Tumor-targeting synthetic adenoviruses and uses thereof

Abstract

To provide tumor-targeting synthetic adenoviruses and uses thereof.SOLUTION: Synthetic adenoviruses with liver-detargeting mutations and expressing an adenovirus type 34 (Ad34) fiber protein, or a chimeric fiber protein with an Ad34 knob domain, are described. The synthetic adenoviruses traffic to sites of tumors. Use of the synthetic adenoviruses for delivering diagnostic or therapeutic transgenes to tumors are also described. Described herein is the finding that a liver-detargeted synthetic adenovirus expressing a fiber protein with an adenovirus type 34 (Ad34) knob domain is capable of homing to sites of tumors.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 433,140, ​​filed December 12, 2016, which is incorporated herein by reference in its entirety.

[0002] Field This disclosure relates to synthetic adenoviruses with chimeric fiber proteins and liver detargeting mutations that traffic to tumor sites. This disclosure further relates to the use of synthetic adenoviruses to express diagnostic or therapeutic transgenes in tumors. [Background technology]

[0003] background Adenovirus (Ad) is a natural multigene expression vehicle. Certain coding regions of the virus, such as the E1, E3 and E4 regions, are not required for replication in culture or can be complemented by available cell lines. Thus, each of these regions can be replaced with a non-viral gene to drive the expression of multiple transgenes from a single virus. There are 68 different human adenovirus serotypes, each with different characteristics. Ad5 has been the main Ad vector used in basic research, gene therapy and oncolytic virotherapy. However, Ad5 has a limited tropism and only infects epithelial cells that have the Coxsackie adenovirus receptor (CAR) receptor for viral uptake. Furthermore, when injected intravenously, Ad5 binds to blood factors that cause Ad5 to be sequestered in the liver, where Ad5 can potentially induce limited inflammation and toxicity. Thus, there remains a need for modified adenovirus vectors that can infect specific cell types after intravenous administration. Summary of the Invention [Means for solving the problem]

[0004] Abstract The discovery described herein is that liver-detargeted synthetic adenoviruses expressing fiber proteins with the Adenovirus type 34 (Ad34) knob domain can be directed to tumor sites. The synthetic adenoviruses can be used to deliver and express diagnostic or therapeutic transgenes in tumor cells, including tumor stromal cells.

[0005] Provided herein is a method for expressing transgene in tumor cells of a subject.In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising transgene, a native or modified capsid that detargets synthetic adenovirus from liver, and Ad34 fiber protein, or a chimeric fiber protein that comprises adenovirus type 5 (Ad5) shaft domain and Ad34 knob domain.The transgene can be, for example, a diagnostic transgene or a therapeutic transgene.

[0006] Also provided herein is a method of diagnosing a subject as having a tumor. In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising a diagnostic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein or a chimeric fiber protein that comprises an Ad5 shaft domain and an Ad34 knob domain. In some examples, the diagnostic transgene is a positron emission tomography (PET) reporter gene. In other examples, the diagnostic transgene encodes a fluorescent protein or an enzyme.

[0007] Further provided herein is a method for treating tumors in a subject.In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising a therapeutic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein or a chimeric fiber protein that comprises an Ad5 shaft domain and an Ad34 knob domain.In some examples, the therapeutic transgene encodes an anti-cancer drug or agent that destroys or kills tumor stromal cells.

[0008] A synthetic adenoviral genome having at least 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:5 is also provided by the present disclosure.

[0009] The above and other objects and features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0010] [Figure 1]AdSyn-CO176 is targeted to pancreatic tumors. (Figure 1A) Overview of the Cre-LoxP KrasG12D / p53 pancreatic tumor model. Mice designated "Kras;p53 / p53" encode the KrasG12D oncogene downstream of a sequence encoding LoxP-stop codon-LoxP. The stop codon blocks expression of KrasG12D in the absence of Cre recombinase. However, in the presence of Cre recombinase, the stop codon is removed, allowing expression of the KrasG12D oncogene. In these same mice, both alleles of the p53 gene are flanked by LoxP sites (LoxP-p53-LoxP). Mice designated "p53 / p53;Cre" have both alleles of the p53 gene flanked by LoxP sites (LoxP-p53-LoxP) and also express a Cre recombinase transgene driven by the pancreatic and duodenal homeobox 1 (Pdx1) promoter. Pdx1 is a gene specifically expressed in pancreatic cells, and therefore both copies of p53 are deleted in pancreatic cells. Crossing between strains produces offspring in which Cre driven by the Pdx1 promoter mediates deletion of both alleles of the tumor suppressor p53 and activation of the mutant KrasG12D in pancreatic cells. Homozygous mice designated "Kras;p53 / p53;Cre" develop pancreatic tumors in 5-7 weeks. (FIG. 1B) AdSyn-CO176, a synthetic virus with a chimeric fiber protein containing the Ad34 knob domain, was intravenously injected into Kras;p53 / p53 and Kras;p53 / p53;Cre mice. 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 minutes, and then scanned for 5 minutes using an IVIS™ imaging system. Kras;p53 / p53 mice had normal pancreas, and the luciferase signal was mainly from the spleen. Kras;p53 / p53;Cre mice had pancreatic tumors, and the signal was mainly from the pancreatic tumor.

[0011] [Diagram 2]AdSyn-CO176-infected pancreatic tumors after tail vein injection in a Cre-mediated genetically engineered heterozygous model. (Figure 2A) Kras;p53 / p53 mice are as described in Figure 1A. Mice designated "p53 / +;Cre" have one wild-type p53 allele and one p53 allele flanked by LoxP sites (LoxP-p53-LoxP). Crossing between these two strains produces offspring in which Cre recombinase driven by the Pdx1 promoter mediates the deletion of a single allele of the tumor suppressor p53 and activation of the mutant KrasG12D in pancreatic cells. These heterozygous mice, designated "Kras;p53 / +;Cre", develop tumors later in life (at 4-9 months of age) due to the fact that they have one wild-type allele of p53. This wild-type allele must be lost or mutated for pancreatic tumors to arise. (FIG. 2B) AdSyn-CO176 was intravenously injected into p53 / +;Cre and Kras;p53 / +;Cre mice (4 months old). 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 minutes, and then scanned for 1 minute using an IVIS™ imaging system. p53 / +;Cre mice had normal pancreases, and the signal was mainly from the spleen. 4-month-old Kras;p53 / +;Cre mice had pancreatic tumors, and the signal was mainly from the tumor and liver.

[0012] [Diagram 3]AdSyn-CO176 can infect and diagnose pancreatic tumors at an early stage after tail vein injection. Heterozygous Kras;p53 / +;Cre mice develop pancreatic tumors at 4-9 months. To test whether AdSyn-CO176 can infect pancreatic tumors at a very early stage of tumor development (before tumors are visible), AdSyn-176 was injected into 2-month-old Kras;p53 / +;Cre mice and luciferase expression was measured. (Figure 3A) AdSyn-CO176 was injected intravenously into 2-month-old Kras;p53 / +;Cre mice. 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 minutes, and scanned for 4 minutes using an IVIS™ imaging system. The pancreas of 2-month-old Kras;p53 / +;Cre mice appeared normal, but luciferase signals were found in this tissue. (Figure 3B) H&E staining showing typical histology of normal pancreas (a) and pancreatic tumor (b). (Figure 3C) H&E staining of pancreas from a 2-month-old Kras;p53 / +;Cre mouse showing that a small portion of the pancreas developed a tumor (shown in polygon). Most of the pancreas appeared normal. This result indicates that AdSyn-CO176 can infect pancreatic tumors at a very early stage.

[0013] [Figure 4] AdSyn-CO176 infects stromal cells in pancreatic tumors. In pancreatic tumors, only 10% of cells are cancer cells, while the remaining 90% are stromal cells. As determined by immunohistochemistry (IHC) and immunofluorescence (IF) staining, cells infected with AdSyn-CO176 are stromal cells. (Figure 4A) IHC staining of pancreatic tumors infected with AdSyn-CO176. CK19 is a marker for tumor cells, while smooth muscle actin (SMA) is a marker for stromal cells. The staining of GFP expressed from AdSyn-CO176 overlapped with SMA staining, indicating that AdSyn-CO176 targets stromal cells. (Figure 4B) IF staining of pancreatic tumors infected with AdSyn-CO176. GFP staining overlapped with SMA staining, confirming AdSyn-CO176 infection of stromal cells.

