Adenovirus having a modified adenovirus hexon protein

Abstract

The present invention discloses a human adenovirus species C having a capsid with a modified adenovirus hexon protein, the modified adenovirus hexon protein having a modified HVR1 region, the modified HVR1 region having the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1). The present invention further discloses the adenovirus of the present disclosure for use in treating or preventing human disease. The present invention further discloses a nucleic acid encoding the modified adenovirus hexon protein. The present invention further discloses the use of an adenovirus according to the present disclosure for transducing mesenchymal stromal cells (MSCs) or tumor cells. The present invention further discloses an in vitro method for transducing MSCs and transduced MSCs obtained by the method. The present invention further discloses the transduced MSCs of the present disclosure for use in treating disease.

Description

Technical Field

[0001] The present invention relates to human adenovirus species C having a capsid with a modified adenovirus hexon protein, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1). The present invention further relates to the adenovirus of the present invention for use in the treatment or prevention of human diseases. The present invention further relates to a nucleic acid encoding a modified adenovirus hexon protein. The present invention further relates to the use of the adenovirus of the present invention for transducing mesenchymal stromal cells (MSCs) or tumor cells. The present invention further relates to an in vitro method for transducing MSCs and the transduced MSCs obtained by that method. The present invention further relates to the transduced MSCs of the present invention for use in the treatment of diseases.

Background Art

[0002] Adenoviruses are non-enveloped viruses belonging to the family Adenoviridae. They have a linear double-stranded DNA genome of approximately 36 kilobases (kb) in size. Currently, 89 different human adenovirus (HAdV) types are known and are classified into 7 species called species A to G. HAdV species C has 6 types: type 1, type 2, type 5, type 6, type 57, and type 89.

[0003] Recombinant adenoviruses may be used in prophylactic or therapeutic settings to introduce nucleic acids, proteins, or other molecules into a patient's cells. However, for example, efficient delivery to tumors after systemic administration of HAdV is still difficult due to several cellular and acellular non-target interactions and sequestration mechanisms.

[0004] Mesenchymal stromal cells (MSCs) are an interesting potential carrier of adenovirus to tumor tissue because they show natural migratory behavior to tumor tissue. However, although adenovirus can infect most types of target cells, MSCs that do not express coxsackievirus and adenovirus receptor (CAR) rarely infect adenovirus in vitro.

[0005] Therefore, new tools and methods are needed to overcome the current limitations of using adenovirus in medicine. As prior art document information related to the invention of this application, there are the following (including documents cited at the international stage after the international filing date and documents cited when entering the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) US Patent Application Publication No. 2013 / 302313 (Patent Document 2) International Publication No. 2016 / 118433 (Patent Document 3) International Publication No. 2016 / 049201 (Non-patent document) (Non-patent Document 1) KRUTZKE L ET AL, "Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, Vol.235, 11 June 2016 (2016-06-11), page 379-392

Summary of the Invention

[0006] In a first aspect, the present invention relates to a human adenovirus species C having a capsid with a modified adenovirus hexon protein, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1).

[0007] In a second aspect, the present invention relates to the adenovirus of the present invention for use in treating or preventing human diseases.

[0008] In a third aspect, the present invention relates to a nucleic acid encoding a modified adenovirus hexon protein of human adenovirus species C, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence of SEQ ID NO: 1.

[0009] In a further aspect, the present invention relates to the use of the adenovirus of the present invention for transducing mesenchymal stromal cells (MSCs) or tumor cells.

[0010] In a further aspect, the present invention relates to an in vitro method for transducing MSCs, said method comprising: contacting a plurality of MSCs with the adenovirus of the present invention and having.

[0011] In a further aspect, the present invention relates to transduced MSCs obtainable by the method of the present invention.

[0012] In a further aspect, the present invention relates to the transduced MSCs of the present invention for use in the treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

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Embodiments for Carrying Out the Invention

[0014] In a first aspect, the present invention relates to a human adenovirus species C having a capsid with a modified adenovirus hexon protein, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1).

[0015] Human adenovirus (HAdV) species C has six types, namely types 1, 2, 5, 6, 57, and 89. The complete nucleotide sequences representing all adenovirus species C prototypes are registered in GenBank and are available: HAdV-C1 (AC_000017.1), HAdV-C2 (AC_000007.1), HAdV-C5 (AC_000008.1), HAdV-C6 (FJ349096.1), HAdV-C57 (HQ003817.1), and HAdV-C89 (MH121097.1).

[0016] Adenoviruses have an icosahedral capsid. The outer shell of the capsid has three main proteins: hexon, penton base, and fiber. These three capsid proteins contribute to most of the activities required for the initial stage of adenovirus infection. The hexon protein of adenovirus occupies most of the outer shell of the capsid, forms 240 homotrimers, and capsidates most of the virus, including the viral genome and related proteins. The fiber proteins protrude from each of the 12 vertices of the icosahedron, and the penton base is located at the bottom of each fiber protein.

[0017] The adenovirus hexon protein has seven hypervariable regions (HVRs) designated as HVR1 to HVR7. The modified adenovirus hexon protein of the present invention has a modified HVR1 region, and the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1). In other words, the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQ (SEQ ID NO: 1). The amino acid sequence of SEQ ID NO: 1 is derived from the wild-type amino acid sequence of HVR1 of the HAdV5 hexon protein by replacing the amino acids over 13 consecutive amino acid residues of the wild-type sequence, namely EEEDDDNEDEVDE (SEQ ID NO: 6), with four consecutive lysine residues. Due to the modified HVR1 region, the modified adenovirus hexon protein is different from each wild-type adenovirus hexon protein.

[0018] The modified HVR1 region can also have additional amino acids located at the N-terminus or C-terminus of SEQ ID NO: 1. Typically, the modified HVR1 region is located at the C-terminus of SEQ ID NO: 1 and has five additional amino acids corresponding to each wild-type amino acid.

[0019] The present invention is based on the discovery that in a modified adenovirus hexon protein having a modified HVR1 region, the modified HVR1 region has the amino acid sequence of SEQ ID NO: 1, resulting in an adenovirus having improved properties. Importantly, no improved properties could be observed with other similar modifications of the HVR1 region.

[0020] The inventors set out to generate six modified (mutant) adenovirus vectors, each having a different type of modified HVR1 region in the hexon protein (Figure 1B). All mutants were designed to reduce the surface negative charge of the viral particles. The mutant named HAdV5-Mut3 has a modified HVR1 region having the sequence of SEQ ID NO: 1 (Figure 1A). The complete sequence of the HVR1 region of the modified hexon protein of HAdV5-Mut3 is DEAATALEINLKKKKQAEQQKTHVF (SEQ ID NO: 43).

[0021] The inventors found that certain modifications of the HVR1 region render the production of mutant viral vectors impossible. The production of the mutant vectors named HAdV5-Mut4, HAdV5-Mut5, and HAdV5-Mut6 was not achievable.

[0022] Furthermore, it was found that the production of three additional modified (mutant) adenovirus vectors named HAdV5-Mut7, HAdV5-Mut8, and HAdV5-Mut9 (Figure 14), each having a different type of modified HVR1 region in the hexon protein, was also impossible.

[0023] Furthermore, the inventors have found that deleting the negatively charged HVR1 loop (mutant HAdV5-ΔHVR1) only slightly reduces the negative surface charge of the viral particles. In the case of mutant HAdV5-Mut2, substituting four aspartic acids with lysine further reduced the negative surface charge. Interestingly, however, HAdV5-Mut3 with the modified HVR1 region of SEQ ID NO:1 was shown to have a significantly reduced negative surface charge compared to HAdV5 wild type, HAdV5-ΔHVR1, and HAdV5-Mut2.

[0024] Surprisingly, the inventors further found that HAdV5-Mut3 showed a significant decrease in the transduction of CAR-negative cells via human coagulation factor X (FX) compared to HAdV5 wild type, HAdV5-ΔHVR1, and HAdV5-Mut2. Binding of FX to HAdV5 mediates the transduction of hepatocytes and induces the sequestration of the particles. This is one of the major obstacles for efficient delivery of HAdV to, for example, tumors after systemic administration of HAdV. The adenovirus of the present invention overcomes this obstacle. On the other hand, HAdV5-ΔHVR1 and HAdV5-Mut2 did not show a decrease in FX binding despite the modification of the HVR1 region being similar to that of HAdV5-Mut3.

[0025] The significant decrease in the binding of FX to HAdV5-Mut3, which consequently reduced the transduction of hepatocytes by HAdV5-Mut3, was an unexpected finding. According to Alba et al. (2009), the interaction between FX and the HAdV5 capsid occurs through binding to HVR5 and HVR7 of the adenovirus hexon protein. In addition, the interactions between FX and HVR3, HVR5, and HVR7 of the adenovirus hexon protein have also been reported. On the other hand, the role of HVR1 in FX binding was not known and not expected. Therefore, it was a surprising discovery that the modification of the HVR1 region of the adenovirus hexon protein affected FX binding. Importantly, the type of modification in the HVR1 region is crucial for affecting FX binding. This is evident from the fact that HAdV5-ΔHVR1 and HAdV5-Mut2 did not show a decrease in FX binding, despite the modification in the HVR1 region being similar to that of HAdV5-Mut3.

[0026] More specifically, it has been found from the inventors' findings that simply introducing the number of 4 or 6 lysine residues may not be sufficient. In HAdV5-Mut4, 5 glutamate residues and 1 aspartic acid residue were substituted with lysine residues. HAdV5-Mut4 was not viable. In HAdV5-Mut2, 4 aspartic acid residues were substituted with lysine residues. However, HAdV5-Mut2 did not show a decrease in FX binding.

[0027] The inventors' findings indicate that a decrease in FX binding was only observed for HAdV5-Mut3 into which 4 continuously arranged lysine residues were introduced. Therefore, the investigation results indicate that it is necessary to introduce 4 consecutive lysine residues.

[0028] Similarly, the inventors have found that simply deleting a stretch of 13 amino acids in the HVR1 region without introducing lysine residues, as in HAdV5-ΔHVR1, is not sufficient to affect FX binding.

