Recombinant canine adenoviruses with heterologous open reading frames and methods of use

Abstract

The identification of sites in the canine adenovirus (CAV) genome that allow for insertion of a heterologous open reading frame (ORF) without significantly altering CAV replication are described. Recombinant CAV genomes having at least one heterologous ORF at selected sites, as well as recombinant CAVs harboring such genomes, are also described. Methods for measuring replication of a recombinant CAV having a genome with one or more heterologous ORFs are further described.

Description

[0001] 7158-105381-02

[0002] RECOMBINANT CANINE ADENOVIRUSES WITH HETEROLOGOUS OPEN READING FRAMES AND METHODS OF USE

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 779,141, filed March 27, 2025, which is herein incorporated by reference in its entirety.

[0005] FIELD

[0006] The present disclosure concerns recombinant canine adenovirus (CAV) genomes containing heterologous open reading frames (ORFs) inserted at specific locations in the CAV genome that do not prevent replication of the recombinant CAV. The recombinant CAV genomes can also include specific gene deletions, such as to increase cargo space for heterologous ORFs.

[0007] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0008] The electronic sequence listing, submitted herewith as an XML file named 7158-105381-02.xml (1,846,570 bytes), created on March 17, 2026, is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Adenoviruses are non-enveloped (80-100 nm) viruses that possess a double-stranded DNA genome encased in a protein capsid with fiber proteins that target infection to specific cellular receptors. Adenoviruses have evolved and adapted to infect humans, monkeys, dogs, mice, chickens, snakes, and pigs. However, the understanding of adenovirus biology is almost exclusively based upon and extrapolated from studies with human subgroup C adenovirus (Ad), particularly Ad5 and Ad2.

[0011] There are two canine adenovirus subtypes, Type 1 (CAV1) and Type 2 (CAV2), with variants arising in each subtype depending on geographical location and genetic differences. Both Type 1 and Type 2 CAVs are significant pathogens in dogs, where they cause infectious canine hepatitis (ICH) and canine infectious respiratory disease complex, respectively. Both types of CAV diseases are preventable through vaccination.

[0012] The development of CAVs as replication-competent live vaccines and oncolytics is highly desirable, with important applications for treating both human and animal diseases. CAV does not replicate in human cells, and as such, could be exploited as a natural vector and vaccine. CAV therapies also have important and critical applications in animals, both as live vaccines to prevent CAV associated diseases, as well as potential development as canine oncolytic therapies. Achieving this goal requires tire identification of the specific CAV genes essential for replication, the establishment of assays for replication, and the incorporation of therapeutic pay loads.7158-105381-02

[0013] SUMMARY

[0014] The present disclosure provides a systematic design and architecture for achieving expression of heterologous open reading frames (ORFs) via distinct mechanisms in different CAV genomic placements, as well as gene deletions to create cargo space, without disrupting recombinant CAV replication. Heterologous ORFs can include, but are not limited to, fluorescent reporters, vaccine antigens, immune modulators, and therapeutic proteins (FIG. 1).

[0015] The data disclosed herein reveal different genomic placements that have distinct expression profiles, which are useful, tunable and customizable for various applications, including but not limited to, the development of imaging-based methods to visualize and quantify viral replication kinetics and the expression of therapeutic ORFs for live multivalent vaccines.

[0016] Provided herein are recombinant CAV genomes that include a first heterologous ORF, and optionally include a second heterologous ORF, a third heterologous ORF and / or a further heterologous ORF, and may further include deletions in one or more CAV genes (e.g., E3-ORF1, E3-ORF2 and / or E4-ORF5).

[0017] In some aspects of the recombinant CAV genome, the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0018] In other aspects, the first heterologous ORF is located 5' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0019] In other aspects, the first heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0020] In other aspects, the first heterologous ORF is located 5' of a DNA polymerase ORF and a selfcleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein die first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame.

[0021] In odier aspects, the first heterologous ORF is located 3' of a DNA polymerase ORF and a selfcleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and die DNA polymerase ORF are operably linked and in the same reading frame.

[0022] In other aspects, die first heterologous ORF is located 3' of an E2A-DNA binding protein (DBP) ORF and the first heterologous ORF is linked to die E2A-DBP ORF.7158-105381-02

[0023] In other aspects, the first heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the pVIII ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame.

[0024] In other aspects, the first heterologous ORF is located 3' of an E3-ORF1 and the first heterologous ORF is linked to the E3-ORF1.

[0025] In other aspects, the genome comprises a complete or partial deletion of an E3-ORF1 coding sequence and the first heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0026] In other aspects, the genome comprises a complete or partial deletion of an E3-ORF1 coding sequence and a complete or partial deletion of an E3-ORF2 coding sequence, and tire first heterologous ORF is inserted at tire site of tire E3-ORF1 deletion.

[0027] Also provided is a method for detecting and quantifying replication of an individual recombinant CAV or a library of recombinant CAVs in a host cell culture. In some aspects, the method includes infecting or transfecting the host cell culture with a recombinant CAV comprising a recombinant CAV genome disclosed herein, wherein the first heterologous ORF encodes a reporter protein; detecting expression of the reporter protein using: (i) an imaging-based system capable of acquiring and analyzing spatially and spectrally resolved image data at different wavelengths, length and time scales, from multiwell plates of recombinant CAV genome reporter gene fluorescence in individual cells, plaques and / or the cell culture area as well as brightfield imaging; and / or (ii) a multi-modal plate reader configured to rapidly measure bulk recombinant CAV genome reporter gene fluorescence intensity and / or luminescence across a plurality of wells in a multi-well format, wherein the signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

[0028] In other aspects, the method includes infecting or transfecting the host cell culture with a recombinant CAV comprising a recombinant CAV genome disclosed herein, wherein the first heterologous ORF encodes a reporter protein; detecting expression of the reporter protein using (i) a plate reader based system that quantifies fluorescence or luminescence from each well of a multi-well plate; or (ii) a plate reader configured or automated to capture fluorescence or luminescence over time, wherein the expression of the reporter protein signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

[0029] Further provided is a method for measuring replication of a recombinant CAV comprising a recombinant CAV genome disclosed herein, wherein the first heterologous ORF encodes a reporter protein. In some aspects, tire method includes (i) transfecting cells with the genome of tire recombinant CAV, or infecting cells with particles of tire recombinant CAV ; (ii) culturing the transfected cell or infected cells for at least 24-72 hours; (iii) measuring reporter gene signal at regular intervals throughout the culturing step, thereby obtaining fluorescence measurements; and (iv) calculating log-slope (day1) from a seini-log plot of fluorescence or intensity signal, normalized to background, versus time in the linear' part of the cu ve, thereby measuring replication of the recombinant CAV.7158-105381-02

[0030] Also provided are kits that include a recombinant CAV genome or recombinant CAV disclosed herein and any combination of cells, cell culture media and / or multi-well plate(s).

[0031] The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Schematic of the systematic approach disclosed herein for engineering canine adenovirus (CAV) genomes to incorporate heterologous open reading frames (ORFs), such as fluorescent reporters, vaccine antigens, immune modulators, and therapeutic proteins, in four different CAV genomic plasmid modules: El, core, E3 and E4. Also disclosed herein and depicted in the figure is combinatorial in vitro assembly and high-throughput (HTP) / high-content screening (HCS) fluorescent-based screening of recombinant CAV libraries.

[0033] FIG.2: ClustalW alignment and phylogenetic tree comparing nucleic acid sequences between canine adenovirus 1 (CAV1), canine adenovirus 2 (CAV2), several human adenovirus members, including the most extensively studied human Ad5 (hAd5), and other animal adenoviruses. CAV2 is most similar to CAV1, with the next closest species being bat and skunk adenoviruses.

[0034] FIG.3: Genomic map of the hAd5 reference genome. The hAd5 genes and transcriptional units are annotated. hAd5 is the most extensively studied member of the adenovirus family and provides a general model for the genes, structure, transcription and functions of Adenoviridae.

[0035] FIG.4: Genome map of the CAV2 Toronto A26 / 61 strain reference genome, which is 31,232 base pairs in length. The nomenclature of protein coding genes follows the accepted schema of transcriptional units, and genes therein, of Adenoviridae, the subgroup C hAd genes and the CAV GENBANK annotation.

[0036] FIGS. 5A-5B: Nucleotide sequence comparison of (FIG. 5A) CAV1, CAV2, and hAd5, with CAV2 used as the reference sequence. There is significant divergence between the nucleotide sequence of hAd5 and CAV1 / CAV2. In contrast, CAV1 and CAV2 have highly similar nucleotide sequences and predicted genes. The most notable difference between CAV1 and CAV2 is that CAV1 has a deletion in the E3 region, which corresponds to E3-ORF2. (FIG. 5B) Nucleotide sequence comparison of CAV1, CAV2, and bat adenovirus, with CAV2 used as the reference sequence. CAV1 and bat adenoviruses have high homology to CAV2, including at genomic junctions chosen to split genomes for CAVSLIC / CAVsembly genomic module plasmids.

[0037] FIG.6: CAV2 ORF conservation, diversity and homology compared to other animal and human Ads. Amino acid similarity of canine ORFs is highest with bat and skunk Ads, which share many of tire same early proteins and are very closely related. As such, many of the genomic placements disclosed for CAV are likely to be conserved and applicable to bat and skunk Ads based on genomic and protein7158-105381-02

[0038] coding sequences homology. In contrast, canine and human Ad ORFs have diverged significantly, and is highest for some of the structural proteins (shaded) that assemble the capsid.

[0039] FIG.7: Schematic showing CAV genome organization (top) and CAVsembly / SLIC genome modules (bottom). Based on the conserved transcriptional and genome organization of CAV1 / CAV2, the CAV genome was split into four genomic modular parts (El, core, E3 and E4), which were cloned into plasmids to facilitate their genetic engineering and creation of variant libraries for systematic assembly. The four CAVsembly / CAVSLIC functional genomic modules allow for manipulation to confer new and engineered properties, can be assembled together to confer wild type virus as well as emergent properties, and are compatible with exchange of genomic modules in adenoviruses across species, including hAd5.

[0040] FIG.8: Schematic of the CAV El genome module, which spans from the left-hand internal terminal repeat (ITR) through the El genes and the TATA box for pIX. The El A and E1B gene products encoded within this module are essential for viral replication. These genes can be deleted for the assembly and production of recombinant non-replicating CAV vectors.

[0041] FIG.9: Schematic of the CAV core genome module, which includes tire early / intermediate genes, pIX and IV A2; the major late promoter (MLP) transcriptional unit that drives the expression of CAV capsid and late proteins, including 52 / 55k, pllla, penton, pVII, pV, pX, pVI, hexon, endoproteinase, 100k, 22k, and 33k (putative 22k / 33k) on the positive strand. On the negative strand, the E2 transcriptional unit drives the expression of the viral DNA replication proteins, including E2A DBP, pTP (terminal protein) and E2B DNA polymerase.

[0042] FIG. 10: Schematic of the CAV E3 genome module. This genomic module includes the MLP transcribed late protein pVIII and the E3 protein coding genes (E3-ORF1 , E3-ORF2 and fiber) on the positive strand, and the U exon on the negative strand. The E3 encoded ORFs are highly variable among adenoviruses.

[0043] FIG. 11: Schematic of the CAV E4 genome module, which includes the right ITR and the E4 transcriptional unit and protein coding genes on the negative strand. In human adenoviruses, the E4 genes play important functions in usurping host cell signaling pathways and function to facilitate viral replication. In CAV, there are five predicted and annotated ORFs, which include from right to left, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5.

[0044] FIG. 12: Illustration of SLIC mediated cloning of a CAV2 viral genome into a self-excising I-Scel plasmid backbone that was used as template and resource for generating recombinant CAVs and CAV genome module plasmids.

[0045] FIG. 13: Schematic showing the construction and synthesis of the CAV El, core, E3 and E4 genome module plasmid constructs that were used as die basis for different placements of heterologous ORFs, as well as deletions and replacements of CAV genes in variant engineered El, core, E3 and E4 libraries that were assembled into recombinant CAV virus genomes and screened for ORF expression and viral replication competence and other properties. CAV El, Core, E3, and E4 genomic modules were7158-105381-02

[0046] generated using geneblocks (gBlock) or cloned from the ATCC CAV2 reference viral genome plasmid, PCMN-1312. The El, E3, and E4 genomic modules were inserted into entry plasmids containing a kanamycin resistance gene and flanking sequences compatible with multisite Gateway assembly, sequence and ligation-independent cloning (SLIC), and Gibson assembly. Silent mutations were engineered into E4 genome sequences that ablate Pact and Bglll sites. The CAV core genomic region was made into a destination plasmid containing an ampicillin resistance gene and flanked on the left and right by either unique Pad and Bglll restriction enzyme sites for assembly with E1 / E3 / E4 modules, and / or Gateway recombination sites with positive and negative selection cassettes. Core plasmid modules for Gateway assembly (core-D) have compatible SSR sites and ccdB cassettes on the left and / or right side, while core modules for SLIC lack these cassettes and were assembled by scarless cloning with Bglll and Pad linearization. The core plasmid serves as tire central backbone for assembling the complete CAV genome by facilitating the integration of the El, E3, and E4 modules through Gateway, SLIC, and / or Gibson assembly.

[0047] FIG. 14: Map of the CAV El module entry plasmid, El-269. The CAV El entry plasmid includes the CAV El genomic module, extending from the right-hand ITR region to include the El A and E1B genes, as well as the TATA box breakpoint before plX. The plasmid backbone has an attLl site followed by an I-Scel restriction site flanking the left ITR, an attL4 site before the plX Kozak sequence, a kanamycin resistance gene and an origin of replication (ori) for bacterial selection.

[0048] FIG. 15: Map of the CAV E3 module entry plasmid CAV E3-522, which includes pVIII through the E3-ORF1 and E3-ORF2 early region immunomodulatory proteins and the fiber gene on the positive strand and the U exon on the negative strand. The plasmid backbone has an attL5 site before pVIII and an attR3 site engineered before putative predicted overlapping fiber and E4 poly A motifs, a kanamycin resistance gene and an ori for bacterial selection.

[0049] FIG. 16: Map of the CAV E4 module entry plasmid, E4-099. The E4 entry plasmid includes the right-hand ITR and the E4 transcriptional unit, encoding the annotated E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4, and E4-ORF5 early viral genes. The E4 module was constructed using three primer extension PCR products amplified from the CAV2 genome. Two silent degenerate base -pair modifications were engineered within the E4 coding region to eliminate Bglll and Pad restriction sites while preserving the encoded amino acid sequences. The E4 plasmid backbone includes a kanamycin resistance gene and an ori for bacterial selection. AttL2 and an I-Scel restriction site flank the right ITR of tire E4 genome module, and an attL3 is present at the fiber and E4 non-coding junction.

[0050] FIG. 17: Plasmid map of the CAV core SLIC module (Core-059), used for tire engineering of CAV core module libraries and recombinant CAV viral genomes described herein. The Core-059 plasmid module includes the CAV2 core genomic region spanning from the ea ly plX gene to the putative late 33k gene. The plasmid backbone has a low-copy pl5A bacterial origin of replication, bacterial transcriptional terminator sequences and ampicillin resistance genes that enable viral genome plasmid propagation and selection in bacteria. There is also an I-Scel expression cassette that includes a7158-105381-02

[0051] mammalian RSV viral promoter that drives the I-Scel restriction enzyme upon plasmid transfection into mammalian (including canine) cells. The mammalian promoter and expression of the I-Scel restriction enzyme specifically in mammalian cells cuts the CAV vaccine plasmid backbone at the I-Scel cleavage sites that flank the left and right assembled CAV genome ITRs linearizing and releasing the CAV genome so that it can be recognized by CAV E2-TP and undergo E2B mediated Type I and II viral genome replication and virion production. The key elements that were engineered to facilitate viral genome fragmentation and scarless module assembly via core module linearization with unique restriction sites were: (1) attRl and attR4 SSR sites on the left hand side of the core module, which have ccdB negative selection and chloramphenicol positive selection cassettes that are recombined out upon successful recombination with an El module; and (2) on the right side of the core module there are attR5 and attR2 sites with ccdB negative selection and chloramphenicol positive selection cassettes that are recombined out upon successful recombination with an E3 module.

[0052] FIG. 18: Map of the core destination plasmid module (Core-074D), used for the assembly of recombinant CAV viral genomes via multi-site SSR recombination described herein. The Core-074D plasmid module includes the CAV2 core genomic region spanning from the early pIX gene to the putative late 33k gene. The plasmid backbone has a low-copy pl5A bacterial origin of replication, bacterial transcriptional terminator sequences and ampicillin resistance genes that enable viral genome plasmid propagation and selection in bacteria. There is also an I-Scel expression cassette. The mammalian promoter and expression of the I-Scel restriction enzyme specifically in mammalian cells cuts the CAV vaccine plasmid backbone at the I-Scel cleavage sites that flank the left and right assembled CAV genome ITRs linearizing and releasing the CAV genome so that it can be recognized by CAV E2-TP and undergo E2B mediated Type I and II viral genome replication and virion production. The key elements that were engineered to facilitate viral genome fragmentation and scarless module assembly via core module linearization with unique restriction sites were: (1) attRl and attR4 SSR sites on the left hand side of the core module, which have ccdB negative selection and chloramphenicol selection cassettes that are recombined out upon successful recombination with an El module; and (2) on the right side of the core module, at the pVIII junction, there are attR5 and attR2 sites with negative and positive selection cassettes in between.

[0053] FIGS. 19A-19B: Schematics showing how the CAV El, E3, E4 and core genome module plasmids are designed and engineered such that they can be assembled via multi-site Gateway recombination and / or scarless assembly methods. (FIG. 19A) Schematic of CAVSLIC, which is a method of assembling CAV genome module plasmids that uses scarless and SLIC based assembly of El, E3 and E4 modules to create a complete CAV genome in two (this example) or three steps. The CAV core genome module plasmid is engineered to have unique restriction sites engineered at left and right hand sides, that are not present or are ablated in the El, E3 or E4 modules. The core module is linearized by restriction enzyme digestion and assembled with a PCR linearized El module (for example) via SLIC / Gibson and overlapping sequences in El and core modules. Following transformation and selection7158-105381-02

[0054] of the El-core macromodule plasmid with E3 and E4 module plasmids, a fully assembled viral genome is generated. The CAVSLIC method results in a “scarless” viral genome product. (FIG. 19B) CAVsembly uses site-specific recombination (e.g., Multisite Gateway recombination). The CAV core module is cloned into a plasmid with ampicillin resistance and made into a Gateway compatible destination (DEST) plasmid whereby the CAV core genome module is flanked on left and / or right with multisite gateway cassettes that have site specific recombination sequences (att sites) and ccdB counterselection cassettes. The El, E3, and E4 genome modules are cloned into entry plasmids that can be combined with, in this example, a “dual-DEST” core module in a standard Multisite Gateway LR reaction. The resultant reaction contains fully assembled CAV genomes. Assembled CAV genomes can then be transformed into bacteria and correct assemblies selected by virtue of ampicillin and successful recombination and replacement of ccdB with CAV genome modules. The virus genome can also be transfected into canine cells as only correctly assembled CAV genomes are replication competent.

[0055] FIG. 20: Schematic of an exemplary 3-step CAVSLIC CAV genome assembly workflow using the order El -core, followed by E4 and then E3. The generic seamless assembly process depicted can be performed using Gibson assembly, SLIC, In-Fusion, restriction enzyme and ligation cloning, or other approaches.

[0056] FIG. 21: Schematic showing scarless CAVSLIC assembly of a wildtype CAV genome from El, core, E3 and E4 modules and inacromodules in the order shown. The generic seamless assembly process depicted can be performed using Gibson assembly, SLIC, In-Fusion, restriction enzyme and ligation cloning, or other approaches.

[0057] FIG. 22: Map of the ASMM-149 El-core assembled macromodule plasmid.

[0058] FIG. 23: Map of the ASMM-150 El-core-E4 assembled macromodule plasmid.

[0059] FIG. 24: Map of the PCMN-1313 whole CAV genome plasmid generated by scarless assembly of El, core, E3 and E4 modules and macromodule intermediates.

[0060] FIG. 25: Schematic showing an exemplary scarless assembly of a recombinant CAV genome, PCMN-1324, that has a modified El plasmid module, El-271, wherein El A was fused to a P2A sequence and operably linked to drive expression of a YPet fluorescent protein fusion. EL271 was assembled with Core, E3 and E4 plasmids to generate PCMN-1324.

[0061] FIG. 26: Map of the El genome module plasmid, El-271, which was engineered to express a heterologous ORF, in this example YPet, via a P2A fusion with El A. Bottom panel shows how YPet was fused to E1A via P2A self-cleaving peptide sequence.

[0062] FIG. 27: Map of the PCMN-1324 whole CAV genome plasmid.

[0063] FIG. 28: Workflow illustrating the process of evaluating recombinant CAV composition heterologous ORF expression and replication competence, starting from plasmid transfection in MDCK or DK cells, leading to plaque formation and cytopathic effects (CPE). The timeline and conditions for detecting productive replication and secondary infection are shown.7158-105381-02

[0064] FIG. 29: Fluorescence microscopy images following transfection of MDCK cells with PCMN-1324 (El A-P2A-YPet) whole genome plasmids. In these and subsequent fluorescence images, fluorescent cells appear as bright cells against a dark background. At 3 days post-transfection (dpt), fluorescent plaques were apparent in the MDCK monolayer, indicating successful transfection, first-round replication, and productive infection of the recombinant CAV genome, as well as YPet fluorescent payload expression. The increase and spread of fluorescent cells across the entire monolayer by day 10 indicates productive log-phase replication. Viral supernatants were harvested at day 13 and used to infect fresh MDCK cells in secondary and tertiary rounds of infection. Productive replication and payload expression was demonstrated by an increase in the number of YPet-positive cells over time.

[0065] FIG.30: Photographs of cesium chloride (CsCl) density gradient purification and characterization of PCMN-1324 virus particles propagated in DK cells. (Left) CsCl gradients of Ad5 virus propagated in human 293 cells are shown for comparison. (Right) Productive CAV particle production is demonstrated by the visible and strong lower higher density white band of concentrated recombinant CAV virus particles and the absence of upper ‘empty’ defective virus particles.

[0066] FIG.31: Plasmid map of E3-612, which was engineered to express a YPet P2A fusion with pVIH.

[0067] FIG.32: Map of the PCMN-1584 whole CAV genome plasmid.

[0068] FIG.33: Plasmid map of El-285, which was engineered to express a YPet P2A fusion with E1B-55K.

[0069] FIG. 34: Map of the PCMN-1422 whole CAV genome plasmid.

[0070] FIG. 35: Fluorescent images from a cell based Incucyte imaging assay of MDCK cells that were transfected with PCMN-1584 (FIG. 32). PCMN-1584 genome plasmid was assembled from a modified Core plasmid module engineered with a YPet-P2A-pVIII fusion (top), or PCMN-1422 (FIG. 34), which was assembled from a modified El module engineered with an ElB55k-P2A-YPet fusion (bottom). At 3 days post transfection, fluorescent plaques were apparent in the MDCK monolayer, indicating successful transfection, first round replication and productive infection of the recombinant CAV genome, as well as YPet fluorescent payload expression. The increase and spread of PCMN-1584 and PCMN1422 by day 10 to the entire monolayer, indicated productive log replication. Viral supernatants were harvested and used to infect MDCK cells in secondary and tertiary rounds of infection. Productive replication and payload expression was demonstrated by an increase in the number of YPet cells over time.

[0071] FIG.36: Schematic showing a 2-step scarless assembly of PCMN-1466 from a modified core-065 plasmid module that was engineered to have a YPet-P2A-DNA polymerase fusion, and then assembled with a wildtype El plasmid module to form the ASMM-176 macromodule plasmid. The ASMM-176 macromodule plasmid was then assembled with E3 and E4 plasmid modules.

[0072] FIG.37A-37B: Maps of (FIG. 37 A) Core-065 and (FIG. 37B) ASMM-176 macromodule plasmids.

[0073] FIG.38: Map of the PCMN-1466 whole CAV genome plasmid.7158-105381-02

[0074] FIG. 39: Images from a fluorescence assay following transfection of MDCK cells with PCMN-1466. MDCK cells were transfected with PCMN-1466 whole genome plasmids. At 10 days post transfection, fluorescent plaques were apparent in the MDCK monolayer, indicating productive infection of the recombinant CAV genome, as well as YPet fluorescent payload expression. Productive replication and payload expression was demonstrated by an increase in the number of YPet cells infected over time.

[0075] FIG. 40: Images from a fluorescence assay following transfection of MDCK cells with PCMN-1467 (YPet-P2A-Hexon) or PCMN-1384 (Fiber-P2A-YPet), which were assembled with a modified core module and YPet-P2A fusions engineered to the late protein coding genes, hexon and fiber, respectively. At 3 days post-transfection, some sparse initially transfected YPet expressing cells were visualized. However, YPet-P2A fusions to hexon and fiber negatively impacted productive virus replication and secondary infection, as evidenced by the failure to observe an increase in virally infected YPet positive cells at later timepoints and upon serial passaging of viral supernatants.

[0076] FIG. 41: Images from a fluorescence assay following transfection of MDCK cells with PCMN-1469 (DBP-P2A-YPet) or PCMN-1468 (YPet-P2A-DBP), which were assembled with a modified core module and N- or C-terminal YPet-P2A fusions to the E2A DNA binding protein (DBP). At 3 days posttransfection, some sparse YPet expressing transfected / infected cells were visualized. However, virus genomes with YPet-P2A fusions to E2A-DBP fiber do not undergo productive replication and secondary infection, as evidenced by loss of, and failure to increase, YPet-fluorescence and virally infected cells at later timepoints and upon serial passaging of viral supernatants.

[0077] FIG. 42: Images showing detection of recombinant CAV plaques by fluorescence. Assembling recombinant CAV genomes with modules that have been engineered to express fluorescent protein reporters (such as mCherry in this example) enables CAV plaques to be visualized unambiguously via fluorescence even at the earliest stages of infection and before the appearance of cytopathic effects in brightfield images.

[0078] FIG. 43: Work-Flows for High Content Fluorescent Imaging, FBVK assays and High Throughput Screening of Recombinant CAVs expressing fluorescent reporters.

[0079] FIG. 44: Schematic of theoretical FBVK data of cells infected with a fluorescent CAV at a single low MOI where log fluorescence intensity is plotted versus time. The interpretation of fluorescent plots is shown with the stage of viral infection and replication kinetics. Plotting semi-log plots of Fluorescence versus Time generates viral replication curves, where the slope in the linear part of the curve yields the viral replication kinetics (Day1). The vertical axis fluorescence intensity provides quantitative measurements that enable fluorescent protein expression at different genomic placements to be compared and evaluated. Furthermore, the plots provide valuable information on primary and secondary infection and stages that may be enhanced or impaired under different conditions.

[0080] FIG. 45: Fluorescence-based viral kinetics (FBVK) assay measuring CAV-2 (ElA-P2A-YPet) replication in MDCK cells. YPet fluorescence (arbitrary units) was quantified over time across a range of MOIs (individual curves). At sufficient MOI, log-phase viral replication curves capturing primary and7158-105381-02

[0081] secondary infection cycles are resolved. The slope of the log-phase growth curve is a measure of the replication rate. The height of the curve reflects the YPet expression levels when operably linked to El A via P2A. A dashed horizontal line indicates the plateau YPet expression level, and the dashed diagonal line indicates the slope (rate of replication).

[0082] FIG. 46: Comparison showing FBVK assay fluorescent curves, productive log replication and YPet expression levels from E1A-P2A fusion in Ad5 (right) and CAV2 (left).

[0083] FIG. 47: FBVK assay comparing culture conditions for recombinant CAV production. MDCK cells were infected with PCMN-1324 (WT CAV2, El A-P2A-YPet) and cultured under four conditions: DMEM + 10% FBS with no media exchange (top left), MEM + 10% FBS with no media exchange (top right), DMEM + 10% FBS with 50% daily media exchange (bottom left), and MEM + 10% FBS with 50% daily media exchange (bottom right). YPet fluorescence was quantified over time across a range of MOIs. The FBVK plots and log-phase slopes demonstrate that optimal conditions for recombinant CAV replication and high yield are MEM with 10% FBS and 50% daily media exchange (bottom right), which produced the steepest replication slopes and highest peak fluorescence.

[0084] FIG. 48: Example showing how fluorescent reporters can be used for antibody neutralization assays and serum factors.

[0085] FIG. 49: Plasmid map of Core-076 that has a DBP-P2A-YPet fusion, which was assembled to create PCMN-1470.

[0086] FIG. 50: Images from a fluorescence assay following transfection of MDCK cells with PCMN-1470, which has a direct DBP-P2A-YPet fusion. At 3 days post-transfection, some transfected cells were visualized. At day 16, the number of YPet positive cells had increased and fluorescent plaques were observed, demonstrating that direct fusions of YPet to E2A-DBP undergo modest DNA replication and secondary infection and spread, but are attenuated relative to wildtype virus.

[0087] FIG. 51: Plasmid map of E3-538, which expresses mCherry as a fusion with the putative predicted E3-ORF1 at the C-terminus and was assembled to create the PCMN-1363 virus.

[0088] FIG. 52: FBVK assays and plots of MDCK or DK cells infected with PCMN-1363. Log slope and fluorescent expression are shown.

[0089] FIG. 53: Schematic showing an exemplary scarless assembly of a recombinant CAV genome, PCMN-1326.

[0090] FIG. 54: Plasmid map of E3-527, which has an E3-ORF1 deletion.

[0091] FIG. 55: Schematic showing scarless assembly of PCMN-1327 using an E3 modified plasmid module, E3-528, wherein E3-ORF2 coding sequences are deleted. An El plasmid module that expresses an ElA-P2A-YPet fluorescent reporter, a wildtype core and E4 module plasmid were assembled with E3-527 in tlie order shown.

[0092] FIG. 56: Plasmid map of E3-528 which has an E3-ORF2 deletion.

[0093] FIG. 57: Schematic showing scarless assembly of PCMN-1328 using an E3 modified plasmid module, E3-529, wherein E3-ORF1 and E3-ORF2 coding sequences are deleted. An El plasmid module7158-105381-02

[0094] that expresses an ElA-P2A-YPet fluorescent reporter, a wildtype core and E4 module plasmid were assembled with E3-529 in the order shown.

[0095] FIG.58: Plasmid map of E3-529, which has an E3-ORF1 and E3-ORF2 deletion.

