Methods and materials for making recombinant viruses

Abstract

This document provides methods and materials for making recombinant viruses. For example, recombination systems including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site) are provided.

Description

[0001] METHODS AND MATERIALS FOR MAKING RECOMBINANT VIRUSES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 529,471, filed on July 28, 2023, and claims the benefit of U.S. Patent Application Serial No. 63 / 643,197, filed on May 6, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2240_SL.xml.” The XML file, created on July 25, 2024, is 83,617 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials for making recombinant viruses (e.g., recombinant adenoviral vectors). For example, recombination systems including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first flippase recognition target (FRT) site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of the first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site) can be used for making recombinant adenoviral vectors.

[0008] BACKGROUND

[0009] Adenoviruses and other DNA viruses have been extensively studied for use as a vector for delivering genetic therapeutics in various in vivo applications, such as gene therapy, vaccines, and oncolytic immunotherapy (Lu et al., Expert Opin. Biol. Then, 22:1359- 1378 (2022); Boucher et al, J. Control. Rel., 327:788-800 (2020); Cots et al., Curr. Gene Ther., 13:370-381 (2013); Jacob-Dolan et \., Annu. Rev. Med., 73:41-54 (2022); and Sakurai et al., Drug Metab. Pharmacokinet., 42: 100432 (2022)). These vectors offer numerous advantages as a tool for therapeutic delivery, including a high coding capacity of from 36 kbp to more than 200 kbp, broad tropisms exhibited by several serotypes for targeting various cell types, the ability to easily scale up viral titers for clinical use, and a relatively stable virion and genome that can be lyophilized and stored for extended periods at ambient temperatures (Lu et al., Expert Opin. Biol. Then, 22:1359-1378 (2022)).

[0010] However, the construction of recombinant viral vectors can be challenging in part because the manipulation of the large DNA genome size of such viruses is complex. Historically, the existing approaches for virus genome manipulation can be categorized into three main methods: direct in vitro modification by restriction enzymes (Stow et al., J. Virol., 37: 171-180 (1981); and Mizuguchi et al., Hum. Gene Ther, 9:2577-2583 (1998)), homologous recombination in mammalian cells (McGrory et al., Virology, 163:614-617 (1988); Ng et al., Hum. Gene Then, 10, 2667-2672 (1999); and Bett et al., Proc. Natl. Acad. Sci. USA, 91 :8802-8806 (1994)), and homologous recombination in bacteria (Chartier et al., J. Virol., 70:4805-4810 (1996); Crouzet et al., Proc. Natl. Acad. Sci. USA, 94: 1414-1419 (1997); He et al., Proc. Natl. Acad. Sci. USA, 95:2509-2514 (1998); and Campos et al, Hum. Gene Then, 15:1125-1130 (2004)). Currently, the bacteria-based homologous recombination method appears to be the primary choice of adenoviral genome manipulation in the field because this method has significant advantages by isolating a single adenoviral backbone clone with nearly no need for tedious plaque purification that is typically required in other methods.

[0011] Despite its strong advantage, the homologous recombination method using recombinant plasmid DNA derived from bacteria has a compelling drawback: a significant reduction of virus rescue efficiency due to the lack of viral proteins. For example, rescue of adenoviral vectors is inefficient due to a lack of the viral terminal protein (TP) in the system. The adenovirus TP is a viral protein that is covalently conjugated on both termini of viral genome DNA and is necessary for efficient viral nuclear localization, DNA anchoring and viral replication (Schaack et al., Genes Dev., 4: 1197-1208 (1990); Webster et al., J. Virol., 71 :6381-6389 (1997); and Al-Wassiti et al., hit. J. Mol. Sci., 22:3310 (2021)). It has been shown that adenoviral DNA without TP can be 100 to 1000-fold less efficient to form infectious plaques by using purified adenoviral DNA treated with or without proteases (Jones et al., Cell, 13: 181-188 (1978); and Miyake et al., ( 1996) Proc. Natl. Acad. Sci. USA, 93 : 1320-1324). To compensate for the low rescue efficiency, the Ad rescue period is typically extended by one to three weeks for the initial propagation to achieve a more robust viral amplification (Luo et al., Nat. Protoc., 2: 1236-1247 (2007); and Wu et al., Gene Then, 21 :629-637 (2014)). As a result, using bacteria-derived recombinant adenoviral DNA lacking TPs greatly extends the overall production time.

[0012] In addition, the reduction of virus rescue efficiency caused by the absence of TPs on plasmid-derived adenoviral DNA also hinders the application for constructing diverse adenoviral libraries. Diverse adenoviral libraries can be a powerful tool for studying adenovirus biology and have translational applications (Lupoid et al., Nucleic Acids Res. , 35:el38 (2007); and Miura et al., Mol. Then, 21 : 139-148 (2013)). A human cDNA library or a genome-wide knockout gRNA library requires at least a le5 number of unique clones in each library because the number of encoded genes is around 20,000 in the human genome, while a random peptide library would require a higher number (le9 for 6-mer random peptides) of diversity to include all possible assemblies. However, it is impractical to yield such a high clone number by using bacteria-derived DNA, which generates less than 50 viral clones per microgram of viral DNA (Elahi et al., Gene Then, 9: 1238-1246 (2002)).

[0013] SUMMARY

[0014] This document provides methods and materials for making recombinant viruses (e.g., recombinant adenoviral vectors). For example, recombination systems provided herein can include (1) a viral vector (e.g., an adenoviral vector) having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a first recombinase site of a first recombinase, (b) a packaging sequence, (e) a recombinase site of a second recombinase, (f) a second recombinase site of the first recombinase, (g) a recombinase site of a third recombinase, and optionally including (c) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide, (d) a promoter, and / or (h) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide; and (2) a donor nucleic acid, where the donor nucleic acid includes (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a gene of interest (GO I)), (c) a recombinase site of the third recombinase (e g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site). In some cases, a recombination system provided herein can be used to make recombinant adenoviral vectors. For example, a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a first recombinase site of a first recombinase (e.g., a first FRT site), (b) a packaging sequence, (c) nucleic acid encoding an El A polypeptide, (d) a promoter, (e) a recombinase site of a second recombinase (e.g., a loxP site), (f) a second recombinase site of the first recombinase (e.g., a second FRT site), (g) a recombinase site of a third recombinase (e.g., an attP site), and (h) nucleic acid encoding an E1B polypeptide; and (2) a donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) nucleic acid that can encode a polypeptide of interest (e.g., a GOI), (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site), can be used to insert the nucleic acid that can encode the polypeptide (e.g., the GOI) into the genome of the adenoviral vector within a cell such that the cell can produce and release recombinant adenoviral vectors containing the nucleic acid (e.g., such that cells infected with the recombinant adenoviral vectors can express that polypeptide of interest).

[0015] The recombination systems provided herein can include using recombinase-mediated selections (e.g., positive selections and negative selections) to integrate a nucleic acid that can encode a polypeptide of interest (e.g., a GOI) into a viral vector (e.g., an adenoviral vector) genome and can include using a negative selection mechanism to remove the packaging signal (or another viral sequence that can regulate viral packaging and / or propagation) from adenoviral vectors that do not contain the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) integrated into their genomes within cells such that at least 5% of the adenoviral vectors produced and released by the cells contain the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) integrated into the genomes (see, e.g., Figure 1A). In some cases, a viral vector (e.g., an adenoviral vector) genome can lack packaging signal (or another viral sequence that can regulate viral packaging and / or propagation) and insertions can be positively selected by re-introducing packaging signal (or another viral sequence that can regulate viral packaging and / or propagation) during recombination. As demonstrated herein, a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a first recombinase site of a first recombinase (e.g., a first FRT site), (b) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (c) nucleic acid encoding an El A polypeptide, (d) a promoter, (e) a recombinase site of a second recombinase (e.g., a loxP site), (f) a second recombinase site of the first recombinase (e.g., a second FRT site) (or a sequence that can be targeted by a targeting sequence (e g., a gRNA such as a CRISPR gRNA)), (g) a recombinase site of a third recombinase (e.g., an attP site), and (h) nucleic acid encoding an E1B polypeptide; and (2) a donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) nucleic acid that can encode a polypeptide of interest (e.g., a GOI), (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site) can be delivered to a cell containing a Cre polypeptide and Bxbl polypeptide such that the Bxbl polypeptide facilitates recombination between attP site in the adenoviral vector and the attB site in the donor nucleic acid to integrate the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) into the adenoviral vector genome, and the Cre polypeptide facilitates recombination between the first loxP site and the second loxP site to remove the second FRT site from the adenovirus, such that the cells produce and release a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) integrated into the adenoviral vector genome (see, e.g., Figure IB). Also as demonstrated herein, a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) integrated into the adenoviral vector genome can be delivered to a cell containing a FLP polypeptide such that the FLP polypeptide facilitates recombination between the first FRT site and the second FRT site, if present, to remove the packaging sequence, such that adenoviral vectors that do not contain a nucleic acid that can encode a polypeptide of interest (e.g., a GO I) integrated into their genomes no longer contain the packaging sequence and are not produced by the cell (see, e.g., Figure 1C).

[0016] Having the ability to produce recombinant viruses (e.g., recombinant adenoviral vectors) as described herein (e.g., using a recombination system provided herein) provides a unique and unrealized opportunity to accelerate generation of recombinant adenoviral vectors (e.g., individual recombinant adenoviral vectors as well as recombinant adenoviral vectors libraries). In addition, using a recombination system provided herein (e g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of the first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) to produce recombinant adenoviral vectors as described herein can circumvent cloning and / or rescue steps resulting in recombinant adenoviral vector production that is less labor-intensive and less time-consuming than traditional methods of recombinant adenoviral vector production.

[0017] In general, one aspect of this document features viral vectors (e.g., adenoviral vectors) having a genome including a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence includes (a), (b), (e), (f), (g), and optionally (c), (d), and (h): (a) a first recombinase site of a first recombinase, (b) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (c) nucleic acid encoding an El A polypeptide, (d) a promoter, (e) a recombinase site of a second recombinase, (f) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (g) a recombinase site of a third recombinase, and (h) nucleic acid encoding an adenovirus E1B polypeptide. The first recombinase site and the second recombinase site can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (g) and optional (h), a polyA sequence. The engineered DNA sequence can include (d), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence.

[0018] In another aspect, this document features isolated nucleic acid including (a), (b), (e), (f), (g), and optionally (c), (d), and (h): (a) a first recombinase site of a first recombinase, (b) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (c) nucleic acid encoding an adenovirus El A polypeptide, (d) a promoter, (e) a recombinase site of a second recombinase, (f) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (g) a recombinase site of a third recombinase, and (h) nucleic acid encoding an adenovirus E1B polypeptide. The first recombinase site and the second recombinase site can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (g) and optional (h), a polyA sequence. The engineered DNA sequence can include (d), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter.

[0019] In another aspect, this document features isolated nucleic acid including (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of the first recombinase. The first recombinase site and the second recombinase site can be loxP sites. The recombinase site of the second recombinase can be an attB site. The isolated nucleic acid can include the multicloning site. The isolated nucleic acid can include the nucleic acid sequence encoding the polypeptide of interest. The isolated nucleic acid can include the MCS and the nucleic acid sequence encoding the polypeptide of interest.

[0020] In another aspect, this document features systems for making a recombinant adenovirus. The systems can include, or consist essentially of, (a) an adenoviral vector having a genome including a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence includes (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii): (i) a first recombinase site of a first recombinase, (ii) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (iii) nucleic acid encoding an adenovirus El A polypeptide, (iv) a promoter, (v) a recombinase site of a second recombinase, (vi) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (vii) a recombinase site of a third recombinase, and (viii) nucleic acid encoding an adenovirus E1B polypeptide; and (b) a donor nucleic acid including (i-b) a first recombinase site of the second recombinase, (ii-b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (iii-b) a recombinase site of the third recombinase, and (iv-b) a second recombinase site of the second recombinase. The first recombinase site and the second recombinase site of the engineered DNA sequence can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (vii) and optional (viii), a polyA sequence. The engineered DNA sequence can include the (iv), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence. The first recombinase site and the second recombinase site of the donor nucleic acid can be loxP sites. The recombinase site of the second recombinase can be an attB site. The donor nucleic acid can include the multicloning site. The donor nucleic acid can include the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include a promotor sequence operably linked to the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include the MCS and the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can be in the form of DNA plasmid. The system can include a population of cells including the first recombinase. The system can include a population of cells including the second recombinase. The system can include a population of cells including the first recombinase and the second recombinase. The first recombinase can be a Cre polypeptide. The second recombinase can be a Bxbl polypeptide. The system can include a population of cells including Cre polypeptide and a Bxbl polypeptide. The system can include a population of cells including the third recombinase. The third recombinase can be a FLP polypeptide. The system can include a population of cells including FLP polypeptide.

[0021] In another aspect, this document features in vitro host cells including (a) an adenoviral vector having a genome including a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence includes (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii): (i) a first recombinase site of a first recombinase, (ii) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (iii) nucleic acid encoding an adenovirus E1A polypeptide, (iv) a promoter, (v) a recombinase site of a second recombinase, (vi) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (vii) a recombinase site of a third recombinase, and (viii) nucleic acid encoding an adenovirus E1B polypeptide. The first recombinase site and the second recombinase site can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (vii) and optional (viii), a polyA sequence. The engineered DNA sequence can include the (iv), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence. The host cell can be a vero cell, a BHK-21 cell, a 293 cell, a A549 cell, a PER.C6 cell, a 911 cell, a HEL299 cell, or a HeLa cell.

[0022] In another aspect, this document features in vitro host cells including nucleic acid including (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of the first recombinase. The first recombinase site and the second recombinase site can be loxP sites. The recombinase site of the second recombinase can be an attB site. The nucleic acid can include the multicloning site. The nucleic acid can include the nucleic acid sequence encoding the polypeptide of interest. The nucleic acid can include the MCS and the nucleic acid sequence encoding the polypeptide of interest. The host cell can be a vero cell, a BHK-21 cell, a 293 cell, a A549 cell, a PER.C6 cell, a 911 cell, a HEL299 cell, or a HeLa cell.

[0023] In another aspect, this document features methods for making nucleic acid encoding a recombinant virus. The methods can include, or consist essentially of, delivering an adenoviral vector and a donor nucleic acid to an in vitro cell, where the adenoviral vector has a genome including a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence includes (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii): (i) a first recombinase site of a first recombinase, (ii) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (iii) nucleic acid encoding an adenovirus El A polypeptide, (iv) a promoter, (v) a recombinase site of a second recombinase, (vi) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (vii) a recombinase site of a third recombinase, and (viii) nucleic acid encoding an adenovirus E1B polypeptide, where the donor nucleic acid includes (A) a first recombinase site of the second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of the third recombinase, and (D) a second recombinase site of the second recombinase, where the cell expresses the second recombinase and the third recombinase, where recombination occurs within the cell to form the nucleic acid encoding the recombinant virus, and where the nucleic acid encoding the recombinant virus includes the first recombinase site of the first recombinase, the packaging sequence, the nucleic acid encoding the El A polypeptide (when present), the promoter (when present), a recombinase site of the second recombinase or a remnant thereof post recombination, the multicloning site or the nucleic acid sequence encoding the polypeptide of interest, the recombinase site of the third recombinase or a remnant thereof post recombination, and the nucleic acid encoding the E1B polypeptide (when present). The nucleic acid encoding the recombinant virus can lack the second recombinase site of the first recombinase. The first recombinase site and the second recombinase site of the engineered DNA sequence can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (vii) and optional (viii), a polyA sequence. The engineered DNA sequence can include the (iv), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence. The first recombinase site and the second recombinase site of the donor nucleic acid can be loxP sites. The recombinase site of the second recombinase can be an attB site. The donor nucleic acid can include the multicloning site. The donor nucleic acid can include the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include a promotor sequence operably linked to the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include the MCS and the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can be in the form of DNA plasmid. The first recombinase can be a FLP polypeptide. The second recombinase can be Cre a polypeptide. The third recombinase can be a Bxbl polypeptide. The cell can be a eukaryotic cell.

[0024] In another aspect, this document features methods for making nucleic acid encoding a recombinant virus. The methods can include, or consist essentially of, (a) delivering an adenoviral vector and a donor nucleic acid to a first in vitro cell, where the adenoviral vector has a genome including a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence includes (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii): (i) a first recombinase site of a first recombinase, (ii) a packaging sequence (or another viral sequence that can regulate viral packaging and / or propagation), (iii) nucleic acid encoding an adenovirus El A polypeptide, (iv) a promoter, (v) a recombinase site of a second recombinase, (vi) a second recombinase site of the first recombinase (or a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA)), (vii) a recombinase site of a third recombinase, and (viii) nucleic acid encoding an adenovirus E1B polypeptide, where the donor nucleic acid includes (A) a first recombinase site of the second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of the third recombinase, and (D) a second recombinase site of the second recombinase, where the first cell expresses the second recombinase and the third recombinase, where recombination occurs within the first cell to form the nucleic acid encoding the recombinant virus, where the nucleic acid encoding the recombinant virus includes the first recombinase site of the first recombinase, the packaging sequence, the nucleic acid encoding the El A polypeptide (when present), the promoter (when present), a recombinase site of the second recombinase or a remnant thereof post recombination, the multicloning site or the nucleic acid sequence encoding the polypeptide of interest, the recombinase site of the third recombinase or a remnant thereof post recombination, and the nucleic acid encoding the E1B polypeptide (when present); and (b) delivering viral vectors (e.g., adenoviral vectors) produced by the first cell to a second in vitro cell, where the second cell expresses the first recombinase, where recombination occurs within the second cell on the adenoviral vector, if present, to reduce the presence of nucleic acid not encoding the recombinant virus. The nucleic acid encoding the recombinant virus can lack the second recombinase site of the first recombinase. The first recombinase site and the second recombinase site of the engineered DNA sequence can be FRT sites. The recombinase site of the second recombinase can be a loxP site. The recombinase site of the third recombinase can be an attP site. The engineered DNA sequence can include, between (vii) and optional (viii), a polyA sequence. The engineered DNA sequence can include the (iv), and the promoter can be a CMV promoter, a RSV promoter, a CAG promoter, or a EFla promoter. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence. The first recombinase site and the second recombinase site of the donor nucleic acid can be loxP sites. The recombinase site of the second recombinase can be an attB site. The donor nucleic acid can include the multicloning site. The donor nucleic acid can include the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include a promotor sequence operably linked to the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can include the MCS and the nucleic acid sequence encoding the polypeptide of interest. The donor nucleic acid can be in the form of DNA plasmid. The first cell can be a eukaryotic cell. The second recombinase can be a Cre polypeptide. The third recombinase can be a Bxbl polypeptide. The second cell can be a eukaryotic cell. The first recombinase can be a FLP polypeptide.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0026] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1E. A schematic of the design of exemplary FastVirus systems. Figure 1A) The components and general workflow for recombinant adenovirus production with the FastAd system. Transfected DNA fragments are incorporated into substrate Ads by Cre and Bxbl recombinases in the first producer cells, followed by a negative selection within the producer cell line expressing FLP. Figure IB) Transgene insertion mechanism by Bxbl recombinase. Figure 1C) Negative selection mechanism: excision of packaging signals by FLP recombinase. Figure ID) The pairing mechanism by loxP sites allows conditional removal of the counter selection marker (the FRT site) in the Cre-Bxbl expressing cell lines. Figure IE) Using a single recombinase (Bxbl) to delete the counterselection FRT site with orthogonal recombinase sites.

[0029] Figures 2A-2D. Schematic of vector backbones and selection mechanisms of an exemplary FastVirus system. Figure 2A) Substrate Ad backbone with the FastAd cassette containing a cytomegalovirus (CMV) promoter and a mTagBFP2 reporter between El A and E1B genes. Figure 2B) Donor plasmid with a loxP-mGreenLantern-attB-loxP cassette. Figure 2C) Negative selection mechanism by the excision of the packaging signal. Because the CMV promoter is excised together with the packaging signal, the loss of mTagBFP2 expression can represent the efficiency of negative selection. Figure 2D) Integration of genes of interest (GO I) by Bxbl and Cre recombinases. After transfection, the plasmid backbone will firstly be excised by Cre, leaving a circular DNA fragment containing one loxP site, the transgene and one attB site. This circular DNA will be integrated by Bxbl, creating a loxP- FRT-attR-loxP unit in the front of the transgene. Sequentially, the FRT site that served as a counter- sei ection marker is deleted by Cre. The final form of integrated Ads is not susceptible to negative selection.

[0030] Figures 3A-3B. A schematic of an alternative integration pathway. Figure 3A) Cre- initiated integration, followed by cleavage of the FRT site by Bxbl. Figure 3B) The Cre- initiated integration pathway in the context of the two-fluorescent protein-based reporter system.

[0031] Figures 4A-H. Evaluation of the efficiency of an exemplary FastVirus system. Figure 4A) 293-FLP cells were established to efficiently cleave the packaging signal. Its efficiency was demonstrated by the loss of CMV promoter that reduced the mTagBFP2 expression of the substrate Ad. Figure 4B) The steps and timeline for transfection of donor DNA plasmid and infection for substrate Ads in 293-Cre+Bxbl cells. Figure 4C) The two-fluorescent protein-based reporter assay to evaluate incorporation efficiency by Cre / lox system only and the FastAd system. Figure 4D) The first passage of incorporated Ads from 293-Cre cells to 293-FLP cells in the reporter assay. Figure 4E) The first passage of incorporated Ads from 293-Cre+Bxbl cells to 293-FLP cells in the reporter assay. Figure 4F) Using Dpnl enzyme to digest methylated plasmid DNA fragments within purified total gDNA to distinguish the amplified mGreenLantem DNA fragments by viral replication. The qPCR primer set was designed to amplify a mGreenLantem region that contains two Dpnl sites. Figure 4G) Relative mGreenLantem copies from DpnI-treated total gDNA between 293, 293-Cre and 293-Cre+Bxbl cells was quantified by qPCR. The relative amount of mGreenLantem was normalized by hexon copies as an internal control. Statistics of relative mGreenLantem copies were performed by one-way ANOVA and Tukey’s multiple comparisons (****p < 0.0001, ns: p > 0.05). Figure 4H) Relative mGreenLantem copies from DpnI- treated total gDNA between 293-Cre+Bxbl and the second passage of 293-FLP cells, normalized with hexon copies in the qPCR assay. Statistics of relative mGreenLantem copies were performed by unpaired t-test. (***p<0.0002).

[0032] Figures 5A-5F. Evaluation of the purity of Ads with or without negative selection. Figure 5A) Scheme of vims preparation. 293, 293-Cre and 293-Cre+Bxbl cells were transfected with mGreenLantem donor plasmid and infected with substrate Ad-mTagBFP2. After 3 days post-infection, freeze-thawed cell lysates were passage onto 293 cells or 293- FLP cells and continue to be amplified by a total of 5 passages to reach the scale of a 10- layer CellSTACK® culture chamber (6,360 cm2). Incorporated Ad pools were purified by CsCl gradients. Figure 5B) Purified Ads and one positive control Ad rescued by the traditional bacteria-based homologous recombination method were used to infect A549 cells at concentrations of 50 viral particles (vps) / cell, 1,000 vps / cell and 10,000 vps / cell to compare the efficiency and degree of contamination of the original substrate Ads. Figure 5C) The purified viral DNA from each group was analyzed by Sanger sequencing with the CMV promoter primer. Sequences shown include a sequence from cells infected with a substrate Ad reference (SEQ ID NO:1), a sequence from Cre-Bxbl-FLP- cells (SEQ ID NO:2), a sequence from Cre+Bxbl-FLP- cells (SEQ ID NO:3), a sequence from Cre+Bxbl+FLP- cells (SEQ ID NO:4), a sequence from Cre+Bxbl+FLP+ cells (SEQ ID NO:5), and a sequence from cells infected with a positive control reference (SEQ ID NO: 6). The sequencing peak views are from the SnapGene Software. Figure 5D) The Sanger sequencing results of purified Ad DNA from Cre+Bxbl incorporation but without the negative selection. Sequences shown include a sequence from cells infected with a substrate Ad reference (SEQ ID NO:7) and a sequence from cells infected with mGreenLantem (SEQ ID NO:8). The nucleotide ratio of the sequencing results was analyzed by the BEAT program. Figure 5E) Relative mGreenLantem copies within purified viral DNA, normalized to hexon copies. Statistics of relative mGreenLantem copies / hexon copies were performed by one-way ANOVA and Tukey's multiple comparisons (****p < 0.0001). Figure 5F) The relative copies of the FastAd cassette (loxP-FRT-attP) in the original substrate Ads when normalized to hexon copies. Statistics of relative mGreenLantem copies / hexon copies were performed by one-way ANOVA and Tukey’s multiple comparisons (**p < 0.002, ***p < 0.0002).

[0033] Figures 6A-6C. Expedited workflow to produce large-scale recombinant viral vectors. Figure 6A) A 10-day workflow for producing high yields of Ads by the FastAd system. Figure 6B) The relative copies of the FastAd cassette (loxP-FRT-attP) in the original substrate CRAds when normalized to hexon copies. Figure 6C) Flow cytometry results confirmed the expression of mOX40L expression on A549 cells at 48 hours post-infection with 1000 vp / cell of CRAd6-mOX40L and CRAd6 empty control.

[0034] Figures 7A-7G. PCR-based approaches to generate DNA donors for an exemplary FastVirus system. Figure 7A) One-step generation of donor DNA fragments for the FastAd system by PCR. Figure 7B) The example of one-step PCR amplification of mGreenLantem as DNA donor with the iProof DNA polymerase. Figure 7C) Evaluation if mGreenLantem PCR products with the FastAd adaptors can be used as DNA donors for the FastAd system. Figure 7D) Two-step PCR approaches for generating the FastAd DNA donors. To avoid the possible primer dimer issues caused by loxP sites within the primer sets, only one loxP site is included in each run of PCR. Figure 7E) The example using Platinum SuperFi I Polymerase in a two-step PCR amplification of influenza NA cDNA for preparation of the FastAd DNA donor. Figure 7F) Western blot analysis of A549 cells infected with 1,000 vps / cell of SC- Ad6-NP made by the NP generated by the two-step PCR. Figure 7G) The relative copies of the FastAd cassette (loxP-FRT-attP) in the original substrate Ads when normalized to hexon copies. Figure 8. Primer dimer formation during one-step PCR with the primer set containing loxP sites. Severe primer dimers were observed with one-step PCR when using the Platinum SuperFi I Polymerase even with optimized annealing temperatures.