[0029] Furthermore, the inventors have found that the adenoviruses of the present invention also overcome another obstacle for efficient delivery of HAdV to tumors, for example, after systemic HAdV administration, namely neutralization by natural IgM antibodies. This finding was unexpected because HAdV5-ΔFX particles, which also show a decrease in FX binding, were almost completely neutralized by natural IgM when cultured with plasma from both humans and mice. Interestingly, however, this effect was not observed when HAdV5-Mut3 was cultured with human or mouse plasma samples, even though HAdV5-Mut3 shows a similar decrease in FX binding as HAdV5-ΔFX. Thus, the inventors have shown that the modified HVR1 region of HAdV5-Mut3 enables vector particles lacking the FX shield to escape from natural IgM. This also prevents IgM-mediated binding of the adenoviruses of the present invention to red blood cells, which is another barrier after systemic, especially intravenous, administration of HAdV.

[0030] Another problem for efficient systemic administration of HAdV is Kupffer cells, macrophages present in the liver. Kupffer cells have scavenger receptors on their cell surface, which bind and capture negatively charged molecules, thereby taking up HAdV. Uptake by mouse macrophages was significantly reduced in all three mutant vectors, HAdV5-ΔHVR1, HAdV5-Mut2, and HAdV5-Mut3, compared to wild-type HAdV5, but the effect was most pronounced in HAdV5-Mut3.

[0031] In summary, the adenoviruses of the present invention overcome multiple obstacles related to systemic administration of HAdV that have been faced hitherto. Therefore, the adenoviruses of the present invention are particularly useful for systemic administration.

[0032] Furthermore, it was further found that the adenoviruses of the present invention have transduction efficiency of tumor cells that is improved or similar as compared to each HAdV wild type. Thus, the adenoviruses of the present invention are also particularly useful for use as oncolytic viruses.

[0033] As used herein, the term "cell transduction" means the process of introducing one or more nucleic acids into a cell using an adenovirus. The adenovirus is typically a recombinant adenovirus produced by an appropriate packaging cell. An important use of cell transduction by recombinant adenoviruses is the viral-mediated delivery of one or more genes to target cells, such as target cells of a patient during gene therapy.

[0034] As used herein, the term "transduction efficiency" means the ratio or percentage of target cells that have been successfully transduced by an adenovirus after cell transduction.

[0035] As used herein, the term "target cell" means a cell that is transduced (infected) by an adenovirus.

[0036] Even though HAdV5 was used in the studies underlying the present invention, the present invention is not limited to HAdV5. The findings of the present inventors are similarly applicable to all HAdV species C having a modified adenovirus hexon protein, where the modified adenovirus hexon protein has a modified HVR1 region having the sequence of SEQ ID NO: 1.

[0037] In a preferred embodiment, the adenovirus is adenovirus type 5. Adenovirus type 5 is also referred to herein as HAdV5 type, HAdV-C5, HAdV-5 or HAdV5. Virus vectors based on HAdV5 type belong to the most commonly used vectors in gene therapy research. These have been extensively studied over the years both preclinically and clinically, especially as oncolytic adenoviruses for cancer treatment. The amino acid sequence of SEQ ID NO: 1 is derived from a part of the wild-type amino acid sequence of the HVR1 region of the HAdV5 type hexon protein.

[0038] In another embodiment, the adenovirus is adenovirus type 1, 2, 6, 57, or 89.

[0039] In a preferred embodiment, the modified adenovirus hexon protein has an unmodified (wild-type) HVR5 and / or HVR7 region.

[0040] In a preferred embodiment, the adenovirus is an adenovirus vector or an oncolytic adenovirus.

[0041] As used herein, the term "adenovirus vector" means a replication-deficient adenovirus vector. Based on adenovirus, there are different replication-deficient vector types. Adenovirus (Ad) vectors usually have deletions of at least the E1A and E1B genes and are thus replication-deficient in human cells. Production is carried out in human complementing cell lines that express the E1A and E1B proteins and in which the E1A and E1B genes are integrated into the chromosome. For example, the delta E1 Ad vector (also called the E1-deleted Ad vector or first-generation Ad vector) is widely used as an experimental tool in preclinical research and development, clinical research, product development in the context of gene therapy or gene vaccination. This vector type is made replication-deficient by removing the E1 region encoding the E1A and E1B proteins. Furthermore, the E3 region may be deleted in this vector type. Second-generation Ad vectors have deletions of the E2 gene and / or the E4 gene in addition to the deletion of the E1 gene (and optionally the E3 region). In high-capacity Ad (HC-Ad) vectors (also called helper-dependent Ad vectors), all viral coding sequences are replaced with the transgene of interest.

[0042] An adenovirus vector usually carries an expression cassette that expresses RNA encoding a protein or non-coding RNA that does not encode a protein, such as small hairpin RNA (shRNA) or microRNA (miRNA), under the control of a promoter sequence.

[0043] As used herein, the term "oncolytic adenovirus" refers to a replication-competent adenovirus. Replication-competent adenoviruses are in clinical development primarily for the treatment of cancer, particularly solid tumors. These viruses grow within tumor cells and destroy the tumor cells. With regard to safe clinical use, tumor-selective replication is thought to reduce or prevent damage to non-tumorigenic healthy tissues. Therefore, it is also referred to as a conditionally replication-competent adenovirus. To achieve tumor-selective activity, many different strategies have been pursued. A common strategy involves deletion of the Rb-binding site of E1A (referred to as delta-24), which results in E2F-dependent replication of the adenovirus in tumor cells but not in non-tumor cells. Other strategies rely on using tumor- or tissue-specific control promoter elements to control the expression of adenovirus genes (such as E1A) that are essential for a particular cell type. Overall, oncolytic adenoviruses are promising tools for cancer treatment.

[0044] As described above, the adenoviruses of the present invention have been found to have a transduction efficiency of tumor cells that is improved or similar compared to each HAdV wild type. Therefore, the adenoviruses of the present invention are particularly useful for use as oncolytic viruses.

[0045] In a preferred embodiment, the adenovirus has a transgene. As used herein, the term "transgene" refers to a gene or genetic material that is non-native to the adenovirus and is delivered to a target cell by transducing the target cell with an adenovirus having the transgene. In a preferred embodiment, the adenovirus has one or several transgenes, preferably two or three transgenes.

[0046] In a preferred embodiment, the capsid has at least one additional capsid modification. The additional capsid modification can be present in different adenovirus capsid proteins such as, for example, a modified adenovirus hexon protein and / or an adenovirus fiber protein, penton base protein or minor capsid protein IX. It is known to introduce several modifications into these proteins in order to obtain adenoviruses with altered properties.

[0047] In a preferred embodiment, the additional capsid modification is a modified adenovirus fiber protein. In a preferred embodiment, the modified adenovirus fiber protein lacks coxsackievirus and adenovirus receptor (CAR) binding due to excision of the CAR binding site of the fiber protein by genetic modification of the viral gene encoding the fiber protein. The lack of CAR binding inhibits the binding of HAdV to erythrocytes, thus preventing the sequestration of viral particles by erythrocytes, especially upon intravenous administration. Thus, by using both a modified adenovirus hexon protein having a modified HVR1 region of SEQ ID NO: 1 and a modified adenovirus fiber protein lacking CAR binding, an adenovirus can be obtained that has no non-target interaction with hepatocytes, no non-target interaction with natural IgM antibodies, and no IgM or CAR-mediated non-target interaction with erythrocytes. Such adenoviruses are particularly useful for systemic administration.

[0048] In a second aspect, the present invention relates to the adenovirus of the present invention for use in treating or preventing human diseases. As described above, the adenovirus of the present invention is particularly useful for systemic administration. Therefore, the adenovirus of the present invention is particularly suitable for use in treating or preventing human diseases.

[0049] In a preferred embodiment, the disease is treated or prevented by gene therapy. As used herein, the term "gene therapy" means the therapeutic delivery of nucleic acids to a patient's cells for treating or preventing a disease. The nucleic acid encodes a therapeutic molecule, such as a therapeutic protein, that is expressed by the transduced cells.

[0050] In a preferred embodiment, the disease is treated or prevented by genetic vaccination. As used herein, the term "genetic vaccination" refers to the delivery of nucleic acids to a subject's cells to generate a protective or therapeutic immunological response for protecting the subject against a disease or treating an existing disease. The nucleic acid encodes an immunogen or antigen that is expressed by the transduced cells and induces an immunological response.

[0051] In a preferred embodiment, the disease is cancer. As described above, oncolytic adenoviruses are promising tools for treating cancer, and the adenoviruses of the present invention are particularly useful for use as oncolytic viruses.

[0052] In a third aspect, the present invention relates to a nucleic acid encoding a modified adenovirus hexon protein of human adenovirus species C, the modified adenovirus hexon protein having a modified HVR1 region, the modified HVR1 region having the sequence of SEQ ID NO: 1.

[0053] In a preferred embodiment, the nucleic acid has the sequence of SEQ ID NO: 2.

[0054] In a further aspect, the present invention relates to the use of an adenovirus according to the present invention for transducing mesenchymal stromal cells (MSCs) or tumor cells.

[0055] Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, are naturally equipped with the ability to migrate to tumors. Therefore, they are interesting carrier cells for delivering adenoviruses to tumor tissues. Transduced MSCs hide virus particles from the sequestration mechanism, increase the virus amount by intracellular virus replication, and transport them to the tumor site where newly generated particles are released. However, with the commonly used wild-type HAdV5 vector, MSCs are hardly transduced. Surprisingly, the inventors have found that when using the adenovirus according to the present invention, the transduction of MSCs by the HAdV vector is significantly promoted. Furthermore, the inventors have found that when transducing MSCs with the adenovirus according to the present invention, the replication of adenovirus in MSCs is improved as compared to the case of transduction with wild-type HAdV. The inventors have further confirmed that the migratory behavior of MSCs is not affected by the transduction of MSCs with the adenovirus according to the present invention. Therefore, the adenovirus according to the present invention is particularly useful for transducing MSCs.

[0056] In a preferred embodiment, the MSC is a human MSC.

[0057] As described above, it has been found that the adenovirus of the present invention has an improved or comparable transduction efficiency of tumor cells as compared to each HAdV wild type. Therefore, the adenovirus of the present invention is also useful for transducing tumor cells.

[0058] In a preferred embodiment, the tumor cell is a human tumor cell.

[0059] In a preferred embodiment, the adenovirus is used in combination with a transduction enhancer for transducing MSCs. The inventors have found that the transduction efficiency of MSCs using the adenovirus according to the present invention is further improved by using a transduction enhancer.

