Patent Grant
11130968
This disclosure concerns the optimal placement of exogenous open reading frames in recombinant adenovirus constructs and use of the recombinant viruses in assays for measuring adenovirus replication kinetics.
Adenovirus serotype 5 (Ad5) is the vector of choice in basic research applications, murine lung cancer models, and human gene therapy trials. Adenoviruses have a stable 36 kb double-stranded DNA genome protected by a protein capsid decorated with Ad fiber protein spikes that target infection to receptors on specific cell types. Adenoviruses do not integrate into host DNA, can be produced to high titers using established protocols, and have proven safety in human gene therapy and cancer applications. Thus, Ad-based vectors have enormous promise for cancer diagnostics and therapies. However, a need exists for a rapid and high-throughput means of evaluating replication kinetics of recombinant adenoviruses designed for clinical and therapeutic use.
Disclosed herein are recombinant adenovirus genomes that include a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence. The recombinant adenovirus genomes and recombinant adenoviruses produced by the disclosed genomes can be used, for example, in assays to measure virus replication kinetics.
Provided herein are recombinant adenovirus genomes that include a heterologous ORF and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF. 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 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.
Further provided herein are recombinant adenoviruses that include a recombinant adenovirus genome disclosed herein.
Also provided are methods for measuring replication kinetics of a 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, both operably linked to and in the same reading frame as 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 particles of the recombinant adenovirus; culturing the transfected cells or infected cells for at least two days; measuring fluorescence at regular intervals throughout the culture period; and calculating log-slope from the fluorescence measurements. The method can be used, for example, to select an appropriate therapeutic adenovirus (such as an oncolytic adenovirus) for treatment of a tumor by obtaining tumor cells (such as from a biopsy) and measuring replication kinetics in the tumor cells of a recombinant adenovirus that corresponds to the therapeutic adenovirus, except that a therapeutic ORF of the therapeutic adenovirus is replaced with an ORF encoding a fluorescent protein. Similarly, the method can be used to select cancer patients that would respond to treatment with a particular therapeutic adenovirus or to identify the most efficacious therapeutic adenovirus for a particular tumor.
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.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three 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. The Sequence Listing is submitted as an ASCII text file, created on Jul. 18, 2018, 609 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NO: 1 is the nucleotide sequence of synthetic adenovirus genome CMBT-379 (YPet-P2A-E1A).
SEQ ID NO: 2 is the nucleotide sequence of synthetic adenovirus genome CMBT-432 (E1A-P2A-YPet).
SEQ ID NO: 3 is the nucleotide sequence of synthetic adenovirus genome CMBT-456 (E1B-55k-P2A-YPet).
SEQ ID NO: 4 is the nucleotide sequence of synthetic adenovirus genome CMBT-499 (E1B-55k-P2A-mCherry).
SEQ ID NO: 5 is the nucleotide sequence of synthetic adenovirus genome CMBT-530 (YPet-P2A-(DNA Poly)).
SEQ ID NO: 6 is the nucleotide sequence of synthetic adenovirus genome CMBT-886 (DBP-P2A-YPet).
SEQ ID NO: 7 is the nucleotide sequence of synthetic adenovirus genome CMBT-403 (YPet-P2A-ADP).
SEQ ID NO: 8 is the nucleotide sequence of synthetic adenovirus genome CMBT-429 (ADP-P2A-YPet).
SEQ ID NO: 9 is the nucleotide sequence of synthetic adenovirus genome PCMN-887 (E3-14.7k-P2A-YPet).
SEQ ID NO: 10 is the nucleotide sequence of synthetic adenovirus genome CMBT-457 (YPet-P2A-E4-ORF2).
SEQ ID NO: 11 is the nucleotide sequence of synthetic adenovirus genome CMBT-633 (mCherry-P2A-E4-ORF2).
SEQ ID NO: 12 is the amino acid sequence of P2A.
SEQ ID NO: 13 is the amino acid sequence of F2A.
SEQ ID NO: 14 is the amino acid sequence of E2A.
SEQ ID NO: 15 is the amino acid sequence of T2A.
SEQ ID NO: 16 is the amino acid sequence of a modified P2A comprising GSG at the N-terminus.
SEQ ID NO: 17 is the amino acid sequence of a modified F2A comprising GSG at the N-terminus.
SEQ ID NO: 18 is the amino acid sequence of a modified E2A comprising GSG at the N-terminus.
SEQ ID NO: 19 is the amino acid sequence of a modified T2A comprising GSG at the N-terminus.
SEQ ID NO: 20 is the nucleotide sequence of synthetic adenovirus genome PCMN-888 (Ad9 E3-15k-P2A-YPet).
SEQ ID NO: 21 is the nucleotide sequence of synthetic adenovirus genome PCMN-889 (Ad34 E3-14.8k-P2A-YPet).
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 cells sorting
FMDV food 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
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
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):e18556, 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 2A peptides 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 comprises Gly-Ser-Gly at the N-terminus to improve cleavage efficiency (SEQ ID NOs: 16-19).
Adenovirus: A non-enveloped virus with a linear, double-stranded DNA genome and an icosahedral capsid. There are currently 68 known serotypes of human adenovirus, which are divided 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 (primarily species B and C), conjunctivitis (species B and D) and/or gastroenteritis (species F and G).
Adenovirus death protein (ADP): A protein synthesized in the late stages of adenovirus infection that mediates lysis of cells and release of adenovirus to infect other cells. ADP is an integral membrane glycoprotein of 101 amino acids that localizes to the nuclear membrane, endoplasmic reticulum and Golgi. ADP was previously named E3-11.6K).
Chimeric: Composed of at least two parts having different origins. In the context of the present disclosure, a “chimeric adenovirus” is an adenovirus having genetic material and/or proteins derived from at least two different serotypes (such as from Ad5 and a second serotype of adenovirus). 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 sequence 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 a second serotype of adenovirus. In another example, a chimeric fiber is composed of an Ad5 tail and a fiber shaft and knob from a second serotype of adenovirus (such as Ad9 or Ad34).
Contacting: Placement in direct physical association; includes both in solid and liquid form.
Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to 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.
Deleted: An adenovirus genome encoding a “deleted” protein (such as the 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.
Deregulation of E2F: Refers to an increase in activity of the E2F transcription factor and downstream target genes, which occurs in nearly all types of human cancer. Deregulation of the 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 in Rb, p107 and p130 tumor suppressors. Rb was the first tumor suppressor to be 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. Cyclin D and CDK4 mutations, gene amplifications or over-expression can also result in deregulated E2F activity in human tumors. In addition E2F is activated by growth factor receptor pathway mutations including EGFR, RTKs, RAS, RAF, PI-3K, PTEN, RAF, MYC. Mutations in the p16INK4a-Cyclin D:cdk4/6-RB-E2F pathway generally occur in a mutually exclusive fashion, so that one ‘hit’ (for example, p16) is unaccompanied by others (for example, Rb mutation or cyclin D:cdk over-expression). However, most current chemotherapies are proliferative poisons that inhibit E2F transcriptional targets, but are also toxic to normal cells and have often devastating iatrogenic complications. As disclosed herein, an alternative therapeutic approach is to use a virus that undergoes selective lytic replication in cancer cell lesions that have deregulated the p16-cyclin D:cdk4-RB-E2F pathway.
DNA-binding protein (DBP): This adenovirus protein binds to single-stranded DNA and RNA, as well as double-stranded DNA. DBP, a 72-kilodalton protein, is essential for replication of adenoviral DNA.
E1A: The adenovirus early region 1A (E1A) gene and polypeptides expressed from the gene. The E1A protein plays a role in viral genome replication by driving cells into the cell cycle. As used herein, the term “E1A protein” refers to the proteins expressed from the E1A gene and the term includes E1A proteins produced by any adenovirus serotype.
E3-RIDα/RIDβ and E3-14.7k: Early-expressed proteins produced from the E3 gene. The E3-RIDα, E3-RIDβ, and E3-14.7k proteins make up the receptor internalization and degradation complex (RID), which localizes to the nuclear membrane and causes the endocytosis and degradation of a variety of receptors including CD95 (FasL receptor), and TNFR1 and 2 (TNF/TRAIL receptors) to protect infected cells from host antiviral responses. The E3-RIDα, E3-RIDβ, and E3-14.7k coding sequences are next to each other, in this order.
E4orf1: An adenovirus protein produced from the E4 gene. The term “E4orf1 protein” includes E4orf1 proteins produced by the E4 gene from any adenovirus serotype.
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.
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 GFP, EGFP, AcGFP1, 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, AmCyan1, Midori-Ishi Cyan, mTurquoise and mTFP1), yellow fluorescent proteins (EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1 and mBanana), orange fluorescent proteins (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 fluorescence proteins (dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1) and DsRed-Monomer) and modified versions thereof.
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.
Heterologous: A heterologous protein or polypeptide refers to a protein or polypeptide derived from a different source or species.
Hexon: A major adenovirus capsid protein.
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.
Modification: A change in the sequence of a nucleic acid or protein sequence. For example, 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 the removal of one or more amino acid residues from the protein sequence. In some embodiments 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. As used herein, “A” or “delta” refer to a deletion. 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 well known in the art. A “modified” protein, nucleic acid or virus is one that has one or more modifications as outlined above.
Neoplasia, malignancy, cancer and tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.” Malignant tumors are also referred to as “cancer.”
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (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 high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. In some cases, lymphomas are considered solid tumors.
Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for 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, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, human papilloma virus (HPV)-infected neoplasias, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastasis).
Oncolytic virus: A virus that selectively kills cells of a proliferative disorder, e.g., cancer/tumor cells. Killing of the cancer cells can be detected by any method, such as determining viable cell count, or detecting cytopathic effect, apoptosis, or synthesis of viral proteins in the cancer cells (e.g., by metabolic labeling, immunoblot, or RT-PCR of viral genes necessary for replication), or reduction in size of a tumor.
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.
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 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 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.
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 the 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 cross-reactivity or its ability to induce an immune response. Examples of conservative substitutions are shown below.
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.
The substitutions which 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 histadyl, 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.
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).
Protein IX (pIX): A minor component of the adenovirus capsid that associates with the hexon protein.
Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is 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 a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.