[0035] Figures 9A-9I. Simple and rapid generation of complex adenovirus libraries by an exemplary FastVirus system. Figure 9A) One-step PCR amplification of an oligo library containing random 15-mer nucleotides by the FastAd adaptor primer set. Figure 9B) Analysis of the Ad library constructed by the PCR products of the oligo library. Purified viral DNA was amplified by PCR for amplicon NGS. Total reads and unique reads were shown. Figure 9C) Percentage of the number of unique reads. Figure 9D) The sequence logo demonstrated the nucleotide ratio in each position of the 15 random nucleotides of the unique reads. Figure 9E) Scheme for two-step PCR amplification of the GeCKO v2 human genome-wide knockout gRNA library as the FastAd DNA donor. Figure 9F) The expedited workflow for transferring the whole gRNA library. Figure 9G) The percentage of unique gRNAs was transferred to Ad by the FastAd system. Figure 9H) The counts of each gRNA in the original lentiviral gRNA library and transferred Ad-gRNA library. Figure 91) The ratio of counts of each unique gRNA between the original lentiviral gRNA library and the Ad-gRNA library.

[0036] Figure 10. A schematic of an exemplary workflow for 6-day production of a recombinant RDAd6-Spike-Omicron adenoviral vector.

[0037] Figures 11A-11B. Quality Control of 6-Day Production of RDAd6-Spike-Omicron. Figure 11 A) Sanger sequencing result revealed that about 70% Ad genomes were integrated with nucleic acid encoding a Spike-Omicron polypeptide (SEQ ID NO:9). Figure 1 IB) The expression of Spike-omicron was verified by western blot.

[0038] Figures 12A-12E. The design logic of the FastAd system. Figure 12A) The T and basic recombinase recognition sites on receiver Ad backbone. T: Adenoviral packaging signal; ITR: Inverted terminal repeat. Figure 12B) The basic recombinase recognition sites on circular donor DNA. Figure 12C) The components and general workflow for recombinant Ad production with the FastAd system. Transfected DNA fragments are inserted into receiver Ads by Cre and Bxbl in the first producer cell line, followed by a negative selection within the second producer cell line expressing FLP. Figure 12D) Insertion of donor DNA mediated by Bxbl and Cre. attL / attR: attachment site left / right. Figure 12E) Negative selection: excision of by FLP.

[0039] Figures 13A-13D. Vector backbones for testing the efficiency of the FastAd system by fluorescent protein reporters. Figure 13 A) Receiver RC-Ad6-mTagBFP2 backbone with the FastAd cassette containing a CMV promoter and a mTagBFP2 reporter between the E1A and E1B genes. Figure 13B) Donor DNA plasmid with a loxP-mGreenLantern-attB-loxP cassette. Figure 13C) Negative selection mechanism by the excision of . The loss of mTagBFP2 expression represents the efficiency of negative selection. Figure 13D) Insertion of mGreenLantem gene by Bxbl and Cre. The final form of integrated Ads is not susceptible to negative selection.

[0040] Figures 14A-14H. Evaluation of FastAd efficiency. Figure 14A) 293-FLP cells were established to remove from non-integrated Ads. Loss of BFP -fluorescence in receiver virus infected 293-FLP cells is indicative for its efficiency. Figure 14B) Timeline for the transfection of donor DNA plasmids and infection of FastAd producer cells with receiver Ads. Figure 14C) The two-fluorescent protein-based reporter system for evaluating the incorporation efficiency of FastAd and direct comparison with the Cre / loxP system. Figure 14D) 293 or 293-FLP cells infected with integrated Ads from 293-Cre cells. Figure 14E) 293 or 293-FLP cells infected with integrated Ads from 293-Cre-Bxbl cells. Figure 14F) The qPCR primer set was designed to amplify an amplicon containing two Dpnl sites within the mGreenLantem gene. Dpnl restriction enzyme selectively digests methylated DNA fragments derived from bacteria, allowing to distinguish them from unmethylated, amplified mGreenLantem DNA sequences produced through viral replication. Figure 14G) Relative mGreenLantem copies from DpnLtreated total gDNA between 293, 293-Cre and 293- Cre+Bxbl cells as quantified by qPCR. The relative amount of mGreenLantem was normalized by Ad-hexon copies as an internal control. Statistics of relative mGreenLantem copies were performed by one-way ANOVA and Tukey's multiple comparisons test (****p < 0.0001, ns: p > 0.05). Figure 14H) Relative mGreenLantem copies from DpnL treated total gDNA samples of 293-Cre+Bxbl and the sample after negative selection, normalized by Ad-hexon copies in the qPCR assay. Statistics of relative mGreenLantem copies were performed by unpaired t-test. (***p<0.0002). Figures 15A-15F. Evaluation of the purity of Ads with and without negative selection. Figure 15 A) Scheme of virus preparation for purity assessment experiments. Figure 15B) Purified Ads and the positive control Ad were used to infect A549 cells at MOIs of 50, 1,000 and 10,000 vp / cell to compare the efficiency and degree of contamination of the original receiver Ads. Figure 15C) The purified viral DNA from each group was analyzed by Sanger sequencing with a primer annealing to the CMV promoter. The sequencing peak views were visualized with SnapGene Software. Figure 15D) Base calling ratio from the sequencing results of purified Ad DNA from the Cre+Bxbl+FLP- group was analyzed by the BEAT software to evaluate integration efficiency. Figure 15E) Relative mGreenLantern copies within purified viral DNA, normalized to Ad-hexon copies. Statistics of relative mGreenLantern copies / hexon copies were performed by one-way ANOVA and Tukey's multiple comparisons test (****p < 0.0001). Figure 15F) Relative abundance of nonintegrated receiver Ad sequences when normalized to hexon copies. Statistics of relative mGreenLantern copies / hexon copies were performed by one-way ANOVA and Tukey's multiple comparisons test (**p<0.002, ***p < 0.0002).

[0041] Figures 16A-16I. PCR-amplified DNA donors and flexible Ad backbones for the FastAd system. Figure 16A) One-step PCR protocol for the generation of donor DNA fragments containing recombinase recognition sites. Figure 16B) Example of one-step PCR amplification of mGreenLantern as DNA donor with the iProof DNA polymerase. Figure 16C) Evaluation of the FastAd system using a PCR-amplified mGreenLantern DNA donor. 293-Cre+Bxbl cells were transfected with or without PCR-amplified loxP-mG-attB-loxP fragments and infected with RC-Ad-mTagBFP2 for flow cytometry analysis at 48 postinfection. Figure 16D) Two-step PCR protocol for generating the FastAd DNA donors. Figure 16E) Representative results of the two-step PCR protocol for the generation of a loxP- NP-attB-loxP DNA donor. Figure 16F) Western blot analysis of Influenza-NP expression in A549 cells infected with 1,000 vp / cell of Fast Ad-generated SC-Ad6-NP at 48 hours postinfection. Figure 16G) CRAd6-mGreenLantern, Figure 16H) CRAd6-mOX40L and Figure 161) CRAd6-mCD40L generated in A549-based FastAd producer cell lines were used to infect A549 cells with 1,000 vp / cell for 48 hours and transgene expression was assessed by flow cytometry. Figures 17A-17C. Expedited workflow for producing recombinant Ads with high titers. Figure 17A) A 10-day workflow of the FastAd system for amplifying recombinant Ads to the scale of a 10-layer CellStack culture chamber. Figure 17B) Relative copies of nonintegrated receiver CRAd6-CMV normalized to Ad-hexon copies to assess the purity of the Ad generated by the two-round negative selection protocol. Figure 17C) Flow cytometry analysis confirmed the expression of mOX40L expression on A549 cells at 48 hours postinfection with 1000 vp / cell of CRAd6-mOX40L.

[0042] Figures 18A-18J. Simple and rapid generation of complex Ad libraries by FastAd. Figure 18A) Generating random 17-mer donor DNA fragments for the FastAd library by one-step PCR amplification of a donor library containing 17-mer random oligonucleotides. Figure 18B) Gel electrophoresis to verify PCR amplification of N17 library donor DNA fragments. Figure 18C) N17 library donor DNA fragments were analyzed by NGS. Total raw reads, mapped reads and unique reads are shown. Figure 18D) Distribution of reads in categories of unique and multiple occurrences in the N 17 library donor DNA fragments. Figure 18E) Sequence logo position-based analysis of nucleotide frequency demonstrates an unbiased distribution in the N17 random nucleotides in donor DNA fragments. Figure 18F) Extracting N17 library from Ad library. Figure 18G) Gel electrophoresis to verify PCR amplification of N17 library donor DNA fragments and N17 libraries from three independent Ad library preparations. Figure 18H) N17 libraries from Ad were analyzed by NGS. Total raw reads, mapped reads and unique reads were shown. Figure 181) Percentage of counts in total mapped sequences from three categories in the N17 Ad library. Figure 18J) The sequence logo demonstrating the nucleotide ratio in each position of the N17 random nucleotides in Ad library.

[0043] Figures 19A-19I. Application of Ad-gRNA library for functional evolution. Figure 19A) Schematic for the two-step PCR amplification of the GeCKO v2 human genome-wide knockout gRNA library as FastAd donor DNA. Figure 19B) The expedited workflow for transferring the whole gRNA library in Ads by FastAd. Figure 19C) gRNA representation in lentiviral plasmid DNA and Ad-gRNA library. Bars represent the number of unique gRNAs with a specific read count. Figure 19D) Comparison of gRNA representation in the plasmid library and Ad-gRNA library. Figure 19E) Ad-gRNA library was subjected to 10 passages in A549-Ctrl cells or A549-SpCas9 cells followed by NGS analysis of the resulting libraries. Figure 19F) Clonal distribution of gRNAs in the input Ad-gRNA library and in Ad-gRNA libraries after 10 passages of evolution. Figure 19G) Comparison of gRNA representation between Ad-gRNA selected in A549-Ctrl cells and A549-SpCas9 cells. Figure 19H) Identification of enriched target genes in Ad-gRNA library after 10 passages in A549- SpCas9 cells. Genes highlighted in red represent previously reported Ad restriction factors. Genes in purple represent the top 4 enriched genes in the Ad-gRNA library evolution experiment. Figure 191) PANTHER GO-SLIM Gene Ontology (GO) for enriched target genes (p-value <0.05 and gRNA count fold change > 1.5) in Ad-gRNA library after 10 passages in A549-SpCas9 cells.

[0044] Figures 20A-20D. Mechanisms of FastAd by Cre, Bxbl and FLP recombinases.

[0045] Figure 20A) Receiver RC-Ad6-BFP backbone with a FRT site on the left of the adenoviral packaging signal ( P), and the FastAd cassette containing a CMV promoter, a loxP site, a second FRT site, a attP site and a BFP reporter. Figure 20B) Donor DNA plasmid with a loxP-GFP-attB-loxP cassette. Figure 20C) Negative selection by the excision of T with FLP recombinase. The loss of BFP expression represents the efficiency of negative selection. Figure 20D) Insertion of GFP gene by Bxbl and Cre. The final integrated Ads is not susceptible to negative selection by FLP.

[0046] Figures 21A-21C. Evaluation of the purity of Ads from FastAd. Figure 21A) Scheme of virus preparation. Purified Ads and the positive control Ad were used to infect A549 cells at multiple of infect (M.O.I.) of 1,000 vp / cell to compare the efficiency and degree of contamination of the original receiver Ads. Figure 21B) Relative GFP copies within purified viral DNA, normalized to hexon copies. Statistics of relative GFP copies / hexon copies were performed by one-way ANOVA and Tukey's multiple comparisons (****p < 0.0001). Figure 21C) The relative copies of non-integrated receiver Ads, normalized to hexon copies. Statistics of relative GFP copies / hexon copies were performed by one-way ANOVA and Tukey's multiple comparisons (**p<0.002, ***p < 0.0002).

[0047] Figures 22A-22C. Expedited workflow for producing recombinant Ads with high titers. Figure 22A) A 10-day workflow of the FastAd system for amplifying recombinant Ads to the scale of a 10-layer CellStack culture chamber. Figure 22B) qPCR for quantification of contamination from receiver Ads. The relative copies of non-integrated receiver CRAd6- CMV were normalized to hexon copies. Figure 22C) Flow cytometry results confirmed the expression of mOX40L expression on A549 cells at 48 hours postinfection with 1000 vp / cell of CRAd6-mOX40L.

[0048] Figure 23. Simple and rapid generation of complex Ad libraries by FastAd. One-step PCR was used to amplify the oligonucleotides containing 17-mer random nucleotides (N17) with up to 1.718 x 1010 variants and to add FastAd recombinase recognition sites to for integration. 5 pg of PCR products were transfected into 293-Cre+Bxbl cells and coinfected with 1000 vp / cell of receiver Ads. Subsequently, the Ad population was amplified for 5 passages in 293-FLP cells. Purified viral DNA were further PCR amplified for Next- Generation Sequencing (NGS) to examine library diversity. Total raw reads, mapped reads and unique reads were shown. The sequence logo demonstrated the nucleotide ratio in each position of the N17 random nucleotides in Ad library.

[0049] Figures 24A-24I. Application of Ad-gRNA library for functional evolution. Figure 24A) Scheme for the two-step PCR amplification of the GeCKO v2 human genome-wide knockout gRNA library as the FastAd donor DNA. Figure 24B) The expedited workflow for transferring the whole gRNA library in Ads by FastAd. Figure 24C) gRNA representation in lentiviral plasmid DNA and Ad-gRNA library. Bars represent the number of unique gRNAs with a specific read count. Figure 24D) Comparison of gRNA representation in the plasmid library and Ad-gRNA library. Figure 24E) Ad-gRNA library was subjected to 10 passages in A549-Ctrl cells or A549-SpCas9 cells. Figure 24F) gRNA representation in the Ad-gRNA library and Ad-gRNA libraries after 10 passages of evolution. Figure 24G) Comparison of gRNA representation between Ad-gRNA selected in A549-Ctrl cells and A549-SpCas9 cells. Figure 24H) Identification of enriched target genes in Ad-gRNA library after 10 passages in A549-SpCas9 cells. Genes in red dots are previously reported Ad restriction factors. Genes in purple dots are top 4 enriched genes in the Ad-gRNA library evolution experiment. Figure 241) PANTHER GO-SLIM Gene Ontology (GO) for enriched target genes (p-value <0.05 and gRNA count fold change > 1.5) in Ad-gRNA library after 10 passages in A549-SpCas9 cells. Figure 25. Validation of individual gene candidates from Ad-gRNA library by CRISPR / Cas9 knockout in A549 cells by reporter assay.

[0050] Figure 26. Validation of individual gene candidates from Ad-gRNA library by CRISPR / Cas9 knockout in A549 cells by titering Ad in each infected cell lines.

[0051] Figure 27. Confirmation of VP54-KO cell clone produced higher viral titer than control cell lines.

[0052] Figure 28. Confirmation of VP54-KO cell clone produced higher viral titer than control cell lines.

[0053] DETAILED DESCRIPTION

[0054] This document provides methods and materials for making recombinant viruses (e.g., recombinant adenoviral vectors). For example, recombination systems provided herein can include (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e g., an engineered DNA sequence) including (a) a first recombinase site of a first recombinase, (b) a packaging sequence, (e) a recombinase site of a second recombinase, (f) a second recombinase site of said first recombinase, (g) a recombinase site of a third recombinase, and optionally including (c) nucleic acid encoding an adenovirus El A polypeptide, (d) a promoter, and / or (h) nucleic acid encoding an adenovirus E1B polypeptide; and (2) a donor nucleic acid, where said donor nucleic acid includes (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), (c) a recombinase site of the third recombinase (e g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site). In some cases, a recombination system provided herein can be used for making recombinant adenoviral vectors. For example, a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e g., an engineered DNA sequence) including (a) a first recombinase site of a first recombinase (e.g., a first FRT site), (b) a packaging sequence, (c) nucleic acid encoding an El A polypeptide, (d) a promoter, (e) a recombinase site of a second recombinase (e.g., a loxP site), (f) a second recombinase site of the first recombinase (e.g., a second FRT site), (g) a recombinase site of a third recombinase (e.g., an attP site), and (h) nucleic acid encoding an E1B polypeptide; and (2) a donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) nucleic acid that can encode a polypeptide of interest (e.g., a GOI), (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site), can be used to insert the nucleic acid that can encode the polypeptide of interest (e.g., the GOI) into the genome of the adenoviral vector within a cell such that the cell can produce and release recombinant adenoviral vectors containing the nucleic acid (e.g., such that cells infected with the recombinant adenoviral vectors can express that polypeptide of interest).

[0055] In some cases, the methods and materials provided herein can be used to produce a population of a single recombinant adenoviral vector. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be used to produce from about IxlO12viral particles of a single recombinant adenoviral vector (e.g., per 6,360 cm2growth area) to about 5xl012viral particles of a single recombinant adenoviral vector (e.g., per 6,360 cm2growth area) (e.g., from about IxlO12to about 4xl012, from about IxlO12to about 3xl012, from about IxlO12to about 2xl012, from about 2xl012to about 5xl012, from about 3xl012to about 5xl012, from about 4xl012to about 5xl012, from about 2xl012to about 4xl012, from about 2xl012to about 3xl012, or from about 3xl012to about 4xl012viral particles of a single recombinant adenoviral vector).

[0056] In some cases, the methods and materials provided herein can be used to produce a collection (e.g., a library) of different recombinant adenoviral vectors. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e g., a second loxP site)) can be used to produce from about IxlO4different recombinant adenoviral vectors to about xlO7different recombinant adenoviral vectors (e.g., from about IxlO4to about xlO6, from about IxlO4to about xlCP, from about IxlO5to about xlO6, from about IxlO4to about xlO5, or from about IxlO5to about xlO6different recombinant adenoviral vectors).

[0057] In some cases, the methods and materials provided herein can accelerate the speed of recombinant adenoviral vector production. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be used to produce one or more recombinant adenoviral vectors in less than 1 month. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be used to produce one or more recombinant adenoviral vectors in from about 6 days to about 1 month (e.g., from about 6 days to about 25 days, from about 6 days to about 20 days, from about 6 days to about 15 days, from about 6 days to about 12 days, from about 6 days to about 10 days, from about 8 days to about 30 days, from about 10 days to about 30 days, from about 12 days to about 30 days, from about 15 days to about 30 days, from about 20 days to about 30 days, from about 25 days to about 30 days, from about 8 days to about 25 days, from about 10 days to about 20 days, from about 12 days to about 15 days, from about 8 days to about 12 days, from about 10 days to about 15 days, from about 12 days to about 20 days, or from about 15 days to about 25 days).

[0058] In some cases, the methods and materials provided herein can be used to make recombinant viruses (e.g., recombinant adenoviral vectors) in vitro. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e g., a second loxP site)) can be contacted with an in vitro cell (e.g., a cell in an in vitro culture) to make recombinant viruses (e.g., recombinant adenoviral vectors) in vitro. In some cases, the methods and materials provided herein can be used to make recombinant viruses (e.g., recombinant adenoviral vectors) in vivo. For example, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be administered to a mammal (e.g., a human) to make recombinant viruses (e.g., recombinant adenoviral vectors) in vivo.

[0059] In some cases, the methods and materials provided herein do not include any cloning steps.

[0060] In some cases, the methods and materials provided herein do not include any rescue steps.

[0061] A recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include any appropriate viral vector (e.g., a viral vector having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)). A viral vector provided herein can have a double-stranded DNA genome. In some cases, a viral vector provided herein can infect dividing cells. In some cases, a viral vector provided herein can infect non-dividing cells. Examples of viral vectors that can be designed to have a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) include, without limitation, adenoviral vectors (e.g., adeno viral vectors based on human species C adenoviruses (HAdVs) such as human species C serotype 6 adenovirus (HAdV-6)), herpes virus vectors (e.g., herpes virus vectors based on herpes simplex viruses such as herpes simplex virus type 1 (HSV-1)), baculoviral vectors (e.g., baculoviral vectors based on Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)), iridovirus vectors (e.g. iridoviral vectors based on Bohle iridovirus), and poxvirus vectors (e.g. poxvirus vectors based on vaccinia viruses such as vaccinia virus Ankara (MV A)).

[0062] A viral vector provided herein (e.g., a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)) can include any appropriate nucleic acid sequence (e.g., engineered DNA sequence) including (a) a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site). In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) can be flanked by a first ITR sequence and a second ITR sequence. In some cases, a first ITR sequence can be directly or indirectly followed by a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site). In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) can be directly or indirectly followed by a second ITR sequence. Examples of ITR sequences that can flank a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein are set forth in Table 1.

[0063] Table 1. Exemplary ITR sequences.

[0064] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can be flanked by a first ITR sequence having a DNA sequence set forth in SEQ ID NO: 10 and a second ITR sequence having DNA sequence set forth in SEQ ID NO: 11.

[0065] When nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein includes one or more FRT sites, the FRT site(s) can be any appropriate FRT site(s). In cases where a nucleic acid sequence (e.g., an engineered DNA sequence) provided herein includes a first FRT site and a second FRT site, the first FRT site and the second FRT site can be the same type of FRT site or can be different types of FRT sites. A FRT site can be derived from any appropriate source. In some cases, a FRT site can be a synthetic FRT site. A FRT site can have any appropriate nucleotide sequence. Examples of FRT sites that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein are set forth in Table 2.

[0066] Table 2. Exemplary FRT sequences.

[0067] A nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can include any appropriate packaging sequence. In some cases, a packaging sequence also can be referred to a psi signal or a psi sequence. A packaging sequence can be derived from any appropriate source. In some cases, a packaging sequence can be a synthetic packaging sequence. A packaging sequence can have any appropriate nucleotide sequence. Examples of packaging sequences that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein are set forth in Table 3.

[0068] Table 3. Exemplary packaging sequences.

[0069] In some cases, a viral sequence that can regulate viral packaging and / or propagation can be used in addition to or in place of a packaging sequence of a nucleic acid sequence (e.g., an engineered DNA sequence) otherwise designed to include a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e g., an attP site) within a viral vector provided herein. Examples of viral sequences that can regulate viral packaging and / or propagation and can be used in addition to or in place of a packaging sequence of a nucleic acid sequence provided herein include, without limitation, poly adenylation signals, splicing signals, viral promoters (e.g., El promoters and major late promoters), and nucleic acid encoding a polypeptide that can promote vial propagation (e.g., viral Illa genes, viral protease (PS) genes, viral El genes, viral E2 genes, and viral E4 genes). In some cases, viral sequences that can regulate viral packaging and / or propagation and can be used in addition to or in place of a packaging sequence of a nucleic acid sequence provided herein can be as described elsewhere (see, e.g., Crosby et al., Virology, 462: 158-165 (2014); and Elahi et al., Gene Therapy, 9( 18): 1238- 1246 (2002)).

[0070] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can include nucleic acid encoding an El A polypeptide. Nucleic acid encoding an El A polypeptide can be any appropriate nucleic acid encoding an El A polypeptide. Nucleic acid encoding an El A polypeptide can encode any appropriate El A polypeptide. Examples of El A polypeptides and nucleic acid sequences encoding an El A polypeptide include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at Gene ID Nos. 2652980, 6870531, and 1460853, and at Accession Nos. NC_001405.1 al locations 467 .1630, AC_000008.1 al locations 560..1545, NC_011203.1 at locations 480..1499, and NC_001460.1 at locations 415.. 1442. In some cases, an El A polypeptide and a nucleic acid sequence that can encode an El A polypeptide can be as set forth in Example 2.

[0071] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can include a promoter. A promoter can be any appropriate promoter. A promoter can be a constitutive promoter or a regulated promoter (e.g., an inducible promoter). A promoter can be from any appropriate source. In some cases, a promoter can be a synthetic promoter. In some cases, a promoter can be a recombinant promoter. In some cases, the promoter is not operably linked to (e.g., does not drive expression of) any component within a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein. Examples of promoters that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein include, without limitation, CMV promoters, RSV promoters, CAG promoters, and EFla promoters.

[0072] When a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein includes one or more loxP sites, the loxP site(s) can be any appropriate loxP site(s). A loxP site can be derived from any appropriate source. In some cases, a loxP site can be a synthetic loxP site. A loxP site can have any appropriate nucleotide sequence. Examples of loxP sites that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein are set forth in Table 4.

[0073] Table 4. Exemplary loxP sequences.

[0074] When a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein includes one or more attP sites, the attP site(s) can be any appropriate attP site(s). An attP site can be derived from any appropriate source. In some cases, an attP site can be a synthetic attP site. In cases where a nucleic acid sequence (e.g., an engineered DNA sequence) provided herein includes a first attP site and a second attP site, the first attP site and the second attP site can be the same type of attP site or can be different types of attP sites. For example, a first attP site and a second attP site can be different types of attP sites that do not cross-react such as a wild type attP site and a GA-mutant attP site. An attP site can have any appropriate nucleotide sequence. Examples of attP sites that can be included in a nucleic acid sequence (e g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein are set forth in Table 5.

[0075] Table 5. Exemplary attP sequences.

[0076] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can include nucleic acid encoding an E1B polypeptide, Nucleic acid encoding an E1B polypeptide can be any appropriate nucleic acid encoding an E1B polypeptide. Nucleic acid encoding an E1B polypeptide can encode any appropriate E1B polypeptide. Examples of E1B polypeptides and nucleic acid sequences encoding an E1B polypeptide include, without limitation, those set forth in the NCBI databases at Gene ID Nos. 2652981, 24271511, 1460854, and 2715936, and at Accession Nos. NC_001405.1 at locations 1669..4061, NC_011202.1 at locations

[0077] 1556..3927, NC_001460.1 al locations 1499..3809, and NC_001454.1 at locations

[0078] 1373..3619. In some cases, an E1B polypeptide and a nucleic acid sequence that can encode an E1B polypeptide can be as set forth in Example 2.

[0079] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein can include one or more additional components. Examples of additional components that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein include, without limitation, nucleic acid sequence spacers, polyadenylation (poly A) signals, barcode nucleic acid sequences, transcription factor binding sites, non-coding RNA sequences such as non-coding RNA promoters. In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) within a viral vector provided herein (e.g., a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) can include a polyA signal between the attP site and nucleic acid encoding an E1B polypeptide.

