coming, Cat#430599) were pre-coated with poly-l-lysine (Sigma, Cat#P4707) by following manufacturer’s instructions. HEK293 cells were seeded on coated 15cm dishes at a density of 1.7-1.9*107cells per dish in 16-18 mL of DMEM (supplemented with 10% FBS and 1% Penicillin / Streptomycin) for achieving a confluency of 85-95% at the time of transfection. The seeded dishes were incubated at 37 °C, 5% CO2 for -24 hours. A ratio of 1 : 1.375 was used of DNA (pg) to PEImax (pg). The plasmid quantities utilized in 24-well plate transfections were increased by a proportional scaling factor of 80 (equivalent increase in surface area) to ascertain the amounts required for 15 cm dish transfections. Appropriate amounts of plasmids used for each transfection were mixed together and serum-free DMEM was used to make final volume of 1 mL (DNA DMEM mix). Plasmid amounts used were as follows: 20.64 pg each of pJD64 (encoding Rep2-Cap9) and pJD36 (encoding AAV genome CMV- luciferase) and either of the three helper plasmids (pDF6 41.6 pg or pJD150 23.04 pg, pJD171 18.8 pg). Appropriate volume of PEImax was taken and serum-free DMEM was used to make final volume of 1 mL (PEImax-DMEM mix). PEImax-DMEM mix was incubated at room temperature for 5 minutes. Following incubation, the mix was added to DNA-DMEM mix to make DNA-PEImax mix and incubated for 15-20 minutes. During the incubation period, media was gently removed from the 15 cm dishes and supplied with fresh 16-18 mL of serum-free DMEM (supplemented with l%Penicillin / Streptomycin). Following incubation, DNA-PEImax mix was added dropwise to the cells. The dishes were processed for AAV purification via static resin binding / capture 72 hours post transfection.

[0201] AAV production and purification 500 mL scale

[0202] HYPERflask scale AAV preps were produced in Gene Transfer Vector Core at Schepens Eye Research Institute. HEK293 cells at 80% confluency from four 15-cm dishes were seeded in a HYPERflask (Westnet, Cat#10030) containing complete DMEM media. The seeded flasks were incubated at 37 °C, 5% CO2 for ~24 hours. After 24 hours, pJD64 (Rep2-Cap9), pJD34 (AAV genome: CMV-mScarlet) and one of three helper plasmids (pALD-X80, pJD150 or pJD171) were mixed at an equimolar ratio in serum-free DMEM to make DNA-DMEM mix. For example, the mass equivalent for pJD64:pJD34:pALD-X80 is 130: 130:260 pg. A ratio of 1: 1.375 was used of DNA (pg) to FectoVIR (pg) was used for transfection. A mixture of serum- free media and FectoVIR (Poly plus, Cat# 120- 100) was prepared and added to the DNA-DMEM mix and allowed to incubate at room temperature for 15 minutes. After 15 minutes, this mixture was added to 500 mL of serum-free media and used to replace the media in the HYPERflasks. The flasks were incubated at 37 °C with 5% CO2 for 72 hours. After 72 hours, cells were treated with 5 M NaCl (Fisher Scientific Cat#BP358-l) to induce lysis. Then, the lysate was treated with benzonase endonuclease (Millipore Sigma, Cat#101697), cell debris pelleted and filtered using a 0.45 pm filter (Thermo, Cat# 16211-068). The lysate was then purified and concentrated using a Vivaflow200 TFF cassette (Sartorius, Cat#VF20P4) with a MWCO of 100 kDa. Following diafiltration, the lysate was loaded into a quick-seal tube, followed by 15 %, 25 %, 40 %, and 55 % iodixanol (Serumwerk, Cat# 1893) solutions. The tube was ultracentrifuged at 69000 rpm for 90 minutes and the 40 % and 55 % layers were extracted. Final buffer exchange and concentration was achieved using an Amicon Ultra-15 centrifugal filter units (Millipore Sigma, Cat# UFC910008) (MWCO of 100 kDa) to reach a final volume of 1.2 mL. AAV purification via static resin binding / capture

[0203] 72 hours post transfection, the supernatant was decanted in a 15 mL falcon tube. Triton- X100 (5 pL per 1 mL of lysate) (Alfa Aesar, Cat#A16046), RNase A (4.2 pg per ImL of lysate) (Millipore, Cat#70856), Turbonuclease (25 U per 1 mL of lysate) (Accelagen, 250 U / pL Cat#N0301M) and 1 % final cone, of Pluronic F-68 (Sigma; Cat#P556) (using 10 % stock Pluronic F-68 cone.) was added to the supernatant and mixed and poured back into the dish and incubated 37°C for 1 hour. The lysate was transferred to a 50 mL falcon tube. The dish was washed with PBS (230 pL per 1 mL of lysate) (Coming, Cat#21-031 -CM) and added to the rest of the lysate. The lysate was centrifuged at 4000xg at 4°C for 30 minutes and then the supernatant was filtered using 0.2 pm filter (Coming, Cat#431229).

