- Email: [email protected]
409. Necrosis and Autophagy, but Not Apoptosis, Are Induced in Macrophages In Vivo upon Interaction with Adenovirus
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II the function of AAPs derived from various serotypes. The current system reported in literature relies on mouse monoclonal antibody (mAb) A20, which reacts specifically with assembled AAV2 particles by recognizing an epitope(s) that is present only in the assembled but not in the unassembled VP proteins. Although the system based on mAbs against intact AAV particles is reliable and straightforward, the availability of such mAbs for serotypes other than AAV2 is currently very limited; therefore, it would not be easy to expand its application to various serotypes. To overcome this limitation, we constructed an AAV capsid assembly reporter, AAV-Rep2, and sought to exploit this reporter to indirectly assess the AAV capsid assembly. AAV-Rep2 carried only the AAV2 rep gene between the two AAV2 inverted terminal repeats; therefore it could express Rep proteins but required the cap gene expression in trans for its viral genomes to be packaged in virion shells. HEK293 cells were co-transfected with pAAV-Rep2 (a plasmid containing the AAV-Rep2 genome), pCMV-AAV9-VP3 (a plasmid expressing only AAV9-VP3) and an adenovirus helper plasmid in the presence or absence of the fourth plasmid expressing AAP2 (the reported AAP derived from AAV2) or AAP9 (a putative AAP derived from AAV9). At 48-hours post-transfection, DNase I-resistant AAV-Rep2 viral genomes were quantified by a dot blot assay. As a result, our new system could identify the functional AAP9 amino-acid sequence and successfully demonstrate the requirement of AAP expression in efficient AAV9 capsid assembly and the ability to complement the function of an AAP derived from one serotype with that from another. Thus, the new system we report here provides a powerful and reliable means to study the function of AAPs derived from various serotypes and serves as a tool for AAV capsid engineering that disrupts the AAP ORFs.
408. Directed Evolution of Adeno-Associated Virus: Construction of an Enriched Random Insertion Library and Artificial Selection To Identify Structure-Function Relationships Justin H. Judd,1 Jonathan Silberg,1,2 Junghae Suh.1 Bioengineering, Rice University, Houston, TX; 2Biochemistry and Cell Biology, Rice University, Houston, TX.
1
Adeno-associated virus (AAV) is a promising clinical therapeutic gene delivery vector due to its unusually high safety profile. Still, virus targeting is a critical parameter in successful gene delivery that has yet to be entirely resolved. Using the crystal structure of AAV2 as a structural guide, rational design has been used to achieve vector targeting through ligand-receptor interactions. However, detailed a priori knowledge of protein structure and function is still elusive, often precluding direct translation of known cell-targeting ligands into the context of the AAV capsid. Directed evolution is an attractive alternative for altering virus tropism for which success has been reported using mutagenic techniques such as error-prone PCR and gene shuffling. Transposon-based random insertion or random domain insertion by DNAse can be used to identify ideal insertion sites for known targeting ligands. However, limitations to these approaches include a high frequency of frameshifted, non-viable mutants and/ or limited crossover diversity, reducing the output of ensuing, often tedious, selection efforts. To address these vector design challenges, we have developed a versatile, enriched, random insertion library using the AAV2 capsid gene (cap) as a scaffold. Briefly, a plasmid carrying cap was randomly linearized, blunted and ligated to a linker containing directional, unique, flanking restriction sites encoding small amino acids. Cap genes with a single insert were isolated and non-viable mutants were removed from the pool. Remaining cap genes - containing a single, in-frame, correctly oriented, randomly inserted linker - were subcloned into an AAV expression vector and the resultant library named pAAV_RaPID (Random Peptide Insertion by DNase). The pAAV_RaPID plasmid library can be used to quickly insert any peptide motif randomly into the AAV cap gene S158
with a high frequency of viable variants and broad crossover which should increase the occurrence of functional AAV variants in directed evolution studies. Additionally, we demonstrate that the carefully selected restrictions sites used in the construction of the pAAV_RaPID library facilitate a novel vector-religation/recombination event, resulting in a high-throughput scanning alanine substitution library with enhanced diversity. Furthermore, we show the utility of this latter library variation in quickly identifying important functional motifs in the AAV2 capsid protein using simple in vitro selection methods.