[0014] [Diagram 5]Glioblastoma infected with AdSyn-CO176 after tail vein injection. Synthetic adenovirus AdSyn-CO176 was injected into glioblastoma-bearing mice by tail vein, and luciferase signals were found in glioblastoma. (Figure 5A) Schematic diagram of Cre-mediated genetically engineered glioblastoma model. Lentivirus was directly injected into the brain of GFAP-Cre mice. Cre recombinase driven by glial fibrillary acidic protein (GFAP) promoter cleaves RFP from lentivirus-encoded gene and induces expression of HRasV12 and GFP mainly in astrocytes. Expression of lentivirus-encoded U6-p53 shRNA knocks down expression of p53 in brain cells that take up the virus. Expression of HRasV12 and knockdown of p53 induces tumor formation in the brain one week after injection. GFP signal is used to show the formation of glioblastoma. (Figure 5B) Saline, AdSyn-CO171, or AdSyn-CO176 were injected by intravenous (IV) administration into GFAP-Cre mice that had received tumor-inducing lentivirus 4 weeks earlier. 48 hours after virus injection, mice were scanned for 1 minute using an IVIS™ imaging system 5 minutes after intraperitoneal injection of luciferin. Luciferase signals were detected in mice infected with AdSyn-CO176 (arrows), whereas no signals were detected in mice treated with saline or injected with AdSyn-CO171. (Figure 5C) Wild-type mice (normal brain) and lentivirus-injected GFAP-Cre mice (bearing brain tumors) were injected with AdSyn-CO171 or AdSyn-CO176. Brain tissue was collected 72 hours after injection of synthetic adenovirus, incubated with luciferin for 5 minutes, and scanned for 5 minutes using an IVIS™ imaging system. Only GFAP-Cre mice injected with tumor-inducing lentivirus showed luciferase signals from AdSyn-CO176, demonstrating that AdSyn-CO176 migrates to brain tissue only when tumors are present (Figure 5D). Brain tissue was also scanned for GFP signals. The GFP signal is used to identify glioblastomas.Both GFAP-Cre mice that received the tumor-inducing lentivirus had GFP signals in the brain, whereas mice that did not receive lentivirus had no detectable GFP. The GFP signal completely overlapped with the luciferase signal in GFAP-Cre mice that received the tumor-inducing lentivirus and AdSyn-CO176.

[0015] [Figure 6] AdSyn-CO176 migration to glioblastomas is driven by tumors, not damage. Injection of tumor-inducing lentivirus leads to transient brain damage at the injection site. Although synthetic adenoviruses (AdSyn-CO171 and AdSyn-CO176) were injected 4 weeks after the first injection of lentivirus, it was still unclear whether AdSyn-CO176 migration to glioblastomas was driven by tumor or damage at the injection site. To answer this question, GFAP-Cre mice were injected with synthetic adenovirus 4 weeks after either no injection, sham injection, or tumor-inducing lentivirus injection. (Figure 6A) GFAP-Cre mice were injected with either Hank's balanced salt solution (HBSS) or tumor-inducing lentivirus. Four weeks later, AdSyn-CO171 was injected intravenously. There was no AdSyn-CO171-derived luciferase signal in the brain in either group of mice. (Figure 6B) GFAP-Cre mice were injected with HBSS or tumor-inducing lentivirus, or GFAP-Cre mice were not injected. Four weeks later, the mice were intravenously injected with AdSyn-CO176. Luciferase signals were detected only in the brains of mice that received tumor-inducing lentivirus, whereas mice that received HBSS or no injection did not produce a signal. These results demonstrate that the specificity of AdSyn-CO176 is driven by tumors but not by damage at the injection site.

[0016] [Figure 7]AdSyn-CO176 can migrate into human glioblastoma xenograft tumors. (Figure 7A) Schematic of human glioblastoma xenograft model. Human glioblastoma U87 cells expressing tdTomato fluorescent protein (U87-tdTomato) were intracranially injected into NOD scid gamma (NSG) mice to give rise to glioblastoma tumors. (Figure 7B) Four weeks after NSG mice received intracranial injections of U87-tdTomato, AdSyn-CO171 and AdSyn-CO176 were intravenously injected into NSG mice by tail vein injection. 48 hours after virus injection, the tissues shown in the panels were collected and incubated with luciferin for 5 minutes, then scanned for 1 minute using an IVIS™ imaging system. Only mice injected with AdSyn-CO176 showed luciferase signals in the brain, which completely overlapped with tdTomato expression.

[0017] [Figure 8]Administration of synthetic adenovirus carrying a therapeutic transgene. (Figure 8A) Schematic diagram of the KPCL (KrasG12D; p53 knockout; Pdx1-Cre; firefly luciferase) mouse model. KPCL mice specifically express KrasG12D in the pancreas and are similar to homozygous "Kras; p53 / p53; Cre" mice, which have the p53 gene knocked out only in the pancreas. However, KPCL mice also specifically express firefly luciferase in the pancreas. Tumor development in KPCL mice is also similar to that for "Kras; p53 / p53; Cre" mice. (Figure 8B) Table showing the mean survival time of KPCL mice (at least 4 mice per treatment group) for each treatment. AdSyn-CO987 is a synthetic adenovirus based on AdSyn-CO176. A herpes simplex virus-1 thymidine kinase (TK) / ganciclovir (GCV) suicide gene was cloned into AdSyn-CO176, replacing the firefly luciferase / GFP gene. Renilla luciferase was also inserted immediately after TK in the genome of AdSyn-CO176. A control virus, AdSyn-CO989, was generated by cloning TK-P2A-Renilla luciferase into AdSyn-CO171 to replace the original firefly luciferase / GFP gene. KPCL mice, 5–6 weeks of age, were injected intravenously via the tail vein with 1 × 106 plaque-forming units (PFU) of the indicated viruses. Two days later, mice were injected intraperitoneally (ip) or intravenously (iv) with GCV. Three control groups were used: AdSyn-CO989+GCV; AdSyn-CO987 followed by saline injection (AdSyn-CO987+saline); and GCV injection only (ip or iv). Treatment with AdSyn-CO987+GCV extended the survival time of mice compared to controls. (Figure 8C) Representative mouse images showing firefly luciferase signal. Firefly luciferase signal (expressed by tumors) was analyzed during treatment to monitor tumor growth. Treatment for mouse Z619R was AdSyn-CO987+saline, which served as a control. Mice Z601R and Z607R were treated with AdSyn-CO987+GCV (ip).The intensity of the firefly luciferase signal increased in the control mouse Z619R (indicating an increase in tumor size), whereas the signal decreased in mice Z601R and Z607R (indicating a decrease in tumor size).

[0018] [Figure 9] Histology of tumors in mice treated with AdSyn-CO987. (Figure 9A) Images of H&E staining of pancreatic tumors. Mice Z655, 1806, Z619 and Z621 were all control mice. Mouse Z655 was treated with GCV only by ip injection; mouse 1806 was treated with GCV only by iv injection; mouse Z619 was treated with AdSyn-CO987+saline; and mouse Z621 was untreated. Mouse Z656 was treated with AdSyn-CO987+GCV iv. Compared to the control, tumors from Z656 had larger areas of necrosis (indicated by arrowheads). (Figure 9B) Representative areas of necrosis in the enlarged tumor of Z656. Areas are also shown in Figure 9A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Sequence Listing (Sequence Listing) The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the presented strand. The sequence listing has been submitted as a 215KB ASCII text file created on November 30, 2017, and is incorporated herein by reference. In the attached sequence listing:

[0020] SEQ ID NO:1 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO171.

[0021] SEQ ID NO:2 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO176.

[0022] SEQ ID NO:3 is the amino acid sequence of the Ad5 hexon.

[0023] SEQ ID NO:4 is the amino acid sequence of Ad5 hexon E451Q.

[0024] SEQ ID NO:5 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO987.

[0025] SEQ ID NO:6 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO989. Detailed Description

[0026] I. Abbreviations [Table A] II. Terms and Methods

[0027] II. Terms and Methods Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V (ISBN 0-19-854287-9), published by Oxford University Press in 1994; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (ISBN 0-632-02182-9), published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1-56081-569-8), published by VCH Publishers, Inc. in 1995. In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0028] Adenovirus: a non-enveloped virus with a linear double-stranded DNA genome and an icosahedral capsid. Currently, 68 serotypes of human adenovirus are known, classified into seven species (species A, B, C, D, E, F, and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (mainly species B and C), conjunctivitis (species B and D), and / or gastroenteritis (species F and G).

[0029] Administration: Providing or giving to a subject an agent, such as a therapeutic agent (e.g., a recombinant virus), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0030] Chimeric: composed of at least two parts of different origin. In the context of this disclosure, a "chimeric adenovirus" is an adenovirus having genetic material and / or proteins from at least two different serotypes (e.g., from Ad5 and an adenovirus of a second serotype). In this context, a "capsid-swapped" adenovirus refers to a chimeric adenovirus in which the capsid proteins are from one serotype of adenovirus and the remaining proteins are from another adenovirus serotype. Similarly, a "chimeric fiber" is a fiber protein having amino acid sequences from at least two different serotypes of adenovirus. For example, a chimeric fiber can be composed of a fiber shaft from Ad5 and a fiber knob from an adenovirus of a second serotype.

[0031] Contacting: To bring into direct physical association and includes both solid and liquid forms.

[0032] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a peptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0033] Detargeted: In the context of this disclosure, a "detargeted" adenovirus is a recombinant or synthetic adenovirus that contains one or more modifications that change the tropism of the virus so that it no longer infects or no longer substantially infects a particular cell or tissue type. In some embodiments, the recombinant or synthetic adenovirus contains a capsid mutation, such as a mutation in the hexon protein (e.g., E451Q). In some embodiments, the recombinant or synthetic adenovirus contains the original capsid from an adenovirus that does not naturally infect or does not substantially infect a particular cell or tissue type. In some embodiments herein, the recombinant or synthetic adenovirus is liver detargeted and / or spleen detargeted.