[0204] 15-50 pL of POROS AAV9 resin (Thermo, Cat#A27354) was conditioned by performing three washes with 4 mL of 0.1 M NaCl (Fisher Scientific Cat#BP358-l). The resin was pelleted after each wash and supernatant discarded by pulse centrifugation (2500xg for 2 minutes 30 seconds). The resin was then resuspended in 1 mL of PBS and 0.001 % Pluronic F68 for equilibration and then pulse centrifuged. The resin was further re-suspended with filtered AAV lysate and incubated by rocking at room temperature for 10-20 minutes followed by pulse centrifugation. The resin was then washed thrice with 1 mL of PBS and pulse centrifuged. AAV was eluted twice by resuspending the resin in 1 mL of 0. 1 M citric acid (pH 2) (Sigma. Cat#C1909). The supernatant each time was collected when pulse centrifuged.

[0205] Purified AAV preps were concentrated using Amicon cassettes (Millipore; Cat#UFC810008). For concentration, the cassettes were first equilibrated with 2 mL of Final formulation buffer (FFB) (1750 pL 1 M NaCl + 50 pL 10 % pluronic-f68 + 48.2 mL PBS lx). Then, purified AAV prep was loaded onto the cassette and centrifuged to concentrate to a volume of 100-200 pL. Three washes of the cassette was performed using 2 mL FFB. The volume of concentrated AAV prep was measured and was kept in the range of 150-200 pL.

[0206] AAV titer determination by qPCR

[0207] AAV genome copy number was assessed by qPCR using luciferase or minCMV or WPRE primers and taqman probes (Table 3). Reference is made to Sanmiguel et al 2019 for a detailed protocol. Crude or purified AAV preps were digested with DNase by mixing following components 5 pL AAV sample, 5 pL lOx (1 %) Pluronic-F68 (Sigma, Cat#P556), 5 pL DNase buffer (Roche, Cat#4716728001), 34 pL Ambion Nuclease free water (Invitrogen, Cat#AM9937) and 1 pL DNase (Roche, Cat#4716728001) and incubating at 37 °C for 1 hour. DNase digested samples were diluted 1000-fold using AAV dilution buffer (lx PCR buffer, lx Sheared Salmon Sperm DNA, 0. 1 % Pluronic F-68. Ambion Nuclease free water). qPCR reaction was setup in 96- well plate (Axygen, Cat#PCR-96-AB-C) by mixing the following components: 2 pL Primerl (JD155 (1 pM) or minCMVfp (9 pM) or WPREfp (9 pM)), 2 pL Primer 2 (JD156 (1.75 pM) or minCMVrp (9 pM) or WPRErp (9 pM)), 2 pL 2.5 pM taqman probe (JD1-104-1 (2.5 pM) or minCMV or WPRE ) 2 pL Ambion Nuclease free water, 10 pL 2x Taqman gene expression master mix (Thermo, Cat#4369016) and 2 pL diluted sample. The plate was centrifuged at 2500g for 30 seconds. QuantStudio Flex system was used for running the qPCR reaction with the following program: 95°C for 10 minutes and then 40 cycles of 95°C for 15 s and 60°C for 60 s. pJD36 (CMV-luciferase) plasmid was linearized and quantified using Qubit for making standard. Standard dilutions ranged from 107copies to 10 copies per reaction. Controls for qPCR included: Dnase positive control, Dnase negative control, Water control for DNase digestion and qPCR steps. Genome copies were calculated as follows:

[0208] Quantity value was calculated by QuantStudio flex software. An alternative way of obtaining this value is to plot the Ct values of the standard at different copy numbers, obtain the slope and calculate the x.

