High-throughput assay for measuring adenovirus replication kinetics

Abstract

To provide a high throughput assay for measuring the replication kinetics of adenovirus.SOLUTION: A recombinant adenovirus genome comprising a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence is described. Recombinant adenovirus genomes and recombinant adenoviruses produced by the disclosed genomes can be used, for example, in high-throughput assays to measure viral replication kinetics. Methods for measuring the replication kinetics of recombinant adenoviruses are also described. In one aspect, a recombinant adenovirus genome is provided comprising a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence, both in the same reading frame and operably linked to an endogenous adenovirus ORF.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 298,649, filed February 23, 2016, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates to the optimal placement of exogenous open reading frames in recombinant adenovirus constructs and the use of the recombinant viruses in assays for measuring adenovirus replication kinetics. [Background technology]

[0003] Adenovirus serotype 5 (Ad5) is the vector of choice for basic research applications, mouse lung cancer models, and human gene therapy trials. Adenoviruses possess a stable 36 kb double-stranded DNA genome protected by a protein capsid surrounded by Ad fiber protein spikes that target infection to receptors on specific cell types. Adenoviruses do not integrate into host DNA, can be produced at high titers using established protocols, and have proven safe for human gene therapy and cancer applications. Therefore, Ad-based vectors hold great promise for cancer diagnosis and therapy. However, there is a need for rapid, high-throughput means to evaluate the replication kinetics of recombinant adenoviruses designed for clinical and therapeutic use. Summary of the Invention [Means for solving the problem]

[0004] The present specification discloses a recombinant adenovirus genome, which comprises heterologous open reading frame (ORF) and self-cleaving peptide coding sequence.The recombinant adenovirus genome and the recombinant adenovirus produced by the disclosed genome can be used in assays such as measuring viral replication kinetics.

[0005] Provided herein is a recombinant adenovirus genome comprising a heterologous ORF and a self-cleaving peptide coding sequence, both in the same reading frame as and operably linked to an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF. In some embodiments, the endogenous ORF is E1B-55k and the heterologous ORF is 3' of E1B-55k; the endogenous ORF is DNA polymerase and the heterologous ORF is 5' of the DNA polymerase; the endogenous ORF is DNA binding protein (DBP) and the heterologous ORF is 3' of DBP; the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5' of ADP; the endogenous ORF is E3-14.7k and the heterologous ORF is 3' of E3-14.7k; or the endogenous ORF is E4-ORF2 and the heterologous ORF is 5' of E4-ORF2.

[0006] Further provided herein is a recombinant adenovirus comprising the recombinant adenoviral genome disclosed herein.

[0007] Also provided are methods for measuring the replication kinetics of recombinant adenovirus. In some embodiments, the genome of the recombinant adenovirus comprises a heterologous ORF encoding a fluorescent protein and a self-cleaving peptide coding sequence, all in the same reading frame and operably linked to an endogenous adenovirus ORF selected from E1B-55k, DNA polymerase, DBP, ADP, E3-14.7k, and E4-ORF2. The self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous adenovirus ORF. In some examples, the method includes transfecting cells with the genome of the recombinant adenovirus or infecting cells with recombinant adenovirus particles, culturing the transfected or infected cells for at least two days, measuring fluorescence at regular intervals during the culture period, and calculating a logarithmic slope from the fluorescence measurements. This method can be used to select suitable therapeutic adenovirus (such as oncolytic adenovirus) for tumor treatment, for example, by obtaining tumor cells (for example, by biopsy) and measuring the replication kinetics of recombinant adenovirus in tumor cells, and this recombinant adenovirus corresponds to therapeutic adenovirus, except that the therapeutic ORF of therapeutic adenovirus is replaced with the ORF encoding fluorescent protein.Similarly, this method can be used to select cancer patients who will respond to treatment with specific therapeutic adenovirus, or to identify the most effective therapeutic adenovirus for specific tumor.

[0008] The above and other objects and features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 is a schematic diagram of an exemplary workflow for testing adenovirus constructs. A whole viral genome plasmid is produced and transfected into suitable cells, such as 293-E4 cells, in a multi-well plate. Once the transfected cells have expanded, they are subjected to freeze / thawing to release viral particles, followed by centrifugation to pellet the cell debris. The supernatant (containing viral particles) is transferred to multiple larger culture plates. Viral particles are harvested from the transfected cells, CsCl-purified, and the infectious viral titer is measured by ELISA. The purified virus is then infected into the desired cell type at a known MOI. At 48 or 72 hours post-infection, adenovirus late proteins, adenovirus genomes, or plaques are measured by Western blot, q-PCR, or plaque assay, respectively.

[0010] [Figure 2] Figure 2 is a schematic diagram illustrating exponential viral growth. Exponential viral growth is required for oncolytic killing of all cells in a tumor. However, in most cases, only a small percentage of tumor cells are infected initially. Therefore, small differences in the number of progeny per replication result in large differences in the total number of particles after only a few replications. A comparison is shown between a virus that produces 3 virions per cycle and a virus that produces 5 virions per cycle. As shown in the graph, after 5 to 6 replications, the viral titers of these two viruses are significantly different.

[0011] [Figure 3]Figure 3 is a schematic diagram showing the workflow of the fluorescence-based viral kinetics (FBVK) assay disclosed herein. A whole viral genome plasmid is produced (e.g., by Adsembly or AdSLIC) and used to transfect the cell type of interest in a multi-well plate. Alternatively, the cells are infected with recombinant adenovirus particles. The adenovirus genome contains at least one open reading frame (ORF) encoding a fluorescent protein at a location within the viral genome that does not substantially change the viral replication kinetics. Fluorescence is monitored over time to calculate the viral replication kinetics.

[0012] [Figure 4A] Figures 4A-4B outline an exemplary kinetic assay environment starting with an adenoviral genome plasmid. This assay does not require knowledge of the initial transfection efficiency. Transfection conditions are selected so that approximately 5-10% of cells are initially transfected. In the example shown, a 48-well plate is used, allowing for the testing of 14 different viral constructs in triplicate, with three mock-infected wells and three wells containing FLUORESBRITE™ beads to compensate for tool sensitivity variations. (Figure 4A) The top half of the 48-well plate contains triplicate cells transfected with six different viral genome plasmids, mock-infected cells, and a blank (FLUORESBRITE™ beads). (Figure 4B) The bottom half of the 48-well plate contains triplicate cells transfected with eight different viral genome plasmids. The multiwell plate is placed in a plate reader (e.g., a TECAN plate reader) for continuous fluorescence monitoring. [Figure 4B]Figures 4A-4B outline an exemplary kinetic assay environment starting with an adenoviral genome plasmid. This assay does not require knowledge of the initial transfection efficiency. Transfection conditions are selected so that approximately 5-10% of cells are initially transfected. In the example shown, a 48-well plate is used, allowing for the testing of 14 different viral constructs in triplicate, with three mock-infected wells and three wells containing FLUORESBRITE™ beads to compensate for tool sensitivity variations. (Figure 4A) The top half of the 48-well plate contains triplicate cells transfected with six different viral genome plasmids, mock-infected cells, and a blank (FLUORESBRITE™ beads). (Figure 4B) The bottom half of the 48-well plate contains triplicate cells transfected with eight different viral genome plasmids. The multiwell plate is placed in a plate reader (e.g., a TECAN plate reader) for continuous fluorescence monitoring.

[0013] [Figure 5] Figure 5 outlines an exemplary kinetic assay setup starting with a recombinant virus. This assay does not require knowledge of virus titer. The recombinant virus is serially diluted and used to infect cells seeded in a multiwell plate. In the example shown, a 96-well plate is used, and each virus is diluted 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900, and 1:218,700, allowing 11 viruses to be tested simultaneously. Four wells are mock infected, and FLUORESBRITE™ beads are placed in four wells to compensate for tool sensitivity and variability. The multiwell plate is then placed in a plate reader (such as a TECAN plate reader) for continuous fluorescence monitoring.