Adenovirus and Other DNA Virus Vectors II 409. Necrosis and Autophagy, but Not Apoptosis, Are Induced in Macrophages In Vivo upon Interaction with Adenovirus Nelson C. Di Paolo,1 Dmitry M. Shayakhmetov.1 Medical Genetics, University of Washington, Seattle, WA.
1
Several homeostatic and pathological cell death mechanisms exist, including apoptosis, pyroptosis, necrosis, and others. We are interested in understanding the cell death pathway induced in macrophages in vivo in response to a model virus, adenovirus (Ad). Our studies in mouse models revealed that splenic methallophilic CD169- and MARCO-positive marginal zone macrophages (MZMø) selectively trap Ad after intravascular inoculation. After interaction with Ad, these cells undergo a rapid, caspase-independent pro-inflammatory cell death that is distinct from classical apoptosis. Staining of spleen sections with TUNEL or activated caspase-3-specific antibody revealed that MZMø undergo a caspase-3-independent type of cell death. However, injection of mice with propidium iodine (PI) revealed numerous cells with PI-positive nuclei in splenic MZ 1h after virus administration, indicating that the plasma membrane integrity was compromised, a hallmark of necrotic type of cell death. Furthermore, we found that Ad entry into MZMø induces formation of LC3-positive autophagosomes. Electron microscopy studies showed that MZMø containing virus particles have complete disorganization of the cytoplasm and swelling and breakdown of mitochondria and other organelles by 4h after Ad challenge. Interestingly, administration of a mutant Ad, Ad-ts1, which is unable to rupture cellular endosomes, showed that both CD169- and MARCO-positive cells survived in the splenic marginal zone after virus injection, suggesting a link between endosome rupture and the induction of necrotic type cell death in macrophages. These results shed light on the unique mechanism of the adenovirus-induced macrophage death, as well as broaden our understanding of the biology of adenovirus that is being intensely studied for vaccination and as cancer therapeutic.
410. Adenovirus-Mediated NIS Expression in Xenografted Prostate Tumors Requires a Minimal Threshold of Viral Dose for Efficient Radioiodine Uptake
Miguel A. Trujillo,1 Michael J. Oneal,2 Samantha McDonough,1 John C. Morris.1 1 Endocrinology, Mayo Clinic Rochester, Rochester, MN; 2 Molecular Medicine, Mayo Clinic Rochester, Rochester, MN.
Prostate cancer (PCa) remains the most common malignancy in men, with an estimated 217,730 new cases and 32,050 deaths in the United States in 2010. Despite new aggressive approaches, there is a high rate of recurrence. Gene therapy/virotherapy (GTV) strategies represent a rational direction for the development of novel therapeutics. However, the parameters required for effective GTV such as viral replication, spread, and gene transfer efficiency are not well validated. Clinical trials have demonstrated extremely limited tumor transduction, and this limitation remains the fundamental Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
408. Directed Evolution of Adeno-Associated Virus: Construction of an Enriched Random Insertion Library and Artificial Selection To Identify Structure-Function Relationships Justin H. Judd,1 Jonathan Silberg,1,2 Junghae Suh.1 Bioengineering, Rice University, Houston, TX; 2Biochemistry and Cell Biology, Rice University, Houston, TX.