[0209] AAV transduction assay

[0210] Transduction efficiency of AAV preps was assessed by measuring luciferase intensity. HEK293 cells were seeded at a density of 2*104cells per well in a 96well plate (Coming, Cat#3904) containing 80 pL of complete DMEM media to have around 60 % confluency. After 22-24 hours, cells in each well were infected with wt-Adenovirus5 (ATCC. Cat#VR-1516) at MOI 20. After 0-2 hours, cells were then infected at 104GC / cell with crude or purified AAV sample. After 48 hours, media was aspirated and 20 pL of 1 x lysis buffer (Promega, Cat#E397A) w as added to each well (diluted from 5 x with ddH2O). The plate w as then frozen at -80°C for 60 minutes and then thawed at 37°C for 15-25 minutes. The plate was then placed at RT for 5 minutes. 10 mL of substrate buffer was prepared by mixing the following components: 4.7 mL ddH2O, 5 mL Tris-HCl (1 M, pH 7.8; Boston Bioproduct, Cat# BBT-78), 30 pL ATP (100 mM; Thermo, Cat#R0441), MgCl2(1 M; Sigma, Cat#M1028-100 mL or Cat#M8266-l KG), lOOx Pierce Firefly Enhancer (Thermo, Cat#16180), 100 pL Luciferin (15 mg / mL; Thermo, Cat#88294). Synergy Hl Hybrid plate reader was used for reading luciferase intensity (relative light units or RLUs) at a gain of 150 after dispensing 100 pL of substrate buffer.

[0211] Capsid ELISA

[0212] AAV9 titration ELISA kit (Progen, Cat#PRAAV9) was used as per manufacturer’s instructions for determining the quantity of total capsids in the sample (AAV vectors produced in 15cm dish). 4 Parameter logistic curve fit from AAT Bioquest (aatbio.com / tools / four-parameter- logistic-4pl-curve-regression-online-calculator) was used for calculating capsids / mL. Percentage of full capsids was determined by diving the GC / mL titer by capsids / mL values.

[0213] Generation of stable cell lines

[0214] HEK293 cells were seeded on 24-well plate at a density of 6.5*104cells per well in 500 pL complete DMEM (supplemented with 10 % FBS and 1 %Penicillin / Streptomycin) for achieving a confluency of 60-70 % at the time of transfection. The seeded plates were incubated at 37 °C, 5 % CO2 for ~24 hours. DNA was transfected using Lipofectamine 2000 (Thermo, Cat#! 1668-019) at a ratio of l(pg) : 3(pL). Appropriate volume of Lipofectamine 2000 was taken and serum-free DMEM was used to make final volume of 50 pL. The DMEM-DNA mix was incubated for 5 minutes before mixing with appropriate amount of Lipo2000-DMEM mix. The DNA-Lipo2000 mix was incubated at RT for 10-20 minutes. 100 pL of DNA-Lipo2000 mix was added dropwise to the respective wells. 300 ng of transfer vector plasmid (pJD179 (3 helper genes) or pJD180 (2 helper genes)) and 100 ng of piggybac transposase plasmid (pTransposase (System biosciences, Cat#P210PA-l)) was transfected for stable integration. Media was changed 24 hours post transfection. Both transfer vectors possess an inducible mCitrine fluorescent protein. Following integration, cells were expanded to 6-well plate or 10 cm dish and transfected with pJD109 plasmid (Efla-ET-pA) in a similar fashion for sorting positive integrants using mCitrine fluorescence.

[0215] Fluorescence based sorting of positive stable cells

[0216] Cells were prepared for sorting in the following manner. Firstly, cells were washed with PBS (lx) and trypsinized by adding 1 :1 mix of PBS and Try psin and incubated for 5-10 minutes at 37 °C and 5 % CO2. Appropriate amount of complete DMEM (3 mL for T-75 flask, 100 pL for 24-well plate) was added to neutralize the trypsin. Cells were detached using a pipette and the suspended cell volume was centrifuged at 500xg for 5 minutes. The supernatant was discarded, and the cells re-suspended in PBS-BSA solution (Sigma, Cat#A7030) (amount depends on the sorting volume requirements) and kept on ice. The re-suspended cells were filtered through a cell strainer (Coming, Cat#352235) before sorting. Gating strategy7for sorting polyclonal cells is shown in FIG. 9B-9E. Excitation laser 488 nm and emission filter 530 / 30 with longpass filter 495 nm and PMT of 300 mV for mCitrine was used for sorting. Cells were recovered DMEM media supplemented with 20 % FBS. Cells were expanded and tested for its capacity to produce AAVs.

[0217] Transmission Electron Microscopy of AAV particles

[0218] AAV vectors produced using different helper plasmids in 15 cm dish and purified via static binding assay were utilized as samples for visualization of viral particles. Firstly, 5 pL of sample was adsorbed onto 200 mesh carbon and formvar coated nickel grids for 2 minutes. Three drops of 0.2 pm filtered distilled water was used to rinse the grid. Then, the grid was stained by transferring onto a 50 pL drop of 0.2 pm filtered 2% aqueous uranyl acetate for 30 seconds. The drop was absorbed off on lens paper and air dried. All grids were imaged using a FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro. Oregon) at 100 kV accelerating voltage, interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Wobum, Massachusetts) for digital TIFF file image acquisition. TEM images of samples were assessed, and digital images captured at 2k x 2k pixel, 16-bit resolution, 98000x magnification. Full and empty particles were counted manually from a set of five images per sample.