[0034] E1A: adenovirus early region 1A (E1A) gene and the polypeptide expressed from this gene. E1A protein plays a role in viral genome replication by making cells enter the cell cycle. As used herein, the term "E1A protein" refers to the protein expressed from the E1A gene, and includes the E1A protein produced by any adenovirus serotype.

[0035] Fiber: The adenovirus fiber protein is a trimeric protein that mediates binding to cell surface receptors. The fiber protein consists of a long N-terminal shaft and a globular C-terminal knob.

[0036] Fusion protein: a protein that contains amino acid sequences derived from at least two different (heterologous) proteins or peptides. Fusion proteins can be produced, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences that code for 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 must not contain internal stop codons. Fusion proteins, especially short fusion proteins, can also be produced by chemical synthesis.

[0037] Glioblastoma: A fast-growing central nervous system tumor that forms from glial tissue in the brain and spinal cord. Glioblastoma usually occurs in adults and affects the brain more frequently than the spinal cord. Glioblastoma is the most common and most aggressive cancer that begins in the brain. Glioblastoma is also known as glioblastoma multiforme (GBM) and grade IV astrocytoma.

[0038] Heterologous: A heterologous protein or gene refers to a protein or gene derived from a different source or species.

[0039] Hexon: The major adenovirus capsid protein. An exemplary hexon sequence from Ad5 is set forth herein as SEQ ID NO: 3. A mutant hexon sequence containing an E451Q substitution is set forth herein as SEQ ID NO: 4.

[0040] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from other biological components, such as other chromosomal and extrachromosomal DNA and RNA, proteins, and cells in the cells or tissues of an organism or the organism itself in which it naturally occurs. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0041] MicroRNA (miRNA or miR): A single-stranded RNA molecule that regulates gene expression in plants, animals, and viruses. Genes encoding microRNAs are transcribed to form primary transcripts, microRNAs (pri-miRNAs), which are processed to form short stem-loop molecules called precursor microRNAs (pre-miRNAs), which are subsequently cleaved to form mature microRNAs. Mature microRNAs are approximately 21-23 nucleotides long and are partially complementary to the 3'UTR of one or more target messenger RNAs (mRNAs). MicroRNAs modulate gene expression by promoting cleavage of target mRNAs or by blocking translation of cellular transcripts. In the context of this disclosure, a "liver-specific microRNA" is a microRNA that is preferentially expressed in the liver, such as a microRNA that is expressed only in the liver or a microRNA that is more prominently expressed in the liver compared to other organs or tissue types. In some embodiments, the microRNA is miR-122. In the context of the present disclosure, a "spleen-specific microRNA" is a microRNA that is preferentially expressed in the spleen, such as a microRNA that is expressed only in the spleen or a microRNA that is more prominently expressed in the spleen compared to other organs or tissue types. In some embodiments, the microRNA is miR-142-3p.

[0042] Modification: A change in the sequence of a nucleic acid or a protein sequence. For example, modifications of an amino acid sequence include, for example, substitution, insertion, and deletion, or a combination thereof. Insertions include amino-terminal and / or carboxyl-terminal fusions, and insertions of single or multiple amino acid residues into a sequence. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some embodiments herein, modifications (such as substitutions, insertions, or deletions) result in a change in function, such as a reduction or enhancement of a specific activity of a protein. As used herein, "Δ" or "delta" refers to a deletion. Substitution modifications are those in which at least one residue is removed and a different residue is inserted in its place. Amino acid substitutions are typically of a single residue, but may occur simultaneously at several different positions. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final mutant sequence. These modifications can be prepared by modification of nucleotides in the DNA encoding the protein, thereby producing a DNA encoding this modification. Techniques for making insertion, deletion, and substitution mutations at predetermined sites in DNA having a known sequence are well known in the art. A "modified" protein, nucleic acid, or virus is one that has one or more of the modifications outlined above.

[0043] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0044] Pancreatic cancer: Cancer that begins in the tissues of the pancreas. Pancreatic cancer typically spreads rapidly and is rarely detected at an early stage, resulting in a poor prognosis for most diagnosed patients. The most common type of pancreatic cancer is pancreatic adenocarcinoma, which accounts for approximately 85% of pancreatic cancer cases.

[0045] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules, or agents (e.g., the synthetic viruses disclosed herein).

[0046] Generally, the nature of the carrier will depend on the specific mode of administration used.For example, parenteral formulations usually contain injectable fluids, which contain pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicles.For solid compositions (e.g., in the form of powder, pill, tablet, or capsule), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0047] Polypeptide, peptide, or protein: A polymer in which the monomers are amino acid residues that 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 residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0048] A conservative substitution in a polypeptide is the substitution of one amino acid residue in a protein sequence for a different amino acid residue that has similar biological properties. Typically, a conservative substitution has little or no effect on the activity of the resulting polypeptide. For example, a protein or peptide that contains one or more conservative substitutions (e.g., not more than one, not more than two, not more than three, not more than four, or not more than five substitutions) retains the structure and function of the wild-type protein or peptide. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes the polypeptide, for example, using standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be easily selected by testing the cross-reactivity of antibodies or the ability of the antibodies to induce an immune response. Examples of conservative substitutions are shown below. [Table B]

[0049] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone, e.g., as a sheet or helix conformation, in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0050] In general, the substitutions predicted to result in the greatest changes to protein properties will be non-conservative, e.g., (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or is substituted by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted for (or is substituted by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or is substituted by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or is substituted by) one having no side chain, e.g., glycine.

[0051] Positron Emission Tomography (PET): an imaging technique used to observe metabolic processes in the body. PET detects pairs of gamma rays emitted indirectly by positron-emitting radionuclides, which are introduced into the body on biologically active molecules. PET reporter genes code for molecules (such as receptors or enzymes) that provide targets for the PET probes, which can then be detected by imaging. PET reporter genes are generally classified into three different groups: (1) reporter genes that code for enzymes that phosphorylate specific PET reporter probes, leading to their intracellular capture; (2) reporter genes that code for protein receptors that can be bound by specific PET reporter probes; and (3) reporter genes that code for protein transporters that transport radionuclide reporter probes into cells expressing the reporter gene (Yaghoubi et al., Theranostics 2(4):374-391, 2012). As used herein, "PET reporter gene" includes any gene that encodes a protein that can interact with a PET reporter probe in a manner that allows the probe to be detected by molecular imaging.Exemplary PET reporter genes include, but are not limited to, herpes simplex virus (HSV) thymidine kinase (TK) and its mutant forms, varicella zoster virus (VSV) TK, human mitochondrial TK and its mutants, mutants of deoxycytidine kinase, dopamine 2 receptor mutants, human estrogen receptor alpha ligand binding domain (hERL), human somatostain receptor subtype 2 (hSSTr2), recombinant human carcinoembryonic antigen (CEA), engineered antibody fragments, humanized membrane-anchored anti-polyethylene glycol (PEG), sodium iodide symporter (NIS), and human norepinephrine transporter (hNET) (see Yaghoubi et al. (2012) for an overview of PET reporter genes and corresponding reporter probes).

[0052] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that alleviates the signs or symptoms of a disease or condition after its development has begun. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0053] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter comprises the necessary nucleic acid sequences near the start site of transcription. Typically, a promoter is located near the gene to be transcribed. A promoter also optionally comprises distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the transcription start site. A "constitutive promoter" is a promoter that is continuously active and is not subject to control by external signals or molecules. In contrast, the activity of an "inducible promoter" is controlled by external signals or molecules (e.g., transcription factors or tetracycline). A "tissue-specific promoter" is a promoter that is substantially active only in a specific tissue or tissues.

[0054] Protein IX (pIX): A minor component of the adenovirus capsid that associates with the hexon protein.

[0055] Purified: The term "purified" is intended as a relative term, without requiring absolute purity. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparations and has been subjected to fractionation to remove various components of the initial preparation, e.g., proteins, cell debris, and other components.

[0056] Recombinant: A recombinant nucleic acid molecule, protein, or virus is one that has a sequence that does not occur in nature or that is created by the artificial combination of two segments of otherwise distinct sequences. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of segments of isolated nucleic acid molecules, e.g., by genetic engineering techniques. The term "recombinant" also includes nucleic acids, proteins, and viruses that have been altered only by the addition, substitution, or deletion of portions of the naturally occurring nucleic acid molecule, protein, or virus.

[0057] Sequence identity: The identity or similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed as the identity or similarity between the sequences. Sequence identity can be measured as a percentage of identity, the higher the percentage, the more identical the sequences. Sequence similarity can be measured as a percentage of similarity (taking into account conservative amino acid substitutions), the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more pronounced when orthologous proteins or cDNAs are from more closely related species (e.g., human and mouse sequences) compared to more distantly related species (e.g., human and C.Elegans sequences).

[0058] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., vol. 2, p. 482, 1981; Needleman and Wunsch, J. Mol. Biol., vol. 48, p. 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444, 1988; Higgins and Sharp, Gene, 73, 237-44, 1988; Higgins and Sharp, CABIOS, 5, 151-3, 1989; Corpet et al., Nuc. Acids Res., 16, 10881-90, 1988; Huang et al., Computer Appls. in the Biosciences, 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio., 24, 307-31, 1994. Altschul et al., J. Mol. Biol., 215, 403-10, 1990, present a detailed discussion of sequence alignment methods and homology calculations.

[0059] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol., vol. 215, pp. 403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI website.

[0060] Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.

[0061] Stroma: Epithelial tissue or tumor support tissue consisting of connective tissue and blood vessels. Stromal cells are cells that make up the stroma, mainly fibroblasts and surrounding cells. Tumor stroma is mainly composed of fibroblasts, extracellular matrix, immune cells, vasculature and basement membrane (Bremnes et al., J Thorac Oncol 6:209-217, 2011). Tumor stromal cells are known to play an important role in cancer growth and progression.

[0062] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

[0063] Synthetic: Produced by artificial means in a laboratory, for example, a synthetic nucleic acid or protein can be chemically synthesized in a laboratory.

[0064] Therapeutic Agent: A chemical compound, such as an antisense compound, an antibody, a peptide or nucleic acid molecule, a small molecule, a recombinant virus or other composition that is capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

[0065] Therapeutically effective amount: A quantity of a particular pharmaceutical or therapeutic agent (e.g., a recombinant virus) that is sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of the agent may depend on several factors, including, but not limited to, the subject or cell being treated, as well as the mode of administration of the therapeutic composition.

[0066] Transgene: A gene inserted into the genome of a different organism (such as a virus). A transgene can also be referred to as a heterologous gene. As used herein, a "diagnostic transgene" refers to any transgene that encodes a detectable product, such as, but not limited to, a fluorescent protein, an enzyme, or a PET reporter. As used herein, a "therapeutic transgene" refers to any transgene that encodes a product that has therapeutic application. In the context of this disclosure, a therapeutic transgene can be, for example, an anti-cancer drug or drug that destroys or kills cells in the tumor stroma.

[0067] U exon: An open reading frame located in the I strand (leftward transcription) between the early E3 region and the fiber gene (Tollefson et al., J Virol, vol. 81 (23), pp. 12918-12926).

[0068] Vector: A nucleic acid molecule that allows the insertion of a foreign nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector can contain a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences that allow the transcription and translation of the inserted gene(s). III. Overview of Some Embodiments

[0069] It is disclosed herein that liver-detargeted synthetic adenovirus expressing fiber protein with Ad34 knob domain can be directed to tumor site.Synthetic adenovirus can be used to deliver and express diagnostic or therapeutic transgenes in tumor cells, including tumor stromal cells, for example.

[0070] Provided herein is a method for expressing a transgene in tumor cells of a subject.In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising a transgene, a native or modified capsid that detargets the synthetic adenovirus from liver, and an Ad34 fiber protein, or a chimeric fiber protein that comprises Ad5 shaft domain and Ad34 knob domain.

[0071] In some embodiments, the transgene is a diagnostic transgene. In some examples, the diagnostic transgene encodes a fluorescent protein, such as, but not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), or orange fluorescent protein (e.g., mOrange). In other examples, the diagnostic transgene encodes an enzyme, such as luciferase. In yet other examples, the diagnostic transgene comprises a PET reporter gene.

[0072] In other embodiments, the transgene is a therapeutic transgene. In some embodiments, the therapeutic transgene encodes an anti-cancer drug. In specific embodiments, the anti-cancer drug is a pro-inflammatory molecule or cytokine, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40 ligand (CD40L), Fms-related tyrosine kinase 3 (FLT3) ligand, interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-12, tumor necrosis factor (TNF)-α, interferon, chemokine, B7-1, intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-3, transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF). In other specific embodiments, the anti-cancer drug is an anti-angiogenic factor, such as an inhibitor of vascular endothelial growth factor (VEGF). In other specific embodiments, the anti-cancer drug is an inhibitor of KRas, such as an siRNA or shRNA inhibitor. In other specific embodiments, the anti-cancer agent is an inhibitor of cytotoxic T-lymphocyte-associated molecule (CTLA)-4, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), carcinoembryonic antigen (CEA), or mucin 1 (MUC1). In some embodiments, the therapeutic transgene encodes an agent that destroys or kills tumor stromal cells. In specific embodiments, the antigen is Rexin-G, herpes simplex virus (HSV) thymidine kinase (TK), p53, TNF-α, Fas / FasL, or diphtheria toxin A.

[0073] Also provided herein is a method of diagnosing a subject as having a tumor. In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising a diagnostic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein or a chimeric fiber protein comprising an Ad5 shaft domain and an Ad34 knob domain.

[0074] In some embodiments, the diagnostic transgene is a PET reporter gene. In some examples, the PET reporter gene is a viral or human thymidine kinase (or a mutant form thereof), a mutant of deoxycytidine kinase, a mutant dopamine 2 receptor, human estrogen receptor alpha ligand binding domain (hERL), human somatostatin receptor subtype 2 (hSSTr2), recombinant human CEA, an engineered antibody fragment, a humanized membrane-anchored anti-polyethylene glycol (PEG), a sodium iodide symporter (NIS), or a human norepinephrine transporter (hNET).

[0075] In other embodiments, the diagnostic transgene encodes a fluorescent protein, in some examples, the fluorescent protein comprises GFP, YFP, CFP, RFP, BFP, or orange fluorescent protein.

[0076] In other embodiments, the diagnostic transgene encodes an enzyme. In one example, the enzyme is luciferase.

[0077] Further provided herein is a method for treating tumors in a subject.In some embodiments, the method comprises administering to a subject a synthetic adenovirus comprising a therapeutic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein, or a chimeric fiber protein that comprises an Ad5 shaft domain and an Ad34 knob domain.

[0078] In some embodiments, the therapeutic transgene encodes an anti-cancer drug. In some embodiments, the anti-cancer drug is a pro-inflammatory molecule or cytokine, such as GM-CSF, CD40L, FLT3, IL-1b, IL-2, IL-4, IL-6, IL-12, TNF-α, interferon, chemokine, B7-1, ICAM-1, LFA-3, TGF-β, PDGF, or EGF. In other embodiments, the anti-cancer drug is an anti-angiogenic factor, such as an inhibitor of VEGF. In other embodiments, the anti-cancer drug is an inhibitor of KRas (such as an siRNA or shRNA inhibitor). In other embodiments, the anti-cancer drug is an inhibitor of CTLA-4, PD-1, CEA, or MUC1. In other embodiments, the therapeutic transgene encodes an agent that destroys or kills tumor stromal cells. In some embodiments, the agent is Rexin-G, HSV-TK, p53, TNF-α, Fas / FasL, or diphtheria toxin A.

[0079] In some embodiments of the methods disclosed herein, the synthetic adenovirus comprises a modified capsid that detargets the virus from the liver. In some examples, the synthetic adenovirus comprises a modified hexon protein, such as the E451Q mutation (shown herein as SEQ ID NO: 4). In other embodiments, the synthetic adenovirus has a native (unmodified) capsid (e.g., a capsid from an adenovirus serotype that does not naturally infect the liver) that detargets the synthetic adenovirus from the liver.

[0080] In some embodiments of the method disclosed herein, the synthetic adenovirus further comprises one or more binding sites, for example, two or three binding sites, for liver-specific microRNA. In some embodiments, the liver-specific microRNA is miR-122. In some embodiments, the one or more binding sites are in the 3'UTR of the transgene.

[0081] In some embodiments of the method disclosed herein, the synthetic adenovirus further comprises one or more binding sites, for example, two or three binding sites, for spleen-specific microRNA.In some embodiments, the spleen-specific microRNA is miR142-3p.In some embodiments, the one or more binding sites are in the 3'UTR of the transgene.

[0082] In some embodiments of the methods disclosed herein, the transgene is regulated by a tissue-specific promoter, such as a promoter active in the pancreas or cells of the central nervous system, hi other embodiments, the transgene is regulated by a tumor-specific promoter.

[0083] In some embodiments of the methods disclosed herein, the synthetic adenovirus is generated from an Ad5 vector genome, hi some examples, the synthetic adenovirus comprises an Ad5 capsid protein and a chimeric fiber protein comprising an Ad5 shaft domain and an Ad34 knob domain.

[0084] In some embodiments of the methods disclosed herein, the tumor is a pancreatic tumor. In other embodiments, the tumor is a glioblastoma. In other embodiments, the tumor is a breast cancer, prostate cancer, gastrointestinal cancer, bone cancer, or melanoma tumor.

[0085] In some embodiments of the methods disclosed herein, the tumor is characterized by a loss of p53 tumor suppressor activity. In some examples, the tumor exhibits a mutation in p53. In some examples, the tumor exhibits a loss of the wild-type p53 allele.

[0086] In some embodiments of the methods disclosed herein, the tumor is characterized by a mutation in a Ras gene, such as KRas, HRas or NRas.In some embodiments of the methods disclosed herein, the tumor is characterized by an alteration or mutation in neurofibromatosis type 1 (NF1), epidermal growth factor receptor (EGFR), BRCA1, BRCA2 or HER2.

[0087] In some embodiments of the methods disclosed herein, the genome of the synthetic adenovirus comprises a nucleotide sequence that 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 SEQ ID NO: 2. In some examples, the genome of the synthetic adenovirus comprises or consists of the nucleotide sequence of SEQ ID NO:2.