[0060] In a preferred embodiment, the transduction enhancer is selected from the group consisting of factor X, spermidine, spermine, hexadimethrine bromide, poly-L-lysine, and lactoferrin.

[0061] In a preferred embodiment, the transduction enhancer is factor X, spermidine, spermine, or hexadimethrine bromide.

[0062] In a more preferred embodiment, the transduction enhancer is factor X.

[0063] In a further aspect, the present invention relates to an in vitro method for transducing MSCs, the method comprising: contacting a plurality of MSCs with the adenovirus according to the present invention and having.

[0064] As described above, the adenovirus of the present invention is particularly useful for transducing MSCs. This opens the way for using MSCs as carriers of adenoviruses to tumor tissues.

[0065] In a preferred embodiment, the plurality of MSCs are further contacted with a transduction enhancer, which is preferably selected from the group consisting of factor X, spermidine, spermine, hexadimethrine bromide, poly-L-lysine, and lactoferrin.

[0066] In a further aspect, the present invention relates to transduced MSCs obtainable by the method of the present invention.

[0067] In a further aspect, the present invention relates to the transduced MSCs of the present invention for use in the treatment of diseases.

[0068] In a preferred embodiment, the disease is a human disease.

[0069] In a preferred embodiment, the disease is treated by gene therapy.

[0070] In a preferred embodiment, the disease is treated by cell therapy. As used herein, the term "cell therapy" means the therapeutic delivery of cell material to a patient for treating a disease. The cell material is generally viable cells that are intact, such as the transduced MSCs that can be obtained by the method of the present invention.

[0071] Further disclosed is an in vivo method for transducing tumor cells, said method comprising a step of contacting a plurality of tumor cells with an adenovirus according to the present invention and having.

[0072] Further disclosed is an in vitro method for transducing tumor cells, said method comprising a step of contacting a plurality of tumor cells with an adenovirus according to the present invention and having.

[0073] As described above, the adenovirus of the present invention is particularly useful for transducing tumor cells.

[0074] In a preferred embodiment, the plurality of tumor cells are further contacted with a transduction enhancer, said transduction enhancer being preferably selected from the group consisting of coagulation factor X, spermidine, spermine, hexadimethrine bromide, poly-L-lysine and lactoferrin. By using the transduction enhancer, the transduction efficiency of tumor cells by the adenovirus of the present invention is further improved.

[0075] Further disclosed are transduced tumor cells that can be obtained by an in vitro method for transducing tumor cells according to the present disclosure.

[0076] Further disclosed is a human adenovirus having a capsid with a modified adenovirus hexon protein of SEQ ID NO: 3.

[0077] The amino acid sequence of the modified adenovirus hexon protein of Array ID No. 3 is derived from the wild-type amino acid sequence of the HAdV5 hexon protein by replacing 13 consecutive amino acid residues in the HVR1 region of the wild-type sequence with four consecutive lysine residues. The modified adenovirus hexon protein of Array ID No. 3 has a modified HVR1 region having the sequence of Array ID No. 1. Accordingly, the presence of the modified adenovirus hexon protein of Array ID No. 3 confers improved properties on the adenovirus hexon. More specifically, as described above, such adenoviruses are particularly useful for systemic administration.

[0078] In a preferred embodiment, the adenovirus is of adenovirus species C, preferably adenovirus type 5.

[0079] Further disclosed is a human adenovirus species C having a capsid with a modified adenovirus hexon protein, said modified adenovirus hexon protein having a modified HVR1 region, said modified HVR1 region having 3 to 8 consecutive lysine or arginine residues.

[0080] The modified HVR1 region having the sequence of Array ID No. 1 has four consecutive lysine residues. It can be expected that the same effects found in adenoviruses having a modified hexon protein will be obtained even when the four consecutive lysine residues are inserted at a position different from the position in Array ID No. 1 of the HVR1 region. Furthermore, it can be expected that the same effects will be obtained when the modified HVR1 region has 3 to 8 consecutive lysine residues. Since arginine residues have properties chemically similar to lysine residues, particularly a positive charge, it can be further expected that the same effects will be obtained when the modified HVR1 region has 3 to 8 consecutive arginine residues.

[0081] In a preferred embodiment, 3 to 8 consecutive lysine or arginine residues are directly adjacent to one or several non-negatively charged amino acid residues. The directly adjacent amino acid residues are amino acid residues located at the N-terminus and / or C-terminus of 3 to 8 consecutive lysine or arginine residues.

[0082] In a preferred embodiment, the modified HVR1 region has 4 to 8 consecutive lysine or arginine residues.

[0083] In a preferred embodiment, the modified HVR1 region has 3 to 8, preferably 4 to 8, consecutive lysine residues.

[0084] In a further preferred embodiment, the modified HVR1 region has 4 consecutive lysine residues.

[0085] In a further aspect, the present disclosure relates to an in vitro method for transducing MSCs, the method comprising: contacting a plurality of MSCs with an adenovirus and a transduction enhancer, wherein the transduction enhancer is selected from the group consisting of factor Xa, spermidine, spermine, hexadimethrine bromide, poly-L-lysine, and lactoferrin, the contacting step and having.

[0086] MSCs that do not express coxsackievirus and adenovirus receptor (CAR) are hardly infected by adenovirus in vitro. The inventors have found that the use of a transduction enhancer significantly promotes the introduction of adenovirus into MSCs. This facilitates the use of MSCs as carriers of adenovirus to tumor tissues. The inventors have further found that the use of a transduction enhancer leads to an improvement in the replication of adenovirus in MSCs, which also benefits the medical use of MSCs as carriers of adenovirus. Furthermore, the inventors have confirmed that the migratory behavior of MSCs is not affected by the use of a transduction enhancer.

[0087] In a preferred embodiment, the adenovirus is a human adenovirus species C, and the adenovirus is preferably adenovirus type 5.

[0088] In a preferred embodiment, the transduction enhancer is factor X, spermidine, spermine or hexadimethrine bromide.

[0089] In a more preferred embodiment, the transduction enhancer is spermidine or spermine. The inventors have found that the enhanced transduction of MSCs by adenovirus is most prominent when spermidine or spermine is used as the transduction enhancer.

[0090] In another more preferred embodiment, the transduction enhancer is factor X. The inventors have found that the expression and subsequent secretion of a therapeutic protein by MSCs transfected with each recombinant adenovirus are significantly enhanced in the presence of factor X as the transduction enhancer.

[0091] In a preferred embodiment, the adenovirus has a transgene.

[0092] sequence The amino acid sequence of HAdV5 hexon is shown according to GenBank AY339865.1, positions 18,842 - 21,700. The hypervariable region 1 (HVR1) of the hexon protein is shown in bold according to Khare et al. 2012. Amino acid sequence of wild-type HAdV5 hexon: MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTV APTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTY FDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEED DDNEDEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTF QPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGIL VKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETPDTHI SYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAG QASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRI IENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKN EIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNT YDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLL GNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGND LRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAA NMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYT YSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVD GEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQP MSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPL IGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHA LDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNA TT (Sequence ID number: 5)

[0093] Amino acid sequence of HVR1 of wild-type HAdV5 hexon: DEAATALEINLEEEDDDNEDEVDEQAEQQKTHVF (Sequence ID number: 42)

[0094] Amino acid sequence of the modified hexon protein of the adenovirus of the present invention, i.e., the amino acid sequence of HAdV5-Mut3 hexon: MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTV APTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTY FDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLKKKK QAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGES QWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLE SQVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEG NSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVV DLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDE LPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFA MEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVV APGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFH IQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIK FDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPAN ATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLD GTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQC NMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDT KYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSI TQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDP MDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 3)

[0095] Amino acid sequence of HVR1 of HAdV5-Mut3 hexon: DEAATALEINLKKKKQAEQQKTHVF (Array ID number: 43)

[0096] The nucleotide sequence of HAdV5 hexon is shown according to GenBank AY339865.1, positions 18,842 - 21,700. The HVR1 of hexon is shown in bold according to Khare et al. 2012. Nucleotides encoding inserted lysine residues are shown in capital letters.

[0097] Nucleotide sequence of wild - type HAdV5 hexon: TIFF0007711959000001.tif214157(Array ID number: 4)

[0098] Nucleotide sequence of HAdV5 - Mut3 hexon: TIFF0007711959000002.tif209157(Array ID number: 2)

[0099] Nucleotide sequence of HVR1 of wild - type HAdV5 hexon: gatgaagctgctactgctcttgaaataaacctagaagaagaggacgatgacaacgaagacgaagtagacgagcaagctgagcagcaaaaaactcacgtattt (Array ID number: 44)

[0100] Nucleotide sequence of HVR1 of HAdV5 - Mut3 hexon: gatgaagctgctactgctcttgaaataaacctaAAAAAGAAAAAGcaagctgagcagcaaaaaactcacgtattt (Array ID number: 45)

[0101] Amino acid sequence of HVR1 of HAdV5 - ΔHVR1 hexon: DEAATALEINLQAEQQKTHVF (Array ID number: 46)

[0102] Amino acid sequence of HVR1 of HAdV5 - Mut2 hexon: DEAATALEINLEEEKKKNEKEVDEQAEQQKTHVF (Array ID number: 47)

[0103] Amino acid sequence of HVR1 of HAdV5-Mut4 hexon: DEAATALKINLKKNKVKQAKQQKTHVF (Array ID number: 48)

[0104] Amino acid sequence of HVR1 of HAdV5-Mut5 hexon: DEAATALEINLKKKKQAEQQKTHVF (Array ID number: 43)

[0105] Amino acid sequence of HVR1 of HAdV5-Mut6 hexon: DEAATALKINLKKNKVKQAKQQKTHVF (Array ID number: 48)

[0106] Amino acid sequence of HVR1 of HAdV5-Mut7 hexon: DEAATALEINLKKKKKKQAEQQKTHVF (Array ID number: 51)

[0107] Amino acid sequence of HVR1 of HAdV5-Mut8 hexon: DEAATALEINLKKKKKKKKQAEQQKTHVF (Array ID number: 52)

[0108] Amino acid sequence of HVR1 of HAdV5-Mut9 hexon: DEAATALEINLGGSGGGSGKKKKKKKKGSGGGSGGQAEQQKTHVF (Array ID number: 53)

[0109] In the HVR1 region of the hexon proteins of human adenovirus type 1 (HAdV1), type 2 (HAdV2), type 6 (HAdV6), and type 57 (HAdV57), the portion replaced with the amino acid sequence of SEQ ID NO: 1 is as follows (aa, amino acid): HAdV1: aa 136 - 173 of the HAdV1 hexon (GenBank: BAG48778.1): EQEEPTQEMAEELEDEEEAEEEEAEEEAEAPQADQKVK (SEQ ID NO: 7) HAdV2: aa 136 - 171 of the HAdV2 hexon (GenBank: CAC67477.1): EQTEDSGRAVAEDEEEEDEDEEEEEEEQNARDQATK (SEQ ID NO: 8) HAdV6: aa 136 - 167 of the HAdV6 hexon (GenBank: BAU36782.1) EQNETAQVDAQELDEEENEANEAQAREQEQAK (SEQ ID NO: 9) HAdV57: aa 136 - 166 of the HAdV57 hexon (GenBank: BBF89158.1): DEDDTQVQVAAEDDQDDDEEEEQLPQQRNGK (SEQ ID NO: 10) HAdV89: aa 136 - 172 of the HAdV89 hexon (GenBank: AZR67181): EQTEDSGRAVAEDEEEEEDEDEEEEEEEQNARDQATK (SEQ ID NO: 49)

[0110] The amino acid sequences of the obtained modified adenovirus hexon proteins are shown below.