[0219] Flow cytometry

[0220] Cells were analyzed on flow cytometer 48 hours post transfection. Transduced HEK293 cells were prepared for flow cytometry7by removing the media and supplying the cells with 1 : 1 mix of PBS lx, pH 7.4 (ThermoFisher, Cat# 10010-015) and Accutase (ThermoFisher, Cat#Al 1105-01) in a total volume of 100 pL. The cells were incubated for 5-8 minutes at 37 °C, 5% CO2. Cells were then re-suspended and analyzed using BD LSR Fortessa II Cell Analyzer (BD Biosciences). The machine was calibrated with Sphero Rainbow Calibration Particles 8- peak beads (Spherotech, Cat#PCP-30-5A) prior to use. The excitation lasers (Ex) and emission filters (Em) used for respective fluorescent protein measurements are as folloyvs: mScarlet (Ex: 488 nm, Em: 610 / 20 nm, longpass filter 595 nm). Photo multiplier tube (PMT) voltage for fluorescent channel was adjusted in a yvay that the mean values for 8-peak beads remained constant across different experiments. For reference, PMT for mScarlet was 450 mV. Bar charts showing the absolute fluorescence units was obtained by analysing the flow cytometry data using FlowJo software (BD Biosciences). The following steps were undertaken for calculating the absolute units, (i) Live cells were gated based on forward scatter area (fsc) versus side-scatter area (ssc) plot, (ii) Within the live cells population, single cells were gated based on forward scatter area versus forward scatter width, (iii) Within the single cells population, mScarlet positive cells were gated based on a negative control (non-transfected) sample such that 99.9 % of cells fell outside of the selected gate, (v) For the mScarlet positive cell population, Flowjo was used to calculate mean value of fluorescence and the frequency of positive cells. Multiplying these two values gives absolute intensity which is a direct measure of the fluorescent protein signal.

[0221] Absolute fluorescent intensity = Mean fluorescence * (% fluorescence positive cells')

[0222] Statistical analysis

[0223] All experimental conditions were repeated at least twice. Biological replicates (n=3) were measured for all the samples / conditions. qPCR of samples was either done in duplicates or triplicates. All error bars represent mean ± standard deviation. MS Excel and Graphpad Prism software were used for analyzing data and making charts.

[0224] Table 1: Plasmids used in the study with its cloning procedure.

[0225] Table 2: Sequence of primers and taqman probes used in the study.

[0226] Table 3: Gene fragments / glbocks used in the study

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] Example 2. E2A, E4orf6 and VA RNA I genes are not enough for AAV production

[0242] Prior studies determined the essential adenoviral components for AAV vector production comprise the E2A, E4orf6 and VA RNA I genes. These genes including regulatory elements make up approximately 4.5kb in size (FIG. 1A). However, commonly utilized helpers like pDeltaF6 and pALD-X80 harbor more extensive adenovirus sequences, spanning 12.5 kb and 15 kb respectively (FIG. 1A, FIG.4D). The surplus content in pDF6 includes complete coding sequences of 11 additional adenoviral genes and partial sequences of 5 others. These supplementary regulatory and coding adenoviral sequences confer no known benefit toward AAV production. Presence of unwanted viral sequence poses a safety risk and increases transfection costs. To understand their significance in the context of AAV production, E2A, E4orf6 and VA RNA I genes were cloned in individual plasmids in such a way that E2A and E4orf6 expression was driven by constitutive CMV promoter while VA RNA I expression was driven by its wild-type pol iii promoter. Production titers were compared via qPCR (24-well plate; production scale 500 pL) using equimolar amounts of the three individual genes versus the commonly used helper plasmid, DF6. Alongside helper plasmids. AAV genome comprising a luciferase gene (pJD36: ITR-CMV-luciferase-ITR) and Rep2-Cap9 (pJD64) were transfected. qPCR assay using a luciferase probe showed that the AAV titer was ~7-fold lower when using E2A, E4orf6, VA RNA I compared to DF6 (FIG. IB). Luciferase intensity measured after transducing freshly seeded HEK293 cells at MOI of 10000 genome copies per cell for the two AAV preparations did not reveal any significant difference (FIG. 6A). Therefore, these three genes do produce AAV but with low efficiency. The lack of comparable production titers upon use of E2A, E4orf6 and VA RNA I pointed towards the need of additional sequence(s) present in DF6 helper plasmid that potentiate production.

