Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

doi: 10.1016/j.omtm.2019.01.003

Figure Lengend Snippet: Recombinant HBoV1 Vector Production and Analysis of Packaging Capacity (A) Plasmids for rAAV/HBoV1 production using either the 4-plasmid (constructs 1–4) or 3-plasmid (constructs 1, 2+3, and 4) transfection protocols. In both systems, the plasmids are co-transfected into HEK293T cells, which are then harvested 72 h later. To release the viral particles, cells are subjected to five freeze-thaw cycles, before free plasmid DNA is digested with benzonase. The resulting crude lysate is purified using iodixanol gradient centrifugation, and the vector-containing 40% phase is collected. (B) Purification of scAAV-YFP/HBoV1 via iodixanol gradient centrifugation. Shown is the distribution of benzonase-resistant particles in the different indicated iodixanol fractions. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan RT-PCR. (C) Production of scAAV-YFP/HBoV1 using the 3- or 4-plasmid transfection protocols. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan real-time PCR. (D) Oversized ssAAV-CRISPR constructs used in this work. Sp Cas9 and gRNA cassettes are expressed from different RNA polymerase II (Pol II) (first column) or Pol III (second column) promoters, respectively. Total genome sizes are shown in the third column. (E) Southern blot analysis of the ssAAV-CRISPR genomes from (D), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were detected with a probe against Sp Cas9. (F) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. (G) Oversized scAAV genomes used in this work. Stuffer sequences from lacZ with the indicated lengths (first column) were inserted to increase the total genome size (second column). (H) Southern blot analysis of the scAAV-YFP genomes from (G), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were labeled with a probe against yfp . (I) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. ss, single-stranded; sc, self-complementary.

Article Snippet: Oversized ssAAV vector constructs expressing Cas9 from Streptococcus pyogenes ( Sp ) were derived from our previously reported Cas9 expression plasmid containing the Sp Cas9 cDNA from the Church lab (Addgene plasmid 41815).

Techniques: Recombinant, Plasmid Preparation, Construct, Transfection, Purification, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, CRISPR, Southern Blot, Isolation, Agarose Gel Electrophoresis, Molecular Weight, Labeling