[0111] The amino acid sequence of the modified adenovirus hexon protein of the adenovirus of the present invention, wherein the adenovirus is the amino acid sequence of adenovirus type 1 (amino acid sequence of HAdV1 - Mut3 hexon): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNSCEWDEAATALEINLKKKKQAEQQKTHVYAQAPLAGEKITANGLQIVSDTQTEGNPVFADPTYQPEPQVGESQWNEAEATASGGRVLKKTTPMKPCYGSYARPTNKNGGQGILVANNQGALESKVEMQFFAPSGTAMNERNAVQPSIVLYSEDVNMETPDTHISYKPSKTDENSKAMLGQQAMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGIGVTDTYQGIKSNGNGNPQNWTKNDDFAARNEIGVGNNFALEINLNANLWRNFLYSNIALYLPDKLKYTPTNVEISPNPNSYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 11)

[0112] The amino acid sequence of the modified adenovirus hexon protein of the adenovirus of the present invention, wherein the adenovirus is the amino acid sequence of adenovirus type 2 (amino acid sequence of HAdV2-Mut3 hexon): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNSCEWDEAATALEINLKKKKQAEQQKTHVYAQAPLSGETITKSGLQIGSDNAETQTKPVYADPSYQPEPQIGESQWNEADANAAGGRVLKKTTPMKPCYGSYARPTNPFGGQSVLVPDEKGVPLPKVDLQFFSNTTSLNDRQGNATKPKVVLYSEDVNMETPDTHLSYKPGKGDENSKAMLGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGIGVTDTYQAIKANGNGSGDNGDTTWTKDETFATRNEIGVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYNPTNVEISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKEYQQVGILHQHNNSGFVGYLAPTMREGQAYPANVPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 12)

[0113] The amino acid sequence of the modified adenovirus hexon protein of the adenovirus of the present invention, wherein the adenovirus is the amino acid sequence of adenovirus type 6 (amino acid sequence of HAdV6-Mut3 hexon): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNSCEWDEAATALEINLKKKKQAEQQKTHVYAQAPLSGIKITKEGLQIGTADATVAGAGKEIFADKTFQPEPQVGESQWNEADATAAGGRVLKKTTPMKPCYGSYARPTNSNGGQGVMVEQNGKLESQVEMQFFSTSTNATNEVNNIQPTVVLYSEDVNMETPDTHLSYKPKMGDKNAKVMLGQQAMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGIGITDTFQAVKTTAANGDQGNTTWQKDSTFAERNEIGVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYNPTNVEISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGIIHQHNNSGFVGYLAPTMREGQAYPANVPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 13)

[0114] The amino acid sequence of the modified adenovirus hexon protein of the adenovirus of the present invention, wherein the adenovirus is the amino acid sequence of adenovirus type 57 (amino acid sequence of HAdV57-Mut3 hexon): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNSCEWDEAATALEINLKKKKQAEQQKTHVYAQAPFAGEAINKNGLQIGTNGAATEGNKEIYADKTYQPEPQIGESQWNEAESSVAGGRVLKKTTPMKPCYGSYARPTNSNGGQGVMVEQNGKLESQVEMQFFSTSVNAMNEANAIQPKLVLYSEDVNMETPDTHLSYKPGKSDDNSKAMLGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGIGVTDTYQAIKATNGNGGATTWAQDNTFAERNEIGVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYNPTNVEISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 14)

[0115] The amino acid sequence of the modified adenovirus hexon protein of the adenovirus of the present invention, wherein the adenovirus is the amino acid sequence of adenovirus type 89 (amino acid sequence of HAdV89-Mut3 hexon): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNSCEWDEAATALEINLKKKKQAEQQKTHVYAQAPLSGETITKSGLQIGSDNAETQAKPVYADPSYQPEPQIGESQWNEADANAAGGRVLKKTTPMKPCYGSYARPTNPFGGQSVLVPDEKGVPLPKVDLQFFSNTTSLNDRQGNATKPKVVLYSEDVNLETPDTHLSYKPGKGDENSKAMLGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGIGVTDTYQAIKANGNGAGDNGNTTWTKDETFATRNEIGVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYNPTNVEISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGIIHQHNNSGFVGYLAPTMREGQAYPANVPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHQPHRGVIETVYLRTPFSAGNATT (Array ID number: 50)

[0116] A further aspect of the present invention will be apparent to those skilled in the art from the description of the accompanying examples, particularly the scientific results.

[0117] Examples Materials and Methods Cell All cells were cultured at 90% humidity, 5% CO2, and 37°C and passaged twice a week at the indicated split ratios. Cells were detached with 0.05% trypsin-EDTA, except for J774A.1 cells that were detached by scraping.

[0118] A549 cells (ATCC#CCL-185; split ratio: 1:8) were grown in Eagle's minimum essential medium (Gibco) supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine.

[0119] J774A.1 cells (ATCC#TIB-67; split ratio: 1:10) were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine.

[0120] SKOV-3 cells (ATCC#HTB-77; split ratio: 1:3) were grown in Roswell Park Memorial Institute 1640 medium (Gibco) supplemented with 5% FBS and 1% penicillin / streptomycin / glutamine.

[0121] UM-SCC-11B cells (cancer cells derived from human head and neck squamous cell carcinoma, provided by the kindness of Professor Dr. Brunner of the University Clinic, split ratio: 1:10) were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin / glutamine.

[0122] MiaPaCa cells (ATCC#CRL-1420, split ratio 1:7 to 1:15) were grown in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Gibco) supplemented with 10% FBS, 1x GlutaMAX™, and 1x penicillin / streptomycin.

[0123] Huh7 cells (JCRB0403, split ratio 1:10) were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% FCS and 1% penicillin / streptomycin / glutamine.

[0124] HepG2 cells (ATCC#HB-8065, split ratio 1:4 to 1:7) were grown in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine.

[0125] Human mesenchymal stromal cells (MSCs) (provided by the kindness of the Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Ulm, Germany and prepared as described by Fekete, Rojewski et al.) were grown in BioWhittaker® Alpha Minimum Essential Medium (Lonza) supplemented with 8% irradiated pooled human platelet lysate (PL) (prepared as described by Fekete, Gadelorge et al., 2012 and provided by the Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Ulm) and 500 units of heparin (Ratiopharm).

[0126] Virus and vector The replication-incompetent human adenovirus type 5 (HAdV5) vector particles carried a CMV promoter-driven enhanced GFP (eGFP) expression cassette subcloned from the pEGFP-N1 plasmid (Clontech 6085-1) inserted in the opposite direction into the deleted E1 region (GenBank ID: AY339865.1, sequences of nt 1-440 and nt 3523-35935).

[0127] The HAdV5-ΔCAR vector particles further carried a point mutation (Y→477A) in the fiber knob, which significantly reduced CAR binding (Kirby et al., 2000).

[0128] The HAdV5-ΔFX vector particles further carried a point mutation (E→451Q) in the hypervariable region (HVR) 7 of the hexon protein, which did not abolish but significantly decreased the binding of factor X of blood coagulation to the vector capsid (Krutzke et al., 2016).

[0129] The HAdV5-ΔHVR1 particles carried a 13-amino acid deletion (ΔEEEDDDNEDVDE (SEQ ID NO: 6); nt 19280-19318) within the negatively charged region of HVR1 of the hexon protein, reducing the negative surface charge of HAdV5 particles (Alemany et al., 2000).

[0130] In the case of HAdV5-Mut3 particles, these 13 amino acids within HVR1 were replaced with four lysine residues (EEEDDDNEDEVDE (SEQ ID NO: 6) → KKKK (SEQ ID NO: 15); nt 19280-19318).

[0131] For HAdV5-Mut2, some of the aspartic acids in this 13-amino acid stretch were replaced with lysine residues (EEEDDDNEDEVDE (SEQ ID NO: 6) → EEEKKKNEKEVDE (SEQ ID NO: 16); nt 19289-19306).

[0132] For HAdV5-Mut4, several negatively charged amino acids within HVR1 were replaced with lysine residues (EINLEEEDDDNEDEVDEQAE (SEQ ID NO: 17) → KINLKKNKVKQAK (SEQ ID NO: 18)).

[0133] For HAdV5-Mut5, several amino acids within HVR1 (EEEDDDNEDEVDE (SEQ ID NO: 6) → KKKK (SEQ ID NO: 15)), several amino acids within HVR5 (STTEAAAGNGDNLTPK (SEQ ID NO: 19) → STTKAAAGNGKNLTPK (SEQ ID NO: 20)), and several amino acids within HVR7 (GGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNF (SEQ ID NO: 21) → GGVINTETLTKVKPKTGQKNGWKKKATEFSDKNEIRVGNNF (SEQ ID NO: 22)) were replaced with lysine residues.