[0096] FIG.59: FBVK assay comparing viral replication kinetics of four recombinant CAV genomes (PCMN-1324, PCMN-1326, PCMN-1327, and PCMN-1328) in MDCK cells. YPet fluorescence was quantified over time across a serial dilution of MOIs. The slope of the log-phase growth curve (dashed line) indicates the replication rate for each virus. PCMN-1324 and PCMN-1326 showed comparable replication slopes (~1.34-2.00 and -1.94, respectively), while PCMN-1327 exhibited attenuated replication kinetics (slopes -0.97-1.02) and PCMN-1328 was intermediate (slope -1.93).

[0097] FIG.60: Photographs showing CsCl gradients of PCMN-1324 harvested from DK cells and PCMN-1327 and PCMN-1328 harvested from MDCK cells.

[0098] FIG.61: Exemplary sequence alignment of the E3 region from human adenovirus 5 (Ad5), human adenovirus 34 (Ad34), canine adenovirus 1 (CAV1), and three strains of canine adenovirus 2 (CAV2), including the Toronto strain, G1 strain, and SH-R2 isolate. The alignment is shown relative to the hAd5 sequence. There is significant sequence divergence (white shading of boxes is not conserved, grey is conserved) evident between the canine adenovirus strains and the human adenoviruses in this genomic region. However, a conserved TATA box and promoter element embedded within the pVIII coding sequence is highly conserved and preserved across all strains. These findings suggest that, although not previously characterized, canine adenoviruses likely utilize an embedded internal promoter within the protein VIII gene to initiate the transcription of downstream E3 open reading frame.

[0099] FIG.62: Schematic showing exemplary modifications and engineering of CAV E3 module and replacement and deletion of E3-ORF1 coding sequences after the pVIII stop codon. A ‘G’ nucleotide was engineered and inserted after the pVIII stop codon and at the -1 position of the ectopic ORF, in this example, mCherry, ATG-Methionine initiation codon, creating a Kozak motif. This engineered Kozak sequence enhances translation initiation and efficiency of the ectopic ORF.

[0100] FIG.63: Plasmid map of E3-532, in which E3-ORF1 is replaced with mCherry (AE3-ORF 1 [mCherry] ) .

[0101] FIG.64: Schematic showing an exemplary scarless assembly of a recombinant CAV genome, PCMN-1331.

[0102] FIG.65: FBVK assays and plots of MDCK or DK cells infected with PCMN-1331. Log slope and fluorescent expression are shown.

[0103] FIG.66: Schematic showing an exemplary scar less assembly of a recombinant CAV genome, PCMN-1332.

[0104] FIG.67: Plasmid map of E3-541, which has a deletion of E3-ORF1 and E3-ORF2 (AE3-ORF1 [mCherry], AE3-ORF2).

[0105] FIG.68: Alignment of the CAV2 (top), CAV1 (middle), and PCMN-1507 (bottom) E3-ORF2 regions at the non-coding junction with the U exon. PCMN-1507 is an exemplary recombinant CAV7158-105381-02

[0106] genome expressing CDV-H (canine distemper virus hemagglutinin) in place of E3-ORF1 coding sequences, while preserving the non-coding sequences between E3-ORF1 and E3-ORF2 that may have a regulatory role, together with sequences at the junction with the U exon on the opposing strand.

[0107] Conserved predicted regulatory elements and motifs are annotated and their structural and spatial arrangements visualized, including a polyadenylation (poly A) motif (AAATAAACT), a 5' splice site upstream of the U exon on the negative strand (ACTTACC), and a splice site (AC) in CAV2. These elements are preserved in engineering deletions of E3-ORF1 and E3-ORF2 to express heterologous ORFs. A non-coding remnant between E3-ORF1 and E3-ORF2 is also preserved, which includes a consensus polyA sequence (ATT AAA) that may contribute to the generation of L5 fiber transcripts, as well as splicing sites that could facilitate fiber gene expression. Alternative designs could employ different consensus motifs for polyA signals and splice sites, or delete the E3-ORF1 / E3-ORF2 junction sequences entirely.

[0108] FIGS. 69A-69B: (FIG. 69 A) Images from a fluorescence assay following transfection with PCMN-1366. MDCK cells were transfected with PCMN-1466 whole genome plasmids. At 10 days post transfection, fluorescent plaques were apparent in the MDCK monolayer, indicating productive infection of the recombinant CAV genome. (FIG. 69B) Photograph showing a CsCl gradient of PCMN-1366 harvested from DK cells.

[0109] FIG.70: Comparison of the pVIII and E3-ORF1 junctions in hAd5 (top), CAV1 (middle), and CAV2 (bottom). In hAd5, the initiation codon (Met) for E3-ORF1, encoding the 12.5 kDa protein, occurs immediately downstream of the pVIII stop codon, with no overlap between reading frames. In contrast, in CAV1 and CAV2, E3-ORF1 has predicted overlapping coding sequences with the C-terminus of pVIII: potential alternative start codons, including methionine residues (MAM), are predicted within an alternative reading frame of the pVIII coding sequence. Arrows indicate the predicted initiation codons for E3-ORF1 in each species, highlighting that in CAV1 and CAV2 the predicted E3-ORF1 start codon overlaps the pVIII coding sequence, whereas in hAd5 die E3-ORF1 start codon is located immediately downstream of the pVIII stop codon. The C-terminal amino acid sequence of pVIII is conserved across all three species as "DGYD" (SEQ ID NO: 117); however, the underlying nucleotide sequence is degenerate, allowing for these alternative start codons in CAV1 and CAV2. This overlap could result in N-terminal extensions, in-frame fusions, or aberrant localization (e.g., if signal peptides are inadvertently translated), which are undesirable for expression of a therapeutic ORF and are an important consideration for engineering ectopic gene placements at diis junction.

[0110] FIG.71: Schematic showing synonymous point mutations engineered into the pVIII sequence of E3-572, which incorporates two silent nucleotide substitutions that do not alter the pVIII amino acid sequence but abolish die alternative ATG start codons in die +2 reading frames.

[0111] FIG.72: Plasmid map of E3-572.

[0112] FIG.73: Plasmid map of the PCMN-1428 viral genome.7158-105381-02

[0113] FIG.74: FBVK assay comparing mCherry expression and replication kinetics of PCMN-1366 and PCMN-1428 in MDCK cells. Two viral input dilutions per construct are shown (curves 1-2: PCMN-1366; curves 3-4: PCMN-1428). PCMN-1428 was engineered to remove the predicted upstream alternative start codons at the pVIII / E3-ORFl junction (see FIG. 70), and shows a 3-4 fold increase in mCherry fluorescence intensity compared to PCMN-1366, which retains the cryptic upstream start codons. Both constructs reach comparable plateau fluorescence levels, but PCMN-1428 achieves higher expression earlier, consistent with more efficient translation initiation from the intended start codon. In addition, the mutation on PCMN1428 appears to confer a gain function for viral replication as evidenced by enhanced secondary and tertiary infection and replication kinetics and log slope.

[0114] FIG.75: Western blot analysis of mCherry molecular weight in PCMN-1366 and PCMN-1428 lysates. Lysates from MDCK cells infected with PCMN-1428 or PCMN-1366 were separated by gel electrophoresis, transferred to a membrane and probed for mCherry expression with anti-mCherry antibodies that were detected using fluorescent secondary antibodies and images scanned and analyzed on LiCor Odyssey. A slightly higher mCherry molecular weight band that is shifted by a few kD was observed in PCMN-1366 versus PCMN-1428, consistent with predicted alternative starts in pVIII and N-terminal amino acid additions in mCherry. PCMN-1428 produces mCherry at the expected size, confirming successful elimination of cryptic alternative translation from ATGs in the pVIII sequence.

[0115] FIG.76: FBVK assay comparing YPet cargo expression and viral replication kinetics of PCMN-1326 and PCMN-1386 in DK cells (top) and MDCK cells (bottom). PCMN-1326 carries YPet as an E1A-P2A fusion with E3-ORF1 deleted (AE3-ORF1), while PCMN-1386 expresses YPet from the E3-ORF1 locus (AE3-ORFl[YPet]). YPet fluorescence was quantified over time across a range of MOIs. In both cell types, PCMN-1386 achieved higher peak YPet fluorescence than PCMN-1326, demonstrating that cargo expression from the engineered E3-ORF1 replacement locus is higher than from the El A-P2A fusion placement. Both constructs replicated productively in DK and MDCK cells, with PCMN-1386 also showing faster replication kinetics.

[0116] FIGS. 77A-77B: Plasmid maps of (FIG. 77 A) E3-542 and (FIG. 77B) E3-543.

[0117] FIG.78: DK and MDCK cells were infected with the indicated viruses, PCMN-1367 and PCMN-1368, and analyzed in an FBVK assay for mCherry fluorescence expression levels and replication kinetics.

[0118] FIG.79: Plasmid map of E3-554.

[0119] FIG.80: Images from a fluorescence assay following transfection of MDCK cells with the PCMN-1389 genome. Fluorescence images were captured at 5 and 9 days post infection. The expression of all three heterologous ORFs from the E3-ORF1 deleted replacement was demonstrated by die coexpression of BFP, YPet and mCherry fluorescent proteins in virally infected cells. In addition, by 9 days, robust viral replication is evident, was demonstrated by the spread of fluorescent signal across the entire monolayer, indicating that PCMN-1 89 is replication-competent and capable of productive viral infection.7158-105381-02

[0120] FIG. 81: Fluorescent images of MDCK cells transfected with PCMN-1375 (El A-P2A-YPet, AE3-ORFl[mCherry], AE3-ORF2) virus genomes. Fluorescent images were captured at timepoints indicated.

[0121] FIG. 82: Fluorescent images of MDCK cells transfected with PCMN-1423 (ElB-55k-P2A-YPet; AE3-ORFl[mCherry], AE3-ORF2) virus genome. Fluorescent images were captured at the timepoints indicated.

[0122] FIG. 83: Summary of the impact on CAV replication of fluorescent ORF expression at different genomic placements, and where indicated, when CAVs were assembled with fluorophores at two different genomic placements.

[0123] FIG. 84: Map of the E4-114 plasmid module in which E4-ORF5 coding sequences were deleted.

[0124] FIG. 85: Map of the CMBT-1501 whole genome plasmid, which was assembled from E4-114, E3-541 and ASMM-173D in a multi-site recombination LR reaction.

[0125] FIG. 86: Fluorescent images of cells transfected with CMBT-1501 at 3 and 15 days posttransfection. The expression of die mCherry reporter in the E3-ORF1 placement was clearly observed, demonstrating productive virus replication and spread from initial transfected cells to the entire monolayer by day 15. These data show that multi-site gateway assembly can be used to assemble recombinant CAV genomes with deletions in CAV ORFs to determine which viral genes are dispensable for replication in cell culture. These data show that E4-ORF5, together with E3-ORF1 and E3-ORF2, are not critical for virus replication (although replication is attenuated compared to wildtype virus), at least in cell culture, and can be deleted to create genomic space for therapeutic payloads or heterologous ORFs.

[0126] FIG. 87: Schematic of canine distemper virus (CDV) antigens.

[0127] FIGS. 88A-88B: Plasmid maps of (FIG. 88A) El -285, a modified El module with an E1B-55k-P2A-YPet fluorescent reporter placement and (FIG. 88B) E3-574, a modified E3 module that expresses a heterologous therapeutic ORF, an immunogenic antigen from canine distemper virus (CDV H), instead of E3-ORF1 and has additional deletion of E3-ORF2 coding sequence. The El and E3 modified plasmids were assembled with a core-74D plasmid and wildtype E4 plasmid in an LR reaction to create the whole genome plasmid CMBT-1460.

[0128] FIG. 89: Map of the Core-074 Dest plasmid.

[0129] FIG. 90: Fluorescent images of MDCK cells transfected with CMBT-1460 virus genome at 6 and 12 days post- transfection. At 6 days post- transfection, virally infected YPet fluorescent expressing cells and plaques were visible, and viral replication and spread was demonstrated by the increase in YPet fluorescence and infected cells and destruction of the entire monolayer by day 12.

[0130] FIG. 91: Fluorescent images of MDCK cells transfected with CMBT-1460 virus genome at 6 and 12 days post- transfection. At 6 days post- transfection, YPet fluorescent expressing cells and plaques were visible, which replicated and spread to infect the entire monolayer by day 12.

[0131] FIGS. 92A-92B: Brightfield and fluorescence imaging of MDCK cells transfected with (FIG.

[0132] 92A) PCMN-1513 or (FIG. 92B) PCMN-1510 expressing CDVH and mCherry reporter. PCMN-15137158-105381-02

[0133] expresses the CDVH antigen as an E1A-P2A fusion. PCMN-1510 expresses CDVH as an E1B-55K-P2A fusion. Shown are representative brightfield and fluorescence microscopy images of MDCK cells at 4 and 11 days post- transfection. Both viruses express mCherry from the E3-ORF1 locus, enabling visualization of virus-infected cells, productive replication and spread. At 4 days, small fluorescent foci were visible. By 11 days, a significant increase in fluorescently labeled cells and associated virus induced cytopathic effect were observed, indicating robust viral replication and spread in both constructs. These results demonstrate that CDVH does not inhibit viral replication, infection or spread.

[0134] FIGS. 93A-93B: Brightfield and fluorescence imaging of MDCK cells transfected with (FIG.

[0135] 93A) PCMN-1514 or (FIG. 93B) PCMN-1511, which express CDVF and an mCherry fluorescent reporter. (FIG. 93A) PCMN-1514 expresses die CDVF protein as an E1A-P2A fusion and mCherry fluorescent reporter from the E3-ORF1 locus. Shown are representative brightfield and fluorescent images of MDCK cells transfected witii PCMN-1514. Fluorescent foci were visible at 4 days, with moderate expansion by day 11, indicating moderate viral replication compared to equivalent constructs expressing CDVH. (FIG. 93B) PCMN-1511 expresses CDVF as an E1B-55K-P2A fusion and mCherry is expressed from the E3-ORF1 locus. Fluorescent foci were observed at 4 days post-infection, and by day 11 , robust spread and increase in number of mCherry labeled fluorescent viral infected cells were apparent, indicating strong viral replication.

[0136] FIG. 94: Fluorescence imaging of MDCK cells transfected with recombinant CAV CMBT-1459 expressing CDVF from the AE3-ORF1 / AE3-ORF2 placement and an ElB-55K-P2A-YPet reporter. At day 4 after transfection of CMBT-1459, initially transfected fluorescent cells were observed. However, by day 11, there was a minimal increase in the number of fluorescent cells, indicating a marked inhibition of productive virus replication and spread. These data indicate that expression of CDVF from the AE3-ORF1 / AE3-ORF2 placement negatively impacts CAV propagation, likely due to fusogenic functions of CDVF at elevated expression levels.

[0137] FIGS. 95A-95B: Brightfield images of MDCK cells transfected with recombinant CAVs expressing either CDVH or CDVF. Brightfield images of MDCK cells transfected with either (FIG. 95A) PCMN-1507 or (FIG. 95B) PCMN-1506, which express CDVH or CDVF, respectively, from the AE3-ORF1 / AE3-ORF2 placement. Initial viral transfected cells and infected plaques were observed at day 4. By 9 days, the MDCK monolayer was almost entirely disrupted in MDCK cells initially transfected with PCMN-1507, consistent with robust viral replication and associated cytopathic effects. In contrast, in PCMN-1506 transfected cells, initial plaques did not expand, and the cell monolayer remained largely intact, indicating that viral spread and replication were significantly impaired.

[0138] FIGS. 96A-96B: Assessment of transgene stability at different genomic placements on serial passaging of recombinant CAV vectors expressing therapeutic ORFs (CDVH or CDVF). (FIG. 96A) Agarose gel electrophoresis of transgene stability from serial passage of PCMN-1506 (left) or PCMN-1507 (right) infected MDCK cells. Viral DNA was isolated and analyzed by PCR to assess retention of the transgene in the viral genome. PCMN-1507 stably maintained the CDVH transgene across 10 serial7158-105381-02

[0139] passages. In contrast, the CDVF transgene was lost from the PCMN-1506 virus population by passage 5, suggesting negative selection at elevated levels of expression. (FIG. 96B) PCR-based assessment of CDVF transgene stability from serial passage in MDCK cells of PCMN-1512 and PCMN-1515, which express CDVF from the E1B-55K-P2A and El A-P2A loci, respectively. In contrast to PCMN-1506, the CDVF transgene was maintained, with no loss by passage 5. These results demonstrate that genomic placement strongly influences transgene stability, and that expression of potentially toxic therapeutic proteins such as CDVF are better tolerated at loci with lower relative expression levels.

[0140] FIG. 97: Expression of a therapeutic transgene fromE3-ORFl with simultaneous deletion of E3-ORF2 and E4-ORF5. Brightfield images of MDCK cells transfected with CMBT-1509, which expresses the therapeutic transgene CDVH from the E3-ORF1 locus and carries deletions of E3-ORF2 and E4-ORF5. Plaques were visible by day 4 post-transfection, and widespread cytopathic effect was observed by day 11, demonstrating robust viral replication and transgene tolerance.

[0141] FIG. 98: Dual therapeutic ORF expression from separate transgene genomic placements in a replication-competent CAV vector. Brightfield images of MDCK cells transfected with PCMN-1519, a recombinant CAV expressing two different therapeutic antigens from distinct genomic loci. The CDV nucleoprotein (NP) is expressed from the E1A-P2A locus, and a second therapeutic protein (CDVH) is expressed from the E3-ORF1 replacement site, with a deletion of E3-ORF2 to increase transgene capacity and expression. At day 4 post- transfection, localized dark regions corresponding to early CAV plaque formation were visible. By day 11, extensive cytopathic effect was observed across the well, confirming robust replication of the dual-antigen vector and complete infection of the monolayer.

[0142] FIG. 99: Schematic showing canine parainfluenza virus (CPiV) antigens.

[0143] FIG. 100: Schematic of an exemplary CAVSLIC assembly of the PCMN-1556 CAV2 CPiV multivalent vaccine genome composition.

[0144] FIG. 101: Map of the E3-595 genomic module plasmid. This E3 module replaces a deleted E3-ORF1 with CPiVF-P2A-CPiVHN and also contains a deletion of E3-ORF2.

[0145] FIG. 102: Plasmid map of the PCMN-1556 CPiV F+HN assembled CAV2 viral vaccine genome.

[0146] FIG. 103: Images showing the replication and cytopathic effects of the PCMN-1556 viral genome in MDCK cells following transfection with the assembled viral genome plasmid. The brightfield microscopy images were taken at 4 and 9 days post-transfection of MDCK cells with the complete CAV vaccine genome plasmid. (Top) At 4 days post- transfection, small clusters (plaque-like) of virus transduced / infected expressing cells were seen. (Bottom) The CPE and cell death (change in refractive index and darker patches) resulting from productive viral replication and spread was apparent in complete destruction of the MDCK monolayer between 4 and 9 days.

[0147] FIG. 104: Immunofluorescence images showing CPiV-HN antigen expression in MDCK cells following infection with PCMN-1556 (CPIF-P2A-CPIHN), alongside a control virus (PCMN-1366, mCherry) and uninfected cells. Cells were fixed 12 hours post-infection and stained with anti-CPiV-HN7158-105381-02

[0148] antibody (CPIV8-4). Fluorescent signal appears as bright cells against a dark background. Top row: DAPI nuclear counterstain, confirming comparable cell density across all conditions. Bottom row: anti-CPiV-HN immunofluorescence. Strong CPiV-HN expression was detected in PCMN-1556-infected cells (left), while neither the control virus PCMN-1366 (center) nor uninfected cells (right) showed signal above background, confirming the specificity of antigen expression from the recombinant CAV platform.

[0149] SEQUENCE LISTING

[0150] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standaid letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0151] SEQ ID NO: 1 is the nucleotide sequence of PCMN-1313.

[0152] SEQ ID NO: 2 is the nucleotide sequence of PCMN-1324.

[0153] SEQ ID NO: 3 is the nucleotide sequence of PCMN-1326.

[0154] SEQ ID NO: 4 is the nucleotide sequence of PCMN-1327.

[0155] SEQ ID NO: 5 is the nucleotide sequence of PCMN-1328.

[0156] SEQ ID NO: 6 is the nucleotide sequence of PCMN-1331.

[0157] SEQ ID NO: 7 is the nucleotide sequence of PCMN-1332.

[0158] SEQ ID NO: 8 is the nucleotide sequence of PCMN-1363.

[0159] SEQ ID NO: 9 is the nucleotide sequence of PCMN-1366.

[0160] SEQ ID NO: 10 is the nucleotide sequence of PCMN-1367.

[0161] SEQ ID NO: 11 is the nucleotide sequence of PCMN-1368.

[0162] SEQ ID NO: 12 is the nucleotide sequence of PCMN-1375.

[0163] SEQ ID NO: 13 is the nucleotide sequence of PCMN-1384.

[0164] SEQ ID NO: 14 is the nucleotide sequence of PCMN-1386.

[0165] SEQ ID NO: 15 is the nucleotide sequence of PCMN-1389.

[0166] SEQ ID NO: 16 is the nucleotide sequence of PCMN-1422.

[0167] SEQ ID NO: 17 is the nucleotide sequence of PCMN-1423.

[0168] SEQ ID NO: 18 is the nucleotide sequence of PCMN-1428.

[0169] SEQ ID NO: 19 is the nucleotide sequence of PCMN-1466.

[0170] SEQ ID NO: 20 is the nucleotide sequence of PCMN-1467.

[0171] SEQ ID NO: 21 is the nucleotide sequence of PCMN-1468.

[0172] SEQ ID NO: 22 is the nucleotide sequence of PCMN-1469.

[0173] SEQ ID NO: 23 is the nucleotide sequence of PCMN-1470.

[0174] SEQ ID NO: 24 is the nucleotide sequence of PCMN-1506.

[0175] SEQ ID NO: 25 is the nucleotide sequence of PCMN-1507.7158-105381-02

[0176] SEQ ID NO 26 is the nucleotide sequence of PCMN-1510.

[0177] SEQ ID NO 27 is the nucleotide sequence of PCMN-1511.

[0178] SEQ ID NO 28 is the nucleotide sequence of PCMN-1512.

[0179] SEQ ID NO 29 is the nucleotide sequence of PCMN-1513.

[0180] SEQ ID NO 30 is the nucleotide sequence of PCMN-1514.

[0181] SEQ ID NO 31 is the nucleotide sequence of PCMN-1515.

[0182] SEQ ID NO 32 is the nucleotide sequence of PCMN-1519.

[0183] SEQ ID NO 33 is the nucleotide sequence of PCMN-1556.

[0184] SEQ ID NO 34 is the nucleotide sequence of PCMN-1584.

[0185] SEQ ID NO 35 is the nucleotide sequence of CMBT-1459.

[0186] SEQ ID NO 36 is the nucleotide sequence of CMBT-1460.

[0187] SEQ ID NO 37 is the nucleotide sequence of CMBT-1501.

[0188] SEQ ID NO 38 is the nucleotide sequence of CMBT-1509.

[0189] SEQ ID NO 39 is the nucleotide sequence of the ASMM-149 macromodule plasmid.

[0190] SEQ ID NO 40 is the nucleotide sequence of the ASMM-150 macromodule plasmid.

[0191] SEQ ID NO 41 is the nucleotide sequence of the ASMM-176 macromodule plasmid.

[0192] SEQ ID NO 42 is the nucleotide sequence of the El-269 plasmid module.

[0193] SEQ ID NO 43 is the nucleotide sequence of the El-271 plasmid module.

[0194] SEQ ID NO 44 is the nucleotide sequence of the El-285 plasmid module.

[0195] SEQ ID NO 45 is the nucleotide sequence of PCMN-1312.

[0196] SEQ ID NO 46 is the nucleotide sequence of the E3-522 plasmid module.

[0197] SEQ ID NO 47 is the nucleotide sequence of the E3-527 plasmid module.

[0198] SEQ ID NO 48 is the nucleotide sequence of the E3-528 plasmid module.

[0199] SEQ ID NO 49 is the nucleotide sequence of the E3-529 plasmid module.

[0200] SEQ ID NO 50 is the nucleotide sequence of the E3-532 plasmid module.

[0201] SEQ ID NO 51 is the nucleotide sequence of the E3-538 plasmid module.

[0202] SEQ ID NO 52 is the nucleotide sequence of the E3-541 plasmid module.

[0203] SEQ ID NO 53 is the nucleotide sequence of the E3-542 plasmid module.

[0204] SEQ ID NO 54 is the nucleotide sequence of the E3-543 plasmid module.

[0205] SEQ ID NO 55 is the nucleotide sequence of the E3-554 plasmid module.

[0206] SEQ ID NO 56 is the nucleotide sequence of the E3-572 plasmid module.

[0207] SEQ ID NO 57 is the nucleotide sequence of the E3-574 plasmid module.

[0208] SEQ ID NO 58 is die nucleotide sequence of the E3-595 plasmid module.

[0209] SEQ ID NO 59 is die nucleotide sequence of the E3-596 plasmid module.

[0210] SEQ ID NO 60 is die nucleotide sequence of tire E3-612 plasmid module.

[0211] SEQ ID NO 61 is the nucleotide sequence of the Core-059 plasmid module.

[0212] SEQ ID NO 62 is the nucleotide sequence of the Core-065 plasmid module.7158-105381-02

[0213] SEQ ID NO: 63 is the nucleotide sequence of the Core-74D plasmid module.

[0214] SEQ ID NO: 64 is the nucleotide sequence of the Core-076 plasmid module.

[0215] SEQ ID NO: 65 is the nucleotide sequence of the E4-099 plasmid module.

[0216] SEQ ID NO: 66 is the nucleotide sequence of the E4-114 plasmid module.

[0217] SEQ ID NO: 67 is an amino acid sequence of CPiVF and P2A.

[0218] SEQ ID NO: 68 is a nucleic acid sequence encoding CPiVF and P2A.

[0219] SEQ ID NO: 69 is an amino acid sequence of CPiVHN and the final proline of P2A.

[0220] SEQ ID NO: 70 is a nucleic acid sequence encoding CPiVHN and final codon of P2A.

[0221] SEQ ID NO: 71 is a nucleic acid sequence of the PCMN-1556 E3 region.

[0222] SEQ ID NOs: 72 and 73 are nucleic acid sequences of modified E4 regions.

[0223] SEQ ID NOs: 74-106 are nucleic acid primer sequences.

[0224] SEQ ID NOs: 107-114 are 2A peptide sequences and variants thereof.

[0225] SEQ ID NO: 115 is the amino acid sequence of a P2A functional motif.

[0226] SEQ ID NOs: 116-117 are peptide sequences.

[0227] DETAILED DESCRIPTION

[0228] I. Abbreviations

[0229] Ad adenovirus

[0230] ARF alternative reading frame

[0231] BFP blue fluorescent protein

[0232] CAV canine adenovirus

[0233] CDV canine distemper virus

[0234] CDVF canine distemper virus fusion protein

[0235] CDVH canine distemper virus hemagglutinin protein

[0236] CDVNP canine distemper virus nucleoprotein

[0237] CPiV canine parainfluenza virus

[0238] CPiVF canine parainfluenza virus fusion protein

[0239] CPiVHN canine parainfluenza virus hemagglutinin-neuraminidase protein DBP DNA binding protein

[0240] FBVK fluorescence-based viral kinetics

[0241] hAd human adenovirus

[0242] HCS high-content screening

[0243] HTP high-throughput

[0244] ITR inverted terminal repeat

[0245] MDCK Madin-Darby canine kidney

[0246] MLP major late promoter

[0247] MRI magnetic resonance imaging7158-105381-02

[0248] ORF open reading frame

[0249] ori origin of replication

[0250] PET positron emission tomography

[0251] SLIC sequence- and ligation-independent cloning

[0252] SSR site-specific recombination

[0253] II. Terms and Methods

[0254] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a modification” includes singular or plural modifications and can be considered equivalent to the phrase “at least one modification.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar- or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0255] 2A peptide: A type of self-cleaving peptide encoded by some RNA viruses, such as picornaviruses. 2A peptides function by making the ribosome skip the synthesis of a peptide bond at the C -terminus of a 2A element, leading to separation between the end of the 2A sequence and the downstream peptide (Kim et al., PLoS One 6(4):el8556, 2011). The "cleavage" occurs between the glycine and proline residues found on the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, the 2 A peptides encoded by Thosea asigna virus (TaV; T2A), equine rhinitis A virus (ERAV; E2A), porcine teschovirus-1 (PTV1; P2A) and foot and mouth disease virus (FMDV; F2A), having tire following sequences:

[0256] P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 107)

[0257] F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 108)

[0258] E2A: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 109)

[0259] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 110)

[0260] In some examples, the 2A peptide is modified to include Gly-Ser-Gly at tire N-terminus to improve cleavage efficiency. The sequences of modified P2A, F2A, E2A and T2A are provided below:

[0261] Modified P2A: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 111)

[0262] Modified F2A: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 112)

[0263] Modified E2 A: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 113)7158-105381-02

[0264] Modified T2A: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 114)

[0265] Antigenic protein: A protein that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.

[0266] Att sites: Site-specific recombination sequences for the multi-site Gateway recombination method. There are four classes of att sites called attB, attP, attL, and attR. attB sites react with attP in a BP clonase reaction, generating attL and attR sites. AttL and attR sites recombine with each other in an LR reaction, regenerating attB and attP sites.

[0267] Canine adenovirus (CAV): A type of virus with a double-stranded linear DNA genome belonging to the genus Mastadenovirus, family Adenoviridae. There are two types of canine adenoviruses - CAV-1 and CAV-2, which cause canine hepatitis and infectious tracheobronchitis, respectively, in canines.

[0268] Canine distemper virus (CDV): A single-stranded RNA virus of the family Paramyxoviridae. CDV is a highly contagious and serious disease that affects the respiratory, gastrointestinal and nervous systems of puppies and dogs. CDV is also found in wildlife, including foxes, wolves, coyotes, raccoons, skunks, mink and ferrets. Infected animals typically develop a watery or pus-like discharge from their eyes, which can be followed by fever, nasal discharge, coughing, lethargy, reduced appetite, and vomiting. If the virus spreads to the nervous system, animals can exhibit circling behavior, head tilt, muscle twitches, convulsions, seizures and / or paralysis. CDV infection can also lead to a thickening or hardening of the footpads. Immunogenic proteins of CDV include, but are not limited to, the CDV fusion protein (CDVF), the CDV hemagglutinin protein (CDVH) and the CDV nucleoprotein (CDVNP).

[0269] Canine parainfluenza virus (CPiV): An enveloped, non-segmented, negative-sense RNA virus of the Paramyxoviridae family. CPiV is a highly contagious virus that causes canine infectious respiratory disease complex (CIRDC). Immunogenic proteins of CPiV include, but are not limited to, the CPiV fusion protein (CPiVF) and the CPiV hemagglutinin-neuraminidase protein (CPiVHN).