[0088] In other embodiments of the methods disclosed herein, the genome of the synthetic adenovirus comprises a nucleotide sequence that 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 SEQ ID NO: 5. In some examples, the genome of the synthetic adenovirus comprises or consists of the nucleotide sequence of SEQ ID NO:5.

[0089] Further provided herein is a synthetic adenoviral genome having 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% identity to SEQ ID NO:2 or SEQ ID NO:5. In some embodiments, the synthetic adenoviral genome comprises or consists of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:5. IV. Synthetic Adenoviruses

[0090] Adsembly, AdSLICr and RapAD technologies allow modular design and production of adenoviruses with unique capabilities (see PCT Publication Nos. WO2012 / 024351 and WO2013 / 138505, which are incorporated herein by reference in their entirety). The ability to design custom viruses with novel functions and characteristics opens up the possibility of expanding the utility of adenoviruses as vehicles for delivering therapeutic proteins by prompting the host to produce proteins in situ. This provides a unique ability to use human proteins that are difficult to manufacture for therapeutic purposes, allowing flexible delivery of most proteins to diseased tissues.

[0091] The specific modifications disclosed herein are described with reference to the genome sequence of Adenovirus 5 (Ad5), but may be used with any adenovirus serotype. Adenovirus is a natural multigene expression vehicle. E1, E3, and E4 regions are not required for replication in culture or can be complemented in available cell lines. Each of these regions has an independent promoter element that can be replaced with a cellular promoter when necessary to drive the expression of multigene products by alternative splicing.

[0092] As disclosed herein, to generate Ad5 expression vectors for in vivo use and transgene delivery, the E1A / E1B genes were deleted and replaced with at least one transgene, in some embodiments, the transgene is an EF1α-driven luciferase-GFP fusion.

[0093] The synthetic adenovirus disclosed herein may further comprise modifications that detarget the virus from the liver.Ad5 hexon can bind to factor X in blood, which may lead to its absorption by the Kuppfer cells of the liver, preventing systemic dissemination and limiting inflammation.To overcome this, as described further below, the synthetic adenovirus is engineered to comprise additional genomic modifications in E1 and core modules that prevent adenovirus uptake and transgene expression in the liver. A. Ad34 Fiber and Chimeric Fiber Proteins for Retargeting

[0094] The fiber protein of Ad5 and many other serotypes has been shown to bind the Coxsackie Adenovirus Receptor (CAR) for cell attachment, while other serotypes have been shown to use CD46 (Gaggar et al., Nat Med 9:1408-1412, 2003), desmoglein 2 (Wang et al., Nat Med 17:96-104, 2011), sialic acid (Nilsson et al., Nat Med 17:105-109, 2011), or others (Arnberg, Trends Pharmacol Sci 33:442-448, 2012). Receptor utilization of many serotypes has not been thoroughly investigated, and CD46 is not thought to be expressed in adult mice. Since the globular knob at the C-terminus of the fiber protein is typically responsible for receptor binding, chimeras were created by replacing the Ad5 fiber knob with that from Ad34 (see Example 1 below). The synthetic virus contained an E1 module containing an E1A / E1B deletion and a luciferase-GFP fusion driven by the EF1α promoter. The synthetic adenovirus also contained a liver-detargeting modification in the hexon protein (E451Q) and a binding site in the 3'UTR of the transgene for a microRNA (miR-122) specifically expressed in the liver to prevent off-target expression of the transgene.

[0095] The data disclosed herein demonstrate the ability to combine modified moieties from other serotypes to improve Ad5-based vectors, allowing rapid assembly of viruses optimized for tumor cell entry. B. Liver Detargeting Modifications

[0096] The natural Ad5 vector only infects the lungs (by inhalation) or liver (by intravenous administration). The Ad5 hexon binds to factor X in the blood, which leads to its uptake by Kuppfer cells in the liver, preventing systemic dissemination and inducing virus-limiting inflammation. To overcome this and allow intravenous delivery of a virus that can travel systemically to tumor sites, a synthetic adenovirus was engineered to contain additional genomic modifications in the E1 and core modules that prevent uptake and expression in the liver.

[0097] In order to prevent virus uptake and sequestration in liver by Ad5 hexon binding to factor X, the virus is engineered with an additional mutation in hexon (E451Q) that prevents liver uptake.For example, AdSyn-CO171 does not accumulate in liver, but can instead target other organs such as spleen and lymph nodes.Therefore, in some embodiments herein, synthetic adenovirus comprises modified hexon protein with E451Q substitution.

[0098] To prevent off-target transgene expression in the liver, the virus was engineered to contain a binding site in the 3' untranslated region (UTR) of the transgene for a microRNA that is specifically expressed in the liver.In certain embodiments, miR122 was selected as a liver-specific microRNA, as its expression and binding site are conserved in both human and mouse liver cells.In some examples, two microRNA binding sites for liver-specific miR122 were inserted into the 3' UTR of the transgene to prevent any residual transgene expression in the liver.

[0099] It is disclosed herein that synthetic adenovirus with miR-122 binding site and hexon mutation does not accumulate in liver, but can target tumor instead.In some embodiments, one or more binding sites for liver-specific microRNA are located in the 3'UTR of transgene.In some examples, liver-specific microRNA is miR-122, miR-30 or miR192.

[0100] Other mutations to the adenovirus hexon gene are contemplated herein to prevent adenovirus accumulation in the liver.For example, synthetic adenoviruses can be detargeted from the liver by replacing the nine hypervariable regions of hexon with those from different serotypes.

[0101] In some examples, the recombinant adenovirus comprises a hexon protein that comprises or consists of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. C. Capsid exchange to avoid neutralizing antibodies

[0102] The majority of the human population already has antibodies that recognize Ad5, which is the serotype most frequently used in research and therapeutic applications. Moreover, when a certain adenovirus serotype is used in patients, new antibodies that recognize the viral capsid will be generated, making repeated administration of the same vector problematic. Therefore, the present disclosure further contemplates exploiting the modularity of natural adenoviruses to create chimeric viruses that can avoid existing neutralizing antibodies. For example, the recombinant adenoviruses disclosed herein may further have a complete "capsid" module exchange (almost 60% of the genome), making them "invisible" to pre-existing antibodies and allowing repeated inoculation.

[0103] In some embodiments, the E1, E3 and E4 regions of the genome are derived from a first adenovirus serotype, and the E2B, L1, L2, L3, E2A and L4 regions of the genome are derived from a second adenovirus serotype, such as Ad34. In some examples, the E1 region of the first adenovirus serotype is modified to encode a pIX protein from the second adenovirus serotype; and / or the E3 region of the first adenovirus serotype is modified to encode a Uexon and fiber protein from the second adenovirus serotype. In certain examples, the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad34. D. Expression of Transgenes for Diagnostic and Therapeutic Applications

[0104] It is disclosed herein that recombinant adenoviruses comprising chimeric fiber proteins with Ad34 knob domains and liver detargeting mutations can target tumors. It is further disclosed that recombinant adenoviruses can express transgenes in tumor tissues, such as tumor stromal cells. In one embodiment, the transgene is a reporter, such as a luciferase-GFP reporter, that allows detection of virus-transduced cells. In another embodiment, the transgene is a therapeutic transgene, such as an anti-cancer molecule. The present disclosure provides synthetic adenoviruses comprising diagnostic or therapeutic transgenes for tumor diagnosis and treatment.

[0105] Provided herein is a method for diagnosing a subject having a tumor by administering to the subject a synthetic adenovirus that comprises a diagnostic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein or a chimeric fiber protein that comprises an Ad5 shaft domain and an Ad34 knob domain.The diagnostic transgene can be, for example, a PET reporter gene, a fluorescent protein, or an enzyme.

[0106] Also provided herein is a method for treating a tumor in a subject by administering to the subject a synthetic adenovirus comprising a therapeutic transgene, a native or modified capsid that detargets the synthetic adenovirus from the liver, and an Ad34 fiber protein, or a chimeric fiber protein that comprises an Ad5 shaft domain and an Ad34 knob domain.The therapeutic transgene can, for example, code for an anti-cancer drug or agent that destroys or kills tumor stromal cells.

[0107] In some embodiments, the transgene is inserted into the E1 or E3 region. Suitable transgene insertion sites are well known in the art (see, e.g., PCT Publication No. WO2012 / 024351).

[0108] The transgene, such as a gene encoding a fluorescent protein, is operably linked to a promoter. In some embodiments, the promoter is a heterologous promoter. In some examples, the promoter is an EF1α promoter. The selection of the promoter is within the capabilities of those skilled in the art. In some cases, the promoter is an inducible promoter or a tissue-specific promoter. An exemplary tissue-specific promoter for expression in pancreatic tissue is Pdx1.

[0109] In some cases, a single promoter is used to regulate the expression of multiple genes, which can be achieved through the use of an internal ribosome entry site (IRES) or a 2A peptide. V. Pharmaceutical Compositions and Administration Thereof

[0110] A composition comprising synthetic adenovirus (or one or more nucleic acids or vectors encoding recombinant adenovirus) is provided herein. The composition is optionally suitable for formulation and administration in vitro or in vivo. Optionally, the composition comprises one or more of recombinant adenoviruses and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22nd Edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012). Pharmaceutically acceptable carriers include materials that are not biologically or otherwise undesirable. That is, the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with other components in the pharmaceutical composition in which it is contained. When administered to a subject, the carrier is optionally selected to minimize the degradation of active ingredients and to minimize adverse side effects in the subject.

[0111] The recombinant virus (or one or more nucleic acids or vectors encoding the recombinant adenovirus) is administered according to known methods, for example, intravenously, for example, by bolus or continuous infusion over an extended period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, intratumoral, or inhalation routes. Administration may be local or systemic. The composition may be administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, intrahepatic, intracranial, nebulization / inhalation, or placement via bronchoscopy. Thus, the composition is administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated.

[0112] In some embodiments, the composition for administration comprises the recombinant adenovirus (or recombinant genome) described herein dissolved in a pharma- ceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and usually free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharma- cetically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., as necessary to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of active agent in these formulations can vary widely and will be selected primarily based on the volume, viscosity, weight, etc. of fluid, depending on the specific mode of administration selected and the needs of the subject.

[0113] Pharmaceutical formulations, particularly pharmaceutical formulations of recombinant viruses, can be prepared by mixing a recombinant adenovirus (or one or more nucleic acids encoding the recombinant adenovirus) having a desired degree of purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers. Such formulations can be lyophilized formulations or aqueous solutions.

[0114] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosage and concentration used.Acceptable carriers, excipients, or stabilizers can be acetates, phosphates, citrates, and other organic acids; antioxidants (e.g., ascorbic acid), preservatives, low molecular weight polypeptides; proteins, such as serum albumin or gelatin, or hydrophilic polymers, such as polyvinylpyrrolidone; and amino acids, monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents; and ionic and nonionic surfactants (e.g., polysorbates); salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants.Recombinant adenovirus (or one or more nucleic acids encoding recombinant adenovirus) can be formulated in any suitable concentration of infectious unit.

[0115] Formulations suitable for oral administration may consist of (a) a liquid solution, such as an effective amount of the recombinant adenovirus suspended in a diluent, such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granules, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, moistening agents, preservatives, flavorings, dyes, disintegrants, and pharma- ceutically compatible carriers. Lozenge forms may contain the active ingredient in a flavoring, such as sucrose, as well as pastilles containing the active ingredient in an inert base, such as emulsions of gelatin and glycerin or sucrose and acacia, gels, and the like, which contain, in addition to the active ingredient, carriers known in the art.

[0116] The recombinant adenovirus (or one or more nucleic acids encoding the recombinant adenovirus), alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0117] Suitable formulations for parenteral administration, for example, by intra-articular (into joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient), and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.In the provided method, the composition can be administered, for example, by intravenous injection, orally, topically, intraperitoneally, intravesically, intratumorally, or intrathecally.Parenteral, intratumoral, and intravenous administration are the preferred methods of administration.The formulations of the compound can be presented in single-dose or multi-dose sealed containers, for example, ampoules and vials.

[0118] Injection solution and suspension can be prepared from the above-mentioned type of sterile powder, granules and tablets.The pharmaceutical preparation is preferably in unit dosage form.In such form, the preparation is further divided into unit doses that contain appropriate amounts of active components.Therefore, the pharmaceutical composition can be administered in various unit dosage forms according to the method of administration.For example, the unit dosage forms suitable for oral administration include, but are not limited to, powder, tablet, pill, capsule and lozenge.

[0119] In some embodiments, the composition comprises at least two different recombinant adenoviruses, such as recombinant adenoviruses encoding different transgenes, hi some examples, the composition comprises two, three, four, five or six different recombinant adenoviruses.

[0120] In therapeutic applications, the recombinant adenovirus or a composition thereof is administered to a subject in an effective amount or effective dose. Single or multiple administrations of the composition may be administered as needed. "Patient" or "subject" includes both humans and other animals, particularly mammals. Thus, the method is applicable to both human therapy and veterinary applications.

[0121] The effective amount of recombinant adenovirus is determined for each individual and is based, at least in part, on the particular recombinant adenovirus used, the size, age, sex, and general health of the individual. For example, for administration to humans, an effective amount of recombinant adenovirus is at least 10 3 Plaque forming units (PFU) of recombinant virus are used, depending on the type, size, and number of growing cells or neoplasms present, e.g., at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 PFU, e.g., about 10 3 ~10 12 PFU of the recombinant virus is used. The effective amount is about 1.0 pfu / kg body weight to about 10 15 pfu / kg body weight (e.g., about 10 2 pfu / kg body weight ~ approx. 10 13 The recombinant adenovirus may be administered in a single dose or in multiple doses (e.g., 2, 3, 4, 6 or more doses). The multiple doses may be administered simultaneously or intermittently (e.g., over a period of days or weeks).

[0122] In some embodiments, the methods provided include administering to a subject one or more therapeutic agents, such as one or more agents for the treatment of cancer, such as pancreatic cancer or glioblastoma.

[0123] Administration of the synthetic adenoviruses disclosed herein carrying therapeutic transgenes may be accompanied by administration of other anti-cancer drugs or therapeutic treatments (such as surgical removal of tumors). Any suitable anti-cancer drug can be administered in combination with the recombinant viruses disclosed herein. Exemplary anti-cancer drugs include, but are not limited to, chemotherapeutic agents such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g., anti-androgens), CDK inhibitors, and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibodies (e.g., biologics) that specifically target cancer cells.

[0124] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkylsulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0125] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0126] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0127] Non-limiting examples of other agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenal cortical suppressants (such as mitotane and aminoglutethimide).

[0128] Non-limiting examples of hormones and antagonists include corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatin, Cisplatin, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, e.g., Docetaxel), Velban, Vincristine, VP-16, but some newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, and Calcitriol.

[0129] Non-limiting examples of immune modulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor, Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor, Genentech).

[0130] Another common treatment for some types of cancer is surgery, such as surgical removal of the cancer or a portion thereof. Another example of a treatment is radiation therapy, such as administering radioactive material or energy (such as external beam light therapy) to the tumor site to eradicate the tumor or help it shrink before surgical removal.

[0131] CDK (cyclin-dependent kinase) inhibitors are drugs that inhibit the function of CDK.Non-limiting examples of CDK inhibitors for use in the provided methods include AG-024322, AT7519, AZD5438, flavopiridol, indisulam, P1446A-05, PD-0332991, and P276-00 (see, for example, Lapenna et al., Nature Reviews, vol. 8, pp. 547-566, 2009).Other CDK inhibitors include LY2835219, palbociclib, LEE011 (Novartis), pan-CDK inhibitor AT7519, seliciclib, CYC065, butyrolactone I, hymenialdisine, SU9516, CINK4, PD0183812, or fascaplysin.

[0132] In some embodiments, the CDK inhibitor is a broad-spectrum inhibitor (such as flavopiridol, olomoucine, roscovitine, kempauron, SNS-032, AT7519, AG-024322, (S)-roscovitine, or R547), while in other embodiments, the CDK inhibitor is a specific inhibitor (such as fascaplysin, ryuvidine, purvalanol A, NU2058, BML-259, SU 9516, PD0332991, or P-276-00).

[0133] The choice of agent and dosage can be easily determined by those skilled in the art based on the given disease being treated.The combination of agents or compositions can be administered either in parallel (e.g., as one mixture), separately but simultaneously (e.g., through separate intravenous lines), or sequentially (e.g., one agent is administered first, followed by the second agent).Thus, the term combination is used to refer to the parallel, simultaneous, or sequential administration of two or more agents or compositions.

[0134] The following examples are provided to illustrate certain specific features and / or embodiments, and are not to be construed as limiting the disclosure to the specific features or embodiments described. EXAMPLES

[0135] Example 1 Synthetic adenovirus expressing Ad5 / Ad34 chimeric fiber protein and liver detargeting modifications The fiber protein of Ad5 and many other serotypes have been shown to bind CAR for cell attachment, while other serotypes have been shown to use CD46 (Gaggar et al., Nat Med 9:1408-1412, 2003), desmoglein 2 (Wang et al., Nat Med 17:96-104, 2011), sialic acid (Nilsson et al., Nat Med 17:105-109, 2011), or others (Arnberg, Trends Pharmacol Sci 33:442-448, 2012). Receptor utilization of many serotypes has not been thoroughly investigated, and CD46 is not thought to be expressed in adult mice.