[0014] [Figure 6A]Figures 6A-6C show schematic diagrams of the Adsembly and AdSLIC techniques for the combinatorial assembly of recombinant adenoviruses. (Figure 6A) The adenoviral genome is divided into four modules—E1, core, E3, and E4. (Figure 6B) Adsembly involves reassembly of the genome using a multisite Gateway reaction. (Figure 6C) AdSLIC utilizes sequence- and ligation-independent cloning (SLIC) to assemble adenoviral modules. [Figure 6B] Figures 6A-6C show schematic diagrams of the Adsembly and AdSLIC techniques for the combinatorial assembly of recombinant adenoviruses. (Figure 6A) The adenoviral genome is divided into four modules—E1, core, E3, and E4. (Figure 6B) Adsembly involves reassembly of the genome using a multisite Gateway reaction. (Figure 6C) AdSLIC utilizes sequence- and ligation-independent cloning (SLIC) to assemble adenoviral modules. [Figure 6C] Figures 6A-6C show schematic diagrams of the Adsembly and AdSLIC techniques for the combinatorial assembly of recombinant adenoviruses. (Figure 6A) The adenoviral genome is divided into four modules—E1, core, E3, and E4. (Figure 6B) Adsembly involves reassembly of the genome using a multisite Gateway reaction. (Figure 6C) AdSLIC utilizes sequence- and ligation-independent cloning (SLIC) to assemble adenoviral modules.

[0015] [Figure 7] Figure 7 is a bar graph showing the natural logarithmic slope (ln-slope) of recombinant adenoviruses encoding fluorescent proteins in the E1 region. Values ​​are shown for the direct fusion construct YPet-E1A, as well as the P2A site-containing constructs YPet-P2A-E1A, E1A-P2A-mCherry, and E1B-55k-P2A-YPet. The YPet-P2A-ADP construct is shown for comparison.

[0016] [Figure 8] FIG. 8 is a schematic representation of the analysis and interpretation of kinetic data from a fluorescence-based viral kinetic assay.

[0017] [Figure 9A] 9A-9C are bar graphs showing the natural logarithmic slope values ​​of recombinant adenoviruses derived from Ad5, Ad9, or Ad34 and containing a heterologous ORF 3' of the E3-14.7k (or its equivalent in Ad9 and Ad34) ORF. Values ​​are shown for Ad5 (E3-14.7k-P2A-YPet, PCMN-887), Ad9 (E3-15k-P2A-YPet, PCMN-888), and Ad34 (E3-14.8k-P2A-YPet, PCMN-889) in 293 cells (FIG. 9A), A549 cells (FIG. 9B), and U2OS cells (FIG. 9C). Values ​​for chimeric viruses containing an Ad5 core (containing E3-14.7k-P2A-YPet) and a fiber shaft / knob derived from either Ad9 (Ad5 / Ad9) or Ad34 (Ad5 / Ad34) are also shown in each figure. [Figure 9B] 9A-9C are bar graphs showing the natural logarithmic slope values ​​of recombinant adenoviruses derived from Ad5, Ad9, or Ad34 and containing a heterologous ORF 3' of the E3-14.7k (or its equivalent in Ad9 and Ad34) ORF. Values ​​are shown for Ad5 (E3-14.7k-P2A-YPet, PCMN-887), Ad9 (E3-15k-P2A-YPet, PCMN-888), and Ad34 (E3-14.8k-P2A-YPet, PCMN-889) in 293 cells (FIG. 9A), A549 cells (FIG. 9B), and U2OS cells (FIG. 9C). Values ​​for chimeric viruses containing an Ad5 core (containing E3-14.7k-P2A-YPet) and a fiber shaft / knob derived from either Ad9 (Ad5 / Ad9) or Ad34 (Ad5 / Ad34) are also shown in each figure. [Figure 9C]9A-9C are bar graphs showing the natural logarithmic slope values ​​of recombinant adenoviruses derived from Ad5, Ad9, or Ad34 and containing a heterologous ORF 3' of the E3-14.7k (or its equivalent in Ad9 and Ad34) ORF. Values ​​are shown for Ad5 (E3-14.7k-P2A-YPet, PCMN-887), Ad9 (E3-15k-P2A-YPet, PCMN-888), and Ad34 (E3-14.8k-P2A-YPet, PCMN-889) in 293 cells (FIG. 9A), A549 cells (FIG. 9B), and U2OS cells (FIG. 9C). Values ​​for chimeric viruses containing an Ad5 core (containing E3-14.7k-P2A-YPet) and a fiber shaft / knob derived from either Ad9 (Ad5 / Ad9) or Ad34 (Ad5 / Ad34) are also shown in each figure. DETAILED DESCRIPTION OF THE INVENTION

[0018] (Sequence Listing) The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the presented strand. The sequence listing has been submitted as a 609KB ASCII text file created on February 21, 2017, and is incorporated herein by reference. In the attached sequence listing:

[0019] SEQ ID NO: 1 is the nucleotide sequence of the synthetic adenoviral genome CMBT-379 (YPet-P2A-E1A).

[0020] SEQ ID NO:2 is the nucleotide sequence of the synthetic adenoviral genome CMBT-432 (E1A-P2A-YPet).

[0021] SEQ ID NO: 3 is the nucleotide sequence of the synthetic adenoviral genome CMBT-456 (E1B-55k-P2A-YPet).

[0022] SEQ ID NO: 4 is the nucleotide sequence of the synthetic adenoviral genome CMBT-499 (E1B-55k-P2A-mCherry).

[0023] SEQ ID NO: 5 is the nucleotide sequence of the synthetic adenovirus genome CMBT-530 (YPet-P2A-(DNA poly)).

[0024] SEQ ID NO: 6 is the nucleotide sequence of the synthetic adenoviral genome CMBT-886 (DBP-P2A-YPet).

[0025] SEQ ID NO: 7 is the nucleotide sequence of the synthetic adenoviral genome CMBT-403 (YPet-P2A-ADP).

[0026] SEQ ID NO: 8 is the nucleotide sequence of the synthetic adenoviral genome CMBT-429 (ADP-P2A-YPet).

[0027] SEQ ID NO: 9 is the nucleotide sequence of the synthetic adenoviral genome PCMN-887 (E3-14.7k-P2A-YPet).

[0028] SEQ ID NO: 10 is the nucleotide sequence of the synthetic adenovirus genome CMBT-457 (YPet-P2A-E4-ORF2).

[0029] SEQ ID NO: 11 is the nucleotide sequence of the synthetic adenoviral genome CMBT-633 (mCherry-P2A-E4-ORF2).

[0030] SEQ ID NO: 12 is the amino acid sequence of P2A.

[0031] SEQ ID NO: 13 is the amino acid sequence of F2A.

[0032] SEQ ID NO: 14 is the amino acid sequence of E2A.

[0033] SEQ ID NO: 15 is the amino acid sequence of T2A.

[0034] SEQ ID NO: 16 is the amino acid sequence of modified P2A containing GSG at the N-terminus.

[0035] SEQ ID NO: 17 is the amino acid sequence of a modified F2A containing GSG at the N-terminus.

[0036] SEQ ID NO: 18 is the amino acid sequence of a modified E2A containing GSG at the N-terminus.

[0037] SEQ ID NO: 19 is the amino acid sequence of a modified T2A containing GSG at the N-terminus.

[0038] SEQ ID NO:20 is the nucleotide sequence of the synthetic adenoviral genome PCMN-888 (Ad9 E3-15k-P2A-YPet).

[0039] SEQ ID NO:21 is the nucleotide sequence of the synthetic adenoviral genome PCMN-889 (Ad34 E3-14.8k-P2A-YPet).

[0040] I. Abbreviations Ad: Adenovirus ADP Adenovirus death protein BFP Blue Fluorescent Protein DBP DNA-binding protein E2A Equine rhinitis A virus 2A ELISA enzyme-linked immunosorbent assay ERAV Equine rhinitis A virus F2A Foot-and-mouth disease virus 2A FACS Fluorescence-Activated Cell Sorting FMDV Foot and mouth disease virus GFP Green Fluorescent Protein MOI Multiplicity of infection OD optical density ORF Open Reading Frame P2A Porcine teschovirus-1 2A pIX Protein IX PTV1 Porcine teschovirus-1 RFP Red Fluorescent Protein SLIC Sequence- and Ligation-Independent Cloning T2A Thosea asigna virus 2A TaV Thosea asigna virus YFP Yellow Fluorescent Protein

[0041] II. Terminology and Methods Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V (ISBN 0-19-854287-9), published by Oxford University Press in 1994; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (ISBN 0-632-02182-9), published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1-56081-569-8), published by VCH Publishers, Inc. in 1995.

[0042] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0043] 2A peptide: A type of self-cleaving peptide encoded by some RNA viruses, such as picornaviruses. 2A peptides function by causing the ribosome to skip peptide bond synthesis at the C-terminus of the 2A element, resulting in separation of the end of the 2A sequence from the downstream peptide (Kim et al., PLoS One, Vol. 6(4), e18556, 2011). Cleavage occurs between the glycine and proline residues found at the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, those encoded by Thosea asigna virus (TaV), equine rhinitis A virus (ERAV), porcine teschovirus-1 (PTV1), and foot-and-mouth disease virus (FMDV), which are set forth herein as SEQ ID NOS: 12-15. In some embodiments, the 2A peptide contains Gly-Ser-Gly at the N-terminus to improve cleavage efficiency (SEQ ID NOS: 16-19).