Recombinant: A recombinant nucleic acid molecule, protein or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques. The term “recombinant” also includes nucleic acids, proteins and viruses that have been altered solely by addition, substitution, or deletion of a portion of the natural nucleic acid molecule, protein or virus.
Replication defects: An adenovirus that exhibits “replication defects” in a non-tumor cell (compared to a tumor cell) refers to an adenovirus that exhibits reduced viral replication in normal cells compared to tumor cells. Replication defects are evidenced by, for example, a lack of viral late protein expression, a reduction in viral DNA synthesis, a reduced ability to induce E2F target genes (e.g. cyclin A and B), a reduced ability to elicit S phase entry and/or a reduced ability to induce cell killing in normal cells compared to tumor cells.
Replication deficient virus: A virus that preferentially inhibits cell proliferation, causes cell lysis, or induces apoptosis (collectively considered killing) in a predetermined cell population with a given phenotype (e.g., tumor cells with a deregulated E2F pathway). Such viruses are unable to or are limited in the ability to reduce or inhibit cell proliferation, cause cell lysis, induce apoptosis, or otherwise replicate in cells that do not have the predetermined cell phenotype (such as normal, non-tumor cells).
Self-cleaving peptides: Peptides that induce the ribosome to skip the synthesis of a peptide bond at the C-terminus, leading to separation of the 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 virus (ERAV) and Thosea asigna virus (TaV).
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).
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. 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.
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.
Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein can be chemically synthesized in a laboratory.
Uexon: An open reading frame located on the 1 strand (leftward transcription) between the early E3 region and the fiber gene (Tollefson et al., J Virol 81(23):12918-12926).
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 referents unless context clearly indicates otherwise. “Comprising A or B” means including A, or B, or A and B. It is further to be understood that 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 description. 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Disclosed herein are recombinant adenovirus genomes that include a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence. The recombinant adenovirus genomes and recombinant adenoviruses produced by the disclosed genomes can be used, for example, in high-throughput assays to measure virus replication kinetics.
Provided herein are recombinant adenovirus genomes that include a heterologous ORF and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF. 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 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 embodiments, the self-cleaving peptide is a 2A peptide or variant thereof. In some examples, the 2A peptide includes 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 additional amino acid sequence (such as GSG) at the N-terminus.
In particular 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 particular 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 particular 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 specific non-limiting examples, the self-cleaving peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 12-19.
In some embodiments, the heterologous ORF encodes a fluorescent protein, such as, but not limited to a green fluorescent protein (GFP) a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a blue fluorescent protein (BFP). Exemplary fluorescent proteins are known in the art and include, but are not limited to, the following:
BFPs—EBFP, EBFP2, Sapphire, T-Sapphire, Azurite, mTagBFP;
Cyan fluorescent proteins—ECFP, mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, mTurquoise, mTFP1;
GFPs—GFP, EGFP, AcGFP1, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, ZsGreen;
YFPs—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
RFPs—mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, tdTomato, E2-Crimson.
In specific non-limiting examples, the YFP is YPet or the RFP is mCherry.
In some embodiments, the recombinant adenovirus genome includes, in the 5′ to 3′ direction: 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 adenovirus 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 adenovirus genome comprises or consists of any one of SEQ ID NOs: 3-7, 9-11, 20 and 21.
In some embodiments, the adenovirus is an adenovirus type 5 (Ad5). In other embodiments, the adenovirus is an Ad2, Ad3, Ad9, Ad11, Ad12 or Ad34. In yet other embodiments, the adenovirus is a chimeric adenovirus, such as, but not limited to, an Ad5/Ad9 or Ad5/Ad34 chimeric adenovirus.
Further provided herein are recombinant adenoviruses that include a recombinant adenovirus genome disclosed herein.
Also provided are methods for measuring replication kinetics of a recombinant adenovirus, such as a recombinant adenovirus disclosed herein. In some embodiments, the genome of the recombinant adenovirus includes a heterologous ORF encoding a fluorescent protein and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF selected from E1B-55k, DNA polymerase, DNA-binding protein (DBP), adenovirus death protein (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 embodiments, the method includes transfecting cells with the genome of the recombinant adenovirus, or infecting cells with particles of the recombinant adenovirus; culturing the transfected cells or infected cells for at least two days; measuring fluorescence at regular intervals throughout the culture period; and calculating log-slope from the fluorescence measurements. In some examples, the cells are cultured in a multi-well plate.
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 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 includes a second heterologous ORF.
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 from any of the tumor types listed above) and the second cell type is a non-tumor cell (such as a normal mammalian cell).
In some embodiments, the transfected cells or infected cells are cultured for at least two days, at least three days, at least four days, at least five days, at least six days or at least 7 days. In some examples, the transfected cells or infected cells are cultured for about 2 days to about 14 days, such as about 4 days to about 12, or about 6 days to about 10 days. In specific non-limiting examples, the transfected cells or infected cells are cultured for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 8, about 9, about 10, about 11, about 12, about 13 or about 14 days.
In some embodiments, fluorescence is measured approximately every 2 minutes, every 4, minutes, every 6 minutes, every 8 minutes, every 10 minutes, every 15 minutes, every 20 minutes, every 30 minutes, every 45 minutes, every 60 minutes, every 90 minutes, or every 120 minutes. In some examples, fluorescence is measured using a fluorescence plate reader, such as a TECAN™ fluorescence plate reader.
In some embodiments of the virus replication kinetics assay, the method includes transfecting cells with the genome of the recombinant adenovirus. In some examples, transfection results in approximately 5-10% of cells transfected.
In other embodiments of the virus replication kinetics assay, the method includes infecting cells with particles of the recombinant adenovirus. In some examples, the cells are infected with serial dilutions of the recombinant adenovirus particles. A suitable number of virus dilutions can be selected by one of skill in the art. In some examples, about 4 to about 24 dilutions of virus are used in the assay, such as about 4 to about 20, about 6 to about 16 or about 8 to about 12 dilutions. In particular 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.
In some embodiments, the method includes selecting an appropriate therapeutic adenovirus for treatment of a patient's tumor by measuring replication kinetics of a recombinant adenovirus in tumor cells obtained from the patient, wherein the recombinant adenovirus corresponds to the therapeutic adenovirus, except that a 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 from a biopsy.
In some embodiments, the method includes selecting a cancer patient that would respond to treatment with a therapeutic adenovirus by measuring replication kinetics of a recombinant adenovirus in tumor cells obtained from the patient, wherein the recombinant adenovirus corresponds to the therapeutic adenovirus, except that a 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 as predicted responders and predicted non-responders to a particular therapeutic adenovirus. In some examples, the therapeutic adenovirus is an oncolytic adenovirus. In some examples, the tumor cells are obtained from a biopsy.
In some embodiments, the method includes identifying the most efficacious therapeutic adenovirus for a patient's tumor by measuring replication kinetics of a panel of recombinant adenoviruses in tumor cells obtained from the patient, wherein the recombinant adenoviruses correspond to candidate therapeutic adenoviruses, except that a therapeutic ORF of the therapeutic adenoviruses is replaced with an ORF encoding a fluorescent protein. In some examples, the therapeutic adenoviruses are oncolytic adenoviruses. In some examples, the tumor cells are obtained from a biopsy.
Further provided herein are kits that include a recombinant adenovirus genome or a recombinant adenovirus disclosed herein; and cells, cell culture media and/or a multi-well plate. In some embodiments, the cells are tumor cells (such as cells from any of the tumor types listed herein). In some embodiments, the cells are non-tumor cells. In some embodiments, the cell culture media is selected such that it provides a high signal-to-background ratio. In some examples, the cell culture media is free of phenol red. In some embodiments, the multi-well plate is a 48-well, a 96-well or a 384-well plate. In particular examples, the multi-well plate is any plate that can be read on a fluorescence plate reader, such as a TECAN™ fluorescence plate reader.
The 36 kb Adenovirus genome is compact, using both the top and bottom strands for coding of various genes. At many locations within the adenovirus genome, both the top and bottom strand are used simultaneously for coding separate genes. The genome size has evolved to be optimal for insertion into its capsid. As a result, the insertion of exogenous genes is limited by the size capacity of the capsid as excessive addition of exogenous nucleic acid leads to incomplete genome loading into the capsid and reduced viral kinetics.
A solution to the challenge presented by the limited available space in the adenovirus genome is to locate exogenous open reading frames (ORFs) as fusion products within native adenovirus ORFs. This strategy makes use of adenovirus promoters, 5′UTRs, and polyA tails already encoded in the genome. However, expression of a fusion between a native adenovirus protein and an exogenous protein can be deleterious to one or both protein functions and lead to a significant decrease in adenovirus replication kinetics.
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 the 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 embodiments disclosed herein, the self-cleaving peptide is a 2A peptide (P2A).
Also disclosed herein is the identification of optimal placement sites for exogenous ORFs within the adenovirus genome. The combination of the self-cleaving peptide sequence and the judicious placement of the exogenous ORF leads to high expression and minimal to no impact on viral kinetics. Further disclosed herein is use of the recombinant adenoviruses expressing exogenous genes in a high throughput assay for measuring viral replication kinetics.
As described in Example 1 below, several sites within the adenovirus genome were identified that upon insertion of a heterologous ORF, did not inhibit adenovirus replication kinetics. In particular, it was determined that a heterologous ORF could be inserted C-terminal to the E1B-55k ORF, N-terminal to the DNA polymerase ORF, C-terminal to the DBP ORF, N-terminal to the ADP ORF, C-terminal to the E3-14.7k ORF or N-terminal to E4-ORF2. In each instance, a self-cleaving peptide sequence (P2A site) was inserted between the adenovirus ORF and the heterologous ORF. Therefore, the present disclosure contemplates the use of the following recombinant adenovirus in assays to measure replication kinetics (where “SC” refers to a sequence encoding a self-cleaving peptide, such as P2A):
E1B-55k-SC-heterologous ORF
heterologous ORF-SC-(DNA polymerase)
DBP-SC-heterologous ORF
heterologous ORF-SC-ADP
E3-14.7k-SC-heterologous ORF
heterologous ORF-SC-E4-ORF2
In some embodiments herein, the self-cleaving peptide is a virally encoded 2A peptide, or a modified version thereof as described further below.
