Modified adenovirus by hexon hypervariable region 7 (HVR7) gene insertion

Abstract

The disclosure relates, inter alia, to recombinant adenoviruses having capsid proteins engineered to include an albumin-binding moiety such that the albumin-binding moiety is present on the outer surface of the recombinant adenovirus capsids. Also described are pharmaceutical compositions comprising the recombinant adenoviruses, and methods of using the recombinant adenoviruses to treat genetic diseases and disorders.

Description

[0001] VCN-008PC / 112492-5208

[0002] MODIFIED ADENOVIRUS BY HEXON HYPERVARIABLE REGION 7 (HVR7) GENE INSERTION

[0003] TECHNICAL FIELD

[0004] The present disclosure relates, inter alia, to recombinant adenoviruses having modified capsid proteins engineered to evade immune surveillance. In particular, the modified capsid proteins include an albuminbinding moiety that is exposed on the outer surface of the adenoviral particles and protects the adenoviral particles from neutralizing antibodies.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims priority to U.S. Provisional Application No. 63 / 684,601 , filed August 19, 2024, which is herein incorporated by reference in its entirety.

[0007] DESCRIPTION OF THE XML FILE SUBMITTED ELECTRONICALLY

[0008] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on August 6, 2025, is named “VCN-008_Sequence_Listing.xml” and is 101 ,268 bytes in size.

[0009] BACKGROUND

[0010] Adenoviruses have been extensively used as gene delivery vectors for gene therapy as well as oncolytic agents for cancer treatment. They exhibit several features that make them suitable for these applications. Namely, their structure and biology has been widely studied which allows for an easy modification of their genome, they are able to infect both replicating and non-replicating cells, and they can easily be produced at high titers for their use in the clinic. In terms of safety, they do not cause life-threatening diseases in humans, and their genome is non-integrative, which prevents for insertional mutagenesis. Clinical trials with adenovirus-based vectors report a good toxicology and safety profile, although the efficacy still needs improvement, especially when the virus is administered systemically.

[0011] For example, their clinical use is restricted by high levels of pre-existing anti-adenovirus neutralizing antibodies (NAbs) in the human population. Many humans have been pre-exposed to human adenovirus serotype 5 (hAd5) and display high serum neutralizing activity against adenoviruses (D'Ambrosio, E., et al., “Neutralizing antibodies against 33 human adenoviruses in normal children in Rome,” J Hyg (Lond), 1982. 89(1): p. 155-161). NAbs not only can neutralize the virus directly, but they can also trigger an innate immune

[0012] DBl / 161420432.1 1 VCN-008PC / 112492-5208 response by complement activation and by docking the virus particles to the Fc receptors of monocytes and neutrophils. Furthermore, vector re-administration increases the levels of anti-adenovirus NAbs, and therefore the neutralization of the adenovirus, so NAbs are especially problematic for patients who require systemic administration of adenovirus vectors for successful treatment. The prevalence of anti-Ad5 NAbs is high in the general population, and the majority of these NAbs target the hypervariable regions (HVRs) of the hexon (Barouch, D.H., et al., “International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations,” Vaccine, 2011. 29(32): p. 5203-5209; Sumida, S.M., et al., “Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein,” J Immunol, 2005. 174(11): p. 7179-7185). Pre-existing anti-Ad5 NAbs are considered a major hurdle for systemic efficacy of hAd5 (Uusi-Kerttula, H., et al., “Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications,” Viruses, 2015. 7(11): p. 6009-6042). The presence of NAbs against the adenovirus capsid inactivates systemically delivered hAd5-based viruses and impairs efficient virus delivery to the tumor. For a systemically delivered oncolytic adenovirus to effectively reach the primary tumor as well as metastases, the adenovirus must circumvent pre-existing immunity.

[0013] Accordingly, there is a need for new gene therapy technologies suitable for systemic administration and capable of evading NAbs.

[0014] SUMMARY

[0015] Therefore, the present disclosure provides, in aspects, recombinant adenovirus capsid proteins comprising an albumin-binding moiety insertion, and recombinant adenovirus genomes comprising a sequence encoding an albumin-binding moiety inserted into the coding region of the capsid protein, wherein the albumin-binding moiety is exposed on the outer surface of the packaged recombinant adenovirus particles, and wherein the recombinant adenovirus particles are able to evade NAbs. In aspects, the present disclosure provides the recombinant adenoviruses for gene therapy or virotherapy. In aspects, the recombinant adenoviruses are oncolytic adenoviruses, and the present disclosure provides use of the oncolytic adenoviruses for the prevention and / or treatment of cancer. In embodiments, the recombinant adenoviruses are shielded against neutralizing antibodies present in the bloodstream and are thus particularly suitable for systemic administration.

[0016] In aspects, the present disclosure provides a modified adenovirus capsid protein comprising an albuminbinding moiety insertion within the capsid’s hexon protein’s seventh hypervariable region (HVR7), wherein

[0017] DBl / 161420432.1 2 VCN-008PC / 112492-5208 when the hexon protein is packaged as a component of an adenovirus particle, the albumin-binding moiety is exposed on the outer surface of the adenovirus particle.

[0018] In embodiments, the albumin-binding moiety is a wild-type albumin-binding domain or a functional variant or fragment thereof.

[0019] In embodiments, the albumin-binding moiety is selected from an albumin-binding domain from streptococcal protein G, an albumin-binding domain from Peptostreptococcus magnus protein PAB, an albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 21), and functionally equivalent variants thereof.

[0020] In embodiments, the albumin-binding moiety comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 21, 22, or 23.

[0021] In embodiments, the albumin-binding moiety comprises a polypeptide having the amino acid sequence of SEQ ID NO: 21, 22, or 23.

[0022] In embodiments, the adenovirus capsid protein is from at least one adenovirus type selected from a Group A adenovirus, a Group B adenovirus, a Group C adenovirus, a Group D adenovirus, a Group E adenovirus, a Group F adenovirus, and a Group G adenovirus.

[0023] In embodiments, the adenovirus capsid protein is from at least one adenovirus type selected from a serotype 1 adenovirus genome, a serotype 2 adenovirus genome, a serotype 3 adenovirus genome, a serotype 4 adenovirus genome, a serotype 5 adenovirus genome, a serotype 6 adenovirus genome, a serotype 7 adenovirus genome, a serotype 8 adenovirus genome, a serotype 9 adenovirus genome, a serotype 10, adenovirus genome, a serotype 11 adenovirus genome, a serotype 12 adenovirus genome, a serotype 13 adenovirus genome, a serotype 14 adenovirus genome, a serotype 15 adenovirus genome, a serotype 16 adenovirus genome, a serotype 17 adenovirus genome, a serotype 18 adenovirus genome, a serotype 19 adenovirus genome, a serotype 20, adenovirus genome, a serotype 21 adenovirus genome, a serotype 22 adenovirus genome, a serotype 23 adenovirus genome, a serotype 24 adenovirus genome, a serotype 25 adenovirus genome, a serotype 26 adenovirus genome, a serotype 27 adenovirus genome, a serotype 28 adenovirus genome, a serotype 29 adenovirus genome, a serotype 30, adenovirus genome, a serotype 31 adenovirus genome, a serotype 32 adenovirus genome, a serotype 33 adenovirus genome, a serotype 34 adenovirus genome, a serotype 35 adenovirus genome, a serotype 36 adenovirus genome, a serotype 37

[0024] DBl / 161420432.1 3 VCN-008PC / 112492-5208 adenovirus genome, a serotype 38 adenovirus genome, a serotype 39 adenovirus genome, a serotype 40, adenovirus genome, a serotype 41 adenovirus genome, a serotype 42 adenovirus genome, a serotype 43 adenovirus genome, a serotype 44 adenovirus genome, a serotype 45 adenovirus genome, a serotype 46 adenovirus genome, a serotype 47 adenovirus genome, a serotype 48 adenovirus genome, a serotype 49 adenovirus genome, a serotype 50, adenovirus genome, a serotype 51 adenovirus genome, a serotype 52 adenovirus genome, a serotype 53 adenovirus genome, a serotype 54 adenovirus genome, a serotype 55 adenovirus genome, a serotype 56 adenovirus genome, a serotype 57 adenovirus genome, a serotype 58 adenovirus genome, a serotype 59 adenovirus genome, a serotype 60, adenovirus genome, a serotype 61 adenovirus genome, a serotype 62 adenovirus genome, a serotype 63 adenovirus genome, a serotype 64 adenovirus genome, a serotype 65 adenovirus genome, a serotype 66 adenovirus genome, and a serotype 67 adenovirus genome.

[0025] In embodiments, the albumin-binding moiety insertion is located in the range of positions 400-450, with reference to any one of SEQ ID NOs: 1-10, or positions corresponding thereto.

[0026] In embodiments, the albumin-binding moiety insertion is located immediately after one or more of the following amino acid residues: (a) hAd5: any one of residues 420-449, with reference to SEQ ID NO: 1; (b) hAd3: any one of residues 413-441, with reference to SEQ ID NO: 2; (c) hAd4: any one of residues 403-433, with reference to SEQ ID NO: 3; (d) hAd7: any one of residues 409-434, with reference to SEQ ID NO: 4; (e) hAd11 : any one of residues 417-445, with reference to SEQ ID NO: 5; (f) hAd16: any one of residues 403- 437, with reference to SEQ ID NO: 6; (g) hAd21 : any one of residues 419-446, with reference to SEQ ID NO: 7; (h) hAd34: 421-448, with reference to SEQ ID NO: 8; and (i) hAd35: any one of residues 421-449, with reference to SEQ ID NO: 9.

[0027] In embodiments, the albumin-binding moiety insertion occurs after an amino acid residue corresponding to one of amino acids V420, 1421, N422, T423, E424, T425, L426, T427, K428, V429, K430, P431, K432, T433, G434, Q435, E436, N437, G438, W439, E440, K441, D442, A443, T444, E445, F446, S447, D448, or K449 of the hAd5 capsid, with reference to SEQ ID NO: 1, or positions corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10.

[0028] In embodiments, the albumin-binding moiety insertion is located after amino acid residue 437 of hAd5, with reference to SEQ ID NO: 1 , or a position corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10.

[0029] DBl / 161420432.1 4 VCN-008PC / 112492-5208

[0030] In embodiments, the N- and / or C-terminus of the albumin-binding moiety is connected to the capsid protein by a linker sequence.

[0031] In embodiments, the linker sequence is substantially comprised of glycines and serines.

[0032] In embodiments, the linker sequence comprises GSGS (SEQ ID NO: 73), SGGTSGSTSGTGST (SEQ ID NO: 17), AGSSTGSSTGPGSTT (SEQ ID NO: 18), GGSGGAP (SEQ ID NO: 19), GGGVEGGG (SEQ ID NO: 20), GGSGGSGGGGSGGGGS (SEQ ID NO: 25), LE, GGGGS (SEQ ID NO: 26), (GGGGS)n (n=1-7) (SEQ ID NO: 27-33), (Gly)8 (SEQ ID NO: 34), (Gly)6 (SEQ ID NO: 35), (EAAAK)n (n=1-3) (SEQ ID NO: 36-38), A(EAAAK)nA (n=2-5) (SEQ ID NO: 39-42), AEAAAKEAAAKA (SEQ ID NO: 43), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 44), PAPAP (SEQ ID NO: 45), KESGSVSSEQLAQFRSLD (SEQ ID NO: 46), EGKSSGSGSESKST (SEQ ID NO: 47), GSAGSAAGSGEF (SEQ ID NO: 48), (XP)n (SEQ ID NO: 49), GGS, (GGS)n (n=2-20) (SEQ ID NO: 50-68), GGGSE (SEQ ID NO: 69), GSESG (SEQ ID NO: 70), GSEGS (SEQ ID NO: 71), or GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 72).

[0033] In embodiments, the linker sequence comprises the sequence GSGS (SEQ ID NO: 73).

[0034] In embodiments, the albumin-binding moiety is further fused to a homing polypeptide or a homing domain thereof.

[0035] In embodiments, the modified capsid protein binds to albumin.

[0036] In aspects, the present disclosure provides a nucleic acid encoding a modified adenovirus capsid protein described herein.

[0037] In aspects, the present disclosure provides a recombinant adenovirus genome comprising a nucleic acid described herein.

[0038] In aspects, the present disclosure provides a recombinant adenovirus vector comprising a modified capsid protein described herein, a nucleic acid described herein, or a recombinant adenovirus genome described herein, and further comprising a therapeutic payload.

[0039] In embodiments, the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule.

[0040] In embodiments, the transgene encodes a therapeutic protein.

[0041] In embodiments, the transgene encoding the therapeutic protein comprises a gene used in gene therapy or in vaccination.

[0042] In embodiments, the recombinant adenovirus vector binds to albumin.

[0043] DBl / 161420432.1 5 VCN-008PC / 112492-5208

[0044] In embodiments, in the presence of human serum albumin, the recombinant adenovirus vector has reduced recognition by anti-adenovirus neutralizing antibodies, as compared to an adenovirus vector that does not comprise an albumin-binding moiety.

[0045] In aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adenovirus vector described herein, or a cell described herein, and a pharmaceutically acceptable carrier.

[0046] In aspects, the present disclosure provides a method of delivering a therapeutic payload to a subject comprising administering an effective amount of a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein to the subject.

[0047] In aspects, the present disclosure provides a method of delivering a therapeutic payload to a subject comprising contacting a cell with a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein, and administering the cell to the subject.

[0048] In embodiments, the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule.

[0049] In embodiments, the transgene encodes a therapeutic protein.

[0050] In embodiments, the method results in long-term expression of the transgene in the subject.

[0051] In aspects, the present disclosure provides a method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising administering an effective amount of a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein to the subject.

[0052] In aspects, the present disclosure provides a method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising contacting a cell with a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein, and administering the cell to the subject.

[0053] In embodiments, the disease or disorder is a genetic disease.

[0054] In embodiments, the method prevents an infectious disease in the subject.

[0055] In embodiments, the method provides a vaccination from an infectious disease caused by an infectious or pathogenic agent.

[0056] In embodiments, the infectious or pathogenic agent is selected from one or more of a virus, a prion, a bacteria, a parasite, a fungi, and a protozoa.

[0057] DBl / 161420432.1 6 VCN-008PC / 112492-5208

[0058] In aspects, the present disclosure provides a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein for use in treating, ameliorating, or preventing a disease or disorder in a subject.

[0059] In aspects, the present disclosure provides a use of a recombinant adenovirus vector described herein, a cell described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for the treating, ameliorating, or preventing a disease or disorder in a subject.

[0060] In embodiments, upon administration to a subject, the recombinant adenovirus vectors have reduced recognition by anti-adenovirus neutralizing antibodies, as compared to adenovirus vectors that do not comprise an albumin-binding moiety.

[0061] In embodiments, the subject is a human.

[0062] In embodiments, the recombinant adenovirus vectors are systemically administered.

[0063] DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 shows the location and sequence of an ABD inserted into HVR7 in the plasmid pAdZICOVIR15FiGFP-H7-ABD. The ABD sequence is displayed in blue text, the GSGS linker DNA sequence is shown in red text, and the HVR7 DNA sequence is displayed in black text.

[0065] Figure 2 shows the results of plaque analysis of ABD oncolytic adenoviruses. The control virus was ICOVIR15GFP and did not contain an ABD insertion. The virus containing the ABD insertion was ICOVIR15GFP-H7ABD (H7 ABD).

[0066] Figure 3 shows a template for titering of ABD GFP viruses using clarified cell supernatants from an infected 15 cm plate of A549 cells.

[0067] Figure 4 shows virus production analysis of ABD adenoviruses. Cells and supernatants were harvested 48 or 72 hours after infection and viral titers determined.