[0136] Adsembly / AdSLIC (see PCT Publication No. WO 2012 / 024351, incorporated herein by reference) was used to generate synthetic adenoviruses with chimeric fiber proteins. Because the globular knob at the C-terminus of the fiber protein is typically responsible for receptor binding, a virus with a chimeric fiber protein was generated by replacing the Ad5 fiber knob with the fiber knob from Ad34 (AdSyn-CO176). The control virus (AdSyn-CO171) contains the Ad5 fiber protein (i.e., both the shaft and knob domains are from Ad5). Both viruses were generated with the same E1 module containing E1A / E1B deletions and a luciferase-GFP fusion driven by the EF1α promoter (Table 1). The recombinant viruses also contain liver-detargeting modifications. The native Ad5 vector only infects the lungs (by inhalation) or liver (by intravenous administration). The Ad5 hexon binds to factor X in the blood, which leads to its uptake by Kuppfer cells in the liver, preventing systemic dissemination and inducing limited inflammation. To overcome this and allow systemic administration to alternative cell types, synthetic adenoviruses were engineered to contain additional genomic modifications in the E1 and core regions that prevent uptake and expression in the liver. Specifically, both viruses contain a binding site in the 3'UTR of the transgene for a microRNA (miR-122) that is specifically expressed in the liver, and an E451Q mutation in the hexon. [Table 1]

[0137] Example 2 Synthetic adenovirus expressing the Ad34 knob domain exhibits tropism for tumor stroma This example describes the finding that AdSyn-CO176, expressing a chimeric fiber protein with the Ad34 knob domain, specifically traffics to the tumor stroma. Pancreatic tumor model

[0138] Figure 1A shows the Cre-LoxP Kras G12D A schematic diagram of the Kras / p53 pancreatic tumor model is shown. Mice designated "Kras;p53 / p53" contain a Kras gene downstream of the sequence encoding LoxP-stop codon-LoxP. G12D It encodes an oncogene. The stop codon is a transcription factor that, in the absence of Cre recombinase, converts the mutant Kras (Kras G12D ) expression. However, in the presence of Cre recombinase, the stop codon is removed, and Kras G12D This allows expression of oncogenes. In these same mice, both alleles of the p53 gene are flanked by LoxP sites (LoxP-p53-LoxP). Mice designated "p53 / p53;Cre" also have both alleles of the p53 gene flanked by LoxP sites (LoxP-p53-LoxP), and they express a Cre recombinase transgene driven by the pancreatic and duodenal homeobox 1 (Pdx1) promoter. Pdx1 is a gene that is specifically expressed in pancreatic cells, and therefore both copies of p53 are deleted in pancreatic cells. By crossing between the strains, Cre driven by the Pdx1 promoter led to the deletion of both alleles of the tumor suppressor p53 in pancreatic cells and the expression of mutant Kras G12D The homozygous mice, designated "Kras;p53 / p53;Cre", develop pancreatic tumors in 5-7 weeks. AdSyn-CO176 was intravenously injected into Kras;p53 / p53 and Kras;p53 / p53;Cre mice. 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 min, and then scanned for 5 min using an IVIS™ imaging system. As shown in Figure 1B, Kras;p53 / p53 mice had normal pancreas, and the luciferase signal was mainly derived from the spleen. In contrast, Kras;p53 / p53;Cre mice had pancreatic tumors, and the signal was mainly derived from the pancreatic tumor.

[0139] Another study was performed in a Cre-mediated genetic manipulation heterozygous model (Figure 2A). Mice designated "p53 / +;Cre" have one wild-type p53 allele and one LoxP-flanked p53 allele (LoxP-p53-LoxP). By crossing between Kras;p53 / p53 and p53;+;Cre strains, Cre recombinase driven by the Pdx1 promoter resulted in the deletion of a single allele of the tumor suppressor p53 in pancreatic cells and the expression of mutant Kras G12D These heterozygous mice, designated "Kras;p53 / +;Cre", develop tumors later in life (at 4-9 months of age) due to the fact that they have one wild-type allele of p53. This wild-type allele must be lost or mutated for pancreatic tumors to arise. AdSyn-CO176 was injected intravenously into 4-month-old p53 / +;Cre and Kras;p53 / +;Cre mice. 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 minutes, and then scanned for 1 minute using an IVIS™ imaging system. p53 / +;Cre mice had normal pancreases, and the signal was primarily derived from the spleen. In contrast, 4-month-old Kras;p53 / +;Cre mice had pancreatic tumors, and the signal was primarily derived from the tumor and liver.

[0140] Heterozygous Kras;p53 / +;Cre mice develop pancreatic tumors in 4-9 months. To test whether AdSyn-CO176 can infect pancreatic tumors at a very early stage of tumor development (before tumors are visible), AdSyn-CO176 was intravenously injected into 2-month-old Kras;p53 / +;Cre mice. 72 hours after virus injection, tissues were collected, incubated with luciferin for 5 min, and scanned for 4 min using an IVIS™ imaging system. Although the pancreas of 2-month-old Kras;p53 / +;Cre mice appeared normal, luciferase signals were found in this tissue (Figure 3A). H&E staining was performed to evaluate the histology of the pancreas after infection with AdSyn-CO176. For comparison, Figure 3B shows typical histology of normal pancreatic tissue and pancreatic tumor tissue. H&E staining of the pancreas of 2-month-old Kras;p53 / +;Cre mice showed that a small portion of the pancreas developed tumors (Figure 3C, indicated by polygons), whereas most of the pancreatic tissue appeared normal. This result indicated that AdSyn-CO176 could infect pancreatic tumors at a very early stage.

[0141] In pancreatic tumors, only 10% of cells are cancer cells, while the remaining 90% are stromal cells. To determine which cell types were targeted by AdSyn-CO176, IHC and IF staining was performed. CK19 is a marker for tumor cells, while smooth muscle actin (SMA) is a marker for stromal cells. IHC staining of pancreatic tumors infected with AdSyn-CO176 showed that GFP expressed from AdSyn-CO176 overlapped with SMA staining, indicating that AdSyn-CO176 targeted stromal cells (Figure 4A). IF staining of pancreatic tumors infected with AdSyn-CO176 also demonstrated that GFP overlapped with SMA staining (Figure 4B), confirming that AdSyn-CO176 infects stromal cells. Glioblastoma model

[0142] Shown in Figure 5A is a schematic diagram of the Cre-mediated genetically engineered glioblastoma model. Lentivirus was directly injected into the brain of GFAP-Cre mice. Cre recombinase driven by the glial fibrillary acidic protein (GFAP) promoter cleaved RFP from the lentivirus-encoded gene and expressed HRas predominantly in astrocytes. V12 and GFP expression. Expression of lentivirus-encoded U6-p53 shRNA knocks down p53 expression in brain cells that take up the virus. V12 Expression of AdSyn-CO171 and knockdown of p53 induces tumor formation in the brain one week after injection. GFP signal is used to show the formation of glioblastoma. Saline, AdSyn-CO171, or AdSyn-CO176 were injected intravenously (IV) into GFAP-Cre mice that received tumor-inducing lentivirus four weeks earlier. 48 hours after virus injection, mice were scanned for one minute using an IVIS™ imaging system, 5 minutes after intraperitoneal injection of luciferin (Figure 5B). Luciferase signal was detected in mice infected with AdSyn-CO176, but not in mice injected with saline or AdSyn-CO171.

[0143] Wild-type mice (normal brain) and GFAP-Cre mice that received tumor-inducing lentivirus injections were injected with AdSyn-CO171 or AdSyn-CO176. Brain tissues were collected 72 hours after the injection of synthetic adenovirus, incubated with luciferin for 5 minutes, and scanned for 5 minutes using an IVIS™ imaging system (Figure 5C). Only GFAP-Cre mice that received tumor-inducing lentivirus showed luciferase signals from AdSyn-CO176. This demonstrates that AdSyn-CO176 migrates to brain tissue only when tumors are present. Brain tissues were also scanned for GFP signals (Figure 5D). GFP signals are used to identify glioblastomas. Both GFAP-Cre mice that received tumor-inducing lentivirus had GFP signals in the brain, whereas no GFP was detected in wild-type mice. The GFP signal completely overlapped with the luciferase signal in GFAP-Cre mice that received tumor-inducing lentivirus and AdSyn-CO176.

[0144] Injection of tumor-inducing lentivirus leads to transient brain damage at the injection site. Although synthetic adenovirus (AdSyn-CO171 and AdSyn-CO176) was injected 4 weeks after the first injection of lentivirus, it was still unclear whether the migration of AdSyn-CO176 to glioblastoma was driven by tumor or injection site damage. To answer this question, GFAP-Cre mice were injected with synthetic adenovirus 4 weeks after either no injection, sham injection, or tumor-inducing lentivirus injection. GFAP-Cre mice were injected with either Hank's balanced salt solution (HBSS) or tumor-inducing lentivirus. After 4 weeks, AdSyn-CO171 was injected intravenously. As shown in Figure 6A, there was no luciferase signal from AdSyn-CO171 in the brain in either mouse group. GFAP-Cre mice were injected with HBSS or tumor-inducing lentivirus, or GFAP-Cre mice were not injected. Four weeks later, the mice were intravenously injected with AdSyn-CO176. As shown in Figure 6B, luciferase signals were only detected in the brains of mice that received tumor-inducing lentivirus, whereas mice that received HBSS or no injection did not produce signals. These results demonstrate that the specificity of AdSyn-CO176 is driven by tumors but not by damage at the injection site. Example 3 AdSyn-CO176 migrates into human glioblastoma tumors in xenograft models

[0145] This example describes the finding that a synthetic adenovirus expressing a chimeric fiber protein with the Ad34 knob domain can target human glioblastoma tumors.