[0243] Example 3. 22k / 33k gene is indispensable and E4orf6 is dispensable for AAV production

[0244] All open reading frames present within the helper plasmids pDF6 and pALD-X80 were identified (FIG. 1A, FIG. 4D) in order to determine which encoded sequences enhance AAV production yields. Two helper plasmids were generated from DF6 that were devoid of either fiber, U exon and pVIII genes or pTP gene (FIG. 1C). Both the helper plasmids had titers and transduction efficiency within 2-fold difference of DF6 benchmarks (FIG. ID). Absence of fiber, U exon, pVIII genes or pTP genes did not have any impact on production. Then, the essential E4orf6 component, residing amongst 6 open reading frames in the E4 locus of DF6 was investigated. Plasmids encoding either just E4orf6 + E4orf6 / 7 or the complete E4 region alongside E2A and VA RNA I helper genes were generated. However, the presence of these supplementary E4 elements did not enhance yields (FIG. IE), despite maintaining transduction efficiency (FIG. IE). The critical E2A gene operates within the adenovirus DNA-binding protein coding region, also housing 100k, 22k and 33k genes. The 22k and 33k genes exhibit an overlapping N-terminus but divergent C-termini and were evaluated in combination. 22k / 33k genes recovered titers to levels observed with DF6 without impacting transduction efficiency (FIG. IF). 100k did not have any positive impact on production titer (FIG. IF). 22k and 33k were also evaluated separately. Individual testing revealed that both 22k and 33k genes enhanced production titers, with higher influence from 22k (FIG. 6B). Augmenting 22k / 33k with E4orf6 / 7, the intact E4, or 100K failed to further improve titers (FIG. 1F-1H). Supplementary adenoviral genes 23k and VA RNA II were also non-contributory (FIG. 6B, FIG. 6C). Negligible yields in control samples lacking helpers or AAV components confirmed assay specificity (FIG. 6D). Collectively, these data spotlight 22k / 33k elements as critical adenoviral supplements to base AAV helper genes (E2A, E4orf6, VA RNA I), with other tested regions conferring no production benefit. This raised the question of whether E2A, E4orf6 and VA RNA I were part of the essential minimal helper gene set or if there was some dispensability. This question was addressed by comprehensively assessing AAV production capacity across all possible genetic combinations draw n from E2A, E4orf6, VA RNA and the newly implicated 22k / 33k locus. Specifically, AAV titers were determined for each of the 16 unique arrangements that can be generated using these four components to identify optimal subsets and dependencies. As expected, single genes resulted in negligible production titer (FIG. 2A). Some of the tw o gene subsets resulted in meaningful titers. E2A and 22k / 33k gave titers 2-3-fold lower than DF6 (FIG. 2A). VA RNA I with 22k / 33k resulted in titers however -7-fold lower than DF6 (FIG. 2A. FIG. 7A). All but one three gene combination set resulted in meaningful titer. The best set comprising E2A. VA RNA 1 and 22k / 33k genes that resulted in titers comparable to DF6. E2A, E4orf6 and 22k / 33k set and VA RNA I, E4orf6 and 22k / 33k set gave titers 2-6-fold lower than DF6 (FIG. 2A, FIG. 7A). As expected, the entire four gene set resulted in titers as observed previously. Overall, the data showed that E4orf6 is dispensable for production. Additionally, DF6 plasmid variant that is devoid of any E4 sequence produced comparable titer to DF6 (FIG. 7B) indicating the dispensability of E4orf6 gene. The new minimal helper gene set for AAV production came out to be: E2A, VA RNA I and 22k / 33k, although E2A+22k / 33k condition also had 2.5-fold lower titers than DF6 (FIG. 2A, FIG. 7A). The data also pointed to the fact that titers dropped the most when 22k / 33k was absent. Transduction efficiency values for the possible combinations of the four helper genes agreed with the titer data (FIG. 7C). With the minimal helper gene set defined, titrations of E2A, E4orf6 and VA RNA I quantities were performed to determine optimal ratios relative to Rep-Cap and genome plasmids for maximizing yields. The titration data showed that equivalent molar helper gene amounts conferred peak titers (FIG. 2B). Augmenting any individual component beyond this equimolar balance failed to further improve production outcomes (FIG. 2B).

[0245] Example 4. Scalable next generation helper plasmids

[0246] Applying the results from the earlier studies, next generation adenovirus helper plasmids were designed to either incorporate the minimal gene set (pJD171 or JD171: E2A, VA RNA I and 22k / 33k) or the four genes set (pJD150 or JD150: E2A, VA RNA I, E4orf6 and 22k / 33k). In the next generation helper plasmid designs, E4orf6, E2A and 22k / 33k genes were driven by a constitutive CMV or Efla promoter. Transcription termination for E2A and 22k / 33k genes utilized either bovine growth hormone (bGH) or SV40 polyadenylation signals, respectively, while the native signal was retained for E4orf6 gene. Wild-type pol III promoter and termination signal w ere utilized for VA RNA I gene. The next generated helper plasmids JD150 (4 genes) and JD171 (3 genes) were 5.2 kb and 7.2 kb, respectively, shorter in size than DF6 (FIG. 3A). The capacity of the new helper genes to produce AAVs was tested in 24-well plate scale. Production titers and transduction efficiency of the new helper plasmids JD150 and JD171 remained comparable to helper plasmids DF6 and ALD-X80 (FIG. 3B, FIG. 8A). Up-scaling production to 15 cm dish (three independent runs, 18 mL volume) demonstrated a similar increase in production titers across the three helper plasmids: JD150, JD171 and DF6 (FIG. 3C, and Table 5). Production in 15cm dish was purified using static binding assay (see Methods) with unbiased capturing of full or empty capsids. Transmission electron microscopy images of the purified AAV preparations using different helpers displayed identical morphology (FIG. 3D). The %full capsids determined by capsid ELISA did not reveal any significant variation remaining within 5-10% range for all the helper plasmids (Table 5). Overall, the next generation helper plasmids demonstrated equivalence to DF6 or pALD-X80 regarding key AAV production attributes including titers, transduction efficiency, and full: empty particle ratios, while maintaining scalability advantages. In summary, these newer helper systems provided reduced size eliminating non-essential adenoviral sequences and lower transfection requirements, conferring potential opportunities for cost reductions in manufacturing.

[0247] Table 5: Genome titers, Capsids / mL and % full capsids data for AAVs made using three helper plasmids DF6, pJD150 or pJD171.

[0248] Example 5. Production independent of serotypes and AAV genomes

[0249] The versatility of the next generation helper plasmids was evaluated through vector production of multiple serotypes (AAV6, AAV8, AAV-ACMdelta (Zabaleta et al)) and recombinant genomes alongside comparative benchmarks. Comparable vector titers were achieved across serotypes when employing either the novel helper constructs JD150 or JD171, or the conventionally utilized DF6 helper (FIG. 4A). Both increased production scale (500 mL) and substitution with an alternate AAV genome (a reporter cassette comprising ITR-flanked mScarlet under a CMV promoter, pJD34) were evaluated concurrently, without either factor impacting the consistent vector titers supported by the novel helper constructs. (FIG. 4B, FIG. 4C. FIG. 4E). Quantitative analysis of transduction efficiency by flow cytometry further exhibited less than 2- fold variances across the panel (FIG. 4F). The data demonstrated the versatile application of these novel helper constructs across adeno-associated viral serotypes and distinct recombinant genome configurations, exhibiting their capacity to support vector production independent of such modular swap-ins.

[0250] Example 6. Inducible helper plasmid for generation of stable helper cell line

[0251] Next, it was tested if helper gene transfection could be circumvented entirely. Since constitutive helper expression imparts toxicity that destabilizes producers, E2A and 22k / 33k were configured to be under a bidirectional tet-responsive promoter for inducible control. In this system, tetracycline transactivator protein (tTA) addition prompted E2A and 22k / 33k expression (FIG. 5A). VA RNA I retained constitutive expression signals. Prior to genomic integration, it was validated that the novel helper construct stringently regulated AAV yields using transient transfection - negligible titers accrued without tTA induction (FIG. 5A, middle), while rTA addition restored DF6 comparable outcomes (FIG. 5A). Luciferase intensity as a proxy of AAV transduction efficiency remained within 2-fold between conditions (FIG. 5A. right). Using piggybac transposon, random integration of two different construct designs was carried out: i) a design containing only inducible E2A and 22k / 33k genes, li) a design containing inducible E2A and 22k / 33k genes and constitutively expressing VA RNA I (FIG. 5B). Following integration, the positive integrants were sorted using fluorescence (FIG. 5B and 9B-9E). The viability7of the integrated cells remained in the range of 85-100% over two months of culturing (FIG. 9A). The sorted polyclonal cells were then tested for their ability to produce AAV vectors at different time points (Day 37, 55 and 66 post integration). For cells integrated with three helper genes, titers remained around lxE9 GC / mL in the presence of inducer across different time points (FIG. 5C, top). Comparable titers within 2-fold difference were observed in the absence of the inducer (FIG. 5C, top). The transduction potential of AAV preparations in the absence of inducer at day 37 was negligible (FIG. 5C, bottom). Titers were better for cells integrated with E2A and 22k / 33k and exogenously supplied with VA RNA I (FIG. 5D, top). Titers were negligible in the absence of inducer (FIG. 5D, top). In the presence of inducer, there was 2-6 fold drop in titer from day 37 to day 66 for these cells (FIG. 5D, top). AAVs were also produced, as expected, when no VA RNA I was exogenously supplied (FIG. 5D. top). Transduction profile was according to expectations, null values in absence of inducer and higher values in the presence of inducer (FIG. 5D, bottom). Overall, this data pointed to the applicability of integrating helper genes in the cells to produce AAV vector. However, the titers remained lower than triple plasmid transient transfection which could be attributed to multiple reasons: constitutive expression of VA RNA I, polyclonal population resulting in heterogeneous expression of helper genes, non- uniform activation of helper genes. Stepwise improvements in this regard should result in comparable assessment metrics.