[0134] For HAdV5-Mut6, several negatively charged amino acids within HVR1 (EINLEEEDDDNEDEVDEQAE (SEQ ID NO: 17) → KINLKKNKVKQAK (SEQ ID NO: 18)), several negatively charged amino acids within HVR5 (STTEAAAGNGDNLTPK (SEQ ID NO: 19) → STTKAAAGNGKNLTPK (SEQ ID NO: 20)), and several negatively charged amino acids within HVR7 (GGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNF (SEQ ID NO: 21) → GGVINTETLTKVKPKTGQKNGWKKKATEFSDKNEIRVGNNF (SEQ ID NO: 22)) were replaced with lysine residues.

[0135] For HAdV5-Mut7, the 13 amino acids of SEQ ID NO: 6 within HVR1 were replaced with 6 lysine residues.

[0136] For HAdV5-Mut8, the 13 amino acids of SEQ ID NO: 6 within HVR1 were replaced with 8 lysine residues.

[0137] For HAdV5-Mut9, the 13 amino acids of SEQ ID NO: 6 within HVR1 were replaced by a stretch of 8 lysine residues flanked on either side by a flexible GS-linker of 24 amino acids, i.e., 8 amino acids selected from glycine and serine.

[0138] For the HAdV5-ΔCAR-Mut3 vector particles, the 13 amino acids of SEQ ID NO: 6 within HVR1 were replaced with 4 lysine residues, and the vector particles further carry a point mutation (Y→477A) in the fiber knob, which significantly reduces CAR binding (Kirby et al., 2000).

[0139] The HAdV5-TSG6 vector carries a cDNA encoding the human tumor necrosis factor (TNF) -stimulated gene 6 (TSG-6) protein (nucleotide accession GenBank: AJ419936.1) under the control of the human CMV promoter in the E1 region instead of the eGFP expression cassette. Since TSG-6 is a protein with anti-inflammatory activity, it can be used as an anti-inflammatory therapeutic agent.

[0140] The replication-competent wild-type adenovirus particles (HAdV5wt) do not have deletions but carry an eGFP expression cassette inserted in the forward direction in the non-coding region between E1A and E1B (positions 1648 / 1649).

[0141] Vector and virus rescue and production The replication-incompetent vectors were produced in E1-transcomplementing N52.E6 cells, and the replication-competent virus was produced in A549 cells.

[0142] Vector and viral DNA were each excised from circular bacmid DNA by SwaI restriction enzyme digestion and then purified by standard procedures. Producer cells (N52.E6 or A549 cells) were transfected with the linear DNA prepared as described above using the gene delivery agent polyethyleneimine (PEI). Vector particles and viral particles were each amplified by performing continuous harvesting and infection cycles with increasing cell numbers. After infecting 2x10 8 ~4x10 8 cells, the cells were harvested 48 hours post-infection, resuspended in 50 mM HEPES, 150 mM NaCl, pH 7.4, and lysed by three consecutive freeze / thaw cycles. The particles were purified by one CsCl step gradient (lower density: 1.41 g / ml, upper density: 1.27 g / ml; 176,000 x g, 4°C for 2 hours) and one continuous CsCl gradient (density: 1.34 g / ml; 176,000 x g, 4°C for 20 hours). The particles were then desalted using a PD-10 size exclusion column (GE Healthcare) and stored at -80°C in buffer containing 10% glycerol (50 mM Hepes, 150 mM NaCl, pH 7.4). The physical titer was determined by measuring the optical density at OD260 nm of the isolated virus / vector DNA.

[0143] Homologous recombination Modifications of the HVR1, HVR5, and HVR7 regions of the HAdV5-ΔHVR1, HAdV5-Mut2, HAdV5-Mut3, HAdV5-Mut4, HAdV5-Mut5, and HAdV5-Mut6 vectors were performed by homologous recombination according to the Bacmid Kit "Counter-Selection BAC Modification Kit Red / ET Recombination" as appropriate. Thus, the bacmid carrying the HAdV5 sequence was transfected into the streptomycin-resistant Escherichia coli (E. coli) strain ElectroMAX™ DH10B™ by electroporation. The bacteria were streaked onto lysogenic broth (LB) agar plates supplemented with chloramphenicol (20 μg / ml) and cultured overnight at 37°C. After confirming streptomycin (50 μg / ml) and chloramphenicol (20 μg / ml) resistance, E. coli was transfected by electroporation using the pRed / ET plasmid included in the kit. The bacteria were streaked onto LB agar plates containing tetracycline (3 μg / ml) and chloramphenicol (20 μg / ml). The plates were incubated at 30°C for 20 hours. The next day, single colonies were selected, cultured overnight at 30°C, and the expression of the recombinant proteins Redα and Redβ from the Red / ET plasmid was induced by L-arabinose and a temperature shift to 37°C. Subsequently, the bacteria were transfected with a polymerase chain reaction (PCR) product carrying the rpsL-neo cassette with homologous arms for the gene region of interest. The bacterial suspension was streaked onto LB agar plates containing tetracycline (3 μg / ml), chloramphenicol (20 μg / ml), and kanamycin (15 μg / ml) and cultured at 30°C for 24 hours or more. Next, clones were selected based on the integrity of the deoxyribonucleic acid (DNA) confirmed by streptomycin sensitivity and restriction enzyme analysis. After the introduction of the rpsL-neo cassette was confirmed, the expression of the Red / ET gene was induced again by L-arabinose and a temperature shift to 37°C. The bacteria were transformed with a second PCR product carrying any of the mutations in HVR1, HVR5, or HVR7, respectively.These second PCR products also carry arms homologous to the target gene region, and successfully recombined clones were selected overnight at 37 °C on LB agar containing chloramphenicol (20 μg / ml) and streptomycin (50 μg / ml). For the expected mutations, single colonies were analyzed by DNA restriction analysis. After large-scale bacmid preparation, positive clones were further verified by sequencing.

[0144] Polymerase chain reaction (PCR) for generating PCR products for homologous recombination For the generation of HAdV5-ΔHVR1, HAdV5-Mut2, HAdV5-Mut3, HAdV5-Mut4, HAdV5-Mut5, and HAdV5-Mut6, the PCR products encoding the respective rpsL-neo cassettes are those with homology arms added adjacent to the target gene region in a complementary manner. The primers used were composed of 74 base pairs (bp) (50 bp complementary to the 5’ of the target gene region and 24 bp complementary to the 3’ of rpsL-neo). In the PCR reaction, 5 μl of Pfx amplification buffer (10x), 1 μl of MgSO4 (50 mM), 2 μl of dNTP’s (10 mM), 1 μl of each primer 10 μM (For HVR1: rpsL-neo fw: caaatccttgcgaatgggatgaagctgctactgctcttgaaataaacctaggcctggtgatgatggcgggatcg (SEQ ID NO: 23), rpsL-neo rev: ccagaataaggcgcctgcccaaatacgtgagttttttgctgctcagcttgtcagaagaactcgtcaagaaggcg (SEQ ID NO: 24), For HVR5: rpsL-neo fw: ggagggcaaggcattcttgtaaagcaacaaaatggaaagcta- gaaagtcaagGGCCTGGTGATGATGGCGGGATCG (Array ID number: 25), rpsL-neo rev: gtctggggtttctatatctacatcttcactgtacaataccactttaggagtcTCAGAA- GAACTCGTCAAGAAGGCG (Array ID number: 26); Regarding HVR7: rpsL-neo fw: catggaactgaagatgaacttccaaattactgctttccactgg- gaggtgtgGGCCTGGTGATGATGGCGGGATCG (Array ID number: 27), rpsL-neo rev: ctccacaggttggcatttagattgatttccatggcaaaattatttccaactcTCAGAA- GAACTCGTCAAGAAGGCG (Array ID number: 28); All primer sequences are shown in the 5’→3’ direction), 0.5 μl of rpsL-neo cassette template and 0.5 μl of Platinum® Pfx DNA Polymerase (2.5 U / μl) were mixed to a final volume of 50 μl. The PCR cycle was performed as follows: 1 cycle: 95 °C for 5 min (initial denaturation); 27 cycles: 95 °C for 45 s (denaturation), 60 °C for 45 s (annealing), 68 °C for 2 min (extension); 1 cycle: 68 °C for 10 min (final extension).

[0145] The second PCR product that replaces the previously inserted rpsL-neo cassette was generated using a synthetic template purchased from Invitrogen GeneArt that carried the desired mutations in HAdV5-ΔHVR1, HAdV5-Mut2, HAdV5-Mut3, HAdV5-Mut4, HAdV5-Mut5, or HAdV5-Mut6. In the PCR reaction, 5 μl of Pfx amplification buffer (10x), 1 μl of MgSO4 (50 mM), 2 μl of dNTP’s (10 mM), 1 μl of each primer 10 μM (HVR1 fw: tttaa gccct actct ggcac tgc (SEQ ID NO: 29), HVR1 rev: CCTTC GACAC CTATT TGAAT ACCC (SEQ ID NO: 30); HVR5 fw: cacaa atgaa aatgg agggc aagg, (SEQ ID NO: 31), HVR5 rev: gtggg catgt aagaa atatg agtg (SEQ ID NO: 32); HVR7 fw: gacag ctatg atcca gatgt tagaa (SEQ ID NO: 33), HVR7 rev: ctcca caggt tggca tttag attg (SEQ ID NO: 34); (all primer sequences are shown in the 5’→3’ direction), 100 ng of template plasmid DNA, and 0.5 μl of Platinum® Pfx DNA polymerase (2.5 U / μl) were mixed to a final volume of 50 μl. The PCR cycle was performed as follows: 1 cycle: 95 °C for 5 min (initial denaturation); 27 cycles: 95 °C for 45 s (denaturation), 55 °C for 45 s (annealing), 68 °C for 2 min (extension); 1 cycle: 68 °C for 10 min (final extension).

[0146] The PCR products were separated by agarose gel electrophoresis on a Tris-acetate-ethylenediaminetetraacetic acid (TAE) gel. The desired band was excised and purified by phenol-chloroform extraction followed by ethanol precipitation.

[0147] The production of HAdV5-Mut7, HAdV5-Mut8, and HAdV5-Mut9 was also carried out in the same procedure.