[0270] CAV core genomic module: A portion of the CAV genome that includes the early / intermediate genes, pIX and IV A2, the major late promoter (MLP) and transcriptional unit that drives the expression of CAV capsid and late proteins, including: 52 / 55k, pllla, penton, pVII, pV, pX, pVI, protease, hexon, 100k, 22k, and 33k (putative) on the positive strand. On the negative strand, the CAV E2 transcriptional unit drives the expression of the viral DNA replication proteins, including E2A DBP, pTP and E2B DNA polymerase (see FIGS. 7 and 9).

[0271] CAV core plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that extends 5’ from the early protein IX to the putative L4-33K gene on the positive strand and from the 3’ direction includes the E2 transcriptional unit and E2 encoded proteins E2A, E2B polymerase and terminal protein genes on the negative strand (see FIG. 6). This region of the genome includes the following ORFs: IX, IVa2, DNA pol, pTP, 52K, pllla, penton, pVII, V, pX, pVI, hexon, protease, DNA7158-105381-02

[0272] binding protein (DBP), 100K, 22K and 33K (22k and 33k are putative). Most of the genes in this module are responsible for viral DNA replication and assembly into a protein capsid.

[0273] CAV El genomic module: A portion of the CAV genome that spans from the left-hand internal terminal repeat (ITR) through the E1B-55K gene and the TATA box for pIX (see FIGS. 7-8). The E1A and E1B gene products encoded within this module are essential for viral replication. These genes can be deleted for the assembly and production of recombinant non-replicating CAV vectors.

[0274] CAV El plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes the left-hand inverted terminal repeat (ITR), and the El A, ElB-19k and ElB-55k ORFs and E1A and E1B transcriptional regulatory sequences and units (see FIGS. 7 and 8). The El genes and promoters may be partially or wholly deleted, mutated, or replaced with heterologous open reading frames (ORFs) to generate replication-incompetent viral vectors for gene delivery, gene therapy and vaccines.

[0275] CAV E3 genomic module: A portion of the CAV genome that includes the MLP transcribed late protein pVIII, tire E3 transcriptional unit and protein coding genes (E3-ORF1 and E3-ORF2), and the fiber gene on the positive strand, and the U exon on the negative strand (see FIGS. 7 and 10).

[0276] CAV E3 plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes pVIII, the E3 promoter and genes, the U-exon, and the fiber gene. This region includes the following ORFs: pVIII, E3-ORF1, E3-ORF2, U exon, and Fiber. pVIII is predicted to be transcribed from the major late promoter. An E3 promoter element is predicted to be embedded within the pVIII coding sequence. Also predicted are E3-ORF1 alternative ATG-methionine start codons at the end of pVIII gene in an alternative reading frame. The E3 genes can be partially or completely deleted to expand packaging capacity, regulate host immune responses, and / or encode heterologous genes and are not essential for virus replication in most adenoviruses.

[0277] CAV E4 genomic module: A portion of the CAV genome that includes the right ITR and the E4 transcriptional unit and protein coding genes on the negative strand. The E4 genes have important functions in usurping host cell signaling pathways and function to facilitate viral replication in human Ads and likely canine Ads. In CAV, there are five predicted and annotated ORFs, which include from right to left, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5 (see FIGS. 7 and 11).

[0278] CAV E4 plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes the right hand ITR, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5.

[0279] CAVsembly: A ‘Multisite Gateway’ based genome assembly method. In some aspects of this assembly method, the CAV core module is cloned into a plasmid with ampicillin resistance and made into a Gateway-compatible destination (DEST) plasmid whereby the CAV core genome module is flanked on the left and / or right with multisite Gateway cassettes that have site-specific recombination sequences (att sites) and ccdB counterselection cassettes. The El, E3, and E4 genome modules are cloned into specialized Multisite Gateway compatible entry plasmids that can be combined with, for example, a “dual-DEST” core module in a standard Multisite Gateway LR reaction. The resultant7158-105381-02

[0280] reaction contains fully assembled CAV genomes. Assembled CAV genomes can then be transformed into bacteria and correct assemblies selected by virtue of ampicillin resistance and successful recombination and replacement of ccdB with CAV genome modules. The virus genome can also be transfected into canine cells as only correctly assembled CAV genomes are replication-competent.

[0281] CAVSLIC: An assembly method of CAV genome module plasmids that uses scarless and sequence ligation independent cloning (SLIC) based assembly of El, E3 and E4 modules to create a complete CAV genome in two or three steps. In some aspects of this assembly method, the CAV core genome module plasmid is engineered to have unique restriction sites engineered at tire left and right hand sides. The core module is linearized by restriction enzyme digestion and assembled with a PCR linearized E3 and E4 module via SLIC / Gibson, generating a macromodule plasmid. Following transformation and selection of core-E3-E4 macromodule plasmids, the El module is added by linearizing it with restriction enzyme digestion and then adding an El module using PCR and then assembly via Gibson / SLIC. This generates fully assembled viral genomes.

[0282] Degenerate variant: A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0283] Deletion: An adenovirus genome that has a “deletion” of an adenovirus ORF (such as an E3-ORF1 or E3-ORF2 deletion) refers to an adenovirus having a complete deletion of the ORF, or a partial deletion of the ORF that results in the absence of expression of a protein sequence encoded by the ORF. In some aspects herein, E3-ORF1 and / or E3-ORF2 are completely deleted. In other aspects, E3-ORF1 and / or E3-ORF2 are partially deleted, such as 50%, 60%, 70%, 80%, 90%, 95% or 99% deleted.

[0284] Embedded gene promoter: A regulatory DNA sequence capable of initiating transcription that is located within the coding region of another gene. This overlapping genetic architecture enables the regulation of an alternative transcriptional unit while existing within the sequence of a distinct, independently transcribed gene. Such embedded promoters may be naturally occurring, facilitating the expression of alternative or overlapping transcripts, and may be exploited or synthetically engineered to enable co-regulation of heterologous genes with applications in gene therapy, vaccines, recombinant protein expression, and viral vector systems.

[0285] Expression timing: Refers to the temporal profile of transgene expression within tire context of CAV infection, replication, or transduction. Expression timing may be early, intermediate, or late relative to the viral life cycle, and is influenced by the genomic locus placement, CAV genomic module, transcriptional and posttranscriptional regulation. For example, transgenes inserted as P2A fusions to early genes, such as El A, are expressed soon after infection, whereas P2A fusions to late genes, such as pVIII late, may occur only after genome replication. Expression timing affects transgene functionality, immunogenicity, toxicity, efficacy and potential impact on viral replication, tolerability, stability and / or host response.7158-105381-02

[0286] Fluorescent protein: A protein that emits light of a certain wavelength when exposed to a particular wavelength of light. Fluorescent proteins include, but are not limited to, green fluorescent proteins (such as YPet, GFP, EGFP, AcGFPl , Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP and ZsGreen), blue fluorescent proteins (such as EBFP, EBFP2, Sapphire, T-Sapphire, Azurite and mTagBFP), cyan fluorescent proteins (such as ECFP, mECFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTurquoise and mTFPl), yellow fluorescent proteins (EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl and mBanana), orange fluorescent proteins (Kusabira Orange, Kusabira Orange2, mOrange, mOrange2 and mTangerine), red fluorescent proteins (mRuby, mApple, mStrawberry, AsRed2, mRFPl, JRed, mCherry, Katushka, HcRedl, mRaspbeny, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, tdTomato and E2-Crimson), orange / red fluorescence proteins (dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (Tl) and DsRed-Monomer) and modified versions thereof. In specific examples herein, the fluorescent protein is YPet, mCherry, Katushka, or blue fluorescent protein (BFP).

[0287] Frame-dependent synonymous mutation: A nucleotide substitution occurring within a single open reading frame (ORF) where the mutation is synonymous, preserving the amino acid sequence in that frame, while simultaneously inducing a non-synonymous mutation in an overlapping ORF, resulting in an amino acid substitution or functional modification in the alternate reading frame. Such mutations leverage the degeneracy of the genetic code to engineer precise genetic modifications in overlapping coding regions, allowing for targeted alteration of one protein sequence while maintaining functional integrity of another in dual-coding sequences.

[0288] Fusion protein: A protein containing amino acid sequence from at least two different (heterologous) proteins or peptides. Fusion proteins can be generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons. Fusion proteins, particularly short fusion proteins, can also be generated by chemical synthesis.

[0289] Gateway® cloning: A cloning method based on the site-specific recombination (SSR) system used by phage A. to integrate its DNA into bacterial chromosomes. This method relies on specific recombination (“atf ’) sites, which can be 25-242 base pairs. In this mediod, a gene of interest is cloned into an “entry vector” using the BP reaction. The gene of interest is then subcloned from the entry vector into a “destination vector” using the LR reaction. The BP clonase enzyme reaction recombines attB sites (flanking the gene of interest) with attP sites (in the donor vector), which generates attL sites. The LR reaction is a recombination reaction between attL and attR sites. See, for example, Reece-Hoyes and Walhout (Cold Spring Harb Protoc, January 2, 2018, pdb.top094912), Katzen (Expert Opin. Drug Discov. 2, 571-589, 2007) and Hartley (Curr Protoc Protein Sci, Chapter 5, unit 5.17, February 2003).

[0290] Genomic cargo space: Refers to the amount of foreign genetic material (e.g., transgene, regulatory element, or reporter gene) that can be inserted into a viral genome without impairing vector7158-105381-02

[0291] viability, packaging efficiency, or replication. The total cargo capacity is determined by die structural constraints of die viral capsid and genomic packaging mechanisms, and may be increased by deleting non-essential viral genes or regulatory regions. Payload capacity influences the number, size, and complexity of therapeutic or reporter sequences that can be encoded in a recombinant viral vector. Genomic cargo space is also referred to as “payload.”

[0292] Genomic insertion site, locus or placement: A specific, defined position in die CAV genome, including a protein coding or intergenic region, diat can accommodate the insertion, deletion, or modification of genetic elements such as transgenes, reporter genes, therapeutic genes, alternative start codons, or Kozak elements. The placements can be 5’ or 3’ to a gene start or stop codon, in die same frame or alternative reading frame. A locus may include a native genomic region, an engineered variant thereof, or a synthetic sequence, regulatory motif derived from, or by replacing and modifying native elements. A locus may consist of wholly endogenous sequences, wholly exogenous sequences, or a combination of both (e.g., partially deleted native coding regions flanked by exogenous regulatory elements or self-cleaving peptides). Different loci may be associated with unique expression profiles, timing, tolerability of transgenes, and effects on vector replication or stability.

[0293] Gibson assembly: A sequence-independent, seamless DNA assembly method that enables the joining of one or more linear DNA fragments in a single reaction. The process is based on the coordinated use of enzymatic activities, including exonuclease, DNA polymerase, and DNA ligase, to facilitate the precise and scarless assembly of DNA molecules. A 5’ exonuclease digests DNA ends, creating single-stranded complementary overhangs. Overlapping regions hybridize via complementary base pairing. A high-fidelity DNA polymerase extends and synthesizes missing nucleotides. A DNA ligase enzyme covalently seals the assembled DNA fragments into a contiguous molecule. Gibson assembly is described in Gibson et al. (Nat Methods 6:343-345, 2009).

[0294] Heterologous: A heterologous protein or polypeptide refers to a protein or polypeptide derived from a different source or species.

[0295] High-content: Refers to the acquisition and analysis of multi-parametric data from biological samples, typically using image-based methods. High-content platforms capture spatial, morphological, and intensity-based information from cells or tissues at the single-cell level, enabling detailed analysis of phenotypes, transgene expression patterns, reporter localization, cytopathic effects, or viral plaque formation. High-content analysis is typically performed using automated imaging systems and software-driven quantification.

[0296] High-throughput: Refers to the ability to perform a large number of experimental conditions or measurements in parallel, often in multi-well plate formats such as 96-, 384-, or 1536-well plates. High-throughput systems enable rapid, scalable quantification of readouts such as fluorescence, luminescence, absorbance, or gene expression using automated equipment. High-throughput methods are commonly used for screening libraries of viral vectors, neutralizing antibodies, or drug candidates.7158-105381-02

[0297] I-Scel: A member of a class of restriction enzymes with a very rare-cutting endonuclease recognition site, which are characterized by a recognition sequence of at least 12 base pairs in length, and in some instances 15-22 base pairs or longer. As such, the recognition site occurs infrequently in natural genomes, thereby enabling site-specific cleavage, linearization, or recombination with minimal off-target activity. Other examples of this class of enzymes with long recognition sites and that could replace I-Scel in the compositions disclosed herein include: I-Ceul, I-Ppol, I-Crel, PI-PspI, Pl-Scel, and other homing endonucleases (e.g., LAGLID ADG and GIY-YIG families). In the context of the present disclosure, a “rare-cutting restriction endonuclease” refers to any of the class of enzymes listed above, including, for example, I-Scel, I-Ceul, I-Ppol, I-Crel, PI-PspI, Pl-Scel, or any other homing endonucleases, such as one from the LAGLID ADG or GIY-YIG family.

[0298] Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, virus or cell) has been substantially separated or purified away from other biological components in the cell or tissue of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include those purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

[0299] Live replication competent virus vaccine: A genetically engineered or attenuated virus that retains the ability to undergo full replication within host cells, in the absence of providing ectopically expressed viral genes, and mimicking the infection dynamics, kinetics and viral particles produced by a wild-type virus while being designed to induce a robust and protective immune response without causing disease. These vaccines enable viral genome replication, transcription, and translation, leading to antigen expression of virally encoded and any genetically engineered heterologous encoded proteins from other pathogens, immune system activation, and durable immunity through both humoral and cellular responses. Replication-competent viral vaccines enhance antigen presentation and elicit stronger immune memory compared to inactivated or subunit vaccines.

[0300] This differs from a non-replicating viral vector, which is genetically modified to lack essential replication genes, thereby providing payload capacity to express heterologous gene expression cassettes that may impair ability to undergo full viral replication analogous to wild type virus in host cells. Nonreplicating vectors require higher doses or booster administrations to achieve sustained immunity, whereas replication-competent vaccines more closely mimic natural infection dynamics, providing enhanced immunogenicity through self-amplifying antigen expression.

[0301] Replication-competent live viral vaccines may be naturally attenuated, recombinant, or codon-modified to balance safety, immunogenicity, and transmission control, making them suitable for mucosal delivery, vector-based vaccine platforms, and infectious disease prevention strategies.7158-105381-02

[0302] Macromodule: A nucleic acid molecule that contains at least two CAV genome modules, such as the El module combined with the core module (El-core macromodule), or the El module combined with both the core module and the E4 module (El-core-E4 macromodule).

[0303] Modification: A change in the sequence of a nucleic acid or protein sequence. Amino acid sequence modifications include, for example, substitutions, insertions and deletions, or combinations thereof. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Deletions are characterized by tire removal of one or more amino acid residues from tire protein sequence (or the removal of one or more nucleotides / ribonucleotides from a nucleic acid sequence). In some aspects herein, the modification (such as a substitution, insertion or deletion) results in a change in function, such as a reduction or enhancement of a particular activity of a protein. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final mutant sequence. These modifications can be prepared by modification of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the modification. Techniques for making insertion, deletion and substitution mutations at predetermined sites in DNA having a known sequence are known. A “modified” protein, nucleic acid or virus is one that has one or more modifications as outlined above.

[0304] Modular assembly: A method or system for constructing a nucleic acid sequence (e.g., a viral genome) from discrete, interchangeable, and pre-defined CAV genomic plasmids (“modules”) that are designed to be functionally and physically compatible with one another. Each module includes a distinct CAV genomic region — that can be independently modified, inserted, or exchanged, to assemble a recombinant genome without requiring redesign of the entire genome.

[0305] Module: One of several parts that is made separately but can be joined with other parts to build a structure. In the context of the present disclosure, a CAV “genomic module” is a defined portion of the CAV genome (see FIG. 7) and a CAV “plasmid module” is a nucleic acid molecule that corresponds to a portion of a CAV genome that is engineered and designed with defined boundaries and standardized interfaces (e.g., overhangs, recombination sites, homology arms, restriction sites) to enable seamless integration and / or recombination with other modules using a common framework to assemble a whole genome structure. The CAV plasmid modules disclosed herein include the El module, tire core module, the E3 module and the E4 module. The assembly of all four genomic plasmid modules produces a complete CAV genome.

[0306] Open reading frame (ORF): A span of DNA sequence that begins with a start codon and ends widi a stop codon. In the context of tire present disclosure, an open reading frame (ORF) sequence refers to a contiguous nucleotide sequence within a viral genome that initiates with a start codon (typically AUG), extends uninterrupted by in-frame stop codons, and encodes a functional viral polypeptide. ORF sequences in viral genomes may be arranged as monocistronic or polycistronic transcripts, with some7158-105381-02

[0307] ORFs overlapping within alternative reading frames, allowing for the compact encoding of multiple viral proteins from a constrained genomic region.

[0308] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0309] Overlapping open reading frame: In the context of the present disclosure, an alternative overlapping reading frame refers to a nucleotide sequence within a genetic element that is encoded in a different reading frame relative to a primary coding sequence and is capable of directing tire translation of a distinct polypeptide. Such alternative ORFs may be located within the same nucleic acid strand but shifted by +1 or -1 nucleotides relative to tire primary ORF, or may exist as dual-coding sequences where a single genomic region encodes functionally distinct proteins depending on the frame utilized during translation. These overlapping reading frames may be endogenous viral ORFs or may be engineered to allow for co-expression of multiple or individual proteins within a compact complex genetic region. The presence of an alternative ORF can contribute to regulatory complexity, functional diversity, and optimization of genetic constructs for use in therapeutics, synthetic biology, vaccine development, and recombinant expression systems.

[0310] Plate reader-based detection method: An optical measurement system that quantifies fluorescence, luminescence, or absorbance from each well of a multi-well plate (e.g., 96-, 384-, or 1536-well formats) without capturing spatial image data. These systems provide endpoint or kinetic measurements of bulk signal intensity, typically using photomultiplier tubes or CCD-based detection, and are suitable for high-throughput screening (HTS) applications where signal intensity is the primary readout. Examples include fluorescence plate readers such as the Tecan Infinite, BioTek Synergy, or Molecular Devices SpectraMax platforms.

[0311] Polyadenylation sequence (poly A): The poly (A) consensus sequence refers to a conserved nucleotide motif found near the 3’ untranslated region (3’ UTR) of mRNAs. This sequence is recognized by the cleavage and polyadenylation machinery, leading to the addition of a poly(A) tail, which is important for mRNA stability, nuclear export, and translation efficiency. Poly(A) consensus motifs include the canonical sequence AAUAAA and the non-canonical sequences AUUAAA (most common alternative), AGUAAA, UAUAAA, AAGAAA, and AACAAA.

[0312] Polycistronic: Refers to an mRNA that encodes two or more proteins. Polycistronic generally refers to an mRNA containing multiple genes whose expression is controlled by a single promoter and single terminator.

[0313] Polypeptide, peptide or protein: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the7158-105381-02

[0314] L-optical isomer or the D-optical isomer can be used. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein. These terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0315] A conservative substitution in a polypeptide is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on tire activity of a resulting polypeptide. For example, a protein or peptide including one or more conservative substitutions (for example no more than 1, 2, 3, 4 or 5 substitutions) retains the structure and function of the wild-type protein or peptide. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be readily selected by testing antibody crossreactivity or its ability to induce an immune response. Examples of conservative substitutions are shown below.

[0316] Original Residue Conservative Substitutions

[0317] Ala Ser

[0318] Arg Lys

[0319] Asn Gin, His

[0320] Asp Glu

[0321] Cys Ser

[0322] Gin Asn

[0323] Glu Asp

[0324] His Asn; Gin

[0325] He Leu, Vai

[0326] Leu He ; Vai

[0327] Lys Arg ; Gin ; Glu

[0328] Met Leu ; He

[0329] Phe Met; Leu; Tyr

[0330] Ser Thr

[0331] Thr Ser

[0332] Trp Tyr

[0333] Tyr Trp ; Phe

[0334] Vai He ; Leu7158-105381-02

[0335] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0336] The substitutions that in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

[0337] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter includes necessary nucleic acid sequences near the start site of transcription. Typically, promoters are located near the genes they transcribe. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor or tetracycline). A “tissue-specific promoter” is a promoter that is only active in a particular tissue or cell (e.g., a musclespecific promoter or a liver-specific promoter).

[0338] Recombinant: When used with reference, e.g., to a cell, virus, nucleic acid, protein, or vector, indicates that the cell, virus, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0339] Replication-competent genome: A recombinant or synthetic viral genome that, when introduced into a permissive host cell, is capable of undergoing a complete primary and secondary viral replication cycle, including genome transcription, protein translation, genome replication, capsid assembly, and production of infectious progeny virions that infect surrounding cells, at levels that are comparable to or within an acceptable threshold (e.g., >50-100%) of wild-type virus replication kinetics and / or that do not require a complementing cell line or helper plasmids to be propagated.

[0340] Scalable genome assembly and screening platform: “Scalable,” as used herein, refers to the capacity of the disclosed genome assembly platform, system, or method to support the efficient construction, modification, and evaluation of multiple viral genomes — individually or in parallel — using a modular and systematic approach. A scalable system is compatible with the incorporation of distinct genome modules or libraries of genetic elements, enabling high-throughput generation of recombinant7158-105381-02

[0341] viral constructs without requiring one-at-a-time designs and assembly for each variant to be tested.

[0342] Scalability includes the ability to adapt the assembly and screening process to varying numbers of constructs, such as precision and scarless assembly of individual viruses and high content imaging assays and screening of large numbers of constructs using recombination based assembly and high throughput platforms, genome sizes within and between Ad species, or configurations without a loss of efficiency, fidelity, or reproducibility.

[0343] Scarless cloning: Cloning methods that do not leave recombination sites or other heterologous nucleotides in the final product. Scarless cloning is also called seamless cloning. Scarless cloning methods include, for example, sequence- and ligation-independent cloning (SLIC) and Gibson assembly. Scarless assembly enzyme site refers to a recognition sequence for an enzyme that enables the joining of DNA fragments without leaving any unintended residual nucleotides (“scar s”) at the junction.

[0344] Self-cleaving peptides: Peptides that induce the ribosome to skip the synthesis of a peptide bond at the C-terminus, leading to separation of tire peptide sequence and a downstream polypeptide. Virally encoded 2A peptides are a type of self-cleaving peptide. Virally encoded 2A peptides include, for example, 2A peptides from porcine teschovirus-1 (PTV1), foot and mouth disease virus (FMDV), equine rhinitis A vims (ERAV) and Thosea asigna virus (TaV).

[0345] Sequence- and ligation-independent cloning (SLIC): A scarless cloning method that allows for assembly of one or more linear nucleic acid fragments (e.g., assembly of a linearized vector and one or more inserts). The fragments to be joined are linearized via restriction enzyme digestion, PCR or gene synthesis and are treated with T4 polymerase. The 3' to 5' exonuclease activity of T4 polymerase produces 3' overhangs on each DNA fragment, allowing for annealing of complementary DNA sequences. In the case of SLIC assembly of plasmid sequences, plasmids are transformed into bacteria where DNA molecules are repaired and ligated and propagated. SLIC is described in Li and Elledge (2012, SLIC: A Method for Sequence- and Ligation-Independent Cloning. In: Peccoud J. (eds) Gene Synthesis. Methods in Molecular Biology (Methods and Protocols), vol 852. Humana Press).

[0346] Sequence identity: The identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).

[0347] Methods of alignment of sequences for comparison are known. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J.7158-105381-02

[0348] Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al.,Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0349] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

[0350] Splicing motifs: In the context of the present disclosure, splicing sequences and regulatory motifs refer to conserved or seini-conserved nucleotide sequences within a nucleic acid molecule that mediate the processing of precursor mRNA (pre-inRNA) into mature mRNA through splicing. These sequences include canonical splice donor, splice acceptor, and branch point sites, which are recognized by the spliceosome to facilitate exon-exon ligation and intron removal.

[0351] A splicing consensus sequence refers to the highly conserved nucleotide pattern found at essential splicing junctions, such as the GT-AG rule for most introns, wherein the 5' splice donor sequence (typically GU in RNA) and the 3' splice acceptor sequence (typically AG in RNA) define exonintron boundaries. Additionally, the branch point sequence (BPS), polypyrimidine tract (PPT), and exon splicing enhancers (ESEs) contribute to splicing efficiency and alternative splicing regulation.

[0352] Degeneracy within splicing sequences refers to the allowance for nucleotide variability at certain positions while maintaining functional recognition by the splicing machinery. This degeneracy enables alternative splicing, exon skipping, and regulatory flexibility while preserving transcript fidelity.

[0353] Regulatory motifs such as splicing silencers (ESS, ISS) and splicing enhancers (ESE, ISE) further modulate exon recognition, affecting gene expression and protein diversity.

[0354] These splicing sequences and regulatory motifs may be engineered or optimized for applications in gene therapy, viral vector design, synthetic biology, and recombinant protein production, enabling precise control over transcript processing, exon inclusion, and alternative splicing events.

[0355] Synthetic: An engineered non-native modification and / or one produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein can be chemically synthesized in a laboratory, Gene block (gBlock) or assembled with PCR amplified genomic modules to create native or modified CAV genome sequence.

[0356] TATA Box: A highly conserved cis-regulatory DNA element found in eukaryotic and viral core promoters, typically positioned 25 to 35 nucleotides upstream of the transcription start site (TSS). The TATA box is characterized by tire consensus sequence TATAWAWR (where W = A / T and R = A / G) and serves as a binding site for tire TATA-binding protein (TBP), a key component of tire transcription factor IID (TFIID) complex, which facilitates RNA polymerase II recruitment and transcription initiation. This sequence element plays an important role in gene expression regulation, influencing transcription7158-105381-02

[0357] efficiency, promoter strength, and chromatin accessibility. Variations in the TATA box sequence can modulate transcriptional output, contributing to differential gene expression patterns. The TATA box may be naturally occurring or synthetically engineered to enhance promoter activity, enable tissuespecific expression, or integrate regulatory elements in recombinant DNA constructs, with applications in gene therapy, vaccine development, synthetic biology, and biopharmaceutical production.

[0358] Therapeutic protein: Any protein capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. Therapeutic proteins include, but are not limited to, antigens (such as antigens from a pathogen or tumor antigens), anti-cancer agents, antibodies (or antigenbinding fragments thereof), or immunostimulatory proteins. For example, therapeutic proteins for treating cancer include proteins drat prevent or inhibit tumor growth, tumor development, or tumor metastasis. In some aspects, tire anti-cancer agent is an immunomodulator (e.g., IL-12, GM-CSF, TRAIL, granzyme B, IFN-cx, IFN-p, a CCL21, or CXCL10), a chemotherapeutic agent, an immunotoxin, or a pro-drug activating enzyme. In some aspects, the antibody is an anti-CTLA4 antibody, an anti-PDl antibody, or an anti-PD-Ll antibody

[0359] Toxic transgene: A protein that exerts a negative effect on cell viability, viral replication, or vector genome maintenance. Toxicity may arise from fusogenic activity, membrane disruption, pro-apoptotic signaling, immune activation, or interference with essential viral or cellular pathways. The impact of a toxic transgene may depend on its expression level, timing, cellular localization, or genomic placement, and such transgenes may require optimization of placement site or additional genetic modification to protein or placement site to achieve tolerable and stable expression in recombinant CAVs upon extended passaging.

[0360] Translational read-through: Translational read-through of multiple open reading frames (ORFs) refers to a molecular mechanism wherein ribosomal decoding continues beyond a canonical stop codon, enabling translation of downstream overlapping or distinct ORFs within a single mRNA transcript. This read-through process may be naturally occurring or engineered through sequence modifications, including stop codon suppression, RNA structural elements, programmed ribosomal frameshifting, or alternative translational signals. The ability to bypass termination codons and extend translation across multiple ORFs allows for the coordinated expression of functionally linked polypeptides, production of polyproteins, or regulatory modulation of gene expression. Such translational control mechanisms are applicable to viral genome engineering, synthetic biology, recombinant protein expression systems, and gene therapy platforms, where precise modulation of translational termination and read- through can enhance therapeutic efficacy or enable novel genetic architectures.

[0361] Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered to a subject for the prevention, amelioration, or treatment of infectious or other types of disease. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion,7158-105381-02

[0362] inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.

[0363] III. Introduction

[0364] The present disclosure describes the engineering of CAV genomic modules and placements that enable expression of one or more ectopic payloads, which are assembled to create replication-competent recombinant CAVs for applications in research, vaccine development, drug discovery, gene therapy and oncolytic therapies. Specifically described herein are different genomic placements with distinct expression levels and timing that enable recombinant CAV replication to be determined and quantitatively compared using reporter genes compatible with both high-content imaging and high throughput optical platforms. The present disclosure also allows for the expression of different therapeutic proteins to be screened and optimized for the development of CAVs for multivalent live vaccines, oncolytics, gene therapy and drug development.

[0365] There are many therapeutic and research applications for which it is desirable to engineer CAVs that are wholly or partially replication competent for expressing and delivering ectopic payloads (e.g., vaccine and oncolytic virus vectors). However, there is a maximum packaging capacity and genome size that can be successfully packaged into Ad capsids, and once this size is exceeded, approximately 105% size of CAV genome, the genome will not be packaged and result in defective virus particles. The latter precludes scale up for GMP manufacturing and can also decrease an effective therapeutic dose if defective particles are substantially greater than therapeutic infectious particles.

[0366] Unlike human adenoviruses, which have been extensively studied and engineered for gene therapy and vaccine delivery, CAVs remain largely uncharacterized, particularly in terms of genomic sites suitable for heterologous gene insertion. Common adenovirus engineering strategies involve El deletions to accommodate ectopic expression cassettes; however, El is essential for replication, and El-deleted vectors require complementing cell lines for propagation. Instead, the present disclosure focuses on engineering heterologous ORF placements that do not preclude replication.

[0367] To create additional ‘cargo’ space for large payloads and / or the expression of multiple payloads, it may be desirable or necessary to delete and / or replace non-essential CAV ORFs with heterologous ORFs. However, tire functional requirements for different CAV ORFs have not been systematically studied and remain largely un characterized. There are several examples of ORFs that are unique to CAVs and / or have no homolog or are poorly conserved in other adenovirus species (FIG. 6).

[0368] The challenges associated with engineering CAVs to express heterologous payloads include: 1. Genome size limitation - The CAV genome (~31 kb) is significantly smaller than hAd (36-38 kb), imposing constaints on payload capacity (FIGS. 3 and 4).

