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A novel method for generating and screening peptides and libraries displayed on adenovirus fiber.

Lupold SE, Kudrolli TA, Chowdhury WH, Wu P, Rodriguez R - Nucleic Acids Res. (2007)

Bottom Line: Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology.The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene.For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urology Institute, Johns Hopkins University School of Medicine, Broadway Research Building 467, 733N Broadway, Baltimore, MD 21205, USA. slupold@jhmi.edu

ABSTRACT
Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

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Methods for site specifically transferring modified fiber gene cassettes into adenoviral plasmid vectors or replicating viral genomes in mammalian cells through unidirectional Cre–lox-mediated recombination. (A) The large viral plasmid vector pFex has two regions for accepting transgene cassettes, the Amp-resistant-E1-region cassette and the SacB-fiber-region cassette. The fiber-region-cassette can be recombined before (bottom) or after E1-region-cassette recombination (top). E1 cassettes are recombined through homologous recombination of adenovirus left-hand and right-hand homology regions in RecA positive BJ5183 coli. Recombinant plasmids are selected according to the newly acquired resistance cassette. On the other hand, fiber cassettes are recombined through half-mutant lox site (see legend: Green, wild-type half site; Red, mutant half site; Black and Gray represent non-compatible spacers) recombination in Cre recombinase expressing coli. Non-compatible spacers (gray center versus black center) prevent intragenic recombination. The resulting recombinant plasmids are selected by growth on sucrose containing plates. Following recombination, the donor plasmid product contains fully mutant lox sites (red circles), where the resulting recombinant vector contains fully wild-type lox sites (green circles); therefore, preventing any further recombination between the shuttles and viral vectors. The final resulting vectors are linearized and transfected into mammalian packaging cells to create viral particles (B) Fiber gene cassettes can be directly shuttled into adenoviral genomes in mammalian cells. E1-cassette-containing pFex vectors, such as AdTrack-pFex, can be pseudotyped (packaged in cells that express wild-type fiber) and used to infect packaging cell lines that express Cre recombinase. Transfection of infected cells with a fiber-gene shuttle results in site specific incorporation of the fiber cassette into replicative adenovirus. Only following recombination into a viral genome can the modified fiber gene be expressed. The resulting recombinant virus will be packaged in the newly synthesized modified-fiber capsids and amplified in other cell lines, such as 911–S11.
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Figure 1: Methods for site specifically transferring modified fiber gene cassettes into adenoviral plasmid vectors or replicating viral genomes in mammalian cells through unidirectional Cre–lox-mediated recombination. (A) The large viral plasmid vector pFex has two regions for accepting transgene cassettes, the Amp-resistant-E1-region cassette and the SacB-fiber-region cassette. The fiber-region-cassette can be recombined before (bottom) or after E1-region-cassette recombination (top). E1 cassettes are recombined through homologous recombination of adenovirus left-hand and right-hand homology regions in RecA positive BJ5183 coli. Recombinant plasmids are selected according to the newly acquired resistance cassette. On the other hand, fiber cassettes are recombined through half-mutant lox site (see legend: Green, wild-type half site; Red, mutant half site; Black and Gray represent non-compatible spacers) recombination in Cre recombinase expressing coli. Non-compatible spacers (gray center versus black center) prevent intragenic recombination. The resulting recombinant plasmids are selected by growth on sucrose containing plates. Following recombination, the donor plasmid product contains fully mutant lox sites (red circles), where the resulting recombinant vector contains fully wild-type lox sites (green circles); therefore, preventing any further recombination between the shuttles and viral vectors. The final resulting vectors are linearized and transfected into mammalian packaging cells to create viral particles (B) Fiber gene cassettes can be directly shuttled into adenoviral genomes in mammalian cells. E1-cassette-containing pFex vectors, such as AdTrack-pFex, can be pseudotyped (packaged in cells that express wild-type fiber) and used to infect packaging cell lines that express Cre recombinase. Transfection of infected cells with a fiber-gene shuttle results in site specific incorporation of the fiber cassette into replicative adenovirus. Only following recombination into a viral genome can the modified fiber gene be expressed. The resulting recombinant virus will be packaged in the newly synthesized modified-fiber capsids and amplified in other cell lines, such as 911–S11.

Mentions: We have generated a novel vector system for modifying the fiber gene region of the widely applied Adenovirus vector, pAdEasy-1 (13). The vector system, outlined in Figure 1, applies an ‘acceptor’ vector, pFex, and fiber ‘donor’ vectors, RP-FBR or RPuc-FBR (depending on required antibiotic). The pFex acceptor vector is a modified version of AdEasy-1, where the fiber gene and upstream TPL splice-acceptor-site (Ad5 nucleotides 31 023–32 768) have been replaced with the Bacillus subtilis (SacB) gene. SacB imparts negative selectivity to bacteria grown in 5% sucrose and thus offers a means to negatively select against non-recombined acceptor vectors (16). Correspondingly, the donor vectors contain the modified fiber gene, TPL splice acceptor site and regions for additional transgene incorporation. Both donor and acceptor gene cassettes are flanked by non-compatible half-mutant lox sites. As delineated by Langer et al. (15), the use of these specialized lox sites results in a unidirectional gene exchange with maintained orientation and lack of alternative recombination events. In the presence of Cre Recombinase, the fiber gene cassette replaces SacB, placing fiber in the natural location of the adenovirus genome. By including the natural TPL splice-acceptor sequence in the fiber shuttles, upstream lox sites are not included in the final spliced fiber transcript.Figure 1.