1
Adeno-associated virus (AAV) is a promising clinical therapeutic gene delivery vector due to its unusually high safety profile. Still, virus targeting is a critical parameter in successful gene delivery that has yet to be entirely resolved. Using the crystal structure of AAV2 as a structural guide, rational design has been used to achieve vector targeting through ligand-receptor interactions. However, detailed a priori knowledge of protein structure and function is still elusive, often precluding direct translation of known cell-targeting ligands into the context of the AAV capsid. Directed evolution is an attractive alternative for altering virus tropism for which success has been reported using mutagenic techniques such as error-prone PCR and gene shuffling. Transposon-based random insertion or random domain insertion by DNAse can be used to identify ideal insertion sites for known targeting ligands. However, limitations to these approaches include a high frequency of frameshifted, non-viable mutants and/ or limited crossover diversity, reducing the output of ensuing, often tedious, selection efforts. To address these vector design challenges, we have developed a versatile, enriched, random insertion library using the AAV2 capsid gene (cap) as a scaffold. Briefly, a plasmid carrying cap was randomly linearized, blunted and ligated to a linker containing directional, unique, flanking restriction sites encoding small amino acids. Cap genes with a single insert were isolated and non-viable mutants were removed from the pool. Remaining cap genes - containing a single, in-frame, correctly oriented, randomly inserted linker - were subcloned into an AAV expression vector and the resultant library named pAAV_RaPID (Random Peptide Insertion by DNase). The pAAV_RaPID plasmid library can be used to quickly insert any peptide motif randomly into the AAV cap gene S158
with a high frequency of viable variants and broad crossover which should increase the occurrence of functional AAV variants in directed evolution studies. Additionally, we demonstrate that the carefully selected restrictions sites used in the construction of the pAAV_RaPID library facilitate a novel vector-religation/recombination event, resulting in a high-throughput scanning alanine substitution library with enhanced diversity. Furthermore, we show the utility of this latter library variation in quickly identifying important functional motifs in the AAV2 capsid protein using simple in vitro selection methods.
Adenovirus and Other DNA Virus Vectors II 409. Necrosis and Autophagy, but Not Apoptosis, Are Induced in Macrophages In Vivo upon Interaction with Adenovirus Nelson C. Di Paolo,1 Dmitry M. Shayakhmetov.1 Medical Genetics, University of Washington, Seattle, WA.
1
Several homeostatic and pathological cell death mechanisms exist, including apoptosis, pyroptosis, necrosis, and others. We are interested in understanding the cell death pathway induced in macrophages in vivo in response to a model virus, adenovirus (Ad). Our studies in mouse models revealed that splenic methallophilic CD169- and MARCO-positive marginal zone macrophages (MZMø) selectively trap Ad after intravascular inoculation. After interaction with Ad, these cells undergo a rapid, caspase-independent pro-inflammatory cell death that is distinct from classical apoptosis. Staining of spleen sections with TUNEL or activated caspase-3-specific antibody revealed that MZMø undergo a caspase-3-independent type of cell death. However, injection of mice with propidium iodine (PI) revealed numerous cells with PI-positive nuclei in splenic MZ 1h after virus administration, indicating that the plasma membrane integrity was compromised, a hallmark of necrotic type of cell death. Furthermore, we found that Ad entry into MZMø induces formation of LC3-positive autophagosomes. Electron microscopy studies showed that MZMø containing virus particles have complete disorganization of the cytoplasm and swelling and breakdown of mitochondria and other organelles by 4h after Ad challenge. Interestingly, administration of a mutant Ad, Ad-ts1, which is unable to rupture cellular endosomes, showed that both CD169- and MARCO-positive cells survived in the splenic marginal zone after virus injection, suggesting a link between endosome rupture and the induction of necrotic type cell death in macrophages. These results shed light on the unique mechanism of the adenovirus-induced macrophage death, as well as broaden our understanding of the biology of adenovirus that is being intensely studied for vaccination and as cancer therapeutic.
410. Adenovirus-Mediated NIS Expression in Xenografted Prostate Tumors Requires a Minimal Threshold of Viral Dose for Efficient Radioiodine Uptake
Miguel A. Trujillo,1 Michael J. Oneal,2 Samantha McDonough,1 John C. Morris.1 1 Endocrinology, Mayo Clinic Rochester, Rochester, MN; 2 Molecular Medicine, Mayo Clinic Rochester, Rochester, MN.
Prostate cancer (PCa) remains the most common malignancy in men, with an estimated 217,730 new cases and 32,050 deaths in the United States in 2010. Despite new aggressive approaches, there is a high rate of recurrence. Gene therapy/virotherapy (GTV) strategies represent a rational direction for the development of novel therapeutics. However, the parameters required for effective GTV such as viral replication, spread, and gene transfer efficiency are not well validated. Clinical trials have demonstrated extremely limited tumor transduction, and this limitation remains the fundamental Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy