Tumor-targeting synthetic adenoviruses and uses thereof

Abstract

Described herein is a synthetic adenovirus that has a liver-detargeting mutation and expresses an adenovirus type 34 (Ad34) fiber protein or a chimeric fiber protein with an Ad34 knob domain. The synthetic adenovirus migrates to tumor sites. The use of the synthetic adenovirus to deliver diagnostic or therapeutic transgenes to tumors is also described. Described herein is the discovery that a liver-detargeted synthetic adenovirus that expresses a fiber protein with an adenovirus type 34 (Ad34) knob domain can be directed to tumor sites.

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 Adenoviruses (Ad) are natural multigene expression vehicles. 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. Therefore, each of these regions can be replaced with a nonviral 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 primary Ad vector used in basic research, gene therapy, and oncolytic virotherapy. However, Ad5 has a limited tropism, infecting only epithelial cells that possess the coxsackie adenovirus receptor (CAR) receptor for viral uptake. Furthermore, upon intravenous injection, Ad5 binds to blood factors that cause Ad5 to be sequestered in the liver, where it can potentially induce limited inflammation and toxicity. Therefore, 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 Described herein is the discovery that liver-detargeted synthetic adenoviruses expressing a fiber protein 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 natural 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 are methods for diagnosing a subject as having a tumor. In some embodiments, the method comprises administering to the 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. 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 are methods for treating tumors in a subject. In some embodiments, the methods include 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 comprising 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 explanation of the drawings]

[0010] [Figure 1]AdSyn-CO176 targets 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 LoxP-stop codon-LoxP coding sequence. 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" contain 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 results in 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 within 5–7 weeks of maturation. (Figure 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 pancreases, and the luciferase signal was primarily from the spleen. Kras;p53 / p53;Cre mice had pancreatic tumors, and the signal was primarily from the pancreatic tumor.

[0011] [Figure 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 develop. (Figure 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] [Figure 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 between 4 and 9 months of age. 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 intravenously injected 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. Although the pancreas of 2-month-old Kras;p53 / +;Cre mice appeared normal, luciferase signals were detected in this tissue. (Figure 3B) H&E staining showing typical histological findings of a normal pancreas (a) and a pancreatic tumor (b). (Figure 3C) H&E staining of the pancreas from a 2-month-old Kras;p53 / +;Cre mouse (shown in the polygon) shows that a small portion of the pancreas developed a tumor. 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, AdSyn-CO176-infected cells are stromal cells. (Figure 4A) IHC staining of a pancreatic tumor infected with AdSyn-CO176. CK19 is a marker for tumor cells, while smooth muscle actin (SMA) is a marker for stromal cells. GFP staining from AdSyn-CO176 overlapped with SMA staining, indicating that AdSyn-CO176 targets stromal cells. (Figure 4B) IF staining of a pancreatic tumor infected with AdSyn-CO176. GFP staining overlapped with SMA staining, confirming AdSyn-CO176 infection of stromal cells.

[0014] [Figure 5]Glioblastoma infected with AdSyn-CO176 after tail vein injection. The synthetic adenovirus AdSyn-CO176 was injected into glioblastoma-bearing mice via the tail vein, and luciferase signals were detected in the glioblastoma. (Figure 5A) 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, cleaves RFP from the lentivirus-encoded gene, inducing HRasV12 and GFP expression primarily in astrocytes. Expression of lentivirus-encoded U6-p53 shRNA knocks down p53 expression in brain cells that take up the virus. HRasV12 expression and p53 knockdown induce tumor formation in the brain one week after injection. GFP signals are used to demonstrate glioblastoma formation. (Figure 5B) Saline, AdSyn-CO171, or AdSyn-CO176 were injected intravenously (IV) into GFAP-Cre mice that had received tumor-inducing lentivirus 4 weeks earlier. Forty-eight hours after virus injection, the 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 brains) and lentivirus-injected GFAP-Cre mice (developing brain tumors) were injected with AdSyn-CO171 or AdSyn-CO176. Brain tissue was collected 72 hours after injection of the 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 in the presence of tumors (Figure 5D). Brain tissue was also scanned for GFP signals. GFP signals were used to identify glioblastomas.Both GFAP-Cre mice receiving tumor-inducing lentivirus had GFP signals in the brain, whereas mice not receiving lentivirus had no GFP. The GFP signals completely overlapped with the luciferase signals in GFAP-Cre mice receiving tumor-inducing lentivirus and AdSyn-CO176.