[0252] The production of viral vectors can be visualized as an isolated synthetic system wherein the cellular factory provides the basic resources. Development of an isolated system requires replacement of native and complex regulation elements with synthetic, controllable elements. By detaching the complex regulation pattern of adenovirus, AAV and cellular genes governing AAV production, a system for efficient AAV production was developed.

[0253] First, a minimal set of adenoviral helper genes for production of AAV vectors was systematically determined. Novel helper gene plasmids were generated, which helper gene plasmids possessed the genes for AAV production. Further, the novel helper plasmids were significantly shorter in size than the currently used helper plasmids, e.g., DF6 and ALD-X80. The novel helper plasmids were comparable in quantitative and qualitative metrics, scalable as well as equivalent at producing different AAV serotypes and AAV genomes. In addition, stable helper cell lines for AAV production were generated. Inducible AAV production using stable helper cell lines with optimal copy number of helper genes can eliminate the need for transfection of at least one plasmid. These improvements remain crucial at reducing manufacturing costs associated with gene therapy treatments as well as increasing safety standards. Integrated cells with three helper genes or two helper genes integrated underperformed in assessment metrics. This could be attributed to multiple reasons: polyclonal population resulting in heterogeneous expression of helper genes, constitutive expression of VA RNA I, requirement of transfecting the inducer. Based on the disclosures herein, design of helper plasmids can be refined and improvements in obtaining homogeneous population of stable cells can be implemented. A person of ordinary skill in the art, in view of the disclosures herein can implement such improvements.

[0254] As described herein, adenovirus 22k and 33k genes have been identified to be contributive towards production of AAV vectors. Both 22k and 33k in the context of adenovirus life cycle have multiple functions (Wu et al 2013). 33k is an alternative splicing factor, 22k gene is involved in regulation of adenovirus death protein, both 22k and 33k are involved in adenovirus packaging and interact with multiple other adenovirus proteins.

Claims

CLAIMSWhat is claimed is:

1. A polynucleotide comprising a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 22k protein comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 130.

2. The polynucleotide of claim 1 , further comprising a nucleic acid sequence encoding an adenovirus E2A protein comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 140.

3. The polynucleotide of claim 1 or 2, wherein the polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus E4 protein.

4. The polynucleotide of any one of claims 1-3, further comprising a nucleic acid sequence encoding an adenovirus VA RNA I comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 99.5%, or 100% identity to SEQ ID NO: 142, an adenovirus VA RNA II comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 99.5%, or 100% identity to SEQ ID NO: 132, or a combination thereof.

5. The polynucleotide of any one of claims 1-3, further comprising a nucleic acid sequence encoding an adenovirus VA RNA I comprising at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 125 nucleotides, at least 127 nucleotides, at least 130 nucleotides, at least 132 nucleotides, at least 134 nucleotides, at least 136 nucleotides, at least 138 nucleotides, at least 140 nucleotides, at least 142 nucleotides, at least 144 nucleotides, at least 146 nucleotides, or at least 148 nucleotides, at least 150 nucleotides, at least 152 nucleotides, at least 154 nucleotides, at least 156 nucleotides, at least 158 nucleotides, or at least 160 nucleotides of SEQ ID NO: 142.

6. The polynucleotide of any one of claims 1-5, further comprising a nucleic acid sequence encoding an adenovirus 33k protein comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 139.

7. A polynucleotide comprising a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding an adenovirus 33k protein comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 131.

8. The polynucleotide of claim 7, further comprising a nucleic acid sequence encoding an adenovirus E2A protein comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 140.

9. The polynucleotide of claim 7 or 8, wherein the polynucleotide does not comprise a nucleic acid sequence encoding an adenovirus E4 protein.

10. The polynucleotide of any one of claims 7-9, further comprising a nucleic acid sequence encoding an adenovirus VA RNA I comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 99.5%, or 100% identity7to SEQ ID NO: 142, an adenovirus VA RNA II comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 99.5%, or 100% identity7to SEQ ID NO: 132, or a combination thereof.

11. The polynucleotide of any one of claims 7-10, further comprising a nucleic acid sequence encoding an adenovirus 22k comprising an amino acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 138.

