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Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

ABSTRACT: Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 10¹¹ infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.

SUBMITTER: Cerqueira C

PROVIDER: S-EPMC5423317 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

Cerqueira Carla C Thompson Cynthia D CD Day Patricia M PM Pang Yuk-Ying S YS Lowy Douglas R DR Schiller John T JT

Molecular therapy. Methods & clinical development 20170419

Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer ...[more]

PMID: 28497074

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Project description:Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/-, KrasG12D+/-/Pten+/-, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.

Dataset Information

Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

Publications

Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

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