[0068] Figure 5 shows cytotoxicity analysis of ICOVIR15GFP control virus (without ABD). Cytotoxicity of A549 cells was determined in the presence (open circles, dotted line) or absence (solid circles, solid line) of HSA. Cell viability curves from three separate experiments are displayed. IC50 was calculated from each curve and displayed below the curve.

[0069] DBl / 161420432.1 7 VCN-008PC / 112492-5208

[0070] Figure 6 shows cytotoxicity analysis of H7ABD (ICOVIR15GFP-H7ABD). Cytotoxicity of A549 cells was determined in the presence (solid squares, dotted line) or absence (solid circles, solid line) of HSA. Cell viability curves from three separate experiments are displayed. IC50 was calculated from each curve and displayed below the curve.

[0071] Figure 7 shows mean IC50 values for control and ABD virus. The IC50 of the indicated viruses, in the absence (HSA-) or presence (HSA+) of HSA, were averaged, and the mean -*7- standard deviations are displayed. Mean IC50 was calculated using the data displayed in Figures 5-7.

[0072] Figure 8 shows a template of a 96-well plate set-up of an evasion of NAbs study in the presence or absence of HSA.

[0073] Figure 9 shows the results of fluorimeter evaluation of GFP-positive cells following A549 cell infection with ABD virus in the presence or absence of HSA and NAbs. GFP-positive cells were quantified using a fluorimeter 48 hours after virus infection. The arrow indicates the Nabs dilution (1 / 1280) where the H7 ABD begins to display increased cytotoxicity in the presence of HSA (Nabs avoidance).

[0074] Figure 10 shows the results of evaluation of GFP-positive cells following A549 cell infection with ABD virus in the presence or absence of HSA and NAbs. GFP-positive cells were visualized via fluorescent microscopy 48 hours after virus infection.

[0075] DETAILED DESCRIPTION

[0076] The present disclosure provides, inter alia, modified adenovirus capsid proteins, nucleic acids encoding the modified adenovirus capsid proteins, adenovirus vector genomes comprising the nucleic acids encoding the modified adenovirus capsid proteins, adenovirus vectors and particles comprising the modified adenovirus capsid proteins, pharmaceutical compositions comprising the adenovirus vectors and particles comprising the modified adenovirus proteins, and methods of using the pharmaceutical compositions to deliver therapeutic payloads and / or treat or prevent diseases or disorders.

[0077] In aspects, the present disclosure provides modified adenovirus capsid proteins comprising an albuminbinding moiety insertion. The present disclosure is based, in part, on the discovery that adenoviruses can be engineered to evade, reduce, or eliminate NAbs by inserting an albumin-binding moiety into the capsid protein such that, upon administration, the adenovirus particles are coated in albumin and are not recognized by the NAbs. The present disclosure also provides methods of delivering therapeutic payloads and / or treating or

[0078] DBl / 161420432.1 8 VCN-008PC / 112492-5208 preventing diseases or disorders by administering adenoviruses disclosed herein or cells contacted with adenoviruses disclosed herein.

[0079] Without wishing to be bound by theory, it is thought that the modified adenovirus capsid proteins comprising an albumin-binding moiety insertion described herein, and which display the albumin-binding moiety on the outer surface of the adenovirus capsid when it is packaged as an adenovirus particle, can be coated with albumin, thus protecting the adenovirus particle from NAbs present in the bloodstream of a subject to which the adenovirus particles are administered.

[0080] Recombinant adenoviruses

[0081] The present disclosure is directed, in part, to adenovirus comprising modified capsid proteins comprising an albumin-binding moiety insertion.

[0082] In embodiments, the term “vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0083] In embodiments, a “recombinant adenovirus vector” is derived from a wild-type genome of an adenovirus, or is an adenovirus that does not appear naturally. In embodiments, “recombinant adenovirus vectors” of the present disclosure are composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' inverted terminal repeats (ITRs). It is this recombinant adenovirus vector which is packaged into a capsid protein (referred to herein as a “particle”) and delivered to a selected target cell for subsequent infection or transduction ex vivo, in vitro, or in vivo. Where a recombinant adenovirus sequence is encapsidated or packaged into an adenovirus particle, the particle can also be referred to as a “recombinant adenovirus vector” or “recombinant adenovirus particle.” In embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue.

[0084] DBl / 161420432.1 9 VCN-008PC / 112492-5208

[0085] In embodiments, the term refers to a double-stranded DNA sequence that, in the presence of appropriate proteins, can be packaged, resulting in a complete adenovirus particle. In embodiments, for this packaging to occur, the sequence complies with the following conditions: exhibit separate adenovirus ITR, one at each of its end points; comprise a packaging signal Psi between both ITRs, located in such a way that the distance between the 5' end of the packaging signal Psi and the 3' end of the ITR closest to it does not exceed the distance that would prevent packaging of the natural adenovirus, a distance that is 200 base pairs in the case of the human serotype 5 adenovirus and which, in embodiments, is approximately equal in the case of other serotypes; the distance between the ends of both ITR is not, in embodiments, greater than 105 percent of the size of the adenovirus genome present in nature to which the proteins which will form the capsid belong.

[0086] In embodiments, a recombinant adenovirus contains one or more modifications with respect to the wild-type. In embodiments, modifications include, but are not limited to, modifications to the adenovirus genome that is packaged in the particle in order to make an infectious virus.

[0087] In embodiments, the adenoviral genome is deficient in at least one gene function required for viral replication, thereby resulting in a “replication-deficient” adenoviral vector. In embodiments, “replication-deficient” means that the adenoviral vector comprises an adenoviral genome that lacks at least one replication-essential gene function (e.g., such that the adenoviral vector does not replicate in typical host cells, e.g., those in a human patient that could be infected by the adenoviral vector in the course of treatment in accordance with the invention). In embodiments, a replication-deficient adenoviral vector comprises an adenoviral genome deficient in at least one replication-essential gene function of one or more regions of the adenoviral genome. In embodiments, the adenoviral vector is deficient in at least one essential gene function of the E4 region or E1 region of the adenoviral genome required for viral replication. In embodiments, in addition to a deficiency in the E1 region, the recombinant adenovirus can also have a mutation in the major late promoter (MLP). In embodiments, the adenoviral vector is deficient in at least one essential gene function of the E1 region and at least part of the E3 region (e.g., an Xba I deletion of the E3 region). In embodiments, with respect to the E1 region, the adenoviral vector can be deficient in (e.g., deleted of) at least part of the E1a region and / or at least part of the E1 b region. In embodiments, the adenoviral vector can comprise a deletion of the entire E1 region and part of the E3 region of the adenoviral genome (i.e., nucleotides 355 to 3,511 and 28,593 to 30,470). In embodiments, a singly-deficient adenoviral vector can be deleted of approximately nucleotides 356 to 3,329 and 28,594 to 30,469 (based on the adenovirus serotype 5 genome). In embodiments, the adenoviral vector genome can be deleted of approximately nucleotides 356 to 3,510 and 28,593 to 30,470

[0088] DBl / 161420432.1 10 VCN-008PC / 112492-5208

[0089] (based on the adenovirus serotype 5 genome), thereby resulting in an adenoviral vector having deletions in the E1, E3, and E4 regions of the adenoviral genome.

[0090] In embodiments, a deficiency in a gene, gene function, or gene or genomic region is a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part. In embodiments, deletion of an entire gene region is not required for disruption of a replication-essential gene function. In embodiments, for the purpose of providing sufficient space in the adenoviral genome for one or more transgenes, removal of a majority of a gene region may be desirable. In embodiments, mutation of genetic material by addition or substitution also is appropriate for disrupting gene function. In embodiments, replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the E1, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., NA-RNA-1 and / or NA-RNA-2).

[0091] In embodiments, modifications allow obtaining replication-deficient virus (i.e., virus that cannot reproduce) by removing a gene from the virus genome that is critical for replication. In embodiments, such modifications include, but are not limited to, deletions known in the art, such as deletions in one or more of the E1 a, E1b, E2a, E2b, E3, and E4 coding regions.

[0092] In embodiments, the adenoviral vector also can have essentially the entire adenoviral genome removed except the ITR and the packaging sequence. Such vectors are known in the art as “gutless” or “helperdependent” adenovirus vectors. In this case the capsid sequence modified to contain an albumin-binding moiety is provided by the helper adenovirus. In embodiments, the 5' or 3' regions of the adenoviral genome comprising ITRs and packaging sequence need not originate from the same adenoviral serotype as the remainder of the viral genome. For example, the 5' region of an adenoviral serotype 5 genome (i.e., the region of the genome 5' to the adenoviral E1 region) can be replaced with the corresponding region of an adenoviral serotype 2 genome (e.g., the Ad5 genome region 5' to the E1 region of the adenoviral genome is replaced with nucleotides 1-456 of the Ad2 genome).

[0093] In embodiments, a recombinant adenovirus is a chimeric adenovirus that is formed by combination of elements from different serotypes. In embodiments, the term “recombinant” also includes replicationconditional adenoviruses, which are viruses that preferentially replicate in certain types of cells or tissues but to a lesser degree or not at all in other types. For example, among the adenoviruses provided herein are adenoviruses that replicate in abnormally proliferating tissue, such as solid tumors and other neoplasms.

[0094] DBl / 161420432.1 11 VCN-008PC / 112492-5208

[0095] These include the viruses disclosed in U.S. Pat. Nos. 5,998,205 and 5,801,029, included herein by reference in their entireties. Such viruses are sometimes referred to as “cytolytic” or “cytopathic” viruses or vectors, and, if they have such an effect on neoplastic cells, are referred to as “oncolytic” viruses or vectors. In embodiments, the adenovirus is a replicative adenovirus. In embodiments, the adenovirus is an oncolytic adenovirus. In embodiments, the adenovirus is a non-replicative adenovirus or a replication-deficient adenovirus. In embodiments, a replication-deficient adenovirus or non-replicating adenovirus is an adenovirus unable to replicate in the target cell that is used in gene therapy as a carrier of genes to target cells with the goal to express a therapeutic gene within the cell, rather than lysis of the cell.

[0096] In aspects, the present disclosure provides a recombinant adenovirus genome comprising modified adenovirus capsid proteins described herein.

[0097] A recombinant adenovirus “genome” refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsidated to form an adenovirus particle.

[0098] In aspects, the present disclosure provides a recombinant adenovirus vector or recombinant adenovirus particle comprising modified adenovirus capsid proteins described herein.

[0099] In embodiments, “adenovirus” refers to any virus that can be categorized as an adenovirus, i.e. any virus pertaining to the Adenoviridae family characterized by being a non-enveloped virus with an icosahedral nucleocapsid containing a double stranded DNA genome. This term includes any adenovirus capable of infecting a human or an animal, including all groups, subgroups, and serotypes that use CAR as receptor for infection of target cells. In embodiments, adenoviruses include, but are not limited to, avian, canine, equine, bovine, ovine, porcine, human, or frog adenovirus. In embodiments, an adenovirus of the present disclosure is a human adenovirus, i.e. an adenovirus capable of infecting humans. In embodiments, a “serotype” is an immunologically different type of adenovirus. There are at least 67 serotypes of human adenovirus that are classified into several subgroups (A to G). In embodiments, the adenovirus vectors, as well as methods and uses thereof, include any adenoviral subgroup or serotype. In embodiments, the adenovirus vector is based upon any adenoviral serotype known in the state of the art including, but not limited to, any of the serotypes listed in Table 1.

[0100] Table 1. Examples of adenoviral subgroups and serotypes

[0101] DBl / 161420432.1 12 VCN-008PC / 112492-5208

[0102] In embodiments, the adenovirus vector is based upon any adenovirus genome, such as a Group A adenovirus, a Group B adenovirus, a Group C adenovirus, a Group D adenovirus, a Group E adenovirus, a

[0103] Group F adenovirus, or a Group G adenovirus. In embodiments, the adenovirus vector is based upon a serotype 1 adenovirus genome, a serotype 2 adenovirus genome, a serotype 3 adenovirus genome, a serotype 4 adenovirus genome, a serotype 5 adenovirus genome, a serotype 6 adenovirus genome, a serotype 7 adenovirus genome, a serotype 8 adenovirus genome, a serotype 9 adenovirus genome, a serotype 10, adenovirus genome, a serotype 11 adenovirus genome, a serotype 12 adenovirus genome, a serotype 13 adenovirus genome, a serotype 14 adenovirus genome, a serotype 15 adenovirus genome, a serotype 16 adenovirus genome, a serotype 17 adenovirus genome, a serotype 18 adenovirus genome, a serotype 19 adenovirus genome, a serotype 20, adenovirus genome, a serotype 21 adenovirus genome, a serotype 22 adenovirus genome, a serotype 23 adenovirus genome, a serotype 24 adenovirus genome, a serotype 25 adenovirus genome, a serotype 26 adenovirus genome, a serotype 27 adenovirus genome, a serotype 28 adenovirus genome, a serotype 29 adenovirus genome, a serotype 30, adenovirus genome, a serotype 31 adenovirus genome, a serotype 32 adenovirus genome, a serotype 33 adenovirus genome, a serotype 34 adenovirus genome, a serotype 35 adenovirus genome, a serotype 36 adenovirus genome, a serotype 37 adenovirus genome, a serotype 38 adenovirus genome, a serotype 39 adenovirus genome, a serotype 40, adenovirus genome, a serotype 41 adenovirus genome, a serotype 42 adenovirus genome, a serotype 43 adenovirus genome, a serotype 44 adenovirus genome, a serotype 45 adenovirus genome, a serotype 46 adenovirus genome, a serotype 47 adenovirus genome, a serotype 48 adenovirus genome, a serotype 49 adenovirus genome, a serotype 50, adenovirus genome, a serotype 51 adenovirus genome, a serotype 52 adenovirus genome, a serotype 53 adenovirus genome, a serotype 54 adenovirus genome, a

[0104] DBl / 161420432.1 13 VCN-008PC / 112492-5208 serotype 55 adenovirus genome, a serotype 56 adenovirus genome, a serotype 57 adenovirus genome, a serotype 58 adenovirus genome, a serotype 59 adenovirus genome, a serotype 60, adenovirus genome, a serotype 61 adenovirus genome, a serotype 62 adenovirus genome, a serotype 63 adenovirus genome, a serotype 64 adenovirus genome, a serotype 65 adenovirus genome, a serotype 66 adenovirus genome, or a serotype 67 adenovirus genome.

[0105] In embodiments, adenovirus vectors can be based on the same group or serotype or variant, ora combination of different ones. In embodiments, a recombinant adenovirus vector based upon a particular serotype genome can be identical to the serotype of the capsid proteins that package the vector. In embodiments, an adenovirus vector genome can be based upon an adenovirus serotype genome distinct from the serotype of the adenovirus capsid proteins that package the vector.

[0106] In embodiments, recombinant adenoviruses of the present disclosure are used to target abnormal cells, for example, any cells which are harmful or otherwise unwanted in vivo. Examples include, but are not limited to, cancer cells, and cells causing autoimmune disease, restenosis, and scar tissue formation.

[0107] In embodiments, adenoviruses of the present disclosure can be selectively distributed in vivo in a given tissue, avoiding or significantly reducing expression in non-target or non-tumor tissue.

[0108] In embodiments, adenoviruses of the present disclosure can have modifications in their genomic sequences that confer selective replication in a cell. In embodiments, in order to direct the expression of the adenovirus to the tissue wherein such expression is needed or to the tumoral tissue to be treated, adenoviruses of the present disclosure can comprise a tissue-specific promoter or a tumor-specific promoter. Thus, in embodiments, adenoviruses of the present disclosure further comprise a tissue-specific promoter or a tumorspecific promoter. In embodiments, the tissue-specific promoter or the tumor-specific promoter comprises promoter sequences to control the expression of one or more genes selected from the group consisting of E1 a, E1b, E2, and E4. In embodiments, the promoter controls the expression of E1a.

[0109] In embodiments, the term “promoter” is used according to its art-recognized meaning. In embodiments, it is intended to mean the DNA region, which may be upstream to the coding sequence of a gene, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site. In embodiments, said promoter controls the viral genes that start the replication.