Self-cleaving peptides are peptides that induce the ribosome to skip the synthesis of a peptide bond at the C-terminus, leading to separation of the peptide sequence and a downstream polypeptide. The use of self-cleaving peptides allows for expression of multiple proteins flanking the self-cleaving peptide from a single ORF. Virally encoded 2A peptides are one type of self-cleaving peptide.
As with other self-cleaving peptides, 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):e18556, 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 2A peptides encoded by Thosea asigna virus (TaV), equine rhinitis A virus (ERAV), porcine teschovirus-1 (PTV1) and foot and mouth disease virus (FMDV), or modified versions thereof
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 show below and are set forth herein as SEQ ID NOs: 12-15.
In some examples, the 2A peptide is modified to include Gly-Ser-Gly at the N-terminus to improve cleavage efficiency. The sequences of modified P2A, F2A, E2A and T2A are shown below and are set forth herein as SEQ ID NOs: 16-19.
In some embodiments, the 2A polypeptide is a variant of a 2A polypeptide disclosed herein. Variants can include polypeptide sequences 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. Variants can include, for example, a deletion of at least one N-terminal amino acid from the 2A polypeptide of any one of SEQ ID NOs: 12-19, for example a deletion of 1, 2, 3, 4 or 5 amino acids, including ranges between any two of the listed values. Variants can include a deletion of at least one C-terminal amino acid from the 2A polypeptide of any one of SEQ ID NOs: 12-19, for example a deletion of 1, 2, 3, 4 or 5 amino acids, including ranges between any two of the listed values. Variants can also include, for example, at least 1, 2, 3, 4 or 5 amino acid substitutions, such as conservative amino acid substitutions.
The critical criteria for assessment of selectively replicating viruses is comparing viral growth kinetics between cancer and normal cells over multiple rounds of replication. Subtle differences in virus replication can be masked at high MOIs. Measuring multiple rounds of virus replication can overcome this problem.
To address the need for a rapid virus kinetics assay, a systematic high throughput screen for viral replication kinetics is needed. Current methods of evaluating virus replication often rely on specific cell lines that have incorporated luciferase or a reporter. However, the activity and level of transgene expression conferred by the encoded reporter measures cell viability, not viral replication per se. Furthermore, adenovirus proteins disrupt global gene expression (such as p300, E2F, CBP, mediator, splicing etc.).
Current methods of assessing adenovirus replication are indirect, insensitive endpoint assays that can only be used in certain cell types; depend on Ad5-specific antibodies; do not measure an entire viral life cycle over multiple rounds; require knowledge of viral titer; cannot use transfection of viral plasmids; do not quantify viral replication; do not predict cell killing; and do not enable comparisons between different subgroups.
Assays currently in use include (1) measuring Ad5 late viral proteins via western blot; (2) measuring adenoviral genomes via q-PCR; (3) plaque assays in specialized and limited cell types; (4) indirectly measuring viral replication using cell viability assays (such as wst-1/mtt); and (5) ELISA using adenovirus-specific antibodies and/or FACS.
Each of these assays has significant disadvantages. The first two methods do not measure the entire viral life-cycle, which includes such steps as viral uptake, gene expression, viral gene replication, capsid assembly, genome loading into the capsid, lysis, spread, and productive secondary infection, thus significantly limiting the utility of these methods.
Plaque assays require specialized cell-lines and efficient viral infection and complementation, which makes it difficult to compare the replication for different Ad serotypes, of which there are 68. In addition, plaque assays require cells to survive an agar overlay, which is only possible with limited cell types. Furthermore, plaque assays are inherently subjective, highly laborious, and provide no insight as to where virus replication is selectively impaired or enhanced (such as at initial infection, gene expression, replication, lysis etc.). Moreover, determination of the proper titer of a capsid-swap virus by methods such as plaque assay or ELISA are not possible because the choice of cell type can effect virus entry. Also, Ad5 antibodies do not recognize fiber swaps employed for altering virus tropism.
In regard to ELISA and FACS assays, these methods depend on using specific antibodies to adenovirus proteins and quantifying titer by detecting antibody binding by FACS or ELISA. However, the antibodies used in traditional assays only recognize specific serotypes and cannot be used to compare viral kinetics or different adenoviruses as they are not recognized by the available antibodies.
As disclosed herein, the incorporation of a fluorescent reporter expressed coincident with one or more viral proteins allows one to measure viral kinetics using methods similar to those used to measure growth of bacteria or yeast. In the methods disclosed herein, fluorescent expression levels are monitored over time and fit to a log growth curve, similar to measuring optical density (OD) of a bacterial or yeast culture to determine log-slope growth rate. 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 the whole-genome plasmid instead of infection with purified virions.
Monitoring of fluorophore expression over time in tissue culture provides a non-invasive, multi-time point measure of viral progression. These measurements provide detailed information regarding the viral kinetics over several rounds of replication and thus include all aspects of the viral life cycle.
The fluorescent-based assay disclosed herein is high throughput and is tolerant to variations in initial virus titer and viral entry. This assay is so tolerant to initial conditions that it is possible to skip virion production and purification and simply use direct transfection of whole-genome plasmids produced by the previously described Adsembly and AdSLIC protocols (see WO2012/024351, which is incorporated by reference herein). Several weeks of time and a large volume of reagents, media, and tissue culture supplies are saved in the process. The assays disclosed herein are an indispensable tool for the rapid and accurate assessment of viral constructs.
In addition, the methods of assessing viral kinetics can be applied to any adenovirus serotype as well as any cell line, and is independent of the starting virus titer, the type of fluorophore selected and the viral protein half-life.
Viral kinetics is determined from the log-slope of measured fluorescence over multiple time points, in some instances covering up to about 10 days. This length of time is often optimal to capture several viral lifecycles, each of which last approximately 48 hours. In some embodiments, fluorescence is measured for at least two days, at least three days, at least four days, at least five days, at least six days or at least 7 days. In some examples, fluorescence is measured for about 2 days to about 14 days, such as about 4 days to about 12, or about 6 days to about 10 days. In specific non-limiting examples, fluorescence is measured for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 8, about 9, about 10, about 11, about 12, about 13 or about 14 days.
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. 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.
Since this assay requires fluorescence measurements made at multiple time points over a period of days, a reference standard must be found that allows normalization across data points. This reference standard must be stable over time, temperature, humidity, and exposure to the excitation radiation used for fluorescence measurements. In some embodiments, the reference standard is background fluorescence from the polystyrene of empty wells. In other embodiments, a commercially available latex bead with embedded fluorophore is the reference standard.
The cell culture media used for the assay disclosed herein ideally provides a high signal-to-background ratio. Factors that lead to high background include phenol red or FBS in the media. Thus, in some embodiments, the culture media used in viral kinetic assays is media free of phenol red. The selection of fluorophore can also be selected to overcome background fluorescence from media. For examples, YPet is 2× brighter than enhanced GFP (eGFP). Thus, in some embodiments, the fluorescent protein is YPet. In other embodiments, the fluorescent protein is mCherry.
The adenovirus genome is organized into several functional groups, labeled E1, E2, E3, E4, and L1-5. The E1 region encodes proteins that control the transcription of all other viral genes and induces S-phase in the host cell. The E2 region encodes proteins that drive viral DNA replication. The E3 region proteins modulate host cell immune response and are dispensable in cell culture. The E4 region contains genes for a disparate set of functions. And the L1-5 region encodes the viral particle structural proteins.
Taking advantage of this natural segregation of functionality, the inventors previously developed a method of recombinant adenovirus assembly that allows quick and easy manipulation of the 36 kb Ad genome by separating it into 4 plasmids, E1, E3, E4, and Core, as shown in
Adsembly and AdSLIC enable the combinatorial in vitro assembly of adenoviruses with novel properties from compatible genomic library parts in 4 hours. Adsembly and AdSLIC provide a common genome design platform that enables synthetic viruses with novel properties to be assembled using four libraries of functional parts (
The Adsembly and AdSLIC technologies enable the modular design and production of adenoviruses with unique capabilities. Developing the capability to design, manufacture, and test viruses in an automated, high-throughput manner will accelerate and expand the development of new viruses for therapeutic, diagnostic, and research studies.
While the cloning step was once the bottleneck for producing new viral constructs, the advent of Adsembly and AdSLIC have made it such that the ability to construct viral genomes has outpaced the ability to test them. An equally high throughput kinetics assay is critical to exploit the full potential and high content assembly of synthetic and personalized viral therapies and diagnostics using the Adsembly and AdSLIC methods.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
This example describes the identification of specific locations within the adenovirus genome where exogenous ORFs can be inserted, along with a self-cleaving peptide sequence, without disrupting virus kinetics.
The insertion of exogenous genes in adenovirus vectors is limited by the size capacity of the adenovirus capsid. Excessive addition of exogenous nucleic acid leads to incomplete genome loading into the capsid and reduced viral kinetics. A solution to the challenge presented by the limited available space in the adenovirus genome is to locate exogenous open reading frames (ORFs) as fusion products within native adenovirus ORFs. This strategy makes use of adenovirus promoters, 5′UTRs, and polyA tails already encoded in the genome. However, expression of a fusion between a native adenovirus protein and an exogenous protein can be deleterious to one or both protein functions and lead to a significant decrease in adenovirus replication kinetics. In fact, studies disclosed herein demonstrate that direct fusion of an exogenous ORF to the adenovirus E1A, DNA polymerase or ADP ORFs significantly inhibits adenovirus replication kinetics. In addition, the inventors previously tried using an internal ribosomal entry site (IRES) to insert exogenous ORFs, which also failed to produce recombinant virus with wild-type kinetics.
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 the 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. The adenovirus constructs generated in this example using the self-cleaving peptide P2A and a fluorescent protein (e.g. YPet, mCherry) as the exogenous ORF.
The table below provides a list of the constructs that were generated and indicates the expression level of the exogenous ORF (low, medium or high) and the level of virus replication kinetics (low, medium or high) in two different cells lines—293-E4 cells and A549 cells.