[0369] 2. Uncharacterized ORFs - Many CAV genes have unknown or poorly conserved functions, making it unclear which genes can be deleted or modified to introduce exogenous sequences while maintaining viral viability and replication (FIG. 6).7158-105381-02

[0370] The present disclosure provides a systematic framework for designing, engineering, and producing CAV genome modules and placements that can be assembled to produce recombinant replication-competent CAVs that express heterologous genes and reporters, and enable quantitative assays of screening of therapeutics and biologies production.

[0371] Disclosed herein are genomic placement sites that permit the production of replication-competent CAVs that express individual or multiple transgenes at different times and levels in a productive infection. It is also disclosed herein that certain CAV genes can be deleted, including E3-ORF1, E3-ORF2 and E4-ORF5, without impacting virus replication. It is also demonstrated that although some CAV ORFs cannot be deleted, they can be linked to a heterologous ORF using a self-cleaving peptide coding sequence. To produce and express multiple heterologous genes, viral ORFs were identified that are dispensable in cell culture for viral replication and can be linked to a heterologous ORF via a selfcleaving peptide sequence. The heterologous ORFs of the exemplary recombinant CAVs disclosed herein encode reporter genes, which also facilitate facile screening of heterologous payload expression profiles and their impact on viral replication, as demonstrated in the Examples. In addition, it is shown herein that multiple different therapeutic proteins can be expressed in CAVs, which is illustrated by the insertion of vaccine proteins from other canine pathogens.

[0372] To determine which CAV genes are dispensable for viral replication in cell culture (and can thus be deleted), a large library of virus variants was assembled and screened. As described in the Examples, genomic placements that are compatible with transgene placements and viral replication were identified and engineered. In particular, it is demonstrated herein that a heterologous ORF can be inserted with a P2A cleavage site 3' of the El A ORF, 3' of the ElB-55k ORF, or 5' of the DNA polymerase ORF and pVIII gene, among others. It is also demonstrated herein that heterologous ORFs can be engineered and expressed without insertion of ectopic promoters by designing them to replace E3-ORF1 coding sequences and engineering additional genetic modifications that enhance the expression a heterologous ORF.

[0373] The present disclosure further details CAV gene deletions to enhance the CAV genome payload expression and capacity. Examples include engineered deletions in the E3-ORF1, E3-ORF2, and E4-ORF5 genes, creating additional genomic payload capacity for the incorporation of therapeutic and antigenic ORFs. The strategic placements, deletions and combinations thereof, enable virus genome compositions that express one or more heterologous ORFS, such as reporter genes and / or therapeutic payloads, that can be produced to high titers without helper plasmids or complementing cell lines.

[0374] Furthermore, it is disclosed herein that these different engineered genomic placements can be used to express reporters, such as different fluorescent proteins, to enable a quantitative high content and high throughput viral replication assay to quantify and compare tire replication kinetics of libraries of CAV variants with different genetic modifications, payloads, cell types, and / or treatments (FIG. 43). The disclosed fluorescent-based viral kinetics (FBVK) assay provides a method for quantitative screening of genetically engineered CAV genomes, optimization of GMP manufacturing processes, and evaluating the7158-105381-02

[0375] impact of pharmaceuticals biologies, antibodies or cell lines on recombinant CAV replication or pathogenicity.

[0376] Additionally, exemplary applications include the expression of therapeutic ORFs, such as antigenic proteins from canine pathogens (e.g., CDV and CPiV), demonstrating potential for live vaccine development. The development of replication competent CAVs that can express multiple heterologous payloads, as disclosed herein, provides an unmet need for the development and enablement of live vaccines and oncolytic viruses.

[0377] IV. Heterologous Open Reading Frame Insertion Sites

[0378] The present disclosure concerns engineering genomic placement sites for expressing heterologous ORFs, such as one or more ORFs encoding fluorescent, immunogenic, diagnostic, or therapeutic proteins. Insertion of heterologous ORFs at the selected CAV genomic sites do not significantly alter replication (such as no more than 20%, no more than 10%, no more than 5%, no more than 1%, or no more than 0.1%, such as 0-5%, 1-5%, 0.1-10%, or 0 to 20%) of the recombinant CAV and results in a replication competent virus, which does not require a complementing cell line or cotransfection of a helper plasmid for replication.

[0379] Provided herein are recombinant CAV genomes that include a first ORF, and optionally include a second heterologous ORF, a third heterologous ORF and / or a further heterologous ORF, and may further include deletions in one or more CAV genes (e.g., E3-ORF1, E3-ORF2 and / or E4-ORF5), as detailed below.

[0380] In some aspects, the first heterologous ORF is located 3' of an E1A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0381] In other aspects, the first heterologous ORF is located 5' of an E1A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0382] In other aspects, the first heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame.

[0383] In other aspects, tire first heterologous ORF is located 5' of a DNA polymerase ORF and a selfcleaving peptide coding sequence is located between tlie first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame.7158-105381-02

[0384] In other aspects, the first heterologous ORF is located 3' of a DNA polymerase ORF and a selfcleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein die first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame.

[0385] In odier aspects, the first heterologous ORF is located 3' of an E2A-DNA binding protein (DBP) ORF and die first heterologous ORF is linked to the E2A-DBP ORF.

[0386] In odier aspects, the first heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between die first heterologous ORF and the pVIII ORF, wherein the first heterologous ORF, die self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame.

[0387] In other aspects, die first heterologous ORF is located 3' of an E3-ORF1 and the first heterologous ORF is linked to the E3-ORF1.

[0388] In other aspects, die genome comprises a complete or partial deletion of an E3-ORF1 coding sequence and the first heterologous ORF is inserted at die site of die E3-ORF1 deletion. In these aspects, the heterologous ORF replaces the E3-ORF1 sequence.

[0389] In other aspects, die genome comprises a complete or partial deletion of an E3-ORF 1 coding sequence and a complete or partial deletion of an E3-ORF2 coding sequence, and the first heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0390] In some aspects, a canine adenovirus that includes a recombinant CAV genome disclosed herein is replication competent.

[0391] In some aspects, the E3 region of the recombinant CAV genome does not include a heterologous promoter. In some examples, the recombinant CAV genome does not include a heterologous promoter (in any region of the genome) and / or does not include a heterologous polyA sequence (in any region of the genome).

[0392] In some examples of the recombinant CAV genomes, the first heterologous ORF is located 3’ of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; and the genome includes a complete or partial deletion of the E3-ORF1 coding sequence.

[0393] In other examples, the first heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the pVIII ORF, wherein the first heterologous ORF, tire self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame; and the genome includes a complete or partial deletion of the E3-ORF1 coding sequence.

[0394] In other examples, the first heterologous ORF is located 3' of an ElB-55k ORF and a selfcleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are7158-105381-02

[0395] operably linked and in the same reading frame; and the genome includes a complete or partial deletion of the E3-ORF1 coding sequence.

[0396] In yet other examples, the first heterologous ORF is located 5' of a DNA polymerase ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame; and the genome includes a complete or partial deletion of the E3-ORF1 coding sequence.

[0397] In some aspects, the recombinant CAV genome further includes a second heterologous ORF. In some examples in which the recombinant CAV genome include two (or more) heterologous ORFs, the first heterologous ORF is located 3' of the E1A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; and the genome includes s a complete or partial deletion of the E3-ORF1 coding sequence and the second heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0398] In other examples, the first heterologous ORF is located 3' of the ElB-55k ORF and a selfcleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; and the genome includes a complete or partial deletion of the E3-ORF1 coding sequence and the second heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0399] In other examples, the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; and the second heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the second heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the second heterologous ORF are operably linked and in the same reading frame.

[0400] In other examples, the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; and the second heterologous ORF is located 5' of a pVIII ORF and a selfcleaving peptide coding sequence is located between the second heterologous ORF and tire pVIII ORF, wherein tire second heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame.

[0401] In yet other examples, the recombinant CAV genome includes a complete or partial deletion of the E3-ORF1 coding sequence and the first heterologous ORF and the second heterologous ORF are inserted at the site of the E3-ORF1 deletion. In specific examples, a self-cleaving peptide coding7158-105381-02

[0402] sequence is located between the first heterologous ORF and the second heterologous ORF, and the first heterologous ORF, the self-cleaving peptide coding sequence the second heterologous ORF are operably linked and in the same reading frame. In other specific examples, a linker sequence is located between the first heterologous ORF and the second heterologous ORF, and the first heterologous ORF, tire linker sequence, and the second heterologous ORF are operably linked and in the same reading frame.

[0403] In some aspects, the recombinant CAV genome further includes a third heterologous ORF. In some examples in which the recombinant CAV genome includes three heterologous ORFs, the genome includes a complete or partial deletion of the E3-ORF1 coding sequence and the third heterologous ORF is inserted at tlie site of the E3-ORF1 deletion. In specific examples, a linker sequence is located between the first heterologous ORF and the second heterologous ORF; a selfcleaving peptide coding sequence is located between the second heterologous ORF and the third heterologous ORF; and the first heterologous ORF, the linker sequence, the second heterologous ORF, the self-cleaving peptide coding sequence, and the third heterologous ORF are operably linked and in the same reading frame.

[0404] In other examples, the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; the second heterologous ORF is located 3' of an ElB-55k ORF and a selfcleaving peptide coding sequence is located between the second heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the second heterologous ORF are operably linked and in the same reading frame; and the third heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between the third heterologous ORF and the pVIII ORF, wherein the third heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame.

[0405] In some aspects, the recombinant CAV genome further includes a fourth heterologous ORF. The fourth heterologous ORF can be inserted at any of the locations described above.

[0406] In some examples, the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF are under the control of native viral post-transcriptional and translational elements.

[0407] In some aspects in which the recombinant CAV genome includes a complete or partial deletion of the E3-ORF1 coding sequence, the deletion excludes a 5’ region of the E3-ORF1 that overlaps with a 3’ region of the pVIII ORF. In some examples, the 5’ region of the E3-ORF1 that overlaps with the 3’ region of the pVIII ORF includes one or more nucleotide substitutions, wherein the one or more nucleotide substitutions eliminate one or two alternative start codons, and wherein the one or more nucleotide substitutions do not result in any amino acid substitutions in tire pVIII protein. In some examples, the first heterologous ORF inserted at the site of the E3-ORF1 deletion comprises a start codon and / or a Kozak consensus sequence.7158-105381-02

[0408] In some aspects, the recombinant CAV genome further includes a complete or partial deletion of the E3-ORF2 coding sequence. In some examples, deletion of the E3-ORF2 coding sequence increases expression by at least 50% of a heterologous ORF inserted at the site of the E3-ORF1 deletion relative to expression in the absence of the E3-ORF2 deletion.

[0409] In some aspects, the recombinant CAV genome further includes a complete or partial deletion of the E4-ORF5 coding sequence. In some examples, a heterologous ORF is inserted at the site of the E4-ORF5 deletion.

[0410] In some aspects, the recombinant CAV genome includes a complete or partial deletion of the E3-ORF1 coding sequence, a complete or partial deletion of the E3-ORF2 coding sequence, and a complete or partial deletion of tire E4-ORF5 coding sequence. These deletions allow for additional cargo space for heterologous ORFs and in some cases, modulate expression of levels of an inserted heterologous ORF (e.g., deletion of E3-ORF2).

[0411] In some aspects of tire recombinant CAV genome, the E3-ORF1 is deleted and replaced with a polycistronic cassette comprising three or more linked heterologous ORFs. In some examples, the E3-ORF1 is deleted and replaced with a polycistronic cassette that includes three or more linked heterologous ORFs, and wherein the genome further includes a complete or partial deletion of E3-ORF2 and / or E4-ORF5. In specific examples, the ORFs are linked using a self-cleaving peptide sequence. The self-cleaving peptide sequence can be, for example, a 2A peptide, such as a 2A peptide selected from a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus (TaV) 2A (T2A) peptide, and variants thereof (such as variants with Gly-Ser-Gly at the N-terminus to improve cleavage efficiency).

[0412] In some aspects of the recombinant CAV genome, the non-coding junctions between E3-ORF1 and E3-ORF2, and between E3-ORF2 and U exon are preserved when the E3-ORF1 and E3-ORF2 coding sequences are completely or partially deleted.

[0413] In some aspects, the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encode a therapeutic protein. In some examples, the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF each encode a different therapeutic protein. In some examples, the therapeutic protein is an antigenic protein, an anti-cancer agent, an immunostimulatory transgene, or an antibody. In specific examples, the antigenic protein is a protein from a pathogen (such as CDV or CPiV or another canine pathogen). In specific examples, the antigenic protein is a cancer antigen. In specific examples, the anticancer agent is an immunomodulator or a chemotherapeutic agent, an immunotoxin, or a pro-drug activating enzyme. In specific examples, the immunostimulatory transgene includes IL-12, GM-CSF, TRAIL, granzyme B, IFN-a, IFN-p, a CCL21, or CXCL10. In other specific examples, die antibody is an anti-CTLA4 antibody, an anti-PDl antibody, or an anti-PD-Ll antibody.7158-105381-02

[0414] In other aspects, the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encode a reporter protein. In some examples, the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or die fourth heterologous ORF each encode a different reporter protein.

[0415] In otiier aspects, the first heterologous ORF, the second heterologous ORF, the diird heterologous ORF and / or the fourth heterologous ORF encode a combination of therapeutic proteins and reporter proteins.

[0416] In some examples in which at least one of the heterologous ORFs encodes a reporter protein, die reporter protein is a fluorescent protein, a luminescent protein, an enzyme, or an imaging reporter for positron emission tomography (PET) or magnetic resonance imaging (MRI). In specific examples, die PET reporter includes HSV1-TK, dopamine D2 receptor (D2R), or human sodium iodide symporter (NTS); or the MRI reporter includes tyrosinase, ferritin, or aquaporin-1 (AQP1). In specific examples, the fluorescent protein is YPet, mCherry, Katushka, Halo-tag, Snap-tag, or blue fluorescent protein (BFP).

[0417] In some aspects of the recombinant CAV genomes disclosed herein, the self-cleaving peptide is a 2A peptide or variant thereof. In some examples, the 2A peptide includes a P2A peptide, a F2A peptide, an E2A peptide or a T2A peptide, or a variant thereof. In particular examples, the 2A peptide variant includes a Gly-Ser-Gly on the N-terminus to improve cleavage efficiency. Exemplary 2A peptides sequences and variants thereof are set forth herein as SEQ ID NOs: 107-114.

[0418] In specific examples, the recombinant CAV genome includes the following features in the 5' to 3' direction: El A-2A-heterologous ORF; heterologous ORF-2A-E1A; ElA-2A-heterologous ORF-2A-heterologous ORF; ElB-55k-2A-heterologous ORF; El B-55k-2A-heterologous ORF-2A-heterologous ORF; heterologous ORF-2A-DNA polymerase; DNA polymerase-2A-heterologous ORF; E2A-DBP-heterologous ORF; E2A-DBP-linker-hetero logons ORF; heterologous ORF-2 A-p VIII; E3-ORF1-heterologous ORF; E3-ORF1 -linker-heterologous ORF; AE3-ORFl-heterologous ORF; AE3-ORF1-heterologous ORF-AE3-ORF2; AE3-ORFl-heterologous ORF-P2A-heterologous ORF; AE3-ORF1-heterologous ORF-linker-heterologous ORF; AE3-ORFl-heterologous ORF-linker-heterologous ORF-AE3-ORF2; AE3-ORF1 -heterologous ORF-linker-heterologous ORF-2A-heterologous ORF-AE3-ORF2; AE3-ORF1 -heterologous ORF-2 A-heterologous ORF-2 A-heterologous ORF-AE3-ORF2; or AE3-ORF1-heterologous ORF-2 A-heterologous ORF-2 A-heterologous ORF-AE3-ORF2, DE4-ORF5. In particular non-limiting examples, the recombinant CAV genome includes: El A-2 A-heterologous ORF and AE3-ORF1; El A-2 A-heterologous ORF, AE3-ORF1 and AE3-ORF2; El A-2 A-heterologous ORF, AE3-ORF1 and AE3-ORF2; AE4-ORF5; heterologous ORF-2A-E1A and AE3-ORF1; heterologous ORF-2A-E1A, AE3-ORF1 and AE3-ORF2; El A-2 A-heterologous ORF and AE3-ORF1 -heterologous ORF; E1A-2A-heterologous ORF, AE3-ORFl-heterologous ORF, and AE3-ORF2; El A-2 A-heterologous ORF, AE3-ORF1 -heterologous ORF, AE3-ORF2 and AE4-ORF5; El A-2 A-heterologous ORF, AE3-ORF1-heterologous ORF-2A-heterologous ORF, AE3-ORF2 and AE4-ORF5; El A-2 A-heterologous ORF and7158-105381-02

[0419] ElB-55K-2A-heterologous ORF; heterologous ORF-2A-E1A and AE3-ORF1 -heterologous ORF; heterologous ORF-2A-E1A, AE3-ORFl-heterologous ORF, and AE3-ORF2; ElB-55k-2A-heterologous ORF and AE3-ORF1; ElB-55k-2A-heterologous ORF, AE3-ORF1 and AE3-ORF2; ElB-55k-2A-heterologous ORF and AE3-ORF1 -heterologous ORF; ElB-55k-2A-heterologous ORF, AE3-ORF1-heterologous ORF, and AE3-ORF2; ElA-2A-heterologous ORF and heterologous ORF-2A-pVIII; El A-2A-heterologous ORF, heterologous ORF-2A-pVIII , AE3-ORF1 and AE3-ORF2; heterologous ORF-2A-pVIII and AE3-ORFl-heterologous ORF; heterologous ORF-2A-pVIII, AE3-ORFl-heterologous ORF, and AE3-ORF2; heterologous ORF-2A-DNA polymerase and AE3-ORF1 -heterologous ORF; or heterologous ORF-2A-DNA polymerase, AE3-ORF1 -heterologous ORF and AE3-ORF2. In the above examples, “2A” represents any 2A peptide or variant thereof. When more than one 2A sequence is present in a single genome, any combination of 2A peptides can be used. Typically, if more than one 2A sequence is included, they are different 2A peptides (e.g., P2A and T2A). In some examples, any of the above-listed viruses further include AE4-ORF5.

[0420] In some examples the nucleotide sequence of the recombinant CAV genome is 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%, or at least 99% percent idential to any one of SEQ ID NOs: 1-38 and 45. In specific non-limiting examples, the nucleotide sequence of the recombinatn CAV genome includes or consists of any one of SEQ ID NOs: 1-38 and 45.

[0421] In some example, the nucleotide sequence of a macromodule plasmid, an El plasmid module, a core plasmid module, an E3 plasmid module, or an E4 plasmid module used to generate a recombinant CAV genome disclosed herein is 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%, or at least 99% percent idential to any one of SEQ ID NOs: 39-44 and 46-66. In specific examples, the nucleotide sequence of a macromodule plasmid, an El plasmid module, a core plasmid module, an E3 plasmid module, or an E4 plasmid module used to generate a recombinant CAV genome disclosed herein includes or consists of any one of SEQ ID NOs: 39-44 and 46-66.

[0422] Also provided herien are compositions that include a recombinant CAV genome disclosed herein. In some aspects, the composition further includes a pharmaceutically acceptable earner.

[0423] Further provided are recombinant CAVs that include a recombinant CAV genome disclosed herein. Compositions that include a recombinant CAV and a pharmaceutically acceptable carrier are also provided.

[0424] Also provided are kits that include a recombinant CAV genome or a recombinant CAV disclosed herein; and cells, cell culture media, and / or a multi-well plate.

[0425] V. CAV Fluorescence-Based Viral Kinetics (FBVK) Assay7158-105381-02

[0426] The CAVSLIC and CAVsembly platforms for assembling large libraries of CAVs are amenable to high throughput techniques, but the screening needs to also have such scalability, which is not provided by any existing methods that have been developed for CAVs.

[0427] There is no systematic platform to quantitatively measure the replication kinetics and properties of CAVs. Antibodies to CAV proteins, and assays thereof, do not measure productive assembly of viral capsids, virus spread and secondary infection. For existing methods, accurate knowledge of the virus titer is critical for producing usable results with die single step growth assay. The titer of the virus to be tested is essential for calculating the conditions of initial infection and the final titer of the virus produced after one life cycle is also required to calculate the virus expansion. Plaque assays have low sensitivity at a low multiplicity of infection (MOI), and are time consuming and laborious.

[0428] To overcome these limitations and enable large libraries of recombinant CAV virus variants to be systematically screened and quantitatively compared, CAV genome modules and genomes were engineered with one or more reporter genes that are linked to CAV viral gene transcription, expression and replication. The examples provided herein include different fluorescent proteins, but other reporters, such as luciferase, could also be used.

[0429] The assays disclosed herein exploit fluorescent proteins that are transcribed instead of and / or translated coincident with viral ORFs, and as such, reflect the endogenous rate of viral gene transcription, splicing, RNA export and translation. In one example, recombinant CAVs were generated with ORFs that are linked to transgene payload expression or fluorescent reporters via self-cleaving 2A protein coding sequences. Using this method, the rate and level of fluorophore expression are directly linked to that of viral proteins. The assays disclosed herein enable every aspect of the CAV life cycle to be interrogated over several rounds in a reporter (such as fluorescent) assay.

[0430] The CAV reporter placements were engineered via P2A linkage to essential viral genes, such as El A, ElB-55k, pVIII, DNA pol, and / or replace the expression of dispensable CAV ORFs, such as E3-ORF1 and / or E4-ORF5. The engineered fhiorophore / reporter gene placements in different CAV modules can be assembled to create recombinant CAVs that can be quantified and compared through the development and application of high content imaging-based platforms and / or high throughput plate reader optical platforms, such as a fluorescence -based viral kinetic (FBVK) assay (FIG. 43). The exemplary fluorescent proteins used included YPet, mCherry and BFP, but are not limited to these examples. YPet is 2.4x brighter than eGFP and detected at wavelengths less sensitive to background fluorescence from the media. The plate based 96-well HTP method extends upon methods and data quantification that had previously been developed for human Ads, and technical details and protocols therein (see also PCT Publication No. WO 2017 / 147265).

[0431] The FBVK assay uses HTP or HCS automated platforms to quantify reporter gene expression, generally every 1-2 hours, over a period of 4-7 days, which enables the exponential rate of CAV replication kinetics to be calculated from the slope of a semi-log plot of fluorescence versus time. This is7158-105381-02

[0432] analogous to optical density (OD) readings of batch phase bacterial cultures and log-phase growth analyses (FIGS. 43-45).

[0433] A method to determine the kinetics of a virus without knowledge of its initial titer would include infecting the cell type of interest such that only a small percentage of cells are initially infected and monitoring the number of infected cells over a period of several virus lifecycles. By infecting or transfecting a small percentage of cells initially (using serial dilutions so that one is likely to be in linear part of curve), primary, secondary, and tertiary replication can be captured (FIG.43).

[0434] A semi-log plot of the exponential growth in number of infected cells versus time would yield a straight line proportional to tlie exponential growth rate. The data is also information rich (FIG. 44). Since log-slope is the only pertinent parameter, this method is robust against variations or errors in initial infection titer and can even be employed with transfection of tlie whole-genome plasmid instead of infection with purified virions. Comparing kinetics between different viral constructs, each with a potentially different fluorophore and signal level, can be addressed by use of log-slope. Taking the slope of the logarithm of the exponential growth in fluorescence signal vs. time results in a single value for each viral construct that can be cross-compared regardless of signal magnitudes or any initial time delay that might occur before exponential growth begins (FIG. 45). This feature of data interpretation makes the assay insensitive to initial starting points. Poor control or even knowledge of initial viral titer has no impact on the log-slope during exponential growth. All that is necessary is an initial infection (or transfection) that results in transduction of a small fraction of cells in the tissue culture dish. The remaining, unaffected cells are available for secondary and tertiary infection.

[0435] The FBVK assay can quantify unambiguously the magnitude of difference in replication kinetics between two viruses / conditions, and also give valuable information as to which parts of the replication process might be defective in viruses with slower rates of replication. In summary, the FBVK assay is high-throughput (e.g., 96-well to 384-well plates can be used), nondestructive, insensitive to initial viral titer, highly sensitive to small differences in viral replication rates, and it captures multiple viral infection cycles.

[0436] The primary reasoning behind the requirement for insensitivity to an accurately known virus titer was the desire for a kinetics assay to skip tlie entire virus production, purification, and titering steps and simply transfect the whole virus genome plasmid into tlie cell type of interest. In some examples, the method includes transfecting cells with the genome of the recombinant CAV or infecting cells with CsCl purified preparations, or virus supernatants.

[0437] Provided herein is a method for detecting and quantifying replication of an individual recombinant CAV or a library of recombinant CAVs in a host cell culture. In some aspects, tlie method includes infecting or transfecting the host cell culture with a recombinant CAV that has a recombinant CAV genome disclosed herein, wherein tlie first heterologous ORF encodes a reporter protein; detecting expression of the reporter protein using: (i) an imaging-based system capable of acquiring and analyzing spatially and spectrally resolved image data at different wavelengths, length and time scales, from multi-7158-105381-02

[0438] well plates of recombinant CAV genome reporter gene fluorescence in individual cells, plaques and / or the cell culture area as well as brightfield imaging; and / or (ii) a multi-modal plate reader configured to rapidly measure bulk recombinant CAV genome reporter gene fluorescence intensity and / or luminescence across a plurality of wells in a multi-well format, wherein the signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

[0439] In other aspects, the method includes infecting or transfecting the host cell culture with a recombinant CAV that includes a recombinant CAV genome disclosed herein, wherein the first heterologous ORF encodes a reporter protein; detecting expression of the reporter protein using (i) a plate reader based system that quantifies fluorescence or luminescence from each well of a multi-well plate; or (ii) wherein the plate reader is configured or automated to capture fluorescence or luminescence over time, wherein the expression of the reporter protein signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

[0440] In some examples of these methods, the reporter protein is a fluorescent reporter protein, such as, but not limited to, blue fluorescent protein (BFP), mNeonGreen, YPet, mCherry, iRFP720, mScarlet, Katushka, HaloTag, or a derivative thereof.

[0441] In some examples, the reporter or fluorescence is detected using a hybrid multi-mode reader. In some examples, the imaging-based detection includes time-lapse imaging to visualize and analyze fluorescent plaque growth and viral infection of cells over time. In specific examples, the imaging-based detection of fluorescent plaques is used to quantify viral titers and fluorescent plaque forming units.

[0442] In some aspects of the disclosed methods, the heterologous ORF is located 3’ of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; or the heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0443] In some examples, the recombinant CAV genome further includes a complete or partial deletion of the E3-ORF1 coding sequence and optionally further includes a complete or partial deletion of the E3-ORF2 coding sequence.

[0444] In some examples, the recombinant CAV further includes a second heterologous ORF, a third heterologous ORF and / or a fourth heterologous ORF. In specific examples, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encodes a therapeutic protein.

[0445] In some examples of the disclosed methods, transduction and / or replication of the recombinant CAV is measured in a plurality of different cell types; transduction and / or replication of the recombinant CAV is compared to replication of second recombinant CAV comprising different genome modifications; replication of the recombinant CAV is compared to replication of an adenovirus from a different species; and / or replication of the recombinant CAV is measured in different culture media and / or in the presence of different drugs.7158-105381-02

[0446] Further provided is a method for measuring replication of a recombinant CAV that includes a recombinant CAV genome disclosed herein, wherein the first heterologous ORF encodes a reporter protein. In some aspects, the method includes (i) transfecting cells with the genome of the recombinant CAV, or infecting cells with particles of the recombinant CAV; (ii) culturing tire transfected cell or infected cells for at least 24-72 hours; (iii) measuring reporter gene signal at regular intervals throughout the culturing step, thereby obtaining fluorescence measurements; and (iv) calculating log-slope (day1) from a semi-log plot of fluorescence or intensity signal, normalized to background, versus ti me in tire linear part of the curve, thereby measuring replication of the recombinant CAV.

[0447] In some aspects, tire heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and tire El A ORF, wherein the E1A ORF, tire self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; or the heterologous ORF is inserted at the site of the E3-ORF1 deletion.

[0448] In some examples, the recombinant CAV genome further includes a complete or partial deletion of the E3-ORF1 coding sequence and optionally further includes a complete or partial deletion of the E3-ORF2 coding sequence.

[0449] In some examples, the recombinant CAV further includes a second heterologous ORF, a third heterologous ORF and / or a fourth heterologous ORF. In specific examples, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encodes a therapeutic protein.

[0450] In some examples of the disclosed method, replication of the recombinant CAV is measured in a plurality of different cell types; replication of the recombinant CAV is compared to replication of one or more recombinant CAVs comprising different genome modifications; replication of the recombinant CAV is compared to replication of an adenovirus from a different species; replication of the recombinant CAV is measured in different culture media and / or in the presence of different drugs; replication of the recombinant CAV is measured in different cell lines; and / or replication of the recombinant CAV is measured in different cell lines in CRISPR genome wide screens. In specific examples, the plurality of cell types includes a first cell type that is a tumor cell and a second cell type that is a non-tumor cell.

[0451] In some aspects, the method includes transfecting cells with the genome of the recombinant CAV. In other aspects, the method includes infecting cells with particles of the recombinant CAV. In some examples, the cells are infected with serial dilutions of the recombinant CAV particles, such as, but not limited to dilutions of 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900 and 1:218,700.

[0452] EXAMPLES

[0453] The following non-limiting examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to diose features exemplified.7158-105381-02

[0454] Example 1: CAV assembly and CAVSLIC system for modular assembly of replication competent CAV genomes that express heterologous antigenic proteins

[0455] To facilitate the construction, engineering and screening of recombinant CAVs that express heterologous payloads, the CAV genome was divided into four CAVsembly / CAVSLIC functional genomic fragments and modules that would be desirable to manipulate to express heterologous fluorescent reporters and / or therapeutic ORFs, and could be modified to confer wild type as well as engineered properties (FIG. 7). Recombinant CAVs generated using CAVsembly or CAVSLIC can be used to quantify viral replication kinetics, infection and properties by expression and placement of fluorescent reporter genes.

[0456] Modular CAV Genome Organization

[0457] Adenoviral genomes have a natural modular-like genomic structure whereby genes cluster in groups of shared transcriptional elements and general protein functions, which are conserved between Ads of different species including hAd5 and CAV1 / CAV2 (FIGS. 3-5). The disclosed methods exploit some of the natural modularity based on (1) native conserved viral gene clusters and organization that are predicted to have shared functional and transcriptional regulation; (2) the estimated frequency of desired engineering within each module; and (3) identification of genomic regions between genomic modules / regions that were predicted to be amenable to engineering and segmentation and tolerate the introduction of additional sequences, such as site- specification recombination (SSR) sites or restriction enzyme sites that facilitate modular assembly but do not negatively impact viral replication competence.