[0015] [Figure 6] The migration of AdSyn-CO176 into glioblastomas is driven by the tumor, not injury. Injection of tumor-inducing lentivirus results in transient brain damage at the injection site. Although synthetic adenoviruses (AdSyn-CO171 and AdSyn-CO176) were injected 4 weeks after the first lentivirus injection, it remained unclear whether AdSyn-CO176 migration into glioblastomas was driven by tumor or injury at the injection site. To address 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 intravenously injected. No AdSyn-CO171-derived luciferase signal was present in the brain in any of the mouse groups. (Figure 6B) GFAP-Cre mice were injected with HBSS or tumor-inducing lentivirus, or no injection was given. 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 signals. 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 diagram of the human glioblastoma xenograft model. Human glioblastoma U87 cells expressing tdTomato fluorescent protein (U87-tdTomato) were intracranially injected into NOD scid gamma (NSG) mice to develop glioblastoma tumors. (Figure 7B) Four weeks after the NSG mice received intracranial injections of U87-tdTomato, AdSyn-CO171 and AdSyn-CO176 were intravenously injected into the tail vein of the NSG mice. 48 hours after virus injection, the tissues shown in the panels were collected, incubated with luciferin for 5 minutes, and 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 a synthetic adenovirus carrying a therapeutic transgene. (Figure 8A) Schematic diagram of the KPCL (KrasG12D; p53 knockout; Pdx1-Cre; firefly luciferase) mouse model. KPCL mice express KrasG12D specifically 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 in "Kras; p53 / p53; Cre" mice. (Figure 8B) Table showing the mean survival time of KPCL mice (at least four mice per treatment group) for each treatment. 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 immediately after TK in the genome of AdSyn-CO176. The 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 the indicated virus at 1 × 10 plaque-forming units (PFU). Two days later, the 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 alone (ip or iv). Treatment with AdSyn-CO987 + GCV extended the survival time of mice compared with controls. (Figure 8C) Images of representative mice showing firefly luciferase signals. Firefly luciferase signals (expressed by tumors) were 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) H&E staining image of pancreatic tumors. Mice Z655, 1806, Z619, and Z621 were all control mice. Mouse Z655 was treated with GCV alone by ip injection; mouse 1806 was treated with GCV alone by iv injection; mouse Z619 was treated with AdSyn-CO987 plus saline; and mouse Z621 received no treatment. Mouse Z656 was treated with AdSyn-CO987 plus GCV iv. Compared to the control, tumors from Z656 had larger areas of necrosis (indicated by arrowheads). (Figure 9B) Representative areas of necrosis in a magnified Z656 tumor. The areas are also shown in Figure 9A. DETAILED DESCRIPTION OF THE INVENTION

[0019] Sequence Listing (Sequence Listing) The nucleic acid and amino acid sequences listed in the accompanying 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 215 KB 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 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. Terminology and Methods

[0027] II. Terminology and Methods Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V (ISBN 0-19-854287-9), published by Oxford University Press in 1994; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (ISBN 0-632-02182-9), published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1-56081-569-8), published by VCH Publishers, Inc. in 1995. 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, there are 68 known serotypes of human adenovirus, classified into seven species (species A, B, C, D, E, F, and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (mainly species B and C), conjunctivitis (species B and D), and / or gastroenteritis (species F and G).

[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 origins. In the context of the present disclosure, a "chimeric adenovirus" is an adenovirus having genetic material and / or proteins derived from at least two different serotypes (e.g., from Ad5 and an adenovirus of a second serotype). In this context, a "capsid-swapped" adenovirus refers to a chimeric adenovirus in which the capsid proteins are derived from one serotype of adenovirus and the remaining proteins are derived from another adenovirus serotype. Similarly, a "chimeric fiber" is a fiber protein having amino acid sequences derived from at least two different serotypes of adenovirus. For example, a chimeric fiber can be composed of a fiber shaft derived from Ad5 and a fiber knob derived 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 the present disclosure, a "detargeted" adenovirus is a recombinant or synthetic adenovirus that contains one or more modifications that alter 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 an 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: The adenovirus early region 1A (E1A) gene and the polypeptide expressed from this gene. The E1A protein plays a role in viral genome replication by causing cells to enter the cell cycle. As used herein, the term "E1A protein" refers to the protein expressed from the E1A gene, and includes the E1A protein produced by any adenovirus serotype.