[0044] Adenovirus: A non-enveloped virus with a linear double-stranded DNA genome and an icosahedral capsid. Currently, there are 68 known serotypes of human adenovirus, classified into seven species (species A, B, C, D, E, F, and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (mainly species B and C), conjunctivitis (species B and D), and / or gastroenteritis (species F and G).

[0045] Adenovirus death protein (ADP): A protein synthesized late during adenovirus infection that mediates cell lysis and release of the adenovirus to infect other cells. ADP is a 101-amino acid integral membrane glycoprotein that localizes to the nuclear envelope, endoplasmic reticulum, and Golgi apparatus. ADP was previously designated E3-11.6K.

[0046] Chimeric: Composed of at least two portions of different origins. In the context of the present disclosure, a "chimeric adenovirus" is an adenovirus having genetic material and / or proteins from at least two different serotypes (e.g., from Ad5 and an adenovirus of a second serotype). In this context, a "capsid-swapped" adenovirus refers to a chimeric adenovirus in which the capsid proteins are derived from one serotype of adenovirus and the remaining proteins are derived from another adenovirus serotype. Similarly, a "chimeric fiber" is a fiber protein having amino acid sequences derived from at least two different serotypes of adenovirus. For example, a chimeric fiber can be composed of a fiber shaft from Ad5 and a fiber knob from an adenovirus of a second serotype. In another example, a chimeric fiber is composed of an Ad5 tail and a fiber shaft and knob from an adenovirus of a second serotype (such as Ad9 or Ad34).

[0047] Contacting: To bring into direct physical association and includes both solid and liquid forms.

[0048] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a peptide that contains 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. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0049] Deleted: An adenovirus genome encoding a "deleted" protein (such as an E4orf1 or E4orf6 / 7 protein) refers to an adenovirus having a complete deletion of the protein coding sequence or a partial deletion that results in the absence of protein expression.

[0050] E2F deregulation refers to increased activity of E2F transcription factors and downstream target genes, which occurs in almost all types of human cancer. Deregulation of E2F pathway activity and transcription can result from a variety of different mutations in any upstream component of the pathway, such as loss-of-function mutations and deletions of Rb, p107, and p130 tumor suppressors. Rb was the first tumor suppressor identified and is absent or mutated in at least one-third of human tumors. In addition, p16 mutations and / or epigenetic silencing can activate E2F in tumor cells. Mutations, gene amplification, or overexpression of cyclin D and CDK4 can also result in deregulation of E2F activity in human tumors. In addition, E2F is activated by mutations in growth factor receptor pathways, including EGFR, RTK, RAS, RAF, PI-3K, PTEN, RAF, and MYC. p16 INK4a Mutations in the cyclin D:cdk4 / 6-RB-E2F pathway generally occur in a mutually exclusive manner, so that one "hit" (e.g., p16) is not accompanied by the other (e.g., Rb mutation or cyclin D:cdk overexpression). However, most current chemotherapeutics inhibit E2F transcriptional targets, but are also toxic to normal cells and are proliferative poisons that often have devastating iatrogenic complications. As disclosed herein, an alternative therapeutic approach is to use viruses that selectively undergo lytic replication in cancer cell lesions with deregulated p16-cyclin D:cdk4-RB-E2F pathway.

[0051] DNA-binding protein (DBP): This adenoviral protein binds to single-stranded DNA and RNA, as well as double-stranded DNA. DBP, a 72 kilodalton protein, is essential for adenoviral DNA replication.

[0052] E1A: The adenovirus early region 1A (E1A) gene and the polypeptide expressed from this gene. The E1A protein plays a role in viral genome replication by causing cells to enter the cell cycle. As used herein, the term "E1A protein" refers to the protein expressed from the E1A gene, and includes the E1A protein produced by any adenovirus serotype.

[0053] E3-RIDα / RIDβ and E3-14.7k: Early-expressing proteins produced by the E3 gene. The E3-RIDα, E3-RIDβ, and E3-14.7k proteins form the receptor internalization and degradation complex (RID), which localizes to the nuclear membrane and induces the endocytosis and degradation of various receptors, including CD95 (FasL receptor) and TNFR1 and 2 (TNF / TRAIL receptors), to protect infected cells from the host's antiviral response. The coding sequences for E3-RIDα, E3-RIDβ, and E3-14.7k are adjacent to each other in this order.

[0054] E4orf1: An adenoviral protein produced by the E4 gene. The term "E4orf1 protein" includes the E4orf1 protein produced by the E4 gene from any adenovirus serotype.

[0055] E4orf6 / 7: A protein encoded by the adenovirus E4 gene. The term "E4orf6 / 7 protein" includes E4orf6 / 7 proteins produced by the E4 gene from any adenovirus serotype.

[0056] Fluorescent protein: A protein that emits light of a specific wavelength when exposed to light of a specific wavelength. Fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, AcGFP1, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, and ZsGreen), blue fluorescent proteins (e.g., EBFP, EBFP2, Sapphire, T-Sapphire, Azurite, and mTagBFP), cyan fluorescent proteins (e.g., ECFP, mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, mTurquoise, and mTFP1), yellow fluorescent proteins (e.g., EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, and mBanana), and orange fluorescent proteins (e.g., Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, and mTangerine), red fluorescent proteins (mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, tdTomato, and E2-Crimson), orange / red fluorescent proteins (dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), and DsRed-Monomer), and modified versions thereof.

[0057] Fusion protein: A protein containing amino acid sequences derived from at least two different (heterologous) proteins or peptides. Fusion proteins can be produced, 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 produce a fusion protein, the nucleic acid sequences must be in the same reading frame and must not contain internal stop codons. Fusion proteins, especially short fusion proteins, can also be produced by chemical synthesis.

[0058] Heterologous: A heterologous protein or polypeptide refers to a protein or polypeptide that is derived from a different source or species.

[0059] Hexon: The major adenovirus capsid protein.

[0060] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from other biological components, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells, in the cells or tissues of an organism or the organism itself, in which the component naturally occurs. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0061] Modification: A change in a nucleic acid sequence or protein sequence. For example, modifications of an amino acid sequence include, for example, substitution, insertion, and deletion, or a combination thereof. Insertions include amino- and / or carboxyl-terminal fusions and the insertion of single or multiple amino acid residues into a sequence. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some embodiments herein, modifications (such as substitutions, insertions, or deletions) result in a change in function, such as a reduction or enhancement of a specific activity of a protein. As used herein, "Δ" or "delta" refers to a deletion. A substitution modification is one in which at least one residue is removed and a different residue is inserted in its place. Amino acid substitutions are typically of a single residue, but may occur simultaneously at several different positions. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final mutant sequence. These modifications can be prepared by modifying nucleotides in the DNA encoding the protein, thereby producing DNA encoding this modification. Techniques for making insertion, deletion, and substitution mutations at predetermined sites in DNA having a known sequence are well known in the art. A "modified" protein, nucleic acid, or virus is one that has one or more of the modifications outlined above.

[0062] Neoplasia, Malignancy, Cancer, and Tumor: A neoplasia is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can produce a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues and / or metastasize are called "malignant." Malignant tumors are also called "cancer."

[0063] Hematological cancer is cancer of blood or bone marrow.Examples of hematological (or hematopoietic) cancer include leukemia, for example, acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.In some cases, lymphoma is considered as solid tumor.

[0064] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, human papillomavirus (HPV)-infected neoplasia, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, and cervical cancer. cancer), testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinomas, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0065] Oncolytic virus: A virus that selectively kills cells with proliferative disorders, such as cancer / tumor cells. Killing of cancer cells can be detected by any method, such as determining the number of viable cells, or detecting cytopathic effects, apoptosis, or viral protein synthesis in cancer cells (for example, by metabolic labeling of viral genes required for replication, immunoblot, or RT-PCR), or reduction in tumor size.

[0066] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it 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.

[0067] Polypeptide, peptide, or protein: A polymer in which the monomers are amino acid residues joined together through amide bonds. When the amino acids are alpha amino acids, either the 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 residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. The term "residue" or "amino acid residue" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide.