[0458] The CAV2 genome, which is 31,232 base pairs in length, was divided into four distinct functional and transcriptional genome modules (FIG. 7, top). The modular design reflects the natural genomic organization of Ads across species, despite sequence-level differences (FIGS. 3-5). Assembly of the CAV2 genome is exemplified herein, but the same methods and systems can be applied to both CAV1 and other CAV strains, which have a conserved viral genome module architecture and organization (FIGS. 5-6). By dividing the genome into functional plasmid modules, this system allows for the screening of combinatorial libraries of viruses that have transgenes and deletions engineered in different genomic regions and transcriptional units.

[0459] The four CAV genome modules comprise the following genes and predicted genes and functions:

[0460] 1. El genome module (FIG. 8):

[0461] This spans from tire left-hand internal terminal repeat (ITR) through tire El genes and the TATA box for pIX. The E1A and E1B gene products encoded within this module are essential for viral replication. El A and E1B gene products play critical roles in transcriptional activation of other early (E) viral genes, cell cycle initiation and preventing apoptosis. These genes are often deleted in nonreplicating CAV vectors in gene therapy applications and replaced by heterologous gene expression cassettes, but require complementary cell lines for virus production.7158-105381-02

[0462] 2. Core genome module (FIG. 9):

[0463] This genomic module includes the early / intermediate genes, pIX and IV A2; the major late promoter (MLP) and transcriptional unit that drives the expression of CAV capsid and late proteins, including: 52 / 55k, pllla, penton, pVII, pV, pX, pVI, hexon, endoproteinase, 100k, 22k, 33k (putative 22k / 33k) on the positive strand. On the negative strand the E2 transcriptional unit drives the expression of the viral DNA replication proteins, including E2A DBP, pTP and E2B DNA polymerase.

[0464] 3. E3 genome module (FIG.10):

[0465] This genomic module includes the MLP transcribed late protein pVIII, the E3 protein coding genes (E3-ORF1 and E3-ORF2) and the CAV fiber gene on the positive strand, and the U exon on the negative strand. The E3 encoded ORFs are highly variable among adenoviruses and diverge within and between species.

[0466] 4. E4 genome module (FIG.11):

[0467] The E4 module includes the right ITR and the E4 transcriptional unit and protein coding genes are on the negative strand. The E4 genes play important functions in usurping host cell signaling pathways and functions to facilitate viral replication in human Ads and likely canine Ads. In CAV there are five predicted and annotated ORFs, which include from right to left, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5. In Ad5 these genes play key roles in hijacking cellular signaling pathways and function to facilitate viral DNA replication and protein synthesis.

[0468] Example 2: Reference genome template for developing CAV assembly modules

[0469] As a template for construction of CAV genomes with heterologous ORFs and the development of quantitative assays for virus biology and therapeutics discovery, CAV2 VR-800, which is the CAV-2 Toronto A26 / 61 strain, was obtained from ATCC. Upon complete genome sequencing, several differences were identified between VR800 and the GenBank reference sequence U77082, which are noted below and are present in the genomic modules used to assemble recombinant CAVs.

[0470] Location Mutation (A26 / 61 -to VR-800) Consequence

[0471] 293bp C-to-G Unknown. Possibly located in E1A promoter 10,509 C-to-T Silent mutation in 52 / 55kDa protein

[0472] 11189-11192 TGTG-to-GTGT C A- to- VS mutation in pllla precursor 14018 C-to-T A-to-V mutation in penton base

[0473] 18408 G-to-A Silent mutation in Hexon

[0474] 19599 T-to-G Silent mutation in Hexon

[0475] 19610 G-to-T G-to-V mutation in Hexon

[0476] 22562 C-to-T R-to-C mutation in lOOkDa protein

[0477] 24058 C-to-A P-to-H mutation in 33kDa protein

[0478] 28627 A-to-T V-to-E mutation in E4-ORF5 on bottom strand7158-105381-02

[0479] CAV2 VR-800 was grown in MDCK cells. Virus was harvested and purified using two-stage CsCl purification. CAV-2 DNA was extracted and cloned into a plasmid backbone via SLIC (see U.S. Patent No. 9,217,160), producing PCMN-1312 (SEQ ID NO: 45; FIG. 12). PCMN-1312 was used as the template for creating CAVsembly and CAVSLIC El, core, E3 and E4 modules.

[0480] Example 3: Generation of CAVsembly / CAVSLIC plasmid modules

[0481] To facilitate the introduction of genome modifications, El, E3 and E4 sizes were designed such that they would be amenable to amplification (generally <10,000bp). The four exemplary genomic modules are designated as El, Core, E3, and E4 (see FIGs 7-11). El, E3 andE4 entry plasmids were engineered to be compatible with both CAVSLIC and CAVsembly whole genome assembly platforms. The El, E3 and E4 modules contain Gateway recombination sites outside of the CAV2 genome sequence, allowing for dual-system use of these modules. Core module plasmids are specific to either CAVSLIC or CAVsembly. The CAVSLIC core module contains restriction enzymes at the module junctions, while tire CAVsembly core module contains Gateway recombination sites at the module junctions.

[0482] CAV core modules for scarless assembly have Pad and Bglll sites engineered at the left and right side of the core module sequences, respectively. These sites enable linearization of the large core module genome sequences for scarless assembly with El modules and E3 / E4 modules in one to three steps, as desired and determined by engineering and screening library goals. The CAVsembly and CAVSLIC systems described herein provide an efficient, flexible, and scalable platform for the assembly of synthetic CAV genomes that express heterologous ORFs and therapeutic payloads.

[0483] Primers used for module construction and genome assembly are shown in Table 1. The CAV core genome plasmid module was constructed via Gibson assembly by combining (1) a 571 bp gBlock™ gene fragment (Integrated DNA Technologies), (2) CAV2 VR-800 (ATCC) DNA digested with BstZ17I / PshAI, and (3) a plasmid backbone with a pl5A origin of replication and ampicillin resistance, amplified by PCR using primers nl and n2. The El, E3 and E4 plasmid modules were constructed using PCR amplification of pCAV2 ATCC VR-800 DNA reference genome as the template using primers ml and m2 (El module), ol and o2 (E3 module), and pl, p2, p3, p4, p5 and p6 (E4 module), and each module was Gibson assembled with a plasmid backbone having a pMBl origin of replication and kanamycin resistance, amplified with PCR primers m3 and m4 (El module), o3 and o4 (E3 module), and p7 and p8 (E4 module). The E4 module was engineered with two silent nucleotide mutations (shown in bold underline below) to eliminate Bglll and Pad restriction sites, while maintaining amino acid sequence. This facilities linearization of the plasmid module by restriction enzyme digestion and assembly of El, E3, E4 plasmids with core virus genome modules by scarless Gibson and SLIC based assembly methods.7158-105381-02

[0484] E4 region 1 (SEQ ID NO: 72)

[0485] agcacccgctctattacagacctcacccacacagcacagtt

[0486] E4 region 2 (SEQ ID NO: 73)

[0487] acgtcagaaatttctttaatcaaagtgcctttaaaatgtgc

[0488] Table 1. PCR primers

[0489] Primer Sequence SEQ ID NO: ml gcagattaccctgttatccctaCATCATCAATAATATACAGGACAAAGAGGTGTG 74 G

[0490] m2 agttgggctgtaaatacacagcCACGTACCGCGCCCCTTTTATAC 75 m3 GCTGTGTATTTACAGCCCAAC 76 m4 TAGGGATAACAGGGTAATCTGC 77 nl cacgtaccgcgccccttttaATTAAGTAATCTTACAGACAAGCTGTGAC 78 n2 caacaaatactgtcaaggactcgagtccggcacagactgagcagatcTATAAACGCAGAAAGG 79 CCCAC

[0491] 01 ATGTCTAAAGAAATACCAACCCC 80 o2 CCGCTCGCGTGTATGAAAAATAAAG 81 o3 gggttggtatttctttagacatCACAACTTTTGTATACAAAGTTGGC 82 o4 tttttcatacacgcgagcggTACAACTTTGTATAATAAAGTTGAACGAG 83 Pl ccaactttgtataataaagttgTAAGGCTGCCGCCTTCAG 84 p2 tgtgggtgaggTCTGTAATAGAGCGGGTGC 85 p3 ctattacagacCTCACCCACACAGCACAG 86 p4 aaaggcactttgATTAAAGAAATTTCTGACGTTGTTAATAATCAC 87 P5 tttctttaatcAAAGTGCCTTTAAAATGTGCAAGAG 88P6 gctgattaccctgttatccctaC ATC ATC A AT AAT AT ACAGGAC AA AG AGG 89CTTTTTTATAATGCCAACTTTGTATAATAAAGTTG

[0492] P7 90P8 TAGGGATAACAGGGTAATCAGC 91

[0493]

[0494] *Upper case letters indicate overlap with template DNA, lower case letters indicate overlap with neighboring regions for Gibson assembly

[0495] Example 4: CAV genome plasmid module assembly via multi-site recombination or scarless cloning methods

[0496] El, E3 and E4 plasmid modules were designed to be dually compatible with both CAVSLIC and CAVsembly genome assembly methods, or combinations thereof. Core plasmid modules were designed to be used with either CAVSLIC or CAVsembly. The El, E3 and E4 modules contain Gateway7158-105381-02

[0497] recombination sites outside of the CAV2 genome sequence and PCR primers for Gibson / SLIC, allowing for dual-system use of these modules (FIGS. 14-16). The CAVSLIC core module contains restriction enzymes, Pad and Bglll, at the module junctions (FIG. 17), while the CAVsembly core module contains att recombination cassettes (FIG. 18).

[0498] CAVsembly is a single-tube reaction requiring just the plasmids and LR Clonase II plus enzyme mix (FIG. 19B). While CAVsembly provides a system to rapidly generate viral genomes from multiple parts in a single in vitro reaction, there are instances where the presence of recombination sites would be undesirable, for example a clinical candidate or due to unknown effect on viral replication or biology. In order to address this, a parallel approach to genome construction was developed that uses tire same modular entry vectors as CAVsembly, but reassembles the whole CAV genome in a seamless fashion (FIG. 19A). The CAV-SLIC system utilizes scarless assembly strategies, such as Gibson assembly, InFusion cloning or SLIC, to create single strand regions that are complementary to each other and facilitate modular genome assembly. Assembly generally begins by linearizing the destination core module plasmid, through restriction enzyme digestion, such as with Pad or Bglll (FIG. 20), or PCR amplification. Linearization via restriction enzymes is more efficient as PCR can introduce errors in a fragment of this size. Also, linearization via Pad or Bglll enables serial assembly of El or E3 / E4 fragments at the 5’ or 3’ ends, respectively. The El, E3, and E4 plasmids modules are then linearized with either restriction enzymes, or PCR-amplified with primers with complementary sequences to core module sequences. Single strand overlaps are generated upon treatment of core and E1 / E3 / E4 fragments with an exonuclease, which are then assembled through complementary sequence annealing and in vivo circular plasmid repair upon transformation into bacteria or in vitro using DNA polymerase / Taq DNA ligase Gibson enzyme mix.

[0499] CAVSLIC of El, E3 and E4 modules with the core module can be performed in any order, and in two, three or four steps, which is dictated by combinatorial complexity of engineering goals and modifications as well as efficiency. The assembly reaction with larger nucleic acid molecules is lower with more than three fragments. Thus, three or two fragment SLIC / Gibson assemblies are generally used herein. As such, it is often desirable to assemble an El and core module first into an El-core macromodule intermediary plasmid, which is subsequently assembled with E3 and / or E4 modules in another Gibson / SLIC reaction (FIG. 20). As shown in FIG. 20, a Pad digested core plasmid is assembled with PCR linearized El modules, which are Gibson / SLIC assembled, transformed into bacteria, selected and purified. The El-Core macromodule plasmid is then digested with Bglll and assembled in a 3-fragment Gibson / SLIC assembly reaction with PCR-linearized E3 and E4 genomic modules.

[0500] These assembly methods are used in the following examples to construct and assemble recombinant CAV genomes with different heterologous ORF placements and / or gene deletions to determine their properties in terms of gene expression and impact on CAV replication.

[0501] Example 5: Modular genome assembly of wild-type CAV2 (PCMN-1313)7158-105381-02

[0502] This example describes CAVSLIC assembly of a recombinant CAV genome from El, core, E3 and E4 modules to produce a wildtype CAV.

[0503] CAVSLIC was used to construct the PCMN-1313 CAV2 wild type virus genome, which was assembled with El, E3, E4 and core module plasmids (FIG. 21), and used as a benchmark and control to determine the impact and properties of heterologous ORF placements, gene deletions and / or replacements or modifications of recombinant CAV genomes. The core module plasmid, Core-059 (SEQ ID NO: 61), was digested with Pad and Gibson assembled with the linear product of the El-269 plasmid (SEQ ID NO: 42), amplified with primers rl and r2. This resulting macromodule plasmid is designated ASMM-149 (FIG. 22; SEQ ID NO: 39). The ASMM-149 macromodule plasmid was digested with Bglll and Gibson assembled with the linear products of die E4-099 plasmid module (SEQ ID NO: 65) amplified with primers t3 and t2, to create ASMM-150 (FIG. 23; SEQ ID NO: 40). The ASMM-150 macromodule plasmid was then digested with Bglll and assembled with the E3-522 plasmid module (SEQ ID NO: 46) amplified with primers si and s2. See Table 2 for primer sequences. Final constructs were selected using ampicillin resistance.

[0504] The left column of Table 2 indicates the logic for assembly with what is a universal set of primers for assembly of CAVSLIC El, E3 and E4 modules to core and with each other in exemplary two or three step assembly schemes, and not peculiar to just this example of PCMN-1313. In this example, there is a three-step assembly used, where the E4 module is added to the El -core to create a second macromodule plasmid, followed by assembly with the E3 module. In other schemas and examples (such as the example shown in FIG. 36), an El-core macromodule is assembled with E3 and E4 modules in a 3-fragment Gibson and single step. In this example, the E4 module is amplified with primers tl and t2.

[0505] The PCMN-1313 genome sequence is set forth as SEQ ID NO: 1, with the corresponding virus genome plasmid map illustrated in FIG. 24.

[0506] Table 2. Primer sequences for assembly of CAVSLIC modules in different orders

[0507] El to core Dest AgcttgtctgtaagattacttaCTTTTTT AT A AT GCC A ACTTT GT A El For (rl) AA

[0508] plasmid CAAAAAAGCAG (SEQ ID NO: 92)

[0509] El to pIX-core El Rev (r2) AB Cacgtaccgcgccccttttatac (SEQ ID NO: 93)

[0510] E3 to core 33k CgagtccggcacagactgagcaATGTCTAAAGAAATACCAACC E3 For (si) AE

[0511] assembly CCTTATATGTGGAG (SEQ ID NO: 94)

[0512] T ttcctgaaggcggcagccttaCCGCTCGCGT GT AT GA A A A AT A E3 to E4 no Bglll E3 Rev (s2) AF

[0513] AAG (SEQ ID NO: 95) AgctttatttttcatacacgcgagcgGTAAGGCTGCCGCCTTCAG E4 to E3 E4 For (tl) AF

[0514] (SEQ ID NO: 96)

[0515] E4 to core Dest GgtgggcctttctgcgtttataaCTTATAATGCCAACTTTGTACAA E4 Rev (t2) AD

[0516] plasmid GAAAGCTG (SEQ ID NO: 97)

[0517]

[0518] 7158-105381-02

[0519] E4 to core 33k E4 to core For TCCGGCACAGACTGAGCAGATCTAAGGCTGCCGCCT with Bglll (t3) AC TCAG (SEQ ID NO: 98)

[0520] E4 to core Dest GgtgggcctttctgcgtttataaCTT AT A ATGCC A ACTTTGT AC A A E4 Rev (t2) AD

[0521] plasmid GAAAGCTG (SEQ ID NO: 97)

[0522]

[0523] Transformation and transfection

[0524] E. coli DHIOb cells were used to transform and amplify the assembled virus genome plasmids, which were selected on ampicillin plates, sequence verified and purified for transfection and production in canine cells. MDCK cells or DK cells (such as those from ATCC) were used for viral genome plasmid transfection and virus propagation. Transfection was performed in 6-well plates, using Lipofectamine 3000 (Thermo Fisher), with 2-2.5 pg DNA and 7.5 pl Lipofectamine 3000 diluted and incubated as per standard manufacturer optimized protocols. Upon transfection into mammalian cells, the RSV promoter drives expression of the I-Scel enzyme, which releases and linearizes the CAV genome at the left and right ITRs, allowing viral genome replication. There was no need to cut, isolate, and purify the CAV genome prior to transfection, facilitating efficient viral production.

[0525] Virus successfully propagated in MDCK cells using MEM10 with 2-10% FBS media was transferred to freshly plated MDCK cells for further viral amplification (FIG. 28). Parental plasmids specified were used to assemble intermediate macromodule plasmids ASMM-149 (FIG. 22), ASMM-150 (FIG. 23), and the complete whole-genome plasmid PCMN-1313 (FIG. 24).

[0526] Example 6: Engineering CAVs that express heterologous ORFs operably linked to the CAV proteins though P2A self-cleaving peptide fusions

[0527] CAVs have significantly diverged from both human and non-human primate adenovirus species (FIGS. 5A-5B). The CAV genome is much smaller than hAds, and genome capacity for expressing payloads is more limited. The present disclosure provides a method for assembling and expressing heterologous genes in recombinant CAVs using self-cleaving peptide sequences that functionally link viral gene expression to heterologous ORFs. This eliminates tire need for insertion of additional sequences for ectopic promoters, UTR, and Poly A sequences, which saves genomic space and prevents any negative impact on viral gene transcription and replication caused by such sequences.

[0528] Unlike traditional methods that rely on internal ribosome entry sites (IRES) or separate promoters, self-cleaving peptides ensure stoichiometric expression as translation occurs from a single transcript and does not require deletions or impair functions of essential viral genes (unlike direct fusions), such as for example El A and E1B genes, which results in replication incompetent vectors. Unlike direct fusions, cleavage releases both the heterologous encoded ORF and viral protein upon translation.

[0529] P2A is a self-cleaving peptide sequence derived from the porcine tescho virus- 1 (PTV-1) that enables the co-expression of multiple proteins from a single open reading frame (ORF) without the need7158-105381-02

[0530] for separate promoters, thereby saving genomic space, if such fusions can be made with CAV genes. When placed between tire two ORFs on a single mRNA, the presence of the self-cleaving peptide sequence leads to ribosome skipping and release of the first protein separate from the second protein. In some aspects disclosed herein, tire self-cleaving peptide is a 2A peptide (P2A). The P2A sequence, along with other members of the “2A-like” peptide family (including T2A, E2A, and F2A), functions by causing ribosomal skipping during translation, leading to the production of multiple discrete proteins from a single transcript. The core functional motif of P2A is a conserved GSGEGR(G / S)L sequence (SEQ ID NO: 115), which mediates cleavage between tire upstream and downstream polypeptides during translation. The cleavage occurs via a ribosomal “stop-go” mechanism rather than through enzymatic hydrolysis, meaning that each protein product retains a small residual C-terminal proline (P) or glycine (G) remnant of the 2A peptide.

[0531] The combination of the self-cleaving peptide sequence and the judicious placement of tire exogenous ORF at specific genomic locations, is shown in the following examples. The incorporation of P2A and related 2A peptides into CAV genomes allows for:

[0532] 1. Co-expression of fluorescent reporters with viral proteins to monitor real-time viral replication kinetics and the development of a CAV FBVK quantitative viral replication assay for HTP and HCS scalable screening of libraries of virus variants (see, e.g., FIGS. 39-48 and 59).

[0533] 2. Generation of multi-gene expression constructs at individual placements and combined with different genomic placements (see, e.g., FIGS. 77-82).

[0534] Considerations for engineering and screening P2A-fusions

[0535] To screen for placements that are compatible with P2A fusions, fluorescent proteins were used as the exemplary class of heterologous ORF and reporter gene. YPet has higher brightness compared to eGFP and a shift in the excitation and emission wavelengths into a region of greatly reduced fluorescence from the media. mCherry also has high quantum yields and little overlap with YPet, enabling spectral discrimination and multiple placements and fluorescent proteins to be quantified and screened in recombinant CAVs. A third fluorophore used was BFP, which also has high spectral discrimination. All of these fluorophores also use common filters on standard laboratory microscopes.

[0536] The fluorescence levels are a direct readout of fluorescent protein expression at different genomic placements and enable quantitative comparisons to be made, between and within recombinant CAVs. The spread of fluorescence from initially infected / transfected cells to surrounding cells demonstrates productive secondary and tertiary infection and production of infectious particles, which can be visualized in live imaging platforms, such as Incucyte, Cytation or plate readers.

[0537] The CAV El, core, E3 and E4 plasmid modules were used to engineer, assemble and screen recombinant CAV genomes with P2A-flurophore fusions at different genomic placements and viral genes, including placements in tire El module (El A and E1B-55K), the core module (DNA polymerase,7158-105381-02

[0538] hexon, DNA binding protein (DBP), and fiber), and the E3 module (pVIII and fiber). The P2A fusions were engineered at the 5' and / or 3' of the CAV viral genes.

[0539] E1 -P2A fusions

[0540] An El plasmid module was engineered to encode a YPet-P2A-fusion with the E1A gene, El-271 (FIG. 26; SEQ ID NO: 43). Recombinant CAV genomes were generated by CAVSLIC assembly of the El-271 El modified plasmid with core, E3 and E4 modules (FIG. 25) using the methods described in the Examples above, to assemble PCMN-1324 (FIG. 27; SEQ ID NO: 2). Small and large-scale general workflows for initial screening, production, purification and characterization of CAVs are described in FIG. 28.

[0541] A high content imaging based method to screen libraries of CAVs for replication (FIG. 43) is illustrated by the example and schema in FIG. 29 and hereafter. In these examples, MDCK cells were transfected with whole genome plasmids with fluorescent reporters engineered at different placements. To enable facile non-destructive screening of a large number of CAV variants, an Incucyte high content imaging system or Cytation Multi-Modal imaging system was used. Fluorescent and / or brightfield images were taken approximately once every few hours over several days (3-11 days) and the number of fluorescence-expressing cells quantified over time. The fluorescent payload expression at the engineered locus was directly determined by visualizing fluorescence. Virus replication from initially transfected cells was demonstrated by a log increase in the number of fluorescent cells due to productive CAV replication, infection and spread to cells in surrounding monolayer. At day 13, cells and virus supernatant were harvested and used to perform secondary infections, where again fluorescent transgene expression and virus replication was further validated.

[0542] Using this scheme, MDCK cells were transfected with PCMN-1324. The average life cycle of CAV2 is between 48-72 hours in MDCK cells. At 3 days post transfection, isolated fluorescent plaques were apparent in the MDCK monolayer, indicating successful transfection as well as YPet fluorescent payload expression from the recombinant CAV genome. By day 10, virus infection had spread from initially transduced cells and plaques to infect the entire monolayer, indicating productive log virus replication (FIG. 29). The increase in virally infected fluorescent cells over time demonstrated productive viral replication and secondary infection and spread from initially transfected cells. Productive replication and payload expression was demonstrated by an increase in the number of YPet-positive cells infected over time.

[0543] FIG. 30 shows cesium chloride (CsCl) density gradients of viral supernatants harvested from large scale preps of PCMN-1424, which were compared to an Ad5 virus, PCMN-1346, produced in human 293 cells as a benchmark. An infectious virus band appears lower in the gradient due to the higher density of genome-filled virions and contains fully infectious viral particles. A defective virus band is found higher in the gradient, representing empty capsids or incomplete virions, indicating they may lack functional genomes, or have packaging defects that render them non-infectious.7158-105381-02

[0544] Productive recombinant CAV virus genome assembly and infectious particle production was demonstrated by a visible and strong lower white band of concentrated recombinant CAV virus particles and the absence of upper unfilled defective virus particles.

[0545] YPet-P2A-pVIII fusions

[0546] An example of a P2A-fusion in the E3 module is with tire late expressed pVIII gene. In this example, an E3 plasmid module was engineered to encode a YPet-P2A-fusion with tire pVIII gene, E3-612 (FIG. 31; SEQ ID NO: 60) and used to assemble the PCMN-1584 viral genome (FIG. 32; SEQ ID NO; 34) using CAVSLIC, similar to methods for previous examples. MDCK cells were transfected with PCMN-1584 and fluorescence images captured and quantified over several days (FIG. 35). The increase in virally infected fluorescent cells over time demonstrated productive viral replication and secondary infection, with spread from initially transfected cells. Notably, the optimal placement of ectopic genes differed between the two platforms: in CAV, N-terminal YPet-P2A fusions to pVIII were functional, whereas in hAd5, N-terminal fusions failed but C-terminal fusions were tolerated. These reciprocal differences highlight the functional and sequence divergence between CAV and hAd5 and underscore that successful ectopic gene placements in one adenovirus platform cannot be reliably predicted from studies in the other.

[0547] ElB-55k-YPet-P2A fusions

[0548] Another example of a P2A-fusion in the El module is with the ElB-55k gene. In this example, an El plasmid module was engineered to encode a YPet-P2A-fusion with ElB-55k, El -285 (FIG. 33; SEQ ID NO: 44) and used to assemble the PCMN-1422 viral genome (FIG. 34; SEQ ID NO: 16) using CAVSLIC. MDCK cells were transfected with PCMN-1422 and fluorescence images captured and quantified over several days (FIG. 35). The increase in virally infected fluorescent cells over time demonstrated productive viral replication and secondary infection and spread from initially transfected cells.

[0549] PCMN-1466: YPet-P2A-E2B-DNA polymerase fusion

[0550] An example of a P2A-heterolous ORF placement in tire core E2 DNA replication gene is illustrated in FIG. 36. E2B DNA polymerase is expressed from the E2 promoter and is essential for CAV genome replication and virus production. The Core-065 plasmid (FIG. 37; SEQ ID NO: 62) was engineered to have a P2A-YPet fusion with E2B polymerase and then assembled via CAVSLIC with an El plasmid to generate ASMM-176 macromodule plasmid (FIGS. 36 and 37; SEQ ID NO: 41), which was then assembled with E3 (si and s2) and E4 (t2 and tl) modules in a 3-fragment SLIC using primers listed in Table 2 in Example 5.

[0551] MDCK cells were transfected with PCMN-1466 whole genome plasmid (FIG. 38; SEQ ID NO: 19). Between 3-10 days post transfection, fluorescent plaques appeared in the MDCK monolayer,7158-105381-02

[0552] indicating successful transfection, and YPet fluorescent payload expression from the P2A fusion with E2B-DNA pol (FIG. 39). PCMN-1466 and YPet fluorescence spread to infect the entire monolayer, indicating productive log replication (FIG. 39). As such, the increase in virally infected fluorescent cells over time demonstrated productive viral replication and secondary infection and spread from initially transfected cells.

[0553] Failed Examples of P2A fusions

[0554] A virus is considered failed when it does not produce plaques within 10 days after transfection. The transfection is attempted two additional times to avoid a false declaration of failure.

[0555] FIGS. 40 and 41 show failed examples of P2A fusions of ORFs to viral genes, including, PCMN- 1467 (YPet-P2A-Hexon), PCMN-1384 (Fiber-P2A-YPet), PCMN-1469 (DBP-P2A-YPet), and PCMN- 1468 (YPet-P2A-DBP). At 3 days after transfection, some sparse initially transfected YPet expressing cells were apparent. However, in contrast to previous examples, these exemplary CAV genome placements and P2A fusion compositions did not undergo productive replication and secondary infection, as evidenced by the lack of YPet-positive cells at later timepoints and on serial passaging of viral supernatants.

[0556] Summary

[0557] Table 3 below summarizes P2A fusions to the CAV genes that were tested and compares results to hAd5 fusions tested previously (U.S. Patent Nos. 10,738,325 and 11,130,968). Neither the successful nor the failed examples of CAV P2A fusions with CAV genes to express heterologous ORFs could have been predicted or expected from studies in hAds. For example, P2A-YPet fusions at the C-terminus of DBP were successful in hAd5, but not in CAV. Furthermore, in CAV, P2A fusions at the N-terminus of pVIII were successful but failed in hAd5, and vice versa.

[0558] Table 3. Comparison of the replication of hAd5 and CAV2 with P2A fusions to fluorescent proteins to viral genes at 10 days post transfection in 293 and MDCK cells, respectively.

[0559] Similar N-terminal / C-terminal genome N-terminal / C-terminal genome between hAd5 structure amendable? addition success? and CAV Canine Ad2 Human Ad5 Canine Ad2 Human Ad5

[0560] E1A Yes / Yes Yes / Yes N-(nd) / C-Yes N-Yes / C-Yes Yes E1B 55K No / Yes No / Yes C-Yes C-Yes Yes pol Yes / No Yes / No N-Yes N-Yes Yes DBP Yes / Yes Yes / Yes N-No / C-No C-Yes No pVIII Yes / No Yes / Yes N-Yes N-No / C-Yes No

[0561] E3-ORF1 No / Yes Yes / Yes CAV specific hAd5 specific N / A

[0562] E3 ORF2 Yes / No Not applicable CAV specific hAd5 specific N / A

[0563]

[0564] fiber No / Yes Yes / Yes C-No C-Yes No7158-105381-02

[0565] The Table compares recombinant CAV replication in MDCK cells within 10 days of transfection and recombinant human Ads in HEK293 cells within 10 days of transfection. Nd-not done / tested

[0566] Example 7: Fluorescent reporters enable the development of scalable HTP and HCS screening of CAV variants, virus replication to be visualized and kinetics quantitatively determined and compared

[0567] This example shows that the reporter gene placements in CAV modules and assembled recombinant CAV genomes, enable high content imaging and high-throughput plate reader optical measurements of transgene expression at different genomic placements, as well as viral replication and cytopathic effects and virus replications kinetics to be quantitatively determined and compared for large libraries of CAV variants.

[0568] High content screening and titering enabled by fluorescent plaque detection

[0569] In contrast to Ad5, CAV infected cells and plaques are not so easily distinguished or counted, in all cell types. Furthermore, recombinant CAVs that express heterologous therapeutic immunogenetic antigens, such as fusogenic proteins from pathogens, for example CDV-F, are very hard to identify as they change the appearance and ability to detect plaques (FIG. 95). However, it is shown herein that CAVsembly / SLIC modules with fluorescent proteins enable CAV plaques and infected cells to be identified and counted at early stages unambiguously and transfection efficiencies, titers, and replication to be determined. This enables high content screening (FIG. 43). Furthermore, by linking fluorescent protein expression to high viral gene expression and amplification, fluorescent signals are readily observable on basic benchtop microscopes (FIG. 42).