[0148] Silver staining 5x10 9 Vector particles were dissolved in 20 μl of PBS with 1x SDS-loading buffer containing β-mercaptoethanol and denatured at 70°C for 5 minutes. Then, the viral proteins were separated by SDS-PAGE (5% stacking gel, 8% separating / running gel). Silver staining was performed to visualize the viral proteins. The proteins were fixed with a fixing buffer (50% methanol, 12% acetic acid, 0.05% formaldehyde 37%) for 30 minutes, washed with a washing buffer (50% ethanol) for 15 minutes, and then pretreated with an equilibration buffer (0.8 mM sodium thiosulfate) for 1 minute. Next, the gel was washed three times with dH2O and incubated with an impregnation buffer (11.78 mM silver nitrate, 0.05% formaldehyde 37%) for 20 minutes. The staining of the proteins was performed by incubating the gel in a developing buffer (0.57 M sodium carbonate, 0.05% formaldehyde 37%, 15.8 μM sodium thiosulfate) for an appropriate time. The development was stopped with a stopping buffer (50% methanol, 12% acetic acid) when the bands of the proteins were very clear but the bands of the markers were almost invisible.

[0149] Zeta potential measurement The zeta potential was measured using a Zetasizer Nano-ZS (Malvern, UK). 2x10 11The vector particles were dialyzed three times (after 2 hours, overnight, and for 6 hours) at 4 °C with gentle stirring against 50 mM HEPES (pH 7.4) using a Slide-A-Lyzer dialysis cassette (3.5K MWCO 0.5 ml, Thermo Fisher Scientific). Subsequently, size-exclusion chromatography using a PD MiniTrap G-25 column (GE Healthcare Life Sciences) was performed according to the manufacturer without a filter to remove glycerol. Since the required volume of a clear disposable zeta cell cuvette (Malvern, UK) was 1 ml, the purified vector particles were filled to 1 ml with 50 mM HEPES (pH 7.4, sterile filtered) (Krutzke et al, 2016). For measurement and analysis with DTS Nano 5.10 software, the following settings were applied: measurement type, zeta potential; sample material, protein RI 1.450, Absor 0.00; sample dispersion medium, water temperature 25 °C, viscosity 0.8872 CP, RI 1.330, dielectric constant 78.5; sample general option, Smoluchowski; sample temperature 25 °C, 2-minute equilibration time at the start of measurement; sample cell, DTS 1060C transparent disposable zeta cell; measurement, automatic minimum 10, maximum 15; number of measurements, 1.

[0150] Transduction of SKOV-3 cells via FX SKOV-3 cells were seeded at a density of 2x10 4 cells / well in 200 μl of serum-containing Roswell Park Memorial Institute 1640 medium. The next day, the cells were washed and provided with 100 μl of serum-free Roswell Park Memorial Institute 1640 medium with or without 8 μg / ml of FX (as a control). The cells were transduced with a pMOI of 1000 (2x10 7 VP). After incubation at 37 °C for 3 hours, the cells were washed and 200 μl of serum-containing Roswell Park Memorial Institute 1640 medium was added. At 72 hours post-transduction, the cells were harvested and eGFP expression was analyzed by flow cytometry.

[0151] Uptake via scavenger receptor J774A.1 cells were seeded in 1 ml of Dulbecco's modified Eagle's medium containing serum at a density of 1 x 10 5 cells / well in a 24-well plate and cultured overnight at 37°C. The next day, the cells were washed and 500 μl of serum-free Dulbecco's modified Eagle's medium was provided. When indicated, polyinosinic acid (Poly-(I)) (30 μg / ml) dissolved in Dulbecco's phosphate-buffered saline was added to the cells, and the cells were incubated at 37°C for 1 hour. Finally, the cells were transduced with 2000 pMOI (1 x 10 8 VP). After incubation at 37°C for 3 hours, the cells were washed twice and then incubated in 1 ml of Dulbecco's modified Eagle's medium containing serum. Twenty-four hours after transduction, 200 μl of Dulbecco's phosphate-buffered saline, 20 μl of proteinase K, and 20 μl of RNase were added per well to harvest the cells. After incubation at room temperature for 2 minutes, an additional 200 μl of AL buffer ("QIAmp DNA Mini Kit") was added, and the mixture was incubated at 56°C for 10 minutes. Subsequently, the DNA of the lysed cells was isolated according to the protocol of the "QIAmp DNA Mini Kit" (Krutzke et al., 2016).

[0152] To quantify the adenovirus content of the isolated total DNA samples, quantitative PCR (qPCR) of the adenovirus E4 gene was performed. The copy number of mouse β-actin was used for normalization to exclude the efficiency of heterologous cell harvesting. To quantify the adenovirus content of the isolated total DNA samples, quantitative PCR (qPCR) of the adenovirus E4 gene was performed. The copy number of mouse β-actin was used for normalization to exclude the efficiency of heterologous cell harvesting. The qPCR reaction consisted of 10 μl of Kapa SYBRE FAST qPCR master mix, 0.4 μl of each primer (10 μM) (for mouse β-actin: fw: caaggagtgcaagaacacag (SEQ ID NO: 35); rev: gccttggagtgtgtattgag (SEQ ID NO: 36); for Ad5 E4: fw: tagacgatccctactgtacg (Sequence ID number: 37); rev: ccggacgtagtcatatttcc (Sequence ID number: 38); All primer sequences are shown in the 5’→3’ direction, and included 2 μl of isolated total DNA in a final volume of 20 μl.

[0153] The PCR cycle was performed as follows: 1 cycle: 95 °C for 10 minutes (initial denaturation); 40 cycles: 95 °C for 30 seconds (denaturation), 60 °C for 30 seconds (annealing), 72 °C for 20 seconds (extension); 1 cycle: 95 °C for 1 minute (denaturation), 55 °C for 30 seconds (annealing); 1 cycle: 95 °C for 30 seconds (final extension).

[0154] Neutralization via IgM A549 cells were seeded in 200 μl of serum-containing minimum essential medium (Gibco) in a 96-well plate. The next day, the indicated viral vector particles were incubated at 37 °C for 10 minutes with PBS or plasma samples at a ratio of 2E6 VP / μl. Naive human and mouse plasma samples were prepared from whole blood by centrifugation at 800 xg for 10 minutes. To maintain complement activity, blood samples were anticoagulated with 100 μg / ml of hirudin (Celgene). The cells were washed and transduced with a pre-incubation viral vector with a pMOI of 1000 in 100 μl of serum-free medium and incubated at 37 °C for 3 hours. Then, the cells were washed and supplemented with 200 μl of serum-containing medium. After incubation at 37 °C for 24 hours, the cells were detached and the expression of eGFP was analyzed by flow cytometry.

[0155] Transduction of MSCs with HAdV5-TSG6 and coagulation factor X (FX) as transduction enhancers 2 - 3x10 5MSCs were seeded into 6-cm dishes with 6 ml of PL containing alpha-MEM Eagle's minimum essential medium (Lonza) and cultured overnight at 37°C. The next day, FX (2 μg / ml) was pre-incubated at 37°C for 20 - 30 minutes at a final volume of 290 μl in alpha-minimum essential medium together with the corresponding amount of TSG-6 expression vector particles HAdV5-TSG6. During the pre-incubation of the vector particles with FX, the cells were washed and provided with 2.7 ml of alpha-minimum essential medium without supplements per 6-cm dish. Finally, the pre-incubated vector particle solution was added to the cells and incubated at 37°C for 3 hours, followed by three washes with alpha-minimum essential medium. The cells were further cultured at 37°C for 72 hours in PL containing alpha-minimum essential medium. The cell culture supernatant was collected, and the concentration of TSG-6 in the cell culture supernatant was analyzed by sandwich TSG-6 ELISA.

[0156] For this purpose, 96-well MaxiSorb Nunc plates were incubated overnight at 4 °C with 100 μl / well of coating solution (1 μg / ml TSG-6 monoclonal mouse IgG Santa Cruz SC-65886 in 0.1 M sodium carbonate / sodium bicarbonate buffer pH 9.6). The next day, the plates were washed three times with 300 μl / well of 0.05% Tween-PBS using an ELISA plate washer "Well Wash Versa", and then incubated for 1 h at room temperature with 300 μl / well of SuperBlock (Thermo Fisher) on a shaker. After another wash, samples of the corresponding diluent or TSG-6 protein standard (R&D 2104-TS-050) were added to the wells at 100 μl / well and incubated for 1 h at room temperature. Again, the plates were washed, and 100 μl / well of detection solution (0.25 μg / ml anti-TSG-6 goat polyclonal IgG R&D BAF2104, PBS) was incubated for 1 h at room temperature on a shaker. 100 μl / well of streptavidin-horseradish peroxidase conjugate (Dako P0397 c = 0.020 μg / ml) was added and the plates were washed again before incubating for 1 h at room temperature on a shaker. Finally, after another washing step, 100 μl / well of detection substrate (1-StepUltra TMB ELISA Substrate Thermo Fisher 34028) was added to each well and the enzyme reaction was initiated in the dark. After 15 min, 100 μl / well of sulfuric acid (2 M) was added to stop the reaction and measurements were taken at 450 nm using an ELISA reader.

[0157] Transduction of tumor cell lines with HAdV5 and HAdV5-Mut3, and HAdV5-ΔCAR-Mut3 and HAdV5-ΔCAR Different tumor cell lines (UM-SCC-11B, MiaPaCa, Huh7, HepG2, A549) were seeded into 200 μl of their respective media in 96-well plates. The next day, the cells were washed and transduced with either HAdV5 or HAdV5-Mut3 at 300 or 1000 pMOI in 100 μL of serum-free medium. After incubation at 37 °C for 2 hours, the cells were washed and 200 μL of fungal medium was added. The cells were harvested 24 hours after transduction and the expression of eGFP was analyzed by flow cytometry.

[0158] The same procedure was applied to the transduction of different tumor cell lines with HAdV5-ΔCAR-Mut3 and HAdV5-ΔCAR at pMOI of 1000.

[0159] Transduction of MSCs with HAdV5 or HAdV5-Mut3 with or without enhancer molecules MSCs were seeded in 1 mL of PL-containing BioWhittaker® alpha minimum essential medium (Lonza) in 24-well plates. The next day, in PL-free medium, with a total volume of 300 μL, the indicated viral vector particles were pre-incubated at 37 °C for 30 minutes with or without an enhancer molecule. As enhancer molecules, factor X (250 - 2000 ng / mL), spermidine (1 - 625 ng / μL), spermine (1 - 1000 ng / μL), Polybrene (0.08 - 81 μg / μL), poly-L-lysine (1 - 25% v / v), and lactoferrin (1 - 1000 μg / mL) were used. Then, the MSCs were washed and 500 μL of PL-free medium was provided. The cells were transduced by adding 300 μL of the pre-incubated viral particles (resulting in the indicated pMOI) and incubated at 37 °C for 2 hours. Then, the cells were washed and 1 mL of PL-containing medium was added. The MSCs were detached 72 hours after transduction and the expression of eGFP was analyzed by flow cytometry.