[0080] In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) that can be included in a viral vector provided herein (e.g., a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) can include (a) a first FRT site, (b) a packaging sequence, (c) nucleic acid encoding an El A polypeptide, (d) a promoter, (e) a loxP site, (f) a second FRT site, (g) an attP site, and (h) nucleic acid encoding an E1B polypeptide.

[0081] A recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include any appropriate donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) a multi cloning site and / or nucleic acid that can encode a polypeptide of interest, (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site). A donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be linear or circular. In some cases, a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e g., a second loxP site)) can be in the form of a vector (e.g., a plasmid such as a DNA plasmid).

[0082] When a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) includes one or more loxP sites, the loxP site(s) can be appropriate loxP site(s). In cases where a donor nucleic acid provided herein includes a first loxP site and a second loxP site, the first loxP site and the second loxP site can be the same type of loxP site or can be different types of loxP sites. A loxP site can be derived from any appropriate source. In some cases, a loxP site can be a synthetic loxP site. A loxP site can have any appropriate nucleotide sequence. Examples of loxP sites that can be included in a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) are set forth in Table 6.

[0083] Table 6. Exemplary loxP sequences.

[0084] When a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) includes a multicloning site, the multicloning site can be any appropriate multicloning site. A multicloning site can be derived from any appropriate source. In some cases, a multi cloning site can be a synthetic multicloning site. A multicloning site can have any appropriate nucleotide sequence. Examples of multicloning sites that can be included in a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) are set forth in Table 7.

[0085] Table 7. Exemplary multicloning sequences. When a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) includes nucleic acid that can encode a polypeptide of interest, the nucleic acid can be any appropriate nucleic acid that can encode a polypeptide of interest. A nucleic acid that can encode a polypeptide of interest can encode any appropriate polypeptide of interest. In some cases, nucleic acid that can encode a polypeptide of interest can encode a therapeutic polypeptide (e.g., an immune modulating polypeptide such as an immune stimulating polypeptide or an immune suppressing polypeptide). For example, nucleic acid that can encode a polypeptide of interest can encode an antigenic polypeptide (e.g., an antigenic polypeptide such as a viral antigen or a tumor antigen that can be used in a polypeptide vaccine). In another example, nucleic acid that can encode a polypeptide of interest can encode one or more gene therapy components (e.g., gene editing components) such as a nuclease. In yet another example, nucleic acid that can encode a polypeptide of interest can encode an anti-cancer polypeptide (e.g., a cell death-inducing polypeptide or a tumor suppressor polypeptide). Examples of polypeptides of interest that can be encoded by nucleic acid that can be included in a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) include, without limitation, therapeutic polypeptides, immunogenic polypeptides designed to produce an immune response within a mammal (e.g., a human) to treat a particular condition, enzymes, and antibodies. In some cases, nucleic acid that can encode a polypeptide of interest can encode one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or more) polypeptides of interest. For example, nucleic acid that can encode a polypeptide of interest can encode a library two or more polypeptides of interest. For example, nucleic acid that can encode a polypeptide of interest can encode a library three or more polypeptides of interest. For example, nucleic acid that can encode a polypeptide of interest can encode a library four or more polypeptides of interest. In some cases, nucleic acid that can encode a polypeptide of interest can encode a collection (e.g., library) of polypeptides of interest. For example, the polypeptides listed in Table 8 can be used as polypeptides of interest. In some cases, in addition to or in place of nucleic acid that can encode a polypeptide of interest, a donor nucleic acid provided herein can include nucleic acid that can encode a non-coding nucleic acid (e.g., non-coding RNAs (ncRNAs) such as microRNAs, small interfering RNAs (siRNAs), piwi -interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNA (snRNAs), short hairpin RNAs (shRNAs)) of interest, and targeting sequences such as guide RNAs (gRNAs; e.g., CRISPR gRNAs). In some cases, in addition to or in place of nucleic acid that can encode a polypeptide of interest, a donor nucleic acid provided herein can include nucleic acid that can encode one or more oncolytic viral genomes (e.g., AVV genomes).

[0086] Table 8. Exemplary polypeptides of interest and non-coding nucleic acids of interest. When a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) includes one or more attB sites, the attB site(s) can be any appropriate attB site(s). An attB site can be derived from any appropriate source. In some cases, an attB site can be a synthetic attB site. In cases where a donor nucleic acid sequence provided herein includes a first attB site and a second attB site, the first attB site and the second attB site can be the same type of attB site or can be different types of attB sites. For example, a first attB site and a second attB site can be different types of attB sites that do not cross-react such as a wild type attB site and a GA-mutant attB site. An attB site can have any appropriate nucleotide sequence. Examples of attB sites that can be included in a donor nucleic acid provided herein (e g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) are set forth in Table 9.

[0087] Table 9. Exemplary attB sequences.

[0088] In some cases, a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include a promoter. When a donor nucleic acid provided herein includes a promoter, the promoter can be any appropriate promoter. A promoter can be a constitutive promoter or a regulated promoter (e.g., an inducible promoter). A promoter can be from any appropriate source. In some cases, a promoter can be a synthetic promoter. In some cases, a promoter can be a recombinant promoter. In cases where the donor nucleic acid provided herein includes nucleic acid that can encode a polypeptide of interest, the promoter can be operably linked to the nucleic acid that can encode a polypeptide of interest. Examples of promoters that can be included in a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector provided herein include, without limitation, CMV promoters, RSV promoters, CAG promoters, and EFla promoters.

[0089] In some cases, a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include (a) a first loxP site, (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest, (c) an attB site, and (d) a second loxP site.

[0090] In some cases, nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector (e.g., an adenoviral vector) provided herein and / or a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) also can include nucleic acid encoding a reporter polypeptide. A nucleic acid encoding a reporter polypeptide can encode any appropriate reporter polypeptide. In some cases, a reporter polypeptide can be a fluorescent polypeptide. In some cases, a reporter polypeptide can be an enzyme. In some cases, a reporter polypeptide can be a polypeptide that provides resistance to a particular antibiotic. In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) within a viral vector (e.g., an adenoviral vector) provided herein can include nucleic acid encoding a first reporter polypeptide, a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include nucleic acid encoding a second reporter polypeptide, and the first reporter polypeptide and the second reporter polypeptide can be different reporter polypeptides. Examples of reporter polypeptides include, without limitation, green fluorescent polypeptides (e.g., mGreenLantem polypeptides), red fluorescent polypeptides, blue fluorescent polypeptides (e.g., mTagBFP2 polypeptides), puromycin N-acetyl-transferase (PAC) polypeptides, amino 3 '-glycosylphosphotransferase (neo) polypeptides, and luciferase polypeptides (e.g., firefly luciferase polypeptides).

[0091] In some cases, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include (1) an adenoviral vector having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including (a) a first loxP site, (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest, (c) an attB site, and (d) a second loxP site.

[0092] In some cases, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include (1) an adenoviral vector having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e g., a wild-type attP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a second recombinase site of the second recombinase where the second recombinase site of the second recombinase does not cross-react with the first recombinase site of the second recombinase (e.g., a GA-mutant attP site); and (2) a donor nucleic acid including (a) a recombinase site of the second recombinase (e.g., wild-type attB site), (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest, and (c) a second recombinase site of the second recombinase where the second recombinase site of the second recombinase does not cross-react with the first recombinase site of the second recombinase (e.g., a GA-mutant attB site).

[0093] Also provided herein are cells (e.g., host cells) containing one or more adenoviral vectors provided herein (e.g., one or more adenoviral vectors each having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)). In some cases, a host cell containing one or more adenoviral vectors provided herein (e.g., one or more adenoviral vectors each having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)) can be an in vitro cell (e.g., a cell in an in vitro culture).

[0094] Also provided herein are cells (e.g., host cells) containing a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)). In some cases, a host cell containing a donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be an in vitro cell (e.g., a cell in an in vitro culture). In some cases, cells that can contain one or more adenoviral vectors provided herein and / or one or more donor nucleic acids provided herein can be from a cell line. In some cases, cells that can contain one or more adenoviral vectors provided herein and / or one or more donor nucleic acids provided herein can be primary cells. Examples of cells (e.g., host cells) that can contain one or more adenoviral vectors provided herein (e.g., one or more adenoviral vectors each having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)) and / or one or more donor nucleic acids provided herein (e.g., a donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) a multicloning site and / or nucleic acid that can encode a polypeptide of interest, (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site)) include, without limitation, vero cells, BHK-21 cells, 293 cells, A549 cells, PER.C6 cells, 911 cells, HEL299 cells, and HeLa cells.

[0095] In some cases, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include cells (e.g., producer cells) that can contain nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases. In some cases, nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can be provided in a viral vector provided herein. In some cases, nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can be provided in a donor nucleic acid provided herein. In some cases, nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can be provided independent of a viral vector provided herein and a donor nucleic acid provided herein. For example, nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can be provided in the form of a vector (e.g., a viral vector or a plasmid such as an expression plasmid).

[0096] Cells (e.g., producer cells) that can contain (e g., can be designed to contain) nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can be any appropriate type of cell. In some cases, a cell (e.g., a producer cell) that can contain nucleic acid encoding a recombinase such that the cell contains one or more (e.g., one, two, three, or more) recombinases can be a eukaryotic cell (e.g., a mammalian cell). For example, a cell (e.g., a producer cell) that can contain nucleic acid encoding a recombinase such that the cell contains one or more (e.g., one, two, three, or more) recombinases can be a human cell, a murine cell, an avian cell, a reptilian cell, an amphibian cell, or a fish cell. Examples of cells (e.g., producer cells) that can contain (e.g., can be designed to contain) nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases include, without limitation, HEK293 cells, A549 cells, and HeLa cells. It will be appreciated that a recombination system provided herein can include (e.g., can be designed to include) a viral vector (e.g., an adenoviral vector) capable of infecting the species of cell(s) (e.g., producer cell(s)) that can contain (e.g., can be designed to contain) nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases.

[0097] Cells (e.g., producer cells) that can contain (e.g., can be designed to contain) nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can contain (e.g., can be designed to contain) nucleic acid encoding any appropriate recombinase. Examples of recombinases include, without limitation, Cre polypeptides, Bxbl polypeptides, FLP polypeptides, vika polypeptides, Dre polypeptides, vCre polypeptides, sCre polypeptides, X-Int polypeptides, R polypeptides, Kw polypeptides, Kd polypeptides, B2 polypeptides, B3 polypeptides, PhiC31 polypeptides, Nm60 polypeptides, Bt24 polypeptides, Si74 polypeptides, Fm04 polypeptides, No67 polypeptides, uCb4 polypeptides, Cbl6 polypeptides, Bm99 polypeptides, PaOl polypeptides, Cs56 polypeptides, Vhl9 polypeptides, Kp03 polypeptides, Me99 polypeptides, Rh64 polypeptides, Ma05 polypeptides, Cc91 polypeptides, Bu30 polypeptides, Sh25 polypeptides, Ma37 polypeptides, Ps78 polypeptides, and Y134 polypeptides. It will be appreciated that cells (e.g., producer cells) that contain nucleic acid encoding a recombinase can contain (e.g., can be designed to contain) nucleic acid encoding a recombinase that can facilitate recombination between recombinase sites present in a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)).

[0098] Examples of Cre polypeptides and nucleic acid sequences encoding a Cre polypeptide include, without limitation, those set forth in the NCBI databases at Accession Nos. YP_006472 and NC_005856. In some cases, a Cre polypeptide and a nucleic acid sequence that can encode a Cre polypeptide can be as set forth in Example 2.

[0099] Examples of Bxbl polypeptides and nucleic acid sequences encoding a Bxbl polypeptide include, without limitation, those set forth in the NCBI databases at Accession Nos. AFM44955.1 and JQ929585.1. In some cases, a Bxbl polypeptide and a nucleic acid sequence that can encode a Bxbl polypeptide can be as set forth in Example 2.

[0100] Examples of FLP polypeptides and nucleic acid sequences encoding a FLP polypeptide include, without limitation, those set forth in the NCBI databases at Accession Nos. AKA09972.1 and P03870. In some cases, a FLP polypeptide and a nucleic acid sequence that can encode a FLP polypeptide can be as set forth in Example 2.

[0101] Cells (e.g., producer cells) that can contain (e g., can be designed to contain) nucleic acid encoding a recombinase such that the cells contain one or more (e.g., one, two, three, or more) recombinases can contain (e.g., can be designed to contain) nucleic acid encoding any appropriate number of recombinases. In some cases, cells (e g., producer cells) can contain (e.g., can be designed to contain) nucleic acid encoding a single recombinase such that the cells contain a single recombinase. In some cases, cells (e.g., producer cells) can contain (e.g., can be designed to contain) nucleic acid encoding two different recombinases such that the cells contain two different recombinases. In some cases, cells (e.g., producer cells) can contain (e.g., can be designed to contain) nucleic acid encoding three different recombinases such that the cells contain three different recombinases. In some cases, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can include a first population of cells (e.g., a first population of producer cells) that contain (e.g., are designed to contain) nucleic acid encoding a Cre polypeptide and nucleic acid encoding a Bxbl polypeptide and a second population of cells (e.g., a second population of producer cells) that contain (e.g., are designed to contain) nucleic acid encoding a FLP polypeptide, such that the first population of cells (e.g., the first population of producer cells) contain both a Cre polypeptide and a Bxbl polypeptide and the second population of cells (e.g., the second population of producer cells) contain a FLP polypeptide.

[0102] In some cases, a nucleic acid sequence that can be targeted by one or more gene editing components can be used in addition to or in place of one or more recombination sites of a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e g., a second loxP site)). Gene editing components that can be used in addition to or in place of one or more recombination sites of a recombination system provided herein can be from any appropriate gene editing system. Examples of such gene editing systems include, without limitation, CRISPR / Cas gene editing systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and homing endonucleases. For example, a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA) can be used in place of a recombination site of a recombination system provided herein, and a nuclease (e.g., a Cas9 polypeptide, a ZFN, a TALEN, or homing endonuclease) can be used in place of a recombinase. Examples of sequences that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA) include, without limitation, protospacer adjacent motif (PAM) sequences (e.g., nucleotide sequences including 5'-NNG-3'; e.g., to be targeted by a Cas9 polypeptide), nucleotide sequences including 5'-TTTV-3' (e.g., to be targeted by a Casl2a polypeptide), nucleotide sequences including 5'-TTTN-3' (e.g., to be targeted by a Casl2a polypeptide), and nucleotide sequences including 5'- TAGGGATAACAGGGTAAT-3' (SEQ ID NO:74; e.g., to be targeted by a I-Scel homing endonuclease). In some cases, sequences that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA) can be as described elsewhere (see, e.g., Gendron et al., Genes (Basel), 12(8): 1204 (2021); and Yang et al., Molecular Therapy-Nucleic Acids, 7:378-386 (2017)). In some cases, the second recombinase site of a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site) of a viral vector (e.g., an adenoviral vector) in a recombination system provided herein can be replaced with a sequence that can be targeted by a targeting sequence (e.g., a gRNA such as a CRISPR gRNA), and the recombination system can include cells (e.g., producer cells) that can contain nucleic acid encoding a nuclease (e.g., a Cas9 polypeptide, a ZFN, a TALEN, or homing endonuclease).

[0103] Also provided herein are methods for making recombinant adenoviral vectors. In some cases, the methods provided herein can be used to produce a population of a single type of recombinant adenoviral vector. In some cases, the methods provided herein can be used to produce a collection (e.g., a library) of different recombinant adenoviral vectors. In some cases, a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) to integrate the nucleic acid that can encode a polypeptide of interest of the donor nucleic acid provided herein into the genome of a viral vector (e.g., an adenoviral vector) provided herein. For example, the Cre polypeptide can facilitate recombination between a first loxP site and a second loxP site of donor nucleic acid provided herein (e.g., a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest, a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) to remove a second recombinase site of the first recombinase (e.g., a second FRT site) from a viral vector provided herein (e.g., a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)). For example, the Bxbl polypeptide can facilitate recombination between an attP site of a viral vector provided herein (e.g., a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)) and an attB site of a donor nucleic acid provided herein (a donor nucleic acid including (a) a first recombinase site of the second recombinase (e.g., a first loxP site), (b) a multi cloning site and / or nucleic acid that can encode a polypeptide of interest, (c) a recombinase site of the third recombinase (e.g., an attB site), and (d) a second recombinase site of the second recombinase (e.g., a second loxP site)) to integrate the nucleic acid that can encode the polypeptide of interest into the adenoviral vector genome. In some cases, when nucleic acid that can encode a polypeptide of interest is integrated into a viral vector (e.g., an adenoviral vector) genome, the nucleic acid that can encode the polypeptide of interest is integrated into the adenoviral vector genome such that the nucleic acid that can encode the polypeptide of interest of the adenoviral vector genome is operably linked to the promoter present in the nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site)) of the adenoviral vector genome. As used herein, “operably linked” refers to positioning of a promoter relative to a nucleic acid encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a recombinant adenoviral vector produced by a recombination system provided herein can have a genome that includes a promoter and nucleic acid encoding a polypeptide of interest. In this case, the promoter can be operably linked to a nucleic acid encoding a polypeptide of interest such that it drives expression of the polypeptide of interest.

[0104] Cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) that have been delivered a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) can produce and release a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome.

[0105] Any appropriate method can be used to deliver a recombination system provided herein (e.g., a recombination system including (1) a viral vector (e.g., an adenoviral vector) having a genome comprising a nucleic acid sequence (e.g., an engineered DNA sequence) including a first recombinase site of a first recombinase (e.g., a first FRT site), a packaging sequence, a recombinase site of a second recombinase (e.g., a loxP site), a second recombinase site of said first recombinase (e.g., a second FRT site), and a recombinase site of a third recombinase (e.g., an attP site); and (2) a donor nucleic acid including a first recombinase site of the second recombinase (e.g., a first loxP site), a multicloning site and / or nucleic acid that can encode a polypeptide of interest (e.g., a GOI), a recombinase site of the third recombinase (e.g., an attB site), and a second recombinase site of the second recombinase (e.g., a second loxP site)) to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide). A viral vector (e.g., an adenoviral vector) of a recombination system provided herein and a donor nucleic acid of a recombination system provided herein can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) together or separately. Examples of methods that can be used to deliver one or more components of a recombination system provided herein to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) include, without limitation, transduction, transfection, and electroporation. In some cases, a viral vector (e.g., an adenoviral vector) provided herein can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) by transduction and a donor nucleic acid provided herein can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) by transfection. In some cases, a viral vector (e.g., an adenoviral vector) provided herein can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) by transduction and a donor nucleic acid provided herein can be delivered to cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) by electroporation.

[0106] In some cases, a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome can be collected from the cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide). For example, cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) that produce a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome can be lysed to generate a cell lysate containing the population of adenoviral vectors, and the population of adenoviral vectors can be collected from the cell lysate. In some cases, cells (e.g., a first population of cells) containing a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) that produce a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome can be lysed by subjecting to the cells to one or more (e.g., one, two, three, four, or more) freeze / thaw cycles.

[0107] A population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome that were collected from the cells (e.g., a first population of cells) that contain a first recombinase (e.g., a Cre polypeptide) and containing a second recombinase (e.g., a Bxbl polypeptide) and that were contacted with a recombination system provided herein can be delivered to cells (e.g., a second population of cells) containing a third recombinase (e.g., FLP polypeptide) to select for recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome. For example, the FLP polypeptide can facilitate recombination between a first FRT site and a second FRT site present in adenoviral vectors that do not contain the nucleic acid that can encode a polypeptide of interest of a doner nucleic acid integrated into their genomes to remove the packaging sequence, such that adenoviral vectors no longer contain the packaging sequence and are not produced by the cell.

[0108] Any appropriate method can be used to deliver a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome to cells (e.g., a second population of cells) containing a third recombinase (e.g., a FLP polypeptide). In some cases, a population of adenoviral vectors where at least some of the adenoviral vectors are recombinant adenoviral vectors having the nucleic acid that can encode the polypeptide of interest integrated into the adenoviral vector genome can be delivered to cells (e.g., a second population of cells) containing a third recombinase (e.g., a FLP polypeptide) by transduction.

[0109] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0110] EXAMPLES

[0111] Example 1: Recombinases-Based Selection System in Producer Cells

[0112] Adenoviral vectors are recognized as highly effective gene delivery systems for in vivo applications. However, traditional methods of cloning recombinant adenoviral vectors are typically labor-intensive and time-consuming. This Example describes the design and generation of the FastVirus system - a technology for the simple and rapid generation of recombinant adenoviral vectors. For example, the FastVirus system uses recombinase-mediated selections (e.g., positive selections and negative selections) within mammalian cells infected with adenovirus substrates.

[0113] Material and Methods

[0114] Construction of donor plasmids and substrate adenoviral vectors

[0115] Bxbl recombinase attB and attP GA-mutant sites were used. Donor plasmid DNA with mGreenLantern-attB site flanked by two loxP sites (pUC57-loxP-mGreenLantern-attB- loxP) was synthesized. The donor plasmid with multiple cloning sites (pUC57-loxP-MCS- attB-loxP) was generated from pUC57-loxP-mGreenLantern-attB-loxP plasmids with restriction enzyme cloning. Other transgenes, including mIL-21, and mOX40L were cloned into pUC57-loxP-MCS-attB-loxP donor plasmid by restriction enzymes.

[0116] Substrate Ad plasmid DNA, RCAd6-FRT-'P-ElA-CMV-loxP-FRT-attP-mTagBFP2- bGHpolyA-ElB-AE3, was sequentially cloned from RCAd6-AE3 adenoviral vector. First, an FRT site was introduced to the left side of the packaging signal ( ) with an FRT-Zeocin- FRT cassette with the homology arms via red recombination. The plasmid was later transformed into FLP-expressing competent cells (DH5 / pCP20) to remove the Zeocin selection marker. Next, RCAd6-FRT-'P-ElA-CMV-loxP-FRT-attP-mTagBFP2-bGHpolyA substrate adenoviral vector plasmid DNA was generated by red recombination with the E1A- CMV-loxP-FRT-attP-mTagBFP2-bGHpolyA-ICeuI-Zeo-IceuI-ElB DNA fragment. The zeocin cassette was later deleted from the Ad plasmid DNA with Iceul digestion and selfligation of the adenoviral plasmid.

[0117] Other formats of substrate adenoviral vectors were generated with similar strategies: RCAd6-FRT-'P-ElA-loxP-FRT-attP-bGHpolyA-ElB-AE3-RSV-mGreenLantem, CRAd6- FRT- -ElA-dl 101 / dl 107-CMV-loxP-FRT-attP-bGHpolyA-ElB-AE3 and SCAd6- FRT- - E 1 A-CMV-loxP-FRT-attP-bGHpoly A-E IB-AIIIa- AE3. Rescue of substrate adenoviral vectors with plasmid DNA

[0118] Adenoviral plasmid DNA was linearized with AsisI restriction enzyme digestion. 5 pg of linearized adenoviral DNA was transfected into a T25 flask of HEK293 cells with Lipofectamine™ 3000 reagent (Invitrogen) following the manufacturer’s instructions. After 2 weeks of initial viral rescue, the substrate Ads were propagated in HEK293 cells for RCAd6 -based backbones, A549 cells for the CRAd6-based backbone, or 293-IIIa cells for the SCAd6-based backbone.

[0119] Ad Purification

[0120] Final propagated infected cell pellets were harvested from a 10-Stack CellSTACK® culture chamber (Corning). The collected cell pellets were re-suspended in 7 mL 20 mM HEPES, pH 7.4 buffer, and lysed with 2 mL of 5% sodium deoxycholate for 30 minutes at room temperature and later 150 pL of DNAse A (10 mg / mL), 15 pL RNAse A (10 mg / mL) and 170 pL 2M MgCh for 30 minutes at 37°C. After centrifuging at 3000 x g for 15 minutes at 4°C, crude supernatants were loaded for two CsCl gradients ultracentrifuge at 26,000 x g and desalted in sucrose buffer (0.5 M sucrose, 20 mM HEPES). The estimated number of viral particles (vps) was measured by OD260 with NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific).

[0121] Lentiviral vectors

[0122] NLS-HA-Bxbl fragment was PCR amplified and was inserted into pLV-EFla-IRES- Puro and pLV-EFla-IRES -Blast lentiviral plasmids to generate pLV-EFla-NLS-HA-Bxbl- IRES-Puro and pLV-EFla-NLS-HA-Bxbl-IRES-Blast lentiviral plasmids, respectively. pLV-EFla-pIHa-lRES-Puro was generated by PCR amplification of the human adenovirus serotype 6 pllla gene and inserted into the pLV-EFla-IRES-Puro vector.

[0123] Production of lentiviruses

[0124] All lentiviruses were produced by co-transfection of 1 pg of pMD.G, 1.5 pg pGGW, and 2.5 pg of pLV-EFla lentiviral vector into HEK293 cells in T25 flask with Lipofectamine™ 3000 reagent. At 16 hours post-transfection, media containing transfection reagents were replaced with fresh Dulbecco’s Modified Eagle Medium (DMEM) (Gibco). Lentiviruses-containing supernatants were harvested at 48 hours post-transfection.

[0125] Cell lines

[0126] HEK293 cells were purchased from Microbix (Toronto, Ontario, Canada). Human A549 lung carcinoma and HeLa cervical carcinoma cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA).

[0127] 293-Cre (116) cell line is a subclone of HEK293 cell lines expressing Cre recombinase. A 293-Cre+Bxbl cell line was generated by lentiviral transduction of 293-Cre cells with pLV-EFla-NLS-HA-Bxbl-IRES-Puro. A 293-Cre+Bxbl-pIIIa cell line was generated by lentiviral transduction of 293-Cre cells with pLV-EF la-NLS-HA-Bxbl-IRES- Blast and pLV-EFla-pIIIa-IRES-Puro lentiviruses. A 293-FLP cell line was generated by lentiviral transduction of HEK293 cells with pLV-EFl-FLP-PGK-Neo lenti virus. A 293FLPo-pIIIa cell line was generated by lentiviral transduction of 293FLPo cell line with pLV-EF 1 a-pIIIa-IRES-Puro lenti virus.

[0128] All stable cell lines were selected in either 1 pg / mL of puromycin, 10 pg / mL of blasticidin, or 400 pg / mL of G418, and later single-cell-cloned with the limiting dilution method.