E1B-55k-P2A-YPet
CMBT-456
3
High
High
High
E1B-55k-P2A-mCherry
CMBT-499
4
High
High
High
YPet-P2A-(DNA Poly)
CMBT-530
5
Medium
High
High
DBP-P2A-YPet
CMBT-886
6
High
High
High
YPet-P2A-ADP
CMBT-403
7
High
High
High
E3-14.7k-P2A-YPet
PCMN-887
9
High
High
High
YPet-P2A-E4-ORF2
CMBT-457
10
Medium
High
High
mCherry-P2A-E4-ORF2
CMBT-633
11
Medium
High
High
Constructs exhibiting “high” replication kinetics (i.e. replication kinetics that are comparable to wild-type adenovirus) in both cell types are considered candidates for use in the virus replication kinetics assays described in Example 2 (candidate constructs are shown in bold).
Comparison of Direct Fusion and Insertion of a P2A Site
Several constructs were generated in which a fluorescent protein was fused directly to an adenovirus ORF. In particular, the following direct fusions were generated: YPet-E1A, YPet-(DNA polymerase) and mCherry-ADP.
YPet-E1A adenovirus exhibited a significant impairment in virus kinetics. Insertion of the P2A site between YPet and E1A (YPet-P2A-E1A) improved virus kinetics, but did not restore virus kinetics to wild-type level. Another construct was then generated to test fusion of P2A and YPet to the C-terminal end of E1A (E1A-P2A-YPet). This construct further improved virus kinetics, but again did not restore kinetics to the level of wild-type adenovirus.
Multiple attempts at transfecting the YPet-(DNA-poly) genome plasmid failed to produce viable virus (no plaques were formed). However, fusion of YPet-P2A to the N-terminus of DNA polymerase (YPet-P2A-(DNA poly)) produced a virus with wild-type kinetics, as shown in the table above.
Finally, the direct fusion of mCherry to ADP (mCherry-ADP) produced a virus with significantly impaired kinetics. However, insertion of the P2A site between the mCherry ORF and the ADP ORF resulted in a virus with wild-type kinetics (mCherry-P2A-ADP). The same result was obtained using a different fluorescent protein; the YPet-P2A-ADP construct exhibited wild-type virus kinetics. However, placement of P2A and the heterologous ORF on the C-terminal side of ADP produced a virus that did not replicate. Thus, for the ADP, the heterologous ORF must be placed at the N-terminus.
Additional Constructs with Wild-Type Virus Kinetics
Evaluation of viral kinetics for constructs having a P2A site and heterologous ORF on the C-terminus of DBP (DBP-P2A-YPet) or the C-terminus of E3-14.7k (E3-14.7k-P2A-YPet), or having a P2A site and heterologous ORF on the N-terminus of E4-ORF2 (YPet-P2A-E4-ORF2 and mCherry-P2A-E4-ORF2) produced viruses with wild-type replication kinetics.
The results of these data demonstrate that at least the following adenovirus genome constructs can be used on the viral replication assays described in Example 2:
E1B-55k-SC-heterologous ORF
heterologous ORF-SC-(DNA polymerase)
DBP-SC-heterologous ORF
heterologous ORF-SC-ADP
E3-14.7k-SC-heterologous ORF
heterologous ORF-SC-E4-ORF2
For use in the virus replication assays disclosed herein, the heterologous ORF encodes a fluorescent protein, such as (but not limited to) YPet or mCherry.
Other Adenovirus Serotypes
Previously described methods of measuring viral kinetics are all highly dependent upon cell-type specific assays and are thus serotype specific due to the divergent tropism of each adenovirus serotype. The adenovirus kinetic assay disclosed herein is not dependent upon any one cell type and so can be extended to serotypes other than Ad5. All adenovirus serotypes contain an ORF equivalent to Ad5 E3-14.7k. Therefore, viruses equivalent to Ad5 E3-14.7k-P2A-YPet (PCMN-887; SEQ ID NO: 9) were generated using Ad9 (containing E3-15k) and Ad34 (containing E3-14.8k): 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 a fiber shaft and knob from either Ad9 or Ad34 were also generated. The four recombinant viruses were then tested in the FBVK assay using 293 cells (
The Adsembly and AdSLIC methods for assembling recombinant adenoviruses provide a means for generating large numbers of recombinant virus genomes and viruses in a short period of time. However, a need exists for a rapid and high-throughput method for evaluating 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 test virus replication kinetics of recombinant adenoviruses (
When starting with a recombinant adenovirus genome, the assay includes transfecting cells with adenovirus genome plasmids (such as those described above in Example 1) and monitoring fluorophore expression over time (
The assay can also be carried out by infecting cells with recombinant virus particles. In this version of the assay, cells are infected with recombinant virus particles and fluorophore expression is monitored over time (
The TECAN™ plate readers also provide incubation functions (maintaining an appropriate temperature as well as CO2 and O2 levels). Data points are taken every 15 minutes to calculate the ln-slope. Using these methods, it is possible to rapidly and efficiently compare the kinetics between a number of different viruses and between different cell types. For example, to evaluate whether particular recombinant adenoviruses could be used therapeutically as oncolytic viruses, this assay could be employed to find viruses that exhibit high replication kinetics in tumor cells, but slow virus kinetics in non-tumor cells. Furthermore, the virus kinetics of the recombinant viruses can be evaluated by infecting or transfecting the tumor cell type of interest in this assay.
Calculating Log-Slope
To measure log-slope, the linear plot of fluorescence intensity versus time is converted to a semi-log plot by taking the natural logarithm of the measured fluorescence intensity at each time point. Since the fluorescence intensity exhibits exponential growth during viral replication, this conversion results in a straight line when plotting ln(fluorescence intensity) vs. time. This straight line is then fit using standard least-squares methods. The resulting slope produced by this fit is the ln-slope of the fluorescence vs. time and thus the ln-slope of the viral growth vs. time. Equations are shown below.
FI(t)=F0eα(t-t
where FI is measured fluorescence intensity, t is time, F0 is the initial fluorescence intensity at time=t0, and α is the ln-slope.
Take natural logarithm of both sides:
ln[FI(t)]=ln[F0eα(t-t
The right hand side is now a linear equation with a ln-slope of α.
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.
This application is a continuation of PCT International Application No. PCT/US2017/019082, filed Feb. 23, 2017, published in English under PCT Article 21(2), which claims the benefit of U.S. Provisional Application No. 62/298,649, filed Feb. 23, 2016. The above-referenced applications are herein incorporated by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5559099 | Wickham et al. | Sep 1996 | A |
| 5670488 | Gregory et al. | Sep 1997 | A |
| 5677178 | McCormick | Oct 1997 | A |
| 5731190 | Wickham et al. | Mar 1998 | A |
| 5801029 | McCormick | Sep 1998 | A |
| 5846782 | Wickham et al. | Dec 1998 | A |
| 5846945 | McCormick | Dec 1998 | A |
| 5856181 | McCormick | Jan 1999 | A |
| 5922315 | Roy | Jul 1999 | A |
| 5945335 | Colosi | Aug 1999 | A |
| 5962311 | Wickham et al. | Oct 1999 | A |
| 5965541 | Wickham et al. | Oct 1999 | A |
| 5972706 | McCormick | Oct 1999 | A |
| 6020172 | Both | Feb 2000 | A |
| 6069134 | Roth et al. | May 2000 | A |
| 6127525 | Crystal et al. | Oct 2000 | A |
| 6133243 | Kirn | Oct 2000 | A |
| 6153435 | Crystal et al. | Nov 2000 | A |
| 6296845 | Sampson et al. | Oct 2001 | B1 |
| 6329190 | Wickham et al. | Dec 2001 | B1 |
| 6410010 | Zhang et al. | Jun 2002 | B1 |
| 6455314 | Wickham et al. | Sep 2002 | B1 |
| 6465253 | Wickham et al. | Oct 2002 | B1 |
| 6475480 | Mehtali et al. | Nov 2002 | B1 |
| 6506379 | Clackson et al. | Jan 2003 | B1 |
| 6506602 | Stemmer | Jan 2003 | B1 |
| 6569677 | Legrand et al. | May 2003 | B1 |
| 6596268 | Coffey et al. | Jul 2003 | B1 |
| 6635466 | Davidson et al. | Oct 2003 | B2 |
| 6635476 | Murphy | Oct 2003 | B1 |
| 6649157 | Coffey et al. | Nov 2003 | B2 |
| 6737234 | Freimuth | May 2004 | B1 |
| 6740525 | Roelvink et al. | May 2004 | B2 |
| 6797702 | Roth et al. | Sep 2004 | B1 |
| 6811774 | Haddada et al. | Nov 2004 | B2 |
| 6824771 | Curiel et al. | Nov 2004 | B1 |
| 6838285 | Farmer et al. | Jan 2005 | B2 |
| 6841540 | Curiel et al. | Jan 2005 | B1 |
| 6849446 | Tikoo et al. | Feb 2005 | B2 |
| 6867022 | Imperiale | Mar 2005 | B1 |
| 6869936 | Vogels et al. | Mar 2005 | B1 |
| 6878549 | Vogels et al. | Apr 2005 | B1 |
| 6905678 | Havenga et al. | Jun 2005 | B2 |
| 6911199 | Vigne et al. | Jun 2005 | B2 |
| 6911200 | Yu et al. | Jun 2005 | B2 |
| 6913922 | Bout et al. | Jul 2005 | B1 |
| 6929946 | Vogels et al. | Aug 2005 | B1 |
| 6951755 | Wickham et al. | Oct 2005 | B2 |
| 6984635 | Schreiber et al. | Jan 2006 | B1 |
| 7001596 | Johnson et al. | Feb 2006 | B1 |
| 7045347 | Graham et al. | May 2006 | B2 |
| 7094398 | Lieber et al. | Aug 2006 | B1 |
| 7094399 | Otto | Aug 2006 | B2 |
| 7109179 | Roth et al. | Sep 2006 | B2 |
| 7157266 | Freimuth et al. | Jan 2007 | B2 |
| 7232899 | Seggem et al. | Jun 2007 | B2 |
| 7235233 | Havenga et al. | Jun 2007 | B2 |
| 7247472 | Wilson et al. | Jul 2007 | B2 |
| 7252817 | Coffey et al. | Aug 2007 | B2 |
| 7252989 | Zhang et al. | Aug 2007 | B1 |
| 7256036 | Legrand et al. | Aug 2007 | B2 |
| 7291498 | Roy et al. | Nov 2007 | B2 |
| 7297542 | Curiel et al. | Nov 2007 | B2 |
| 7306793 | Haddada et al. | Dec 2007 | B2 |
| 7332337 | van Es et al. | Feb 2008 | B2 |
| 7344711 | Bonastre et al. | Mar 2008 | B2 |
| 7344872 | Gao et al. | Mar 2008 | B2 |
| 7364727 | Li et al. | Apr 2008 | B2 |
| 7410954 | Davidson et al. | Aug 2008 | B2 |
| 7456008 | Lindholm et al. | Nov 2008 | B2 |
| 7473418 | Yu et al. | Jan 2009 | B2 |
| 7482156 | Arroyo et al. | Jan 2009 | B2 |
| 7491508 | Roy et al. | Feb 2009 | B2 |
| 7510868 | Harden et al. | Mar 2009 | B2 |
| 7589069 | Wold et al. | Sep 2009 | B1 |
| 7611868 | Monaci et al. | Nov 2009 | B2 |
| 7741099 | Havenga et al. | Jun 2010 | B2 |
| 7749493 | Havenga et al. | Jul 2010 | B2 |
| 7754201 | Wang et al. | Jul 2010 | B2 |
| 7906113 | Bout et al. | Mar 2011 | B2 |
| 7943373 | Fujiwara et al. | May 2011 | B2 |
| 7951585 | Ke | May 2011 | B2 |
| 7968333 | Yu et al. | Jun 2011 | B2 |
| 8105574 | Wilson et al. | Jan 2012 | B2 |
| 8168168 | Fueyo et al. | May 2012 | B2 |
| 8231880 | Roy et al. | Jul 2012 | B2 |
| 8470310 | Roy et al. | Jun 2013 | B2 |
| 8524219 | Roy et al. | Sep 2013 | B2 |
| 8603459 | Wilson et al. | Dec 2013 | B2 |
| 8685387 | Roy et al. | Apr 2014 | B2 |
| 8715642 | Kochanek et al. | May 2014 | B2 |
| 8765146 | Bruder et al. | Jul 2014 | B2 |
| 8765463 | Harden et al. | Jul 2014 | B2 |
| 8815563 | Davis et al. | Aug 2014 | B2 |
| 8834863 | Roy et al. | Sep 2014 | B2 |
| 8846031 | Roy et al. | Sep 2014 | B2 |
| 8865182 | Mayall et al. | Oct 2014 | B2 |
| 8920813 | Bruder et al. | Dec 2014 | B2 |
| 8940290 | Roy et al. | Jan 2015 | B2 |
| 8974777 | Cascallo et al. | Mar 2015 | B2 |
| 9017672 | Yu et al. | Apr 2015 | B2 |
| 9018182 | Koh et al. | Apr 2015 | B2 |
| 9056090 | Colloca et al. | Jun 2015 | B2 |
| 9061055 | Fueyo et al. | Jun 2015 | B2 |
| 9133483 | Wilson et al. | Sep 2015 | B2 |
| 9163261 | Kollipara et al. | Oct 2015 | B2 |
| 9187733 | O'Shea et al. | Nov 2015 | B2 |
| 9200041 | Lieber et al. | Dec 2015 | B2 |
| 9206238 | Roy et al. | Dec 2015 | B2 |
| 9217159 | Roy et al. | Dec 2015 | B2 |
| 9217160 | O'Shea et al. | Dec 2015 | B2 |
| 9267153 | Curiel | Feb 2016 | B2 |
| 9315827 | Wang et al. | Apr 2016 | B2 |
| 9359618 | Roy et al. | Jun 2016 | B2 |
| 9382551 | Roy et al. | Jul 2016 | B2 |
| 9410129 | Ranki et al. | Aug 2016 | B2 |
| 9476061 | Baker et al. | Oct 2016 | B2 |
| 9493745 | Lee et al. | Nov 2016 | B2 |
| 9555089 | Shiratsuchi et al. | Jan 2017 | B2 |
| 9593346 | Roy et al. | Mar 2017 | B2 |
| 9597363 | Roy et al. | Mar 2017 | B2 |
| 9682133 | Crystal et al. | Jun 2017 | B2 |
| 9688727 | Lieber et al. | Jun 2017 | B2 |
| 9714435 | Dicks et al. | Jul 2017 | B2 |
| 9718863 | Colloca et al. | Aug 2017 | B2 |
| 9790519 | Wei et al. | Oct 2017 | B2 |
| 9885090 | O'Shea et al. | Feb 2018 | B2 |
| 9913866 | O'Shea et al. | Mar 2018 | B2 |
| 10016470 | Bonastre et al. | Jul 2018 | B2 |
| 10034905 | Seymour et al. | Jul 2018 | B2 |
| 10046067 | Yun et al. | Aug 2018 | B2 |
| 10066215 | Lee et al. | Sep 2018 | B2 |
| 10071126 | Kumon et al. | Sep 2018 | B2 |
| 10077430 | Lee et al. | Sep 2018 | B2 |
| 10080774 | Fueyo et al. | Sep 2018 | B2 |
| 10113182 | Roy et al. | Oct 2018 | B2 |
| 10149873 | Roy et al. | Dec 2018 | B2 |
| 10150798 | Lieber et al. | Dec 2018 | B2 |
| 10155930 | Holm | Dec 2018 | B2 |
| 10232053 | Hicklin et al. | Mar 2019 | B2 |
| 10272162 | McVey et al. | Apr 2019 | B2 |
| 10294493 | Wang et al. | May 2019 | B2 |
| 10316065 | Carrió et al. | Jun 2019 | B2 |
| 10376549 | Shayakhmetov et al. | Aug 2019 | B2 |
| 10391183 | Fueyo-Margareto et al. | Aug 2019 | B2 |
| 10501757 | Roy et al. | Dec 2019 | B2 |
| 10538744 | Holm | Jan 2020 | B2 |
| 10544192 | Colloca et al. | Jan 2020 | B2 |
| 10604549 | Alemany Bonastre et al. | Mar 2020 | B2 |
| 10611803 | Lieber et al. | Apr 2020 | B2 |
| 10617729 | Dobbins | Apr 2020 | B2 |
| 10738325 | O'Shea et al. | Aug 2020 | B2 |
| 20010039046 | Yeh et al. | Nov 2001 | A1 |
| 20020037274 | Williams et al. | Mar 2002 | A1 |
| 20020086411 | Holm et al. | Jul 2002 | A1 |
| 20020001063 | Young et al. | Aug 2002 | A1 |
| 20020142989 | Alemany et al. | Oct 2002 | A1 |
| 20020151069 | Korokhov | Oct 2002 | A1 |
| 20020168343 | Curiel et al. | Nov 2002 | A1 |
| 20020187128 | Imperiale | Dec 2002 | A1 |
| 20020193327 | Nemerow | Dec 2002 | A1 |
| 20020193328 | Ketner | Dec 2002 | A1 |
| 20030001713 | Havenga et al. | Jan 2003 | A1 |
| 20030021768 | Shen | Jan 2003 | A1 |
| 20030027338 | Freimuth | Feb 2003 | A1 |
| 20030073072 | Havenga et al. | Apr 2003 | A1 |
| 20030082146 | van Es | May 2003 | A1 |
| 20030082150 | Falleur et al. | May 2003 | A1 |
| 20030082811 | Orlando et al. | May 2003 | A1 |
| 20030092162 | Shankara et al. | May 2003 | A1 |
| 20030095989 | Irving et al. | May 2003 | A1 |
| 20030099615 | Tikoo | May 2003 | A1 |
| 20030099619 | Wickham et al. | May 2003 | A1 |
| 20030104625 | Cheng et al. | Jun 2003 | A1 |
| 20030138405 | Fueyo et al. | Jul 2003 | A1 |
| 20030143730 | Blanche et al. | Jul 2003 | A1 |
| 20030166286 | Wickham et al. | Sep 2003 | A1 |
| 20030170899 | McVey et al. | Sep 2003 | A1 |
| 20030175244 | Curiel et al. | Sep 2003 | A1 |
| 20030175245 | Brough et al. | Sep 2003 | A1 |
| 20030215948 | Kaleko et al. | Nov 2003 | A1 |
| 20030219899 | Korokhov | Nov 2003 | A1 |
| 20030220284 | Yotnda et al. | Nov 2003 | A1 |
| 20040002060 | Kaleko et al. | Jan 2004 | A1 |
| 20040038205 | Van Raaij et al. | Feb 2004 | A1 |
| 20040001023 | Schughart et al. | May 2004 | A1 |
| 20040091456 | Nakai et al. | May 2004 | A1 |
| 20040146489 | Yu et al. | Jul 2004 | A1 |
| 20040175362 | Curiel et al. | Sep 2004 | A1 |
| 20040185555 | Emini et al. | Sep 2004 | A1 |
| 20040191222 | Emini et al. | Sep 2004 | A1 |
| 20040191761 | Routes | Sep 2004 | A1 |
| 20040213764 | Wold et al. | Oct 2004 | A1 |
| 20040219516 | Bennett et al. | Nov 2004 | A1 |
| 20040219543 | Wirtz | Nov 2004 | A1 |
| 20040265277 | Holm | Dec 2004 | A1 |
| 20050032045 | Tikoo et al. | Feb 2005 | A1 |
| 20050036989 | Shen et al. | Feb 2005 | A1 |
| 20050079158 | Zhou et al. | Apr 2005 | A1 |
| 20050095231 | Curiel et al. | May 2005 | A1 |
| 20050095705 | Kadan et al. | May 2005 | A1 |
| 20050169891 | Vogels et al. | Aug 2005 | A1 |
| 20050181507 | Havenga et al. | Aug 2005 | A1 |
| 20050186178 | Ennist | Aug 2005 | A1 |
| 20050201936 | Wold et al. | Sep 2005 | A1 |
| 20050201978 | Lipton | Sep 2005 | A1 |
| 20050232900 | Vogels et al. | Oct 2005 | A1 |
| 20050238622 | Axelrod et al. | Oct 2005 | A1 |
| 20050260162 | Fueyo et al. | Nov 2005 | A1 |
| 20050271622 | Zhou et al. | Dec 2005 | A1 |
| 20050277193 | Wickham et al. | Dec 2005 | A1 |
| 20050287120 | Fisher et al. | Dec 2005 | A1 |
| 20060002893 | Vigne et al. | Jan 2006 | A1 |
| 20060034804 | Gregory et al. | Feb 2006 | A1 |
| 20060099178 | Holm | May 2006 | A1 |
| 20060104953 | Havenga et al. | May 2006 | A1 |
| 20060140910 | Gregory et al. | Jun 2006 | A1 |
| 20060147420 | Fueyo et al. | Jul 2006 | A1 |
| 20060182718 | Roth et al. | Aug 2006 | A1 |
| 20060211115 | Roy et al. | Sep 2006 | A1 |
| 20060228334 | Calatrava et al. | Oct 2006 | A1 |
| 20060257370 | Hermiston et al. | Nov 2006 | A1 |
| 20060281090 | Lieber et al. | Dec 2006 | A1 |
| 20060286121 | Gall et al. | Dec 2006 | A1 |
| 20060292122 | Hermiston et al. | Dec 2006 | A1 |
| 20060292682 | Hawkins et al. | Dec 2006 | A1 |
| 20070003923 | Nemerow | Jan 2007 | A1 |
| 20070110719 | Holm | May 2007 | A1 |
| 20070202080 | Yun et al. | Aug 2007 | A1 |
| 20070202524 | Murphy | Aug 2007 | A1 |
| 20070253932 | Gregory et al. | Nov 2007 | A1 |
| 20070254357 | Gregory et al. | Nov 2007 | A1 |
| 20070292396 | Fueyo et al. | Dec 2007 | A1 |
| 20070292954 | Elledge | Dec 2007 | A1 |
| 20080069836 | Nabel et al. | Mar 2008 | A1 |
| 20080089864 | Bonastre et al. | Apr 2008 | A1 |
| 20080108129 | Pitcovski et al. | May 2008 | A1 |
| 20080112929 | Kovesdi et al. | May 2008 | A1 |
| 20080118470 | Ennist et al. | May 2008 | A1 |
| 20080124360 | Seggern | May 2008 | A1 |
| 20080213220 | Fisher et al. | Sep 2008 | A1 |
| 20080242608 | Bonni et al. | Oct 2008 | A1 |
| 20080247996 | Yu et al. | Oct 2008 | A1 |
| 20080254059 | Bett et al. | Oct 2008 | A1 |
| 20090074810 | Roy et al. | Mar 2009 | A1 |
| 20090111144 | Bebbington | Apr 2009 | A1 |
| 20090202565 | Labow et al. | Aug 2009 | A1 |
| 20090232800 | Holm | Sep 2009 | A1 |
| 20090280089 | Benihoud et al. | Nov 2009 | A1 |
| 20090311219 | Bonastre et al. | Dec 2009 | A1 |
| 20100008977 | Boulikas et al. | Jan 2010 | A1 |
| 20100034774 | Vogels et al. | Feb 2010 | A1 |
| 20100047208 | Ke | Feb 2010 | A1 |
| 20100075951 | Cardin et al. | Mar 2010 | A1 |
| 20100075998 | Vanotti et al. | Mar 2010 | A1 |
| 20100098668 | Seth | Apr 2010 | A1 |
| 20100151576 | Li et al. | Jun 2010 | A1 |
| 20100233125 | Tagawa | Sep 2010 | A1 |
| 20100272753 | Ketner et al. | Oct 2010 | A1 |
| 20100292166 | Lee et al. | Nov 2010 | A1 |
| 20100310554 | Holm | Dec 2010 | A1 |
| 20100311145 | Holm | Dec 2010 | A1 |
| 20110053249 | Bonastre et al. | Mar 2011 | A1 |
| 20110059135 | Kovesdi et al. | Mar 2011 | A1 |
| 20110086063 | Crystal et al. | Apr 2011 | A1 |
| 20110104788 | Baker et al. | May 2011 | A1 |
| 20110189234 | Van Beusechem et al. | Aug 2011 | A1 |
| 20110256524 | Lee et al. | Oct 2011 | A1 |
| 20110275093 | Holm | Nov 2011 | A1 |
| 20110286999 | Holm | Nov 2011 | A1 |
| 20120020924 | Nakai et al. | Jan 2012 | A1 |
| 20120039877 | Holm | Feb 2012 | A1 |
| 20120207711 | Fueyo et al. | Aug 2012 | A1 |
| 20130058897 | Lee et al. | Mar 2013 | A1 |
| 20130101557 | Yun et al. | Apr 2013 | A1 |
| 20130231267 | O'Shea et al. | Sep 2013 | A1 |
| 20130243729 | O'Shea et al. | Sep 2013 | A1 |
| 20130243731 | Dias et al. | Sep 2013 | A1 |
| 20130323205 | Diaconu et al. | Dec 2013 | A1 |
| 20130345295 | Wang et al. | Dec 2013 | A1 |
| 20140023619 | Kosai et al. | Jan 2014 | A1 |
| 20140199688 | Mizuguchi et al. | Jul 2014 | A1 |
| 20140294890 | Ketner et al. | Oct 2014 | A1 |
| 20140341857 | Bressy et al. | Nov 2014 | A1 |
| 20140348791 | Barouch et al. | Nov 2014 | A1 |
| 20140377294 | Fueyo-Margareto et al. | Dec 2014 | A1 |
| 20140377295 | Ertl et al. | Dec 2014 | A1 |
| 20150005397 | O'Shea et al. | Jan 2015 | A1 |
| 20150017127 | O'Shea et al. | Jan 2015 | A1 |
| 20150071881 | Bonastre et al. | Mar 2015 | A1 |
| 20150086579 | Mayall et al. | Mar 2015 | A1 |
| 20150202324 | Hemminki et al. | Jul 2015 | A1 |
| 20150232880 | Hemminki et al. | Aug 2015 | A1 |
| 20150246949 | Lieber et al. | Sep 2015 | A1 |
| 20150352203 | Wilson et al. | Dec 2015 | A1 |
| 20150374766 | O'Shea et al. | Dec 2015 | A1 |
| 20160017294 | Reid et al. | Jan 2016 | A1 |
| 20160051603 | Roy et al. | Feb 2016 | A1 |
| 20160053235 | O'Shea et al. | Feb 2016 | A1 |
| 20160082100 | Ranki et al. | Mar 2016 | A1 |
| 20160090574 | Fisher et al. | Mar 2016 | A1 |
| 20160102295 | Roy et al. | Apr 2016 | A1 |
| 20160143967 | Fueyo-Margareto et al. | May 2016 | A1 |
| 20160208287 | Hemminki et al. | Jul 2016 | A1 |
| 20160244783 | Roy et al. | Aug 2016 | A1 |
| 20160289645 | Tufaro et al. | Oct 2016 | A1 |
| 20170035818 | Seymour et al. | Feb 2017 | A1 |
| 20170073647 | Fisher et al. | Mar 2017 | A1 |
| 20170080069 | Cerullo et al. | Mar 2017 | A1 |
| 20170096646 | Roy et al. | Apr 2017 | A1 |
| 20170137786 | Hemminki et al. | May 2017 | A1 |
| 20170183636 | Roy et al. | Jun 2017 | A1 |
| 20170190752 | Holm | Jul 2017 | A1 |
| 20170202893 | O'Shea et al. | Jul 2017 | A1 |
| 20170252443 | Holm | Sep 2017 | A1 |
| 20170314044 | Davydova et al. | Nov 2017 | A1 |
| 20170348405 | Shiratsuchi et al. | Dec 2017 | A1 |
| 20180000966 | Dicks et al. | Jan 2018 | A1 |
| 20180051301 | Rentschler et al. | Feb 2018 | A1 |
| 20180072809 | Hemminki et al. | Mar 2018 | A1 |
| 20180100164 | Wei et al. | Apr 2018 | A1 |
| 20180104288 | Galili et al. | Apr 2018 | A1 |
| 20180163190 | Gerardy-Schahn et al. | Jun 2018 | A1 |
| 20180216081 | Colloca et al. | Aug 2018 | A1 |
| 20180221423 | O'Shea et al. | Aug 2018 | A1 |
| 20180318365 | Yeung et al. | Nov 2018 | A1 |
| 20180346929 | Kosai et al. | Dec 2018 | A1 |
| 20180355374 | O'Shea et al. | Dec 2018 | A1 |
| 20180355379 | O'Shea et al. | Dec 2018 | A1 |
| 20180369417 | Yun et al. | Dec 2018 | A1 |
| 20190055522 | Holm | Feb 2019 | A1 |
| 20190062395 | Merchant et al. | Feb 2019 | A1 |
| 20190070233 | Yeung et al. | Mar 2019 | A1 |
| 20190093085 | Tufaro et al. | Mar 2019 | A1 |
| 20190136204 | Reid et al. | May 2019 | A1 |
| 20190142967 | Hicklin et al. | May 2019 | A1 |
| 20190175716 | Gilbert et al. | Jun 2019 | A1 |
| 20190183946 | Bonastre et al. | Jun 2019 | A1 |
| 20190201462 | Tufaro et al. | Jul 2019 | A1 |
| 20190201551 | Curiel | Jul 2019 | A1 |
| 20190233845 | Maloveste et al. | Aug 2019 | A1 |
| 20190247452 | Lan et al. | Aug 2019 | A1 |
| 20190269794 | McVey et al. | Sep 2019 | A1 |
| 20190275092 | Tufaro et al. | Sep 2019 | A1 |
| 20190275093 | Aboody et al. | Sep 2019 | A1 |
| 20190300905 | Ammendola et al. | Oct 2019 | A1 |
| 20190314523 | O'Shea et al. | Oct 2019 | A1 |
| 20190314525 | O'Shea et al. | Oct 2019 | A1 |
| 20190345204 | Carrió et al. | Nov 2019 | A1 |
| 20190350992 | Cascallo Piqueras et al. | Nov 2019 | A1 |
| 20190352616 | Reid et al. | Nov 2019 | A1 |
| 20190352669 | Reid et al. | Nov 2019 | A1 |
| 20190374589 | Suzuki et al. | Dec 2019 | A1 |
| 20190388487 | Shayakhmetov et al. | Dec 2019 | A1 |
| 20200014798 | Hicklin et al. | Jan 2020 | A1 |
| 20200032223 | Reid et al. | Jan 2020 | A1 |
| 20200078415 | Reid et al. | Mar 2020 | A1 |
| 20200095560 | Holm | Mar 2020 | A1 |
| 20200102352 | Colloca et al. | Apr 2020 | A1 |
| Number | Date | Country |
|---|---|---|
| 1330715 | Jan 2002 | CN |
| 13 80420 | Nov 2002 | CN |
| 102191245 | Sep 2011 | CN |
| 0689447 | Apr 1999 | EP |
| 0931830 | Mar 2001 | EP |
| 0760675 | Aug 2001 | EP |
| 1167533 | Jan 2002 | EP |
| 1284294 | Feb 2003 | EP |
| 1413586 | Apr 2004 | EP |
| 1196616 | Dec 2004 | EP |
| 1185279 | Jan 2005 | EP |
| 0851769 | Feb 2005 | EP |
| 0861329 | Mar 2005 | EP |
| 1181382 | Mar 2005 | EP |
| 1121137 | Jul 2005 | EP |
| 0991763 | Sep 2005 | EP |
| 1294918 | Oct 2005 | EP |
| 0889969 | Nov 2005 | EP |
| 1498129 | Nov 2005 | EP |
| 1593742 | Nov 2005 | EP |
| 0920524 | Dec 2005 | EP |
| 1307573 | Jan 2006 | EP |
| 0978566 | May 2006 | EP |
| 0778889 | Jul 2006 | EP |
| 1070118 | Oct 2006 | EP |
| 1214098 | Nov 2006 | EP |
| 1230378 | Jun 2007 | EP |
| 1550722 | Jun 2007 | EP |
| 1187919 | Nov 2007 | EP |
| 0863987 | Jan 2008 | EP |
| 0920514 | Jan 2008 | EP |
| 1159438 | Jul 2008 | EP |
| 1266022 | Oct 2008 | EP |
| 1678193 | Dec 2008 | EP |
| 1054064 | Dec 2009 | EP |
| 2012822 | Jan 2010 | EP |
| 1816204 | Oct 2010 | EP |
| 1749098 | Dec 2010 | EP |
| 1799836 | Dec 2010 | EP |
| 1816205 | Aug 2011 | EP |
| 1818408 | Aug 2011 | EP |
| 1409748 | Oct 2011 | EP |
| 1180932 | Jan 2012 | EP |
| 1466001 | Apr 2012 | EP |
| 1743041 | Jun 2012 | EP |
| 1446479 | Aug 2012 | EP |
| 1649028 | Aug 2012 | EP |
| 1990418 | Aug 2012 | EP |
| 2311499 | Aug 2012 | EP |
| 1636370 | Apr 2014 | EP |
| 1767642 | Apr 2014 | EP |
| 1689445 | Feb 2015 | EP |
| 2350269 | Sep 2015 | EP |
| 2403951 | Sep 2015 | EP |
| 2643465 | May 2016 | EP |
| 2428229 | Aug 2016 | EP |
| 2459716 | Aug 2016 | EP |
| 2220241 | Sep 2016 | EP |
| 2325298 | Oct 2016 | EP |
| 2379586 | Nov 2016 | EP |
| 2220242 | Dec 2016 | EP |
| 2774985 | Dec 2016 | EP |
| 2163260 | Mar 2017 | EP |
| 2580234 | Mar 2017 | EP |
| 2798069 | Mar 2017 | EP |
| 2855685 | Mar 2017 | EP |
| 2900818 | Jun 2017 | EP |
| 2301582 | Jul 2017 | EP |
| 3049520 | Jul 2017 | EP |
| 1453543 | Aug 2017 | EP |
| 2463362 | Nov 2017 | EP |
| 2558481 | Dec 2017 | EP |
| 2682459 | Dec 2017 | EP |
| 2714916 | Jan 2018 | EP |
| 2391638 | Jun 2018 | EP |
| 2563919 | Jun 2018 | EP |
| 2971008 | Jul 2018 | EP |
| 2606137 | Aug 2018 | EP |
| 2855669 | Oct 2018 | EP |
| 2986311 | Nov 2018 | EP |
| 3145537 | Dec 2018 | EP |
| 2654786 | Feb 2019 | EP |
| 3280798 | Jun 2019 | EP |
| 3029144 | Jul 2019 | EP |
| 3150706 | Jul 2019 | EP |
| 2809788 | Sep 2019 | EP |
| 3071697 | Oct 2019 | EP |
| 3274363 | Oct 2019 | EP |
| 3460052 | Oct 2019 | EP |
| 2005-525779 | Sep 2005 | JP |
| 2008-517627 | May 2008 | JP |
| 2010-527324 | Aug 2010 | JP |
| 2011-524904 | Sep 2011 | JP |
| WO 9618418 | Jun 1996 | WO |
| WO 9854346 | Dec 1998 | WO |
| WO 9855641 | Dec 1998 | WO |
| WO 9944423 | Sep 1999 | WO |
| WO 0003029 | Jan 2000 | WO |
| WO 0022137 | Apr 2000 | WO |
| WO 0042208 | Jul 2000 | WO |
| WO 0102431 | Jan 2001 | WO |
| WO 01004282 | Jan 2001 | WO |
| WO 0121217 | Mar 2001 | WO |
| WO 0123004 | Apr 2001 | WO |
| WO 0190392 | Nov 2001 | WO |
| WO 0198513 | Dec 2001 | WO |
| WO 0246372 | Jun 2002 | WO |
| WO 03064666 | Aug 2003 | WO |
| WO 03076605 | Sep 2003 | WO |
| WO 2003092579 | Nov 2003 | WO |
| WO 03104467 | Dec 2003 | WO |
| WO 2004018627 | Mar 2004 | WO |
| WO 2004031357 | Apr 2004 | WO |
| WO 2005001103 | Jan 2005 | WO |
| WO 2005023848 | Mar 2005 | WO |
| WO 2005030261 | Apr 2005 | WO |
| WO 2005065348 | Jul 2005 | WO |
| WO 2005075506 | Aug 2005 | WO |
| WO 2005107474 | Nov 2005 | WO |
| WO 2005113781 | Dec 2005 | WO |
| WO 2005117993 | Dec 2005 | WO |
| WO 2006086357 | Aug 2006 | WO |
| WO 2006119449 | Nov 2006 | WO |
| WO 2007124065 | Nov 2007 | WO |
| WO 2008095168 | Aug 2008 | WO |
| WO 2008150496 | Dec 2008 | WO |
| WO 2009065800 | May 2009 | WO |
| WO 2010024483 | Mar 2010 | WO |
| WO 2010037027 | Apr 2010 | WO |
| WO 2011133040 | Oct 2011 | WO |
| WO 2012003287 | Jan 2012 | WO |
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| Entry |
|---|
| Finke, et al. (2004) “Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles”, Journal of Virology, 78(22): 12333-43 (Year: 2004). |
| Minskaia, et al. (2013) “Protein Coexpression Using FMDV 2A: Effect of ‘Linker’ Residues”, BioMed Research International, vol. 2013, Article ID 291730, 12 pages. |
| Ono, et al. (2005) “Noninvasive Visualization of Adenovirus Replication with a Fluorescent Reporter in the E3 Region”, Cancer Research, 65(22): 10154-58. |
| Liu, et al. (2006) “Oncolytic adenoviral vector carrying the cytosine deaminase gene for melanoma gene therapy”, Cancer Gene Therapy, 13: 845-55. |
| Mohr (2005) “To replicate or not to replicate: achieving selective oncolytic virus replication in cancer cells through translational control”, Oncogene, 24: 7697-709. |
| Chopra (Dec. 9, 2007; Updated Jan. 2, 2008) “Recombinant Adenovirus with Enhanced Green Fluorescent Protein”, In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013), 5 pages as printed. |
| Funston et al., “Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping,”J Gen Virol 89:389-396, 2008. |
| Funston et al., “Expression of Heterologous Genes in Oncolytic Adenoviruses using Picornaviral 2A Sequences that Trigger Ribosome Skipping,” J. Gen. Virol., vol. 89:389-396, 2008. |
| Ketzer et al., “Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses,” Nucleic Acids Res 40(21):e167 (10 pages), 2012. |
| Kim et al., “High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice,” PLoS ONE, vol. 64:e18556, 2011. |
| Pelka et al., “Adenovirus E1A Directly Targets the E2F/DP-1 Complex,” J. Virol., vol. 85:8841-8851, 2011. |
| Szymczak et al., “Correction of Multi-Gene Deficiency in vivo using a Single ‘self-cleaving’ 2A Peptide-Based Retroviral Vector,” Nature Biotech., vol. 22:589-594, 2004. |
| Tan et al., “Coexpression of double or triple copies of the rabies virus glycoprotein gene using a ‘self-cleaving’ 2A peptide-based replication-defective human adenovirus serotype 5 vector,” Biologicals 38:586-593, 2010. |
| Alba et al., “Gutless adenovirus: last-generation adenovirus for gene therapy,” Gene Ther., vol. 12:S18-S27, 2005. |
| Alba et al., “Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer,” Blood, vol. 114(5): 965-971, 2009. |
| Alonso et al., “Combination of the oncolytic adenovirus ICOVIR-5 with chemotherapy provides enhanced anti-glioma effect in vivo,” Cancer Gene Ther., vol. 14:756-761,2007. |
| Barton, et al., “Second-Generation Replication-Competent Oncolytic Adenovirus Armed with Improved Suicide Genes and ADP Gene Demonstrates Greater Efficacy without Increased Toxicity”, Molecular Therapy, 2006, 13(2):347-356. |
| Batzer et al., “Enhanced evolutionary PCR using oligonucleotides with inosine at the 3′-terminus”, Nucleic Acid Research, 1991, 19(18):5081. |
| Bauerschmitz et al., “Tissue-Specific Promoters Active in CD44+CD24-1low Breast Cancer Cells,” Cancer Res., vol. 68(14):5533-5539, 2008. |
| Bayle et al., “Rapamycin Analogs with Differential Binding Specificity Permit Orthogonal Control of Protein Activity,” Chem Biol., vol. 13:99-107, 2006. |
| Behar et al., “Llama Single-Domain Antibodies Directed against Nonconventional Epitopes of Tumor-Associated Carcinoembryonic Antigen Absent from Nonspecific Cross-Reacting Antigen,” FEBS J., vol. 276:3881-3893, 2009. |
| Belousova et al., “Modulation of Adenovirus Vector Tropism via Incorporation of Polypeptide Ligands into the Fiber Protein,” J Virol., vol. 76(17):8621-8631, 2002. |
| Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66: 1-19. |
| Bett et al., “DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309,” Virus Res., vol. 39: 75-82, 1995. |
| Binkowski et al., “Ligand-Regulated Peptides: A General Approach for Modulating Protein-Peptide Interactions with Small Molecules,” Chem. Biol., vol. 12: 847-855, 2005. |
| Bradshaw et al., “Biodistribution and inflammatory profiles of novel pen ton and hexon double-mutant serotype 5 adenoviruses,” J Control Release 164(3): 394-402, 2012. |
| Bremnes et al., “The Role of Tumor Stroma in Cancer Progression and Prognosis,” J. Thorac. Oncol., vol. 6:209-217, 2011. |
| Card et al., “MicroRNA silencing improves the tumor specificity of adenoviral transgene expression,” Cancer Gene Ther., vol. 19: 451-459, 2012. |
| Chen et al., “Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue,” Proc Natl Acad Sci USA, 92:4947-4951, 1995. |
| Cheo et al., “Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones,” Genome Res., vol. 14:2111-2120, 2004. |
| Chong et al., “A System for Small-Molecule Control of Conditionally Replication-Competent Adenoviral Vectors,” Mol. Ther., vol. 5(2): 195-203, 2002. |
| Doronin et al., “Overexpression of ADP (E3-11.6K) Protein Increases Cell Lysis and Spread of Adenovirus,” Virology 305: 378-387, 2003. |
| Doronin et al., “Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein,” J. Virol., vol. 74:6147-6155, 2000. |
| Evans et al., “Relocalization of the Mrel1-Rad50-Nbs1 Complex by the Adenovirus E4 ORF3 Protein Is Required for Viral Replication”, Journal of Virology, 2005, 79(10):6207-6215. |
| Extended European Search Report dated Dec. 11, 2013 for European Application No. 11818698.0, 10 pages. |
| Extended European Search Report for European Application No. 13760821.2, dated Sep. 30, 2015. |
| Fang et al., “An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo,” Mol. Ther., vol. 15:1153-1159, 2007. |
| Frese et al., “Selective PDZ protein-dependent stimulation of phosphatidylinositol 3-kinase by the adenovirus E4-ORF1 oncoprotein,” Oncogene 22: 710-721, 2003. |
| Fuerer et al., “Adenoviruses with Tcf binding sites in multiple early promoters show enhanced selectivity for tumour cells with constitutive activation of the wnt signalling pathway,” Gene Ther 9:270-281, 2002. |
| Gall et al., “Construction and Characterization of Hexon-Chimeric Adenoviruses: Specification of Adenovirus Serotype,” J Virol 72(12): 10260-10264, 1998. |
| Gibson et al., “Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases,” Nature Meth., vol. 6:343-360, 2009. |
| Glasgow et al., “A Strategy for Adenovirus Vector Targeting with a Secreted Single Chain Antibody,” PLoS One, vol. 4:e8355, 2009. |
| Havenga et al., “Novel Replication-Incompetent Adenoviral B-group Vectors: High Vector Stability and Yield in PER.C6 Cells,” J. Gen. Viral., vol. 87:2135-2143, 2006. |
| Hawkins et al., “Gene delivery from the E3 region of replicating human adenovirus: evaluation of the E3B region,” Gene Therapy 8, 1142-1148, 2001. |
| Heise et al., “An Adenovirus E1A Mutant that Demonstrates Potent and Selective Systemic Anti-Tumoral Efficacy,” Nat Med. 6: 1134-1139, 2000. |
| Helin et al., “Heterodimerization of the Transcription Factors E2F-1 and DP-1 is required for Binding to the Adenovirus E4 (ORF6/7) Protein,” J. Virol., 68:5027-5035, 1994. |
| Henikoff et al., “Amino acid substitution matrices from protein blocks”, Proc. Natl. Acad. Sci. USA, 1992, 89:10915-10919. |
| Hernandez-Aya et al. “Targeting the Phosphatidylinositol 3-Kinase Signaling Pathway in Breast Cancer”, The Oncologist, 16, pp. 404-414, 2011. |
| Holm et al., “Multidrug-resistance Cancer Cells Facilitate El-independent Adenovirus Replication: Impact for Cancer Gene Therapy,” Cancer Res 64:322-328, 2004. |
| International Preliminary Report on Patentability and Written Opinion dated Feb. 19, 2013 for International Application No. PCT/US2011/048005, 5 pages. |
| International Search Report dated Mar. 23, 2012 for International Application No. PCT/US2011 /048005, 6 pages. |
| International Search Report and Written Opinion for PCT/US2019/026626, dated Jun. 24, 2019 (12 pages). |
| Javier, “Cell polarity proteins: common targets for tumorigenic human viruses,” Oncogene 27:7031-7046, 2008. |
| Johnson et al., “Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents,” Cancer Cell 1:325-337, 2012. |
| Kim, “Clinical research results with dl1520 (Onyx-015, a replication-selective adenovims for the treatment of cancer: what have we learned?”, Gene Therapy, 2001, 8(2):89-98. |
| Kovesdi et al., “Role of an Adenovirus E2 Promoter Binding Factor in E1A Mediated Coordinate Gene Control,” Proc Nat Acad Sci USA, vol. 84: 2180-2184, 1987. |
| Leicher et al., “Coexpression of the KCNA3B Gene Product with Kv1 .5 Leads to a Novel A-type Potassium Channel*”, The Journal of Biological Chemistry, 1998, 273(52):35095-35101. |
| Leppard et al., “Adenovirus type 5 E4 Orf3 protein targets promyelocytic leukaemia (PML) protein nuclear domains for disruption via a sequence in PML isoform II that is predicted as protein as a protein interaction site of bioinformatics analysis”, Journal of General Virology, 2009, 90(1):95-104. |
| Li et al., “Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC,” Nat Methods, vol. 4(3):251-256, 2007. |
| Lopez et al., “A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice,” Mol. Ther., vol. 20(12):2222-2233, 2012. |
| McCormick, “Cancer Gene Therapy: Fringe or Cutting Edge?,” Nature Rev. Cancer, vol. 1:130-141,2001. |
| Murakami et al., “Chimeric Adenoviral Vectors Incorporating a Fiber of Human Adenovirus 3 Efficiently Mediate Gene Transfer into Prostrate Cancer Cells,” The Prostate, vol. 70:362-376, 2009. |
| NCBI Accession No. CVl 10986, Jan. 11, 2011, 3 pages. |
| Nevels et al., “The Adenovirus E4orf6 can Promote E1A/E1B-induced Focus Formation by Interfering with p53 Tumor Suppressor Function,” Proc. Natl. Acad. Sci. USA, vol. 94:1206-1211, 1997. |
| Office Action and Search Report from China Application No. 2013 80014047.7, dated Aug. 5, 2015 (English translation). |
| O'Shea et al., “Adenoviral Proteins Mimic Nutrient/Growth Signals to Activate the mTOR Pathway for Viral Replication,” EMBO J., vol. 24:1211-1221, 2005. |
| O'Shea et al., “Adenovirus Overrides Cellular Checkpoints for Protein Translation,” Cell Cycle 4(7):883-888, 2005. |
| O'Shea et al., “DNA Tumor Viruses—the Spies who Lyse Us,” Curr. Opin. Genet. Dev., vol. 15:18-26, 2005. |
| O'Shea et al., “Viruses—seeking and destroying the tumor program,” Oncogene, vol. 24: 7640-7655, 2005. |
| Pearson et al., “Improved tools for biological sequence comparison”, Proc. Natl. Acad. Sci. USA, 1988, 85:2444-2448. |
| Rossolini et al., “Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information”, Mol. Cell. Probes, 1994, 8:91-98. |
| Roy et al., “Rescue of chimeric adenoviral vectors to expand the serotype repertoire,” J Virol Methods, vol. 14:41-21, 2007. |
| Shapiro et al., “Recombinant Adenoviral Vectors Can Induce Expression of p73 via the E4-orf6/7 Protein,” J Virol., vol. 80(11):5349-5360, 2006. |
| Shepard et al., “E4orfl3 Is Necessary for Enhanced S-Phase Replication of Cell Cycle-Restricted Subgroup C Adenoviruses,” J Virol 77(15):8593-8595, 2003. |
| Smith et al., “Comparison of Biosequences”, Advances in Applied Mathematics, 1981,2:482-489. |
| Soria et al., “Heterochromatin silencing of p53 target genes by a small viral protein”, Nature, 2010, 466(7310): 1076-1083. |
| Stanton et al. “Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function” Bio Techniques 45: 659-668 (Dec. 2008). |
| Ullman et al., “Adenovirus E4 ORF3 Protein Inhibits the Interferon-Mediated Antiviral Response”, Journal of Virology, 2007, 81(9):47 44-4752. |
| Verheije et al., “Retargeting of Viruses to Generate Oncolytic Agents,” Adv. Virol., vol. 2012:1-15,2012. |
| Volk et al., “Enhanced Adenovirus Infection of Melanoma Cells by Fiber-Modification,” Cancer Biol Ther., vol. 2(5): 511-515, 2003. |
| Waehler et al., “Engineering targeted viral vectors for gene therapy,” Nat Rev Genet., vol. 8(8):573-587, 2007. |
| Wang et al., “Identification of Specific Adenovirus E1A-N-Terminal Residues Critical to the Binding of Cellular Proteins and to the Control of Cell Growth,” J. Virol., vol. 67:476-488, 1993. |
| Warram et al., “A Genetic Strategy for Combined Screening and Localized Imaging of Breast Cancer,” Mol Imaging Biol., vol. 13:452-461, 2011. |
| Whyte et al., “Association between an Oncogene and an Anti-Oncogene: the Adenovirus ElA Proteins Bind to the Retinoblastoma Gene Product,” Nature, vol. 334:124-129, 1988. |
| Yaghoubi et al., “Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications,” Theranostics, vol. 2:374-391, 2012. |
| Yount et al., “Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model,” J. Virol., vol. 74: 10600- 10611, 2000. |
| Number | Date | Country | |
|---|---|---|---|
| 20180355374 A1 | Dec 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62298649 | Feb 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2017/019082 | Feb 2017 | US |
| Child | 16109513 | US |