[0110] In embodiments, the term “tissue-specific” means that the promoter that the gene essential for replication is operably linked to functions specifically in that tissue, or a “target tissue,” so that replication proceeds in a

[0111] DBl / 161420432.1 14 VCN-008PC / 112492-5208 target tissue. In embodiments, this can occur by the presence in a target tissue, and not in non-target tissues, of positive transcription factors that activate the promoter. In embodiments, this can also occur by the absence of transcription inhibiting factors that normally occur in non-target tissues and prevent transcription as a result of the promoter. Thus, in embodiments, when transcription occurs, it proceeds into the gene essential for replication such that in a target tissue, replication of the vector and its attendant functions occur.

[0112] In embodiments, tissue specificity is particularly relevant with respect to targeting an abnormal counterpart of a particular tissue type while avoiding the normal counterpart of the tissue, or avoiding surrounding tissue of a different type than the abnormal tissue, while treating the abnormal tissue. In embodiments, the promoter is “tumor-specific,” and it functions specifically or primarily in tumoral tissues. For example, in embodiments, recombinant adenoviruses of the present disclosure are useful for treating metastases to the liver. One example is colon cancer, which often metastasizes into the liver. It has been found that even when colon cancer metastasizes into the liver, the CEA promoter is active in the cells of the metastases but not in normal liver cells. Accordingly, in embodiments, normal human adult liver should not support replication of a virus that has viral genes essential for replication linked to the colon cancer CEA-specific promoter, and replication should occur in the primary cancer cells. Another example is the alpha-fetoprotein promoter, which is active only in hepatocellular carcinoma. Another example is the tyrosinase promoter, which is active only in melanoma and not in normal skin cells. In embodiments, in each case, replication is expected in the abnormal but not the normal cells. In embodiments, the promoter is a tissue-specific promoter selected from alphafetoprotein promoter, DE3 promoter, tyrosinase promoter, carcinoembryonic antigen (CEA) promoter, surfactant protein promoter. E2F promoter, telomerase hTERT promoter, prostate-specific antigen promoter, COX-2 promoter, albumin gene promoter, the core promoter of hepatitis virus, the promoter of the globulin- binding protein which binds to thyroxine, and ErbB2 promoter. In embodiments, the promoter is selected from the group consisting of a E2F promoter, a telomerase hTERT promoter, a tyrosinase promoter, a prostatespecific antigen promoter, an alpha-fetoprotein promoter, and a COX-2 promoter.

[0113] In embodiments, adenoviruses of the present disclosure are particularly useful for the treatment of cancer. All tumors are potentially amenable to treatment with adenoviruses of the present disclosure. Tumor types include, but are not limited to, hematopoietic, pancreatic, neurologic, hepatic, gastrointestinal tract, endocrine, biliary tract, sinopulmonary, head and neck, soft tissue sarcoma and carcinoma, dermatologic, reproductive tract, and the like. In embodiments, tumors for treatment are those with a high mitotic index relative to normal tissue. In embodiments, tumors for treatment are solid tumors.

[0114] DBl / 161420432.1 15 VCN-008PC / 112492-5208

[0115] In embodiments, adenoviruses of the present disclosure are oncolytic adenoviruses. In embodiments, “oncolytic adenovirus” refers to any adenovirus that is able to replicate or that is replication-competent in a tumor cell, even without selectivity. In embodiments, the therapeutic action of oncolytic adenoviruses is based on the capability to replicate and to lyse the tumor cell to be eliminated. The death of the tumor cells can be detected by any method of the state of the art, including but not limited to, determining the number of viable cells, the cytopathic effect, the apoptosis of tumor cells, the synthesis of viral proteins in tumor cells (for example, by metabolic labelling and / or Western blot of viral proteins or PCR with reverse transcription of the viral genes needed for replication), or the reduction in the size of the tumor.

[0116] In embodiments, another strategy to achieve selective replication in tumors is the deletion of viral functions that are necessary for replication in normal cells but that are not needed in tumor cells. This includes, for example, the deletion of early E1 Afunctions which block the retinoblastoma (pRB) pathway. Other viral genes that interact directly with pRB such as E4 and E4orf6 / 7 are candidates to be deleted in order to achieve selective replication in tumor cells. In embodiments, another modification described to achieve selective replication in tumors is the deletion of adenoviral genes coding for the virus-associated RNAs (VA-RNAs). These RNAs block the antiviral activity of interferon and their deletion results in adenoviruses that are sensitive to interferon inhibition. Due to the characteristic truncation in the interferon pathway in tumor cells such adenoviruses replicate normally in tumors. In embodiments, adenoviruses of the present disclosure further comprises mutations in one or more genes selected from the group consisting of E 1a, E 1b, E4, and VA-RNAs. In embodiments, mutations in one or more genes selected from the group consisting of E1 a, E1b, E4, and VA-RNAs allows adenoviruses of the present disclosure to achieve selective replication in tumors. In embodiments, the mutations are in E1a. In embodiments, the mutation in E1a is a deletion of some amino acids of the E1 A protein affecting the interaction of E1 A with pRB. In embodiments, the mutation in E1a is a deletion of the amino acids 121-129 of the polypeptide chain (A24 deletion).

[0117] In embodiments, “selective replication” means that the adenovirus has replication efficiency in tumor cells higher than in normal cells, in embodiments, the adenovirus has replication efficiency in tumor cells about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400- fold, about 450-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold or more higher than in normal cells.

[0118] DBl / 161420432.1 16 VCN-008PC / 112492-5208

[0119] In embodiments, “replication” refers to the duplication of adenoviral vectors that occurs at the level of nucleic acid or at the level of infectious viral particle. In the case of DNA viruses, replication at the nucleic acid level is DNA replication. However, replication also includes the formation of infectious DNA viral particles. Replication of an adenovirus can be assayed by well-known techniques. In embodiments, assays for replication of an adenoviral vector in a cell involve detecting a polynucleotide, virions, or infective virus. A variety of well-known methods that can be used for this purpose involve determining the amount of a labelled substrate incorporated into a polynucleotide during a given period in a cell. When replication involves a DNA polynucleotide, 3H-thymidine often is used as the labelled substrate, in this case, the amount of replication is determined by separating DNA of the vector from the bulk of cellular DNA and measuring the amount of tritium incorporated specifically into vector DNA. Replication of a polynucleotide vector also may be detected by lysing or permeating cells to release the polynucleotide, then isolating the polynucleotide and quantitating directly the DNA or RNA that is recovered. Polynucleotide replication also may be detected by quantitative PCR using primers that are specific for the assayed polynucleotide. Virions may be assayed by electron microscope counting techniques well known to the art, e.g., by isolating the virions and determining protein and nucleic acid content, and by labelling viral genomic polynucleotides or virion proteins and determining the amount of virion from the amount of polynucleotide or protein.

[0120] In embodiments, another strategy to achieve selective replication in tumors is the modification of the virus capsid proteins implied in the infection of the host cell to target the adenovirus to a receptor present in a tumor cell. The modification of the capsid proteins that the virus uses to infect the cells may also be used to increase infectivity of the adenovirus (i.e. increasing the entry of the virus in the cell). Targeting adenovirus to the tumor can also be achieved with bifunctional ligands that bind to the virus in one end and to the tumor receptor in the other. In embodiments, adenoviruses of the present disclosure further comprises capsid modifications to increase their infectivity or to target them to a receptor present in a tumor cell. In embodiments, the modification of the capsid is the insertion of an RGD motif (Arginine-Glycine-Asparagine motif) into the HI loop of the adenoviral fiber protein knob. This insertion allows the adenovirus to use integrins to dock in the cell and not only to internalize as it is the case with wild-type adenovirus. The use of integrins as cellular receptors of the virus increases the infectivity and the oncolytic potency. In embodiments, the oncolytic adenovirus has the capsid modified by means of a replacement of the KKTK heparan sulphate binding domain in the adenovirus fiber shaft with the domain RGDK (N. Bayo et al. Human Gene Therapy 2009, 20:1214-21).

[0121] DBl / 161420432.1 17 VCN-008PC / 112492-5208

[0122] In embodiments, another strategy to increase infectivity of target cells with adenoviruses is the replacement of a portion of the fiber with the homologous portion from a different serotype. In embodiments, the fiber shaft and knob of human adenoviruses derived from serotype 5 is replaced with the fiber shaft and knob of human serotype 3 or 35 adenoviruses. The obtained recombinant adenoviruses with genomes derived from different serotypes are known in the art as chimeric adenoviruses. In embodiments, adenoviruses of the present disclosure further comprises a chimeric capsid derived from different adenovirus serotypes. In embodiments, the modification of the capsid is the substitution of part of the fiber gene with the homologous part from a different adenovirus serotype to form a chimeric adenovirus.

[0123] In embodiments, the oncolytic adenovirus is a tumor-selective replicating adenovirus characterized by containing a deletion of some amino acids of the E1 A protein affecting the interaction of E1A with pRB. In embodiments, the E1A protein has a deletion of the amino acids 121-129 of the polypeptide chain (A24 deletion). In embodiments, the oncolytic adenovirus is a tumor-selective replicating adenovirus characterized by containing an insertion of four E2F binding sites and one Sp1 binding site in the endogenous promoter of E1a to control the expression of E1a. In embodiments, the oncolytic adenovirus is a tumor-selective replicating adenovirus characterized by containing the insertion of the RGD peptide in the adenoviral fiber to increase the infectivity of the virus. In embodiments, the oncolytic adenovirus is a tumor-selective replicating adenovirus characterized by containing a mutant version of the E1A protein where amino acids 121-129 of the polypeptide chain have been deleted (A24 deletion) affecting the interaction of E1a with pRB, the insertion of four E2F binding sites and one Sp1 binding site in the endogenous promoter of E1a to control the expression of E1 a, and the insertion of the RGD peptide in the adenoviral fiber to increase the infectivity of the virus. Said modifications may be present in combination in the same adenovirus or in isolation.

[0124] In embodiments, the genome of the adenovirus can also contain a heterologous gene that encodes a therapeutic protein such that the heterologous gene is expressed within an infected cell.

[0125] Modified Capsid Protein

[0126] In embodiments, the recombinant adenovirus is modified by insertion of a heterologous sequence on the outer surface of the adenoviral capsid protein. In embodiments, the heterologous sequence encodes for an albumin-binding moiety.

[0127] DBl / 161420432.1 18 VCN-008PC / 112492-5208

[0128] In embodiments, the terms “modified” or “variant” mean that a nucleic acid or protein deviates from a reference or parental sequence. A modified or variant protein refers to a protein sequence which has been altered compared to a reference (e.g., wild-type) or parental sequence. Modified and variant sequences can therefore have substantially the same, greater, or less activity or function than a reference or parental sequence, but can retain at least partial activity or function of the reference or parental sequence. A nucleic acid coding sequence can be genetically modified to encode a modified or variant protein.

[0129] In embodiments, an adenovirus particle comprises a capsid that encloses the viral DNA. In embodiments, “capsid” refers to the protein shell of a virus formed by subunits named capsomers that may be pentagonal or hexagonal. In embodiments, an adenoviral capsid has an icosahedral shape, which has 20 equilateral triangular faces. In embodiments, most of the capsid is formed by the hexon protein, and each vertex has a complex formed by penton base and fiber protein. In embodiments, the terms “capsid protein” and “hexon protein” are used interchangeably.

[0130] In embodiments, the term “adenoviral hexon protein” or “hexon protein” (formerly referred to as “protein II”) refers to the major structural capsid protein found in adenoviruses that self-associates to form trimers, each in the shape of a hexagon. 240 hexon trimers are assembled to provide an adenoviral capsid. The hexon protein is essential for virus capsid assembly, determination of the icosahedral symmetry of the capsid and integrity of the capsid. The major structural features of the hexon protein are shared by adenoviruses across serotypes, but the hexon protein differs in size and immunological properties between serotypes. In embodiments, the term “hexon protein” or “capsid protein” encompasses the hexon or capsid protein of any adenovirus, including, but not limited to, those having sequences of SEQ ID NO: 1 (hAd5), SEQ ID NO: 2 (hAd3), SEQ ID NO: 3 (hAd4), SEQ ID NO: 4 (hAd7), SEQ ID NO: 5 (hAd11 ), SEQ ID NO: 6 (hAd6), SEQ ID NO: 7 (hAd21), SEQ ID NO: 8 (hAd34), or SEQ ID NO: 9 (hAd35), or variants thereof. In embodiments, the term includes all the natural variants of hexon protein that appear naturally in other subgroups or serotypes. in embodiments, a “modified adenovirus hexon protein” or a “modified adenovirus capsid protein” means that the capsid protein has an amino acid modification compared the reference or parental unmodified adenovirus capsid protein. In embodiments, a modification of a capsid protein is an amino acid substitution. In embodiments, a modification of a capsid protein is an insertion, e.g., a peptide insertion.

[0131] In embodiments, a modified capsid protein retains at least part of a function or activity of unmodified reference or parental capsid protein. The function or activity of an adenovirus capsid protein includes the ability to package the adenovirus vector genome into productive viral particles that are able to transduce cells and in

[0132] DBl / 161420432.1 19 VCN-008PC / 112492-5208 turn able introduce the heterologous sequence in the vector genome in the transduced cells for expression. Modified adenovirus capsid proteins can exhibit different characteristics or have improvements compared to a reference or parental adenovirus capsid protein. When comparing the different characteristics or improvements of a modified adenovirus capsid protein, it is appropriate to compare it to the reference or parental adenovirus capsid protein. In the case that the parental adenovirus capsid protein has already been modified, a further modification of the parental adenovirus capsid protein as set forth herein, for example a peptide insertion as set forth herein, the comparison is to the parental modified adenovirus capsid protein prior to the peptide insertion.

[0133] In embodiments, adenovirus capsid modifications include amino acid substitutions and peptide insertions. Non-limiting examples of amino acid substitutions include substituting 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250 or more amino acid residues. Non-limiting examples of peptide insertions include 2 or more contiguous / adjacent amino acid residues inserted into adenovirus capsids, for example, 2-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more amino acid residues.

[0134] In embodiments, a peptide insertion has a length of about 5 amino acids to about 70 amino acids. In embodiments, a peptide insertion has a length of about 10 amino acids to about 60 amino acids. In embodiments, a peptide insertion has a length of about 20 amino acids to about 55 amino acids. In embodiments, a peptide insertion has a length of about 30 amino acids to about 50 amino acids.

[0135] In embodiments, modified adenovirus capsid proteins and nucleic acids encoding the capsid proteins exhibit less than 100% sequence identity to a reference or parental adenovirus serotype such as Group A, Group B, Group C, Group D, Group E, Group F, or Group G, or serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67. In embodiments, a modified adenovirus capsid protein comprises or consists of a sequence at least about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9% identical to a reference or parental adenovirus capsid protein, such as Group A, Group B, Group C, Group D, Group E, Group F, or Group G, or serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55,

[0136] DBl / 161420432.1 20 VCN-008PC / 112492-5208

[0137] 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67., as well as variants of Group A, Group B, Group C, Group D, Group E, Group F, and Group G, or serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67..

[0138] In embodiments, recombinant adenoviruses provided herein comprise capsid components from one or more of Group A, Group B, Group C, Group D, Group E, Group F, or Group G, or serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9,

[0139] 10. 11. 12. 13. 14. 15. 16. 17. 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, or 67, or other recombinant adenovirus particles, or combinations of two or more thereof. In embodiments, recombinant adenovirus particles comprise a capsid protein at least about 80% or more identical, e.g., about 85%, about 85%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, etc., i.e. up to about 100% identical, to e.g., hexon protein sequence of an adenovirus capsid selected from Group A, Group B, Group C, Group D, Group E, Group F, or Group G, or serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0140] 18. 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67, or a derivative, modification, or pseudotype thereof.