[0129] Evaluation ofFastAd Efficiency with Flow Cytometry

[0130] For evaluating the efficiency and the dynamics ofFastAd transgene insertion with the two fluorescent proteins system: substrate RCAd6-mTagBFP2 and mGreenLantem donor plasmid transfection, cells were seeded in T25 flasks at 95% confluency before transfection. First, 5 pg of donor pUC57-loxP-mGreenLantern-attB-loxP was transfected into cells. After 16 hours post-transfection, purified substrate RCAd6-mTagBFP2 was added to cells with the concentration of 1000 vps / cell. After 48 hours post-infection, cells were harvested and fixed with 2% paraformaldehyde PBS solution. Fixed cells were analyzed with BD LSR II Flow Cytometer (BD). For analyzing the FastAd efficiency in the 1stand 2ndpassage of integrated Ads in 293-FLP cells, infected cells at 72 hours post-infection were harvested and went through 3 freeze-thaw cycles. The total crude cell lysate supernatant then was transferred to the next passages of 293-FLP for analysis at 48 hours post-infection. Flow cytometry data was analyzed and visualized by FlowJo (FlowJo LLC).

[0131] Real-Time PCR (Quantitative PCR, qPCR)

[0132] Total crude genomic DNA (gDNA) was purified from cells with QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s instructions. To distinguish mGreenLantern donor plasmids and mGreenLantern fragments that were amplified by viral replication from crude gDNA, gDNA were treated with Dpnl restriction enzymes for 2 hours in 37°C. Two Dpnl sites are inside the amplicon of mGreenLantern detection for the SYBR Green qPCR primer set (Table 10).

[0133] Ad viral DNA (vDNA) was purified with PureLink™ Viral RNA / DNA Mini Kit (Invitrogen). To distinguish the original substrate Ads and transgene-incorporated Ads, SYBR Green qPCR probes were designed to amplify the loxP-FRT-attP region (Table 10), which is expected only in the original substrate Ads.

[0134] To detect the recombination of Ad5 sequences from 293 cells to purified Ad6 genomes, a TaqMan® probe and primer sets were designed in the position of Ad5 that is corresponding to the insertion site of the loxP-FRT-attP cassette in Ad6 substrate viruses (Table 10).

[0135] The delta-delta (AA) Ct method was used to calculate the relative DNA copies between samples (normalized to the hexon region that is conserved between Ad5 and Ad6 sequences).

[0136] Sanger Sequencing to Evaluate Integration Efficiency

[0137] CMV primer was used for Sanger sequencing (Genewiz / Azenta) (Table 10). The percentage of original substrate Ad and integrated Ad was analyzed with Beats.

[0138] Detection of Transgenes Expression by Flow Cytometry

[0139] Fast-CRAd6-mOX40L transgene expressions were analyzed in A549 cells at 48 hours post-infect with 1000 vps / cell. Cells were harvested and fixed with 2% paraformaldehyde PBS solution. For surface markers, cells were directly stained with anti-mOX40L-PE (clone: RM134L) (BioLegend) antibody. For Fast-CRAd6-mIL-21, infected A549 cells were treated with monensin at 10 pg / mL for 5 hours before harvest. At 48 hours post-infection, cells were harvested, fixed with 2% paraformaldehyde PBS solution, permeabilized with BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit (BD Biosciences), and stained with anti- mIL-21-PE (#IC594P, R&D systems) antibody.

[0140] Detection of Transgenes Expression by Western Blot

[0141] A549 cells infected with 1000 vps / cell of Fast-SCAd6-NP were harvested at 48 hours post-infection. Cells were lysed with RIPA buffer and cell lysates were collected and quantified with BCA assay. Anti-Influenza NP antibody (#PA5-32242, Invitrogen) and anti- 0-Actin-HRP antibody (sc-47778) (Santa Cruz Biotechnology) were used for probing western blots.

[0142] Generation of Individual Transgene Donors or Pooled Library Donors by PCR

[0143] One-step PCR product of mGreenLantern to generate 5’-loxP-overhang and 3’-attB- loxP overhang was amplified by iProof™ High-Fidelity DNA Polymerase (Bio-Rad) in GC buffer + 3% DMSO (Table 10).

[0144] To overcome the loxP primer-dimer issue, two-step PCR was demonstrated with Platinum™ SuperFi™ PCR Master Mix (Invitrogen) for NP. For 15 random-mer (Thermo Fisher Scientific) and GecKO human gRNA library (Addgene pooled Library #1000000048, #1000000049). Platinum™ SuperFi™ PCR Master Mix was used for the first round of PCR and Taq polymerase (New England Biolabs) was used for the second round of PCR.

[0145] Next Generation Sequencing (NGS) and Data Analysis

[0146] Pooled random 15-mer, GecKO human gRNA libraries integrated into FastAd were amplified using PCR with Platinum™ SuperFi™ PCR Master Mix (Invitrogen) with primer sets (Table 10). Library preparation and DNA fragmentation was conducted by custom service by Genewiz / Azenta. Libraries were sequenced by Illumina NovaSeq 6000 (2x150 bp) with an estimated 2.45 million pair-end reads per library for amplicon.

[0147] Statistics

[0148] All statistical analyses were performed by GraphPad Prism 7.0 (GraphPad Software). Table 10. Oligonucleotides

[0149]

[0150]

[0151] RESULTS

[0152] Design concepts and mechanisms for the Fast Ad system

[0153] The FastAd system was designed to utilize “infectious adenoviruses” as an initial substrate material from which producer cells can generate recombinant adenoviral vectors. The system includes two distinct producer cell lines: one specifically designed for the insertion of genes of interest (GOI), and the other tailored for the removal of nonincorporated adenovirus substrates through a negative selection mechanism (Figure 1A).

[0154] To achieve GOI incorporation, a synthetic recombinase-based DNA integration method involving Bxbl was utilized (Figure IB). Bxbl is a large serine recombinase derived from bacteriophages that recognizes a minimal 48-bp attP site (attachment Phage) and a cognate, minimal 38-bp attB site (attachment Bacterial), enabling unidirectional and stable DNA integration. Unlike other tyrosine recombinases, such as Cre and FLP, Bxbl offers the advantage of integrating DNA fragments regardless of their DNA lengths and providing irreversible DNA integration (Merrick et al., ACS Synth. Biol., 7:299-310 (2018)). A negative selection mechanism was introduced by utilizing the Ad packaging signal flanked by two FRT sites (Figure 1C). The same packaging signal flanked by FRT sites was used as a genetic tag to mark non-integrated substrate Ads for deletion by passaging in FLP-expressing cell line.

[0155] To establish a conditional selection mechanism for integrated Ads, a design was employed where the negative selection tag, represented by one FRT site on the right, is removed upon the occurrence of a GOI insertion event. To achieve this, a loxP site was incorporated within both the donor DNA and substrate Ads as an orthogonal genetic pair for conditional deletion of the FRT site (Figure ID). In the absence of any integration events, the packaging signal and the loxP site flanked by FRT sites in the substrate Ad genomes are cleaved together in negative selection FLP-expressing cells. However, in Bxbl and Cre coexpressing cells, when DNA integration events take place, the integrated Ads have an intermediate state where the FRT site on the right side of the genomes is flanked by two loxP sites and subsequently cleaved (Figure ID). Consequently, during the passaging of the mixed Ad pool in FLP-expressing cells, Ads harboring GOI insertions can evade negative selection while non-integrated substrates are removed.

[0156] Testing and benchmarking the efficiency of Fast Ad system

[0157] To assess the efficiency and dynamics of the FastAd system, a two-fluorescent protein-based reporter system was used as a starting point. A FastAd substrate backbone incorporating a FastAd integration cassette was constructed. This cassette consists of a CMV promoter, loxP-FRT-attP sites, an mTagBFP2 reporter, and a bGH polyadenylation signal, all positioned between the El A and E1B genes (Figure 2A). At the same time, a donor plasmid containing a mGreenLantern reporter gene and an attB site flanked by two loxP sites was constructed (Figure 2B).

[0158] When the substrate Ads entered FLP-expressing cells, the packaging signal, E1A gene, and the CMV promoter which drives mTagBFP2 expression, were cleaved (Figure 2C). Hence, the loss of mTagBFP2 expression serves as a reporter for the removal of packaging signals from non-integrated Ads. On the other hand, when mGreenLantern donor plasmids were transfect into Cre and Bxbl -expressing producer cells, the donor plasmid backbone (antibiotics resistance genes and origin of replication) flanked by two loxP sites were removed by Cre recombinase, resulting in a clean, circular loxP-mGreenLantern-attB DNA donor (Figure 2D). Subsequently, the circular DNA donor fragment was inserted into the substrate Ad backbones by Bxbl recombinase with the cognate attB site (donor DNA) and the attP site (substrate Ad backbone), generating an intermediate Ad with an FRT site flanked by two loxP sites. The FRT site then was cleaved by Cre recombinase, producing final integrated Ads that express the mGreenLantern reporter and cease mTagBFP2 expression because of monocistronic translation in mammalian cells (Figure 2D). Alternatively, the donor DNA can also be integrated by Cre recombinase first, followed by Bxbl recombinase cleaving the FRT sites with the attP / attB pair (Figure 3A and 3B). Because Cre recombinase alone can also integrate mGreenLantern into the Ad backbone by alternative integration pathway (Figure 3B), not only can the efficiency of the FastAd system be evaluated by assessing the intensity and percentage of mTagBFP2 and mGreenLantern in infected cells, but the efficiency of the FastAd system also can be benchmarked against the commonly used Cre-only integration system in adenoviruses (Yamamoto et al., Mol. Pharm., 11 : 1069-1074 (2014)).

[0159] To assess the efficiency of negative selection, 293 cells or negative selection producer 293-FLP cells were infected with 1,000 vps per cell of substrate Ad-mTagBFP2. After 48 hours post-infection, it was observed that 99.6% of the 293 cells exhibited positive expression of mTagBFP2, while only 1.34% of the cells in 293-FLP cells showed mTagBFP2 positivity (Figure 4 A), demonstrating the high efficiency of negative selection.

[0160] The mGreenLantern donor plasmids were transfected into three cell lines: 293, 293- Cre, and 293-Cre+Bxbl producer cells, in order to compare the integration efficiency in the presence or absence of Cre recombinase, and in the presence or absence of both Cre and Bxbl recombinases (Figure 4B). The results demonstrated a substantial shift of cells in the 293-Cre+Bxbl groups towards the mGreenLantern fluorescence channel, accounting for a total of 66.6%. In contrast, the 293-Cre groups exhibited a moderate shift (35.24% total), while the negative control 293 cells displayed a minimal shift, representing the background signal (7.93% total) (Figure 4C). Notably, a distinct population exclusively expressing mGreenLantern (30.3%) emerged in the 293-Cre+Bxbl producer cells, which was significantly higher than the percentages observed in the 293-Cre (2.54%) and 293 cells (0.19%) (Figure 4C). It was also observed that the mGreenLantern plasmid-only transfection in both 293-Cre or 293-Cre+Bxbl cells had higher background signals (about 18%) than 293 cells, implying Cre recombinase may increase the rate of random integration of loxP flanked mGreenLantern gene into the host cell genomes.

[0161] During negative selection, it is important to note that Cre recombinase alone cannot remove the FRT site from the substrate Ad backbone (Figure 3B). As a result, when the infected 293-FLP cells underwent the first passage of the mixed Ad pool derived from 293- Cre cells, a significant loss of both mTagBFP2 and mGreenLantern signals was observed compared to infection in non-negative selection 293 cells (Figure 4D). Conversely, when the infected 293-FLP cells underwent the first passage of the mixed Ad pool derived from 293- Cre+Bxbl cells, only mGreenLantern expression was detected (Figure 4E). These findings indicate that the majority of Ads in the population became integrated with mGreenLantern after negative selection. While flow cytometry analysis allowed assessment of the efficiency of incorporation at the cellular level, it did not provide information regarding the DNA copy level. To address this, total gDNA was extracted from the transfected / infected cells and quantitative PCR (qPCR) analysis was performed to detect the amplification of mGreenLantern DNA fragments. In order to distinguish between the amplified mGreenLantern DNA fragments in Ad genomes and transfected donor plasmid sequences, the qPCR amplicon was designed with two Dpnl sites in the middle. Subsequently, the gDNA was digested with Dpnl restriction enzymes, which specifically recognize methylated adenosine of 5'-GATC-3' DNA sequences derived from bacteria but not DNA replicates in mammalian cells (Figure 4F).

[0162] These findings revealed a significant (approximately 50-fold) increase in the number of mGreenLantern copies within Ad genomes produced by 293-Cre+Bxbl cells compared to 293-Cre cells (Figure 4G). Despite the moderate mGreenLantern signal observed in flow cytometry analysis, the mGreenLantern fragments exhibited minimal amplification in 293- Cre cells, indicating that these fragments were primarily shuffling between adenovirus genomes without undergoing viral replication. Furthermore, the negative selection process further increased the ratio of mGreenLantern fragment copies to hexon copies, indicating the efficient removal of non-integrated Ads (Figure 4H).

[0163] Evaluating the purity of Ad vDNA from the Fast Ad system

[0164] To evaluate the efficiency of the FastAd system in producing recombinant adenoviruses on a larger scale, 293, 293-Cre, and 293-Cre+Bxbl cells in T25 flasks were transfected with donor plasmids and infected with substrate Ad-mTagBFP2. The cultures were then amplified through five passages in either 293 cells or 293-FLP cells until reaching the scale of a 10-layer CellSTACK® culture chamber (6,360 cm2). Subsequently, the viruses were purified using CsCl gradients (Figure 5A).

[0165] To assess the purity of the purified Ads, the two-fluorescent protein-based reporter system was employed by infecting A549 cells with different concentrations (50 vps / cell, 1,000 vps / cell, and 10,000 vps / cell) of the purified Ads. Ads integrated with both Cre and Bxbl recombinases exhibited the highest mGreenLantern reporter activity compared to Ads integrated with 293-Cre alone (Figure 5B). Following negative selection, 99.5% of the infected cells with 10,000 vps / cell of purified Ads from 293-Cre+Bxbl cells exclusively expressed mGreenLantern and were negative for mTagBFP2. In addition, a control adenoviral vector was generated with the same mGreenLantern integration arrangement using the traditional cloning method in bacteria. The reporter signals from this control vector were comparable to those obtained with the FastAd system (Figure 5B).

[0166] Furthermore, to obtain more quantitative measurements of mGreenLantern integration efficiency and compare the purity of the Ads, viral DNA was purified from each group for Sanger sequencing to assess the distribution of integrated and non-integrated Ads (Figure 5C). Ads in the integration steps that grew in 293 or 293-Cre cells displayed Sanger sequencing peaks identical to the reference non-integrated substrate Ad. Without negative selection, only about 5-14% of Ads in the pool were integrated from 293-Cre+Bxbl cells (Figure 5D). However, negative selection after integration in 293-Cre+Bxbl cells completely shifted the population to mGreenLantern-integrated Ads, underscoring the involvement of negative selection in removing non-integrated Ads. Meanwhile, qPCR analysis of purified viral DNA confirmed the amount of amplified mGreenLantern copies was 1000-fold higher than the amount from 293-Cre cells or 10-fold higher than from 293-Cre-Bxbl cells without negative selectin (Figure 5E). The low presence of the loxP-FRT-attP cassette after the negative selection was also confirmed (Figure 5F).

[0167] Expedite the Production Time of Recombinant Ads by FastAd System

[0168] Having established an in vivo Ad cloning by the FastAd system within 5 passages in 293-FLP negative selection cells, the number of passages striking a balance between Ad purity and production time was determined. The Ad population in 293-Cre+Bxbl cells was about 5-14% of integrated Ads. In the first passage of the 293-FLP negative selection cell, although the packaging signals of non-integrated Ads were cleaved, this 86 - 95 % of nonintegrated Ads still replicated and competed for the resource within cells for amplification of DNA, leading to a low number of integrated Ads that were packaged. Expedited Ad production was tested by growing Ads in one passage of integration and 2 passages for the negative selection. The whole production took 10 days from beginning to end (Figure 6A). The system was tested by incorporating an immune-stimulatory gene, murine OX40L (rnOX40L), into an oncolytic CRAd6 backbone. The yield was IxlO12vp, which is enough for 100 doses in oncolytic studies. Less than 0.08% of the original substrate Ad (loxP-FRT- attP cassette) was found in the final purified Ad (Figure 6B), and expression of mOX40L in A549 cells was confirmed (Figure 6C).

[0169] Generation of Fast Ad Donors with PCR

[0170] To further simplify the generation of GOI donors and to remove the need for any cloning steps in bacteria, PCR approaches were used to generate the GOI DNA fragments flanked by FastAd integration recombinase recognition sites (loxP-GOLattB-loxP) with primer overhangs (Figure 7A). It was found the mGreenLantern target gene could be amplified by 1-step PCR amplification with primer sets having loxP site and attB-loxP site overhangs by iProof DNA polymerase in GC buffer and 3% DMSO (Figure 7B), while severe primer dimers were observed using Platinum SuperFi I Polymerase even with optimized annealing temperature (Figure 8). By transfecting the PCR-amplified loxP-mG- attB-loxP fragment into 293-Cre+Bxbl cells, it was also observed that at least 45% total shift of infected cells to the mGreenLantern channel (Figure 7C).

[0171] A two-step PCR method that reduced primer dimer problems caused by full complementary 34-bp loxP sites on the forward and the reverse primer was also developed (Figure 7D). By separately PCR amplifying GOI with only one primer containing loxP sites at a time, influenza NP protein was successfully amplified with FastAd integration adaptors by Platinum SuperFi I Polymerase (Figure 7E). The NP PCR product was transfected into 293-Cre-Bxb l-pIIIa cells for integration into a single-cycle format of FastAd substrate (SCAd6- FRT- -E 1 A-CMV-loxP-FRT-attP-bGHpolyA-E l B-AIIIa-AE3) and later it was passaged in 293-FLP-pIIIa cells for negative selection. After purification, the NP protein expression was confirmed by western blot (Figure 7F), and the non-integrated substrate Ads were lower than 2% than the original substrate Ad stock (Figure 7G).

[0172] Overall, these data indicate that a PCR approach can be used to quickly generate DNA donors to accelerate the speed of recombinant adenovirus production. Evaluation of Complex Adenoviral Libraries Generated by FastAd system with Next Generation Sequencing (NGS)

[0173] Finally, the efficiency of the FastAd system for generating adenoviral libraries was assessed. To accomplish this, the previously described one-step PCR method was employed to amplify an oligo template containing random 15-mer nucleotides (Figure 9A). Following purification of the PCR products, the PCR products were transfected into a T25 flask of 293- Cre+Bxbl cells and subsequently amplified in 293-FLP cells. Later, the 15-mer region was amplified from the purified viral DNA and submitted for next-generation sequencing. The sample yielded a total of 3.15039e7 reads, with 2.6899e+7 (85.4%) reads successfully matching the random 15-mer sequences (Figure 9B). It is important to note that some unmatched sequences were attributed to the presence of spiked-in PhiX phage genome sequences during NGS, intended to enhance sequencing quality. Transfection on a T25 flask scale yielded 3.44039e6 unique sequences (Figure 9B), making it by far the largest adenoviral library confirmed by NGS. Most of the unique sequences had fewer than 10 reads in the library (Figure 9C). While the sequence logo data suggested a potential AT -bias in the initial oligo template (Figure 9D), which is a common issue for random nucleotide synthesis due to the varying coupling efficiencies of different nucleotides, an AT-bias in the library can only lead to an underestimation of its library size, rather than an overestimation.

[0174] To illustrate in another example, the two-step PCR method was employed to transfer the GeCKo v2 human genome-wide knockout gRNA library from lentiviral vectors into the adenoviral backbone (Figure 9E). To highlight the rapid procedure to construct the adenoviral library and the achievement of a high titer of viruses, the PCR products were transfected into 6X T225 flasks and cells were infected with substrate Ad, followed by only 2 passages of viruses in 293-FLP cells for CsCl purification (Figure 9F). By performing NGS on both the lentiviral gRNA library (PCR template) and the Ad-gRNA library, the results demonstrate that the Ad-gRNA library captured 99.9866% of the gRNAs present in the original PCR template (Figure 9G). Furthermore, the distribution of unique reads was nearly identical between the original lentiviral vector PCR template and the Ad-gRNA library (Figure 9H). The majority of the corresponding unique gRNA number ratio (unique reads # of Ad-gRNA library / unique reads # of PCR template) is near 1 (Figure 91), indicating an unbiased transfer of gRNA library to Ad backbone by the FastAd system. These data highlighted the valuable utility of the FastAd system for inserting existing or novel libraries into adenoviral vectors, facilitating functional selection or direct evolution library construction.

[0175] Example 2: FastAd: A Versatile Toolkit for Rapid Generation of Single Adenoviruses or Diverse Adenoviral Vector Libraries

[0176] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[0177] MATERIAL AND METHODS

[0178] Construction of Donor DNA Plasmids and Receiver Adenoviruses

[0179] The Bxbl recombinase GA-mutant variant sites were utilized for the attachment of bacteria (attB) site and the attachment of phage (attP) site (Jusiak et al., ACS Synth Biol, 8: 16-24 (2019)). A donor plasmid DNA was generated containing the cDNA of green fluorescent protein (GFP), mGreenLantem (Campbell et al., Proc Natl Acad Sci USA, 117: 30710-30721 (2020)) and an attB site flanked by two loxP sites (pUC57-loxP- mGreenLantern-attB-loxP) (Supplementary Sequences). Similar donor plasmids were constructed with other transgenes including murine OX40L (mOX40L), murine CD40L (mCD40L) and influenza nucleoprotein (NP).

[0180] A series of receiver Ad plasmid were generated to produce replication-competent Ad (RC-Ad), single-cycle Ad (SC-Ad) and conditionally-replicating Ads (CRAds). For example, RC-Ad6-FRT-'P-ElA-CMV-loxP-FRT-attP-mTagBFP2-bGHpolyA-ElB-AE3, was derived from the RC-Ad6-AE3 Ad vector (34). An FRT site was introduced on the left side of its packaging signal (T) with an FRT-Zeocin-FRT cassette via red recombination (Campos & Barry, Hum. Gene Ther., 15: 1125-1130 (2004)). This plasmid was transformed into FLP- expressing competent cells (DH5 / pCP20) to excise the Zeocin selection marker (Campos & Barry, Hum. Gene Ther., 15: 1125-1130 (2004)). Next, RC-Ad6-FRT-T-ElA-CMV-loxP- FRT-attP-mTagBFP2-bGHpolyA-ElB receiver Ad plasmid (RC-Ad6-mTagBFP2) was created by red recombination with a synthetic CMV-loxP-FRT-attP-mTagBFP2-bGHpolyA- ICeuI-Zeo-ICeuI DNA fragment (Supplementary Sequences). This Zeocin cassette was eliminated from the Ad plasmid DNA via ICeuI digestion and self-ligation of the Ad plasmid.

[0181] Additional formats of receiver Ad vectors were generated using similar methodologies: RC-Ad6-FRT-T'-ElA-loxP-FRT-attP-bGHpolyA-ElB-AE3-RSV- mGreenLantern (RC-Ad6-empty-RSV-mGreenLantem), CRAd6-FRT-'P-El A-dl 101 / dl 107- CMV-loxP-FRT-attP-bGHpolyA-ElB-AE3 (CRAd6-CMV) (Doronin et al., J. Virol., 74:6147-6155 (2000)) and SC-Ad6- FRT-'P-ElA-CMV-loxP-FRT-attP-bGHpolyA-ElB- AIIIa-AE3 (SC-Ad6-CMV) (Crosby & Barry, Virology, 462-463: 158-165 (2014)).

[0182] Rescue of Receiver Ads Using Plasmid DNA

[0183] Ad plasmid DNA was linearized by the restriction enzyme AsiSI (New England Biolabs) at 37 °C for 4 hours. 5 pg of this linearized DNA was transfected into a T25 flask grown with 293 cells at 95% confluency using Lipofectamine™ 3000 (Invitrogen), following the manufacturer’s protocol. After an initial viral rescue period of 2 weeks, cell lysates were generated by three freeze-thaw cycles in a dry ice-ethanol bath and the crude viruses were serially passaged in 293 cells for RC-Ad6-based backbones, in A549 cells for the CRAd6- based backbone, or in 293-IIIa cells for SC-Ad6-based backbones.

[0184] Purification of Recombinant Ads via CsCl Gradients

[0185] Large scale receiver viruses were purified from approximately 109cells in 10-layer CellSTACK culture chambers (Coming). These cell pellets were resuspended in 7 m of 20 mM HEPES buffer at pH 7.4 and lysed with 2 mb of 5% sodium deoxycholate for 30 minutes at room temperature, followed by treatment with 150 pl of DNAse I (10 mg / mL) (Sigma- Aldrich), 15 pL of PureLink™ RNAse A (10 mg / mL) (Invitrogen), and 170 pL of 2M MgCh for an additional 30 minutes at 37°C. After centrifugation at 3000 xg for 15 minutes at 4°C, the crude supernatants were subjected to two subsequent CsCl gradients ultracentrifugation runs at 26,000 xg. Next, the purified viruses were desalted with PD-10 desalting columns (Cytiva) in sucrose buffer (0.5 M sucrose, 20 mM HEPES). The estimated concentration of viral particles (vp) per mL was determined by measuring the OD260 using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific). Plasmids for Generating Stable Cell Lines

[0186] Plasmids required for stable transfection (pUC57-EFla-NLS-Cre-IRES-BSD, pUC57-EFla-HA-Bxbl-IRES-Neo, and pUC57-EFla-FLP-IRES-BSD) were synthesized by the Custom Gene Synthesis Service (GenScript).

[0187] The plasmids pLV-EFla-IRES-Puro (Addgene plasmid # 85132) and pLV-EFla- IRES-Blast (Addgene plasmid # 85133) were obtained from Dr. Tobias Meyer’s lab. The plasmid pLV-EFl-FLP-PGK-Neo was obtained from Dr. Javier Alcudia’s lab (Addgene plasmid # 108544). Additionally, the plasmid pCAG-NLS-HA-Bxbl (Addgene plasmid # 51271) was obtained from Dr. Pawel Pelczar’s lab, and lentiCas9-Blast (Addgene plasmid # 52962) was obtained from Dr. Feng Zhang’s lab.