[0570] The Fluorescence-Based Viral Kinetics (FBVK) assay

[0571] The FBVK assay (see also PCT Publication No. WO 2017 / 147265) enables high-throughput, and quantitative measurement of CAV replication kinetics across species, cell types, and genetic variants for the first time (FIGS. 43-47). The assay overcomes the limitations of existing methods and the lack of an unambiguous and sensitive quantitative nondestructive assay for productive viral replication over several life cycles.

[0572] Table 4. Comparison with Existing Methods and Assays

[0573] Method Measures Limitations

[0574] qPCR Viral genomes Does not measure infectivity or secondary spread

[0575] Western Blot Late viral proteins Single time point; labor-intensive Plaque Assay Infectious virus Requires soft agar; time-consuming ELISA Viral proteins Requires virus-specific antibodies

[0576]

[0577] 7158-105381-02

[0578] MTT / WST-1 Cell viability Indirect; affected by bystander effects Assay

[0579] FBVK Assay Real-time, quantitative High-throughput, non-destructive, applicable replication kinetics to any adenovirus

[0580] To overcome these limitations and enable CAV replication to be quantified and compared to CAVs with different heterologous ORFs, a FBVK assay was developed (see also PCT Publication No. WO 2017 / 147265).

[0581] The FBVK assay is high-throughput (e.g., 96-well to 1534- well plates can be used), nondestructive, insensitive to initial viral titer, highly sensitive to small differences in viral replication rates, and captures multiple viral infection cycles. All that is necessary is an initial infection (or transfection) that results in transduction of a small fraction of cells in the tissue culture dish. The remaining, unaffected cells are available for secondary and tertiary infection. This is accomplished by performing serial dilutions of virus supernatants or transfecting cells such that less than 10% of the cells are initially transduced (FIG. 44). This enables virus replication and amplification, as measured by fluorescence intensity increases over time, to be quantified over several days.

[0582] Quantifying fluorescence every few hours enables the exponential rate of viral replication to be calculated from the slope of a semi-log plot of fluorescence versus time (FIG. 44). This is analogous to optical density (OD) readings of batch phase bacterial cultures and log-phase growth analyses. By infecting a small percentage of cells initially, primary, secondary, and tertiary replication can be captured (FIG. 45). A semi-log plot of exponential fluorescence (or fluorescent cells) yields a straight line proportional to the exponential virus replication rate. The FBVK assay has tire ability to not only assess the magnitude of difference in replication kinetics between two viruses, even those from different species (see FIG. 46) but also give valuable information as to which parts of the replication process might be defective in viruses with slower rates of replication (FIG. 45). Taking the slope of die logaridrm of die exponential growdi in fluorescence signal vs. time results in a single value for each viral construct diat can be cross-compared regaidless of signal magnitudes or any initial time delay tiiat might occur before exponential growth begins. This feature of data interpretation makes die assay insensitive to initial starting points. Poor control or even knowledge of initial viral titer has no impact on the log-slope during exponential growth.

[0583] The CAV fluorescent replication kinetics assay enables large libra ies of CAV variants to be screened by incorporating El, core, E3 or E4 modules with fluorescent proteins into assembled libraries of viruses. The resulting virus genomes can then be transfected into cells or viral supernatants used to robustly and quantitatively determine virus replication titers in a high throughput format and high accuracy.

[0584] This enables high-throughput, nondestructive measurements of viral replication in 96- or 384-well plate formats, which do not require knowledge of starting titers and can be sued in any cell type, and7158-105381-02

[0585] is highly sensitive to small differences in viral replication rates. Unlike traditional single-step growth curves, this method is insensitive to initial titer variations, allowing direct comparison between different viral constructs, cell lines and across species (see following examples, but not limited to these).

[0586] Example Applications of fluorescent ORF placements and CA V module and genome composition for the quantification, comparison and screening of CAVs

[0587] Comparing Viral Gene Expression and Replication of Recombinant CAVs

[0588] In the example shown in FIG. 45 with PCMN-1324, it was demonstrated that CAV fluorescent protein placements enable the quantification of recombinant CAVs replication kinetics, life cycle and gene expression to be determined.

[0589] FBVK protocol

[0590] The assay can be performed with either recombinant CAV genome plasmids or recombinant adenovirus particles as the starting material. When starting with a recombinant adenovirus genome, the assay includes transfecting cells with adenovirus genome plasmids and monitoring fluorophore expression over time (FIGS. 43 and 45). Transfection conditions are selected such that about 5-10% of the cells are initially transfected. Cells that are not initially transfected are available for secondary infection by virus particles produced from the initial transfection. Log-slope is used as a measure of kinetics based on secondary, tertiary, and quaternary, etc. infections, thus it is not necessary to know the percentage of cells that are initially transfected. FIG. 43 shows an exemplary virus-based kinetics assay. Multiwell plates are used with 3 "blank" wells with FLUORESBRITE beads, which compensate for sensitivity drift. Triplicates are used for each virus construct. Once cells are transfected, the plate is placed in a TECAN plate reader for continuous fluorescence monitoring or automated robotics system that enables plate readings to be made at least one every hour. The data collected is used to calculate Inslope for each construct.

[0591] The assay can also be carried out by infecting cells with recombinant virus panicles. In this version of the assay, cells arc infected with recombinant vims particles and monitored over time (FIG.

[0592] 45). As with the genome plasmid version of the assay, it is not necessary to know the exact titer of the starting vims stock. Typically, a dilution series is used for initial infection, such as a dilution series ranging from 1 :100 to 1 :218,700, as shown in FIG. 5. A dilution of 1:100 generally leads to infection of all cells, whereas a dilution of 1:218,700 generally leads to initial infection of very few cells, number of different viruses and between different cell types.

[0593] Calculating log-slope

[0594] To measure log-slope, the linear plot of fluorescence intensity versus time is converted to a semilog plot by taking the natural logarithm of the measured fluorescence intensity at each time point. Since7158-105381-02

[0595] the fluorescence intensity exhibits exponential growth during viral replication, this conversion results in a straight line when plotting hi(fluorescence intensity) vs. time. This straight line is then fit using standard least-squares methods. The resulting slope produced by this fit is the In-slope of the fluorescence vs. time and thus the In-slope of the viral growth vs. time. In FIG. 45, using PCMN-1324 as an example, it is shown that this enables viral replication kinetics to be quantified as well as the timing and level of fluorescent transgene expression over the time course of several days.

[0596] Example 8: Comparing viral replication kinetics and characteristics of CAVs and hAds

[0597] In FIG. 46 it is shown that log slope enables direct comparisons of CAV replication kinetics to that of Ad5. This enables for the first time the expression and transcription dynamics and output of El A expression to be compared between Ads of different species, CAV and Ad5. The expression levels of E1A are higher in CAVs than Ad5 (y axis). Also, viral replication kinetics and log slope can be compared. Ad5 has faster kinetics than CAV, and the main contribution appears to be in the lag phase, where Ad5 is more lytic. This may reflect the species specific and serotype expression of the ADP death protein in the E3 region of Ad5 versus CAVs, which do not have this protein.

[0598] Example 9: Applications of Fluorescent Reporter Placement in Canine Adenovirus (CAV) for Screening and Optimization of Virus Production and replication in different conditions

[0599] The reporter gene placements and compositions described in this disclosure enable quantitative comparisons of viral replication kinetics over several days and life cycles, and can be utilized for a range of basic and applied research applications, including but not limited to vaccine development, therapeutic vector manufacturing, neutralizing antibodies, oncolytic virus therapies and antiviral drug screening.

[0600] In one representative example, a recombinant CAV with an El A-P2A-YPet reporter module, PCMN-1324 (FIG. 47; SEQ ID NO: 2), was used to infect MDCK cells under different cell culture conditions (FIG. 47). The purpose of this experiment was to identify optimal conditions for the small and large-scale production of recombinant CAV viruses, such as those intended for clinical or therapeutic use. The experimental variables included (1) the use of different types of basal cell culture media and (2) the frequency of media exchange (e.g., static versus daily replacement). In the example shown, MDCK cells were infected with PMCN-1324, and cultured in DMEM or MEM, with or without a daily media change. The results showed that MEM with daily media exchange was far superior to die other conditions tested, and enabled logarithmic virus replication, higher expression levels of El A (as measured by die P2A fused YPet fluorescence intensity) and superior virus production and infection (FIG. 47).

[0601] These exemplary data show die platform is scalable and supports die use of fluorescent CAV modules and viruses for optimizing manufacturing parameters for C AV-based viral vectors. Importantly, while die cunent example utilized a fluorescent reporter, it is contemplated that any quantifiable7158-105381-02

[0602] reporter — such as luciferase, P-galactosidase, or other enzymatic readouts — could be substituted depending on tire desired sensitivity or detection method.

[0603] This approach can be adapted to high-throughput screening formats, to screen different cell lines, either naturally derived or genetically engineered, for their ability to support high-titer virus production under Good Manufacturing Practice (GMP)-compatible conditions (FIG. 43). In a related aspect, this same platform can be used to evaluate small molecule inhibitors, antiviral compounds, or immunomodulatory drugs by quantifying viral replication in the presence of such agents. Using a fluorescence-based viral kinetics (FBVK) assay, dose-response curves can be generated in real time, allowing the determination of IC50 values, cytotoxicity thresholds, and other pharmacologic parameters relevant to preclinical drug screening. Similarly. The same platform can be used in CRISPR / RNAi based knockdown or cDNA overexpression screens in cell lines to identify genes that enhance or decrease CAV replication or pathogenicity.

[0604] Thus, tire integration of reporter genes into strategic positions within the CAV genome enables a broad range of functional assays that support both basic virology and the development of clinically relevant viral therapeutics. These tools provide a robust, quantitative, and high-throughput means to interrogate viral biology, optimize production workflows, and evaluate candidate therapeutics across diverse cellular contexts.

[0605] Example 10: Neutralizing antibodies

[0606] Another example is the utility of fluorescent proteins in CAV compositions for neutralizing antibody assays to determine if human or animal subjects that are naive or vaccinated or T cell mediated responses to recombinant CAV infected fluorescent cells in co-culture assays. In this aspect (see FIG. 48), CAVs with fluorescent ORFs are incubated with FBS control or serum from patients to determine if serum components, prevent infection and replication in canine cell lines, such as MDCK or DK cells.

[0607] Example 11: Screening and determining the effects of CAV gene deletions and therapeutic transgenes on CAV replication

[0608] As is shown in several examples below, CAV modules with fluorescent reporters can also be used to assemble recombinant CAVs with gene deletions and / or heterologous ORFs / therapeutic ORFs to systematically quantify and compare tire effects of different genetic modifications on CAV replication kinetics with high sensitivity and accuracy. This can be used to identify placements for therapeutic ORFs, such as vaccine antigens (see examples in FIGS. 92-94) as well as genetic compositions for development of oncolytic viruses, which conditionally replicate in canine tumor versus normal cells.

[0609] The ability to express reporter genes in CAVs that have wild type virus replication, represents a fundamental advancement in quantifying CAV replication kinetics, offering a real-time, high-throughput, high content imaging and broadly applicable platform for viral vector development, vaccine production, and antiviral screening. By optimizing the placement of fluorescent reporters and utilizing imaging and7158-105381-02

[0610] kinetic analysis, robust, scalable, and quantitative comparisons and interrogation of large libraries of CAV variants is enabled.

[0611] Example 12. Direct heterologous ORF fusions to CAV genes, DBP and E3-ORF1

[0612] In addition to testing heterologous ORFs with P2A fusions, direct fusions to viral proteins were also tested. Although these are not as flexible in their applications as P2A fusions, where the heterologous ORF is operably linked but not a protein fusion to tire CAV ORF protein from which it is transcribed and translated as it is cleaved by ribosome, direct fusions can be useful as fluorescent reporters of viral replication or for example if an engineered heterologous ORF functions as a fusion or T cell epitope.

[0613] Two exemplary placements are shown whereby protein fusions with CAV ORFs are tolerated and where the assembled viruses are still replication competent, albeit attenuated compared to wild type CAV or comparison to other placements, such as several of the P2A examples.

[0614] PCMN-1470: E2A-DBP-YPet Fusion

[0615] FIG. 41 shows that P2A fusions with E2A DBP failed. However, direct protein fusions with E2A-DBP were also explored. A core module, Core-076 (FIG. 49; SEQ ID NO: 64), was engineered with a C-terminus fusion of YPet to E2A-DBP, and assembled with wildtype El, E3 and E4 modules via CAVSLIC, using methods described in previous examples. The assembled CAV genome, PCMN-1470 (SEQ ID NO: 23), was used to transfect MDCK cells (FIG. 50). At 3 days post transfection, some sparse green fluorescent cells were observed. By day 16, initially transfected cells had expanded to form large plaques. Therefore, PCMN-1470 was able to undergo productive secondary infection and infect surrounding cells, but was attenuated compared to other CAV module placements (for example, E1A-P2A-YPet fusions, FIGS. 35 and 39) and has slower replication kinetics.

[0616] PCMN-1363: E3-ORFl-GS-mCherry fusion

[0617] In this example it is shown that heterologous ORF fluorescent reporters can be expressed as fusions with the putative E3-ORF1 encoded gene. In this example, a modified E3 plasmid module, E3-538 (SEQ ID NO: 51), was engineered to delete the predicted E3-ORF1 stop codon and instead an mCherry fluorescent reporter gene was fused to the C-terminus of E3-ORF1, separated by a GS linker, to facilitate independent protein folding and functionality (FIG. 51). E3-538 was assembled with El, core and E4 modules to assemble PCMN-1363 (SEQ ID NO: 8). MDCK and DK cells were infected PCMN-1363 and virus replication kinetics quantified and compared in an FBVK assay (FIG. 52). PCMN-1363 replicated better in DK cells than MDCK cells but had a log slope that was almost 25-50% reduced compared to wildtype virus (and compared to CAVs with replacement of E3-ORF1 as described in examples below).7158-105381-02

[0618] Example 13. Examples of CAV genes that can be deleted to increase payload space or placements for engineering and expressing heterologous ORFs

[0619] The CAV genome is smaller than Ad5 and pay load capacity for heterologous encoded ORFs and fusions is limited. Therefore, it was desirable to delete CAV genes to A) increase cargo size of heterologous ORF payloads expressed at any placement and B) provide potential placements where heterologous ORFs or expression cassettes could be engineered.

[0620] E3 gene deletion examples

[0621] The annotation of CAV genes in the NCBI database is largely hypothetical, relying on computational predictions rather than experimental validation. The E3 genomic region and predicted ORFs encoded therein are the most divergent, with no homologs in Ad5 (see FIG. 6).

[0622] In the NCBI reference genome annotations, the CAV E3-ORF1 and E3-ORF2 are annotated as putative and lacking experimental validation.

[0623] To determine if deletions of putative E3-ORF1 and E3-ORF2 genes impact CAV virus replication, E3 plasmid modules were engineered with deletions of E3-ORF1 (E3-527; SEQ ID NO: 47), E3-ORF2 (E3-528; SEQ ID NO: 48) or both (E3-529; SEQ ID NO: 49), which were assembled with an El-271 plasmid module (SEQ ID NO: 43) that has an ElA-P2A-YPet reporter, to create PCMN-1326 (SEQ ID NO: 3), PCMN-1327 (SEQ ID NO: 4), and PCMN-1328 (SEQ ID NO: 5) (FIGS. 53-59) and Table 5.

[0624] Table 5. FBVK Log slopes and virus replication kinetics in MDCK cells for recombinant CAVs with E3-ORF1 / 2 deletions

[0625] Virus SEQ ID NO: Virus genotype Log-slope (day1) PCMN-1324 2 ElA-P2A-YPet 2.00 PCMN-1326 3 ElA-P2A-YPet, AE3-ORF1 1.94 PCMN-1327 4 ElA-P2A-YPet, AE3-ORF2 0.97 PCMN-1328 5 ElA-P2A-YPet, AE3-ORF1, AE3-ORF2 1.93

[0626]

[0627] MDCK cells were infected with either PCMN-1324, PCMN-1326, PCMN-1327 or PCMN-1328. Viruses were serially diluted from high to low in a 96-well plate format and fluorescence was monitored over several days in a plate reader. Semi-log plots of YPet fluorescence versus time were plotted and log slopes calculated in the linear part of curve (FIG. 59).

[0628] These data show that viruses with E3-ORF1 deletions have wildtype virus kinetics and are dispensable for viral replication, at least in cell culture. In contrast, the deletion of E3-ORF2 had a significant impact on viral replication kinetics, and log slope is half that of wildtype virus or viruses with deletions in E3-ORF1. The FBVK curves indicate that deletions of E3-ORF2 impact secondary CAV virus lysis and spread, which is reminiscent of E3-ADP in hAd5. However, strikingly, deletions of E3-7158-105381-02

[0629] ORF1 together with E3-ORF2 restore virus replication kinetics and enable wild type virus replication kinetics. This fortuitous and unanticipated discovery also creates an additional 1.5 kb of genomic space for engineering heterologous ORFs in CAVs that replicate better than CAVs with a deletion of E3-ORF2 alone.

[0630] Payload CAV2 ORF(s) Modification Virus Replication

[0631] size (bp) E3-ORF1 Deletion PCMN-1326 Yes 360

[0632] E3-ORF2 Deletion PCMN-1327 Yes 1095

[0633] E3-ORF1 and E3- Deletion PCMN-1328 Yes 1413

[0634] ORF2

[0635]

[0636] Example 14: Genetic modifications, design considerations and engineering of E3 genomic placements

[0637] Genomic payloads that exceed a critical threshold, approximately 3 kb, can result in defective viruses, which are unable to package the recombinant genome within the capsid.

[0638] As such, disclosed herein and in this example is an approach that harnesses the viral transcriptional architecture and CAV gene deletions to express heterologous ORFs.

[0639] The CAV E3 genes are not well characterized but can be deleted and still maintain virus replication similar to wild type virus kinetics (FIG. 59). Previous studies have randomly deleted E3 sequences to insert ectopic promoters in the CAV E3 region to drive the expression of heterologous genes. However, it has been previously shown in human Ad5 that although deletions of E3 maintain Ad replication, placing ectopic promoters to express payloads in this genomic location can result in a severe defect in viral replication and / or select for loss of the transgene and payload (WO 2022 / 010949). Placing a strong unregulated ectopic promoter in the E3 genomic region results in defects in late capsid protein expression, which can be attributed to unregulated transcriptional interference with major late promoter encoded L4 and L5 transcripts. These data are also corroborated by Suzuki et al. in a non-replicating Ad5 vector (Suzuki et al., Clin Cancer Res 7(1): 120-126, 2001). In HEK293-E4 cells, Suzuki et al. found that an exogenous gene employing the EFl a promoter, placed in the E3 region, led to greatly reduced virus particle yield. Given the conserved viral genome organization between Ads across species, placing promoters in the CAV E3 region is also expected to lead to a loss in virus replication and likely select for genome instability and loss of transgene expression.

[0640] FIG. 61 presents a sequence alignment of the pVIII sequence from human adenovirus 5 (Ad5), human adenovirus 34 (Ad34), canine adenovirus 1 (CAV1), and three strains of canine adenovirus 2 (CAV2), specifically the Toronto strain, G1 strain, and SHR2 isolate. The alignment is displayed relative to the Ad5 reference sequence, with the consensus sequence shown on the top line. The alignment reveals sequence divergence between the canine adenovirus strains and the human adenoviruses. However,7158-105381-02

[0641] despite these differences, a conserved TATA box and promoter element remain is predicted to be preserved in CAV genomes, including both CAV1 and CAV2 isolates. While this promoter activity in CAV1 and CAV2 has not been previously characterized or demonstrated, the conservation of key regulatory elements strongly suggests its functional relevance. As such, the present disclosure sought to test and engineer heterologous ORFs in the E3 region without inserting ectopic promoter or regulatory sequences that might interfere with viral replication but instead harness a putative conserved native viral promoter architecture identified and predicted herein.

[0642] To optimize hanslation efficiency of heterologous encoded ORFs, an exemplary optimal Kozak sequence motif (FIG. 62) was engineered by inserting a G nucleotide immediately after the pVIII stop codon, at the -1 position relative to, in this example, the mCherry start codon (ATG), and deleting E3-ORF1 sequences, GAGAGC after the PVIII stop and before a putative ATG-methionine start codon. This engineered deletion of specific E3-ORF1 sequences and engineered Kozak consensus sequence, is predicted to enhance ribosomal recognition and Panslation efficiency of a heterologous ORF at this idealized placement. Notably, a Kozak consensus sequence is absent in Ad5 E3 12.5k, further highlighting its unique design for CAV-based platforms.

[0643] Example 15: E3-ORF1 deletion and replacement with mCherry

[0644] Incorporating these design considerations (FIG. 62), mutations were engineered in E3-532 (FIG.

[0645] 63; SEQ ID NO: 50), and E3-ORF1 was replaced with mCherry to assemble PCMN-1331 (FIG. 64; SEQ ID NO: 6). The non-coding sequences at the junction between the end of E3-ORF1 and start of the E3-ORF2 were retained as they may have important predicted regulatory motifs that function in transgene expression and viral replication (FIG. 68). These sequences include putative polyadenylation (Poly-A) signals and splicing motifs.

[0646] FIG. 65 shows an FBVK assay of MDCK cells infected with PCMN-1331. These data demonstrate that E3-ORF1 coding sequences can be engineered and modified and replaced with that of a heterologous ORF, such as in this example mCherry, and achieve high levels of heterologous ORF expression and good virus replication kinetics. It is shown that the engineered synthetic features result in higher fluorescence levels (which is a measurement of mCherry expression) compared to when mCherry is expressed as fusion with E3-ORF1 (FIG. 59) by almost two orders of magnitude. The novel and optimized design harnesses and improves upon tire native viral transcription / translation architecture, and does not require the insertion of ectopic promoters or sequences.

[0647] Failed example E3-ORF2 deletion and replacement

[0648] In contrast to E3-ORF1, replacing E3-ORF2 coding sequences with mCherry failed, possibly from disrupting viral splicing / translation. Here the deletion and replacement of E3-ORF2 with a heterologous ORF (mCherry) completely killed virus replication. Table 6 shows that irrespective of7158-105381-02

[0649] different combinations tested, viruses that replace E3-ORF2 with mCherry cannot be produced, even when E3-ORF1 is also deleted (Table 6).

[0650] Tabic 6. Replication of CAVs with deletions in E3-ORF1 and / or E3-ORF2

[0651] Virus Composition Replication?

[0652] PCMN-1331 AE3-ORF 1 [mCherry ] Yes

[0653] PCMN-1366 AE3-ORFl[mCherry], AE3-ORF2 Yes

[0654] PCMN-1332 AE3-ORF2[mCherry] No

[0655] PCMN-1369 AE3-ORF1, AE3-ORF2 [mCherry] No

[0656] PCMN-1350 ElA-P2A-YPet, AE3-ORF2[mCheny] No

[0657]

[0658] It was also determined whether E3-ORF1 could be replaced with mCherry with additional deletions of E3-ORF2. The E3-541 plasmid (SEQ ID NO: 52) has mCherry in place of E3-ORF1 (as before) but also has a deletion of the E3-ORF2 protein coding sequences (FIG. 67). PCMN-1366 was assembled using E3-541 and El, core and E4 modules. MDCK cells transfected with PCMN-1366 express mCherry fluorescence and the virus was replication competent, as evidenced by an increase in mCherry infected cells between days 3-9 (FIG. 69 A) and characteristic banding pattern of high density infectious particles in CsCl gradient (FIG. 69B).

[0659] Example 16: Mutations in alternative ATG start codons in pVIII for expression of a heterologous ORF at pVIII / / E3-ORFl engineered sequences

[0660] While developing CAV constructs for therapeutic transgene expression, a potential interfering mechanism was identified whereby alternative start codons, present in alternative reading frames (ARFs) of the protein VIII coding sequence in both CAV1 and CAV2, may initiate translation upstream of the inserted heterologous ORF. This phenomenon could result in N-terminal extensions, in-frame fusions, and aberrant localization (e.g., if signal peptides are inadvertently translated), undesirable for expression or functions of a heterologous ORF.

[0661] FIG. 70 shows an exemplary sequence alignment of tire Protein- VIII (pVIII) and E3 open reading frame 1 (E3-ORF1) junctions from human adenovirus 5 (hAd5), canine adenovirus 1 (CAV1), and canine adenovirus 2 (CAV2). In hAd5, tire initiation codon for E3-ORF1, encoding the 12.5 kilodalton protein, occurs immediately downstream of the pVIII stop codon. However, in CAV1 and CAV2, potential alternative start codons are predicted within an alternative reading frame at tire C- terminus of pVIII. The C-terminal amino acid sequence of pVIII is conserved across all strains as “DGYD” (SEQ ID NO: 117). In an alternative reading frame, this degeneracy in CAVs could result in translation starting at alternative start codons that could result in undesired N-terminal peptide fusions to engineered transgenes at tire E3-ORF1 placement that have unintended functional consequences and unpredictable effects. To address this, E3-572 (FIGS. 71 and 72; SEQ ID NO: 56) was engineered, which7158-105381-02

[0662] incorporates two silent nucleotide substitutions that do not alter the pVIII amino acid sequence but abolish the alternative ATG start codons in the +2 reading frames. E3-572 was assembled to create the PCMN-1428 virus (FIG. 73; SEQ ID NO: 18).

[0663] Evaluation of expression and replication of engineered pVIII / E3-ORFl placement in PCMN-1428 versus PCMN-1366

[0664] The performance of PCMN-1428 was compared to PCMN-1366. In FIG. 74, mCherry fluorescence intensity is plotted against time (in hours post-infection). MDCK cells were infected with two different dilutions of either PCMN-1428 or PCMN-1366. High-content imaging was used to collect fluorescence data at 1-2 hour intervals. The results show that mCherry expression in PCMN-1428 is 3-4 times higher than in PCMN-1366 across both dilutions. Additionally, PCMN-1428 exhibits enhanced replication kinetics, indicating that the engineered mutations do not compromise, and may actually improve, virus fitness.

[0665] Western Blot confirms improved protein fidelity

[0666] FIG. 75 shows a Western blot analysis comparing the size of mCherry protein expressed in lysates from PCMN-1428- and PCMN-1366-infected cells. The blot reveals that mCherry expressed from PCMN-1366 migrates at a higher molecular weight, consistent with an N-terminal extension due to the predicted unintended upstream initiation from ATGs in pVIII. In contrast, mCherry from recombinant PCMN-1428 migrates lower at the expected size, confirming that elimination of alternative start codons restores translational fidelity of the engineered placement and heterologous ORFs.

[0667] Conclusion

[0668] These findings support the conclusion that translational events, resulting from cryptic alternative start codons in pVIII, which are not conserved in hAds, can interfere with the optimal expression and function of heterologous genes in recombinant viral genomes. The engineered synonymous mutations described herein provide a novel and effective solution, enabling accurate translation initiation without alternative start codons and N-terminal extensions of heterologous ORFs that have been engineered to exploit native transcriptional sequences.

[0669] Example 17: Heterologous ORF expression timing and levels is tunable by the different genomic placements

[0670] This example illustrates the application of the disclosed system by comparing the expression of an identical heterologous ORF, YPet, inserted at two different locations within the CAV genome. Specifically, YPet was expressed either as a P2A-linked fusion to the E1A gene (PCMN-1326; SEQ ID NO: 3) or by deleting and replacing E3-ORF1 with YPet (PCMN-1386; SEQ ID NO: 14).7158-105381-02

[0671] FIG. 76 presents a side-by-side comparison of YPet fluorescence levels and FBVK viral replication kinetics assay of MDCK and DK cells infected with either PCMN-1326 or PCMN-1386. YPet is almost two orders of magnitude higher when expressed at the E3-ORF1 deleted locus design compared to P2A-fusions with E1A. The log slope is the same, indicating that these differences reflect the properties of the different E1A versus E3-ORF1 genomic placements to express heterologous ORFs.

[0672] Another example is shown in Table 7. Expression levels of YPet are approximately 50-fold higher when inserted at the E3-ORF1 heterologous ORF placement compared to a P2A-ElB-55k fusion. Furthermore, P2A fusion to E1A versus ElB-55k results in a two-fold higher expression level of YPet.

[0673] Table 7. YPet expression from different genomie locations

[0674] E3- YPet expression level from FBVK assay in DK cells E1A E1B ORF1

[0675] PCMN-1375 ElA-P2A-YPet, AE3-ORF1 [mCherry], AE3- 230

[0676] ORF2

[0677] PCMN-1423 ElB55k-P2A-Ypet; AE3-ORF1 [mCherry], AE3- 95

[0678] ORF2

[0679] PCMN-1386 AE3-ORFl[YPet] (NOTE: virus contains E3- 4530

[0680] ORF2)

[0681]

[0682] This example confirms that different genomic loci within the CAV genome exhibit reproducible and predictable expression properties, which can be harnessed to modulate the expression level of a given transgene (e.g., lower from E1A-P2A, higher from E3-ORF1). These properties are particularly valuable for developing recombinant CAVs for diverse applications, including multivalent vaccine vectors, gene therapy platforms, and reporter viruses for research or screening.

[0683] Example 18: The expression levels of a heterologous ORF that replaces E3-ORF1 is increased by deleting E3-ORF2 in cis

[0684] PCMN-1367 (SEQ ID NO: 10) and PCMN-1368 (SEQ ID NO: 11) were assembled and compared to evaluate the impact of single versus double E3-ORF deletions to heterologous ORF expression at the E3-ORF 1 placement. Both viruses express mCherry fused via a glycine-serine (GS) linker to blue fluorescent protein (BFP) in place of E3-ORF1 coding sequences. PCMN-1368 carries an additional deletion of the E3-ORF2 gene, while PCMN-1367 retains E3-ORF2.

[0685] Expression levels of both mCherry and BFP are significantly enhanced in PCMN-1368 compared to PCMN-1367, indicating that deletion of E3-ORF2 provides a more favorable transcriptional or post- transcriptional environment for heterologous ORF expression from the engineered replacement of heterologous ORFs at the E3-ORF1 locus. The log-slope of replication — as calculated from fluorescence7158-105381-02

[0686] intensity over time — demonstrates that PCMN-1368 replicates more efficiently than PCMN-1367. These data suggest that removal of E3-ORF2 not only enhances expression but also improves viral fitness, possibly by relieving interference or competition between tire inserted cassette and residual E3 gene products.

[0687] The ability to compare placements across El, E3, Core, and E4 has revealed that each locus supports distinct functional outcomes in the context of transgene expression. For instance:

[0688] • The E3-ORF1 locus supports the highest expression levels, especially when E3-ORF2 is also deleted.