[0141] In embodiments, recombinant adenovirus particles comprise a hexon protein at least about 80% or more identical, e.g., about 85%, about 85%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, etc., i.e. up to about 100% identical, to e.g., hexon protein sequence of an adenovirus capsid selected from SEQ ID NO: 1 (hAd5), SEQ ID NO: 2 (hAd3), SEQ ID NO: 3 (hAd4), SEQ ID NO: 4 (hAd7), SEQ ID NO: 5 (hAd11), SEQ ID NO: 6 (hAd6), SEQ ID NO: 7 (hAd21), SEQ ID NO: 8 (hAd34), or SEQ ID NO: 9 (hAd35), or a derivative, modification, or pseudotype thereof.

[0142] In embodiments, these recombinant adenoviruses may comprise a genome comprising a transgene encoding a therapeutic protein.

[0143] In embodiments, a modified adenovirus capsid protein has about 1, about 2, about 3, about 4, about 5, about 5-10, about 10-15, about 15-20, or more amino acid substitutions. In embodiments, a modified adenovirus capsid protein has a peptide insertion length of 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-50, or 50- 60 amino acids.

[0144] DBl / 161420432.1 21 VCN-008PC / 112492-5208

[0145] In embodiments, the reference or parental adenovirus capsid protein has an amino acid sequence of any one of SEQ ID NOs: 1-10, as shown in Table 2 below, or a variant thereof.

[0146] Table 2. Adenovirus capsid sequences

[0147] DBl / 161420432.1 22 VCN-008PC / 112492-5208

[0148] DB1 / 161420432.1 23 VCN-008PC / 112492-5208

[0149] DB1 / 161420432.1 24 VCN-008PC / 112492-5208

[0150] DB1 / 161420432.1 25 VCN-008PC / 112492-5208

[0151] In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety in a position such that the albumin-binding moiety is exposed on the outer surface of the capsid protein when it is packaged as a recombinant adenovirus vector or particle.

[0152] In embodiments, the recombinant adenovirus vector or particle described herein is substantially covered or coated with host serum albumin, e.g., when administered to a subject. In embodiments, the recombinant adenovirus particle or vector described herein is shielded from NAbs, e.g., when administered to a subject. In embodiments, the recombinant adenovirus vector or particle described herein is self-coated with albumin. In embodiments, the recombinant adenovirus vector or particle described herein has an increased ability to evade the immune system when administered to a subject, as compared to adenovirus vectors that do not comprise an albumin-binding moiety. In embodiments, the recombinant adenovirus vector or particle described herein demonstrates reduced recognition by anti-adenovirus neutralizing antibodies, as compared to adenovirus vectors that do not comprise an albumin-binding moiety. In embodiments, the expression “outer surface of the hexon protein” or “outer surface of the capsid protein” or “outer surface of the adenovirus particle” refers to the regions of the hexon protein that are exposed on the surface of the capsid. In embodiments, in order to know if the albumin-binding moiety of the present disclosure has been introduced in the inner part or in the outer surface of the adenoviral hexon protein, an assay for detecting of binding to human serum albumin may be performed as disclosed in the experimental section of this patent application (for example, an ELISA assay) or an in vitro neutralization assay. In embodiments, if human serum albumin is capable of binding to the adenovirus, then the albumin-binding moiety has been introduced in the outer surface of the adenoviral hexon protein.

[0153] DBl / 161420432.1 26 VCN-008PC / 112492-5208

[0154] It has been reported that Loop 1 (L1) and Loop 2 (L2) of hexon protein are exposed on the outside of the viral capsomere structure. L1 contains six hypervariable regions (HVRs), i.e. HVR1 to HVR6 and L2 contains the seventh hypervariable region (HVR7).

[0155] In embodiments, the term “hypervariable region” or “HVR” refers to a region varying in length and sequence between adenoviral serotypes forming part of surfaced exposed loops. There are seven hypervariable regions of the adenoviral hexon for each subunit of the trimer (Biere B and Schweiger B. J Clin Virol 2010; 47(4):366-371). In embodiments, the nomenclature used for the HVRs is as disclosed in Crawford-Miksza and Schnurr (Crawford-Miksza and Schnurr. 1996. Virology, 224(2):357-367).

[0156] In embodiments, an albumin-binding moiety described herein is inserted into a site in VR VII.

[0157] In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety immediately after one or more of the amino acid residues as set forth in Table 3 below, or positions corresponding thereto.

[0158] Table 3. Summary of insertion sites

[0159] DBl / 161420432.1 27 VCN-008PC / 112492-5208

[0160] In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety within (a) 420-449 of hAd5 capsid amino acid sequence (SEQ ID NO: 1), or positions corresponding thereto; (b) 413- 441 of hAd3 capsid amino acid sequence (SEQ ID NO: 2) , or positions corresponding thereto; (c) 403-433 of hAd4 capsid amino acid sequence (SEQ ID NO: 3) , or positions corresponding thereto; (d) 409-434 of hAd7 capsid amino acid sequence (SEQ ID NO: 4) , or positions corresponding thereto; 1 417-445 of hAd 11 capsid amino acid sequence (SEQ ID NO: 5) , or positions corresponding thereto; (f) 403-437 of hAd16 capsid amino acid sequence (SEQ ID NO: 6) , or positions corresponding thereto; (g) 419-446 of hAd21 capsid amino acid sequence (SEQ ID NO: 7) , or positions corresponding thereto; (h) 421-448 of hAd34 capsid amino acid sequence (SEQ ID NO: 8) , or positions corresponding thereto; or (i) 421-449 of hAd35 capsid amino acid sequence (SEQ ID NO: 9) , or positions corresponding thereto. In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety after an amino acid residue corresponding to one of amino acids V420, 1421, N422, T423, E424, T425, L426, T427, K428, V429, K430, P431, K432, T433, G434, Q435, E436, N437, G438, W439, E440, K441, D442, A443, T444, E445, F446, S447, D448, or K449 of the hAd5 capsid, or positions corresponding thereto.

[0161] In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety within the L2 loop of an adenovirus capsid protein. In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety after amino acid residue 437 of hAd5, or positions corresponding thereto, or the corresponding site within any other adenovirus capsid protein.

[0162] In embodiments, the modified capsid protein comprises an insertion of an albumin-binding moiety within any region within the capsid protein that results in the albumin-binding moiety being presented on the outer surface of the capsid, and that does not interfere with capsid assembly, genome packaging, or infectivity.

[0163] In embodiments, albumin is not conjugated to the capsid protein. In embodiments, the albumin-binding moiety is not conjugated to the capsid protein.

[0164] Albumin-Binding Moiety

[0165] In embodiments, the albumin-binding moiety is directly attached to the capsid protein, i.e. the N- and C- terminus of the albumin-binding moiety are linked directly to the capsid protein. In embodiments, the albuminbinding moiety is connected to the capsid protein by means of a linker sequence, i.e., the N- an / or C-terminus of the albumin-binding moiety is connected to the capsid protein by a linker sequence.

[0166] DBl / 161420432.1 28 VCN-008PC / 112492-5208

[0167] In embodiments, the term “linker sequence” refers to an amino acid sequence that acts as a hinge region between the capsid protein and the albumin-binding moiety, providing space between both elements and assuring that the secondary structure of the capsid protein is not affected by the presence of the albuminbinding moiety and vice versa. In embodiments, the linker is of any length that allows both elements (i.e., the capsid protein and the albumin-binding moiety) to move independently from one another while maintaining the three-dimensional form of the individual elements. In embodiments, the linker sequence is a flexible linker peptide with a length of 31 amino acids or less. In embodiments, the linker sequence comprises less than 10 amino acids, less than 5 amino acids, less than 4 amino acids or 2 amino acids. In embodiments, the linker sequence comprises 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. In embodiments, the linker is a polyglycine linker. In embodiments, the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines). In embodiments, the linker is or comprises (Gly4Ser)n, where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8. In embodiments, the linker sequence is or comprises SGGTSGSTSGTGST (SEQ ID NO: 17), AGSSTGSSTGPGSTT (SEQ ID NO: 18), GGSGGAP (SEQ ID NO: 19), GGGVEGGG (SEQ ID NO: 20), GGSGGSGGGGSGGGGS (SEQ ID NO: 25), LE, GGGGS (SEQ ID NO: 26), (GGGGS)n (n=1-7) (SEQ ID NO: 27-33), (Gly)8 (SEQ ID NO: 34), (Gly)6 (SEQ ID NO: 35), (EAAAK)n (n=1-3) (SEQ ID NO: 36-38), A(EAAAK)nA (n=2-5) (SEQ ID NO: 39-42), AEAAAKEAAAKA (SEQ ID NO: 43), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 44), PAPAP (SEQ ID NO: 45), KESGSVSSEQLAQFRSLD (SEQ ID NO: 46), EGKSSGSGSESKST (SEQ ID NO: 47), GSAGSAAGSGEF (SEQ ID NO: 48), and (XP)n (n=4- 10), with X designating any amino acid, e.g., Ala, Lys, or Glu (SEQ ID NO: 49). In embodiments, the linker is GGS or (GGS)n (n=2-20) (SEQ ID NO: 50-68). In embodiments, the linker is one or more of GGGSE (SEQ ID NO: 69), GSESG (SEQ ID NO: 70), GSEGS (SEQ ID NO: 71), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 72), and a linker of randomly placed G, S, and E every 4 amino acid intervals. These sequences have been used for binding designed coiled coils to other protein domains (Muller, K.M. et al. Meth. Enzymology, 2000, 328:261-281). In embodiments, the linker sequence is or comprises the sequence GSGS (SEQ ID NO: 73). Other linkers known in the art can alternatively be used. See, e.g., Reddy Chichili, VP., Kumar, V., and Sivaraman, J. (2013). Linkers in the structural biology of protein-protein interactions. Protein Science 22(2): 153-67).

[0168] In embodiments, recombinant adenoviruses of the present disclosure have an albumin-binding moiety on the outer surface of the capsid protein, such that the capsid of the adenovirus can be coated with albumin.

[0169] DBl / 161420432.1 29 VCN-008PC / 112492-5208

[0170] In embodiments, the term “albumin” refers to a member of the albumin family proteins that are water-soluble globular proteins, moderately soluble in concentrated salt solutions and experiencing heat denaturation. Albumins are commonly found in blood plasma. Serum albumin is produced by the liver, is dissolved in blood plasma and is the most abundant blood protein in mammals. Particularly, the term “serum albumin” refers to a globular protein that in humans is encoded by the ALB gene (UniGene Hs. 418167). Human serum albumin protein is the protein defined by the sequence of the Uniprot database with accession number P02768 dated 19 March 2014 (SEQ ID NO: 74).

[0171] SEQ ID NO: 74 (human serum albumin)

[0172] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP EVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGK ASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADL AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLY EYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVSDRV TKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLK AVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0173] In embodiments, the term “albumin-binding moiety” refers to any amino acid sequence capable of binding to albumin, i.e. having albumin-binding affinity. In embodiments, the albumin-binding moiety is capable of binding serum albumin. In embodiments, the albumin-binding moiety is capable of binding human serum albumin. The term “albumin-binding moiety” includes, but is not limited to, naturally-occurring albumin-binding domains (ABDs), such as ABDs present in bacterial proteins, and albumin-binding sequences from synthetic peptides. In embodiments, the albumin-binding moiety is selected from an albumin-binding domain from streptococcal protein G, an albumin-binding domain from Peptostreptococcus magnus protein PAB, an albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 21) and functionally equivalent variants thereof. In embodiments, the albumin-binding domain is from streptococcal protein G.

[0174] In embodiments, the term “albumin-binding domain” refers to any region from a naturally occurring protein which is capable of binding albumin with sufficient specificity so as to ensure protection of a recombinant adenovirus comprising the modified capsid protein comprising the albumin-binding domain insertion from neutralizing antibodies. In embodiments, the albumin-binding domain is any albumin-binding domain known

[0175] DBl / 161420432.1 30 VCN-008PC / 112492-5208 in the art, including, e.g., those described in Johansson et al., “Structure, specificity, and mode of interaction for bacterial albumin-binding modules,” J Biol Chem. 2002 Mar 8;277(10):8114-20, incorporated herein by reference in its entirety.

[0176] In embodiments, the recombinant adenovirus vector or particle described herein is substantially covered or coated with host serum albumin, e.g., when administered to a subject. In embodiments, a recombinant adenovirus particle or vector described herein is shielded from NAbs. In embodiments, a recombinant adenovirus vector or particle described herein is self-coated with albumin. In embodiments, a recombinant adenovirus vector or particle described herein has an increased ability to evade the immune system with administered to a subject, as compared to adenovirus vectors that do not comprise an albumin-binding moiety. In embodiments, a recombinant adenovirus vector or particle described herein has reduced recognition by anti-adenovirus neutralizing antibodies, as compared to adenovirus vectors that do not comprise an albumin-binding moiety.

[0177] In embodiments, the term “albumin-binding domain from streptococcal protein G,” or “ABD from streptococcal protein G” refers to a domain that consists of 46 amino acid residues forming a three-helix bundle (Kraulis P.J. et al. FEBS Lett, 1996; 378: 190-4), and binds with high affinity to both human and mouse albumin, but not to bovine albumin (Konig T. and Skerra A. J Immunol Methods, 1998; 218:73-83). There are multiple albumin-binding domains in streptococcal protein G. In embodiments, the albumin-binding moiety is the albumin-binding domain 3 from streptococcal protein G. In embodiments, the sequence of the albuminbinding domain 3 from streptococcal protein G is SEQ ID NO: 22.

[0178] In embodiments, the term “albumin-binding domain from Peptostreptococcus magnus protein PAB” refers to the albumin-binding domain from protein PAB of Finegoldia magna (formerly known as Peptostreptococcus magnus) known as the “GA module” that is capable of binding albumin (Lejon S et al. 2004. J Biol Chem 279:42924-42928). Protein PAB of Finegoldia magna is the protein defined by the sequence of the GenBank database with accession number CAA54857.1 dated 9 September 2004 (SEQ ID NO: 23).

[0179] SEQ ID NO: 23 (albumin-binding domain from Peptostreptococcus magnus protein PAB)

[0180] LKNAKEDAIAELKKAGITSDFYFNAINKAKTVEEVNALKNEILK

[0181] In embodiments, the term “albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 21)” refers to peptides that bind albumin derived from phage clones RA and SA as disclosed in Dennis MS et al. (J Biol Chem. 2002. 277:35035-35043).

[0182] DBl / 161420432.1 31 VCN-008PC / 112492-5208

[0183] In embodiments, the albumin-binding moiety insertion is sequence of contiguous amino acids from an albumin-binding moiety or a fragment or variant thereof. In embodiments, the albumin-binding moiety to be inserted is long enough to retain a particular biological function, characteristic, or feature of the albuminbinding moiety from which it is derived. In embodiments, the albumin-binding moiety to be inserted is short enough to allow the capsid protein to form a coat, similarly or substantially similarly to the reference or parental capsid protein without the insertion.

[0184] In embodiments, the albumin-binding moiety is a functionally equivalent variant of an albumin-binding moiety. The term “functionally equivalent variant, refers to any polypeptide derived from an albumin-binding moiety by insertion, deletion or substitution of one or more residues, and which substantially maintains the ability to interact with albumin. In embodiments, a polypeptide is considered a functionally equivalent variant of an albumin-binding moiety if binds to albumin with an affinity of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the albumin-binding affinity of the albumin-binding domain of SEQ ID NO: 22. In embodiments, a polypeptide is considered a functionally equivalent variant of an albumin-binding moiety if it is capable of neutralizing antibodies at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% as efficiently as the albumin-binding domain of SEQ ID NO: 22. Suitable functional variants are those showing a degree of identity with respect to the reference or parental albumin-binding moiety of at least about 25% amino acid sequence identity, such as at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity.

[0185] In embodiments, functionally equivalent variants of albumin-binding moieties can be derivatives of the albumin-binding moieties. The term “derivatives” includes, but is not limited to, albumin-binding domains from bacteria modified to increase their affinity to albumin, as those disclosed in Johansson MU. et al. (J Biol Chem. 2002. 277:8114-8120), Jonsson A. et al. (Protein Eng Des Sei. 2008. 21 : 515-527) and Linhult M. et al. (Protein Sci. 2002. 11 :206-213). For example, a derivative may be the modified streptococcal G ABD ABD035 disclosed in Jonsson A. et al. (Protein Eng Des Sei. 2008. 21 : 515-527).

[0186] In embodiments, the albumin-binding moiety has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at

[0187] DBl / 161420432.1 32 VCN-008PC / 112492-5208 least about 98%, or at least about 99%) sequence identity to one or more of SEQ ID NOs: 21, 22, or 23, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11 , or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications) relative to SEQ ID NOs: 21, 22, or 23.

[0188] In embodiments, the amino acid modifications are amino acid mutations or amino acid substitutions. In embodiments, the amino acid substitutions are conservative and / or non-conservative substitutions. In embodiments, the amino acid modifications are amino acid truncations of two or more amino acids (e.g., about 2 to about 30, or about 2 to about 25, or about 2 to about 20, or about 2 to about 15, or about 2 to about 10, or about 2 to about 5 amino acids).

[0189] “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0190] In embodiments, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices.

[0191] In embodiments, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0192] In embodiments, the albumin-binding moiety has at least about 75% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albumin-binding moiety has at least about 80% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albumin-binding moiety has at least about 85% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albumin-binding moiety has at least about 90% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albumin-binding moiety has at least about 95% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albuminbinding moiety has at least about 97% identity to one or more of SEQ ID NOs: 21, 22, or 23. In embodiments, the albumin-binding moiety has at least about 99% identity to one or more of SEQ ID NOs: 21, 22, or 23.

[0193] DBl / 161420432.1 33 VCN-008PC / 112492-5208

[0194] In embodiments, the albumin-binding moiety has about 1 to about 15 amino acid modifications. In embodiments, the albumin-binding moiety has about 1 to about 10 amino acid modifications. In embodiments, the albumin-binding moiety has about 1 to about 5 amino acid modifications. In embodiments, the albuminbinding moiety has about 1 , or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications. In embodiments, the amino acid modifications are selected from substitutions and deletions.

[0195] In embodiments, the albumin-binding moiety is selected from Table 4 below, or a variant thereof.

[0196] Table 4: Albumin-binding moieties

[0197] In embodiments, the albumin-binding moiety is fused to another heterologous polypeptide. In embodiments, the albumin-binding moiety is fused to a homing or targeting polypeptide that directs the recombinant adenoviruses to certain cells and / or tissues of interest. In embodiments, albumin-binding moiety is fused to a homing protein or a homing domain thereof, or a targeting protein or targeting domain thereof. In embodiments, a “homing domain” or “targeting domain” refers to a domain or protein that preferentially or selectively targets a particular cell type, including cell matrix of a particular cell type, tissue type, organ, tumor type, etc., over other cells, tissues, organs, or tumors. In embodiments, a peptide from a homing protein or domain that is fused to the albumin-binding moiety inserted into the modified capsid protein herein directs the recombinant adenovirus capsid, particle, or vector to target the particular cell type, tissue type, organ, tumor type, etc., or to promote uptake and / or integration of the recombinant adenovirus genome. Examples of homing proteins or domains include, but are not limited to, neural tissue-homing domains, axonemal or cytoplasmic dynein-homing domains, bone-homing domains, kidney-homing domains, muscle-homing domains, endothelial cell-homing domains, retinal cell-homing domains, domains that target particular cellular receptors, such as integrin receptor-binding domains and transferrin receptor-binding domains, tumor celltargeting domains, targeting peptides from other viruses, etc. In embodiments, the terms “homing” and

[0198] DBl / 161420432.1 34 VCN-008PC / 112492-5208

[0199] “targeting” are used interchangeably. These peptides may also or alternatively promote recombinant adenovirus cell uptake, transduction and / or genome integration in cells of the target tissue.

[0200] Examples of homing peptides or homing domains used in embodiments herein include, but are not limited to, those Tables 1A-1 B of W02020206189, incorporated by reference herein in its entirety, and include at least 4, 5, 6, 7, 8, 9, 10, 11, 12, or more contiguous amino acid portions thereof that have the functional attribute of the peptide. See also, e.g., Laakkonen and Vuorinen, 2010, “Homing peptides as targeted delivery vehicles,” Integrative Biology, 2:326-337.

[0201] Therapeutic Payload

[0202] In embodiments, the recombinant adenovirus vector or particle further comprises a therapeutic payload. In embodiments, the therapeutic payload is a small molecule, a heterologous protein, or a heterologous gene or transgene.

[0203] In embodiments, the genome of the recombinant adenovirus further contains a heterologous gene or transgene that encodes a therapeutic protein, such that the heterologous gene is expressed within a cell that has been infected or transduced by the recombinant adenovirus .

[0204] In embodiments, a “therapeutic protein” refers to a protein that would be expected to provide some therapeutic benefit when expressed in a given cell. The therapeutic gene inserted can be any gene used in gene therapy or in vaccination. In embodiments, the heterologous gene is used in cancer gene therapy.

[0205] In embodiments, the recombinant adenovirus comprises one or more genes encoding therapeutic proteins inserted in the genome of the recombinant adenovirus . In embodiments, the genes are used in gene therapy or in vaccination.

[0206] In embodiments, a gene encoding a therapeutic protein is a gene used in cancer gene therapy. Examples of genes used in cancer gene therapy include, but are not limited to, prodrug-activating genes, tumorsuppressor genes, genes encoding anti-tumor interfering RNAs, and immunostimulatory genes.

[0207] In embodiments, a gene encoding a therapeutic protein is a gene used in gene therapy. In embodiments, the term “gene used in gene therapy” refers to a gene that can be used as a drug to prevent or treat a genetic or acquired disease or condition by delivering the gene encoding the therapeutic protein into a subject’s cells. As the person skilled in the art understands, the term gene therapy involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene, or using DNA that encodes a therapeutic protein. For

[0208] DBl / 161420432.1 35 VCN-008PC / 112492-5208 example, the DNA can encode any enzyme, antibody, hormone, cytokine, receptor, or polypeptide of therapeutic value. Any gene that can be used to treat a disease that is suitably treated by gene therapy can be inserted in the recombinant adenovirus genome of the recombinant adenovirus of the present disclosure. Examples of genes used in gene therapy include, but are not limited to, genes coding for enzymes, antibodies, cytokines, blood derivatives, hormones, interleukins, interferons, TNF, growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors, including BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc.; apolipoproteins, including ApoAl, ApoAIV, ApoE, etc.; dystrophin or a minidystrophin; tumor-suppressor genes, including p53, Rb, RaplA, DCC, k-rev, etc.; genes coding for factors involved in coagulation, including factors VII, VIII, IX, etc.; prodrug-activating genes, including thymidine kinase, cytosine deaminase, etc.; genes encoding anti-tumor interfering RNAs, e.g., genes encoding siRNAs capable of inhibiting the activity of factors that compromise MHC class I presentation, block complement, inhibit IFNs and IFN-induced mechanisms, inhibit NK cell based killing, or down regulate the immune response (e.g., IL-10, TGF-Beta), or RNAs expressing chemokines, cytokines, or metalloproteases; and all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.). In embodiments, the therapeutic gene is an antisense gene or sequence whose expression in the target cell enables gene expression or transcription of cellular mRNAs to be controlled.

[0209] In embodiments, a gene encoding a therapeutic protein is a gene used in vaccination. In embodiments, the term “gene used in vaccination” refers to a gene coding for an antigenic peptide that is capable of generating an immune response in an animal or human for the purpose of preventive or therapeutic vaccine production. Examples of genes used in vaccination include, but are not limited to, those encoding peptides specific to the Epstein-Barr virus, the HIV virus, the hepatitis B virus, the pseudorabies virus, and tumor-specific peptides.

[0210] In embodiments, additional genes encoding therapeutic proteins useful in accordance with the present disclosure include, but are not limited to, GAA (acid alpha-glucosidase) for treatment of Pompe disease; TPP1 (tripeptidyl peptidase-1) for treatment of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), ATP7B (copper transporting ATPase2) for treatment of Wilson’s disease; alpha galactosidase for treatment of Fabry disease; ASS1 (arginosuccinate synthase) for treatment of citrullinemia type 1 ; beta- glucocerebrosidase for treatment of Gaucher disease type 1; beta-hexosaminidase A for treatment of Tay Sachs disease; SERPING1 (Cl protease inhibitor; Cl esterase inhibitor) for treatment of hereditary angioedema (HAE); glucose-6-phosphatase for treatment of glycogen storage disease type I (GSDI); erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for

[0211] DBl / 161420432.1 36 VCN-008PC / 112492-5208 treatment of various immune disorders, viral infections and cancer; an interleukin (IL), including any one of IL-I through IL-36, and corresponding receptors, for treatment of various inflammatory diseases or immunodeficiencies; a chemokine, including chemokine (C-X-C motif) ligand 5 (CXCL5) for treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for treatment of immune disorders such as Crohn’s disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for treatment of various human inflammatory diseases; macrophage colony stimulating factor (M-CSF) for treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for treatment of epithelial tissue damage; chemokines such as monocyte chemoattractant protein-1 (MCP-I) for treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for treatment of various immune disorders; alpha 1 -antitrypsin for treatment of emphysema or chronic obstructive pulmonary disease (COPD); alpha-L-iduronidase for treatment of mucopolysaccharidosis I (MPS I); ornithine transcarbamoylase (OTC) for treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for treatment of phenylketonuria (PKU); lipoprotein lipase for treatment of lipoprotein lipase deficiency; apolipoproteins for treatment of apolipoprotein (Apo) A-l deficiency; low- density lipoprotein receptor (LDL-R) fortreatment of familial hypercholesterolemia (FH); albumin for treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for treatment of homocystinuria; branched chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl CoA carboxylase; methylmalonyl-CoA mutase; glutaryl CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); and dystrophin.

[0212] In embodiments, therapeutic proteins encoded by the additional genes that are useful in accordance with the present disclosure include, but are not limited to, antibodies, b-globin, a-globin, spectrin, a metal transporter (ATP7A or ATP7), sulfamidase, arylsulfatase A (cerebroside-sulfatase; ARSA), hypoxanthine guanine phosphoribosyl transferase, b-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a, transforming growth factor b, a cytokine, a-interferon, b-interferon, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor

[0213] DBl / 161420432.1 37 VCN-008PC / 112492-5208 necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-I, NF1, Von Hippel- Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRI, glucokinase, guanylate cyclase 2D (LCA- GUCY2D), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (choroideremia), LCA 5 (LCA-lebercilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1 C (Usher’s syndrome 1 C), X-linked retinitis pigmentosa GTPase, MER proto-oncogene tyrosine kinase (MERTK), ABCA4, DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (achromatopsia), PKD- I or PKD-2 (polycystic kidney disease), a sulfatase, N-acetylglucosamine-1 -phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, and a sphingolipid activator protein.

[0214] In embodiments, the heterologous gene is used in cancer gene therapy. The insertion of a therapeutic gene in the genome of the oncolytic adenovirus generates an “armed oncolytic adenovirus” that increase the cytotoxicity of oncolytic adenovirus towards tumor cells. For example, said heterologous gene can produce the death of the tumor cell, activate the immune system against the tumor, inhibit the angiogenesis, eliminate the extracellular matrix, induction of the apoptosis, among others.

[0215] Methods of Making Recombinant Adenoviruses

[0216] In embodiments, to construct a recombinant adenovirus to be used in the present disclosure, any of the methods of construction of genetically modified recombinant adenoviruses known in the field of gene therapy and virotherapy using recombinant adenoviruses are used. The most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the recombinant adenoviruses region to be modified, and then carrying out homologous recombination in bacteria with a plasmid containing the rest of the viral genome. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.

[0217] In embodiments, nucleic acids encoding the components of the present disclosure can be incorporated into adenoviruses as is known in the art, and depending on the host cells, used to produce recombinant adenovirus vectors or recombinant adenovirus particles comprising the modified capsid proteins of the present disclosure. Generally, the nucleic acids are operably linked to any number of regulatory elements (e.g., promoters, enhancers, origins of replication, selectable markers, ribosomal binding sites, inducers, etc.).

[0218] DBl / 161420432.1 38 VCN-008PC / 112492-5208

[0219] The person skilled in the art knows how to modify and propagate adenoviruses. For example, in embodiments, recombinant adenoviruses of the composure are propagated and amplified following standard methods in the field of adenoviral vectors, e.g., as disclosed in Chilion and Bosch (2014), Adenovirus. Methods and Protocols, 3rdedition, Methods in Molecular Biology, vol. 1089, Springer Protocols, Humana Press; and Alemany and Zhang (1999) Oncolytic adenoviral vectors, Totowa, N.J.: Humana Press.

[0220] In embodiments, the method used for propagation is infection of a cell line that allows for the replication of adenovirus. In embodiments, the cell lines used are HEK-293 and A549 cell lines.

[0221] In embodiments, the propagation is carried out as follows: A549 cells are seeded on cell culture plates and infected with, e.g., 100 viral particles per cell; two days later the cytopathic effect evidences the viral production when cells detach forming “grape-like” clusters; the cells are harvested in tubes; after centrifugation at, e.g., 1000 g for 5 minutes, the cell pellet is frozen and thawed three times to break the cells; the resulting cell extract is centrifuged at, e.g., 1000 g for 5 minutes and the supernatant containing the virus is layered onto a cesium chloride gradient and centrifuged at, e.g., 35000 g for 1 hour; the band of virus obtained from the gradient is collected and layered again onto another gradient of cesium chloride and centrifuged at, e.g., 35000 g for 16 hours; the virus band is collected and dialyzed against, e.g., PBS-10% glycerol; the dialyzed virus is aliquoted and kept at -80° C; the quantification of the number of viral particles and plaque-forming units is carried out following standard protocols. In embodiments, phosphate buffered saline (PBS) with 5% glycerol is used for the storage of the adenovirus. In embodiments, other formulations that improve the stability of the virus and have been described are used for the storage of the adenovirus.

[0222] In embodiments, a recombinant adenovirus used in the present disclosure is propagated and amplified in cell lines normally used in the field of gene therapy and virotherapy such as the lines HEK-293 (Reference number: ATCC CRL-1573) and A549 (Reference number: ATCC CCL185).ln embodiments, the cell is a mammalian cell. In embodiments, the mammalian cell is a human cell. In embodiments, the cell is an insect cell. In embodiments, the cell is immortalized. In embodiments, the cell is a Chinese hamster ovary (CHO), baby hamster kidney (BHK), human embryonic kidney (HEK293T) cells, Vero cell, or Spodoptera frugiperda 9 (Sf9) cell. In embodiments, the CHO cell is a CHO-K1, CHO-DHB11, CHO-DXB1, CHO-S, or CHO-DG44 cell. In embodiments, a CHO cell comprises or is selected from CHO-K1 (ATCC CCL-61) cells, SURE CHO- M cells (derivative of CHO-K1), or baby hamster kidney cells (BHK, ATCC CCL-10). In embodiments, the Vero cell comprises or is selected from Vero, Vero 76 and Vero E6. In embodiments, the cell is, without

[0223] DBl / 161420432.1 39 VCN-008PC / 112492-5208 limitations, human cervical carcinoma cells (HELA, ATCC CCL-2), 293 (ATCC CRL-1573), 3T3 (ATCC CCL- 163), or monkey kidney CV1 line (ATCC CCL-70).

[0224] Compositions and Administration

[0225] The present disclosure also relates to a pharmaceutical composition containing a recombinant adenovirus vector or recombinant adenovirus particle of the present disclosure.