Table 1

[0160] The MSCs were seeded in 1 mL of PL-containing BioWhittaker® Alpha Minimum Essential Medium (Lonza) in 24-well plates. The next day, in PL-free medium, with a total volume of 300 μL, the indicated viral vector particles were pre-incubated with or without enhancer molecules at 37 °C for 30 minutes. As enhancer molecules, Factor X (4 fg / viral particle), spermidine (500 fg / viral particle), polybrene (18 fg / viral particle) were used in combination with HAdV5wt. HAdV5-Mut3wt was used without enhancer. Then, the MSCs were washed and provided with 500 μL of PL-free medium. The cells were infected by adding 300 μL of pre-incubated viral particles (resulting in a pMOI of 300 or 1000) and incubated at 37 °C for 2 hours. Then, the cells were washed and 1 mL of PL-containing medium was added. Cells and supernatants were harvested and lysed at 24, 48, 72, and 96 hours post-infection. MSC lysates were used for re-infection of A549 cells that had been pre-seeded in 24-well plates 24 hours earlier. Four hours after re-infection, A549 DNA was isolated using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Using the resulting A549 DNA samples, infectious adenoviruses produced by MSCs were quantified by real-time quantitative PCR (qPCR). For qPCR analysis, a part of the adenovirus E4 transcription unit was amplified (Forward primer: TAGACGATCCCTACTGTACG (SEQ ID NO: 37); Reverse primer: GGAAATATGACTACGTCCGG (SEQ ID NO: 39); (All primer sequences are shown in the 5’→3’ direction). For normalization, β-actin was also analyzed (Forward primer: GCTCCTCCTGAGCGCAAG (SEQ ID NO: 40); Reverse primer: CATCTGCTGGAAGGTGGACA (SEQ ID NO: 41); (All primer sequences are shown in the 5’→3’ direction).

[0161] Kapa SYBR FAST qPCR Universal Master Mix (PEQLAB Biotechnologie) was used according to the manufacturer's protocol.

[0162] Migration assay of untransduced MSCs and transduced MSCs To evaluate MSC migration (untransduced or transduced with HAdV5 or HAdV5-Mut3), a Boyden chamber assay was performed. For this purpose, MSCs were seeded in 3 mL of PL-containing BioWhittaker® α-Minimum Essential Medium (Lonza) in 6-well plates. The next day, in PL-free medium, in a total volume of 300 μL, the labeled virus vector particles were pre-incubated at 37 °C for 30 min with or without enhancer molecules. As enhancer molecules, factor X, spermidine, spermine, polybrene, poly-L-lysine and lactoferrin were used. Thereafter, the MSCs were washed and 3000 μL of PL-free medium was provided. The cells were transduced (to the indicated pMOI) by adding 300 μL of pre-incubated virus particles and incubated at 37 °C for 2 h. Thereafter, the cells were washed and 1 mL of PL-containing medium was added. Four hours after transduction, the MSCs were detached and seeded into Transwell inserts (pore size 8 μM, 1x10 4 cells per Transwell). The Transwells were placed on a 24-well plate seeded with 1x10 5 UM-SCC-11B cells the previous day. As a control, MSC migration towards UM-SCC-11B culture medium (DMEM containing 3% FBS) was analyzed. Eighteen hours after seeding the MSCs into the Transwells, the Transwells were washed with PBS and the cells were fixed with ice-cold methanol. Thereafter, the MSCs were stained with 10 μg / mL of 4’,6-diamidino-2-phenylindole (DAPI) and 1% Triton-X diluted in PBS. Migrated cells were quantified by counting the nuclei stained with DAPI.

[0163] Transduction of MSCs with HAdV5, HAdV5-Mut3, HAdV5-ΔCAR-Mut3, HAdV5-ΔHVR1 or HAdV5-Mut2 Transduction of MSCs with a pMOI of 1000 was performed as described in the section "Transduction of MSCs with HAdV5 or HAdV5-Mut3 with or without enhancer molecule", except that the MSCs were detached and eGFP expression was analyzed by flow cytometry 24 hours after transduction. No enhancer molecule was used.

[0164] Statistical analysis Results are shown as mean ± standard deviation. Statistical analysis was performed using an unpaired two-sample (Welch) Student's t-test or Wilcox test. Calculations were performed using RStudio-software Version 2.15.0 or GraphPad Prism Version 6.07. A P value ≤ 0.05 was considered statistically significant.

[0165] Results Production and characterization of mutant vectors Figure 1 shows the alignment of the amino acid sequences of HVR1 of HAdV5 wild-type and HAdV5-Mut3 hexon protein (Figure 1A), and HVR1, HVR5 and HVR7 of HAdV5 wild-type, HAdV5-ΔHVR1, HAdV5-Mut2, HAdV5-Mut3, HAdV5-Mut4, HAdV5-Mut5, HAdV5-Mut6 (Figure 1B). Negatively charged amino acids are depicted in bold. Inserted lysine residues are shown in italics.

[0166] Notably, the production of the mutant vectors HAdV5-Mut4, HAdV5-Mut5, HAdV5-Mut6 was not feasible as no viral rescue occurred upon transfection of the bacmid DNA cut in N52.E6 cells, indicating that this vector is non-viable.

[0167] Figure 14 shows the amino acid sequence alignment of HVR1 of HAdV5 wild type and HAdV5-Mut7, HAdV5-Mut8, and HAdV5-Mut9 hexon proteins. The inserted lysine residues are depicted in bold. Additionally, the inserted amino acids are shown in italics.

[0168] The production of the mutant vectors HAdV5-Mut7, HAdV5-Mut8, and HAdV5-Mut9 was not feasible because virus rescue did not occur even when the cut bacmid DNA was transfected into N52.E6 cells, indicating that each of these vectors could not survive and functional virus particles could not be formed.

[0169] Figure 2 shows the nucleotide sequence alignment encoding hypervariable region 1 of HAdV5 wild type and HAdV5-Mut3 hexon proteins. The nucleotides encoding the inserted lysine residues are shown in capital letters.

[0170] The integrity of the viral proteins was confirmed by silver staining (Figure 3). As a control, unmodified vector particles (HAdV5) were used to reveal the molecular weight of the hexon protein, which was 108 kDa. By silver staining, the deletion of the negative loop of HVR1 in HAdV5-ΔHVR1 and the mutation in the HVR1 region in HAdV5-Mut3 were confirmed. All of these modifications resulted in a visible decrease in the molecular weight of the hexon protein, respectively. On the other hand, the hexon protein with lysine inserted instead of aspartic acid (HAdV5-Mut2) showed the same molecular weight as the unmodified hexon protein as expected.

[0171] In zeta potential measurements to determine the surface charge of vector particles, a negative surface charge of -22.8 mV was revealed for the control vector HAdV5, which was consistent with the literature (Figure 4). Interestingly, when the negatively charged HVR1 loop was deleted (HAdV5-ΔHVR1), the negative surface charge of the particles was not statistically significant and only slightly decreased (-19.4 mV). In the case of HAdV5-Mut2, when aspartic acid was substituted with lysine, the surface charge further decreased (-17.33 mV). However, interestingly, HAdV5-Mut3 showed a statistically significant decrease in surface charge compared to HAdV5, HAdV5-ΔHVR1, and HAdV5-Mut2 (-8.1 mV; compared to HAdV5: p < 4.303x10-5; compared to HAdV5-ΔHVR1: p < 2.738x10-4; compared to HAdV5-Mut2: p < 1.139x10-7).

[0172] HAdV5-Mut3 shows significantly less transduction of CAR-negative SKOV-3 cells via FX than wild-type HAdV5 (HAdV5), HAdV5-Mut2, and HAdV5-ΔHVR1. When human coagulation factor X (FX) binds to HAdV5, it mediates the transduction of hepatocytes, which in turn induces the encapsulation of the particles. The binding residues associated with FX are located within HVR5 and HVR7 of the hexon protein (Alba et al., 2009). To exclude cell entry via CAR, the inventors used CAR-negative SKOV-3 cells (Figure 5) and analyzed whether FX promotes the transduction of mutant vectors into cells. HAdV5-ΔFX vector particles are known to exhibit significantly reduced FX binding due to point mutations within HVR7 (Krutzke et al., 2016) and were used as a control. Transduction of cells via FX by HAdV5-ΔFX was significantly reduced compared to HAdV5 in the presence of FX (p < 3.686 x 10-5). Surprisingly, a similar effect was also observed with HAdV5-Mut3, which showed a statistically significant decrease in cell transduction compared to HAdV5 in the presence of FX (p < 1.135 x 10-5). Interestingly, the transduction efficiency of HAdV5-ΔHVR1 via FX was not significantly reduced compared to HAdV5. Additionally, introduction of a lysine residue instead of an aspartic acid residue into HVR1 (HAdV5-Mut2) was shown not to reduce uptake via FX. From the above results, it was found that neither HAdV5-ΔHVR1 nor HAdV5-Mut2 showed a decrease in FX binding ability, while HAdV5-Mut3 showed a significant decrease in FX binding ability comparable to that of HAdV5-ΔFX.

[0173] Scavenger receptor-mediated uptake of HAdV5-Mut3 is significantly reduced compared to wild-type HAdV5 (HAdV5). The main recipients of systemically administered HAdV5 are macrophages resident in the liver, called Kupffer cells (Alemany et al., 2000; Khare et al., 2012). These cells are known to have scavenger receptors that bind and capture negatively charged molecules on the cell surface. HAdV5 exhibits an overall negative surface charge, which is mainly due to a stretch of negatively charged amino acids within HVR1 (Khare et al., 2012). To reduce the negative surface charge of the particles, the inventors either deleted the negatively charged HVR1 loop (HAdV5-ΔHVR1), inserted lysine instead of aspartic acid within the stretch (HAdV5-Mut2), or replaced the entire stretch with four lysine residues (HAdV5-Mut3) (Figure 4). As a result, it was revealed that the uptake of all three mutant vectors, HAdV5-ΔHVR1, HAdV5-Mut2, and HAdV5-Mut3, by mouse macrophages was significantly decreased compared to the control vector HAdV5 (HAdV5-ΔHVR1 p<0.001462; HAdV5-Mut2 p<0.001219; HAdV5-Mut3 p<0.001241) (Figure 6). This effect was more pronounced with HAdV5-Mut3. The uptake of HAdV5 particles by macrophages was significantly inhibited in the presence of polyinosinic acid (Poly-(I)). Since Poly-(I) binds to and saturates the scavenger receptor, the viral uptake mechanism via the scavenger receptor was confirmed. Pre-incubation of the cells with Poly-(I) did not affect the uptake of HAdV5-ΔHVR1 and HAdV5-Mut2 by macrophages. Interestingly, pre-incubation of the cells with Poly-(I) increased the uptake of HAdV5-Mut3 particles by macrophages.