[0188] The NLS-HA-Bxb 1 fragment was PCR amplified and subsequently inserted into the lentiviral plasmids pLV-EFla-IRES-Puro and pLV-EFla-IRES-Blast, resulting in the generation of the pLV-EFla-NLS-HA-Bxbl-IRES-Puro and pLV-EFla-NLS-HA-Bxbl- IRES-Blast lentiviral plasmids, respectively. Furthermore, the plasmid pLV-EFla-pIIIa- IRES-Puro was created by PCR amplification of the human Ad serotype 6 pllla gene, followed by its insertion into the pLV-EFla-IRES-Puro vector.

[0189] Production of Lentiviruses

[0190] Lentiviruses were generated through co-transfection of 1 pg of pMD.G, 1.5 pg pGGW, and 2.5 pg of the pLV-EFla lentiviral vector into 293 cells in T25 flasks using Lipofectamine™ 3000 reagent. 16-hours incubation post-transfection, the media containing the transfection reagents were replaced with fresh Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) containing 10% (v / v) Fetal Bovine Serum (FBS) and IX Penicillin- Streptomycin (Gibco). Lentivirus-containing supernatants were harvested at the 48-hour time point post-transfection and syringe filtered through a PVDF membrane with a pore size of 0.45 pm.

[0191] Cell Lines

[0192] Human 293 cells were procured from Microbix (Toronto, Ontario, Canada). Human A549 lung carcinoma and Human HeLa cervical carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The 293-Cre (116) cell line, a derivative of the 293 cell line expressing Cre recombinase, was provided by Dr. Philip Ng (Palmer & Ng, Mol. Ther., 8:846-852 (2003)). The 293-Cre+Bxbl cell line was generated by lentiviral transduction of 293-Cre cells with LV-EFla-NLS-HA-Bxbl-IRES-Puro. 293-Cre+Bxbl-pIIIa cell line was generated by transducing 293-Cre cells sequentially with the LV-EFla-NLS-HA-Bxbl-IRES-Blast and LV-EFla-pIIIa-IRES-Puro lentiviruses. The 293-FLP cell line was created by lentiviral transduction of 293 cells with LV-EFl-FLP-PGK-Neo lentivirus. 293FLPo-pIIIa cell line was derived by transducing the 293FLPo cell line with the pLV-EFla-pIIIa-IRES-Puro lentivirus.

[0193] The A549-Cre+Bxbl cell line was generated through sequential transfection of linearized pUC57-EFla-NLS-Cre-IRES-BSD and pUC57-EFla-HA-Bxbl-IRES-Neo plasmids. The A549-FLP cell line was constructed by transfection of linearized pUC57- EFla-FLP-IRES-BSD plasmid. Control A549 cells were transduced with the LV-EFla- IRES-Blast construct. The A549-SpCas9 cell line was established by lentiviral transduction with lentiCas9-Blast.

[0194] All stable cell lines were selected using 1 pg / ml of puromycin, 10 pg / mL of blasticidin, or 400 pg / mL of G418, followed by single-cell cloning using the limiting dilution method.

[0195] All cell lines were cultured in complete growth medium consisting of DMEM supplemented with 10% (v / v) heat-inactivated FBS and IX penicillin-streptomycin.

[0196] Evaluation of Fast Ad Efficiency Using Flow Cytometry

[0197] The efficiency and dynamics of FastAd was assessed with two fluorescent proteins provided from receiver Ad RC-Ad6-mTagBFP2 and from an mGreenLantern donor plasmid. Cells were seeded in a T25 flask at 95% confluency and 5 pg of the donor plasmid pUC57- loxP-mGreenLantern-attB-loxP was transfected into the cells using Lipofectamine as per the manufacturer’s instructions. 16 hours later, the cells were infected with the receiver virus, RC-Ad6-mTagBFP2, at a multiplicity of infection (MOI) of 1,000 vp / cell. Cells were harvested at 48 hours post-infection and fixed in 2% (w / v) paraformaldehyde in Phosphate Buffered Saline (PBS) (Coming). The fixed cells were analyzed for their mGreenLantern and mTagBFP2 fluorescence by flow cytometry using a LSR II Flow Cytometer (BD). To assess negative selection by FLP, viruses from infected 293-Cre-Bxbl cells were harvested at 72 hours post-infection and subjected to three freeze-thaw cycles. This lysate was used to infect 293-FLP cells, and after one passage of 48 hours, these cells were analyzed as described.

[0198] Real-Time PCR (Quantitative PCR, qPCR)

[0199] Genomic DNA (gDNA) was extracted from cells using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Ad viral DNA (vDNA) was isolated using the PureLink™ Viral RNA / DNA Mini Kit (Invitrogen). SYBR™ Green PCR Master Mix (Applied Biosystems) was used for all qPCR experiments with QuantStudio™ 3 Real- Time PCR System (Applied Biosystems). 4 ng of gDNA or vDNA were used as input template for each 20 pL qPCR reaction. The cycling condition: initial denaturation of templates with 95 °C for 10 minutes, followed by 40 cycles of 95 °C denaturation for 15 seconds and 60 °C annealing and extension for 45 seconds. AACt method was used for relative quantification by using hexon gene as the reference gene. To discern non-integrated receiver Ads from integrated Ads, qPCR oligonucleotides (Thermo Fisher Scientific) were synthesized to amplify the loxP-FRT-attP region, which is exclusively present in the original receiver Ads. To differentiate transfected mGreenLantern donor plasmids and mGreenLantern fragments amplified by viral replication from crude gDNA, Dpnl restriction enzyme was used to specifically digest methylated bacterial plasmid DNA while sparing unmethylated adenoviral DNA. 60 units of Dpnl were added to 3 pg of gDNA for 2 hours at 37°C. A qPCR primer set targeting the region containing two Dpnl sites within the mGreenLantern cDNA was used for qPCR.

[0200] Sanger Sequencing to Evaluate Integration Efficiency

[0201] Sanger sequencing was performed using the CMV primer (Genewiz / Azenta), allowing for sequencing of modified regions downstream the CMV promoter on Ad genomes. The analysis of the percentage of original receiver Ad and integrated Ad was conducted using BEAT (Xu et al., CRISPR J., 2:223-229 (2019)). Generation of Individual Transgene DNA Donors or Pooled Library Donors by PCR 5’-loxP-overhang and 3’-attB-loxP overhangs were generated by a one-step PCR for mGreenLantern, 15 random-mer (N15) library (custom-oligo synthesis by Thermo Fisher Scientific), and 17 random-mer (N17) library (custom-oligo synthesis by Integrated DNA Technologies, IDT) using iProof™ High-Fidelity DNA Polymerase (Bio-Rad) in GC buffer supplemented with 3% DMSO. To address primer dimer issues associated with loxP on the oligos when using general PCR reagents (Platinum™ SuperFi™ PCR Master Mix, Invitrogen), a two-step PCR method was implemented. The PCR template, RD-Ad5-NP was obtained from Dr. Richard Vile’s lab and GecKO v2 human gRNA library (Addgene pooled Library #1000000049) was obtained from Dr. Feng Zhang’s lab (Sanjana et al., Nat. Methods, 11 :783-784 (2014)).

[0202] Detection of Transgene Expression by Flow Cytometry

[0203] The expression of mGreenLantern, mOX40L, and mCD40L from CRAd6 was assessed in A549 cells at 48 hours post-infection with 1000 vp / cell. Following infection, cells were harvested and fixed using a 2% paraformaldehyde PBS solution. For surface markers, cells were directly stained with anti-mOX40L-PE (clone: RM134L) and anti-mCD40L-APC (clone: MR1) antibodies (BioLegend). The samples were analyzed by BD LSR II Flow Cytometer (BD).

[0204] Detection of Transgene Expression by Western Blot

[0205] A549 cells infected with 1000 vp / cell of SC-Ad6-NP were harvested at 48 hours postinfection. Following this, cells were lysed using RIPA buffer, and the protein concentration of collected cell lysates were quantified using Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific) by following manufacturer’s protocol. Western blot analysis was performed using anti -Influenza NP antibody (#PA5-32242, Invitrogen) and anti-P-Actin- HRP antibody (sc-47778, Santa Cruz Biotechnology) for probing.

[0206] Workflow for Producing Recombinant Ads using FastAd

[0207] For the 18-day production process, 293-Cre+Bxbl cells in a T25 flask at 95% confluency were transfected with 5 pg of donor DNA plasmids or PCR products. At 16 hours post-transfection, cells were infected with 1,000 vps / cell of receiver RC-Ad6-mTagBFP2. After 3 days post-infection, cells were harvested, and freeze-thawed cell lysates were passaged onto 293-FLP cells. The culture was then amplified through a total of five passages, with each passage incubated for 72 hours. This process was repeated to scale up to a 10-layer CellStack culture chamber (6,360 cm2). For SC-Ad6-based receiver Ads, 293-Cre+Bxbl- pllla and 293-FLP-pIIIa were used. For CRAd6-based receiver in A549-based cell lines, the process is identical except for 2-3 more rounds of amplification required in A549-FLP cells.

[0208] For the expedited 9-10-day production, 293-Cre+Bxbl cells in three T225 flasks at 95% confluency were transfected with 45 pg of donor DNA plasmids or PCR products per flask. At 8 hours post-transfection, cells were subsequently infected with 1,000 vps / cell of receiver CRAd6-CMV or RC-Ad6-empty-RSV-mGreenLantem. Then, after 48-72 hours of incubation, 293-Cre+Bxbl cells were harvested, freeze-thawed three times, and then passaged onto 293-FLP cells for two more rounds of negative selection and amplified to a scale of 10-layer CellStack culture chamber.

[0209] Evolution of Ad-gRNA Library in A549-SpCas9 cells

[0210] To select Ads carrying gRNA targeting host restriction factors, four T225 flasks of A549-Ctrl or A549-SpCas9 cells were infected with the Ad-gRNA library at a dose of 50 vps / cell. After 96 hours post-infection, cells were harvested and resuspended in 40 mL of 20 mM HEPES buffer (pH 7.4) and subjected to three freeze-thaw cycles. Following centrifugation at 3000 x g for 5 minutes, 1 mL of 1:40 diluted supernatant was used to infect the next four T225 flasks. This process was repeated up to passage 9, with subsequent infections of six T225 flasks. At passage 10, a 10-layer CellStack culture chamber was infected to allow for CsCl gradient purification.

[0211] Next Generation Sequencing (NGS) and Data Analysis

[0212] Pooled random 15-mer (N15) and 17-mer (N17) sequences, as well as GecKO human gRNA libraries, in the Ad backbones were PCR amplified using iProof™ High-Fidelity DNA Polymerase (Bio-Rad) in GC buffer with 3% DMSO. The primer sets specifically recognize Ad backbones to ensure the fragments are integrated. Library preparation and DNA fragmentation were processed by Genewiz / Azenta. The libraries were sequenced using the Illumina NovaSeq 6000 platform (2x150 bp), yielding an estimated 2.45 million paired- end reads per library for the amplicon.

[0213] For analyzing NGS data obtained from random nucleotide barcoded N15 and N17 libraries, custom Python scripts were developed for the analysis. Additionally, for gRNA library selection analysis, we utilized the Model-based Analysis of Genome-wide CRISPR- Cas9 Knockout (MAGeCK) method (Li et al., Genome Biol., 15:554 (2014)). Gene Ontology (GO)-term enrichment was conducted by PANTHER (Thomas et al., Protein Sci, 31 : 8-22 (2022); and Mi et al., Nucleic Acids Res., 47:D419-D426 (2019)).

[0214] Delta Visualization and Statistics

[0215] General graphing and statistical analyses were performed by GraphPad Prism 10 (GraphPad Software). Flow cytometry data was analyzed and visualized using FlowJo version 10.10.0 (FlowJo LLC). Sanger sequencing results were visualized by SnapGene software (World Wide Web at “snapgene” dot “com”). Sequence Logo for DNA nucleotide distributions within N15 and N17 randomers were generated in Python.

[0216] RESULTS

[0217] FastAd Strategy

[0218] Fast Ad was designed to circumvent the laborious and time-consuming process of cloning / rescuing adenoviral vectors. This method facilitates the direct insertion of recombinant DNA into TP-intact infectious receiver Ads within infected mammalian cells using Bxbl, Cre, and FLP recombinases. While Cre mediates recombination between loxP sites and FLP acts on FRT sites, Bxbl, a large serine recombinase, recognizes minimal 48-bp attP (attachment Phage) and 38-bp attB (attachment Bacterial) sites. Unlike the other two recombinases, Bxbl mediates irreversible and unidirectional integration of donor DNA (Ghosh et al., Mol. Cell, 12: 1101-1111 (2003); and Merrick et al., ACS Synth. Biol., 7:299- 310 (2018)), thus promoting the formation of integrated Ads, which contrasts with the reversible nature of the Cre / loxP system.

[0219] Receiver Ads were designed to contain a landing site for recombination of incoming donor DNA containing a loxP, FRT, and an attP site (Figure 12A). To negatively select against Ads that escape incoming donor DNA, a second FRT site was placed at the 5’ end of the adenoviral packaging signal (T). DNA donor shuttles contain a gene of interest (GOI) flanked by one LoxP sites and one attB site (Figure 12B). Producer cells were generated to express Cre and Bxbl or FLP to act upon these cassettes (Figure 12C).

[0220] FastAd recombination is initiated by transfection of donor DNA followed by infection with the receiver Ad in the Cre and Bxbl expressing cells (Figure 12C). When the Bxbl -mediated DNA integration events take place, integrated Ads enter an intermediate state wherein the second FRT site (towards the 3’ end of the Ad genomes) is flanked by two loxP sites. The recombinase activity of Cre favors deleting this second FRT site on the genome (Figure 12D). Successfully recombined viruses that are passaged onto FRT cells are unaffected and continue to grow. In contrast, if no GOI is inserted, FLP will delete the Ad packaging signal (T), generating a viral genome that cannot be packaged or propagated (Figure 12E). Viruses are then serially amplified in the FLP-expressing cell line to eliminate all non-integrated receiver viruses containing two FRT sites.

[0221] Testing and Benchmarking the Efficiency of FastAd

[0222] As a proof-of-concept, a receiver Ad backbone (RC-Ad6-mTagBFP2) was constructed with an FRT site to the left of and, at the same time, incorporated a FastAd integration cassette between the Ad E1A and E1B genes. This cassette consists of a CMV promoter, loxP-FRT-attP sites, an mTagBFP2 blue fluorescent protein reporter gene, and a bovine growth hormone (bGH) polyadenylation signal (Figure 13A). For donor DNA, a plasmid was constructed containing a mGreenLantern GFP reporter gene and an attB site, both flanked by two loxP sites (Figure 13B).

[0223] When the receiver Ads enter FLP-expressing cells, , El A gene, and CMV promoter responsible for driving mTagBFP2 expression, will be deleted (Figure 13C). Therefore, loss of BFP expression marks the elimination of from Ads that escape the initial recombination. On the other hand, when mGreenLantern donor DNA plasmids are provided into the Cre and Bxbl -expressing producer cell line, the backbone sequences of donor DNA plasmid (antibiotic resistance gene and origin of replication) that are flanked by two loxP sites will be removed by Cre. This process yields a ready-to-integrate, circular loxP-mGreenLantern-attB donor DNA fragment (Figure 13D). When this circular DNA donor fragment is inserted into the receiver Ad backbones by Bxbl, this integration generates an intermediate Ad with the second FRT site flanked by two loxP sites. The second FRT site is subsequently eliminated by Cre, resulting in the desired GFP-expressing recombinant Ad that resists FLP (Figure 13D).

[0224] Alternatively, at lower efficiency, the donor DNA can also undergo integration by Cre, followed by Bxbl cleaving the FRT sites with the attP / attB pair. Because of this alternative integration pathway, one can not only evaluate the efficiency of the FastAd system by assessing the intensity and percentage of mTagBFP2 and mGreenLantern in infected cells, but also compare its efficiency to that only mediated by the Cre / loxP Ad integration system (Yamamoto et al., Mol. Pharm.. 11 : 1069-1074 (2014)).

[0225] To assess the efficiency of negative selection, 293 and 293-FLP cells were infected with 1,000 viral particles (vps) per cell of the receiver virus, RC-Ad6-mTagBFP2. After 48 hours, it was observed that 99.6% of the 293 cells were positive for BFP, while only 1.34% of 293-FLP were BFP-positive, emphasizing the negative selection efficiency of non-inserted receiver Ads (Figure 14A).

[0226] To test the efficiency of donor DNA insertion 293, 293-Cre, and 293 -Cre+Bxbl cell lines were transfected with or without mGreenLantern donor plasmids. 16 hours later, these cells were infected with receiver RC-Ad6-mTagBFP2 or left untreated. Flow cytometry analysis 48 hours after infection demonstrated a substantial increase in GFP-positive cells to 66.6% in 293-Cre+Bxbl cells (Figure 14C). In contrast, 293-Cre had a less substantial increase in GFP positivity (35.24%). The negative control 293 cells displayed only minimal GFP fluorescence, representing the background signal (7.93% total) (Figure 14C). Significantly, a distinct population exclusively expressing mGreenLantern (30.3%) was observed in the 293-Cre+Bxbl producer cells, a percentage markedly higher than that observed in the 293-Cre (2.54%) and 293 cells (0.19%) (Figure 14C). This observation indicates that the mGreenLantern DNA fragments have successfully been integrated into receiver Ads even before the transcription of mTagBFP2 from the backbone started.

[0227] In the negative selection scenario, it is crucial to recognize that Cre alone lacks the capability to remove the second FRT site from the receiver Ad backbone. When 293-FLP cells were infected with the mixed Ad population produced from 293-Cre cells, there was a notable decrease in both mTagBFP2 and mGreenLantern when compared to infection in 293 cells (Figure 14D). In contrast, when 293-FLP were infected with the Ad population derived from infected 293-Cre+Bxbl cells, no BFP signal from the non-integrated receiver virus was observed, and only mGreenLantern expression from integrated Ads was detectable (Figure 14E). These findings suggest that genes of interest are efficiently knocked into the receiver virus in 293-Cre-Bxbl cells, while elimination of non-integrated receiver viruses is reliably achieved in 293-FLP cells.

[0228] Flow cytometry assesses the efficiency of transgene incorporation functionally, but it does not provide information on the state of the recombination site at the DNA level. To address this, genomic DNA (gDNA) from the transfected / infected cells were analyzed by qPCR to detect mGreenLantern DNA fragments. To distinguish the amplified mGreenLantern DNA fragments in Ad genomes from those in transfected donor plasmids, the qPCR amplicon was designed to comprise two Dpnl restriction endonuclease sites. When this DNA is treated with Dpnl, methylated DNA derived from bacteria (Wilson, V.G. (2012) Cell culture assay for transient replication of human and animal papillomaviruses. Curr. Protoc. Microbiol., Chapter 14, Unitl4B 11) is digested, while unmethylated viral and mammalian DNA stays intact (Figure 14F). By this approach, a 50-fold increase in the number of mGreenLantern copies within Ad genomes was observed in 293-Cre+Bxbl cells when compared to 293-Cre cells (Figure 14G). Despite the moderate mGreenLantern expression observed in flow cytometry from Cre-only cells (Figure 14C), only minimal amounts of mGreenLantern DNA were amplified from these cells (Figure 14G). This implies that these fragments were primarily shuffling between Ad genomes without undergoing viral replication in 293-Cre cells. These results also revealed that FLP increased the ratio of mGreenLantern DNA copies to Ad hexon DNA copies in 293-Cre+Bxbl cells, emphasizing the efficient removal of non-integrated Ads by negative selection with FLP (Figure 14H).

[0229] Evaluating the Purity of Recombinant Ads Produced by the Fast Ad System

[0230] To evaluate the purity of the final recombinant Ad preps from the FastAd system, the viruses were amplified through five passages in either 293 cells or 293-FLP cells until reaching the scale of a 10-layer CellStack culture chamber (6,360 cm2) over the course of 18 days. These viruses were purified on CsCl gradients. A549 cells were infected at varying MOIs (50 vp / cell, 1,000 vp / cell, and 10,000 vp / cell) of the purified Ads and flow cytometry was performed to detect BFP and GFP (Figure 15A). Consistent with earlier findings, Ads grown in 293-Cre-Bxbl cells had the highest mGreenLantem reporter activity when compared to other cells (Figure 15B). Importantly, 99.5% of cells infected with 10,000 vps / cell of purified Ads from the 293-Cre+Bxbl plus negation selection (Cre+Bxbl+FLP+) group were exclusively positive for GFP and negative for BFP. The level of GFP expression was similar to that evoked by a positive control Ad vector, where an identical mGreenLantem integration was achieved by traditional recombination in bacteria (Figure 15B).

[0231] To obtain more quantitative information on mGreenLantem integration efficiency and compare the distribution of integrated and non-integrated Ads in the purified vims stock, viral DNA was analyzed by Sanger sequencing (Figure 15C). Ads from Cre Bxbl’FLP" and Cre+Bxbl FLP' groups displayed sequencing peaks identical to the reference non-integrated receiver Ad, indicating no integration into the receiver Ad above the detection limit of Sanger sequencing. In contrast, 5-14% of Ads from the Cre+Bxbl+FLP' group contained mGreenLantem (Figure 15D) after decomposition of the Sanger sequencing result by BEAT software (Xu et al., CRISPR J., 2:223-229 (2019)). Significantly, the Ad population raised in the Cre+Bxbl+FLP+system completely shifted to mGreenLantern-integrated Ads (Figure 15C), highlighting the performance of the negative selection system in removing nonintegrated Ads. qPCR of viral DNA confirmed that the integration success-rate in the Cre+Bxbl+FLP+group was 10-fold higher than in viral DNA from Cre+Bxbl+FLP’ group and 1000-fold higher than in viral DNA from the Cre+Bxbl FLP‘ group (Figure 15E). The prevalence of receiver Ad sequences (loxP-FRT-attP cassette) after the negative selection was also confirmed to be extremely low (<0.0004%) in the Cre+Bxbl+FLP+group (Figure 15F), indicating the FastAd system can yield nearly pure recombinant vims after five negative selection passages. Flexible Forms of Donor DNA and Ad Backbones for the FastAd System

[0232] To further simplify the process for generating donor DNA and eliminate steps typically required for cloning of donor DNA plasmids in bacteria, oligos were designed with overhangs containing the recombinase recognition sites to form a donor DNA with the structure of loxP-GOLattB-loxP (Figure 16A). Initially, this approach generated problems with loxP primer dimers. This problem was avoided by amplification of the mGreenLantern fragment (loxP-mGreenLantern-attB-loxP) in a one-step PCR using iProof DNA polymerase in GC buffer supplemented with 3% DMSO (Figure 16B). When this PCR product was transfected as DNA donor into 293-Cre+Bxbl cells and co-infected with RC-Ad6- mTagBFP2, at least 45% of cells expressed mGreenLantern (Figure 16C). This indicated that a PCR product could serve as a FastAd DNA donor.

[0233] A two-step PCR method was also developed to reduce the loxP primer dimer problem by separately PCR amplifying the GDI with only one loxP primer at a time (Figure 16D). For example, the influenza nucleoprotein (NP) cassette (loxP-NP-attB-loxP) was amplified by a two-step PCR with a standard PCR master mix (Platinum SuperFi PCR Master Mix) (Figure 16E). To demonstrate that FastAd can be used to engineer other types of Ad vectors, NP was knocked into a pIIIA-deleted single-cycle adenovirus (SC-Ad) (Crosby & Barry, Virology, 462-463: 158-165 (2014)). To do this, the PCR-amplified NP cassette was transfected into 293-Cre+Bxbl-pIIIa cells and then infected with a receiver virus (SC-Ad6-CMV). After five passages in 293-FLP-pIIIa cells for negative selection and CsCl gradient purification, SC- Ad6-NP was demonstrated to express influenza NP by western blot after infection of A549 cells (Figure 16F).

[0234] Conditionally-replicating adenoviruses (CRAds) are designed to infect and kill cancer cells while sparing normal cells. Some CRAds achieve this by having mutations like dll 101 / dl 107 in their El A protein to block interactions with pRB or p53 (Doronin et al., J. Virol., 74:6147-6155 (2000)). While CRAds can also be modified by FastAd, doing so in 293 cells risks recombination of wild type El, which is stably integrated into these cells’ genome, to replace the mutant El in the virus. To mitigate this risk, A549-Cre+Bxbl and A549-FLP cells were generated. FastAd was successfully applied in these cells to rescue CRAd6 with mGreenLantern (Figure 16G), OX40L (Figure 16H), and CD40L (Figure 161) transgenes. Each GOI donor was transfected into A549-Cre+Bxbl, rescued with a CRAd6 receiver virus followed by negative selection in A549-FLP cells (Figure 16G, 16H, and 161).

[0235] Rapid Production of Recombinant Ads by FastAd

[0236] In theory, increasing the number of passages in negative selection cells would lead to an increasingly higher purity of recombinant Ads, while taking more time to rescue the virus. In practice, achieving 100% purity in recombinant Ad preparations may not always be necessary for addressing certain scientific questions, especially with minor contamination by a receiver Ad lacking a transgene, therefore only accounting for negligible effects. After successfully producing high titer of recombinant Ads by the FastAd system within 18 days after five passages of negative selection in 293-based cell lines (Figure 15A), the next goal was to determine the optimal number of passages of negative selection to find the goldilocks zone between purity, yield, and production time.

[0237] Based on earlier results, 5-14% of GOI integrated viruses and 86-95 % of nonintegrated viruses were observed prior to FLP-dependent negative selection (Figure 15D). While the of non-integrated Ads can be eliminated upon passage into the FLP-expressing cell line, these -deleted Ad genomes can still replicate their DNA and compete for resources with the desired GOI integrated Ad during the first round of negative selection. This competition may lead to a reduced number of the desired recombinant Ad being packaged and hence result in lower viral titers. To circumvent this potential loss in a one- round negative selection, a minimum of two rounds of negative selection with FLP- expressing cells was selected and tested in a rescue of CRAd6 containing an OX40L transgene cassette. Integration and two rounds of negative selection shortened the time to produce large-scale CsCl-purified virus to 10 days from start with Donor DNA to finish (Figure 17A). Under this accelerated protocol, the total yield of CRAd6 was IxlO12vp after CsCl gradient purification, which equates to approximately 50-100 oncolytic virus doses for typical mouse studies (Doronin et al., J. Virol., 74:6147-6155 (2000)). CRAd6-mOX40L produced by this accelerated 10-day rescue protocol was 99.92% pure, with less than 0.08% of the original receiver Ads remaining in the final purified Ad preparation (Figure 17B). Flow cytometry analysis confirmed that CRAd6-mOX40L expressed mOX40L protein after infection of A549 cells (Figure 17C). This data demonstrates the ability to go from donor DNA to large amounts of purified recombinant adenovirus in only 10 days using the FastAd system.