[0146] The U87-tdTomato cell line is a human glioblastoma cell line that expresses the tdTomato fluorescent protein as a reporter to allow monitoring of tumor growth. U87-tdTomato cells are injected intracranially into NSG mice to give rise to glioblastoma tumors, which typically take 4-8 weeks to give rise to tumors (Figure 7A). This glioblastoma xenograft model was used to determine whether AdSyn-CO176 can migrate into human glioblastoma tumors. Four weeks after mice received an intracranial injection of U87-tdTomato cells, AdSyn-CO171 (SEQ ID NO: 1) and AdSyn-CO176 (SEQ ID NO: 2) were injected intravenously into NSG mice by tail vein injection. 48 hours after virus injection, liver, spleen and brain tissues were collected, incubated with luciferin for 5 min, and then scanned for 1 min using an IVIS™ imaging system. As shown in Figure 7B, only mice injected with AdSyn-CO176 showed luciferase signals in the brain, which completely overlapped with tdTomato expression. Thus, these results demonstrate that AdSyn-CO176, but not AdSyn-CO171, can migrate into human glioblastoma tumors. Example 4 Synthetic adenoviruses targeting tumor stroma and expressing therapeutic transgenes reduce tumor size in animal models

[0147] This example demonstrates that a synthetic adenovirus expressing a chimeric fiber protein with an Ad34 knob domain and a therapeutic payload can track to the tumor stroma and reduce tumor size.

[0148] A study was carried out to determine whether a therapeutic transgene could be incorporated into AdSyn-CO176 (SEQ ID NO: 2) to allow for the treatment of tumors. To carry out this study, KPCL (Kras G12D We used a KPCL (p53 knockout; Pdx1-Cre; firefly luciferase) mouse model (Figure 8A). G12DKPCL mice specifically express p53 and are similar to homozygous "Kras;p53 / p53;Cre" mice, which have the p53 gene knocked out only in the pancreas. However, KPCL mice also specifically express firefly luciferase in the pancreas. Tumor development in KPCL mice is also similar to that for "Kras;p53 / p53;Cre" mice.

[0149] Two further synthetic adenoviruses were generated: AdSyn-CO987 (SEQ ID NO: 5) and AdSyn-CO989 (SEQ ID NO: 6). AdSyn-CO987 is a synthetic adenovirus based on AdSyn-CO176. The herpes simplex virus-1 thymidine kinase (TK) / ganciclovir (GCV) suicide gene was cloned into AdSyn-CO176, replacing the firefly luciferase / GFP gene. Renilla luciferase was also inserted into the genome of AdSyn-CO176 immediately after TK. The control virus AdSyn-CO989 was generated by cloning TK-P2A-Renilla luciferase into AdSyn-CO171 to replace the original firefly luciferase / GFP gene.

[0150] KPCL mice were inoculated with 1 × 10 AdSyn-CO987 or AdSyn-CO989 at 5–6 weeks of age. 6 PFU was injected intravenously via the tail vein. Two days later, mice were injected ip or iv with GCV. Three control groups were used: AdSyn-CO989+GCV; AdSyn-CO987 followed by saline injection (AdSyn-CO987+saline); and GCV injection only (ip or iv). Figure 8B provides a table showing the mean survival time of KPCL mice for each treatment group. Treatment with AdSyn-CO987+GCV extended the survival time of mice compared to controls.

[0151] The firefly luciferase signal expressed by tumor was analyzed during treatment to monitor tumor growth. The results are shown in Figure 8C. The treatment for mouse Z619R was AdSyn-CO987+saline, which served as control. Mice Z601R and Z607R were treated with AdSyn-CO987+GCV (ip). The intensity of firefly luciferase signal increased in control mouse Z619R (indicating increased tumor size), while the signal decreased in mice Z601R and Z607R (indicating reduced tumor size).

[0152] Pancreatic tumor histology was also evaluated by H&E staining (Figure 9A). Mice Z655, 1806, Z619 and Z621 were all control mice. Mice Z655 were treated with GCV only by ip injection; mice 1806 were treated with GCV only by iv injection; mice Z619 were treated with AdSyn-CO987+saline; and mice Z621 were untreated. Mice Z656 were treated with AdSyn-CO987+GCV iv. Compared to controls, tumors from Z656 had larger areas of necrosis (Figure 9B).

[0153] In view of the numerous possible embodiments to which the principles of this disclosure may be applied, it is to be recognized that the illustrated embodiments are merely examples of the invention and are not to be taken as limiting the scope of the invention, which is rather defined by the following claims, and the inventors therefore claim as their invention all that comes within the scope and spirit of these claims. The present invention provides, for example, the following items. (Item 1) 1. A method for expressing a transgene in a tumor cell of a subject, comprising: The transgene, Native or modified capsids that detarget synthetic adenoviruses from the liver, and Adenovirus type 34 (Ad34) fiber protein, or a chimeric fiber protein containing the adenovirus type 5 (Ad5) shaft domain and the Ad34 knob domain administering to the subject the synthetic adenovirus comprising: (Item 2) 2. The method of claim 1, wherein the transgene is a diagnostic transgene. (Item 3) 3. The method of claim 2, wherein the diagnostic transgene encodes a fluorescent protein. (Item 4) 4. The method of claim 3, wherein the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), a red fluorescent protein (RFP), a blue fluorescent protein (BFP), or an orange fluorescent protein. (Item 5) 3. The method of claim 2, wherein the diagnostic transgene encodes an enzyme. (Item 6) 6. The method of claim 5, wherein the enzyme is luciferase. (Item 7) 3. The method of claim 2, wherein the diagnostic transgene comprises a positron emission tomography (PET) reporter gene. (Item 8) 2. The method of claim 1, wherein the transgene is a therapeutic transgene. (Item 9) 9. The method of claim 8, wherein the therapeutic transgene encodes an anticancer drug. (Item 10) 9. The method of claim 8, wherein the therapeutic transgene encodes an agent that destroys or kills tumor stromal cells. (Item 11) 1. A method for diagnosing a subject as having a tumor, comprising: Diagnostic transgenes, Native or modified capsids that detarget synthetic adenoviruses from the liver, and Adenovirus type 34 (Ad34) fiber protein, or a chimeric fiber protein containing the adenovirus type 5 (Ad5) shaft domain and the Ad34 knob domain administering to the subject the synthetic adenovirus comprising: (Item 12) 12. The method of claim 11, wherein the diagnostic transgene comprises a positron emission tomography (PET) reporter gene. (Item 13) 12. The method of claim 11, wherein the diagnostic transgene encodes a fluorescent protein. (Item 14) Item 14. The method of item 13, wherein the fluorescent protein comprises green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), or orange fluorescent protein. (Item 15) 12. The method of claim 11, wherein the diagnostic transgene encodes an enzyme. (Item 16) 16. The method of claim 15, wherein the enzyme is luciferase. (Item 17) 1. A method of treating a tumor in a subject, comprising: Therapeutic transgenes, Native or modified capsids that detarget synthetic adenoviruses from the liver, and Adenovirus type 34 (Ad34) fiber protein, or a chimeric fiber protein containing the adenovirus type 5 (Ad5) shaft domain and the Ad34 knob domain administering to the subject the synthetic adenovirus comprising: (Item 18) 18. The method of claim 17, wherein the therapeutic transgene encodes an anticancer drug. (Item 19) 18. The method of claim 17, wherein the therapeutic transgene encodes an agent that destroys or kills tumor stromal cells. (Item 20) 20. The method of any one of items 1 to 19, wherein the synthetic adenovirus comprises a modified capsid that detargets the synthetic adenovirus from the liver. (Item 21) 21. The method of claim 20, wherein the synthetic adenovirus comprises a modified hexon protein. (Item 22) 23. The method of claim 21, wherein the modified hexon protein comprises an E451Q mutation. 23. The method of any one of items 1 to 22, wherein the synthetic adenovirus further comprises one or more binding sites for liver-specific microRNAs. (Item 24) 24. The method of claim 23, wherein the liver-specific microRNA is miR-122. (Item 25) 25. The method of claim 23 or 24, wherein the one or more binding sites are in the 3'UTR of the transgene. (Item 26) 26. The method of any one of items 1 to 25, wherein the synthetic adenovirus further comprises one or more binding sites for spleen-specific microRNA. (Item 27) Item 28. The method according to item 26, wherein the spleen-specific microRNA is miR142-3p. 28. The method of claim 26 or 27, wherein the one or more binding sites are in the 3'UTR of the transgene. (Item 29) 29. The method of any one of items 1 to 28, wherein expression of the transgene is regulated by a tissue-specific promoter. (Item 30) 30. The method of any one of items 1 to 29, wherein the synthetic adenovirus is derived from an Ad5 vector genome. (Item 31) 31. The method of claim 30, wherein the synthetic adenovirus comprises an Ad5 capsid protein and a chimeric fiber protein comprising an Ad5 shaft domain and an Ad34 knob domain. (Item 32) 32. The method of any one of items 1 to 31, wherein the tumor is a pancreatic tumor. (Item 33) 32. The method of any one of items 1 to 31, wherein the tumor is a glioblastoma. (Item 34) 34. The method of any one of items 1 to 33, wherein the genome of the synthetic adenovirus comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:5. (Item 35) 35. The method of claim 34, wherein the genome of the synthetic adenovirus comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:5. (Item 36) A synthetic adenovirus genome comprising a nucleotide sequence at least 95% identical to SEQ ID NO:2 or SEQ ID NO:5. (Item 37) 37. The synthetic adenovirus genome of item 36, comprising SEQ ID NO:2 or SEQ ID NO:5.

Claims

[Claim 1] The invention as depicted in the drawings.

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