[0068] A conservative substitution in a polypeptide is the replacement of one amino acid residue in a protein sequence with a different amino acid residue that has similar biological properties. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. For example, a protein or peptide containing one or more conservative substitutions (e.g., no more than one, no more than two, no more than three, no more than four, or no more than five 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 encoding the polypeptide using standard procedures, such as site-directed mutagenesis or PCR. In one example, such variants can be easily selected by testing antibody cross-reactivity or the ability of the antibody to induce an immune response. Examples of conservative substitutions are provided below. [Table 1]

[0069] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone, e.g., as a sheet or helix conformation, in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains.

[0070] In general, substitutions predicted to result in the greatest changes in protein properties will be non-conservative, such as (a) a hydrophilic residue, e.g., seryl or threonyl, substituting (or being substituted by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline substituting (or being substituted by) any other residue; (c) a residue with an electropositive side chain, e.g., lysyl, arginyl, or histadyl, substituting (or being substituted by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue with a bulky side chain, e.g., phenylalanine, substituting (or being substituted by) one without a side chain, e.g., glycine.

[0071] Promoter: A region of DNA that directs / initiat- es the transcription of a nucleic acid (e.g., a gene). A promoter comprises necessary nucleic acid sequences near the start site of transcription. Typically, a promoter is located near the gene to be transcribed. A promoter also optionally contains distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the transcription start site. A "constitutive promoter" is a promoter that is continuously active and is not subject to control by external signals or molecules. In contrast, the activity of an "inducible promoter" is controlled by external signals or molecules (e.g., transcription factors or tetracycline).

[0072] Protein IX (pIX): A minor component of the adenovirus capsid that associates with the hexon protein.

[0073] Purified: The term "purified" does not require absolute purity and is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, e.g., proteins, cellular debris, and other components.

[0074] Recombinant: A recombinant nucleic acid molecule, protein, or virus is one that has a sequence that does not occur in nature or that is created by the artificial combination of two segments of otherwise distinct sequence. This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of segments of isolated nucleic acid molecules, e.g., by genetic engineering techniques. The term "recombinant" also includes nucleic acids, proteins, and viruses that have been altered only by the addition, substitution, or deletion of portions of a naturally occurring nucleic acid molecule, protein, or virus.

[0075] Replication deficiency: Adenovirus that exhibits "replication deficiency" in non-tumor cells (compared to tumor cells) refers to an adenovirus that exhibits reduced viral replication in normal cells compared to tumor cells.Replication deficiency is manifested, for example, by the lack of viral late protein expression in normal cells, reduced viral DNA synthesis, reduced ability to induce E2F target genes (e.g., cyclin A and B), reduced ability to induce S phase entry, and / or reduced ability to induce cell killing, compared to tumor cells.

[0076] Replication-deficient virus: A virus that preferentially inhibits cell proliferation, causes cell lysis, or induces apoptosis (collectively considered killing) in a given cell population with a given phenotype (e.g., tumor cells with deregulated E2F pathway). Such viruses are unable to reduce or inhibit cell proliferation, cause cell lysis, induce apoptosis, or replicate in cells that do not otherwise have the given cellular phenotype (such as normal, non-tumor cells) or have limited ability to do so.

[0077] Self-cleaving peptides: These peptides induce ribosomes to skip peptide bond synthesis at the C-terminus, resulting in the separation of the peptide sequence from the downstream polypeptide. Virus-encoded 2A peptides are a type of self-cleaving peptide. Examples of virus-encoded 2A peptides include those derived from porcine teschovirus-1 (PTV1), foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), and Thosea asigna virus (TaV).

[0078] Sequence identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as the identity or similarity between the sequences. Sequence identity can be measured as a percentage of identity, with the higher the percentage, the more identical the sequences. Sequence similarity can be measured as a percentage of similarity (taking into account conservative amino acid substitutions), with the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more pronounced when orthologous proteins or cDNAs are derived from more closely related species (e.g., human and mouse sequences) than from more distantly related species (e.g., human and C. Elegans sequences).

[0079] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988); Higgins and Sharp, Gene, 73, 237-44 (1988); Higgins and 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) present a detailed discussion of sequence alignment methods and homology calculations.

[0080] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol., 215:403-10, 1990) is available from multiple sources, including the National Center for Biological Information (NCBI) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0081] Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.

[0082] Subject: A living multi-cellular vertebrate organism, a category that includes human and non-human mammals.

[0083] Synthetic: Produced by artificial means in a laboratory, for example, a synthetic nucleic acid or protein may be chemically synthesized in a laboratory.

[0084] U exon: An open reading frame located between the early E3 region and the fiber gene in the 1 strand (leftward transcription) (Tollefson et al., J Virol, Vol. 81 (No. 23), pp. 12918-12926).

[0085] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. "Comprising A or B" means including A, B, or A and B. It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0086] III. Overview of the Embodiments The present specification discloses a recombinant adenovirus genome, which comprises heterologous open reading frame (ORF) and self-cleaving peptide coding sequence.The recombinant adenovirus genome and the recombinant adenovirus produced by the disclosed genome can be used in high-throughput assays, for example, to measure viral replication kinetics.

[0087] Provided herein is a recombinant adenovirus genome comprising a heterologous ORF and a self-cleaving peptide coding sequence, both in the same reading frame as and operably linked to an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF. In some embodiments, the endogenous ORF is E1B-55k and the heterologous ORF is 3' of E1B-55k; the endogenous ORF is DNA polymerase and the heterologous ORF is 5' of the DNA polymerase; the endogenous ORF is DNA binding protein (DBP) and the heterologous ORF is 3' of DBP; the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5' of ADP; the endogenous ORF is E3-14.7k and the heterologous ORF is 3' of E3-14.7k; or the endogenous ORF is E4-ORF2 and the heterologous ORF is 5' of E4-ORF2.

[0088] In some embodiments, the self-cleaving peptide is a 2A peptide or a variant thereof. In some examples, 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. In particular examples, the P2A peptide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 16. In some examples, the 2A peptide variant comprises an additional amino acid sequence (e.g., GSG) at the N-terminus.

[0089] In certain examples, the F2A peptide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 17. In certain examples, the E2A peptide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18. In certain examples, the T2A peptide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19. In certain non-limiting examples, the self-cleaving peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 12-19.

[0090] In some embodiments, the heterologous ORF encodes a fluorescent protein, such as, but not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), or blue fluorescent protein (BFP). Exemplary fluorescent proteins are known in the art and include, but are not limited to: BFP-EBFP, EBFP2, Sapphire, T-Sapphire, Azurite, mTagBFP, Cyan fluorescent protein-ECFP, mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, mTurquoise, mTFP1, GFP-GFP, EGFP, AcGFP1, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, ZsGreen, YFP-EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Orange fluorescent proteins - Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, mTangerine, Orange or red fluorescent proteins - dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, and RFP-mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, tdTomato, E2-Crimson.

[0091] In specific, non-limiting examples, the YFP is YPet or the RFP is mCherry.

[0092] In some embodiments, the recombinant adenoviral genome comprises, from 5' to 3', E1B-55K-P2A-YPet, E1B-55K-P2A-mCherry, YPet-P2A(DNA polymerase), DBP-P2A-YPet, YPet-P2A-ADP, E3-14.7k-P2A-YPet, YPet-P2A-E4-ORF2, or mCherry-P2A-E4-ORF2. In some examples, the nucleotide sequence of the recombinant adenoviral genome is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:3-7, 9-11, 20, and 21. In specific, non-limiting examples, the nucleotide sequence of the recombinant adenoviral genome comprises or consists of any one of SEQ ID NOs: 3-7, 9-11, 20, and 21.

[0093] In some embodiments, the adenovirus is adenovirus type 5 (Ad5). In other embodiments, the adenovirus is Ad2, Ad3, Ad9, Ad11, Ad12, or Ad34. In still other embodiments, the adenovirus is a chimeric adenovirus, such as, but not limited to, an Ad5 / Ad9 or an Ad5 / Ad34 chimeric adenovirus.

[0094] Further provided herein is a recombinant adenovirus comprising the recombinant adenoviral genome disclosed herein.

[0095] Also provided is a method for measuring the replication kinetics of recombinant adenovirus, such as the recombinant adenovirus disclosed herein.In some embodiments, the genome of the recombinant adenovirus comprises a heterologous ORF encoding a fluorescent protein and a self-cleaving peptide coding sequence, all of which are operably linked to an endogenous adenovirus ORF selected from E1B-55k, DNA polymerase, DNA-binding protein (DBP), adenovirus death protein (ADP), E3-14.7k, and E4-ORF2, in the same reading frame.The self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous adenovirus ORF.In some embodiments, the method includes transfecting cells with the genome of the recombinant adenovirus or infecting cells with recombinant adenovirus particles, culturing the transfected or infected cells for at least 2 days, measuring fluorescence at regular intervals during the culture period, and calculating the logarithmic slope from the fluorescence measurements. In some examples, the cells are cultured in multi-well plates.