[0035] Fiber: The adenoviral 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 containing amino acid sequences derived from at least two different (heterologous) proteins or peptides. Fusion proteins can be produced, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To produce a fusion protein, the nucleic acid sequences must be in the same reading frame and must not contain internal stop codons. Fusion proteins, especially short fusion proteins, can also be produced by chemical synthesis.

[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, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells, in the cells or tissues of an organism or the organism itself, in which the component naturally occurs. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[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, called 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 in length and are partially complementary to the 3'UTRs 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 that is more significantly 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 significantly 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 a nucleic acid sequence or protein sequence. For example, modifications of an amino acid sequence include, for example, substitution, insertion, and deletion, or a combination thereof. Insertions include amino- and / or carboxyl-terminal fusions and the insertion of single or multiple amino acid residues into a sequence. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some embodiments herein, modifications (such as substitutions, insertions, or deletions) result in a change in function, such as a reduction or enhancement of a specific activity of a protein. As used herein, "Δ" or "delta" refers to a deletion. A substitution modification is one in which at least one residue is removed and a different residue is inserted in its place. Amino acid substitutions are typically of a single residue, but may occur simultaneously at several different positions. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final mutant sequence. These modifications can be prepared by modifying nucleotides in the DNA encoding the protein, thereby producing DNA encoding this modification. Techniques for making insertion, deletion, and substitution mutations at predetermined sites in DNA having a known sequence are well known in the art. A "modified" protein, nucleic acid, or virus is one that has one or more of the modifications outlined above.

[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 if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0044] Pancreatic cancer: Cancer that begins in the tissues of the pancreas. Pancreatic cancer typically spreads rapidly and is rarely detected in its early stages, 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: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. See Remington's Pharmaceutical Sciences by E.W. 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 particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid as a vehicle, which contains pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in the form of powder, pill, tablet, or capsule), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition 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 joined together through amide bonds. When the amino acids are alpha amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. The term "residue" or "amino acid residue" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide.

[0048] A conservative substitution in a polypeptide is the replacement of one amino acid residue in a protein sequence with a different amino acid residue that has similar biological properties. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. For example, a protein or peptide containing one or more conservative substitutions (e.g., no more than one, no more than two, no more than three, no more than four, or no more than five substitutions) retains the structure and function of the wild-type protein or peptide. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence encoding the polypeptide using standard procedures, such as site-directed mutagenesis or PCR. In one example, such variants can be easily selected by testing antibody cross-reactivity or the ability of the antibody to induce an immune response. Examples of conservative substitutions are provided below. [Table 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 chains.

[0050] In general, substitutions predicted to result in the greatest changes in protein properties will be non-conservative, such as (a) a hydrophilic residue, e.g., seryl or threonyl, substituting (or being substituted by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline substituting (or being substituted by) any other residue; (c) a residue with an electropositive side chain, e.g., lysyl, arginyl, or histadyl, substituting (or being substituted by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue with a bulky side chain, e.g., phenylalanine, substituting (or being substituted by) one without a side chain, e.g., glycine.

[0051] Positron Emission Tomography (PET): An imaging technique used to observe metabolic processes in the body. PET detects pairs of gamma rays indirectly emitted by positron-emitting radionuclides, which are introduced into the body on biologically active molecules. PET reporter genes encode molecules (such as receptors or enzymes) that provide targets for PET probes, which can then be detected by imaging. PET reporter genes are generally classified into three distinct groups: (1) reporter genes encoding enzymes that phosphorylate specific PET reporter probes, leading to their intracellular capture; (2) reporter genes encoding protein receptors that can be bound by specific PET reporter probes; and (3) reporter genes encoding 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" refers to any gene encoding 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, mutant deoxycytidine kinase, dopamine 2 receptor mutants, human estrogen receptor alpha ligand binding domain (hERL), human somatostatin 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 the 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 / initiat- es the transcription of a nucleic acid (e.g., a gene). A promoter comprises necessary nucleic acid sequences near the start site of transcription. Typically, a promoter is located near the gene to be transcribed. A promoter also optionally contains distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the transcription start site. A "constitutive promoter" is a promoter that is continuously active and is not subject to control by external signals or molecules. In contrast, the activity of an "inducible promoter" is controlled by external signals or molecules (e.g., transcription factors or tetracycline). A "tissue-specific promoter" is a promoter that is substantially active only in a specific tissue(s).

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

[0055] Purified: The term "purified" does not require absolute purity and is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, e.g., proteins, cellular debris, and other components.