A novel method for generating and screening peptides and libraries displayed on adenovirus fiber.

Lupold SE, Kudrolli TA, Chowdhury WH, Wu P, Rodriguez R - Nucleic Acids Res. (2007)

Methods for site specifically transferring modified fiber gene cassettes into adenoviral plasmid vectors or replicating viral genomes in mammalian cells through unidirectional Cre–lox-mediated recombination. (A) The large viral plasmid vector pFex has two regions for accepting transgene cassettes, the Amp-resistant-E1-region cassette and the SacB-fiber-region cassette. The fiber-region-cassette can be recombined before (bottom) or after E1-region-cassette recombination (top). E1 cassettes are recombined through homologous recombination of adenovirus left-hand and right-hand homology regions in RecA positive BJ5183 coli. Recombinant plasmids are selected according to the newly acquired resistance cassette. On the other hand, fiber cassettes are recombined through half-mutant lox site (see legend: Green, wild-type half site; Red, mutant half site; Black and Gray represent non-compatible spacers) recombination in Cre recombinase expressing coli. Non-compatible spacers (gray center versus black center) prevent intragenic recombination. The resulting recombinant plasmids are selected by growth on sucrose containing plates. Following recombination, the donor plasmid product contains fully mutant lox sites (red circles), where the resulting recombinant vector contains fully wild-type lox sites (green circles); therefore, preventing any further recombination between the shuttles and viral vectors. The final resulting vectors are linearized and transfected into mammalian packaging cells to create viral particles (B) Fiber gene cassettes can be directly shuttled into adenoviral genomes in mammalian cells. E1-cassette-containing pFex vectors, such as AdTrack-pFex, can be pseudotyped (packaged in cells that express wild-type fiber) and used to infect packaging cell lines that express Cre recombinase. Transfection of infected cells with a fiber-gene shuttle results in site specific incorporation of the fiber cassette into replicative adenovirus. Only following recombination into a viral genome can the modified fiber gene be expressed. The resulting recombinant virus will be packaged in the newly synthesized modified-fiber capsids and amplified in other cell lines, such as 911–S11.
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Figure 1: Methods for site specifically transferring modified fiber gene cassettes into adenoviral plasmid vectors or replicating viral genomes in mammalian cells through unidirectional Cre–lox-mediated recombination. (A) The large viral plasmid vector pFex has two regions for accepting transgene cassettes, the Amp-resistant-E1-region cassette and the SacB-fiber-region cassette. The fiber-region-cassette can be recombined before (bottom) or after E1-region-cassette recombination (top). E1 cassettes are recombined through homologous recombination of adenovirus left-hand and right-hand homology regions in RecA positive BJ5183 coli. Recombinant plasmids are selected according to the newly acquired resistance cassette. On the other hand, fiber cassettes are recombined through half-mutant lox site (see legend: Green, wild-type half site; Red, mutant half site; Black and Gray represent non-compatible spacers) recombination in Cre recombinase expressing coli. Non-compatible spacers (gray center versus black center) prevent intragenic recombination. The resulting recombinant plasmids are selected by growth on sucrose containing plates. Following recombination, the donor plasmid product contains fully mutant lox sites (red circles), where the resulting recombinant vector contains fully wild-type lox sites (green circles); therefore, preventing any further recombination between the shuttles and viral vectors. The final resulting vectors are linearized and transfected into mammalian packaging cells to create viral particles (B) Fiber gene cassettes can be directly shuttled into adenoviral genomes in mammalian cells. E1-cassette-containing pFex vectors, such as AdTrack-pFex, can be pseudotyped (packaged in cells that express wild-type fiber) and used to infect packaging cell lines that express Cre recombinase. Transfection of infected cells with a fiber-gene shuttle results in site specific incorporation of the fiber cassette into replicative adenovirus. Only following recombination into a viral genome can the modified fiber gene be expressed. The resulting recombinant virus will be packaged in the newly synthesized modified-fiber capsids and amplified in other cell lines, such as 911–S11.
Mentions: We have generated a novel vector system for modifying the fiber gene region of the widely applied Adenovirus vector, pAdEasy-1 (13). The vector system, outlined in Figure 1, applies an ‘acceptor’ vector, pFex, and fiber ‘donor’ vectors, RP-FBR or RPuc-FBR (depending on required antibiotic). The pFex acceptor vector is a modified version of AdEasy-1, where the fiber gene and upstream TPL splice-acceptor-site (Ad5 nucleotides 31 023–32 768) have been replaced with the Bacillus subtilis (SacB) gene. SacB imparts negative selectivity to bacteria grown in 5% sucrose and thus offers a means to negatively select against non-recombined acceptor vectors (16). Correspondingly, the donor vectors contain the modified fiber gene, TPL splice acceptor site and regions for additional transgene incorporation. Both donor and acceptor gene cassettes are flanked by non-compatible half-mutant lox sites. As delineated by Langer et al. (15), the use of these specialized lox sites results in a unidirectional gene exchange with maintained orientation and lack of alternative recombination events. In the presence of Cre Recombinase, the fiber gene cassette replaces SacB, placing fiber in the natural location of the adenovirus genome. By including the natural TPL splice-acceptor sequence in the fiber shuttles, upstream lox sites are not included in the final spliced fiber transcript.Figure 1.

Bottom Line: Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology.The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene.For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urology Institute, Johns Hopkins University School of Medicine, Broadway Research Building 467, 733N Broadway, Baltimore, MD 21205, USA. slupold@jhmi.edu

ABSTRACT
Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

Show MeSH
Related in: MedlinePlus