[0238] Rapid Generation of Complex Adenoviral Libraries by FastAd

[0239] The ability of FastAd to rapidly generate complex genetic libraries in adenoviral vectors, a task unachievable following conventional protocols for Ad cloning and rescue, was next evaluated. To quantitatively assess the library size, oligonucleotides containing 17-mer random nucleotides (N17) with up to 1.718 x IO10variants were synthesized. One-step PCR was used to amplify the N17 sequences and to add FastAd recombinase recognition sites for integration. To benchmark the future Ad library, this PCR product was first subjected to Next-Generation Sequencing (NGS) to examine library diversity. (Figure 18A and 18B). Of the total 4.35 x io7output NGS reads, 3.95 x io7reads mapped to the N17 oligonucleotide, with 3.81 x io7reads occurring only once (Figure 18C). As expected, the N17 library had a high diversity, with 96.59% of all sequences being unique (Figure 18D), while maintaining an unbiased nucleotide distribution (Figure 18E).

[0240] This N17 PCR product library was transfected into a T25 flask of 293-Cre+Bxbl cells and infected with a receiver virus. This population was passaged 5 times in 293-FLP cells, and the virus was purified from a 10-layer CellStack on CsCl gradients. The 17-mer region was amplified from purified viral DNA from three independent amplifications and subjected to NGS analysis (Figure 18F and 18G). The average number of unique reads from the Ad-N17 libraries was 3.01 x 106from an average of 4.13x 107mapped reads (Figure 18H).

[0241] Although the diversity of N17 reads was reduced compared to the original PCR template (Figure 181), the nucleotide distribution remained unbiased after integration into Ad (Figure 18J). In another pilot study utilizing a 15-mer random library from a different vendor, comparable outcomes were observed.

[0242] This demonstrated that the process generated a highly diverse genetic library with approximately 1 unique viral clone generated in each cell of the T25 flask. This efficiency was in striking contrast with conventional Ad construction methods, that typically yield less than 50 clones per pg of transfected DNA (Elahi et al., Gene Ther., 9:1238-1246 (2002)).

[0243] Comprehensive Capture of a Genome -Wide CRISP R gRNA by Fast Ad

[0244] The GeCKO v2 human genome-wide knockout gRNA lentiviral library contains

[0245] 119,418 unique gRNAs (Sanjana et al., Nat. Methods, 11 :783-784 (2014)). This library was used as a template to generate a FastAd gRNA donor population by PCR (Figure 19A). This donor population was transfected into three T225 flasks of 293-Cre+Bxbl cells followed by infection with RC-Ad receiver virus and only 2 passages of viruses in 293-FLP cells (Figure 19B). By this approach, a FastAd gRNA library was generated in only 9 days. NGS analysis of the PCR-amplified gRNAs from the original lentiviral gRNA library and the newly raised Ad-gRNA library demonstrated that the Ad-gRNA library captured 99.97% of the original gRNAs (Table 11). Furthermore, the distribution of unique gRNA counts was nearly identical between the original lentiviral library and the Ad-gRNA library (Figure 19C) as evidenced by a Spearman’s rank correlation coefficient of 0.8529 between the gRNA library from lentiviral vectors and the Ad-gRNA library (where r = 1 indicates the highest positive correlation) (Figure 19D). Thus, a complex gRNA library was rapidly transferred into adenoviral vectors by FastAd.

[0246] Functional Selection of the FastAd gRNA Library to Identify Novel Ad-Host Interactions

[0247] Since the gRNA sequences were encoded by Ads within the Ad-gRNA library, it was expected that Ads carrying gRNAs targeting antiviral host genes will likely increase in their clone counts in SpCas9-expressing cells, as the suppressor function of host genes is ablated for better virus propagation (King et al., Cell Host Microbe, 31 : 1552-1567 el558 (2023)). This Ad-gRNA library was passaged in human A549 cells, as well as in A549 cells expressing SpCas9, to identify host genes that might inhibit Ad life cycle. Interestingly, gRNAs in the libraries, whether from A549-Ctrl or A549-SpCas9 cells, exhibited right- skewed distributions towards higher clone counts at the tenth passage (P10) (Figure 19F). Although the data shows that the distribution of Ad-gRNA library can drift overtime during passaging even without the expression of SpCas9, we indeed observed a low correlation (Spearman’s r = 0.4647) between the two libraries (Figure 19G), suggesting that expression of SpCas9 was altering the selective pressure on the gRNA population.

[0248] Table 11. Mapped gRNA sequences counts and coverage in lentiviral library and Ad library.

[0249] Total mapped Unique gRNA Percentage of Percentage of gRNA NGS sequences all unique gRNA reads (Depth) identified gRNA captured by sequences Fast Ad

[0250] Expected # of unique N / A 1.1946E+05 100.00% N / A gRNA gRNA in lentiviral 9.7071E+06 1.1942E+05 99.96% N / A vectors gRNA in Ad-gRNA 9.0973E+06 1.1938E+05 99.93% 99.97% library

[0251] Using the sample from A549-Ctrl cells as the control group and the sample from A549-SpCas9 cells as the experimental group in the MAGeCK analysis (Li et al., Genome Biol., 15:554 (2014)), several host genes were identified to be enriched in the A549-SpCas9 group (Figure 19H). Genes previously identified as restriction factors for Ads, including APOBEC3A (Gottig et al., mBio, 14:e0347822 (2023)), BMI1 (Na et al., Virology, 456- 457:227-237 (2014)), hnRNPA2Bl (Wang et al., Nuclear hnRNPA2Bl initiates and amplifies the innate immune response to DNA viruses. Science, 365 (2019)), RAD 18 (Lloyd et al., PLoS Pathog., 2:e40 (2016)), and STAT1 (Sohn & Hearing, J. Virol, 85:7555-7562 (2011)), were observed, suggesting that the library was properly sampling the genome space. While gRNAs against these previously reported Ad-interacting genes were found to be enriched, the top four targeted genes (with the lowest p-values and the highest fold changes in gRNA counts) were not previously reported to be associated with Ad restriction (Figure 19H). Among those unreported genes, ZNF500 has been documented to activate the p53- p21-E2F4 signaling axis (Ma et al., Cancer Sci., 114:4237-4251 (2023)), suggesting that knocking out the ZNF500 gene could potentially suppress p53 and enhance cellular proliferation, thereby promoting more robust Ad replication (Li et al., J. Virol., 85:7976-7988 (2011)). TDP1 is involved in DNA repair mechanisms in cells (Pommier et al., DNA Repair (Amst), 19: 114-129 (2014)). While the role of TDP1 in the Ad life cycle has not yet been specifically reported, DNA repair mechanisms such as the Mrel l-Rad50-NBSl (MRN) complex have been shown to inhibit Ad replication (Weitzman et al., Oncogene, 24:7686- 7696 (2005)). gRNAs against vacuolar protein sorting-associated protein 54 (VPS54) were also enriched. VPS54 is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in intracellular vesicle transport (Wei et al., Cell Rep., 19:2823- 2835 (2017)). While a direct relationship to Ad is unclear, it is possible that VPS54 could impact endosomal trafficking during the Ad life cycle. Finally, gRNAs targeting coiled-coil domain-containing 184 (CCDC184) were also highly enriched in the Ad gRNA library. At present, it is unclear what the function of CCDC184 is and how it might be related to Ad biology.

[0252] The top 565 gene target candidates that enriched in the A549-SpCas9 group were selected based on having a p-value smaller than 0.05 and a gRNA count fold change greater than 1.5. These were subjected to PANTHER GO-slim analysis for Gene Ontology (GO) term enrichment (Thomas et al., Protein Sci, 31 :8-22 (2022); and Mi et al., Nucleic Acids Res., 47:D419-D426 (2019)). Based on this analysis, enriched terms included immune system process (G0:0002376) and biological processes involved in interspecies interaction between organisms (G0:0044419) (Figure 191). Nine target gene candidates were identified in the biological processes involved in interspecies interaction between organisms category (G0:0044419) (Table 12), and some of them were not previously reported to be associated with Ads. Overall, these results demonstrate rapid production and utilization and application of an Ad-gRNA library by FastAd to uncover novel host restriction factors.

[0253] Table 12. gRNA targeted gene candidates in the biological processes involved in interspecies interaction between organisms (G0:0044419) in PANTHER GO-slim.

[0254] Gene Name Full Name Protein Function Role in Ad life cycle

[0255] DNA dC-dU-editing RNA processing Inhibition (Gottig et al.,

[0256] APOBEC3A enzyme APOBEC-3A factor mBio, 14:e0347822 (2023))

[0257] Interleukin

[0258] IL36G Interleukin-36 gamma superfamily Not yet described

[0259] No effect (Espert et al.,

[0260] J. Biol. ('hem.,

[0261] Interferon-stimulated gene 278: 16151-16158

[0262] ISG20 20 kDa protein Exoribonuclease (2003)

[0263] Inhibition or Enhancement (Pietrantoni et al., Antimicrob. Agents Chemother. , 47:2688- 2691 (2003); and Johansson et al., J.

[0264] Transfer / carrier Virol., 81 :954-963

[0265] LTF Lactotransferrin protein (2007))

[0266] Membrane

[0267] Regenerating islet-derived trafficking regulatory

[0268] REG3G protein 3 -gam ma protein Not yet described

[0269] Ret finger protein-like 4A- Ubiquitin-protein

[0270] RFPL4AL1 like protein 1 ligase Not yet described

[0271] Innate immune

[0272] Signal transducer and responses ((Sohn & activator of transcription 1- Rel homology Hearing, J. Virol,

[0273] STAT1 alpha_beta transcription factor 85:7555-7562 (2011))

[0274] Tripartite motif-containing Ubiquitin-protein

[0275] TRIM64B protein 64B ligase Not yet described Tripartite motif-containing Ubiquitin-protein

[0276] TRIM64C protein 64C ligase Not yet described

[0277] In conclusion, FastAd represents a significant advance in adenoviral vector construction, offering enhanced efficiency, speed, and versatility. It not only shortens virus production timelines to just 10 days or less while maintaining high purity and titer, but also facilitates the generation of complex genetic libraries with unique sequences at a millionscale for genetic research and screening.

[0278] Data Availability

[0279] The raw data for NGS have been deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) with the accession number PRJNA 1102000.

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[0295] 15. Schaack, J., Ho, W.Y., Freimuth, P. and Shenk, T. (1990) Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA. Genes Dev, 4, 1197-1208.

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[0297] 17. ALWassiti, H.A., Thomas, D.R., Wagstaff, K.M., Fabb, S.A., Jans, D.A., Johnston, A.P. and Pouton, C.W. (2021) Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus. Ini J Mol Sci, 22.

[0298] 18. Jones, N. and Shenk, T. (1978) Isolation of deletion and substitution mutants of adenovirus type 5. Cell, 13, 181-188.

[0299] 19. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C. and Saito, I. (1996) Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl AcadSci USA, 93, 1320-1324.

[0300] 20. Luo, J., Deng, Z.L., Luo, X., Tang, N., Song, W.X., Chen, J., Sharff, K.A., Luu, H.H., Haydon, R.C., Kinzler, K.W. et al. (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc, 2, 1236-1247.

[0301] 21. Wu, N., Zhang, H., Deng, F., Li, R., Zhang, W., Chen, X., Wen, S., Wang, N., Zhang, J., Yin, L. et al. (2014) Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in -293 packaging cells. Gene Ther, 21, 629-637.

[0302] 22. Planas, D., Staropoli, I., Michel, V., Lemoine, F., Donati, F., Prot, M., Porrot, F., Guivel-Benhassine, F., Jeyarajah, B., Brisebarre, A. et al. (2024) Distinct evolution of SARS- CoV-2 Omicron XBB and BA.2.86 / JN.1 lineages combining increased fitness and antibody evasion. Nat Commun, 15, 2254.

[0303] 23. Lupoid, S.E., Kudrolli, T.A., Chowdhury, W.H., Wu, P. and Rodriguez, R. (2007) A novel method for generating and screening peptides and libraries displayed on adenovirus fiber. Nucleic Acids Res, 35, el38. 24. Miura, Y., Yamasaki, S., Davydova, J., Brown, E., Aoki, K., Vickers, S. and Yamamoto, M. (2013) Infectivity-selective oncolytic adenovirus developed by high- throughput screening of adenovirus-formatted library. Mol Ther, 21, 139-148.

[0304] 25. Nov, Y. (2012) When second best is good enough: another probabilistic look at saturation mutagenesis. Appl Environ Microbiol, 78, 258-262.

[0305] 26. Hatanaka, K., Ohnami, S., Yoshida, K., Miura, Y., Aoyagi, K., Sasaki, H., Asaka, M., Terada, M., Yoshida, T. and Aoki, K. (2003) A simple and efficient method for constructing an adenoviral cDNA expression library. Mol Ther, 8, 158-166.

[0306] 27. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. and Phipps, M.L. (1997) Construction of adenovirus vectors through Cre-lox recombination. J Virol, 71, 1842-1849.

[0307] 28. Hillgenberg, M., Hofmann, C., Stadler, H. and Loser, P. (2006) High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries. J Virol, 80, 5435-5450.

[0308] 29. Ng, P., Beauchamp, C., Evelegh, C., Parks, R. and Graham, F.L. (2001) Development of a FLP / frt system for generating helper-dependent adenoviral vectors. Mol Ther, 3, 809- 815.

[0309] 30. Renaut, L., Bernard, C. and D'Halluin, J.C. (2002) A rapid and easy method for production and selection of recombinant adenovirus genomes. J Virol Methods, 100, 121- 131.

[0310] 31. Zeng, M., Smith, S.K., Siegel, F., Shi, Z., Van Kampen, K.R., Elmets, C.A. and Tang, D.C. (2001) AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination. Biotechniques, 31, 260-262.

[0311] 32. Wu, C., Nerurkar, V.R., Yanagihara, R. and Lu, Y. (2008) Effective modifications for improved homologous recombination and high-efficiency generation of recombinant adenovirus-based vectors. J Virol Methods, 153, 120-128.

[0312] 33. Yang, Y., Chi, Y., Tang, X., Ertl, H.C.J. and Zhou, D. (2016) Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly. Curr Protoc Mol Biol, 113, 16 26 11-16 26 18.

[0313] 34. Choi, E.W., Seen, D.S., Song, Y.B., Son, H.S., Jung, N.C., Huh, W.K., Hahn, J.S., Kim, K., Jeong, J.Y. and Lee, T.G. (2012) AdHTS: a high-throughput system for generating recombinant adenoviruses. J Biotechnol, 162, 246-252.

[0314] 35. Okada, T., Ramsey, W.J., Munir, J., Wildner, O. and Blaese, R.M. (1998) Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex. Nucleic Acids Res, 26, 1947-1950.

[0315] 36. Ni, N., Deng, F., He, F„ Wang, H., Shi, D , Liao, J., Zou, Y., Wang, H., Zhao, P., Hu, X. et al. (2021) A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology. Mol Ther Oncolytics, 23, 602-611.

[0316] 37. Robert, M.A., Zeng, Y., Raymond, B., Desfosse, L , Mairey, E., Tremblay, J.P., Massie, B. and Gilbert, R. (2012) Efficacy and site-specificity of adenoviral vector integration mediated by the phage phiC31 integrase. Hum Gene Ther Methods, 23, 393-407.

[0317] 38. Rosewell, A., Vetrini, F. and Ng, P. (2011) Helper-Dependent Adenoviral Vectors. J Genet Syndr Gene Ther, Suppl 5.

[0318] 39. Alba, R., Hearing, P., Bosch, A. and Chillon, M. (2007) Differential amplification of adenovirus vectors by flanking the packaging signal with attB / attP-PhiC31 sequences: implications for helper-dependent adenovirus production. Virology, 367, 51-58. 40. Durrant, M.G., Fanton, A., Tycko, J., Hinks, M., Chandrasekaran, S.S., Perry, N.T., Schaepe, J., Du, P.P., Lotfy, P., Bassik, M.C. et al. (2023) Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat Biotechnol, 41, 488-499.

[0319] Example 3: FastAd: A System for Rapid Generation of Single Adenoviruses or Complex Adenoviral Vector Libraries

[0320] Ads are potent in vitro and in vivo gene therapy vectors with uses in basic biology research and in translational applications as genetic vaccines, gene therapies, and oncolytic viruses. Traditional method (bacterial homologous recombination) for constructing and rescuing recombinant Ads are time-consuming and inefficient, with greater than two months production time and yielding less than 50 viral clones / pg of transfected recombinant DNA. Current methods cannot produce AD libraries with over one million unique clones, limiting their novel utility for both basic Ad biology and translational applications.

[0321] The design logic is shown in Figure 20.

[0322] FastAd was 100-fold more efficient than the Cre / loxP system, and yielded nearly pure Ads with negative selection. See, Figure 21.

[0323] Ad production time was shortened down to 10 days. See, Figure 22.

[0324] FastAd generated complex Ad libraries with over 3 million unique clones from just a T25 flask of transfection. See, Figure 23.

[0325] An exemlarly application of FastAd library: Ad-gRNA library for functional evolution is shown in Figure 24.

[0326] FastAd yields high-titer recombinant Ads in 10 days, significantly reducing production time compared to trasitional methods (greater than 2 months).

[0327] FastAd creates libraries with over 3 million unique clones, potentially the most complex libranry in Ad, confirmed by NGS.

[0328] Functional evolution using the Ad-gRNA library in A549-SpCas9 cells reveals novel host restriction factors, demonstrating the value of FastAd not only in applications of Ad vectors but also foundational Ad biology research. Example 4: FastAd: A System for Rapid Generation of Single Adenoviruses or Complex Adenoviral Vector Libraries

[0329] Validation of individual gene candidates from Ad-gRNA library by CRISPR / Cas9 knockout in A549 cells by reporter assay. A549 cells were transduced with lentiviral vectors carrying SpCas9 and gRNA targeting VPS54, MRPS15, C3orf62, FRZB, CYP2J2, B4GALNT3, ZNF500, C12orf68, RRRP7A, IQCF6, STAT3, RBMX, CYBB, RAET1L, ATP6V1V1, PTPRG, KLGL42, ATP2A2, CWNPW, KLHL9, METRN, FACE, TDP1, CARDIO and AAVS1 control. Individual cell lines were infected with 1000 vp / cells of SC- Ad6-GFP-Luc. 24 hours-post infection, luciferase activity was measured for evaluating Ad replication. See, Figure 25.

[0330] Validation of individual gene candidates from Ad-gRNA library by CRISPR / Cas9 knockout inA549 cells by titering Ad in each infected cell lines. Each gene candidates identified in Ad-gRNA evolution were knockout through lentiviral transduction. Pooled transduced cells were infected with RC-Ad6-mGreenLantern at 1000 vp / cells. After 48 hours post-infection, cell lysates were freeze-thawed and supernatant were titered by flow cytometry of newly infected A549 cells. See, Figure 26.

[0331] Confirmation ofVP54-KO cell clone produced higher viral titer than control cell lines. A549 cells transduced with lentiviral gRNA vector targeting AAVS1, C3orf62, VPS54 were single-cell-isolated and used to compare viral titer after infection as described by flow cytometry. See, Figure 27.

[0332] Confirmation ofVP54-KO cell clone produced higher viral titer than control cell lines. A549 cells transduced with lentiviral gRNA vector targeting AAVS1, C3orf62, VPS54 were single-cell-isolated and used to compare viral titer after infection. Large scale viral preparation with CsCl gradient purification demonstrated that VPS54-KO cells produced higher titer in Ad production. See, Figure 28. Example 5: Exemplary Sequences

[0333] Exemplary E1A sequences

[0334] E1A-13S polypeptide sequence (SEQ ID NO:60):

[0335] MRHI ICHGGVI TEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLDVTAPEDPNEEAV SQI FPESVMLAVQEGIDLFTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTC HEAGFPPSDDEDEEGEEFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSP VSEPEPEPEPEPEPARPTRRPKLVPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIHPWP LCPIKPVAVRVGGRRQAVECIEDLLNESGQPLDLSCKRPRP

[0336] E1A-12S polypeptide sequence (SEQ ID NO:61):

[0337] MRHI ICHGGVI TEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLDVTAPEDPNEEAV SQI FPESVMLAVQEGIDLFTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTC HEAGFPPSDDEDEEGPVSEPEPEPEPEPEPARPTRRPKLVPAILRRPTSPVSRECNSSTDSC DSGPSNTPPEIHPWPLCPIKPVAVRVGGRRQAVECIEDLLNESGQPLDLSCKRPRP

[0338] Nucleic acid encoding an El A polypeptide (SEQ ID NO:62):

[0339] TATTTATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCTC CTCCGAGCCGCTCCGACACCGGGACTGAAAATGAGACATATTATCTGCCACGGAGGTGTTAT TACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATC TTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACG GCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGAGTCTGTAATGTTGGC GGTGCAGGAAGGGATTGACTTATTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTC ACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAAC CTTGTGCCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGACGA GGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGT CTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATG AGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAAATTATGGGCAGTGGGTGATAGAGTGG TGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTTTGTA TTGTGATTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGC CTGCAAGACCTACCCGGCGTCCTAAATTGGTGCCTGCTATCCTGAGACGCCCGACATCACCT GTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCC TGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGC GTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGTCTGGGCAACCTTTGGACTTG AGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGGTTAACGCCTTT G T T T G C T GAAT GAG T T GAT G T AAG T T T AAT AAAG G G T GAGAT AAT G T T T A Exemplary E1B sequences

[0340] E1B-19K polypeptide sequence (SEQ ID NO:63):

[0341] MFNLHGVLNGAGLKGYIMRRGLILVTSDLMEAWECLEDFSAVRNLLEQSSNSTSWFWRFLWG SSQAKLVCRIKEDYKWEFEELLKSCGELFDSLNLGHQALFQEKVIKTLDFSTPGRAAAAVAF LS FIKDKWSEETHLSGGYLLDFLAMHLWRAWRHKNRLLLLSSVRPAI IPTEEQQQEEARRR RRQEQS PWNPRAGLDPRE

[0342] E1B-55K polypeptide sequence (SEQ ID NO:64):

[0343] MERRNPSERGVPAGFSGHASVESGGETQESPATWFRPPGNNTDGGATAGGSQAAAAAGAEP MEPESRPGPSGMNWQVAELFPELRRILT INEDGQGLKGVKRERGASEATEEARNLTFSLMT RHRPECVTFQQIKDNCANELDLLAQKYS IKQLTTYWLQPGDDFEEAIRVYAKVALRPDCKYK I SKLVNIRNCCYI SGNGAEVEIDTEDRVAFRCSMINMWPGVLGMDGWIMNVRFTGPNFSGT VFLANTNLILHGVS FYGFNNTCVEAWTDVRVRGCAFYCCWKGWCRPKSRAS IKKCLFERCT LGILSEGNSRVRHNVASDCGCFMLVKSVAVIKHNMVCGNCEDRASQMLTCSDGNCHLLKT IH VASHSRKAWPVFEHNILTRCSLHLGNRRGVFLPYQCNLSHTKILLEPESMSKVNLNGVFDMT MKIWKVLRYDETRTRCRPCECGGKHIRNQPVMLDVTEELRPDHLVLACTRAEFGSSDEDTD

[0344] Nucleic acid encoding an E1B polypeptide (SEQ ID NO:65):

[0345] ATGTTTAACTTGCATGGCGTGTTAAATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGG GCTAATCTTGGTTACATCTGACCTCATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTG TGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGC TCCTCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCT TTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAG AGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTT TTGAGTTTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGA TTTTCTGGCCATGCATCTGTGGAGAGCGGTGGTGAGACACAAGAATCGCCTGCTACTGTTGT CTTCCGTCCGCCCGGCAATAATACCGACGGAGGAGCAACAGCAGGAGGAAGCCAGGCGGCGG CGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAATGTT GTACAGGTGGCTGAACTGTTTCCAGAACTGAGACGCATTTTAACCATTAACGAGGATGGGCA GGGGCTAAAGGGGGTAAAGAGGGAGCGGGGGGCTTCTGAGGCTACAGAGGAGGCTAGGAATC T AC TTTTAGCTTAAT GAC C AGAC AC C G T C C T GAG TGTGTTACTTTT C AGC AGAT T AAG GAT AATTGCGCTAATGAGCTTGATCTGCTGGCGCAGAAGTATTCCATAAAGCAGCTGACCACTTA CTGGCTGCAGCCAGGGGATGATTTTGAGGAGGCTATTAGGGTATATGCAAAGGTGGCACTTA GGCCAGAT T GCAAGTACAAGAT TAGCAAAC T T GTAAATAT CAGGAAT T GT T GC TACAT T T CT GGGAACGGGGCCGAGGTGGAGATAGATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGAT AAATATGTGGCCGGGGGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTGAGGTTTACTG GTCCCAATTTTAGCGGTACGGTTTTCCTGGCCAATACCAATCTTATCCTACACGGTGTAAGC TTCTATGGGTTTAACAATACCTGTGTGGAAGCCTGGACCGATGTAAGGGTTCGGGGCTGTGC CTTTTACTGCTGCTGGAAGGGGGTGGTGTGTCGCCCCAAAAGCAGGGCTTCAATTAAGAAAT

[0346] GCCTGTTTGAAAGGTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGGGTGCGCCACAAT

[0347] GTGGCCTCCGACTGTGGTTGCTTTATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAACAT

[0348] GGTGTGTGGCAACTGCGAGGACAGGGCCTCTCAGATGCTGACCTGCTCGGACGGCAACTGTC

[0349] ACTTGCTGAAGACCATTCACGTAGCCAGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCAC AACATACTGACCCGCTGTTCCTTGCATTTGGGTAACAGGAGGGGGGTGTTCCTACCTTACCA ATGCAATTTGAGTCACACTAAGATATTGCTTGAGCCCGAGAGCATGTCCAAGGTGAACCTGA ACGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACCCGCACC AGGTGCAGACCCTGCGAGTGTGGCGGTAAACATATTAGGAACCAGCCTGTGATGCTGGATGT

[0350] GACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTA GC GAT GAAGAT ACAGAT T GA

[0351] Exemplary Cre sequences

[0352] Cre polypeptide sequence (SEQ ID NO:66):

[0353] MGPKKKRKVMSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWA

[0354] AWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKT IQQHLGQLNMLHRRSGLPRPSDSNAVSL VMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEI ARIRVKDI SRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWI SVSGVADDPNNYLF CRVRKNGVAAP S T S QL S TRALE G I FEATHRL I YGAKDDS GQRYLAWS GHS ARVGAARDMAR AGVS IPE IMQAGGWTNVNIVMNYIRTLDSETGAMVRLLEDGD

[0355] Nucleic acid encoding an Cre polypeptide (SEQ ID NO: 67):

[0356] ATGGGACCCAAGAAGAAGAGGAAGGTGATGAGTAATTTGCTCACAGTGCACCAGAATCTCCC

[0357] CGCCCTGCCTGTTGACGCTACGTCCGACGAGGTGAGGAAGAACCTGATGGACATGTTCCGGG

[0358] ACCGGCAGGCCTTCTCCGAACACACCTGGAAGATGCTGCTGTCCGTGTGCAGAAGCTGGGCT GCATGGTGCAAGCTGAATAACCGCAAATGGTTCCCCGCAGAGCCCGAGGACGTGCGTGACTA CCTGCTCTACCTCCAGGCTCGCGGACTGGCAGTGAAGACCATCCAGCAGCACCTGGGCCAAC TGAACATGCTGCACAGACGGTCTGGTCTGCCCCGGCCGTCTGATAGCAACGCCGTGTCGCTG GTGATGAGGCGCATCCGCAAGGAGAACGTGGACGCGGGAGAGCGTGCGAAGCAGGCCCTTGC

[0359] GTTCGAGCGGACCGACTTTGACCAGGTGCGGTCCCTGATGGAAAACTCCGACCGCTGTCAGG ACATCAGAAACCTGGCATTCCTGGGCATCGCGTACAATACCCTGCTGAGGATCGCGGAAATC GCCAGGATTCGCGTGAAGGACATTTCGCGCACCGATGGAGGCCGAATGCTGATTCACATCGG CCGCACCAAGACCTTGGTGTCCACTGCCGGGGTCGAGAAGGCGCTGTCACTCGGCGTCACCA

[0360] AGCTAGTCGAGCGCTGGATCTCTGTCAGTGGCGTTGCCGATGATCCAAACAACTACCTCTTC

[0361] TGCAGAGTGAGGAAGAACGGCGTCGCCGCGCCGAGCGCCACCAGCCAGCTGTCCACGAGGGC

[0362] TCTGGAGGGGATATTTGAGGCTACCCACAGACTGATCTACGGCGCCAAAGACGACAGCGGCC AGCGATATCTCGCTTGGAGCGGCCATAGCGCCCGCGTAGGCGCCGCCCGCGACATGGCCCGC GCCGGGGTGAGCATCCCTGAGATCATGCAGGCCGGGGGGTGGACAAACGTGAACATCGTGAT GAACTATATCCGCACGCTGGACAGCGAAACTGGTGCCATGGTGCGCCTGTTAGAAGACGGTG ATT GA

[0363] Exemplary Bxbl sequences

[0364] Bxbl polypeptide sequence (SEQ ID NO:68):

[0365] MPKKKRKVGSRALWIRLSRVTDATTSPERQLESCQQLCAQRGWDWGVAEDLDVSGAVDPF DRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRS IRHLQQLVHWAEDHKKLWSATEAHFDTT TPFAAWIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPD PVQRERILEVYHRWDNHEPLHLVAHDLNRRGVLSPKDYFAQLQGREPQGREWSATALKRSM I SEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRAKPAVSTPSLLLRV LFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTVAMAEWDAFCEEQVLDLLGDAERL EKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEA RPSGWEWRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRT IDFGDLQEYEQHLR LGSWERLHTGMS

[0366] Nucleic acid encoding an Bxbl polypeptide (SEQ ID NO:69):

[0367] ATGCCTAAAAAGAAGAGAAAAGTGGGCAGCAGAGCCCTGGTGGTGATTCGGCTGAGCCGGGT GACCGACGCCACAACCAGCCCCGAGAGACAGCTGGAAAGCTGTCAGCAGCTTTGTGCCCAGC GGGGATGGGACGTCGTCGGAGTGGCCGAAGATCTGGATGTGTCCGGCGCTGTGGACCCTTTC GACAGAAAGCGGAGACCCAATCTGGCCAGATGGCTCGCCTTTGAGGAACAGCCTTTCGACGT AATCGTGGCCTACAGAGTTGATAGACTGACCAGATCTATCCGGCACCTGCAGCAGCTCGTCC ATTGGGCCGAAGATCACAAGAAACTGGTGGTGTCCGCCACCGAGGCCCACTTCGATACAACC ACACCTTTTGCCGCTGTGGTCATCGCCCTGATGGGCACCGTGGCTCAAATGGAACTGGAGGC TATCAAGGAACGAAACCGGAGCGCCGCTCATTTCAACATCAGAGCCGGCAAGTATCGGGGCA GCCTGCCTCCTTGGGGCTACCTGCCTACCAGAGTGGATGGAGAGTGGCGGCTGGTTCCTGAC CCCGTGCAGAGAGAGAGAATCCTGGAAGTGTACCACAGAGTGGTGGACAACCACGAGCCTCT GCACCTGGTGGCCCACGATCTGAACAGAAGAGGCGTGCTGAGCCCTAAGGACTACTTCGCCC AGCTGCAAGGCAGAGAACCACAGGGCAGAGAGTGGTCCGCAACCGCCCTCAAGCGGAGCATG ATCAGCGAGGCCATGCTGGGCTACGCCACACTGAATGGAAAAACCGTGCGGGACGATGACGG CGCCCCTCTGGTGCGGGCCGAACCCATCCTGACACGGGAACAGCTGGAAGCACTGAGAGCTG AGCTGGTGAAGACCAGCAGAGCCAAGCCCGCCGTGTCAACACCTAGCCTGCTGCTGAGAGTG CTGTTCTGCGCCGTGTGCGGCGAGCCTGCCTATAAGTTCGCCGGCGGCGGACGGAAGCACCC TAGGTACCGCTGCAGAAGCATGGGCTTCCCCAAGCACTGCGGCAACGGCACCGTGGCTATGG CCGAGTGGGATGCTTTTTGCGAGGAACAAGTGCTGGACCTGCTGGGGGACGCCGAGCGGCTG GAGAAGGTGTGGGTGGCCGGCAGCGATTCTGCCGTGGAACTGGCCGAGGTGAACGCCGAGCT GGTCGACCTGACCTCCCTGATCGGCTCTCCAGCTTACAGAGCTGGCAGCCCTCAGAGAGAAG CCCTGGACGCCAGAATCGCCGCCCTGGCCGCCAGGCAGGAGGAACTGGAGGGCCTGGAGGCC CGGCCTAGCGGCTGGGAGTGGAGAGAGACAGGCCAGAGATTCGGCGACTGGTGGCGGGAACA GGACACCGCCGCTAAGAACACCTGGCTGAGGTCTATGAACGTGCGCCTGACATTCGACGTGA GAGGAGGCCTGACCCGGACCATCGACTTCGGCGATCTGCAGGAGTACGAGCAGCACCTGAGA CTGGGAAGCGTGGTCGAGAGACTCCACACAGGCATGTCTTGA

[0368] Exemplary FLP sequences

[0369] FLP polypeptide sequence (SEQ ID NO:70):

[0370] MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMI THNGTAIK RATEMSYNT I I SNSLSFDIVNKSLQFKYKTQKAT ILEASLKKLIPAWEFTI I PYNGQKHQSD I TDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGES IWE I TEKILNSFEYTSRFTKTKTL YQFLFLATFINCGRFSDIKNVDPKS FKLVQNKYLGVI IQCLVTETKTSVSRHI YFFSARGRI DPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPI FAIKNG PKSHIGRHLMTS FLSMKGLTELTNWGNWSDKRASAVARTTYTHQI TAIPDHYFALVSRYYA YDPISKEMIALKDETNPIEEWQHIEQLKGSAEGS IRYPAWNGI ISQEVLDYLSSYINRRI

[0371] Nucleic acid encoding an FLP polypeptide (SEQ ID N0:71):

[0372] ATGGCCCCTAAGAAGAAAAGAAAGGTGATGAGCCAGTTTGATATCCTGTGTAAAACCCCTCC AAAAGTGCTGGTCAGACAGTTCGTGGAAAGATTCGAGAGACCTAGCGGCGAAAAGATCGCCT CATGCGCCGCTGAACTGACATACCTGTGCTGGATGATCACCCACAACGGCACAGCCATTAAG CGGGCCACCTTTATGAGCTACAACACCATCATTAGCAACAGCCTGAGCTTCGACATCGTGAA CAAGAGCCTGCAATTTAAGTACAAGACGCAGAAAGCCACAATCCTGGAAGCTAGCCTGAAGA AGCTGATCCCTGCTTGGGAATTCACCATCATCCCATATAATGGCCAGAAGCACCAGAGCGAT ATTACAGACATCGTGTCTTCTCTGCAGCTGCAGTTCGAGTCTAGCGAGGAAGCCGACAAGGG AAATTCTCATTCTAAGAAGATGCTGAAGGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGAGA T C AC AGAGAAGAT C C T GAAC AG C T T T GAG TAG AC C AG CCGGTTTAC C AAGAC C AAGAC AC T G TACCAGTTCCTGTTCCTGGCTACCTTCATCAACTGCGGCAGATTCAGCGATATCAAGAATGT GGACCCTAAGTCCTTCAAGCTGGTCCAGAACAAATACCTGGGAGTGATCATCCAATGCCTGG TTACAGAGACCAAGACCAGCGTGTCCCGCCACATCTACTTCTTCTCTGCTAGAGGCCGGATC GACCCCCTGGTGTACCTCGACGAGTTCCTGAGAAACTCTGAACCTGTGCTGAAGAGAGTGAA CCGGACCGGCAATAGCTCCAGCAACAAGCAGGAATACCAGCTGCTGAAGGACAACCTGGTGC GGAGCTACAACAAGGCCCTGAAAAAAAATGCCCCTTACCCCATCTTCGCCATCAAGAACGGC C C T AAGAG C C AC AT C GG C AGAC AC C T GAT GAC TTCCTTCCT C AGC AT GAAAG GAC T C AC C GA GCTGACCAACGTGGTGGGCAACTGGTCCGATAAGAGGGCCTCCGCCGTGGCCAGAACCACAT

[0373] ACACCCACCAAATCACCGCCATCCCCGACCACTACTTCGCCCTCGTGTCCAGATACTACGCC TACGACCCCATCAGCAAGGAAATGATCGCCCTGAAAGATGAGACAAACCCCATTGAGGAATG GCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAAGGCAGCATCCGGTATCCTGCCTGGAACG GAATCATCAGCCAGGAGGTGCTGGACTACCTGAGCTCTTATATCAACAGACGGATCTGA loxP-mGreenLantern-HSV TKpolyA-attB (GA)-loxP (SEQ ID NO: 72): mGreenLantern in bold / italic font, loxP sites are in bold font, HSV TKpolyA in underlined font, and attB (GA-mutant) site in highlighted font.

[0374] ATAACTTCGTATAGCATACATTATACGAAGTTATCCTAGGACCGGTGCCACCATGGTGTCTA AAGGCGAGGAGCTGTTCACCGGCGTCGTGCCCATCCTCGTGGAGCTGGATGGCGACGTGAAC GGACACAAGTTCAGTGTGAGGGGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACACT GAAGTTTATCTGCACCACTGGAAAACTGCCCGTGCCTTGGCCTACACTGGTGACCACCCTGG GCTACGGAGTGGCCTGCTTCGCCAGATATCCAGATCATATGAAGCAGCACGACTTCTTTAAG TCCGCCATGCCCGAGGGCTATGTGCAGGAAAGAACCATTAGCTTCAAGGATGACGGGACCTA TAAAACAAGAGCAGAGGTGAAGTTCGAGGGAGATACCCTGGTGAATCGGATCGTGCTGAAGG GCATCGACTTTAAGGAGGACGGCAACATTCTGGGCCACAAGCTGGAGTACAACTTCAATAGC CACAAGGTGTACATCACCGCCGATAAGCAGAAGAACGGCATTAAGGCAAACTTTAAGACCCG GCACAACGTTGAGGATGGCGGAGTGCAGCTGGCCGACCATTACCAGCAGAACACCCCCATCG GAGACGGCCCTGTGCTGCTGCCCGATAACCACTACCTGTCCCACCAGAGCAAGCTGAGCAAA GACCCCAATGAAAAAAGAGACCACATGGTGCTGAAGGAAAGGGTGACCGCCGCCGGAATCAC CCA TGACA TGGA TGAGCTGTACAAGTGAG TACTACGCGTCTC GAGCGGCAATAAAAAGACAG AATAAAACGCACGGTGTTGGGTCGTTTGTTCAC TAG TAG GAG C T C AC C GC C AT T G T C G G C

[0375] GGGAT C TATAAC T TCGTATAGCATACAT TATACGAAGT TAT

[0376] RCAd6-FRT-T-ElA-loxP-FRT-attP-bGHpolyA-ElB (modified left arm of Ad6 genome) (SEQ ID NO:73):

[0377] ITR is the first section of highlighted font, FRT sites are in underlined font, the E1A gene is the first bold / italic font, the CMV promoter is the second section of highlighted font, the loxP site is the first section of bold font, the attP (GA-mutant) is the second section of bold font, the mTagBFP2 gene is the second bold / italic font, the bGH polyA signal is the third section of highlighted font, and the E1B gene is the third bold / italic font.

[0378] GTGTGATGTTGTAAGTGTGGCGGAACACATGTAAGCGCCGGATGTGGTAAAAGTGACGTTTT

[0379] TGGTGTGCGCCGGTGTACACGGCGACGATGCTTCTGATCGCGGCCGCGATATCGCTAGCGAA GTTCCTATTCTCTAGAAAGTATAGGAACTTCG GAT C C T C T AG G T C GAT AG C T GAAG T GAG AATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCAAGTAATATTTGG CCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTCTGTGTTACTCATA GCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGT GTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCT GACGCGCAGTGTATTTATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGT AGAGTTTTCTCCTCCGAGCCGCTCCGACACCGGGACTGAAAATGAGACATATTATCTGCCAC GGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACT GGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATT TAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGAGTCT GTAATGTTGGCGGTGCAGGAAGGGATTGACTTATTCACTTTTCCGCCGGCGCCCGGTTCGCC GGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTT CTATGCCAAACCTTGTGCCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCC AGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCA CGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGC TTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAAATTATGGGCAGTGGG TGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAA AGAATTTTGTATTGTGATTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCC AGAACCGGAGCCTGCAAGACCTACCCGGCGTCCTAAATTGGTGCCTGCTATCCTGAGACGCC CGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCT AACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAG AGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGTCTGGGCAAC CTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGG

[0380] AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCGGCCGCATT liiiiliiiil C T AGGC C TCTTCTCGTTCCT CAT CAC G T AT ACATAAC T TCGTATAGCATAC AT TATACGAAGT TATAG C G C T GAAG TTCCTATTCTC T AGAAAG TAT AG GAAC T T CAC C G G T C GTGGTTTGTCTGGTCAACCACCGCGGACTCAGTGGTGTACGGTACAAACCCCTCGAGCTAGC GCCACCATGGTGTCAAAGGGCGAGGAGCTGATCAAGGAGAACATGCATATGAAGCTGTACAT GGAGGGCACCGTGGATAATCACCACTTTAAGTGTACTTCCGAGGGGGAAGGAAAACCTTACG AGGGAACCCAGACCATGCGCATCAAAGTGGTGGAAGGAGGCCCTCTGCCTTTCGCATTCGAT ATCCTGGCTACTTCTTTTCTGTATGGGTCCAAGACCTTTATCAACCACACCCAGGGCATCCC TGATTTCTTCAAGCAGAGCTTTCCTGAGGGCTTCACATGGGAGAGGGTTACAACATACGAGG ACGGAGGCGTGCTGACCGCCACTCAGGATACCAGCCTGCAGGATGGCTGCCTGATCTATAAT GTGAAGATCAGGGGAGTGAACTTCACCAGCAATGGGCCAGTGATGCAGAAGAAGACCCTGGG CTGGGAAGCCTTCACAGAAACACTCTACCCAGCCGATGGCGGCCTGGAGGGACGGAACGACA TGGCCCTGAAGCTGGTGGGCGGCTCCCACCTGATCGCCAACGCCAAGACCACTTACAGAAGC AAGAAGCCCGCCAAGAACCTGAAAATGCCAGGGGTGTATTACGTGGACTACCGCCTGGAAAG AATCAAGGAGGCCAATAATGAGACCTACGTGGAGCAGCACGAGGTGGCCGTGGCCCGCTATT GCGACCTGCCCTCCAAGCTGGGCCACAAACTGAACTAAAC TAG T CAAT T GG T T AAC T AT AAC

[0381] GCCTTTGTTTGCT GAAT GAG T T GAT G T AAG T T T AAT AAAG G G T GAGAT AATGTTTAACTTGC ATGGCGTGTTAAATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCTAATCTTGGTT ACATCTGACCTCATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCT GGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCCTCCCAGGCAA AGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGT GGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAA GACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAA AGGATAAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATG CATCTGTGGAGAGCGGTGGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCC GGCAATAATACCGACGGAGGAGCAACAGCAGGAGGAAGCCAGGCGGCGGCGGCGGCAGGAGC AGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAATGTTGTACAGGTGGCTG AACTGTTTCCAGAACTGAGACGCATTTTAACCATTAACGAGGATGGGCAGGGGCTAAAGGGG GTAAAGAGGGAGCGGGGGGCTTCTGAGGCTACAGAGGAGGCTAGGAATCTAACTTTTAGCTT AATGACCAGACACCGTCCTGAGTGTGTTACTTTTCAGCAGATTAAGGATAATTGCGCTAATG AGCTTGATCTGCTGGCGCAGAAGTATTCCATAAAGCAGCTGACCACTTACTGGCTGCAGCCA GGGGATGATTTTGAGGAGGCTATTAGGGTATATGCAAAGGTGGCACTTAGGCCAGATTGCAA GTACAAGATTAGCAAACTTGTAAATATCAGGAATTGTTGCTACATTTCTGGGAACGGGGCCG AGGTGGAGATAGATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGGCCG GGGGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTGAGGTTTACTGGTCCCAATTTTAG CGGTACGGTTTTCCTGGCCAATACCAATCTTATCCTACACGGTGTAAGCTTCTATGGGTTTA ACAATACCTGTGTGGAAGCCTGGACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGC TGGAAGGGGGTGGTGTGTCGCCCCAAAAGCAGGGCTTCAATTAAGAAATGCCTGTTTGAAAG GTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGGGTGCGCCACAATGTGGCCTCCGACT GTGGTTGCTTTATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAACATGGTGTGTGGCAAC TGCGAGGACAGGGCCTCTCAGATGCTGACCTGCTCGGACGGCAACTGTCACTTGCTGAAGAC CATTCACGTAGCCAGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCACAACATACTGACCC GCTGTTCCTTGCATTTGGGTAACAGGAGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGT CACACTAAGATATTGCTTGAGCCCGAGAGCATGTCCAAGGTGAACCTGAACGGGGTGTTTGA CATGACCATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCT GCGAGTGTGGCGGTAAACATATTAGGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTG AGGCCCGATCACTTGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATAC AGATTGA

[0382] Example 6: Exemplary Embodiments

[0383] Embodiment 1. A viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (a), (b), (e), (f), (g), and optionally (c), (d), and (h):

[0384] (a) a first recombinase site of a first recombinase,

[0385] (b) a packaging sequence, (c) nucleic acid encoding an adenovirus early region 1A (El A) polypeptide,

[0386] (d) a promoter,

[0387] (e) a recombinase site of a second recombinase,

[0388] (f) a second recombinase site of said first recombinase,

[0389] (g) a recombinase site of a third recombinase, and

[0390] (h) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

[0391] Embodiment 2. The viral vector of embodiment 1, wherein said first recombinase site and said second recombinase site are FRT sites.

[0392] Embodiment 3. The viral vector of any one of embodiments 1-2, wherein said recombinase site of said second recombinase is a loxP site.

[0393] Embodiment 4. The viral vector of any one of embodiments 1-3, wherein said recombinase site of said third recombinase is an attP site.

[0394] Embodiment 5. The viral vector of any one of embodiments 1-4, wherein said engineered DNA sequence comprises, between said (g) and said optional (h), a polyA sequence.

[0395] Embodiment 6. The viral vector of any one of embodiments 1-5, wherein said engineered DNA sequence comprises (d), and said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

[0396] Embodiment 7. The viral vector of any one of embodiments 1-6, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

[0397] Embodiment 8. The viral vector of any one of embodiments 1-6, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence. Embodiment 9. The viral vector of any one of embodiments 1-8, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

[0398] Embodiment 10. The viral vector of any one of embodiments 1-8, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

[0399] Embodiment 11. The viral vector of any one of embodiments 1-10, wherein said viral vector is an adenoviral vector.

[0400] Embodiment 12. An isolated nucleic acid comprising (a), (b), (e), (f), (g), and optionally

[0401] (c), (d), and (h):

[0402] (a) a first recombinase site of a first recombinase,

[0403] (b) a packaging sequence,

[0404] (c) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,

[0405] (d) a promoter,

[0406] (e) a recombinase site of a second recombinase,

[0407] (f) a second recombinase site of said first recombinase,

[0408] (g) a recombinase site of a third recombinase, and

[0409] (h) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

[0410] Embodiment 13. The isolated nucleic acid of embodiment 12, wherein said first recombinase site and said second recombinase site are FRT sites.

[0411] Embodiment 14. The isolated nucleic acid of any one of embodiments 12-13, wherein said recombinase site of said second recombinase is a loxP site.

[0412] Embodiment 15. The isolated nucleic acid of any one of embodiments 12-14, wherein said recombinase site of said third recombinase is an attP site. Embodiment 16. The isolated nucleic acid of any one of embodiments 12-15, wherein said engineered DNA sequence comprises, between said (g) and said optional (h), a polyA sequence.

[0413] Embodiment 17. The isolated nucleic acid of any one of embodiments 12-16, wherein said engineered DNA sequence comprises said (d), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

[0414] Embodiment 18. An isolated nucleic acid comprising (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of said first recombinase.

[0415] Embodiment 19. The isolated nucleic acid of embodiment 18, wherein said first recombinase site and said second recombinase site are loxP sites.

[0416] Embodiment 20. The isolated nucleic acid of any one of embodiments 18-19, wherein said recombinase site of said second recombinase is an attB site.

[0417] Embodiment 21. The isolated nucleic acid of any one of embodiments 18-19, wherein said isolated nucleic acid comprises said multicloning site.

[0418] Embodiment 22. The isolated nucleic acid of any one of embodiments 18-19, wherein said isolated nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest.

[0419] Embodiment 23. The isolated nucleic acid of any one of embodiments 18-19, wherein said isolated nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest. Embodiment 24. A system for making a recombinant adenovirus, wherein said system comprises:

[0420] (a) a viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):

[0421] (i) a first recombinase site of a first recombinase,

[0422] (ii) a packaging sequence,

[0423] (iii) nucleic acid encoding an adenovirus early region 1 A (El A) polypeptide,

[0424] (iv) a promoter,

[0425] (v) a recombinase site of a second recombinase,

[0426] (vi) a second recombinase site of said first recombinase,

[0427] (vii) a recombinase site of a third recombinase, and

[0428] (viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide; and

[0429] (b) a donor nucleic acid comprising (i-b) a first recombinase site of said second recombinase, (ii-b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (iii-b) a recombinase site of said third recombinase, and (iv-b) a second recombinase site of said second recombinase.

[0430] Embodiment 25. The system of embodiment 24, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites.

[0431] Embodiment 26. The system of any one of embodiments 24-25, wherein said recombinase site of said second recombinase is a loxP site.

[0432] Embodiment 27. The system of any one of embodiments 24-26, wherein said recombinase site of said third recombinase is an attP site. Embodiment 28. The system of any one of embodiments 24-27, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

[0433] Embodiment 29. The system of any one of embodiments 24-28, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

[0434] Embodiment 30. The system of any one of embodiments 24-29, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

[0435] Embodiment 31. The system of any one of embodiments 24-29, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

[0436] Embodiment 32. The system of any one of embodiments 24-31, wherein said engineered DNA sequence is directly followed by said second ITR sequence. Embodiment 33. The system of any one of embodiments 24-31, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

[0437] Embodiment 34. The system acid of any one of embodiments 24-33, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites.

[0438] Embodiment 35. The system of any one of embodiments 24-34, wherein said recombinase site of said second recombinase is an attB site.

[0439] Embodiment 36. The system of any one of embodiments 24-35, wherein said viral vector is an adenoviral vector. Embodiment 37. The system of any one of embodiments 24-36, wherein said donor nucleic acid comprises said multicloning site.

[0440] Embodiment 38. The system of any one of embodiments 24-36, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest.

[0441] Embodiment 39. The system of any one of embodiments 24-36, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

[0442] Embodiment 40. The system of any one of embodiments 24-39, wherein said donor nucleic acid is in the form of DNA plasmid.

[0443] Embodiment 41. The system of any one of embodiments 24-40, comprising a population of cells comprising said first recombinase.

[0444] Embodiment 42. The system of any one of embodiments 24-40, comprising a population of cells comprising said second recombinase.

[0445] Embodiment 43. The system of any one of embodiments 23-40, comprising a population of cells comprising said first recombinase and said second recombinase.

[0446] Embodiment 44. The system of any one of embodiments 24-40, wherein said first recombinase comprises a Cre polypeptide.

[0447] Embodiment 45. The system of any one of embodiments 24-40, wherein said second recombinase comprises a Bxbl polypeptide. Embodiment 46. The system of any one of embodiments 24-40, comprising a population of cells comprising Cre polypeptide and a Bxbl polypeptide.