[0226] It will be understood that the compositions used in the present disclosure are used in a pharmaceutically acceptable presentation. This means that any recombinant adenovirus vector or recombinant adenovirus particle described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

[0227] In embodiments, the pharmaceutical composition containing an recombinant adenovirus vector or recombinant adenovirus particle of the present disclosure contains a pharmaceutically acceptable excipient, diluent or carrier. Examples of suitable pharmaceutical carriers etc. are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose.

[0228] Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, intrathecal, intratumoral, intraocular, intravitreal, intraarterial, topical, intradermal, etc., administration. The route of administration, of course, depends, inter alia, on the kind of vector contained in the pharmaceutical composition.

[0229] The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of infection or disease, general health and other drugs being administered concurrently.

[0230] In embodiments, doses range from at least about 1x108vector genomes per kilogram (vg / kg) of the weight of the subject, or more, for example, about 1x109, about 1x1010, about 1x1011, about 1x1012, about 1x1013, about 1x1014, or more, vg / kg of the weight of the subject, to achieve a therapeutic effect.

[0231] DBl / 161420432.1 40 VCN-008PC / 112492-5208

[0232] In embodiments, the recombinant adenovirus vectors or recombinant adenovirus particles may be administered, for example, more than once daily (e.g., about two, about three, about four, about five, about six, about seven, about eight, about nine, or about ten times per day), about once per day, about every other day, about every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year.

[0233] In embodiments, the recombinant adenovirus vectors or recombinant adenovirus particles may be administered alone, or in combination with one or more compound, agent, drug, treatment, or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. In embodiments, combination compositions and treatments include second actives, such as, biologies and immunosuppressive agents. In embodiments, such compounds, agents, drugs, treatments, or other therapeutic regimens or protocols can be administered or performed prior to, substantially contemporaneously with, or following any other method or use of the present disclosure.

[0234] In embodiments, the recombinant adenovirus vectors or recombinant adenovirus particles are administered in conjunction with one or more immunosuppressive agents prior to, substantially at the same time as, or after administering the recombinant adenovirus vectors or recombinant adenovirus particles. In embodiments, one or more immunosuppressive agents are administered about 1-12, about 12-24 or about

[0235] 24-48 hours, or about 2-4, about 4-6, about 6-8, about 8-10, about 10-14, about 14-20, about 20-25, about

[0236] 25-30, about 30-50, or more than about 50 days following administration of the recombinant adenovirus vectors or recombinant adenovirus particles. In embodiments, an immunosuppressive agent is an antiinflammatory agent. In embodiments, an immunosuppressive agent is a steroid. In embodiments, an immunosuppressive agent is cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab, or a derivative thereof.

[0237] In embodiments, adenovirus empty capsid (i.e., adenovirus lacking a heterologous nucleic acid) can be delivered to the subject prior to administration of recombinant adenovirus vectors or recombinant adenovirus particles described herein.

[0238] In embodiments, subjects are tested for an immune response, e.g., anti-adenovirus NAbs after treatment, and optionally monitored for a period of time after treatment for anti-adenovirus NAbs. Subjects developing anti-adenovirus NAbs can be treated with an immunosuppressive agent, or can be administered one or more additional amounts of recombinant adenovirus vector.

[0239] DBl / 161420432.1 41 VCN-008PC / 112492-5208

[0240] In embodiments, the recombinant adenovirus vectors or recombinant adenovirus particles described herein provide a therapeutic effect without an immunosuppressive agent. In embodiments, the therapeutic effect is sustained for a period of time, e.g., about 2-4, about 4-6, about 6-8, about 8-10, about 10-14, about 14-20, about 20-25, about 25-30, or about 30-50 days or more, for example, about 50-75, about 75-100, about 100- 150, about 150-200 days, or more, without administering an immunosuppressive agent.

[0241] In embodiments, administration of the recombinant adenovirus vectors or recombinant adenovirus particles described herein does not result in an immune response, e.g., production of anti-adenovirus NAbs. In embodiments, in the presence of human serum albumin, the recombinant adenovirus vectors or recombinant adenovirus particles described herein have reduced recognition by anti-adenovirus neutralizing antibodies, as compared to an adenovirus vector that does not comprise an albumin-binding moiety.

[0242] In embodiments, recombinant adenoviruses described herein are administered before having been bound to serum albumin, and they bind to albumin in the blood of the subject to which they are administered. In embodiments, recombinant adenoviruses described herein are coated with albumin before being administered.

[0243] Methods of Treatment or Prevention / Payload Delivery / Gene Transfer

[0244] The present disclosure also relates to methods of using the pharmaceutical compositions containing a recombinant adenovirus vector or recombinant adenovirus particle of the present disclosure.

[0245] In aspects, the present disclosure provides a method of treating, ameliorating or preventing a disease or disorder in a subject, comprising administering an effective amount the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the recombinant adenovirus vector or recombinant adenovirus particle of any one of the embodiments and / or aspects disclosed herein, cells contacted with the recombinant adenovirus vector or recombinant adenovirus particle of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein to the subject.

[0246] In embodiments, the present compositions are used in gene transfer methods to provide therapy for inherited diseases. In embodiments, the present compositions are used in gene transfer methods to provide therapy for inherited diseases involving deficiency states. In embodiments, the gene transfer methods are used to bring a normal gene into affected tissues for replacement therapy. In embodiments, the gene transfer

[0247] DBl / 161420432.1 42 VCN-008PC / 112492-5208 methods are used to create animal models for the disease using antisense mutations. In embodiments, the present compositions are used in gene transfer methods to provide therapy for inherited diseases involving unbalanced states. In embodiments, the gene transfer methods are used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. In embodiments, the present compositions and methods permit the treatment of genetic diseases. In embodiments a genetic disease is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe. In embodiments, the present compositions and methods use site-specific integration of nucleic sequences to cause mutations or to correct defects.

[0248] In aspects, the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the recombinant adenovirus vector or recombinant adenovirus particle of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein for use in treating, ameliorating or preventing a patient with a disease or disorder.

[0249] In aspects, the present disclosure provides use of the nucleic acid of any one of the embodiments and / or aspects disclosed herein, the recombinant adenovirus vector or recombinant adenovirus particle of any one of the embodiments and / or aspects disclosed herein, or the pharmaceutical composition of any one of the embodiments and / or aspects disclosed herein in the manufacture of a medicament for the treating, ameliorating or preventing of a disease or disorder.

[0250] In embodiments, the methods provide a means for delivering heterologous nucleic acid sequences into a broad range of host cells, including both dividing and non-dividing cells. In embodiments, the recombinant adenovirus vectors, recombinant adenovirus particles, other reagents, methods, and pharmaceutical compositions of the present disclosure are useful in a method of administering a protein, peptide or therapeutic nucleic acid to a subject in need thereof, as a method of treatment. In embodiments, the subject may be in need of the protein or peptide because the subject has a deficiency of the protein or peptide, or because the production of the protein or peptide in the subject may impart some therapeutic effect, as a method of treatment or otherwise.

[0251] In embodiments, the methods may be employed to deliver any foreign nucleic acid with a biological effect to treat or ameliorate the symptoms associated with any disorder related to gene expression. In embodiments, the disease or disorder is a genetic disease or disorder. In embodiments, the disease or disorder is an inherited disease or disorder. Illustrative disease states include, but are not limited to: cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood coagulation

[0252] DBl / 161420432.1 43 VCN-008PC / 112492-5208 disorders, AIDs, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher’s disease, Hurler’s disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, retinal degenerative diseases (and other diseases of the eye), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like.

[0253] In embodiments, the methods may be employed to deliver nucleic acids encoding monoclonal antibodies or fragments thereof that are known to provide beneficial biological effects to treat or ameliorate the symptoms associated with cancers, infectious diseases, and autoimmune diseases such as rheumatoid arthritis.

[0254] In embodiments, the present compositions or methods reduce or prevent neutralization of the recombinant adenovirus vectors or recombinant adenovirus particles by NAbs, e.g., as compared to effects observed with recombinant adenoviruses lacking an albumin-binding moiety. In embodiments, the present compositions or methods reduce or prevent the triggering of an innate immune response by the recombinant adenovirus vectors or particles, e.g., as compared to effects observed with recombinant adenoviruses lacking an albumin-binding moiety. In embodiments, the present compositions or methods reduce or prevent the triggering of an innate immune response by the recombinant adenovirus vectors or particles, e.g., as compared to effects observed with recombinant adenoviruses lacking an albumin-binding moiety, by reducing or preventing complement activation and / or docking of the virus particles to the Fc receptors of monocytes and neutrophils.

[0255] In embodiments, the present compositions or methods allow re-administration of the recombinant adenovirus vectors or particles, e.g., that is not tolerated when using an recombinant adenovirus lacking an albuminbinding moiety. In embodiments, the present compositions or methods reduce or prevent adverse events of re-administration of the recombinant adenovirus vectors or particles, e.g., as observed with an recombinant adenovirus lacking an albumin-binding moiety. In embodiments, the present compositions or methods allow dose-fractionation, dose escalation, and the like, of the recombinant adenovirus vectors or particles, e.g., as would not be suitable with an recombinant adenovirus lacking an albumin-binding moiety.

[0256] Kits

[0257] The present disclosure provides kits that can simplify the administration of any agent described herein. An illustrative kit of the present disclosure comprises any composition described herein in unit dosage form. In

[0258] DBl / 161420432.1 44 VCN-008PC / 112492-5208 one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agentforthe administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the present disclosure and an effective amount of another composition, such those described herein.

[0259] Definitions

[0260] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0261] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50%” covers the range of 45% to 55%.

[0262] An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disorder of interest.

[0263] As used herein, something is “decreased” if a read-out of activity and / or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in embodiments, activity is decreased and some downstream read-outs will decrease but others can increase.

[0264] Conversely, activity is “increased” if a read-out of activity and / or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7- fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

[0265] DBl / 161420432.1 45 VCN-008PC / 112492-5208

[0266] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0267] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

[0268] As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0269] In various embodiments, the “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested)). In embodiments, the subject is a human.

[0270] In embodiments, the subject has pre-existing immunity to adenovirus. In embodiments, the subject has antiadenovirus NAbs. In embodiments, the subject is likely to develop NAbs to non-engineered recombinant adenovirus, e.g., a recombinant adenovirus lacking an albumin-binding moiety.

[0271] It will also be understood that, although the terms first, second, etc., may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject. Furthermore, the terms “subject,” “user,” and “patient” are used interchangeably herein.

[0272] DBl / 161420432.1 46 VCN-008PC / 112492-5208

[0273] In embodiments, methods of the disclosure are useful in treatment a human subject. In embodiments, the human may be referred to as a patient. In embodiments, the human is a female. In embodiments, the human is a male.

[0274] In embodiments, the human has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

[0275] The amount of each component in the compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g., recombinant adenoviruses described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating or preventing the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0276] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population) or the maximum tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and

[0277] DBl / 161420432.1 47 VCN-008PC / 112492-5208 therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays or measurements or methane production in stool samples. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0278] In embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0279] As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and / or compositions for use and / or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

[0280] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials, similar or equivalentto those described herein, can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

[0281] This disclosure is further illustrated by the following non-limiting examples.

[0282] EXAMPLES

[0283] Example 1 : Evaluation of ABD insertion into Hexon HVR7 of Adenovirus

[0284] The viral capsid of hAd5 was modified by inclusion of an albumin-binding domain (ABD) within a hypervariable region (HVR) of the capsid hexon. Without wishing to be bound by theory, it is thought that the ABD binds to albumin present in the blood allowing the virus to self-coat with albumin and effectively shield

[0285] DBl / 161420432.1 48 VCN-008PC / 112492-5208 itself from circulating NAbs. The ABD was derived from ABD3 of streptococcal protein G bacterial cell surface receptor and was inserted into hexon HVR7. The ABD binds to human and murine albumin. The ABD-human albumin interaction was demonstrated to circumvent NAbs from different sources and tumor targeting was observed in the presence of high levels of NAbs in vivo (Mato-Berciano, A., et al., “Oncolytic adenovirus with hyaluronidase activity that evades neutralizing antibodies: VCN-11 ,” J Control Release, 2021. 332: p. 517- 528; Rojas, L.A., et al., “Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery,” Journal of Controlled Release, 2016. 237: p. 78-88).

[0286] Part A. Construction of oncolytic adenoviruses encoding the reporter gene, green fluorescent protein (GFP), containing ABD variants in hexon HVR7

[0287] Oncolytic adenoviruses were generated encoding the ABD inserted into hexon HVR7. The ABD, a 46 amino acid domain originating from the streptococcal protein G bacterial cell surface receptor, was inserted centrally into HVR7 flanked by glycine, serine, glycine, serine (GSGS) linkers (DNA sequence of 5’ linker: GGCAGCGGATCC (SEQ ID NO: 78) and 3’ linker: GGGTCTGGTAGC (SEQ ID NO: 79)).

[0288] The amino acid sequence of the ABD is:

[0289] NH3-LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP-COOH (SEQ ID NO: 23)

[0290] The DNA sequence of the ABD is:

[0291] 5’-

[0292] CTGGCCGAGGCTAAGGTGCTTGCGAACCGGGAACTAGACAAATACGGTGTTTCTGATTATTACAAGAA TTTGATTAACAATGCCAAAACCGTCGAGGGCGTAAAGGCTCTGATCGACGAAATACTTGCGGCCCTAC CC-3’ (SEQ ID NO: 80)

[0293] The pAdZ system (Stanton RJ, et al., “Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function,” Biotechniques, 2008. 45: p.659-662) was used to introduce the rpslneo cassette into HVR7 of the pAdZICOVIRI 5Fi-GFP backbone followed by replacement of the repslneo cassette with the ABD to generate the bacterial artificial chromosome (BAC):

[0294] • pAdZICOVIRI 5FIGFP-H7-ABD: This plasmid encodes a tumor-selective human adenovirus that expresses GFP after fiber and encodes the ABD in the HVR7 of the hexon.

[0295] The BAC was verified to be correct by restriction enzyme digestion and sequencing of the modified region. Plasmid DNA was scaled-up and used for calcium phosphate transfection of HEK-293 cells. Cells were monitored for the appearance of cytopathic effect (CPE) indicating the presence of virus and by fluorescence

[0296] DBl / 161420432.1 49 VCN-008PC / 112492-5208 microscopy for GFP-expressing cells as all viruses encode GFP. Once CPE was observed, within 4-9 days after transfection, cells were collected, freeze-thawed, centrifuged, and cell extract was used for virus amplification in A549 cells and subsequent cell extracts were used for plaque assay analysis.

[0297] The HVR7 DNA sequence within the plasmid pAdZICOVIR15FiGFP-H7-ABD is from bp 20264 to 20386 bp with the ABD plus linkers inserted centrally (Figure 1).

[0298] Part B. Plaque assay of ABD virus

[0299] A549 cells were plated in 6-well plates and 1 / 10 serial dilutions of the cell extracts, ranging from 101to 107, were used to infect each well of the plates. A total of 100 uL of cell extract dilution was used to infect each well for 4 hours at 37°C, after which media was aspirated, cells washed with PBS, and overlayed with a mixture of cell culture media and agarose. Cells were incubated until the appearance of plaques within 6-10 days (Figure 2). Plaque analysis demonstrates that all viruses are viable.

[0300] At least three plaques from each virus were collected in separate 1.5 mL microcentrifuge tubes containing 500 uL of cell culture media and stored at -80°C.

[0301] Part C. Amplification and GFP titering of ABD-encoding GFP viruses

[0302] Frozen plaques were subjected to three freeze / thaw cycles, centrifuged to remove cell debris, and clarified supernatants were used to infect A549 cells seeded in 6 well plates. Infected cells were incubated for 3-4 days until full CPE was observed, after which cells were collected, subjected to 3 freeze / thaw cycles, and centrifuged, and clarified supernatants were used to infect A549 cells plated on 10 cm plates. The cycle was repeated once again, and cell supernatants were used to infect a 15 cm plate. Clarified supernatants from the 15 cm plate were used for GFP titering.

[0303] For GFP-expressing viruses, 1 / 10 serial dilutions of the cell supernatants derived from the 15 cm plates were prepared and used to infect HEK-293 cells plated in 96-well dishes (Figure 3). Cells were infected with 100ul / well of the cell supernatant dilutions, incubated for 36 hrs at 37°C, and GFP-positive cells were quantified by fluorescence microscopy. GFP titers displayed as transducing units (TU) / mL are shown in Table 5. These data demonstrate that the ABD virus is viable and able to be propagated.

[0304] Table 5. Titers of ABD GFP-expressing adenoviruses. The control virus, ICOVIR15FiGFP, does not contain an ABD.

[0305] DBl / 161420432.1 50 VCN-008PC / 112492-5208

[0306] Part D. Viral production of ABD virus

[0307] A549 cells were seeded in triplicate in 24 well tissue culture plates (1e5 cells / well) in 500 uL of DMEM + 5% FBS and incubated for 16 hrs at 37°C in 5% CO2. The next day, media was removed and infected with titered cell extract at 20 TU / cell for an expected 100% cell infection efficiency. Plates were incubated for 4 hrs at 37°C in 5% CO2, media was removed, cells were washed 3 times with 500 uL of PBS, and fresh media was added. Plates were incubated for an additional 48 or 72 hrs, after which cells and media were harvested. Cells and media were subjected to three freeze / thaw cycles, and centrifuged, and clarified supernatants were collected.

[0308] Clarified supernatants were used to quantify GFP positive cells as described in Example 3 (Figure 3). ABD GFP-expressing virus showed an increase in viral titer (GFP-positive cells) between 48 hrs and 72 hrs indicating that the ABD virus propagates effectively (Figure 4). Compared to the control GFP-expressing virus without an ABD (ICOVIR15-GFP), ABD virus titers at 72 hrs were not significantly different (Figure 4, Table 6).

[0309] Table 6. Virus titers from virus production assay at 72 hrs.

[0310] Part E. Cytotoxicity of the ABD virus in the presence or absence of human serum albumin (HSA)

[0311] A549 cells (3e4 cells / well) were plated in 96 well plates and infected with serial dilutions of the GFP- expressing viruses with the highest viral concentration of 1000 TU / cell. Serial dilutions of the virus were prepared in tissue culture media (DMEM + 5% FBS). HSA was added to the media where indicated at 1 mg / mL. Cells were maintained at 37°C in 5% CO2. At day 5 postinfection, wells were washed once with PBS and stained for total protein content using a commercial bicinchoninic acid assay (Pierce Biotechnology), incubated for 30 minutes, and then absorbance was read at 540 nm. As the virus-mediated CPE causes cell

[0312] DBl / 161420432.1 51 VCN-008PC / 112492-5208 detachment, the amount of protein remaining in each well corresponds to the number of attached or living cells. The TU per cell required to reduce to half the amount of protein per well, compared to uninfected cells was estimated in dose response curves by standard nonlinear regression. These values correspond to the 50% inhibition or IC50 values.

[0313] As expected, the control virus, ICOVIR15GFP, displayed similar cytotoxicity in the absence or presence of HSA, as this virus does not have an ABD and does not bind HSA (Figures 5 and 7, Table 7). In contrast, ICOVIR15GFP-H7ABD (H7ABD) displayed lower cytotoxcity (higher IC50) in the presence of HSA (Figures 6 and 7, Table 7) related to the binding of albumin.

[0314] Table 7. Mean IC50 of the indicated viruses in the absence or presence of HSA. Fold change in IC50 in the presence of HSA compared to without HSA is displayed.

[0315] Part F. Evasion of adenovirus neutralizing antibodies (NAbs) in the presence or absence of HSA

[0316] To assess the ability of viruses containing the ABD to evade NAbs in the presence of HSA, a study was performed where A549 cells were seeded in 96-well white opaque plates (Perkin Elmer) at 3e4 cells / well in the presence or absence of 1 mg / mL HSA. The next day cells were infected with the indicated control or ABD GFP-expressing viruses at 1 TU / cell in the absence or presence of NAbs at dilutions ranging from 1 / 160 to 1 / 81920 for 1h at room temperature (Figure 8). The fluorescence signal from GFP-positive cells was quantified 48 hrs later via fluorimeter (Figure 9). This analysis was performed in triplicate wells of each plate and the entire study was repeated three times. In parallel, cells were plated on clear-bottom 96-well plates and treated identically to the conditions displayed in Figure 8, to allow the direct visualization of GFP-positive cells via microscopy (Figure 10). As expected, the % GFP-positive cells were no different with and without HSA for the control virus without an ABD. The virus was effectively neutralized with increasing NAb concentrations. . In contrast, the H7ABD virus rendered more GFP-positive cells (infected cells) when albumin was present compared to the amount of GFP-positive cells when albumin was absent (Figure 9). This was observed for NAb dilutions between 1280 to 40960. This indicated that H7ABD virus was able to evade neutralizing antibodies beginning ata NAbs dilution of 1 / 1280, the NAbs dilution where the two curves,

[0317] DBl / 161420432.1 52 VCN-008PC / 112492-5208 with and without HSA, diverge. Direct visualization of GFP-positive cells via microscopy displayed a similar pattern that was observed via fluorimeter analysis (Figure 10).

[0318] These data demonstrate that inclusion of the ABD in H7 of the adenovirus hexon allows the virus to evade NAbs in the presence of HSA. Notably, inclusion of the ABD in H7 minimally affects virus infection in the presence of HSA while mediating protection from NAbs.

[0319] Example 2: Testing the function of ABD in the modified adenovirus vectors

[0320] In vivo: The ABD-modified adenovirus will be tested in the passive immunization model. Seven -week-old C57BL6 female will be immunized with an intraperitoneal injection of wild-type adenovirus (Ad5) and one week later, boosted with an intravenous administration of Ad5. One week later, mice will be sacrificed and serum collected.

[0321] Subcutaneous tumors will be established in Athymic nu / nu mice by injection of 1 x 107SKmel28 cells into both flanks of each mouse. When tumors reach 150 mm3mice will be randomized and passively immunized with an intraperitoneal injection of naive or anti-Ad5 neutralizing mouse serum. The next day, mice will be treated intravenously with PBS, the parental virus AdZICOVIR15FiGFP (without the ABD) and AdZICOVIR15FiGFP-H7-ABD, the ABD-containing adenovirus. The next day, tumors will be collected and analyzed for GFP expression by immunohistochemistry.

[0322] Both the parental and ABD-containing adenovirus will infect tumor cells, as indicated by the presence of GFP- positive tumor cells in naive (non-passively immunized mice). In contrast, in passively immunized mice, only the ABD-containing adenovirus will infect tumor cells, resulting in GFP-positive tumors. The parental, non- ABD-containing adenovirus will be neutralized by the anti-Ad5 antibodies present in the blood of passively immunized mice, and no GFP-positive cells will be observed in tumors.

[0323] EQUIVALENTS

[0324] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention

[0325] DBl / 161420432.1 53 VCN-008PC / 112492-5208 pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0326] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0327] INCORPORATION BY REFERENCE

[0328] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0329] The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0330] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

[0331] EMBODIMENTS

[0332] Various additional embodiments of the disclosure are provided by the following enumerated embodiments, which can be combined in any number and in any combination.

[0333] Embodiment 1. A modified adenovirus capsid protein comprising an albumin-binding moiety insertion within the capsid’s hexon protein’s seventh hypervariable region (HVR7), wherein when the hexon protein is packaged as a component of an adenovirus particle, the albumin-binding moiety is exposed on the outer surface of the adenovirus particle.

[0334] Embodiment 2. The modified adenovirus capsid protein of embodiment 1 , wherein the albumin-binding moiety is a wild-type albumin-binding domain or a functional variant or fragment thereof.

[0335] Embodiment 3. The modified adenovirus capsid protein of embodiment 1 or 2, wherein the albumin-binding moiety is selected from an albumin-binding domain from streptococcal protein G, an albumin-binding domain from Peptostreptococcus magnus protein PAB, an albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 21), and functionally equivalent variants thereof.

[0336] DBl / 161420432.1 54 VCN-008PC / 112492-5208

[0337] Embodiment 4. The modified adenovirus capsid protein of embodiment 3, wherein the albumin-binding moiety comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 21, 22, or 23.

[0338] Embodiment 5. The modified adenovirus capsid protein of embodiment 4, wherein the albumin-binding moiety comprises a polypeptide having the amino acid sequence of SEQ ID NO: 21, 22, or 23.

[0339] Embodiment 6. The modified adenovirus capsid protein of any one of embodiments 1-5, wherein the adenovirus capsid protein is from at least one adenovirus type selected from a Group A adenovirus, a Group B adenovirus, a Group C adenovirus, a Group D adenovirus, a Group E adenovirus, a Group F adenovirus, and a Group G adenovirus.

[0340] Embodiment 7. The modified adenovirus capsid protein of embodiment 6, wherein the adenovirus capsid protein is from at least one adenovirus type selected from a serotype 1 adenovirus genome, a serotype 2 adenovirus genome, a serotype 3 adenovirus genome, a serotype 4 adenovirus genome, a serotype 5 adenovirus genome, a serotype 6 adenovirus genome, a serotype 7 adenovirus genome, a serotype 8 adenovirus genome, a serotype 9 adenovirus genome, a serotype 10, adenovirus genome, a serotype 11 adenovirus genome, a serotype 12 adenovirus genome, a serotype 13 adenovirus genome, a serotype 14 adenovirus genome, a serotype 15 adenovirus genome, a serotype 16 adenovirus genome, a serotype 17 adenovirus genome, a serotype 18 adenovirus genome, a serotype 19 adenovirus genome, a serotype 20, adenovirus genome, a serotype 21 adenovirus genome, a serotype 22 adenovirus genome, a serotype 23 adenovirus genome, a serotype 24 adenovirus genome, a serotype 25 adenovirus genome, a serotype 26 adenovirus genome, a serotype 27 adenovirus genome, a serotype 28 adenovirus genome, a serotype 29 adenovirus genome, a serotype 30, adenovirus genome, a serotype 31 adenovirus genome, a serotype 32 adenovirus genome, a serotype 33 adenovirus genome, a serotype 34 adenovirus genome, a serotype 35 adenovirus genome, a serotype 36 adenovirus genome, a serotype 37 adenovirus genome, a serotype 38 adenovirus genome, a serotype 39 adenovirus genome, a serotype 40, adenovirus genome, a serotype 41 adenovirus genome, a serotype 42 adenovirus genome, a serotype 43 adenovirus genome, a serotype 44 adenovirus genome, a serotype 45 adenovirus genome, a serotype 46 adenovirus genome, a serotype 47 adenovirus genome, a serotype 48 adenovirus genome, a serotype 49 adenovirus genome, a serotype 50, adenovirus genome, a serotype 51 adenovirus genome, a serotype 52 adenovirus genome, a serotype 53 adenovirus genome, a serotype 54 adenovirus genome, a serotype 55 adenovirus genome, a serotype 56

[0341] DBl / 161420432.1 55 VCN-008PC / 112492-5208 adenovirus genome, a serotype 57 adenovirus genome, a serotype 58 adenovirus genome, a serotype 59 adenovirus genome, a serotype 60, adenovirus genome, a serotype 61 adenovirus genome, a serotype 62 adenovirus genome, a serotype 63 adenovirus genome, a serotype 64 adenovirus genome, a serotype 65 adenovirus genome, a serotype 66 adenovirus genome, and a serotype 67 adenovirus genome.

[0342] Embodiment 8. The modified adenovirus capsid protein of any one of embodiments 1-7, wherein the albumin-binding moiety insertion is located in the range of positions 400-450, with reference to any one of SEQ ID NOs: 1-10, or positions corresponding thereto.

[0343] Embodiment 9. The modified adenovirus capsid protein of any one of embodiments 1-7, wherein the albumin-binding moiety insertion is located immediately after one or more of the following amino acid residues:

[0344] (a) hAd5: any one of residues 420-449, with reference to SEQ ID NO: 1;

[0345] (b) hAd3: any one of residues 413-441, with reference to SEQ ID NO: 2;

[0346] (c) hAd4: any one of residues 403-433, with reference to SEQ ID NO: 3;

[0347] (d) hAd7: any one of residues 409-434, with reference to SEQ ID NO: 4;

[0348] (e) hAd11 : any one of residues 417-445, with reference to SEQ ID NO: 5;

[0349] (f) hAd 16: any one of residues 403-437, with reference to SEQ ID NO: 6;

[0350] (g) hAd21 : any one of residues 419-446, with reference to SEQ ID NO: 7;

[0351] (h) hAd34: 421-448, with reference to SEQ ID NO: 8; and

[0352] (i) hAd35: any one of residues 421-449, with reference to SEQ ID NO: 9.

[0353] Embodiment 10. The modified adenovirus capsid protein of embodiment 9, wherein the albumin-binding moiety insertion occurs after an amino acid residue corresponding to one of amino acids V420, 1421, N422, T423, E424, T425, L426, T427, K428, V429, K430, P431 , K432, T433, G434, Q435, E436, N437, G438, W439, E440, K441, D442, A443, T444, E445, F446, S447, D448, or K449 of the hAd5 capsid, with reference to SEQ ID NO: 1 , or positions corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10.

[0354] Embodiment 11. The modified adenovirus capsid protein of embodiment 10, wherein the albumin-binding moiety insertion is located after amino acid residue 437 of hAd5, with reference to SEQ ID NO: 1 , or a position

[0355] DBl / 161420432.1 56 VCN-008PC / 112492-5208 corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10.

[0356] Embodiment 12. The modified adenovirus capsid protein of any one of embodiments 1-11, wherein the N- and / or C-terminus of the albumin-binding moiety is connected to the capsid protein by a linker sequence.

[0357] Embodiment 13. The modified adenovirus capsid protein of embodiment 12, wherein the linker sequence is substantially comprised of glycines and serines.

[0358] Embodiment 14. The modified adenovirus capsid protein of embodiment 13, wherein the linker sequence comprises GSGS (SEQ ID NO: 73), SGGTSGSTSGTGST (SEQ ID NO: 17), AGSSTGSSTGPGSTT (SEQ ID NO: 18), GGSGGAP (SEQ ID NO: 19), GGGVEGGG (SEQ ID NO: 20), GGSGGSGGGGSGGGGS (SEQ ID NO: 25), LE, GGGGS (SEQ ID NO: 26), (GGGGS)n (n=1-7) (SEQ ID NO: 27-33), (Gly)8 (SEQ ID NO: 34), (Gly)6 (SEQ ID NO: 35), (EAAAK)n (n=1-3) (SEQ ID NO: 36-38), A(EAAAK)nA (n=2-5) (SEQ ID NO: 39- 42), AEAAAKEAAAKA (SEQ ID NO: 43), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 44), PAPAP (SEQ ID NO: 45), KESGSVSSEQLAQFRSLD (SEQ ID NO: 46), EGKSSGSGSESKST (SEQ ID NO: 47), GSAGSAAGSGEF (SEQ ID NO: 48), (XP)n (SEQ ID NO: 49), GGS, (GGS)n (n=2-20) (SEQ ID NO: 50-68), GGGSE (SEQ ID NO: 69), GSESG (SEQ ID NO: 70), GSEGS (SEQ ID NO: 71), or GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 72).

[0359] Embodiment 15. The modified adenovirus capsid protein of embodiment 14, wherein the linker sequence comprises the sequence GSGS (SEQ ID NO: 73).

[0360] Embodiment 16. The modified adenovirus capsid protein of any one of embodiments 1-15, wherein the albumin-binding moiety is further fused to a homing polypeptide or a homing domain thereof.

[0361] Embodiment 17. The modified adenovirus capsid protein of any one of embodiments 1-16, wherein the modified capsid protein binds to albumin.

[0362] Embodiment 18. A nucleic acid encoding the modified adenovirus capsid protein of any one of embodiments 1-17.

[0363] Embodiment 19. A recombinant adenovirus genome comprising the nucleic acid of embodiment 18.

[0364] Embodiment 20. A recombinant adenovirus vector comprising the modified capsid protein of any one of embodiments 1-17, the nucleic acid of embodiment 18, or the recombinant adenovirus genome of embodiment 19, and further comprising a therapeutic payload.

[0365] DBl / 161420432.1 57 VCN-008PC / 112492-5208

[0366] Embodiment 21. The recombinant adenovirus vector of embodiment 20, wherein the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule.

[0367] Embodiment 22. The recombinant adenovirus vector of embodiment 21, wherein the transgene encodes a therapeutic protein.

[0368] Embodiment 23. The recombinant adenovirus vector of embodiment 22, wherein the transgene encoding the therapeutic protein comprises a gene used in gene therapy or in vaccination.

[0369] Embodiment 24. The recombinant adenovirus vector of any one of embodiments 19-23, wherein the recombinant adenovirus vector binds to albumin.

[0370] Embodiment 25. The recombinant adenovirus vector of any one of embodiments 19-24, wherein, in the presence of human serum albumin, the recombinant adenovirus vector has reduced recognition by antiadenovirus neutralizing antibodies, as compared to an adenovirus vector that does not comprise an albuminbinding moiety.

[0371] Embodiment 26. A cell comprising the recombinant adenovirus vector of any one of embodiments 19-25.

[0372] Embodiment 27. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant adenovirus vector of any one of embodiments 19-25, or the cell of embodiment 26, and a pharmaceutically acceptable carrier.

[0373] Embodiment 28. A method of delivering a therapeutic payload to a subject comprising administering an effective amount of the recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27 to the subject.

[0374] Embodiment 29. A method of delivering a therapeutic payload to a subject comprising contacting a cell with the recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27, and administering the cell to the subject.

[0375] Embodiment 30. The method of embodiment 28 or 29, wherein the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule.

[0376] Embodiment 31. The method of embodiment 30, wherein the transgene encodes a therapeutic protein.

[0377] Embodiment 32. The method of embodiment 30 or 31, wherein the method results in long-term expression of the transgene in the subject.

[0378] DBl / 161420432.1 58 VCN-008PC / 112492-5208

[0379] Embodiment 33. A method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising administering an effective amount of the recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27 to the subject.

[0380] Embodiment 34. A method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising contacting a cell with the recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27, and administering the cell to the subject.

[0381] Embodiment 35. The method of embodiment 33 or 34, wherein the disease or disorder is a genetic disease.

[0382] Embodiment 36. The method of embodiment 33 or 34, wherein the method prevents an infectious disease in the subject.

[0383] Embodiment 37. The method of embodiment 36, wherein the method provides a vaccination from an infectious disease caused by an infectious or pathogenic agent.

[0384] Embodiment 38. The method of embodiment 37, wherein the infectious or pathogenic agent is selected from one or more of a virus, a prion, a bacteria, a parasite, a fungi, and a protozoa.

[0385] Embodiment 39. The recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27 for use in treating, ameliorating, or preventing a disease or disorder in a subject.

[0386] Embodiment 40. Use of the recombinant adenovirus vector of any one of embodiments 19-25, the cell of embodiment 26, or the pharmaceutical composition of embodiment 27 in the manufacture of a medicament for the treating, ameliorating, or preventing a disease or disorder in a subject.

[0387] Embodiment 41. The method or use of any one of embodiments 28-40, wherein upon administration to a subject, the recombinant adenovirus vectors have reduced recognition by anti-adenovirus neutralizing antibodies, as compared to adenovirus vectors that do not comprise an albumin-binding moiety.

[0388] Embodiment 42. The method or use of any one of embodiments 28-41 , wherein the subject is a human.

[0389] Embodiment 43. The method or use of any one of embodiments 28-42, wherein the recombinant adenovirus vectors are systemically administered.

[0390] DBl / 161420432.1 59

Claims

VCN-008PC / 112492-5208CLAIMSWhat is claimed is:

1. A modified adenovirus capsid protein comprising an albumin-binding moiety insertion within the capsid’s hexon protein’s seventh hypervariable region (HVR7), wherein when the hexon protein is packaged as a component of an adenovirus particle, the albumin-binding moiety is exposed on the outer surface of the adenovirus particle.

2. The modified adenovirus capsid protein of claim 1 , wherein the albumin-binding moiety is a wild-type albumin-binding domain or a functional variant or fragment thereof.

3. The modified adenovirus capsid protein of claim 1 or 2, wherein the albumin-binding moiety is selected from an albumin-binding domain from streptococcal protein G, an albumin-binding domain from Peptostreptococcus magnus protein PAB, an albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 21), and functionally equivalent variants thereof, optionally wherein the albumin-binding moiety comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 23, 21, or 22, optionally wherein the albumin-binding moiety comprises a polypeptide having the amino acid sequence of SEQ ID NO: 23, 21, or 22.

4. The modified adenovirus capsid protein of any one of claims 1-3, wherein the adenovirus capsid protein is from at least one adenovirus type selected from a Group C adenovirus, a Group A adenovirus, a Group B adenovirus, a Group D adenovirus, a Group E adenovirus, a Group F adenovirus, and a Group G adenovirus, optionally wherein the adenovirus capsid protein is from at least one adenovirus type selected from a serotype 5 adenovirus genome, a serotype 1 adenovirus genome, a serotype 2 adenovirus genome, a serotype 3 adenovirus genome, a serotype 4 adenovirus genome, a serotype 6 adenovirus genome, a serotype 7 adenovirus genome, a serotype 8 adenovirus genome, a serotype 9 adenovirus genome, a serotype 10, adenovirus genome, a serotype 11 adenovirus genome, a serotype 12 adenovirus genome, a serotype 13 adenovirus genome, a serotype 14 adenovirus genome, a serotype 15 adenovirus genome, a serotype 16 adenovirus genome, a serotype 17 adenovirus genome, a serotype 18 adenovirus genome, a serotype 19 adenovirus genome, a serotype 20, adenovirus genome, a serotype 21 adenovirus genome, a serotype 22 adenovirus genome, a serotype 23 adenovirus genome, a serotype 24 adenovirus genome, aDBl / 161420432.1 60VCN-008PC / 112492-5208 serotype 25 adenovirus genome, a serotype 26 adenovirus genome, a serotype 27 adenovirus genome, a serotype 28 adenovirus genome, a serotype 29 adenovirus genome, a serotype 30, adenovirus genome, a serotype 31 adenovirus genome, a serotype 32 adenovirus genome, a serotype 33 adenovirus genome, a serotype 34 adenovirus genome, a serotype 35 adenovirus genome, a serotype 36 adenovirus genome, a serotype 37 adenovirus genome, a serotype 38 adenovirus genome, a serotype 39 adenovirus genome, a serotype 40, adenovirus genome, a serotype 41 adenovirus genome, a serotype 42 adenovirus genome, a serotype 43 adenovirus genome, a serotype 44 adenovirus genome, a serotype 45 adenovirus genome, a serotype 46 adenovirus genome, a serotype 47 adenovirus genome, a serotype 48 adenovirus genome, a serotype 49 adenovirus genome, a serotype 50, adenovirus genome, a serotype 51 adenovirus genome, a serotype 52 adenovirus genome, a serotype 53 adenovirus genome, a serotype 54 adenovirus genome, a serotype 55 adenovirus genome, a serotype 56 adenovirus genome, a serotype 57 adenovirus genome, a serotype 58 adenovirus genome, a serotype 59 adenovirus genome, a serotype 60, adenovirus genome, a serotype 61 adenovirus genome, a serotype 62 adenovirus genome, a serotype 63 adenovirus genome, a serotype 64 adenovirus genome, a serotype 65 adenovirus genome, a serotype 66 adenovirus genome, and a serotype 67 adenovirus genome.

5. The modified adenovirus capsid protein of any one of claims 1-4, wherein the albumin-binding moiety insertion is located in the range of positions 400-450, with reference to any one of SEQ ID NOs: 1-10, or positions corresponding thereto.

6. The modified adenovirus capsid protein of any one of claims 1-4, wherein the albumin-binding moiety insertion is located immediately after one or more of the following amino acid residues:(a) hAd5: any one of residues 420-449, with reference to SEQ ID NO: 1;(b) hAd3: any one of residues 413-441, with reference to SEQ ID NO: 2;(c) hAd4: any one of residues 403-433, with reference to SEQ ID NO: 3;(d) hAd7: any one of residues 409-434, with reference to SEQ ID NO: 4;(e) hAd11 : any one of residues 417-445, with reference to SEQ ID NO: 5;(f) hAd 16: any one of residues 403-437, with reference to SEQ ID NO: 6;(g) hAd21 : any one of residues 419-446, with reference to SEQ ID NO: 7;(h) hAd34: 421-448, with reference to SEQ ID NO: 8; andDBl / 161420432.1 61VCN-008PC / 112492-5208(i) hAd35: any one of residues 421-449, with reference to SEQ ID NO: 9.

7. The modified adenovirus capsid protein of claim 6, wherein the albumin-binding moiety insertion occurs after an amino acid residue corresponding to one of amino acids N437, V420, 1421, N422, T423, E424, T425, L426, T427, K428, V429, K430, P431, K432, T433, G434, Q435, E436, G438, W439, E440, K441, D442, A443, T444, E445, F446, S447, D448, or K449 of the hAd5 capsid, with reference to SEQ ID NO: 1, or positions corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10, optionally wherein the albumin-binding moiety insertion is located after amino acid residue 437 of hAd5, with reference to SEQ ID NO: 1, or a position corresponding thereto, or the corresponding site within any other adenovirus capsid protein, with reference to any one of SEQ ID NOs: 1-10.

8. The modified adenovirus capsid protein of any one of claims 1-7, wherein the N- and / or C-terminus of the albumin-binding moiety is connected to the capsid protein by a linker sequence, optionally wherein the linker sequence is substantially comprised of glycines and serines, optionally wherein the linker sequence comprises GSGS (SEQ ID NO: 73), SGGTSGSTSGTGST (SEQ ID NO: 17), AGSSTGSSTGPGSTT (SEQ ID NO: 18), GGSGGAP (SEQ ID NO: 19), GGGVEGGG (SEQ ID NO: 20), GGSGGSGGGGSGGGGS (SEQ ID NO: 25), LE, GGGGS (SEQ ID NO: 26), (GGGGS)n (n=1-7) (SEQ ID NO: 27-33), (Gly)8 (SEQ ID NO: 34), (Gly)6 (SEQ ID NO: 35), (EAAAK)n (n=1-3) (SEQ ID NO: 36-38), A(EAAAK)nA (n=2-5) (SEQ ID NO: 39- 42), AEAAAKEAAAKA (SEQ ID NO: 43), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 44), PAPAP (SEQ ID NO: 45), KESGSVSSEQLAQFRSLD (SEQ ID NO: 46), EGKSSGSGSESKST (SEQ ID NO: 47), GSAGSAAGSGEF (SEQ ID NO: 48), (XP)n (SEQ ID NO: 49), GGS, (GGS)n (n=2-20) (SEQ ID NO: 50-68), GGGSE (SEQ ID NO: 69), GSESG (SEQ ID NO: 70), GSEGS (SEQ ID NO: 71), or GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 72), optionally wherein the linker sequence comprises the sequence GSGS (SEQ ID NO: 73).

9. The modified adenovirus capsid protein of any one of claims 1-8, wherein the albumin-binding moiety is further fused to a homing polypeptide or a homing domain thereof.

10. The modified adenovirus capsid protein of any one of claims 1 -9, wherein the modified capsid protein binds to albumin.

11. A nucleic acid encoding the modified adenovirus capsid protein of any one of claims 1-10.

12. A recombinant adenovirus genome comprising the nucleic acid of claim 11 .DBl / 161420432.1 62VCN-008PC / 112492-520813. A recombinant adenovirus vector comprising the modified capsid protein of any one of claims 1-10, the nucleic acid of claim 11, or the recombinant adenovirus genome of claim 12, and further comprising a therapeutic payload, optionally wherein the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule, optionally wherein the transgene encodes a therapeutic protein, optionally wherein the transgene encoding the therapeutic protein comprises a gene used in gene therapy or in vaccination.

14. The recombinant adenovirus vector of claim 13, wherein the recombinant adenovirus vector binds to albumin.

15. The recombinant adenovirus vector of claim 13 or 14, wherein, in the presence of human serum albumin, the recombinant adenovirus vector has reduced recognition by anti-adenovirus neutralizing antibodies, as compared to an adenovirus vector that does not comprise an albumin-binding moiety.

16. A cell comprising the recombinant adenovirus vector of any one of claims 13-15.

17. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant adenovirus vector of any one of claims 13-15, or the cell of claim 16, and a pharmaceutically acceptable carrier.

18. A method of delivering a therapeutic payload to a subject comprising administering an effective amount of the recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17 to the subject, optionally wherein the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule, optionally wherein the transgene encodes a therapeutic protein, optionally wherein the method results in long-term expression of the transgene in the subject.

19. A method of delivering a therapeutic payload to a subject comprising contacting a cell with the recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17, and administering the cell to the subject, optionally wherein the therapeutic payload comprises a transgene, therapeutic protein, and / or small molecule, optionally wherein the transgene encodes a therapeutic protein, optionally wherein the method results in long-term expression of the transgene in the subject.

20. A method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising administering an effective amount of the recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17 to the subject, optionally wherein (i) the diseaseDBl / 161420432.1 63VCN-008PC / 112492-5208 or disorder is a genetic disease, or (ii) the method prevents an infectious disease in the subject, optionally wherein the method provides a vaccination from an infectious disease caused by an infectious or pathogenic agent, optionally wherein the infectious or pathogenic agent is selected from one or more of a virus, a prion, a bacteria, a parasite, a fungi, and a protozoa.

21. A method of treating, ameliorating, or preventing a disease or disorder in a subject, comprising contacting a cell with the recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17, and administering the cell to the subject, optionally wherein (i) the disease or disorder is a genetic disease, or (ii) the method prevents an infectious disease in the subject, optionally wherein the method provides a vaccination from an infectious disease caused by an infectious or pathogenic agent, optionally wherein the infectious or pathogenic agent is selected from one or more of a virus, a prion, a bacteria, a parasite, a fungi, and a protozoa.

22. The recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17 for use in treating, ameliorating, or preventing a disease or disorder in a subject.

23. Use of the recombinant adenovirus vector of any one of claims 13-15, the cell of claim 16, or the pharmaceutical composition of claim 17 in the manufacture of a medicament for the treating, ameliorating, or preventing a disease or disorder in a subject.

24. The method or use of any one of claims 18-23, wherein upon administration to a subject, the recombinant adenovirus vectors have reduced recognition by anti-adenovirus neutralizing antibodies, as compared to adenovirus vectors that do not comprise an albumin-binding moiety.

25. The method or use of any one of claims 18-24, wherein the subject is a human.

26. The method or use of any one of claims 18-25, wherein the recombinant adenovirus vectors are systemically administered.

27. A modified adenovirus capsid protein comprising an albumin-binding moiety insertion within the capsid’s hexon protein’s seventh hypervariable region (HVR7), wherein when the hexon protein is packaged as a component of an adenovirus particle, the albumin-binding moiety is exposed on the outer surface of the adenovirus particle, wherein the albumin-binding moiety comprises an amino acid sequence of SEQ ID NO: 23, and wherein the albumin-binding moiety insertion is located after amino acid residue 437 of hAd5, with reference to SEQ ID NO: 1.DBl / 161420432.1 64

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