[0174] HAdV5-Mut3 vector particles with disrupted FX binding escape neutralization by natural IgM. Others, including the inventors, have shown that FX shields adenovirus type 5 vector particles from neutralization by natural IgM antibodies (Krutzke et al., 2016). Since the inventors confirmed that HAdV5-Mut3 shows significantly reduced FX binding (Figure 5), they analyzed whether these particles are more susceptible to neutralization by human and mouse IgM. Therefore, the inventors incubated vector particles with verifiable Ad-naïve human and mouse plasma samples from different donors and mouse strains, and subsequently analyzed A549 cell transduction (Figure 7). Control HAdV5 vector particles that bind FX normally (Figure 5) transduced A549 cells with the same efficiency as when incubated with PBS in the presence of human or mouse plasma. On the other hand, pre-incubation of HAdV5-ΔFX particles with reduced binding to FX (Figure 5) was almost completely neutralized by natural IgM upon incubation with plasma from both human and mouse. However, interestingly, although HAdV5-Mut3 shows a reduction in FX binding equivalent to that of HAdV5-ΔFX (p < 5 x 10-11 compared to each pre-incubation of HAdV5-ΔFX), the inventors did not observe this effect (Figure 5). Thus, the inventors show that vector particles lacking FX shielding can escape from natural IgM due to the introduced Mut3 mutation.

[0175] Significant enhancement of the secretion of the therapeutic protein TSG-6 encoded by HAdV5 by MSCs transduced with HAdV5-TSG6 in the presence of factor X as a transduction enhancer. In the presence of factor X (FX), the expression and subsequent secretion of TSG-6 as a representative therapeutic protein by MSCs were found to be significantly promoted (Figure 8). The concentration of TSG-6 in the supernatant of cells transduced at pMOI 900 in the presence of FX was approximately three-fold compared to the concentration of TSG-6 in the supernatant of cells transduced at pMOI 20,000 in the absence of FX.

[0176] Using HAdV5-Mut3 improves or maintains transduction of some tumor cell lines compared to wild-type HAdV5 (HAdV5). When comparing HAdV5-Mut3 with HAdV5, it was found that the transduction efficiency into some tumor cell lines was improved or maintained, suggesting the merit of using HAdV5-Mut3 as an oncolytic virus (Figure 9). For example, the proportion of eGFP-positive UM-SCC-11B cells can increase by approximately 20% to approximately 75% when using HAdV5-Mut3 compared to HAdV5 (pMOI 300).

[0177] Pre-incubation of wild-type HAdV5 (HAdV5) with an enhancer or the use of HAdV5-Mut3 significantly promoted the transduction of MSCs. When MSCs were transduced with HAdV5 alone, almost no eGFP expression was detected, but all enhancer molecules and the mutant HAdV5-Mut3 viral vector were statistically significantly improved up to more than 800-fold in mean fluorescence intensity (MFI) (Figure 10). Surprisingly, transduction with HAdV5-Mut3 resulted in high eGFP expression (MFI increased by more than 600-fold compared to HAdV5 transduction) without using enhancer molecules.

[0178] The combination of HAdV5-Mut3 and enhancer molecules such as spermidine (500 fg / viral particle), spermine (1250 fg / viral particle), and factor X (4 fg / viral particle) was also examined (Figure 12). As a result, it was found that cell transduction by HAdV5-Mut3 was slightly (3-fold) enhanced by the enhancer molecules.

[0179] Improved adenovirus replication in MSCs by using HAdV5wt pre-incubated with an enhancer or by using HAdV5-Mut3wt. It was found that not only HAdV5-Mut3wt but also enhancer molecules improved adenovirus replication compared to infection with HAdV5wt alone. In particular, HAdV5-Mut3wt was shown to have a maximum of approximately 5x10 3 infectious particles / MSC after 48 hours, independent of the pMOI used. Only the combination of spermidine and HAdV5wt with a pMOI of 1000 had a similarly high virus yield (~4.7x10 3It becomes (virus particle / MSC). With all other combinations of HAdV5wt and enhancers, infectious adenovirus particles accumulated over time (presumably due to a certain reinfection of initially uninfected MSCs), and did not show a detectable peak.

[0180] Introduction of MSCs does not suppress migration to UM-SCC-11B cells. It was found that the migration of MSCs to UM-SCC-11B cells was significantly increased compared to the single culture medium (Figure 13). Surprisingly, it was found that the migration to UM-SCC-11B cells was not inhibited even when the cells were transduced with HAdV5 or HAdV5-Mut3. None of the tested enhancing molecules showed a negative effect.

[0181] Using HAdV5-Mut3 (HAdV-5-M3) improves or maintains transduction of some tumor cell lines compared to wild-type HAdV5 (HAdV-5), and using HAdV5-ΔCAR-Mut3 (HAdV-5-ΔCAR-M3) improves transduction of some tumor cell lines compared to HAdV5-ΔCAR (HAdV-5-ΔCAR). When comparing HAdV5-Mut3 with HAdV5, it was found that the transduction efficiency of some tumor cell lines was improved or maintained (see also Figure 15 and Figure 9).

[0182] Also, when using HAdV5-ΔCAR-Mut3, it was found that the transduction efficiency of some tumor cell lines was significantly improved compared to HAdV5-ΔCAR (Figure 15). This indicates that HAdV5-ΔCAR-Mut3 enables CAR-independent transduction of tumor cells.

[0183] Utilizing HAdV5-Mut3 (HAdV-5-M3) and HAdV5-ΔCAR-Mut3 (HAdV-5-ΔCAR-M3) significantly improves the transduction of MSCs, while transduction of MSCs does not occur when using HAdV5-ΔHVR1 (HAdV-5-ΔHVR1) and HAdV5-Mut2 (HAdV-5-M2). It was found that MSCs were efficiently transduced by HAdV5-Mut3 and HAdV5-ΔCAR-Mut3 vectors, while almost no eGFP expression was detected when transduced by wild-type HAdV5 (HAdV-5) (see also Figure 16 and Figure 10 for HAdV5-Mut3 and HAdV5).

[0184] Furthermore, it was found that MSCs were not transduced by HAdV5-ΔHVR1 and HAdV5-Mut2 (Figure 16).

[0185] References Alba, R. et al. Identification of coagulation factor (F)X binding sites on the adeno-virus serotype 5 hexon: effect of mutagenesis on FX interactions and gene trans-fer. Blood 2009;114:965-971. Alemany, R. et al. Blood clearance rates of adenovirus type 5 in mice. J. Gen. Vi-rol. 2000;81:2605-2609. Fekete N., Gadelorge M. et al. Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesnchymal stromal cells: production process, content and identification of active components. Cytotherapy 2012;14:540-554. Fekete N., Rojewski M.T. et al.. GMP-Compliant Isolation and Large-Scale Expan-sion of Bone Marrow-Derived MSC. PLOS ONE 2012;7:e43255. Khare, R. et al. Identification of Adenovirus Serotype 5 Hexon Regions That In-teract with Scavenger Receptors. J. Virol. 2012;86:2293-2301. Kirby, I. et al. Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein. J. Virol. 2000;74:2804-2813. Krutzke, L. et al. Substitution of blood coagulation factor X-binding to Ad5 by posi-tion-specific PEGylation: Preventing vector clearance and preserving infectivity. J. Controlled Release 2016;235:379-392.

Claims

1. A human adenovirus species C having a capsid with a modified adenovirus hexon protein, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence DEAATALEINLKKKKQAEQQQ (SEQ ID NO: 1).

2. The adenovirus according to claim 1, wherein the adenovirus is adenovirus type 5.

3. The adenovirus according to claim 1 or 2, wherein the adenovirus is an adenovirus vector or an oncolytic adenovirus.

4. The adenovirus according to any one of claims 1 to 3, wherein the adenovirus has a transgene.

5. The adenovirus according to any one of claims 1 to 4, wherein the capsid has at least one additional capsid modification.

6. The adenovirus according to claim 5, wherein the additional capsid modification is a modified adenovirus fiber protein.

7. The adenovirus according to any one of claims 1 to 6 for use in the treatment or prevention of human diseases.

8. A nucleic acid encoding a modified adenovirus hexon protein of human adenovirus species C, wherein the modified adenovirus hexon protein has a modified HVR1 region, and the modified HVR1 region has the sequence of SEQ ID NO:

1.

9. The nucleic acid according to claim 8, wherein the nucleic acid has the sequence of SEQ ID NO:

2.

10. The adenovirus according to any one of claims 1 to 6 for use in transducing mesenchymal stromal cells (MSCs) or tumor cells.

11. The adenovirus according to claim 10, wherein the adenovirus is used in combination with a transduction enhancer for transducing MSCs.

12. The adenovirus according to claim 11, wherein the transduction enhancer is selected from the group consisting of factor X, spermidine, spermine, hexadimethrine bromide, poly-L-lysine, and lactoferrin.

13. An in vitro method for transducing MSCs, the method comprising the step of contacting the adenovirus according to any one of claims 1 to 6 with a plurality of MSCs.

14. The method according to claim 13, wherein the plurality of MSCs are further contacted with a transduction enhancer.

15. The method according to claim 14, wherein the transduction enhancer is selected from the group consisting of factor X, spermidine, spermine, hexadimethrine bromide, poly L-lysine, and lactoferrin.

16. MSCs transduced with the adenovirus according to any one of claims 1 to 6, which have the modified adenovirus hexon protein of the adenovirus.

17. The transduced MSCs according to claim 16 for use in the treatment of diseases.

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