[0689] • The E1A-P2A locus supports moderate, early-phase expression, suitable for transgenes that benefit from early kinetics.

[0690] • The E1B-55K locus supports lower expression, which may be desirable for payloads that would otherwise inhibit viral replication if overexpressed.

[0691] • Core genes (e.g., polymerase) exhibit stringent tolerance to insertions and must be engineered with care, as demonstrated by non-viable combinations in other examples.

[0692] These examples show that placements at different genomic loci in CAV genome have distinct, reproducible and predictable expression properties, which can be harnessed to modulate the expression levels of any given transgene.

[0693] Thus, discovery of these distinct expression profiles provides a rational design framework for the development of customized viral vectors with optimized and tunable transgene expression based on the placement location and design. For example, transgenes that may be toxic to viral replication or whose overexpression could inhibit production can be expressed from lower-expressing modules (e.g., E1B-55K), reducing the risk of transgene deletion or negative selection during viral propagation or large-scale manufacturing.

[0694] These findings enable rational design of recombinant CAV vectors for tailored expression of heterologous genes, supporting both basic research and applied uses, including multivalent vaccine development, therapeutic protein delivery, and combinatorial gene expression platforms.

[0695] Example 19: Recombinant CAV genomes that express three heterologous ORFs from single placement

[0696] The following examples illustrate the use of the disclosed modular genome placements and assembly platform for tire generation of CAVs encoding two or more heterologous ORFs.

[0697] This example describes a recombinant CAV designated PCMN-1389 (AE3-ORFl[mCherry-GS-BFP-P2A-YPet], which demonstrates the successful expression of three distinct heterologous ORFs. Using the E3-554 plasmid (FIG. 79; SEQ ID NO: 55), in which E3-ORF1 was replaced with a synthetic cassette encoding mCherry, fused via a glycine- serine (GS) linker to blue fluorescent protein (BFP), and further linked to YPet via a P2A self-cleaving peptide. E3-ORF2 was also deleted, increasing available genome space for the heterologous cassette. The total size of the inserted payload was 2,289 base pairs.7158-105381-02

[0698] FIG. 80 shows images of MDCK cells transfected with the PCMN-1389 genome (SEQ ID NO: 15). The upper panel shows cells that have blue, green, and red fluorescence signals corresponding to BFP, YPet, and mCherry expression, respectively. This confirms successful simultaneous expression of all three ORFs from the E3-ORF1 engineered placement. By 9 days post-transfection, robust viral replication is evident, as demonstrated by the increase in fluorescent cells.

[0699] This example also demonstrates that the E3 region of the CAV genome can be engineered to accommodate multi-cistronic heterologous payloads of substantial size (up to at least ~2.3 kb) while maintaining viral replication competency. It further shows that multiple protein products can be expressed in a single transcriptional unit using combinatorial linkers, such as GS linkers for fusions and P2A for post-translational cleavage, although other linkers such as T2A, F2A, or synthetic cleavage sequences may be used.

[0700] This strategy can be extended beyond fluorescent reporters to include therapeutic transgenes, adjuvants, or vaccine antigens (FIGS. 100-104), enabling the development of multivalent CAV-based vaccines or complex gene therapy vectors capable of expressing multiple functional payloads from a single engineered locus. The modular nature of the assembly system allows such configurations to be rapidly prototyped, screened, and validated using standard molecular biology tools and fluorescencebased replication assays.

[0701] Example 20: Modular and combinatorial expression of multiple heterologous ORFs from distinct genomic locations in replication-competent CAV constructs

[0702] The following examples demonstrate that multiple heterologous ORFs can be expressed simultaneously from distinct, engineered genomic loci within the same recombinant assembled CAV genome, enabling a flexible and modular system for the design of recombinant viruses with tailored multi-gene expression levels and timing characteristics, which is locus dependent. These examples demonstrate the ability to assemble and evaluate CAV vectors in which fluorescent reporters (used here as model transgenes) are inserted into various native loci — either individually or in combination — such that the resulting recombinant viruses remain replication-competent and exhibit predictable and specific patterns and properties for gene expression.

[0703] The ability to place heterologous ORFs into multiple independent genomic modules allows a recombinant CAV to express, for example, a fluorescent reporter from one locus, while simultaneously expressing a therapeutic payload or vaccine antigen from another locus. This modular strategy supports the development of multivalent vaccine vectors, for example, viruses engineered to co-express antigens from canine distemper virus (CDV) and canine parainfluenza virus (CPiV) in a single viral composition, hi addition, it allows the distinct properties of the different genomic placements to be tuned and customized to tire transgene and therapeutic ORF. For example, if a therapeutic ORF function is noxious to CAV replication at elevated expression levels at the E3-ORF1 placement, it may still be tolerated at The ElB-55k-P2A placement where expression is lower (FIGS. 91-96).7158-105381-02

[0704] Combinatorial placement examples

[0705] The following combinations are examples of combinatorial ORF placements. PCMN-1375 has an ElA-P2A-YPet fusion and AE3-ORFl[mCherry], AE3-ORF2. PCMN-1423 was assembled from an ElB-55K-P2A-YPet plasmid and has AE3-ORFl[mCherry], AE3-ORF2. MDCK cells were transfected with PCMN-1375 (SEQ ID NO: 12) or PCMN-1423 (SEQ ID NO: 17) and fluorescence images were captured at the time points indicated in FIGS. 81 and 82.

[0706] Both of these examples i II nstrate the successful expression of different heterologous ORFs from two distinct placements in the same recombinant CAV genome, while preserving replication and fitness, as evidenced by tire increase in fluorescent vitally infected cells over time. Importantly, the successful combinations validate that certain insertions are compatible, even when approaching the upper limits of genome packaging (-105% of wild- type CAV genome size).

[0707] Negative example

[0708] Although not all possible combinations were tested (FIG. 83) a combination was identified that was not tolerated. For example, when an engineered CAV genome containing a YPet-P2A insertion within the viral DNA polymerase (Pol) gene was combined with an E3-ORFl-mCherry replacement, the resulting construct was non-viable (Table 8).

[0709] Table 8. Replication of CAVs with combinations of heterologous ORF insertions

[0710] Replication with Replication with heterologous ORF CAV ORF

[0711] heterologous ORF and other inserts

[0712] E1A Yes (ElA-P2A-YPet) Yes (E3-ORF1 [mCherry])

[0713] ElB-55k Yes (ElB55k-P2A-YPet) Yes (E3-ORF1 [mCherry])

[0714] Yes (YPet-P2A-DNA

[0715] DNA polymerase No (E3-ORF1 [mCherry])

[0716] polymerase)

[0717] Yes (ElB-55k-P2A-YPet)

[0718] E3-ORF1 Yes (AE3-ORF1 [mCherry])

[0719] Yes (ElA-P2A-YPet)

[0720]

[0721] These findings establish that expression of multiple heterologous ORFs from different genomic placements in the CAV genome is feasible. The use of modular genome plasmids that can be engineered to express heterologous ORFs at different genomic placements, genes, timing levels, and their combinatorial assembly using CAVSLIC and CAVsembly enables tire rapid generation and testing of such combinations, facilitating the development of customized, multivalent CAV vectors for therapeutic, prophylactic, or research applications.7158-105381-02

[0722] Example 21: Individual and combinatorial deletions of CAV genes that create additional payload cargo space and are compatible with virus replication

[0723] This example shows that multiple deletions of CAV genes can be engineered in one or more modules to create additional cargo space for the expression payloads in recombinant CAV genomes.

[0724] This example describes CMBT-1501 (FIG. 85; SEQ ID NO: 37), which was assembled using a multi-site LR reaction from E3-541 (SEQ ID NO: 52), which expresses mCherry instead of E3-ORF1 and has an additional deletion of E3-ORF2 sequences. Furthermore, E4-ORF5 protein coding sequences were deleted in the E4 genome plasmid module, E4-114 (FIG. 84; SEQ ID NO: 66).

[0725] MDCK cells were transfected with CMBT-1501 and mCherry fluorescence was visualized and quantified at 3 and 15 days post-transfection. As shown in FIG. 86, mCherry fluorescence was observed at 3 days post-transfection and virus spread and infection to the monolayer is observable by 15 days posttransfection. These results demonstrate that deletion of CAV E4-ORF5 together with E3-ORF1 and E3-ORF2 is still compatible with virus replication, and creates an additional 2.2kb in cargo space for expression of heterologous ORFs, which can be combined with these CAV gene deletions.

[0726] Example 22: Recombinant CAVs containing therapeutic heterologous ORFs

[0727] Vaccination plays a crucial role in safeguarding dogs from severe, highly transmissible, and potentially life-threatening diseases. Canine adenoviruses (Type 1 and Type 2 - CAV1 and CAV2), canine parainfluenza virus (CPiV) and the canine distemper virus (CDV) are significant pathogens in dogs (FIGS. 87 and 99), capable of causing fatal diseases and life-threatening symptoms in unvaccinated animals. Consequently, the development of effective and improved vaccines that protect animals from adenovirus, parainfluenza and distemper viruses is essential not only for the individual health of the dog but also for controlling the spread of these diseases within the canine population and potentially zoonotic transmission.

[0728] Additionally, many major viral pathogens of dogs (including CAV1, CAV2, and CDV) are transmitted to dogs by oral or respiratory routes. Thus, a vaccine that elicits potent mucosal immunity, for example anti-viral IgA immunity, may offer better protection than a vaccine in which the primary immunity is driven by systemic IgG. Licensed vaccines for canine distemper virus, canine parvovirus (CPV), and canine adenoviruses Type 1 and Type 2 are generally administered parenterally, and fail to elicit potent mucosal immunity to combat viruses like CAV1, CAV2, or CDV. There are no existing licensed vaccines that are delivered orally (and thus should elicit a mucosal immune response) for CAV1, CAV2, or CDV. Furthermore, the use of an oral vaccine obviates tire need for painful and invasive needle-based administration. Taken together, there is a need for improved vaccines that elicit mucosal immunity and provide vaccine administration conveniences.

[0729] To develop live vaccines that can be administered via multiple routes and that induce protective immunity against life threatening diseases, the engineering and assembly of recombinant modified canine7158-105381-02

[0730] adenovirus genomes that are replication competent and express heterologous CDV (FIG. 87) or CPiV antigens (FIG. 99) from the CAV transcriptional architecture would be highly desirable.

[0731] To determine if therapeutic ORFs, in this case immunogenic proteins from other canine pathogens, can be expressed in the placements shown in examples above to create live replication competent viruses, the following plasmids were engineered to a assemble recombinant CAV genomes. In addition, the examples show that the assembly of viruses with both a fluorescent reporter module and a therapeutic ORF enables the identification of the impact of heterologous ORFs on viral replication and the determination of optimal therapeutic compositions.

[0732] Assembly and screening of recombinant CAV genomes that express heterologous and therapeutic ORFs in one or more CAV genome modules

[0733] This example shows CMBT-1460 (FIG. 90; SEQ ID NO: 36), which was assembled in an LR reaction from a Core-74D plasmid (FIG. 89; SEQ ID NO: 63), the El-285 (SEQ ID NO: 44) and E3-574 (SEQ ID NO: 57) plasmid modules (FIG. 88), and wildtype E4 plasmid in an LR reaction to create the whole genome plasmid. The El-285 plasmid is a modified El module with an ElB-55k-P2A-YPet fluorescent reporter placement. The E3-574 plasmid is a modified E3 module that expresses a heterologous therapeutic ORF, an immunogenic antigen from canine distemper virus (CDV H), instead of E3-ORF1 and also has an additional deletion of E3-ORF2 coding sequences. The El and E3 modified plasmids were assembled with a core-74D plasmid and wildtype E4 plasmid in an LR reaction to create the whole genome plasmid CMBT-1460 (FIG. 90). For assembly, module plasmids were combined into a single tube. Gateway LR Clonase II plus enzyme mix (Thermo Fisher) was added and the reaction was incubated at room temperature for 8-16 hours. Clonase enzyme was inactivated with proteinase K at 37°C for 10 minutes. The reaction can be transformed into bacteria for genome screening or directly transfected into mammalian cells to reconstitute virus. In this example, the reaction and DNA were transformed into E. coli DHIOb cells and selected using ampicillin resistance.7158-105381-02

[0734] MDCK cells were transfected with CMBT-1460 and YPet fluorescence was visualized and quantified at 6 and 12 days post-transfection (FIG .91). The increase in the number of YPet expressing and infected cells between day 6 and 12 demonstrate productive virus replication and secondary infection spread. These data demonstrate that expression of a heterologous therapeutic ORF, in this case a vaccine antigen from a different canine pathogen, in a CAV modified E3 module can be assembled to create a replication competent virus using multi-site gateway recombination. This assembly scheme is useful to rapidly screen and enable tire development of therapeutic recombinant CAVs, such as, in this example, live virus vaccine candidate placements and genome compositions. Once an optimal composition has been identified, the same genomic modules can be scarlessly assembled to generate CAV genomes for screening and production as clinical candidate development leads.

[0735] Comparative expression and stability of different therapeutic ORFs in distinct genomic placements within recombinant CAV

[0736] In previous examples, it was demonstrated that heterologous ORFs, including examples of fluorescent reporters and vaccine antigens, can be inserted into several different genomic placements, within the CAV genome. It was also discovered in screening that different genomic placements have distinct properties and differential relative expression levels and timing. For example, the AE3-ORF1 [transgene] AE3-ORF2 placement has the highest expression levels, E1A-P2A intermediate levels and ElB-55k-P2A lowest levels, when compared to each other. Fluorescent reporter expression is innocuous to CAV replication. However, the expression of certain therapeutic proteins, for example, a protein from another pathogen, or an immunotoxin or cytokine, may have intrinsic protein functions that could inhibit CAV replication, especially if expressed at higher levels. In this example, it is demonstrated that screening optimal placements for potentially CAV-noxious proteins can be predicted by fluorescent reporters in the same virus compositions. It is also demonstrated that if a transgene inhibits virus replication, it can be unstable and select for viruses that delete the transgene on extended passaging. However, the same transgene if expressed in a different placement at a lower expression level can be stable and as such amenable to further development and exploration as a clinical candidate. These aspects are demonstrated by the examples below.

[0737] In the previous example (FIG. 91), it was shown that CMBT-1460 (AE3-ORFl[nFLAG-CDVH] AE3-ORF2) with an ElB-55k-P2A-YPet reporter exhibits strong virus replication in MDCK cells, as evidenced by an increase in fluorescent cells. Similarly, PCMN-1513 (SEQ ID NO: 29) and PCMN-1510 (SEQ ID NO: 26), which express CDVH as an E1A-P2A or an ElB-55k-P2a fusion, respectively, also show strong virus replication (FIG. 92).

[0738] Replication of PCMN-1514 (SEQ ID NO: 30) and PCMN-1511 (SEQ ID NO: 27), which express CDVF as an E1A-P2A or an ElB-55k-P2A fusion, respectively, was tested in MDCK cells. PCMN-1511 showed strong virus replication and an increase in fluorescent cells over time (FIG. 93B). PCMN-1514 showed moderate virus replication and an increase in the number of fluorescently labeled7158-105381-02

[0739] cells over time, with a slower plaque expansion and lower numbers of fluorescent infected cells over same time period (FIG. 93B).

[0740] Further studies tested CMBT-1459 (SEQ ID NO: 35), AE3-ORF1[CDVF] AE3-ORF2 with an ElB-55K-P2A-YPet reporter. In contrast to CMBT-1460, which expresses CDVH at same genomic placement (FIG. 91), there is a minimal or no increase in the number of fluorescently labeled cells between 4 and 9 days (FIG. 94) and even by day 16, only small plaques were labeled.

[0741] Furthermore, FIG. 95 compares PCMN-1506 (SEQ ID NO: 24) and PCMN-1507 (SEQ ID NO: 25), which express CDVF or CDVH, respectively, at the E3-ORF1 / (AE3-ORF2) replacement. Small plaques were visible at day 4 for both viruses. By day 9 post transfection, virus replication, associated CPE and complete disruption of MDCK monolayer was demonstrated for PCMN-1507 (FIG. 95 A). In contrast, PCMN-1506, showed much slower growth and minimal expansion of initial plaques by day 9 (FIG. 95B).

[0742] Consistent with, and predicted by, the inhibition of virus replication, using PCR and sequencing, it was demonstrated that upon serial passaging, PCMN-1506 virus mutants that have deleted the CDVF transgene are selected and dominate the population (FIG. 96A). In contrast, PCMN-1507, which expresses CDVH at same placement does not inhibit virus replication and the transgene is stably maintained. In FIG. 96B, it is shown that in PCMN-1512 (SEQ ID NO: 28) and PCMN-1515 (SEQ ID NO: 31), which express CDVF as E1B-55K-P2A and E1A-P2A fusions, respectively, the CDVF transgene is stably maintained, in contrast to PCMN-1507.

[0743] Conclusions

[0744] These examples demonstrate that the different genomic placements disclosed herein have differential properties for the expression of heterologous reporters and therapeutic transgenes. Genomic placements that drive higher expression, such as E3-ORF1 with E3-ORF2 deletion, may be ideal for some applications and therapeutic ORFs, but for proteins with functions that may be noxious to CAV replication, lower levels and later expression may work better. In these cases, lower expression at placements, such as E1A-P2A or ElB-55k-P2A, or as a P2A fusion to a late protein, such as pVIII, may be optimal.

[0745] The present disclosure teaches methods of screening and optimization by assembling viruses that have fluorescent reporters and the therapeutic ORF expressed at different genomic placements (FIGS. 88-96). Screening using fluorescent surrogates is novel, generalizable, and critical for the development of multivalent or live viral vaccine platforms. Furthermore, it underscores that the different heterologous ORF placements disclosed herein, have distinct properties with respect to the timing and levels of expression of a transgene, which may be important to successfully and stably express specific therapeutic proteins, such as the CDVF protein (as one example).

[0746] Due to its fusogenic activity, CDVF has the potential to interfere with virus replication, which depends on its relative expression levels and timing in distinct genomic placements, possibly through7158-105381-02

[0747] premature cell fusion and death. The extent to which virus replication and spread was inhibited directly correlates with, and is predicted by, the relative expression levels at the different placements: E3-ORF1 (AE3-ORF2)»> E1A-P2A» ElB-55k-P2A

[0748] Importantly, it is demonstrated herein that these outcomes can be determined by incorporating fluorescent reporter-based screening, enabling rapid screening and assessment of the potential viability of different CAV compositions prior to large-scale production. This approach can be used to screen new vaccine antigen compositions or therapeutic ORFs across different CAV insertion sites to identify optimal placements that maximize expression while preserving replication competency and genetic stability — key requirements for scalable vaccine vector manufacturing under GMP conditions.

[0749] Example 23: Creating genomic cargo space in the CAV genome for expression of therapeutic transgenes by deleting CAV genes, E3-ORF1, E3-ORF2 and E4-ORF5, dispensable for CAV replication in cell culture

[0750] This example demonstrates that a recombinant canine adenovirus (CAV) can be engineered to express a therapeutic transgene, such as in this example, a vaccine antigen, by replacing E3-ORF1 coding sequences and other engineered modifications as described in previous examples (FIGS. 58, 62 and 71), while incorporating additional genomic deletions of E3-ORF2 protein coding sequences and E4-ORF5 to increase payload capacity and expression of transgene at E3-ORF1 replacement.

[0751] A CAV genome was assembled in an LR reaction (as per methods described for example CMBT-1460 above) in which the therapeutic antigen CDVH, from canine distemper virus (CDV), was inserted at the E3-ORF1 locus. To create additional cargo space and modulate transgene expression, the genome also included deletions of E3-ORF2 and E4-ORF5. The resulting virus was designated CMBT-1509 (SEQ ID NO: 38).

[0752] CMBT-1509 was transfected into MDCK cells and monitored for plaque formation and cytopathic effect using brightfield microscopy. By day 4 post-transfection, clear CAV plaques were visible, indicated by localized changes in cell refractive index. By day 11, the virus had spread throughout tire culture well, leading to complete monolayer lysis (FIG. 97).

[0753] These results confirm that expression of a therapeutic transgene such as CDVH is compatible with robust CAV replication, and that deletion of non-essential ORFs such as E3-ORF2 and E4-ORF5 does not impair viral fitness.

[0754] This strategy allows for efficient packaging of therapeutic payloads, which may be larger (> 3-4 kb) and exceed packaging capacity of CAV capsid, necessitating additional deletions of CAV genes to provide genomic capacity for therapeutic gene pay loads.7158-105381-02

[0755] Example 24: Multigene expression of therapeutic ORFS from different genomic placements in same replication-competent CAV vector

[0756] This example demonstrates that two distinct therapeutic antigens can be stably and simultaneously expressed from two different heterologous ORF placements (described in previous examples) within tire same CAV genome, without impairing viral replication. This design supports the development of multivalent live vaccine vectors or oncolytic viruses, which benefit from the enhanced immunogenicity characteristic of live replication competent virus platforms.

[0757] A recombinant CAV genome, designated PCMN-1519 (SEQ ID NO: 32), was assembled as described in previous examples. A first therapeutic antigen, tire nucleoprotein (NP) from CDV, was inserted at die E1A-P2A placement. A second drerapeutic protein, CDV-H, from CDV, was inserted by replacing the endogenous E3-ORF1 coding sequence and E3-ORF2 protein coding sequence was deleted to (i) provide additional genomic space and (ii) enhance expression of the transgene at the E3 locus. The assembled genome was transfected into MDCK cells, and viral replication was monitored by brightfield microscopy (FIG. 98). By day 4 post-transfection, small, dense regions corresponding to early plaque formation were observed. By day 11, complete cytopathic effect (CPE) was evident across the monolayer, consistent with widespread viral replication and cell lysis.

[0758] These results demonstrate that a replication-competent CAV can express two distinct heterologous therapeutic antigens from separate genomic placements, without compromising virus replication or spread. This strategy enables the development and design of multivalent as well as ‘live’ replicating CAV therapeutics, including vaccines, combining multiple antigens to improve immune response breadth and vaccination durance.

[0759] Example 25: Construction of PCMN-1556 expressing multivalent vaccine antigens as a polycistronic construct at engineered E3-ORF1 placement with additional deletions of E3-ORF2 Canine parainfluenza virus (CPiV), like CDV, is a negative-sense, single stranded RNA virus (FIG. 99). This example describes the production of the recombinant CAV PCMN-1556 (SEQ ID NO: 33), which contains a deletion of E3-ORF1 and E3-ORF2, a first heterologous ORF encoding the fusion protein of canine parainfluenza virus (CPiVF) and a second heterologous ORF encoding the hemagglutinin-neuraminidase (HN) protein of CPiV (CPiVHN), separated by a P2A coding sequence, inserted into E3-ORF1 (AE3-ORFl[CPiVF-P2A-CPiVHN], AE3-ORF2).

[0760] The CPiVF and CPiVHN protein sequences are encoded by DNA sequences of 1653 bp and 1695 bp, respectively. The amino acid sequences are based on the D008 challenge strain obtained from the USDA. Alignment with public NCBI sequences shows the CPiVF amino acid sequence matches the amino acid sequence described in GenBank accession AFE48526, from the fusion protein of mammalian rubulavirus 5 (GenBank accession JQ743328). The CPiVF gene was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using the Integrated DNA Technologies DNA codon optimization tool.7158-105381-02

[0761] CPiVF + P2A amino acid sequence (SEQ ID NO: 67; P2A sequence shown in bold) MGTIIQFLVVSCLLAGAGSLDPAALMQIGVIPTNVRQLMYYTEASSAFIVVKLMPTIDSPISGCNIT SISSYNATVTKLLQPIGENLETIRNQLIPTRRRRRFAGVVIGLAALGVATAAQVTAAVALVKANK NAAAILNLKNAIQKTNTAVADVVQATQSLGTAVQAVQDHINSVVSPAITAANCKAQDAIIGSILN LYLTELTTIFHNQITNPALSPITIQALRILLGSTLPTVVEKSFNTQISAAELLSSGLLTGQIVGLDLTY MQMVIKIELPTLTVQPATQIIDLATISAFINNQEVMAQLPTRVIVTGSLIQAYPASQCTITPNTVYC RYNDAQVLSDDTMACLQGNLTRCTFSPVVGSFLTRFMLFDGIVYANCRSMLCKCMQPAAVILQ PSSSPVTVIDMYKCVSLQLDNLRFTITQLANVTYNSTIKLETSQILPIDPLDISQNLAAVNKSLSDA LQHLAQSDTYLSAITSATTTSVLS1MAICLGSLGL1L1ILLSVVVWKLLT1VTANRNRMENFVYHNS AFHHSRSDLSEKNQPATLGTRGSGATNFSLLKQAGDVEENPG CPiVF + P2A DNA sequence (SEQ ID NO: 68; P2A coding sequence shown in bold) atggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattcccaccaa cgtcagacagctcatgtattataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaatattacatcaa taagttcttataatgctacagtcacgaaacttctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaagaagaagaagacga tttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggctaacaagaacgctgcag ctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctgggtacggcagttcaagctgttcag gaccacatcaatagcgttgtgtctccggccattaccgccgcgaactgtaaggcacaggatgcaattataggaagtatccttaacctctacctcactgaact cacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatcttgcttggctcaacccttccgacggttgtgg aaaagtcttttaacacgcagataagtgctgcagaactgcttagctctggtttgcttacaggtcaaatagtcggactggatctgacctatatgcagatggtgat taaaatcgagctgcctaccctgactgtccagccagcaacccaaattatcgaccttgcaactataagcgcctttatcaacaaccaagaagtcatggctcagt tgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtacaattacgccgaacacggtgtattgcagatacaacgacgc acaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgttcagtccggtcgtggggtctttcctcacccgcttcatgttgtttg atggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctgcggttatactccagccctccagcagcccggtgactgtgatcgat atgtataagtgcgtttcacttcagcttgacaacctgaggttcacaataacccaattggcgaacgtgacttataattctacgattaaacttgagactagccagat ccttccgatcgatcctctggatataagccaaaacctggcggcagtgaataaaagtctcagtgatgccttgcagcacctggcccaatcagacacgtatcttt ccgctataacctcagccacgacaacaagtgttctttccataatggcaatctgtcttggtagtctgggtctcatactcattatactgcttagcgttgtggtctgga agctgcttacgattgtcaccgcaaatcgcaatcgaatggagaactttgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaac cagcctgctactctcgggactcgcggaagcggagctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggc Alignment with public NCBI sequences showed that the CPiVHN amino acid sequence was most similar to GenBank accession AFE48527, from the hemagglutinin-neuraminidase protein of mammalian rubulavirus 5 (GenBank accession JQ743328) with differences in amino acids 288 and 447. The CPiVHN gene was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Cams familiaris using the Integrated DNA Technologies DNA codon optimization tool.

[0762] CPiVHN amino acid sequence + final proline of P2A (SEQ ID NO: 69) PMVAEDAPVRGTCRVLFRTTTLIFLCTLLALSISILYESLITQKQIMSHAGSTGSNSRLGSITDLLN NILSVANQIIYNSAVALPLQLDTLESTLLTAIKSLQTSDKLEQNCSWGAALINNNRYINGINQFYFS IAEGRNLTLGPLLNIPSFIPTATTPEGCTRIPSFSLTKTHWCYTHNVILNGCQDHVSSNQFVSMGIIE PTSAGFPSFRTLKTLYLSDGVNRKSCSISTVPGGCMMYCFVSTQPERDDYFSTAPPEQRIIIMYYN DTIVERIINPPGVLDVWATLNPGTGSGVYYLGWVLFPIYGGVIKDTSLWNNQANKYFIPQMVAA LCSQNQATQVQNAKSSYYSSWFGNRMIQSGILACPLQQDLTNECLVLPFSNDQVLMGAEGRLY MYGDSVYYYQRSNSWWPMTMLYKVTITFTNGQPSAISAQNVPTQQVPRPGTGDCSATNRCPGF CLKGVYADAWLLTNPSSTSTFGSEATFTGSYLNAATQRINPTMYIANNTQIISSQQFGSSGQEAA YSHTTCFRDTGSVMVYCIYIIELSSSLLGQFQIVPFIRQVTLS CPiVHN DNA sequence + final codon of P2A (SEQ ID NO: 70)7158-105381-02

[0763] ccaatggtcgctgaggatgcgccagttaggggaacctgtcgcgtgctcttccgaaccaccacgctcatattcctctgcaccttgcttgcgctttcaatctca atcttgtatgagtcacttataacacagaaacaaattatgtctcatgctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttgaataatatt cttagtgtggcgaaccagattatctataactcagcggttgcgctccctctccagttggatactctggagagcaccctgttgactgctattaagtctcttcaaac cagtgacaagctcgagcagaactgtagctggggcgcggctctgattaataacaacagatatattaacggcataaatcaattttatttctccattgccgaagg tagaaacctgaccctgggacctttgctcaatatccctagcttcataccaacggcgaccacccctgagggctgtactcgcattccatcctttagtcttaccaa aacacactggtgctacacccataatgttatccttaatggatgccaagatcacgtctcatcaaaccagttcgtctctatgggtataatcgaacccaccagcgc tggcttcccatcatttcgaacattgaagacgctttatctttccgatggtgtcaaccgaaagtcctgctctatttctactgtgccgggagggtgcatgatgtact gctttgtttccacgcagcctgaacgagacgattattttagtacggctccgccggagcaacggataataatcatgtactataacgatacgatagtggaaaga ataatcaatcctcctggtgttctcgacgtgtgggcgacgttgaatcctggaaccggatctggcgtctattatcttggatgggttctgtttcctatttacggcgg agttattaaagacacaagcttgtggaataatcaagctaataaatatttcattccacaaatggtcgcagccttgtgttcccaaaaccaggcgacccaagttca aaacgcaaaaagttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggcctgcccgttgcagcaggacttgacgaacgaatgtct ggtccttcctttttccaacgaccaagttctgatgggggctgagggtagactttacatgtacggagattccgtttactactaccagcggagcaattcttggtgg ccgatgaccatgttgtataaagtcacaatcactttcactaatgggcagccgagcgccatttccgctcagaatgtccccacccaacaagttcctcgaccagg tacaggcgattgtagtgcgactaacagatgccctggtttttgccttaagggcgtttacgctgacgcatggttgttgacaaatccatctagtacgagtacttttg ggagcgaggccacattcacgggctcatacctgaatgctgcaacgcaacgcataaaccctacaatgtatatcgccaacaacacgcaaataattagtagcc aacagtttggatcatctgggcaggaggcagcctacagtcatacgacgtgtttccgggacaccgggtctgtgatggtgtattgtatctacataatagagttga gtagttcactcttggggcagttccagatcgtcccattcattaggcaagtgacgctctct

[0764] Construction of PCMN-1556 is illustrated in FIG. 100. The E3 module was first modified to remove E3-ORF2 using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. CPiVF and CPiVHN DNA sequences were then inserted into E3-ORF1 using Gibson assembly with DNA amplified using primer-extension PCR with primers bl and b2 and PCR with primers cl and c2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers dl and d2. The P2A sequence was introduced in primers b2 and cl. Included in this insert is the upstream pVIII-E3-ORFl DNA sequence that was modified to remove potential E3-ORF1 start codons while maintaining tire pVIII coding sequence. CPiVF and CPiVHN are linked via a P2A self-cleaving peptide. The efficiency of P2A cleavage will determine the amount of linked and unlinked CPiVF and CPiVHN. Cleaved P2A results in the residual peptide GSGATNFSLLKQAGDVEENPG (SEQ ID NO: 116) on the C-terminus of CPiVF, and a residual proline on the N-terminus of CPiVHN (residue 1 of SEQ ID NO: 69). The sequence of the modified E3 region of PCMN-1556 (SEQ ID NO: 71) is shown below.