12. The polynucleotide of any one of claims 1-11. comprising a further regulatory7sequence.

13. The polynucleotide of claim 12, wherein the further regulatory sequence is selected from a promoter, a polyadenylation / termination signal, and combinations thereof.

14. The polynucleotide of any one of claims 1-13, wherein the promoter is a constitutive promoter or an inducible promoter.

15. The polynucleotide of claim 14, wherein the constitutive promoter is a CMV promoter or an EFla promoter.

16. The polynucleotide of claim 14 or 15, wherein the constitutive promoter is operably linked to the nucleic acid encoding the E2A protein.

17. The polynucleotide of claim 13, wherein the promoter is a pol Til promoter.

18. The polynucleotide of claim 17, wherein the pol III promoter is operably linked to the adenovirus VA RNA I, adenovirus VA RNA II. or combination thereof.

19. The polynucleotide of claim 14, wherein the inducible promoter is operably linked to the nucleic acid encoding the 22k protein, the nucleic acid encoding the 33k protein, and / or the nucleic acid encoding an E2A protein.

20. The polynucleotide of claim 13, wherein the termination signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or a pol III termination signal.

21. A polynucleotide comprising adenovirus helper genes for packaging adeno-associated virus (AAV), which polynucleotide comprises no more than 5300 nucleotides.

22. The polynucleotide of claim 21, wherein the adenovirus helper genes for packaging AAV comprise an adenovirus 22k gene comprising a nucleic sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 138.

23. The polynucleotide of claim 21, wherein the adenovirus helper genes for packaging AAV comprise an adenovirus 33k gene comprising a nucleic acid sequence that has 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 99.5%, or 100% identity to SEQ ID NO: 139.

24. The polynucleotide of any one of claims 21-23, wherein the adenovirus helper genes for packaging AAV further comprise an adenovirus E2A gene comprising a nucleic acid sequence that has at least 85%. at least 86%. at least 87%. at least 88%. at least 89%. atleast 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 99.5%, or 100% identity to SEQ ID NO: 140.

25. The polynucleotide of claim 21-24, wherein the adenovirus helper genes for packaging AAV further comprise an adenovirus VA RNA I comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, 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 99.5%. or 100% identity to SEQ ID NO: 142, an adenovirus VA RNA II comprising a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 99.5%, or 100% identity to SEQ ID NO: 132, or a combination thereof.

26. The polynucleotide of any one of claims 21-25, wherein the polynucleotide further comprises a regulatory sequence.

27. The polynucleotide of claim 26, wherein the regulatory sequence is selected from a promoter, a termination signal, and combinations thereof.

28. The polynucleotide of claim 27, wherein the promoter is a constitutive promoter or an inducible promoter.

29. The polynucleotide of claim 28, wherein the constitutive promoter is a CMV promoter or an EFla promoter.

30. The polynucleotide of claim 28 or 29, wherein the inducible promoter is operably linked to the adenovirus 22k gene, the adenovirus 33k gene, and / or the adenovirus E2A gene.

31. The polynucleotide of claim 27, wherein the promoter is a pol III promoter.

32. The polynucleotide of claim 31, wherein the pol III promoter is operably linked to the VA RNA I, VA RNA II, or combination thereof.

33. The polynucleotide of any one of claims 27-32, wherein the termination signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or a pol III termination signal.

34. The polynucleotide of any one of claims 1-33, wherein at least one nucleic acid sequence of any one of claims 1-20 is present in a cellular genome and / or at least one of the adenovirus helper genes of any one of claims 21-34 is present in a cellular genome.

35. An adeno-associated virus (AAV) packaging system comprising a polynucleotide of any one of claims 1-34 and a nucleic acid sequence comprising an inverted terminal repeat and encoding a gene of interest, a nucleic acid sequence encoding an AAV replication protein (Rep) and a nucleic acid sequence encoding an AAV capsid protein (Cap).

36. The AAV packaging system of claim 35, not comprising any additional adenovirus helper gene encoding nucleic sequences.

37. An AAV packaging system consisting essentially of a polynucleotide of claims 21-34, a nucleic acid sequence comprising an inverted terminal repeat and encoding a gene of interest, a nucleic acid sequence encoding an AAV replication protein (Rep) and a nucleic acid sequence encoding an AAV capsid protein (Cap).

38. A vector comprising a polynucleotide of any one of claims 1-34.

39. A cell comprising a polynucleotide of any one of claims 1-34. a packaging system of any one of claims 35-37, or a vector of claim 38.

40. A method of preparing an adeno-associated virus (AAV), the method comprising introducing into a cell a polynucleotide of any one of claims 1-34, a packaging system of any one of claims 35-37, or a vector of claim 38; culturing the cell under conditions such that an AAV is generated in the cell; and isolating the AAV.

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