[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 sequence. This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of segments of isolated nucleic acid molecules, e.g., by genetic engineering techniques. The term "recombinant" also includes nucleic acids, proteins, and viruses that have been altered only by the addition, substitution, or deletion of portions of a naturally occurring nucleic acid molecule, protein, or virus.

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

[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., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988); Higgins and Sharp, Gene, 73, 237-44 (1988); Higgins and Sharp, CABIOS, 5, 151-3 (1989); Corpet et al., Nuc. Acids Res., 16, 10881-90 (1988); Huang et al., Computer Appls. in the Biosciences, 8, 155-65 (1992); and Pearson et al., Meth. Mol. Bio., 24, 307-31 (1994). Altschul et al., J. Mol. Biol., 215, 403-10 (1990) present a detailed discussion of sequence alignment methods and homology calculations.

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

[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, primarily fibroblasts and pericytes. Tumor stroma is primarily 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: A living multi-cellular vertebrate organism, 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 may be chemically synthesized in a laboratory.

[0064] Therapeutic Agent: A chemical compound, such as an antisense compound, antibody, peptide or nucleic acid molecule, small molecule, 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 particular pharmaceutical or therapeutic agent (e.g., a recombinant virus) 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 with therapeutic application. In the context of the present disclosure, a therapeutic transgene can be, for example, an anti-cancer drug or agent that destroys or kills cells in the tumor stroma.

[0067] U exon: An open reading frame located between the early E3 region and the fiber gene in the 1 strand (leftward transcription) (Tollefson et al., J Virol, Vol. 81 (No. 23), pp. 12918-12926).

[0068] Vector: A nucleic acid molecule that allows the insertion of 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] Disclosed herein is that liver-detargeted synthetic adenoviruses expressing fiber proteins with the Ad34 knob domain can be directed to tumor sites. The synthetic adenoviruses can be used, for example, to deliver and express diagnostic or therapeutic transgenes in tumor cells, including tumor stromal cells.

[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 the liver, and an Ad34 fiber protein, or a chimeric fiber protein comprising an Ad5 shaft domain and an 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 examples, the therapeutic transgene encodes an anti-cancer drug. In specific examples, 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 examples, the anti-cancer drug is an anti-angiogenic factor, such as an inhibitor of vascular endothelial growth factor (VEGF). In other specific examples, the anti-cancer drug is an inhibitor of KRas (e.g., 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 are methods for diagnosing a subject as having a tumor. In some embodiments, the method comprises administering to the 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 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, which in some examples 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 are methods for treating tumors in a subject. In some embodiments, the method comprises 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 comprising an Ad5 shaft domain and an Ad34 knob domain.

[0078] In some embodiments, the therapeutic transgene encodes an anti-cancer drug. In some examples, 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 examples, the anti-cancer drug is an anti-angiogenic factor, such as an inhibitor of VEGF. In other examples, the anti-cancer drug is an inhibitor of KRas (e.g., an siRNA or shRNA inhibitor). In other examples, 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 disrupts or kills tumor stromal cells. In some examples, 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 an E451Q mutation (shown herein as SEQ ID NO: 4). In other embodiments, the synthetic adenovirus has an original (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 methods disclosed herein, the synthetic adenovirus further comprises one or more binding sites, for example, two or three binding sites, for a liver-specific microRNA. In some examples, the liver-specific microRNA is miR-122. In some examples, the one or more binding sites are in the 3'UTR of the transgene.

[0081] In some embodiments of the methods 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 are synthetic adenoviral genomes 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 examples, the synthetic adenoviral genome comprises or consists of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 5. IV. Synthetic Adenovirus

[0090] The technologies of Adsembly, AdSLICr, and RapAD allow for the 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 usefulness of adenoviruses as vehicles for delivering therapeutic proteins by prompting the host to produce proteins in situ. This provides the unique ability to use human proteins that are difficult to produce for therapeutic purposes, allowing for the 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 can be used for any adenovirus serotype. Adenovirus is a natural multigene expression vehicle. The E1, E3, and E4 regions are not required for replication in culture, or can be complemented by 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 through 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 can further comprise modifications that allow virus to detarget from liver.Ad5 hexon can bind to factor X in blood, which can lead to its absorption by the Kupfer cell of liver, preventing systemic dissemination and limiting inflammation.To overcome this, as further described below, synthetic adenovirus is engineered to comprise additional genome modifications in E1 and core module, which prevent adenovirus from being taken up and transgene expression in 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 to 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. Because the globular knob at the C-terminus of the fiber protein is typically responsible for receptor binding, a chimera was created by replacing the Ad5 fiber knob with one 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 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 for the rapid assembly of viruses optimized for tumor cell entry. B. Liver Detargeting Modification

[0096] The native Ad5 vector infects only 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 Kupfer cells in the liver, preventing systemic dissemination and inducing virus-limiting inflammation. To overcome this and enable intravenous delivery of a virus capable of systemic transport 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 the liver due to 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 the liver, but can instead target other organs such as the spleen and lymph nodes.Therefore, in some embodiments herein, synthetic adenovirus comprises a 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 specifically expressed in the liver. In certain embodiments, miR122 was selected as a liver-specific microRNA because 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] Disclosed herein is that synthetic adenoviruses with miR-122 binding sites and hexon mutations do not accumulate in the liver, but can instead target tumors. In some embodiments, one or more binding sites for liver-specific microRNAs are located in the 3'UTR of transgene. In some examples, the 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 comprising or consisting 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, the serotype most frequently used in research and therapeutic applications. Furthermore, when a specific 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 (approximately 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 particular 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 a chimeric fiber protein with an Ad34 knob domain and a liver-detargeting mutation can target tumors. It is further disclosed that the recombinant adenoviruses can express a transgene in tumor tissue, for example, in tumor stromal cells. In one example, the transgene is a reporter, such as a luciferase-GFP reporter, that allows detection of cells transduced by the virus. In another example, 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 with tumors 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 tumors 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 comprising an Ad5 shaft domain and an Ad34 knob domain.The therapeutic transgene can, for example, encode 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] A 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 a 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 internal ribosome entry sites (IRES) or 2A peptides. V. Pharmaceutical Compositions and Their Administration

[0110] A composition comprising a synthetic adenovirus (or one or more nucleic acids or vectors encoding a recombinant adenovirus) is provided herein. The composition is optionally suitable for in vitro or in vivo formulation and administration. Optionally, the composition comprises one or more recombinant adenoviruses and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22nd ed., 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 harmful 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 degradation of the active ingredient and 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, e.g., by bolus or continuous infusion over an extended period of time, by intramuscular, intraperitoneal, intrabrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, intratumoral, or inhalation routes. Administration can be local or systemic. The compositions can be administered topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, or intradermally. The compositions can be administered via any of several routes, including intrahepatic, intracranial, nebulized / inhaled, or via bronchoscopic placement. Thus, the compositions are 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 a recombinant adenovirus (or recombinant genome) described herein dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable material. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, as needed to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, depending on the particular 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 pharmaceutically 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 acetate, phosphate, citrate, 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, e.g., an effective amount of recombinant adenovirus suspended in a diluent, e.g., 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, flavors, dyes, disintegrants, and pharmaceutically compatible carriers. Lozenge forms may contain the active ingredient in a flavoring, e.g., sucrose, as well as pastilles containing the active ingredient in an inert base, e.g., an emulsion of gelatin and glycerin or sucrose and acacia, a gel, or the like, which contain carriers known in the art in addition to the active ingredient.

[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. 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, intraarticular (intra-articular), 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, thickeners, 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 administration, intratumoral administration, and intravenous administration are preferred administration methods.The compound formulation can be presented in a sealed container, such as an ampule or vial, in single or multiple doses.

[0118] Injection solution and suspension can be prepared from the above-mentioned kind of sterile powder, granule and tablet.Pharmaceutical preparation is preferably in unit dosage form.In this form, preparation is further divided into unit doses that contain appropriate amount of active ingredients.Therefore, pharmaceutical composition can be administered in various unit dosage forms according to the method of administration.For example, the unit dosage form suitable for oral administration includes but is 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 its composition 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 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., approximately 10 3 ~10 12 PFU of the recombinant virus is used. The effective dose is about 1.0 pfu / kg body weight to about 10 15 pfu / kg body weight (e.g., approximately 10 2 pfu / kg body weight ~ approx. 10 13 pfu / kg body weight). The recombinant adenovirus is administered in a single dose or in multiple doses (e.g., 2, 3, 4, 6, or more doses). The multiple doses can be administered simultaneously or intermittently (e.g., over a period of several days or weeks).

[0122] In some embodiments, the methods provided include administering to the 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 a therapeutic transgene may be accompanied by the 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 antimitotics, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, antihormones (e.g., antiandrogens), 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), alkyl sulfonates (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, for example, 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 adrenocortical 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, hydrair, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, e.g., docetaxel), velban, vincristine, VP-16, although 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 immunomodulatory factors 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 phototherapy) to the tumor site to eradicate the tumor or help it shrink before surgical removal.

[0131] CDK (cyclin-dependent kinase) inhibitor is a drug that inhibits the function of CDK.Non-limiting examples of CDK inhibitors that can be used 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 drug and dosage can be easily determined by those skilled in the art based on the given disease being treated.The combination of drug or composition can be administered either in parallel (for example, as a mixture), separately but simultaneously (for example, through separate intravenous lines), or sequentially (for example, one drug is administered first, and then the second drug is administered).Therefore, the term combination is used to refer to the parallel administration, simultaneous administration, or sequential administration of two or more drugs 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. [Example]

[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). The 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). A control virus (AdSyn-CO171) contains the Ad5 fiber protein (i.e., both the shaft and knob domains are derived from Ad5). Both viruses were generated using 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. Native Ad5 vectors infect only 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 Kupfer cells in the liver, preventing systemic dissemination and inducing limited inflammation. To overcome this and enable 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 a transgene for a microRNA (miR-122) 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, which expresses a chimeric fiber protein with the Ad34 knob domain, traffics specifically 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 The stop codon encodes an oncogene. In the absence of Cre recombinase, the stop codon is converted to a mutant Kras (Kras G12D However, in the presence of Cre recombinase, the stop codon is removed, resulting in the expression of Kras G12D This allows the 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 specifically expressed in pancreatic cells, and therefore both copies of p53 are deleted in pancreatic cells. By crossing strains, Cre driven by the Pdx1 promoter resulted in the deletion of both alleles of the tumor suppressor p53 and the expression of mutant Kras in pancreatic cells. G12D This gene mediates activation of the Kras;p53 / p53;Cre gene, resulting in the development of progeny. Homozygous mice, designated "Kras;p53 / p53;Cre," developed pancreatic tumors within 5–7 weeks. AdSyn-CO176 was intravenously injected into Kras;p53 / p53 and Kras;p53 / p53;Cre mice. 72 h after viral injection, tissues were collected, incubated with luciferin for 5 minutes, and then scanned for 5 minutes using an IVIS™ imaging system. As shown in Figure 1B, Kras;p53 / p53 mice had normal pancreases, and the luciferase signal was primarily derived from the spleen. In contrast, Kras;p53 / p53;Cre mice developed pancreatic tumors, and the signal was primarily 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 develop. AdSyn-CO176 was intravenously injected 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, with the signal primarily originating from the spleen. In contrast, 4-month-old Kras;p53 / +;Cre mice developed pancreatic tumors, with the signal primarily originating from the tumor and liver.

[0140] Heterozygous Kras;p53 / +;Cre mice develop pancreatic tumors between 4 and 9 months of age. 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 minutes, and scanned for 4 minutes using an IVIS™ imaging system. Although the pancreas of 2-month-old Kras;p53 / +;Cre mice appeared normal, luciferase signals were detected 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 from a 2-month-old Kras;p53 / +;Cre mouse showed that a small portion of the pancreas developed tumors (Figure 3C, indicated by the polygon), whereas most of the pancreatic tissue appeared normal. This result indicated that AdSyn-CO176 can 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 were performed. CK19 is a marker for tumor cells, while smooth muscle actin (SMA) is a marker for stromal cells. IHC staining of AdSyn-CO176-infected pancreatic tumors showed that GFP expressed by AdSyn-CO176 overlapped with SMA staining, indicating that AdSyn-CO176 targeted stromal cells (Figure 4A). IF staining of AdSyn-CO176-infected pancreatic tumors also demonstrated that GFP overlapped with SMA staining (Figure 4B), confirming that AdSyn-CO176 infects stromal cells. Glioblastoma model

[0142] Figure 5A shows a schematic diagram of the Cre-mediated gene-engineered glioblastoma model. Lentivirus was injected directly 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, resulting in the expression of HRas primarily in astrocytes. V12 Expression of lentivirally 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 induce tumor formation in the brain one week after injection. GFP signal was used to demonstrate glioblastoma formation. Saline, AdSyn-CO171, or AdSyn-CO176 was intravenously (IV) injected into GFAP-Cre mice that had 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 brains) and GFAP-Cre mice injected with tumor-inducing lentivirus were injected with AdSyn-CO171 or AdSyn-CO176. Brain tissue was collected 72 hours after injection of the synthetic adenovirus, incubated with luciferin for 5 minutes, and scanned for 5 minutes using an IVIS™ imaging system (Figure 5C). Only GFAP-Cre mice injected with tumor-inducing lentivirus showed luciferase signals from AdSyn-CO176. This demonstrates that AdSyn-CO176 migrates to brain tissue only in the presence of tumors. Brain tissue was also scanned for GFP signals (Figure 5D). The GFP signal was 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 results in transient brain damage at the injection site. Synthetic adenoviruses (AdSyn-CO171 and AdSyn-CO176) were injected 4 weeks after the initial lentivirus injection, but it remained unclear whether AdSyn-CO176 migration into glioblastomas was driven by tumor or injection site damage. To address 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. Four weeks later, AdSyn-CO171 was intravenously injected. As shown in Figure 6A, no luciferase signal derived from AdSyn-CO171 was present in the brain in any of the mouse groups. GFAP-Cre mice were injected with HBSS or tumor-inducing lentivirus, or no injection was given. Four weeks later, the mice were intravenously injected with AdSyn-CO176. As shown in Figure 6B, luciferase signals were detected only in the brains of mice that received tumor-inducing lentivirus, whereas no signals were observed in mice that received HBSS or no injection. 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 a xenograft model

[0145] This example describes the finding that synthetic adenoviruses expressing chimeric fiber proteins 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, allowing for tumor growth monitoring. U87-tdTomato cells were intracranially injected into NSG mice to develop glioblastoma tumors, which typically take 4–8 weeks to develop (Figure 7A). This glioblastoma xenograft model was used to determine whether AdSyn-CO176 could migrate into human glioblastoma tumors. Four weeks after the mice received intracranial injections of U87-tdTomato cells, AdSyn-CO171 (SEQ ID NO: 1) and AdSyn-CO176 (SEQ ID NO: 2) were intravenously injected into NSG mice via tail vein injection. Forty-eight hours after virus injection, liver, spleen, and brain tissues were collected, incubated with luciferin for 5 minutes, and then scanned for 1 minute using an IVIS™ imaging system. As shown in Figure 7B, only mice injected with AdSyn-CO176 exhibited luciferase signals in the brain, which completely overlapped with tdTomato expression. Therefore, these results demonstrate that AdSyn-CO176, but not AdSyn-CO171, can translocate into human glioblastoma tumors. Example 4 Synthetic adenoviruses that target tumor stroma and express 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 conducted to determine whether a therapeutic transgene could be incorporated into AdSyn-CO176 (SEQ ID NO: 2) to enable tumor treatment. To perform this study, KPCL (Kras G12D We used a KPCL mouse model (p53 knockout; Pdx1-Cre; firefly luciferase) (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 in "Kras;p53 / p53;Cre" mice.

[0149] Two additional synthetic adenoviruses, AdSyn-CO987 (SEQ ID NO: 5) and AdSyn-CO989 (SEQ ID NO: 6), were generated. 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 immediately after TK into the genome of AdSyn-CO176. 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, the mice were injected i.p. or i.v. with GCV. Three control groups were used: AdSyn-CO989 + GCV; AdSyn-CO987 followed by saline injection (AdSyn-CO987 + saline); and GCV injection only (i.p. or i.v.). 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 tumors was analyzed during treatment to monitor tumor growth. The results are shown in Figure 8C. Mouse Z619R was treated with 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 control mouse Z619R (indicating an increase in tumor size), while the signal decreased in mice Z601R and Z607R (indicating a decrease in tumor size).

[0152] The histology of pancreatic tumors was also evaluated by H&E staining (Figure 9A). Mice Z655, 1806, Z619, and Z621 were all control mice. Mouse Z655 was treated with GCV alone by ip injection; mouse 1806 was treated with GCV alone by iv injection; mouse Z619 was treated with AdSyn-CO987 plus saline; and mouse Z621 received no treatment. Mouse Z656 was treated with AdSyn-CO987 plus GCV iv. Compared with the 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 recognized that the illustrated embodiments are merely examples of the invention and are not to be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. 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 tumor cells 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 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 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 item 1, wherein the transgene is a therapeutic transgene. (Item 9) 9. The method of claim 8, wherein the therapeutic transgene encodes an anti-cancer 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 of 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 anti-cancer 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) 22. The method of claim 21, wherein the modified hexon protein comprises an E451Q mutation. (Item 23) 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 microRNAs. (Item 27) 27. The method of claim 26, wherein the spleen-specific microRNA is miR142-3p. (Item 28) 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 adenoviral genome comprising a nucleotide sequence at least 95% identical to SEQ ID NO:2 or SEQ ID NO:5. (Item 37) 37. The synthetic adenoviral genome of item 36, comprising SEQ ID NO: 2 or SEQ ID NO: 5.

Claims

1. 1. A composition for expressing a therapeutic transgene in tumor stromal cells of a subject, comprising: the therapeutic transgene inserted into the E1 region, encoding an anti-cancer drug; Native or modified capsids that detarget synthetic adenoviruses from the liver, and Adenovirus type 34 (Ad34) fiber protein or a chimeric fiber protein comprising an adenovirus type 5 (Ad5) shaft domain and an Ad34 knob domain and the synthetic adenovirus comprising the modified capsid comprises a modified hexon protein; The composition, wherein the tumor is a pancreatic tumor or a glioblastoma.

2. 1. A composition for treating a tumor in a subject, comprising: a therapeutic transgene inserted into the E1 region, encoding an anti-cancer drug; Native or modified capsids that detarget synthetic adenoviruses from the liver, and Adenovirus type 34 (Ad34) fiber protein or a chimeric fiber protein comprising an adenovirus type 5 (Ad5) shaft domain and an Ad34 knob domain the synthetic adenovirus comprising the compound, which migrates to tumor stromal cells, the modified capsid comprises a modified hexon protein; The composition, wherein the tumor is a pancreatic tumor or a glioblastoma.

3. 3. The composition of claim 1, wherein the anticancer agent is selected from the group consisting of a pro-inflammatory molecule or cytokine, an anti-angiogenic factor, an inhibitor of KRas, and an inhibitor of CTLA-4, PD-1, CEA, or MUC1.

4. 4. The composition of any one of claims 1 to 3, wherein the synthetic adenovirus comprises a modified capsid that detargets the synthetic adenovirus from the liver.

5. The composition of claim 4, wherein the modified hexon protein comprises an E451Q mutation.

6. 6. The composition of any one of claims 1 to 5, wherein the synthetic adenovirus further comprises one or more binding sites for liver-specific microRNAs.

7. The composition of claim 6, wherein the liver-specific microRNA is miR-122.

8. 8. The composition of claim 6 or claim 7, wherein the one or more binding sites are in the 3'UTR of the transgene.

9. 9. The composition of any one of claims 1 to 8, wherein the synthetic adenovirus further comprises one or more binding sites for spleen-specific microRNAs.

10. The composition of claim 9, wherein the spleen-specific microRNA is miR142-3p.

11. 11. The composition of claim 9 or claim 10, wherein the one or more binding sites are in the 3'UTR of the transgene.

12. 12. The composition of any one of claims 1 to 11, wherein expression of the transgene is regulated by a tissue-specific promoter.

13. 13. The composition of any one of claims 1 to 12, wherein the synthetic adenovirus is derived from an Ad5 vector genome.

14. 14. The composition of claim 13, wherein the synthetic adenovirus comprises an Ad5 capsid protein and a chimeric fiber protein comprising an Ad5 shaft domain and an Ad34 knob domain.

15. The composition of any one of claims 1 to 14, wherein the tumor is a pancreatic tumor.

16. The composition of any one of claims 1 to 14, wherein the tumor is a glioblastoma.

17. 17. The composition of any one of claims 1 to 16, wherein the genome of the synthetic adenovirus comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

18. 18. The composition of claim 17, wherein the genome of the synthetic adenovirus comprises the nucleotide sequence of SEQ ID NO:

5.

19. A synthetic adenovirus having a genome comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

20. 20. The synthetic adenovirus of claim 19, having a genome comprising the nucleotide sequence of SEQ ID NO:

5.

21. 17. The composition of any one of claims 3 to 16, wherein the pro-inflammatory molecule or cytokine is selected from the group consisting of 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), and epidermal growth factor (EGF).

22. The composition according to any one of claims 3 to 16, wherein the anti-angiogenic factor is an inhibitor of vascular endothelial growth factor (VEGF).

23. 23. The composition of any one of claims 1-16 or 21-22, wherein the therapeutic transgene is operably linked to an EF1α promoter.

24. The composition of any one of claims 1 to 16 or 21 to 23, wherein the synthetic adenovirus is a capsid-swapped adenovirus.

25. The composition of any one of claims 1 to 18 or 21 to 24, wherein the composition is for intravenous administration to the subject.

26. The composition of any one of claims 1 to 18 or 21 to 25, which reduces tumor size in the subject.

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