[0765] Table 9. PCR primers for insertion of donor DNA

[0766] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 99 a2 acaagCGGCGGCGATGAAGAAGC 100 bl tctgtcgacggctacgactgagATGGGGACAATTATTCAGTTCTTG 101 b2 caccagcctgcttcagcaggctgaagttagtagctccgcttccGCGAGTCCCGAGAGTAG 102 cl cctgctgaagcaggctggtgacgtcgaggagaatcctggcccaATGGTCGCTGAGGATGC 103 c2 acaagtttaataatattatctaAGAGAGCGTCACTTGCCTAATG 104 dl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 105 d2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 106

[0767]

[0768] 7158-105381-02

[0769] PCMN-1556 E3 region (SEQ ID NO: 71)

[0770] Modified sequence (bold), CPiVF and CP iVHN insertions (italics), P2A insertion (bold italics), location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gagatggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattccc accaacgtcagacagctcatgtattataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaat attacatcaataagttcttataatgctacagtcacgaaacttctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaag aagaagaagacgatttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggct aacaagaacgctgcagctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctggg tacggcagttcaagctgttcaggaccacatcaatagcgttgtgtctccggccattaccgccgcgaactgtaaggcacaggatgcaattataggaagt atccttaacctctacctcactgaactcacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatctt gcttggctcaacccttccgacggttgtggaaaagtcttttaacacgcagataagtgctgcagaactgcttagctctggtttgcttacaggtcaaatagtc ggactggatctgacctatatgcagatggtgattaaaatcgagctgcctaccctgactgtccagccagcaacccaaattatcgaccttgcaactataa gcgcctttatcaacaaccaagaagtcatggctcagttgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtaca attacgccgaacacggtgtattgcagatacaacgacgcacaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgt tcagtccggtcgtggggtctttcctcacccgcttcatgttgtttgatggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctg cggttatactccagccctccagcagcccggtgactgtgatcgatatgtataagtgcgtttcacttcagcttgacaacctgaggttcacaataacccaat tggcgaacgtgacttataattctacgattaaacttgagactagccagatccttccgatcgatcctctggatataagccaaaacctggcggcagtgaat aaaagtctcagtgatgccttgcagcacctggcccaatcagacacgtatctttccgctataacctcagccacgacaacaagtgttctttccataatggc aatctgtcttggtagtctgggtctcatactcattatactgcttagcgttgtggtctggaagctgcttacgattgtcaccgcaaatcgcaatcgaatggaga actttgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaaccagcctgctactctcgggactcgcggaagcggagcta ctaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggcccaatggtcgctgaggatgcgccagttaggggaacctgtcgcgtg ctcttccgaaccaccacgctcatattcctctgcaccttgcttgcgctttcaatctcaatcttgtatgagtcacttataacacagaaacaaattatgtctcat gctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttgaataatattcttagtgtggcgaaccagattatctataactcagcggttg cgctccctctccagttggatactctggagagcaccctgttgactgctattaagtctcttcaaaccagtgacaagctcgagcagaactgtagctggggc gcggctctgattaataacaacagatatattaacggcataaatcaattttatttctccattgccgaaggtagaaacctgaccctgggacctttgctcaata tccctagcttcataccaacggcgaccacccctgagggctgtactcgcattccatcctttagtcttaccaaaacacactggtgctacacccataatgttat ccttaatggatgccaagatcacgtctcatcaaaccagttcgtctctatgggtataatcgaacccaccagcgctggcttcccatcatttcgaacattgaa gacgctttatctttccgatggtgtcaaccgaaagtcctgctctatttctactgtgccgggagggtgcatgatgtactgctttgtttccacgcagcctgaac gagacgattattttagtacggctccgccggagcaacggataataatcatgtactataacgatacgatagtggaaagaataatcaatcctcctggtgtt ctcgacgtgtgggcgacgttgaatcctggaaccggatctggcgtctattatcttggatgggttctgtttcctatttacggcggagttattaaagacacaa gcttgtggaataatcaagctaataaatatttcattccacaaatggtcgcagccttgtgttcccaaaaccaggcgacccaagttcaaaacgcaaaaag ttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggcctgcccgttgcagcaggacttgacgaacgaatgtctggtccttcctt tttccaacgaccaagttctgatgggggctgagggtagactttacatgtacggagattccgtttactactaccagcggagcaattcttggtggccgatga ccatgttgtataaagtcacaatcactttcactaatgggcagccgagcgccatttccgctcagaatgtccccacccaacaagttcctcgaccaggtaca ggcgattgtagtgcgactaacagatgccctggtttttgccttaagggcgtttacgctgacgcatggttgttgacaaatccatctagtacgagtacttttgg gagcgaggccacattcacgggctcatacctgaatgctgcaacgcaacgcataaaccctacaatgtatatcgccaacaacacgcaaataattagta gccaacagtttggatcatctgggcaggaggcagcctacagtcatacgacgtgtttccgggacaccgggtctgtgatggtgtattgtatctacataata gagttgagtagttcactcttggggcagttccagatcgtcccattcattaggcaagtgacgctctcttagataatattattaaacttgttttacagctaccacc ataatgcgcttcagcttcttcatcgccgccgcttgtcaaataaacttacctaatttttgctaagacgtctgggtcctgcgtttct

[0771] The CPiVF-P2A-CPiVHN E3 plasmid is designated E3-595 (FIG. 101) and has the sequence of SEQ ID NO: 58.

[0772] CAV-SLIC was used to construct the PCMN-1556 whole viral genome vaccine composition that expresses CPiVF and CPIVHN from the endogenous viral E3 transcriptional architecture in place of the CAV E3-ORFs.

[0773] The ASMM-149 El-core macromodule plasmid (SEQ ID NO: 39) was digested with Bglll and Gibson assembled with the linear product of E4-099 (SEQ ID NO: 65) amplified with primers tl and t2 (Example 5, Table 3) to create the El-core-E4 macromodule plasmid ASMM-150 (SEQ ID NO: 40).7158-105381-02

[0774] The ASMM-150 plasmid was then digested with Bglll and Gibson assembled with the linear product of the CPiVF E3-596 plasmid (SEQ ID NO: 59) amplified with si and s2 primers. See Table 2 for the rl, r2, si, s2, tl and t2 primer sequences. Final constructs were selected using ampicillin resistance. A map of the PCMN-1556 virus genome plasmid is shown in FIG. 102.

[0775] E. coli DHIOb cells were used to transform and amplify tire shuttle vector and assembled virus genome. MDCK cells (such as from ATCC) were used as the host cell line for construct transfection and virus production. Transfection was done using Lipofectamine 3000 using 2 (ig DNA per 6-well. Virus successfully grown in MDCK cells with MEM 10 media was transferred to freshly plated MDCK cells for virus propagation.

[0776] MDCK cells were transfected with PCMN-1556 (FIG. 103) and brightfield images were taken on day 4 and day 9:

[0777] 1. Plaque Formation:

[0778] Distinct plaques were observed within 4 days post-transfection, confirming viral replication and cytopathic effects consistent with productive infection.

[0779] 2. Progressive Spread of Infection:

[0780] Infection with replication-competent CAV vaccine particles led to secondary and tertiary rounds of infection, resulting in extensive monolayer disruption and widespread cytopathic effects across the culture. At 9 days post initial transfection, the CAV CPiV vaccine particles produced initially had spread to and killed the entire MDCK monolayer.

[0781] Recombinant CAV vaccines express CPiV payloads in infected cells

[0782] To confirm and validate expression of vaccine antigens from recombinant CAV vaccine candidates, MDCK cells were infected with PCMN-1556 virus supernatants. PCMN-1366, which does not express CPiV antigens, was used as a negative control for background staining (FIG. 104).

[0783] Cells were fixed with paraformaldehyde 12 hours post infection, permeabilized with 0.1% Triton X-100, blocked with 5% normal goat serum and incubated overnight with primary antibodies. Cells were washed and then stained for 1 hour at room temperature with secondary antibodies and washed. Nuclei were counterstained with DAPI. Cells were imaged using fluorescence.

[0784] Antibodies used to detect CPiV antigens

[0785] CPIV8-4 mAb hybridoma supernatant Lot 2898-093-19Augl4 (Elanco 070720) against CPiV HN at 1:160 dilution. Anti-CPI 1:1000 CPI Capture Antibody chicken antiserum Lot 2626-42- 11 Jun08 (Elanco 070720).

[0786] As shown in FIG. 104, expression of the CPiV HN is clearly demonstrated and specific to PCMN-1556 infected cells, but not PCMN-1366 infected cells or non-infected cells.7158-105381-02

[0787] Since CPiVHN is expressed as a P2A fusion with CPiVF as the first heterologous antigen, detection of CPiVHN in PCMN-1556 infected cells demonstrates that CPiVF is also expressed and that the P2A fusion performs as designed and is validated by these data.

[0788] Implications for Vaccine Development

[0789] These findings demonstrate that the CAV-based vaccine compositions described herein are replication-competent, capable of amplifying in infected cells, and able to propagate efficiently across cell cultures. This productive replication is critical for ensuring robust antigen expression, immune stimulation, and effective vaccine performance.

[0790] The ability to observe progressive plaque formation, cytopathic spread, and monolayer clearance provides a quantifiable and reproducible method for assessing the functional integrity of recombinant CAV vaccine candidates, ensuring their suitability for clinical and veterinary applications.

[0791] It will be apparent that tire precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

7158-105381-02CLAIMS1. A recombinant canine adenovirus (CAV) genome, comprising a first heterologous open reading frame (ORF), wherein:the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame;the first heterologous ORF is located 5' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in tire same reading frame;the first heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein tire ElB-55k ORF, tire self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame;the first heterologous ORF is located 5' of a DNA polymerase ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame;the first heterologous ORF is located 3' of a DNA polymerase ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the DNA polymerase ORF are operably linked and in the same reading frame;the first heterologous ORF is located 3' of an E2A-DNA binding protein (DBP) ORF and the first heterologous ORF is linked to the E2A-DBP ORF;the first heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between tire first heterologous ORF and tire pVIII ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame;the first heterologous ORF is located 3' of an E3-ORF1 and the first heterologous ORF is linked to the E3-ORF1;the genome comprises a complete or partial deletion of an E3-ORF1 coding sequence and the first heterologous ORF is inserted at the site of the E3-ORF1 deletion; orthe genome comprises a complete or partial deletion of an E3-ORF1 coding sequence and a complete or partial deletion of an E3-ORF2 coding sequence, and the first heterologous ORF is inserted at the site of tire E3-ORF1 deletion.7158-105381-022. The recombinant CAV genome of claim 1, wherein a canine adenovirus comprising the recombinant CAV genome is replication competent.

3. The recombinant CAV genome of claim 1 or claim 2, wherein the E3 region does not comprise a heterologous promoter.

4. The recombinant CAV genome of any one of claims 1-3, wherein the recombinant CAV genome does not comprise a heterologous promoter and / or a heterologous polyA sequence.

5. The recombinant CAV genome of anyone of claims 1-4, wherein:the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence.

6. The recombinant CAV genome of any one of claims 1-4, wherein:the first heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the pVIII ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence.

7. The recombinant CAV genome of any one of claims 1-4, wherein:the first heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence.

8. The recombinant CAV genome of any one of claims 1-4, wherein:the first heterologous ORF is located 5' of a DNA polymerase ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the DNA polymerase ORF, wherein the first heterologous ORF, the self-cleaving peptide coding sequence and tlie DNA polymerase ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence.7158-105381-029. The recombinant CAV genome of any one of claims 1-8, further comprising a second heterologous ORF.

10. The recombinant CAV genome of claim 9, wherein:the first heterologous ORF is located 3' of the El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence and the second heterologous ORF is inserted at the site of the E3-ORF1 deletion.

11. The recombinant CAV genome of claim 9, wherein:the first heterologous ORF is located 3' of the ElB-55k ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe genome comprises a complete or partial deletion of the E3-ORF1 coding sequence and the second heterologous ORF is inserted at the site of the E3-ORF1 deletion.

12. The recombinant CAV genome of claim 9, wherein:the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the El A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe second heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the second heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the second heterologous ORF are operably linked and in the same reading frame.

13. The recombinant CAV genome of claim 9, wherein:the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; andthe second heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between the second heterologous ORF and tine pVIII ORF, wherein the second7158-105381-02heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in the same reading frame.

14. The recombinant CAV genome of claim 9, wherein the genome comprises a complete or partial deletion of the E3-ORF1 coding sequence and the first heterologous ORF and the second heterologous ORF are inserted at the site of the E3-ORF1 deletion.

15. The recombinant CAV genome of claim 14, wherein a self-cleaving peptide coding sequence is located between the first heterologous ORF and the second heterologous ORF, and the first heterologous ORF, the self-cleaving peptide coding sequence the second heterologous ORF axe operably linked and in the same reading frame.

16. The recombinant CAV genome of claim 14, wherein a linker sequence is located between the first heterologous ORF and the second heterologous ORF, and the first heterologous ORF, the linker sequence, and the second heterologous ORF are operably linked and in the same reading frame.

17. The recombinant CAV genome of any one of claims 1-16, further comprising a third heterologous ORF.

18. The recombinant CAV genome of claim 17, wherein the genome comprises a complete or partial deletion of the E3-ORF1 coding sequence and the third heterologous ORF is inserted at the site of the E3-ORFl deletion.

19. The recombinant CAV genome of claim 18, wherein:a linker sequence is located between the first heterologous ORF and the second heterologous ORF;a self-cleaving peptide coding sequence is located between the second heterologous ORF and the third heterologous ORF; andthe first heterologous ORF, the linker sequence, the second heterologous ORF, the self-cleaving peptide coding sequence, and the third heterologous ORF are operably linked and in the same reading frame.

20. The recombinant CAV genome of claim 17, wherein:the first heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the self-cleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame;7158-105381-02the second heterologous ORF is located 3' of an ElB-55k ORF and a self-cleaving peptide coding sequence is located between the second heterologous ORF and the ElB-55k ORF, wherein the ElB-55k ORF, the self-cleaving peptide coding sequence and the second heterologous ORF are operably linked and in the same reading frame; andthe third heterologous ORF is located 5' of a pVIII ORF and a self-cleaving peptide coding sequence is located between tire third heterologous ORF and the pVIII ORF, wherein the third heterologous ORF, the self-cleaving peptide coding sequence and the pVIII ORF are operably linked and in tire same reading frame.

21. The recombinant CAV genome of any one of claims 1-20, further comprising a fourth heterologous ORF.

22. The recombinant CAV genome of any one of claims 1-21, wherein the first heterologous ORF, tire second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF are under the control of native viral post-transcriptional and translational elements.

23. The recombinant CAV genome of any one of claims 1-22, wherein the partial deletion of the E3-ORF1 coding sequence excludes a 5’ region of the E3-ORF1 that overlaps with a 3’ region of the pVIII ORF.

24. The recombinant CAV of claim 23, wherein the 5’ region of the E3-ORF1 that overlaps with the 3’ region of the pVIII ORF comprises one or more nucleotide substitutions, wherein the one or more nucleotide substitutions eliminate one or two alternative start codons, and wherein the one or more nucleotide substitutions do not result in any amino acid substitutions in the pVIII protein.

25. The recombinant CAV genome of claim 23 or claim 24, wherein the first heterologous ORF inserted at tire site of die E3-ORF1 deletion comprises a start codon and / or a Kozak consensus sequence.

26. The recombinant CAV genome of any one of claims 1-25, further comprising a complete or partial deletion of the E3-ORF2 coding sequence.

27. The recombinant CAV of claim 26, wherein deletion of the E3-ORF2 coding sequence increases expression by at least 50% of a heterologous ORF inserted at die site of die E3-ORF1 deletion relative to expression in the absence of the E3-ORF2 deletion.7158-105381-0228. The recombinant CAV genome of any one of claims 1-27, further comprising a complete or partial deletion of the E4-ORF5 coding sequence.

29. The recombinant CAV of claim 28, comprising a complete or partial deletion of the E3-ORF1 coding sequence, a complete or partial deletion of the E3-ORF2 coding sequence, and a complete or partial deletion of the E4-ORF5 coding sequence.

30. The recombinant CAV of claim 28 or claim 29, wherein a heterologous ORF is inserted at the site of the E4-ORF5 deletion.

31. The recombinant CAV genome of any one of claims 1-30, wherein the E3-ORF1 is deleted and replaced with a polycistronic cassette comprising three or more linked heterologous ORFs.

32. The recombinant CAV genome of any one of claims 1-31, wherein the E3-ORF1 is deleted and replaced with a polycistronic cassette comprising three or more linked heterologous ORFs, and wherein the genome further comprises a complete or partial deletion of E3-ORF2 and / or E4-ORF5.

33. The recombinant CAV genome of claim 31 or claim 32, wherein the ORFs are linked using a self-cleaving peptide sequence.

34. The recombinant CAV of claim 33, wherein the self-cleaving peptide sequence is a 2A peptide comprising a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2 A (T2A) peptide, or a variant thereof.

35. The recombinant CAV of any one of claims 26-34, wherein the non-coding junctions between E3-ORF1 and E3-ORF2, and between E3-ORF2 and U exon are preserved when the E3-ORF1 and E3-ORF2 coding sequences are completely or partially deleted.

36. The recombinant CAV genome of any one of claims 1-35, wherein the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encode a therapeutic protein.

37. The recombinant CAV genome of claim 36, wherein the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF each encode a different therapeutic protein.7158-105381-0238. The recombinant CAV genome of claim 36 or claim 37, wherein the therapeutic protein is an antigenic protein, an anti-cancer agent, an immunostimulatory transgene, or an antibody.

39. The recombinant CAV genome of claim 38, wherein the antigenic protein is a protein from a pathogen.

40. The recombinant CAV genome of claim 38, wherein the antigenic protein is a cancer antigen.

41. The recombinant CAV genome of claim 38, wherein the anti-cancer agent is an iinmunomodulator or a chemotherapeutic agent, an immunotoxin, or a pro-drug activating enzyme.

42. The recombinant CAV genome of claim 38, wherein the immunostimulatory transgene comprises interleukin (IL)- 12, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme B, interferon (IFN)-a, interferon (IFN)-p, C-C motif chemokine ligand 21 (CCL21), or C-X-C motif chemokine ligand 10 (CXCL10).

43. The recombinant CAV genome of claim 38, wherein the antibody is an anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) antibody, an anti-programmed cell death protein 1 (PD1) antibody, or an anti-programmed death-ligand 1 (PD-L1) antibody.

44. The recombinant CAV genome of any one of claims 1-35, wherein the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encode a reporter protein.

45. The recombinant CAV genome of claim 44, wherein the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF each encode a different reporter protein.

46. The recombinant CAV of any one of claims 1-35, wherein the first heterologous ORF, the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encode a combination of therapeutic proteins and reporter proteins.

47. The recombinant CAV genome of any one of claims 44-46, wherein the reporter protein is a fluorescent protein, a luminescent protein, an enzyme, or an imaging reporter for positron emission tomography (PET) or magnetic resonance imaging (MRI).7158-105381-0248. The recombinant CAV of claim 47, wherein:the PET reporter comprises herpes simplex virus 1 (HSVl)-thymidine kinase (TK), dopamine D2 receptor (D2R), or human sodium iodide symporter (NIS); orthe MRI reporter comprises tyrosinase, ferritin, or aquaporin-1 (AQP1).

49. The recombinant CAV genome of any one of claims 47, wherein the fluorescent protein is YPet, mCherry, Katushka, Halo- tag, Snap-tag, or blue fluorescent protein (BFP).

50. The recombinant CAV genome of any one of claims 1-49, wherein the self-cleaving peptide is a 2A peptide or variant thereof.

51. The recombinant CAV genome of claim 50, wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.

52. The recombinant CAV genome of any one of claims 1-51, comprising in the 5' to 3’ direction:El A-2A-hetero logons ORF;heterologous ORF-2A-E1A;ElA-2A-hetero logons ORF-2 A-heterologous ORF;E1B-55k-2A-heterologous ORF;ElB-55k-2A-heterologous ORF-2A-heterologous ORF;heterologous ORF-2A-DNA polymerase;DNA polymerase-2A-heterologous ORF;E2A-DBP-heterologous ORF;E2A-DBP-linker-heterologous ORF;heterologous ORF-2A-pVIII;E3-ORF1 -heterologous ORF;E3-ORF1 -linker-heterologous ORF;AE3-ORFl-heterologous ORF;AE3-ORF1 -heterologous ORF-AE3-ORF2;AE3-ORFl-heterologous ORF-P2A-heterologous ORF;AE3-ORF1 -heterologous ORF-linker-heterologous ORF;AE3-ORFl-heterologous ORF-linker-heterologous ORF-AE3-ORF2;AE3-ORFl-heterologous ORF-linker-heterologous ORF-2A-heterologous ORF-AE3-ORF2; AE3-ORFl-heterologous ORF-2A-heterologous ORF-2A-heterologous ORF-AE3-ORF2; or7158-105381-02AE3-ORFl-heterologous ORF-2A-heterologous ORF-2A-heterologous ORF-AE3-ORF2, DE4- ORF5.

53. The recombinant CAV genome of claim 52, comprising:ElA-2A-heterologous ORF and AE3-ORF1;ElA-2A-hetero logons ORF, AE3-ORF1 and AE3-ORF2;El A-2A-heterologous ORF, AE3-ORF1 and AE3-ORF2; AE4-ORF5heterologous ORF-2A-E1A and AE3-ORF1;heterologous ORF-2A-E1A, AE3-ORF1 and AE3-ORF2;ElA-2A-heterologous ORF and AE3-ORF1 -heterologous ORF;ElA-2A-hetero logons ORF, AE3-ORFl-heterologous ORF, and AE3-ORF2;ElA-2A-heterologous ORF, AE3-ORF1 -heterologous ORF, AE3-ORF2 and AE4-ORF5;El A-2A-hetero logons ORF, AE3-ORF1 -heterologous ORF-2A-heterologous ORF, AE3-ORF2 and AE4-ORF5;ElA-2A-hetero logons ORF and E1B— 55K-2A-heterologous ORF;heterologous ORF-2A-E1A and AE3-ORF1 -heterologous ORF;heterologous ORF-2A-E1A, AE3-ORFl-heterologous ORF, and AE3-ORF2;ElB-55k-2A-heterologous ORF and AE3-ORF1;ElB-55k-2A-heterologous ORF, AE3-ORF1 and AE3-ORF2;ElB-55k-2A-heterologous ORF and AE3-ORF1 -heterologous ORF;ElB-55k-2A-heterologous ORF, AE3-ORFl-heterologous ORF, and AE3-ORF2;ElA-2A-heterologous ORF and heterologous ORF-2A-pVIII;ElA-2A-hetero logons ORF, heterologous ORF-2A-pVIII , AE3-ORF1 and AE3-ORF2; heterologous ORF-2A-pVIII and AE3-ORF1 -heterologous ORF;heterologous ORF-2A-pVIII, AE3-ORF1 -heterologous ORF, and AE3-ORF2; heterologous ORF-2A-DNA polymerase and AE3-ORFl-heterologous ORF; or heterologous ORF-2A-DNA polymerase, AE3-ORFl-heterologous ORF and AE3-ORF2.

54. The recombinant CAV genome of claim 52 or claim 53, further comprising AE4-ORF5.

55. The recombinant CAV genome of any one of claims 1-54, comprising the nucleotide sequence of any one of SEQ ID NOs: 1-23, 32-34 and 37.

56. A composition comprising the recombinant CAV genome of any one of claims 1-55 and a pharmaceutically acceptable carrier.7158-105381-0257. A recombinant CAV comprising the recombinant CAV genome of any one of claims 1- 55.

58. A composition comprising the recombinant CAV of claim 57 and a pharmaceutically acceptable carrier.

59. A method for detecting and quantifying replication of an individual recombinant CAV or a library of recombinant CAVs in a host cell culture, comprising:infecting or transfecting the host cell culture with a recombinant CAV comprising the recombinant CAV genome of claim 1, wherein the first heterologous ORF encodes a reporter protein;detecting expression of the reporter protein using:(i) an imaging-based system capable of acquiring and analyzing spatially and spectrally resolved image data at different wavelengths, length and time scales, from multi-well plates of recombinant CAV genome reporter gene fluorescence in individual cells, plaques and / or the cell culture area as well as brightfield imaging; and / or(ii) a multi-modal plate reader configured to rapidly measure bulk recombinant CAV genome reporter gene fluorescence intensity and / or luminescence across a plurality of wells in a multi-well format,wherein the signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

60. A method for detecting and quantifying replication of an individual recombinant CAV or a library of recombinant CAVs in a host cell culture, comprising:infecting or transfecting the host cell culture with a recombinant CAV comprising the recombinant CAV genome of claim 1, wherein the first heterologous ORF encodes a reporter protein;detecting expression of the reporter protein using:(i) a plate reader based system that quantifies fluorescence or luminescence from each well of a multi-well plate; or(ii) wherein the plate reader is configured or automated to capture fluorescence or luminescence over time,wherein the expression of the reporter protein signal correlates with viral genome transgene expression upon transfection, infection, replication or spread in the culture.

61. The method of claim 59 or claim 60, wherein the reporter protein is a fluorescent reporter protein comprising blue fluorescent protein (BFP), mNeonGreen, YPet, mCherry, iRFP720, mScarlet, Katushka, HaloTag, or a derivative thereof.7158-105381-0262. The method of any one of claims 59-61, wherein the reporter or fluorescence is detected using a hybrid multi-mode reader.

63. The method of any one of claims 59-62, wherein the imaging-based detection comprises time-lapse imaging to visualize and analyze fluorescent plaque growth and viral infection of cells over time.

64. The method of claim 63, wherein tire imaging-based detection of fluorescent plaques is used to quantify viral titers and fluorescent plaque forming units.

65. The method of any one of claims 59-64, wherein:the heterologous ORF is located 3' of an El A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the E1A ORF, wherein the E1A ORF, the selfcleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; orthe heterologous ORF is inserted at the site of tire E3-ORF1 deletion.

66. The method of any one of claims 59-65, wherein the recombinant CAV genome further comprises a complete or partial deletion of the E3-ORF1 coding sequence and optionally further comprises a complete or partial deletion of the E3-ORF2 coding sequence.

67. The method of any one of claims 59-66, wherein the recombinant CAV further comprises a second heterologous ORF, a third heterologous ORF and / or a fourth heterologous ORF.

68. The method of claim 67, wherein the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encodes a therapeutic protein.

69. The method of any one of claims 59-68, wherein:transduction and / or replication of the recombinant CAV is measured in a plurality of different cell types;transduction and / or replication of the recombinant CAV is compared to replication of second recombinant CAV comprising different genome modifications;replication of the recombinant CAV is compared to replication of an adenovirus from a different species;replication of the recombinant CAV is measured in different culture media and / or in the presence of different drugs.7158-105381-0270. A method for measuring replication of a recombinant CAV comprising the recombinant CAV genome of claim 1, wherein the first heterologous ORF encodes a reporter protein, the method comprising:(i) transfecting cells with the genome of the recombinant CAV, or infecting cells with particles of the recombinant CAV;(ii) culturing the transfected cell or infected cells for at least 24-72 hours;(iii) measuring reporter gene signal at regular intervals throughout tire culturing step, thereby obtaining fluorescence measurements; and(iv) calculating log-slope (day1) from a semi-log plot of fluorescence or intensity signal, normalized to background, versus time in tire linear part of tire curve, thereby measuring replication of the recombinant CAV.

71. The method of claim 70, wherein:the heterologous ORF is located 3' of an E l A ORF and a self-cleaving peptide coding sequence is located between the first heterologous ORF and the El A ORF, wherein the E1A ORF, the selfcleaving peptide coding sequence and the first heterologous ORF are operably linked and in the same reading frame; orthe heterologous ORF is inserted at the site of the E3-ORF1 deletion.

72. The method of claim 70 or claim 71 , wherein the recombinant CAV genome further comprises a complete or partial deletion of the E3-ORF1 coding sequence and optionally further comprises a complete or partial deletion of the E3-ORF2 coding sequence.

73. The method of any one of claims 70-72, wherein the recombinant CAV further comprises a second heterologous ORF, a third heterologous ORF and / or a fourth heterologous ORF.

74. The method of claim 73, wherein the second heterologous ORF, the third heterologous ORF and / or the fourth heterologous ORF encodes a therapeutic protein.

75. The method of any one of claims 70-74, wherein:replication of the recombinant CAV is measured in a plurality of different cell types; replication of the recombinant CAV is compared to replication of one or more recombinant CAVs comprising different genome modifications;replication of the recombinant CAV is compared to replication of an adenovirus from a different species;replication of the recombinant CAV is measured in different culture media and / or in the presence of different drugs;7158-105381-02replication of the recombinant CAV is measured in different cell lines; and / orreplication of the recombinant CAV is measured in different cell lines in CRISPR genome wide screens.

76. The method of claim 75, wherein the plurality of cell types includes a first cell type that is a tumor cell and a second cell type that is a non-tumor cell.

77. The method of any one of claims 70-76, comprising transfecting cells with the genome of the recombinant CAV.

78. The method of any one of claims 70-76, comprising infecting cells with particles of the recombinant CAV.

79. The method of claim 78, wherein tire cells are infected with serial dilutions of tire recombinant CAV particles.

80. The method of claim 79, comprising dilutions of 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900 and 1:218,700.

81. A kit comprising:(i) the recombinant CAV genome of any one of claims 1-55, or the recombinant CAV of claim 57; and(ii) cells, cell culture media, and / or a multi-well plate.

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