[0448] Embodiment 47. The system of any one of embodiments 24-46, comprising a population of cells comprising said third recombinase.

[0449] Embodiment 48. The system of embodiment 47, wherein said third recombinase comprises a FLP polypeptide.

[0450] Embodiment 49. The system of any one of embodiments 24-46, wherein said system comprises a population of cells comprising FLP polypeptide.

[0451] Embodiment 50. An in vitro host cell comprising (a) a viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):

[0452] (i) a first recombinase site of a first recombinase,

[0453] (ii) a packaging sequence,

[0454] (iii) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,

[0455] (iv) a promoter,

[0456] (v) a recombinase site of a second recombinase,

[0457] (vi) a second recombinase site of said first recombinase,

[0458] (vii) a recombinase site of a third recombinase, and

[0459] (viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

[0460] Embodiment 51. The host cell of embodiment 50, wherein said first recombinase site and said second recombinase site are FRT sites.

[0461] Embodiment 52. The host cell of any one of embodiments 50-51, wherein said recombinase site of said second recombinase is a loxP site. Embodiment 53. The host cell of any one of embodiments 50-52, wherein said recombinase site of said third recombinase is an attP site.

[0462] Embodiment 54. The host cell of any one of embodiments 50-53, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

[0463] Embodiment 55. The host cell of any one of embodiments 50-54, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

[0464] Embodiment 56. The host cell of any one of embodiments 50-55, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

[0465] Embodiment 57. The host cell of any one of embodiments 50-55, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

[0466] Embodiment 58. The host cell of any one of embodiments 50-57, wherein said engineered DNA sequence is directly followed by said second ITR sequence. Embodiment 59. The host cell of any one of embodiments 50-57, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

[0467] Embodiment 60. The host cell of any one of embodiments 50-58, wherein said viral vector is an adenoviral vector.

[0468] Embodiment 61. An in vitro host cell comprising nucleic acid comprising (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence

[0469] Ill encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of said first recombinase.

[0470] Embodiment 62. The host cell of embodiment 61, wherein said first recombinase site and said second recombinase site are loxP sites.

[0471] Embodiment 63. The host cell of any one of embodiments 61-62, wherein said recombinase site of said second recombinase is an attB site.

[0472] Embodiment 64. The host cell of any one of embodiments 61-62, wherein said nucleic acid comprises said multicloning site.

[0473] Embodiment 65. The host cell of any one of embodiments 61-62, wherein said nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest.

[0474] Embodiment 66. The host cell of any one of embodiments 61-62, wherein said nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

[0475] Embodiment 67. The host cell of any one of embodiments 50-66, wherein said host cell is a vero cell, a BHK-21 cell, a 293 cell, aA549 cell, a PER.C6 cell, a 911 cell, a HEL299 cell, or a HeLa cell.

[0476] Embodiment 68. A method for making nucleic acid encoding a recombinant virus, wherein said method comprises delivering a viral vector and a donor nucleic acid to an in vitro cell, wherein said viral vector comprises a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):

[0477] (i) a first recombinase site of a first recombinase, (ii) a packaging sequence,

[0478] (iii) nucleic acid encoding an adenovirus early region 1 A (El A) polypeptide,

[0479] (iv) a promoter,

[0480] (v) a recombinase site of a second recombinase,

[0481] (vi) a second recombinase site of said first recombinase,

[0482] (vii) a recombinase site of a third recombinase, and

[0483] (viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide, wherein said donor nucleic acid comprises (A) a first recombinase site of said second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of said third recombinase, and (D) a second recombinase site of said second recombinase, wherein said cell expresses said second recombinase and said third recombinase, wherein recombination occurs within said cell to form said nucleic acid encoding said recombinant virus, and wherein said nucleic acid encoding said recombinant virus comprises said first recombinase site of said first recombinase, said packaging sequence, said nucleic acid encoding said El A polypeptide (when present), said promoter (when present), a recombinase site of said second recombinase or a remnant thereof post recombination, said multicloning site or said nucleic acid sequence encoding said polypeptide of interest, said recombinase site of said third recombinase or a remnant thereof post recombination, and said nucleic acid encoding said E1B polypeptide (when present).

[0484] Embodiment 69. The method of embodiment 68, wherein said nucleic acid encoding said recombinant virus lacks said second recombinase site of said first recombinase.

[0485] Embodiment 70. The method of any one of embodiments 68-69, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites. Embodiment 71. The method of any one of embodiments 68-70, wherein said recombinase site of said second recombinase is a loxP site.

[0486] Embodiment 72. The method of any one of embodiments 68-71, wherein said recombinase site of said third recombinase is an attP site.

[0487] Embodiment 73. The method of any one of embodiments 68-72, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

[0488] Embodiment 74. The method of any one of embodiments 68-73, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

[0489] Embodiment 75. The method of any one of embodiments 68-74, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

[0490] Embodiment 76. The method of any one of embodiments 68-74, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

[0491] Embodiment 77. The method of any one of embodiments 68-76, wherein said engineered DNA sequence is directly followed by said second ITR sequence. Embodiment 78. The method of any one of embodiments 68-76, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

[0492] Embodiment 79. The method acid of any one of embodiments 68-78, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites. Embodiment 80. The method of any one of embodiments 68-79, wherein said recombinase site of said second recombinase is an attB site.

[0493] Embodiment 81. The method of any one of embodiments 68-80, wherein said viral vector is an adenoviral vector.

[0494] Embodiment 82. The method of any one of embodiments 68-81, wherein said donor nucleic acid comprises said multicloning site.

[0495] Embodiment 83. The method of any one of embodiments 68-81, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest.

[0496] Embodiment 84. The method of any one of embodiments 68-81, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

[0497] Embodiment 85. The method of any one of embodiments 68-84, wherein said donor nucleic acid is in the form of DNA plasmid.

[0498] Embodiment 86. The method of any one of embodiments 68-85, wherein said first recombinase comprises a FLP polypeptide.

[0499] Embodiment 87. The method of any one of embodiments 68-86, wherein said second recombinase comprises Cre a polypeptide. Embodiment 88. The method of any one of embodiments 68-87, wherein said third recombinase comprises a Bxbl polypeptide. Embodiment 89. The method of any one of embodiments 68-88, wherein said cell is a eukaryotic cell.

[0500] Embodiment 90. A method for making nucleic acid encoding a recombinant virus, wherein said method comprises:

[0501] (a) delivering a viral vector and a donor nucleic acid to a first in vitro cell, wherein said viral vector comprises a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):

[0502] (i) a first recombinase site of a first recombinase,

[0503] (ii) a packaging sequence,

[0504] (iii) nucleic acid encoding an adenovirus early region 1 A (El A) polypeptide,

[0505] (iv) a promoter,

[0506] (v) a recombinase site of a second recombinase,

[0507] (vi) a second recombinase site of said first recombinase,

[0508] (vii) a recombinase site of a third recombinase, and

[0509] (viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide, wherein said donor nucleic acid comprises (A) a first recombinase site of said second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of said third recombinase, and (D) a second recombinase site of said second recombinase, wherein said first cell expresses said second recombinase and said third recombinase, wherein recombination occurs within said first cell to form said nucleic acid encoding said recombinant virus, wherein said nucleic acid encoding said recombinant virus comprises said first recombinase site of said first recombinase, said packaging sequence, said nucleic acid encoding said El A polypeptide (when present), said promoter (when present), a recombinase site of said second recombinase or a remnant thereof post recombination, said multicloning site or said nucleic acid sequence encoding said polypeptide of interest, said recombinase site of said third recombinase or a remnant thereof post recombination, and said nucleic acid encoding said E1B polypeptide (when present); and

[0510] (b) delivering viral vectors produced by said first cell to a second in vitro cell, wherein said second cell expresses said first recombinase, wherein recombination occurs within said second cell on said viral vector, if present, to reduce the presence of nucleic acid not encoding said recombinant virus.

[0511] Embodiment 91. The method of embodiment 90, wherein said nucleic acid encoding said recombinant virus lacks said second recombinase site of said first recombinase.

[0512] Embodiment 92. The method of any one of embodiments 90-91, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites.

[0513] Embodiment 93. The method of any one of embodiments 90-92, wherein said recombinase site of said second recombinase is a loxP site.

[0514] Embodiment 94. The method of any one of embodiments 90-93, wherein said recombinase site of said third recombinase is an attP site.

[0515] Embodiment 95. The method of any one of embodiments 90-94, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

[0516] Embodiment 96. The method of any one of embodiments 90-95, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter. Embodiment 97. The method of any one of embodiments 90-96, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

[0517] Embodiment 98. The method of any one of embodiments 90-96, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

[0518] Embodiment 99. The method of any one of embodiments 90-98, wherein said engineered DNA sequence is directly followed by said second ITR sequence. Embodiment 100. The method of any one of embodiments 90-98, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

[0519] Embodiment 101. The method acid of any one of embodiments 90-100, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites.

[0520] Embodiment 102. The method of any one of embodiments 90-101, wherein said recombinase site of said second recombinase is an attB site.

[0521] Embodiment 103. The method of any one of embodiments 90-102, wherein said viral vector is an adenoviral vector.

[0522] Embodiment 104. The method of any one of embodiments 90-103, wherein said donor nucleic acid comprises said multicloning site.

[0523] Embodiment 105. The method of any one of embodiments 90-104, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest. Embodiment 106. The method of any one of embodiments 90-105, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

[0524] Embodiment 107. The method of any one of embodiments 90-106, wherein said donor nucleic acid is in the form of DNA plasmid.

[0525] Embodiment 108. The method of any one of embodiments 90-107, wherein said first cell is a eukaryotic cell.

[0526] Embodiment 109. The method of any one of embodiments 90-108, wherein said second recombinase comprises a Cre polypeptide.

[0527] Embodiment 110. The method of any one of embodiments 90-108, wherein said third recombinase comprises a Bxbl polypeptide.

[0528] Embodiment 111. The method of any one of embodiments 90-110, wherein said second cell is a eukaryotic cell. Embodiment 112. The method of any one of embodiments 90-105, wherein said first recombinase comprises a FLP polypeptide.

[0529] OTHER EMBODIMENTS

[0530] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (a), (b), (e), (f), (g), and optionally (c),(d), and (h):(a) a first recombinase site of a first recombinase,(b) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(c) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,(d) a promoter,(e) a recombinase site of a second recombinase,(f) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(g) a recombinase site of a third recombinase, and(h) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

2. The viral vector of claim 1, wherein said first recombinase site and said second recombinase site are FRT sites.

3. The viral vector of any one of claims 1-2, wherein said recombinase site of said second recombinase is a loxP site.

4. The viral vector of any one of claims 1-3, wherein said recombinase site of said third recombinase is an attP site.

5. The viral vector of any one of claims 1-4, wherein said engineered DNA sequence comprises, between said (g) and said optional (h), a poly A sequence.

6. The viral vector of any one of claims 1-5, wherein said engineered DNA sequence comprises (d), and said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFl a promoter.

7. The viral vector of any one of claims 1-6, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

8. The viral vector of any one of claims 1-6, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

9. The viral vector of any one of claims 1-8, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

10. The viral vector of any one of claims 1-8, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

11. The viral vector of any one of claims 1-10, wherein said viral vector is an adenoviral vector.

12. The viral vector of any one of claims 1-11, wherein said engineered DNA sequence comprises said packaging sequence.

13. The viral vector of any one of claims 1-11, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

14. The viral vector of claim 13, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

15. The viral vector of any one of claims 1-14, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

16. The viral vector of any one of claims 1-14, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

17. The viral vector of claim 16, where said targeting sequence is a guide RNA.

18. An isolated nucleic acid comprising (a), (b), (e), (f), (g), and optionally (c), (d), and (h):(a) a first recombinase site of a first recombinase,(b) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(c) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,(d) a promoter,(e) a recombinase site of a second recombinase,(f) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(g) a recombinase site of a third recombinase, and(h) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

19. The isolated nucleic acid of claim 18, wherein said first recombinase site and said second recombinase site are FRT sites.

20. The isolated nucleic acid of any one of claims 18-19, wherein said recombinase site of said second recombinase is a loxP site.

21. The isolated nucleic acid of any one of claims 18-20, wherein said recombinase site of said third recombinase is an attP site.

22. The isolated nucleic acid of any one of claims 18-21, wherein said engineered DNA sequence comprises, between said (g) and said optional (h), a polyA sequence.

23. The isolated nucleic acid of any one of claims 18-22, wherein said engineered DNA sequence comprises said (d), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

24. The isolated nucleic acid of any one of claims 18-23, wherein said engineered DNA sequence comprises said packaging sequence.

25. The isolated nucleic acid of any one of claims 18-23, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

26. The isolated nucleic acid of claim 25, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

27. The isolated nucleic acid of any one of claims 18-26, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

28. The isolated nucleic acid of any one of claims 18-26, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

29. The isolated nucleic acid of claim 28, where said targeting sequence is a guide RNA.

30. An isolated nucleic acid comprising (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of said first recombinase.

31. The isolated nucleic acid of claim 30, wherein said first recombinase site and said second recombinase site are loxP sites.

32. The isolated nucleic acid of any one of claims 30-31, wherein said recombinase site of said second recombinase is an attB site.

33. The isolated nucleic acid of any one of claims 30-31, wherein said isolated nucleic acid comprises said multicloning site.

34. The isolated nucleic acid of any one of claims 30-31, wherein said isolated nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest.

35. The isolated nucleic acid of any one of claims 30-31, wherein said isolated nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

36. A system for making a recombinant adenovirus, wherein said system comprises:(a) a viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):(i) a first recombinase site of a first recombinase,(ii) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(iii) nucleic acid encoding an adenovirus early region 1 A (El A) polypeptide,(iv) a promoter,(v) a recombinase site of a second recombinase,(vi) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(vii) a recombinase site of a third recombinase, and(viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide; and(b) a donor nucleic acid comprising (i-b) a first recombinase site of said second recombinase, (ii-b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (iii-b) a recombinase site of said third recombinase, and (iv-b) a second recombinase site of said second recombinase.

37. The system of claim 36, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites.

38. The system of any one of claims 36-37, wherein said recombinase site of said second recombinase is a loxP site.

39. The system of any one of claims 36-38, wherein said recombinase site of said third recombinase is an attP site.

40. The system of any one of claims 36-39, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

41. The system of any one of claims 36-40, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

42. The system of any one of claims 36-41, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

43. The system of any one of claims 36-41, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

44. The system of any one of claims 36-43, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

45. The system of any one of claims 36-43, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

46. The system acid of any one of claims 36-45, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites.

47. The system of any one of claims 36-46, wherein said recombinase site of said second recombinase is an attB site.

48. The system of any one of claims 36-47, wherein said viral vector is an adenoviral vector.

49. The system of any one of claims 36-48, wherein said engineered DNA sequence comprises said packaging sequence.

50. The system of any one of claims 36-48, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

51. The system of claim 50, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

52. The system of any one of claims 36-51, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

53. The system of any one of claims 36-51, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

54. The system of claim 53, where said targeting sequence is a guide RNA.

55. The system of any one of claims 36-54, wherein said donor nucleic acid comprises said multi cloning site.

56. The system of any one of claims 36-54, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest.

57. The system of any one of claims 36-54, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

58. The system of any one of claims 36-57, wherein said donor nucleic acid is in the form of DNA plasmid.

59. The system of any one of claims 36-58, comprising a population of cells comprising said first recombinase.

60. The system of any one of claims 36-58, comprising a population of cells comprising said second recombinase.

61. The system of any one of claims 36-58, comprising a population of cells comprising said first recombinase and said second recombinase.

62. The system of any one of claims 36-58, wherein said first recombinase comprises a Cre polypeptide.

63. The system of any one of claims 36-58, wherein said second recombinase comprises a Bxbl polypeptide.

64. The system of any one of claims 36-58, comprising a population of cells comprising Cre polypeptide and a Bxbl polypeptide.

65. The system of any one of claims 36-64, comprising a population of cells comprising said third recombinase.

66. The system of claim 65, wherein said third recombinase comprises a FLP polypeptide.

67. The system of any one of claims 36-64, wherein said system comprises a population of cells comprising FLP polypeptide.

68. An in vitro host cell comprising (a) a viral vector comprising a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i),(ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):(i) a first recombinase site of a first recombinase,(ii) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(iii) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,(iv) a promoter,(v) a recombinase site of a second recombinase,(vi) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(vii) a recombinase site of a third recombinase, and(viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide.

69. The host cell of claim 68, wherein said first recombinase site and said second recombinase site are FRT sites.

70. The host cell of any one of claims 68-69, wherein said recombinase site of said second recombinase is a loxP site.

71. The host cell of any one of claims 68-70, wherein said recombinase site of said third recombinase is an attP site.

72. The host cell of any one of claims 68-71, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

73. The host cell of any one of claims 68-72, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

74. The host cell of any one of claims 68-73, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

75. The host cell of any one of claims 68-73, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

76. The host cell of any one of claims 68-75, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

77. The host cell of any one of claims 68-75, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

78. The host cell of any one of claims 68-77, wherein said viral vector is an adenoviral vector.

79. The host cell of any one of claims 68-78, wherein said engineered DNA sequence comprises said packaging sequence.

80. The host cell of any one of claims 68-78, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

81. The host cell of claim 80, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

82. The host cell of any one of claims 68-81, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

83. The host cell of any one of claims 68-81, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

84. The host cell of claim 83, where said targeting sequence is a guide RNA.

85. An in vitro host cell comprising nucleic acid comprising (a) a first recombinase site of a first recombinase, (b) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (c) a recombinase site of a second recombinase, and (d) a second recombinase site of said first recombinase.

86. The host cell of claim 85, wherein said first recombinase site and said second recombinase site are loxP sites.

87. The host cell of any one of claims 85-86, wherein said recombinase site of said second recombinase is an attB site.

88. The host cell of any one of claims 85-86, wherein said nucleic acid comprises said multicloning site.

89. The host cell of any one of claims 85-86, wherein said nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest.

90. The host cell of any one of claims 85-86, wherein said nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

91. The host cell of any one of claims 68-90, wherein said host cell is a vero cell, a BHK- 21 cell, a 293 cell, a A549 cell, a PER.C6 cell, a 911 cell, a HEL299 cell, or a HeLa cell.

92. A method for making nucleic acid encoding a recombinant virus, wherein said method comprises delivering a viral vector and a donor nucleic acid to an in vitro cell, wherein said viral vector comprises a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):(i) a first recombinase site of a first recombinase,(ii) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(iii) nucleic acid encoding an adenovirus early region 1 A (E1A) polypeptide,(iv) a promoter,(v) a recombinase site of a second recombinase,(vi) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(vii) a recombinase site of a third recombinase, and(viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide, wherein said donor nucleic acid comprises (A) a first recombinase site of said second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of said third recombinase, and (D) a second recombinase site of said second recombinase, wherein said cell expresses said second recombinase and said third recombinase, wherein recombination occurs within said cell to form said nucleic acid encoding said recombinant virus, and wherein said nucleic acid encoding said recombinant virus comprises said first recombinase site of said first recombinase, said packaging sequence or said viral sequence that can regulate viral packaging and / or propagation, said nucleic acid encoding said El A polypeptide (when present), said promoter (when present), a recombinase site of said second recombinase or a remnant thereof post recombination, said multicloning site or said nucleic acid sequence encoding said polypeptide of interest, said recombinase site of said third recombinase or a remnant thereof post recombination, and said nucleic acid encoding said E1B polypeptide (when present).

93. The method of claim 92, wherein said nucleic acid encoding said recombinant virus lacks said second recombinase site of said first recombinase.

94. The method of any one of claims 92-93, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites.

95. The method of any one of claims 92-94, wherein said recombinase site of said second recombinase is a loxP site.

96. The method of any one of claims 92-95, wherein said recombinase site of said third recombinase is an attP site.

97. The method of any one of claims 92-96, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

98. The method of any one of claims 92-97, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

99. The method of any one of claims 92-98, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

100. The method of any one of claims 92-98, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

101. The method of any one of claims 92-100, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

102. The method of any one of claims 92-100, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

103. The method acid of any one of claims 92-102, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites.

104. The method of any one of claims 92-103, wherein said recombinase site of said second recombinase is an attB site.

105. The method of any one of claims 92-104, wherein said viral vector is an adenoviral vector.

106. The method of any one of claims 92-105, wherein said engineered DNA sequence comprises said packaging sequence.

107. The method of any one of claims 92-105, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

108. The method of claim 107, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

109. The method of any one of claims 92-108, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

110. The method of any one of claims 92-108, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

111. The method of claim 110, where said targeting sequence is a guide RNA.

112. The method of any one of claims 92-111, wherein said donor nucleic acid comprises said multi cloning site.

113. The method of any one of claims 92-111, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest.

114. The method of any one of claims 92-111, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

115. The method of any one of claims 92-114, wherein said donor nucleic acid is in the form of DNA plasmid.

116. The method of any one of claims 92-115, wherein said first recombinase comprises a FLP polypeptide.

117. The method of any one of claims 92-116, wherein said second recombinase comprises Cre a polypeptide.

118. The method of any one of claims 92-117, wherein said third recombinase comprises a Bxbl polypeptide.

119. The method of any one of claims 92-118, wherein said cell is a eukaryotic cell.

120. A method for making nucleic acid encoding a recombinant virus, wherein said method comprises:(a) delivering a viral vector and a donor nucleic acid to a first in vitro cell, wherein said viral vector comprises a genome comprising a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (i), (ii), (v), (vi), (vii), and optionally (iii), (iv), and (viii):(i) a first recombinase site of a first recombinase,(ii) a packaging sequence or a viral sequence that can regulate viral packaging and / or propagation,(iii) nucleic acid encoding an adenovirus early region 1A (E1A) polypeptide,(iv) a promoter,(v) a recombinase site of a second recombinase,(vi) a second recombinase site of said first recombinase or a sequence that can be targeted by a targeting sequence,(vii) a recombinase site of a third recombinase, and(viii) nucleic acid encoding an adenovirus early region IB (E1B) polypeptide,wherein said donor nucleic acid comprises (A) a first recombinase site of said second recombinase, (B) a multicloning site or a nucleic acid sequence encoding a polypeptide of interest, (C) a recombinase site of said third recombinase, and (D) a second recombinase site of said second recombinase, wherein said first cell expresses said second recombinase and said third recombinase, wherein recombination occurs within said first cell to form said nucleic acid encoding said recombinant virus, wherein said nucleic acid encoding said recombinant virus comprises said first recombinase site of said first recombinase, said packaging sequence or said viral sequence that can regulate viral packaging and / or propagation, said nucleic acid encoding said E1A polypeptide (when present), said promoter (when present), a recombinase site of said second recombinase or a remnant thereof post recombination, said multicloning site or said nucleic acid sequence encoding said polypeptide of interest, said recombinase site of said third recombinase or a remnant thereof post recombination, and said nucleic acid encoding said E1B polypeptide (when present); and(b) delivering viral vectors produced by said first cell to a second in vitro cell, wherein said second cell expresses said first recombinase, wherein recombination occurs within said second cell on said viral vector, if present, to reduce the presence of nucleic acid not encoding said recombinant virus.

121. The method of claim 120, wherein said nucleic acid encoding said recombinant virus lacks said second recombinase site of said first recombinase.

122. The method of any one of claims 120-121, wherein said first recombinase site and said second recombinase site of said engineered DNA sequence are FRT sites.

123. The method of any one of claims 120-122, wherein said recombinase site of said second recombinase is a loxP site.

124. The method of any one of claims 120-123, wherein said recombinase site of said third recombinase is an attP site.

125. The method of any one of claims 120-124, wherein said engineered DNA sequence comprises, between said (vii) and said optional (viii), a polyA sequence.

126. The method of any one of claims 120-125, wherein said engineered DNA sequence comprises said (iv), and wherein said promoter is selected from the group consisting of a CMV promoter, a RSV promoter, a CAG promoter, and a EFla promoter.

127. The method of any one of claims 120-126, wherein said first ITR sequence is directly followed by said engineered DNA sequence.

128. The method of any one of claims 120-126, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.

129. The method of any one of claims 120-128, wherein said engineered DNA sequence is directly followed by said second ITR sequence.

130. The method of any one of claims 120-128, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.

131. The method acid of any one of claims 120-130, wherein said first recombinase site and said second recombinase site of said donor nucleic acid are loxP sites.

132. The method of any one of claims 120-131, wherein said recombinase site of said second recombinase is an attB site.

133. The method of any one of claims 120-132, wherein said viral vector is an adenoviral vector.

134. The method of any one of claims 120-133, wherein said engineered DNA sequence comprises said packaging sequence.

135. The method of any one of claims 120-133, wherein said engineered DNA sequence comprises said viral sequence that can regulate viral packaging and / or propagation.

136. The method of claim 135, wherein said viral sequence that can regulate viral packaging and / or propagation is selected from the group consisting of a poly adenylation signal, a splicing signal, a viral promoter, and nucleic acid encoding a polypeptide that can promote vial propagation.

137. The method of any one of claims 120-136, wherein said engineered DNA sequence comprises said second recombinase site of said first recombinase.

138. The method of any one of claims 120-136, wherein said engineered DNA sequence comprises said sequence that can be targeted by said targeting sequence.

139. The method of claim 138, where said targeting sequence is a guide RNA.

140. The method of any one of claims 120-139, wherein said donor nucleic acid comprises said multi cloning site.

141. The method of any one of claims 120-140, wherein said donor nucleic acid comprises said nucleic acid sequence encoding said polypeptide of interest, or wherein said donor nucleic acid comprises a promotor sequence operably linked to said nucleic acid sequence encoding said polypeptide of interest.

142. The method of any one of claims 120-141, wherein said donor nucleic acid comprises said MCS and said nucleic acid sequence encoding said polypeptide of interest.

143. The method of any one of claims 120-142, wherein said donor nucleic acid is in the form of DNA plasmid.

144. The method of any one of claims 120-143, wherein said first cell is a eukaryotic cell.

145. The method of any one of claims 120-144, wherein said second recombinase comprises a Cre polypeptide.

146. The method of any one of claims 120-144, wherein said third recombinase comprises a Bxbl polypeptide.

147. The method of any one of claims 120-146, wherein said second cell is a eukaryotic cell.

148. The method of any one of claims 120-147, wherein said first recombinase comprises a FLP polypeptide.

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