[0096] In some embodiments, the endogenous ORF is E1B-55k and the heterologous ORF is 3' of E1B-55k, the endogenous ORF is DNA polymerase and the heterologous ORF is 5' of the DNA polymerase, the endogenous ORF is DNA binding protein (DBP) and the heterologous ORF is 3' of DBP, the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5' of ADP, the endogenous ORF is E3-14.7k and the heterologous ORF is 3' of E3-14.7k, or the endogenous ORF is E4-ORF2 and the heterologous ORF is 5' of E4-ORF2. In some examples, the recombinant adenovirus further comprises a second heterologous ORF.

[0097] In some embodiments, the replication kinetics of the recombinant adenovirus is measured in a first cell type and a second cell type, in some examples, the first cell type is a tumor cell (such as derived from any of the tumor types listed above) and the second cell type is a non-tumor cell (such as a normal mammalian cell).

[0098] In some embodiments, the transfected or infected cells are cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some examples, the transfected or infected cells are cultured for about 2 days to about 14 days, e.g., about 4 days to about 12 days, or about 6 days to about 10 days. In specific, non-limiting examples, the transfected or infected cells are cultured for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0099] In some embodiments, fluorescence is measured approximately every 2, 4, 6, 8, 10, 15, 20, 30, 45, 60, 90, or 120 minutes. In some examples, fluorescence is measured using a fluorescence plate reader, such as a TECAN™ fluorescence plate reader.

[0100] In some embodiments of the viral replication kinetics assay, the method includes transfecting cells with a recombinant adenoviral genome, in some examples, transfection results in approximately 5-10% of the cells being transfected.

[0101] In other embodiments of the viral replication kinetics assay, the method includes infecting cells with recombinant adenovirus particles. In some examples, cells are infected with serial dilutions of the recombinant adenovirus particles. A suitable number of virus dilutions can be selected by one skilled in the art. In some examples, about 4 to about 24 dilutions of the virus are used in the assay, e.g., about 4 to about 20, about 6 to about 16, or about 8 to about 12 dilutions. In specific examples, at least 4, at least 5, about 6, about 7, or at least 8 dilutions are used in the assay. In specific, non-limiting examples, the dilutions are 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900, and 1:218,700.

[0102] In some embodiments, the method includes selecting a therapeutic adenovirus appropriate for treating a tumor in a patient by measuring the replication kinetics of the recombinant adenovirus in tumor cells obtained from the patient, wherein the recombinant adenovirus corresponds to the therapeutic adenovirus except that the therapeutic ORF of the therapeutic adenovirus is replaced with an ORF encoding a fluorescent protein. In some examples, the therapeutic adenovirus is an oncolytic adenovirus. In some examples, the tumor cells are obtained by biopsy.

[0103] In some embodiments, the method includes selecting cancer patients who will respond to treatment with a therapeutic adenovirus by measuring the replication kinetics of a recombinant adenovirus in tumor cells obtained from the patient, where the recombinant adenovirus corresponds to the therapeutic adenovirus except that the therapeutic ORF of the therapeutic adenovirus is replaced with an ORF encoding a fluorescent protein. This method can be used, for example, to stratify cancer patients into predicted responders and predicted non-responders to a particular therapeutic adenovirus. In some embodiments, the therapeutic adenovirus is an oncolytic adenovirus. In some embodiments, the tumor cells are obtained by biopsy.

[0104] In some embodiments, the method includes identifying the therapeutic adenovirus most effective for the patient's tumor by measuring the replication kinetics of a panel of recombinant adenoviruses in tumor cells obtained from the patient, where the recombinant adenovirus corresponds to the therapeutic adenovirus candidate except that the therapeutic ORF of the therapeutic adenovirus is replaced with an ORF encoding a fluorescent protein. In some examples, the therapeutic adenovirus is an oncolytic adenovirus. In some examples, the tumor cells are obtained by biopsy.

[0105] Further provided herein is a kit comprising a recombinant adenovirus genome or recombinant adenovirus disclosed herein and cells, cell culture medium, and / or a multi-well plate. In some embodiments, the cells are tumor cells (such as cells derived from any of the tumor types listed herein). In some embodiments, the cells are non-tumor cells. In some embodiments, the cell culture medium is selected to provide a high signal-to-background ratio. In some examples, the cell culture medium is phenol red-free. In some embodiments, the multi-well plate is a 48-well, 96-well, or 384-well plate. In certain examples, the multi-well plate is any plate that can be read with a fluorescent plate reader, such as a TECAN™ fluorescent plate reader.

[0106] IV. Optimal Placement of Exogenous ORFs The 36 kb adenovirus genome is small and uses both the top and bottom strands to encode various genes. At many locations within the adenovirus genome, both the top and bottom strands are used simultaneously to encode separate genes. The size of the genome has evolved to optimize its insertion into the capsid. As a result, the insertion of exogenous genes is limited by the size capacity of the capsid, because the addition of excessive exogenous nucleic acid results in incomplete genome loading into the capsid and reduced viral kinetics.

[0107] A solution to the problem presented by the limited space available in the adenovirus genome is to position an exogenous open reading frame (ORF) as a fusion product within the native adenovirus ORF. This strategy utilizes the adenovirus promoter, 5'UTR, and poly(A) tail already encoded in the genome. However, expression of a fusion between a native adenovirus protein and an exogenous protein can be detrimental to the function of one or both proteins, resulting in a significant decrease in adenovirus replication kinetics.

[0108] The present disclosure provides a solution to this problem by using a self-cleaving peptide sequence placed between the native ORF and the exogenous ORF.When placed between two ORFs on a single mRNA, the presence of the self-cleaving peptide sequence causes ribosome skipping, and the first protein is released separately from the second protein.In some embodiments disclosed herein, the self-cleaving peptide is 2A peptide (P2A).

[0109] Also disclosed herein is the identification of the optimal placement site of exogenous ORF in adenovirus genome.By combining self-cleaving peptide sequence and good placement of exogenous ORF, high expression is achieved and the influence on virus dynamics is minimal or not at all.Further disclosed herein is the use of recombinant adenovirus expressing exogenous gene in high-throughput assay for measuring virus replication dynamics.

[0110] As described in Example 1 below, several sites within the adenovirus genome were identified where insertion of a heterologous ORF did not inhibit adenovirus replication kinetics. Specifically, it was determined that the heterologous ORF could be inserted C-terminally to the E1B-55k ORF, N-terminally to the DNA polymerase ORF, C-terminally to the DBP ORF, N-terminally to the ADP ORF, C-terminally to the E3-14.7k ORF, or N-terminally to the E4-ORF2. In each case, a self-cleaving peptide sequence (P2A site) was inserted between the adenovirus ORF and the heterologous ORF. Thus, the present disclosure contemplates the use of the following recombinant adenoviruses in assays for measuring replication kinetics ("SC" refers to the sequence encoding the self-cleaving peptide, such as P2A): E1B-55k-SC-Heterogeneous ORF Heterologous ORF-SC-(DNA polymerase) DBP-SC-heterogeneous ORF Heterogeneous ORF-SC-ADP E3-14.7k-SC-Heterogeneous ORF Heterogeneous ORF-SC-E4-ORF2

[0111] In some embodiments herein, the self-cleaving peptide is a virally encoded 2A peptide, or a modified version thereof as further described below.

[0112] V. Self-cleaving peptide sequences A self-cleaving peptide is a peptide that induces the ribosome to skip the synthesis of a peptide bond at the C-terminus, resulting in the separation of the peptide sequence from the downstream polypeptide. The use of a self-cleaving peptide allows the expression of multiple proteins flanked by the self-cleaving peptide from a single ORF. The 2A peptide, encoded by viruses, is one example of a self-cleaving peptide.

[0113] Like other self-cleaving peptides, 2A peptides function by causing the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element, resulting in separation of the end of the 2A sequence from the downstream peptide (Kim et al., PLoS One, Vol. 6(4), e18556, 2011). The "cleavage" occurs between the glycine and proline residues found at the C-terminus of the 2A peptide. Exemplary 2A peptides include Thosea These include, but are not limited to, the 2A peptides encoded by Tasigna virus (TaV), equine rhinitis A virus (ERAV), porcine teschovirus-1 (PTV1), and foot-and-mouth disease virus (FMDV), or modified versions thereof.

[0114] In particular examples herein, the 2A peptide comprises PTV1 2A (P2A), FMDV 2A (F2A), ERAV 2A (E2A), or TaV 2A (T2A), the sequences of which are shown below as SEQ ID NOs: 12-15 and described herein. P2A:ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12) F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 13) E2A: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14) T2A:EGRGSLLTCGDVEENPGP (SEQ ID NO: 15)

[0115] In some examples, the 2A peptide is modified to include Gly-Ser-Gly at the N-terminus to improve cleavage efficiency. The sequences of the modified P2A, F2A, E2A, and T2A are shown below as SEQ ID NOs: 16-19 and described herein. Modified P2A: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 16) Modified F2A: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 17) Modified E2A: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 18) Modified T2A: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19)

[0116] In some embodiments, the 2A polypeptide is a variant of a 2A polypeptide disclosed herein. A variant may include a polypeptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to a wild-type or modified 2A polypeptide disclosed herein. A variant may include, for example, a 2A polypeptide of any one of SEQ ID NOS: 12-19, in which at least one N-terminal amino acid has been deleted, e.g., 1, 2, 3, 4, or 5 amino acids (including any two of the recited values) have been deleted. A variant may include a 2A polypeptide of any one of SEQ ID NOS: 12-19, in which at least one C-terminal amino acid has been deleted, e.g., 1, 2, 3, 4, or 5 amino acids (including any two of the recited values) have been deleted. A variant may also contain at least one, two, three, four, or five amino acid substitutions, eg, conservative amino acid substitutions.

[0117] VI. Methods for Monitoring Viral Dynamics in Tissue Culture An important criterion for evaluating selectively replicating viruses is to compare viral growth kinetics between cancer cells and normal cells over multiple rounds of replication. Small differences in viral replication may be masked at high MOIs. Measuring multiple rounds of viral replication can overcome this issue.

[0118] To address the need for fast viral kinetic assays, systematic high-throughput screening of viral replication kinetics is necessary. Current methods for assessing viral replication often rely on specific cell lines engineered with luciferase or reporters. However, the activity and level of transgene expression conferred by the encoded reporter measures cell viability, not viral replication itself. Furthermore, adenoviral proteins disrupt global gene expression (e.g., p300, E2F, CBP, mediators, splicing, etc.).

[0119] Current methods to assess adenovirus replication are indirect and insensitive endpoint assays that can only be used in certain cell types, rely on Ad5-specific antibodies, do not measure the entire viral life cycle at multiple times, require knowledge of viral titer, cannot use viral plasmid transfection, do not quantify viral replication, are not predictive of cell killing, and do not allow comparisons across different subgroups.

[0120] Currently used assays include (1) measurement of Ad5 late viral proteins by Western blot, (2) measurement of adenovirus genome by q-PCR, (3) plaque assays in specialized and limited cell types, (4) indirect measurement of viral replication using cell viability assays (e.g., wst-1 / mtt), and (5) ELISA and / or FACS using adenovirus-specific antibodies.

[0121] Each of these assays has significant drawbacks: the first two methods do not measure the entire viral life cycle, including steps such as viral uptake, gene expression, viral gene replication, capsid assembly, genome loading into capsids, lysis, spread, and productive secondary infection, thus significantly limiting the usefulness of these methods.

[0122] Plaque assays require specialized cell lines and efficient viral infection and complementation, making it difficult to compare replication across the 68 different Ad serotypes. Additionally, plaque assays require cells to tolerate the agar overlay, which is only possible with limited cell types. Furthermore, plaque assays are inherently subjective and laborious, and do not provide insight into cases where viral replication is selectively impaired or enhanced (e.g., primary infection, gene expression, replication, lysis, etc.). Furthermore, determining the correct titer of capsid-exchanged viruses by methods such as plaque assays or ELISA is not possible because the choice of cell type can affect viral entry. Furthermore, Ad5 antibodies do not recognize the fiber exchanges used to alter viral tropism.

[0123] Regarding ELISA and FACS assays, these methods rely on the use of antibodies specific to adenovirus proteins and quantifying titers by detecting antibody binding by FACS or ELISA.However, the antibodies used in conventional assays only recognize specific serotypes, and cannot be used to compare viral kinetics or different adenoviruses, because they are not recognized by available antibodies.

[0124] As disclosed herein, by incorporating a fluorescent reporter that is simultaneously expressed with one or more viral proteins, it is possible to measure viral dynamics using a method similar to that used to measure bacterial or yeast growth.In the method disclosed herein, the fluorescent expression level is monitored over time and fitted to a logarithmic growth curve, similar to measuring the optical density (OD) of bacterial or yeast culture to determine the logarithmic slope growth rate.Because the logarithmic slope is the only relevant parameter, this method is robust to fluctuations or errors in the initial infectious titer, and can even be used by transfection of whole genome plasmids instead of infection with purified virions.

[0125] Monitoring fluorophore expression over time in tissue culture provides a non-invasive, multi-time point measure of viral progression. These measurements provide detailed information about viral dynamics over multiple rounds of replication, thus encompassing all aspects of the viral life cycle.

[0126] The fluorescence-based assay disclosed herein is high-throughput and tolerant to variations in initial virus titer and virus entry.This assay is so tolerant to initial conditions that it can skip virion production and purification, and simply use the direct transfection of the whole genome plasmid produced by the previously described Adsembly and AdSLIC protocol (see International Publication No. 2012 / 024351, which is incorporated herein by reference).This process saves weeks of time, a large amount of reagents, medium, and tissue culture supplies.The assay disclosed herein is an essential tool for fast and accurate evaluation of virus constructs.

[0127] In addition, the method for assessing viral kinetics can be applied to any adenovirus serotype as well as any cell type, and is independent of the starting viral titer, the type of fluorophore selected, and the half-life of viral proteins.

[0128] Viral kinetics are determined from the logarithmic slope of fluorescence measured over multiple time points, in some cases over a period of up to about 10 days. This length of time is often optimal for capturing multiple viral life cycles, each lasting approximately 48 hours. In some embodiments, fluorescence is measured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some examples, fluorescence is measured for about 2 days to about 14 days, e.g., about 4 days to about 12 days, or about 6 days to about 10 days. In certain non-limiting examples, fluorescence is measured for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0129] Comparison of kinetics between different viral constructs, each with potentially different fluorophores and signal levels, can be addressed through the use of logarithmic slopes. By obtaining the logarithmic slope of the exponential growth of the fluorescent signal versus time, a single value is obtained for each viral construct, and this value can be cross-compared regardless of the magnitude of the signal or any initial time delay that may occur before exponential growth begins. This property of data interpretation makes the assay insensitive to early starting points. Malregulation or even understanding of the initial viral titer does not affect the logarithmic slope during exponential growth. Only an initial infection (or transfection) is required, resulting in the transduction of a small percentage of cells in the tissue culture dish. The remaining uninfected cells are available for secondary and tertiary infections.

[0130] Because this assay requires fluorescence measurements at multiple time points over a period of several days, a reference standard must be found that allows normalization across data points. This reference standard must be stable to the time, temperature, humidity, and exposure to the excitation radiation used in the fluorescence measurements. In some embodiments, the reference standard is background fluorescence from polystyrene in empty wells. In other embodiments, commercially available latex beads with embedded fluorophores are the reference standard.

[0131] The cell culture medium used in the assays disclosed herein ideally provides a high signal-to-background ratio. Factors that result in a high background include phenol red or FBS in the medium. Therefore, in some embodiments, the culture medium used in the viral kinetics assay is a phenol red-free medium. The fluorophore selection can also be selected to overcome background fluorescence from the medium. For example, YPet is two times brighter than enhanced GFP (eGFP). Therefore, in some embodiments, the fluorescent protein is YPet. In other embodiments, the fluorescent protein is mCherry.

[0132] VI. Adsembly and AdSLIC The adenovirus genome is organized into several functional groups labeled E1, E2, E3, E4, and L1-5. The E1 region encodes proteins that regulate the transcription of all other viral genes and induce S phase in host cells. The E2 region encodes proteins that drive viral DNA replication. Proteins in the E3 region modulate the immune response of host cells and are nonessential in cell culture. The E4 region contains genes with different functions. Finally, the L1-5 region encodes structural proteins for the viral particle.

[0133] Taking advantage of this natural segregation of functionality, we have previously developed a method of recombinant adenovirus assembly that allows for rapid and easy manipulation of the 36 kb Ad genome by separating it into four plasmids: E1, E3, E4, and core, as shown in Figure 6A (Adsembly and AdSLIC, see WO 2012 / 024351, which is incorporated herein by reference). Due to their more streamlined size, these smaller plasmids are easier to manipulate using standard techniques.

[0134] Adsembly and AdSLIC enable the in vitro combinatorial assembly of adenoviruses with novel properties from compatible genomic library segments within 4 hours. Adsembly and AdSLIC provide a general genome design platform that allows the assembly of synthetic viruses with novel properties using a library of four functional segments (Figure 6A). These segment libraries can be reassembled in all possible combinations using either multiple site-specific recombination sites (Adsembly, Figure 6B) or sequence-independent seamless cloning (AdSLIC, Figure 6C).

[0135] Adsembly and AdSLIC's technologies enable the modular design and production of adenoviruses with unique capabilities. Developing the ability to design, manufacture, and test viruses in an automated, high-throughput manner will accelerate and scale the development of new viruses for therapeutic, diagnostic, and research studies.

[0136] The cloning step was once a bottleneck in the production of new viral constructs, but with the advent of Adsembly and AdSLIC, the ability to assemble viral genomes has surpassed the ability to test them. Comparably, high-throughput kinetic assays are crucial to unlocking the full potential and high-content assembly of synthetic personalized viral therapeutics and diagnostics using Adsembly and AdSLIC methods.

[0137] The following examples are provided to illustrate certain specific features and / or embodiments, and are not to be construed as limiting the disclosure to the specific features or embodiments described. [Example]

[0138] Example 1: Identifying the optimal location of an exogenous ORF in the adenovirus genome This example describes the identification of specific locations within the adenovirus genome where an exogenous ORF can be inserted along with a self-cleaving peptide sequence without disrupting viral dynamics.

[0139] The insertion of exogenous genes into adenovirus vectors is limited by the size capacity of the adenovirus capsid. Excessive addition of exogenous nucleic acids results in incomplete genome loading into the capsid and reduced viral kinetics. A solution to the problem presented by the limited space available in the adenovirus genome is to position an exogenous open reading frame (ORF) as a fusion product within the original adenovirus ORF. This strategy utilizes the adenovirus promoter, 5'UTR, and polyA tail already encoded in the genome. However, the expression of a fusion between an original adenovirus protein and an exogenous protein may be harmful to the function of one or both proteins, resulting in a significant decrease in adenovirus replication kinetics. In fact, the research disclosed herein demonstrates that direct fusion of an exogenous ORF with the ORF of adenovirus E1A, DNA polymerase, or ADP significantly inhibits adenovirus replication kinetics. In addition, we have previously attempted to insert exogenous ORFs using internal ribosome entry sites (IRES), but this has also failed to produce recombinant viruses with wild-type kinetics.

[0140] This example describes a solution to this problem by using a self-cleaving peptide sequence placed between the native adenovirus ORF and the exogenous ORF. When placed between two ORFs on a single mRNA, the presence of the self-cleaving peptide sequence causes ribosome skipping, releasing the first protein separately from the second protein. The adenovirus construct generated in this example uses the self-cleaving peptide P2A and a fluorescent protein (e.g., YPet, mCherry) as the exogenous ORF.

[0141] The table below provides a list of the constructs generated and indicates the expression levels (low, moderate, or high) of the exogenous ORF and the viral replication kinetics (low, moderate, or high) in two different cell lines, 293-E4 and A549 cells. [Table 2]

[0142] Constructs exhibiting "high" replication kinetics (i.e., replication kinetics comparable to wild-type adenovirus) in both cell types were considered candidates for use in the viral replication kinetics assay described in Example 2 (candidate constructs are shown in bold).

[0143] Comparison of direct fusion and P2A site insertion Several constructs were generated in which fluorescent proteins were directly fused to adenoviral ORFs, specifically the following direct fusions were generated: YPet-E1A, YPet (DNA polymerase), and mCherry-ADP.

[0144] The YPet-E1A adenovirus exhibited a significant impairment in viral kinetics. Inserting a P2A site between YPet and E1A (YPet-P2A-E1A) improved viral kinetics, but did not restore viral kinetics to wild-type levels. Another construct was then generated to test the fusion of P2A and YPet to the C-terminus of E1A (E1A-P2A-YPet). This construct further improved viral kinetics, but again, did not restore kinetics to the level of wild-type adenovirus.

[0145] Several attempts to transfect the YPet-(DNA-poly) genome plasmid failed to produce viable virus (no plaques formed). However, fusing YPet-P2A to the N-terminus of DNA polymerase (YPet-P2A-(DNA-poly)) produced virus with wild-type kinetics, as shown in the table above.

[0146] Finally, direct fusion of mCherry to ADP (mCherry-ADP) produced viruses with significantly impaired kinetics. However, inserting a P2A site between the mCherry and ADP ORFs yielded viruses with wild-type kinetics (mCherry-P2A-ADP). The same results were obtained using a different fluorescent protein; the YPet-P2A-ADP construct exhibited wild-type viral kinetics. However, placing P2A and a heterologous ORF C-terminal to ADP produced viruses that did not replicate. Therefore, for ADP, the heterologous ORF must be placed N-terminally.

[0147] Additional constructs with wild-type viral kinetics Figure 7 shows a comparison of the natural logarithmic slopes of five different constructs: YPet-E1A, YPet-P2A-E1A, E1A-P2A-mCherry, E1B-55k-P2A-YPet, and YPet-P2A-ADP. As noted above, direct fusion of YPet to E1A produced viruses with significantly impaired kinetics. Adding a P2A site to either the N-terminus (YPet-P2A-E1A) or C-terminus (E1A-P2A-mCherry) improved viral kinetics, but not to wild-type levels. However, inserting a P2A site and a heterologous ORF into the C-terminus of E1B-55k (E1B-55k-P2A-YPet) or the N-terminus of ADP (YPet-P2A-ADP) generated recombinant viruses with wild-type viral kinetics.

[0148] Evaluation of the viral kinetics of constructs with a P2A site and a heterologous ORF at the C terminus of DBP (DBP-P2A-YPet) or E3-14.7k (E3-14.7k-P2A-YPet) or at the N terminus of E4-ORF2 (YPet-P2A-E4-ORF2 and mCherry-P2A-E4-ORF2) yielded viruses with wild-type replication kinetics.

[0149] The results of these data demonstrate that at least the following adenoviral genome constructs can be used in the viral replication assay described in Example 2: E1B-55k-SC-Heterogeneous ORF Heterologous ORF-SC-(DNA polymerase) DBP-SC-heterogeneous ORF Heterogeneous ORF-SC-ADP E3-14.7k-SC-Heterogeneous ORF Heterogeneous ORF-SC-E4-ORF2

[0150] For use in the viral replication assays disclosed herein, the heterologous ORF encodes a fluorescent protein, such as (but not limited to) YPet or mCherry.

[0151] Other adenovirus serotypes All previously described methods for measuring viral kinetics are highly dependent on cell type-specific assays and are therefore serotype-specific due to the diverse tropism of each adenovirus serotype. The adenovirus kinetics assay disclosed herein does not depend on any one cell type and can therefore be extended to serotypes other than Ad5. All adenovirus serotypes contain ORFs equivalent to Ad5 E3-14.7k. Therefore, Ad9 (containing E3-15k) and Ad34 (containing E3-14.8k) were used to generate viruses equivalent to Ad5 E3-14.7k-P2A-YPet (PCMN-887, SEQ ID NO: 9): PCMN-888 (Ad9 E3-15k-P2A-YPet, SEQ ID NO: 20) and PCMN-889 (Ad34 E3-14.8k-P2A-YPet, SEQ ID NO: 21). Chimeric viruses containing the Ad5 core and fiber shaft and knob from either Ad9 or Ad34 were also generated. Four recombinant viruses were then tested in FBVK assays using 293 cells (Figure 9A), A549 cells (Figure 9B), and U2OS cells (Figure 9C). All four recombinant viruses exhibited high levels of YPet expression, and insertion of the exogenous ORF had minimal impact on viral kinetics.

[0152] Example 2: Methods for assessing adenovirus replication kinetics The Adsembly and AdSLIC methods for assembling recombinant adenoviruses provide a means for generating large numbers of recombinant viral genomes and viruses in a short time. However, there is a need for a rapid, high-throughput method for evaluating the replication kinetics of recombinant adenoviruses designed for clinical and therapeutic use. This example describes a fluorescence-based viral kinetics assay that can be used to examine the viral replication kinetics of recombinant adenoviruses (Figure 3). This assay can be performed using either recombinant adenovirus genome plasmids or recombinant adenovirus particles as starting materials.

[0153] When starting with a recombinant adenoviral genome, the assay involves transfecting cells with an adenoviral genome plasmid (such as that described above in Example 1) and monitoring fluorophore expression over time (Figures 4A-4B). Transfection conditions are selected so that approximately 5-10% of the cells are initially transfected. Cells not initially transfected are available for secondary infection with viral particles produced by the initial transfection. The logarithmic slope is used as a measure of kinetics based on secondary, tertiary, and quaternary infections (and so on), so it is not necessary to know the percentage of cells initially transfected. Figures 4A and 4B show an exemplary virus-based kinetic assay starting with a recombinant adenoviral genome plasmid. In this example, a 48-well plate is used, allowing for simultaneous testing (in triplicate) of 14 different viral constructs. The top half of the 48-well plate (Figure 4A) contains six different viruses, three mock-infected wells, and three "blank" wells with FLUORESBRITE™ beads to compensate for tool sensitivity variations. The bottom half of the 48-well plate (Figure 4B) contains triplicate wells of eight different viral constructs. Once the cells are transfected, the plate is placed in a TECAN™ plate reader for continuous fluorescence monitoring. The collected data is used to calculate the natural logarithmic slope of each construct (Figure 8).

[0154] This assay can also be performed by infecting cells with recombinant viral particles. In this version of the assay, cells are infected with the recombinant viral particles and fluorophore expression is monitored over time (Figure 5). As with the genomic plasmid version of the assay, it is not necessary to know the exact titer of the starting viral stock. Typically, a dilution series ranging from 1:100 to 1:218,700 is used for initial infection, as shown in Figure 5. A dilution of 1:100 generally results in infection of all cells, while a dilution of 1:218,700 generally results in initial infection of very few cells. In this example, a 96-well plate is used, and 11 different viral constructs are tested simultaneously at eight different dilutions (1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900, and 1:218,700). The plate also contains four wells of mock-infected cells and four wells of FLUORESBRITE™ beads. Once the cells are infected, the plate is placed in a TECAN™ plate reader for continuous fluorescence monitoring. The collected data is used to calculate the natural logarithmic slope of each construct (Figure 8).

[0155] The TECAN™ plate reader also provides an incubation function (maintaining the appropriate temperature, CO2, and O2 levels). Data points are acquired every 15 minutes, and the natural logarithmic slope is calculated. These methods allow for fast and efficient comparison of kinetics between many different viruses and different cell types. For example, to evaluate whether a particular recombinant adenovirus can be used therapeutically as an oncolytic virus, this assay can be used to find viruses that exhibit high replication kinetics in tumor cells but slow viral kinetics in non-tumor cells. Furthermore, the viral kinetics of recombinant viruses can be evaluated by infecting or transfecting tumor cell types of interest in this assay.

[0156] Calculating the logarithmic slope To measure the logarithmic slope, the linear plot of fluorescence intensity versus time is converted to a semi-logarithmic plot by taking the natural logarithm of the fluorescence intensity measured at each time point. Because fluorescence intensity exhibits exponential growth during viral replication, this conversion results in a straight line when the natural logarithm (fluorescence intensity) is plotted against time. This line is then fitted using standard least squares methods. The resulting slope produced by this fit is the natural logarithmic slope of fluorescence versus time, and therefore the natural logarithmic slope of viral growth versus time. The formula is shown below. [ka] where FI is the measured fluorescence intensity, t is time, F0 is the initial fluorescence intensity at time = t0, and α is the natural logarithmic slope. Taking the natural logarithm of both sides: [ka] The right hand side becomes a linear equation with a natural logarithmic slope of α.

[0157] In view of the numerous possible embodiments to which the principles of the present disclosure may be applied, it should be recognized that the illustrated embodiments are merely examples of the present disclosure and should not be taken as limiting the scope of the present disclosure. Rather, the scope of the present disclosure is defined by the following claims. The inventors therefore claim all that comes within the scope and spirit of these claims. The present invention provides, for example, the following items. (Item 1) A recombinant adenoviral genome comprising a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence, both in the same reading frame as and operably linked to an endogenous adenoviral ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF; and the endogenous ORF is E1B-55k and the heterologous ORF is 3' of E1B-55k; the endogenous ORF is a DNA polymerase and the heterologous ORF is 5' to the DNA polymerase; the endogenous ORF is a DNA binding protein (DBP) and the heterologous ORF is 3' to the DBP; the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5' to ADP; the endogenous ORF is E3-14.7k and the heterologous ORF is 3' of E3-14.7k; or A recombinant adenoviral genome, wherein the endogenous ORF is E4-ORF2 and the heterologous ORF is 5' to E4-ORF2. (Item 2) 2. The recombinant adenovirus genome of item 1, wherein the self-cleaving peptide is a 2A peptide or a variant thereof. (Item 3) 3. The recombinant adenovirus genome of item 2, 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. (Item 4) 4. The recombinant adenovirus genome according to Item 3, wherein the amino acid sequence of the self-cleaving peptide is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 12 to 19. (Item 5) 4. The recombinant adenovirus genome according to Item 3, wherein the self-cleaving peptide comprises any one of the amino acid sequences of SEQ ID NOs: 12 to 19. (Item 6) 6. The recombinant adenovirus genome of any one of items 1 to 5, wherein the heterologous ORF encodes a fluorescent protein. (Item 7) 7. The recombinant adenovirus genome of item 6, wherein the fluorescent protein is green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), or blue fluorescent protein (BFP). (Item 8) 8. The recombinant adenovirus genome of item 7, wherein the YFP is YPet or the RFP is mCherry. (Item 9) From 5' to 3' E1B-55K-P2A-YPet, E1B-55K-P2A-mCherry, YPet-P2A-(DNA polymerase), DBP-P2A-YPet, YPet-P2A-ADP, E3-14.7k-P2A-YPet, YPet-P2A-E4-ORF2, or 9. The recombinant adenoviral genome of any one of items 1 to 8, comprising mCherry-P2A-E4-ORF2. (Item 10) 10. The recombinant adenovirus genome of any one of items 1 to 9, comprising a nucleotide sequence of any one of SEQ ID NOs: 3 to 7, 9 to 11, 20, and 21. (Item 11) 11. A recombinant adenovirus comprising the recombinant adenovirus genome of any one of items 1 to 10. (Item 13) A method for measuring the replication kinetics of a recombinant adenovirus comprising the recombinant adenovirus genome according to item 1, wherein the heterologous ORF encodes a fluorescent protein, and the method comprises the steps of: (i) transfecting a cell with the genome of said recombinant adenovirus or infecting a cell with particles of said recombinant adenovirus; (ii) culturing the transfected or infected cells for at least 2 days; (iii) measuring fluorescence at regular intervals during the incubation period; (iv) calculating a logarithmic slope from the fluorescence measurements, thereby measuring the replication kinetics of the recombinant adenovirus. (Item 14) 14. The method of claim 13, wherein the recombinant adenovirus further comprises a second heterologous ORF. (Item 15) 15. The method of claim 13 or 14, wherein the cells are cultured in a multi-well plate. (Item 16) 16. The method of any one of items 13 to 15, wherein the replication kinetics of the recombinant adenovirus is measured in a first cell type and a second cell type. (Item 17) 17. The method of claim 16, wherein the first cell type is a tumor cell and the second cell type is a non-tumor cell. (Item 18) 18. The method of any one of items 13 to 17, wherein fluorescence is measured approximately every 10 minutes, every 15 minutes, every 30 minutes, every 60 minutes, or every 120 minutes. (Item 19) 19. The method of any one of items 13 to 18, wherein fluorescence is measured in a TECAN™ fluorescence plate reader. (Item 20) 20. The method of any one of items 13 to 19, comprising transfecting a cell with the genome of the recombinant adenovirus. (Item 21) 21. The method of item 20, wherein the transfection results in transfecting approximately 5-10% of the cells. (Item 22) 20. The method of any one of items 13 to 19, comprising a step of infecting a cell with the recombinant adenovirus particle. (Item 23) 23. The method of claim 22, wherein the cells are infected with serial dilutions of the recombinant adenovirus particles. (Item 24) 24. The method of claim 23, including dilutions of 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24,300, 1:72,900, and 1:218,700. (Item 25) (i) a recombinant adenovirus genome according to any one of items 1 to 10 or a recombinant adenovirus according to item 11; (ii) cells, cell culture medium, and / or multiwell plates; Includes a kit.

Claims

[Claim 1] The method described in the specification.

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