1. Volume 14, Issue 1, Pages 97-106 (July 2006)
Metabolic Biotinylation Provides a Unique Platform for the Purification and Targeting of Multiple AAV Vector Serotypes 
Gregory S. Arnold, A. Kate Sasser, Matthew D. Stachler, Jeffrey S. Bartlett 
Molecular Therapy 
Volume 14, Issue 1, Pages (July 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Particle titers and infectious titers of BAP-modified AAV vectors. DNase-resistant particle (DRP) titers were determined by real-time PCR assay [18] and are expressed as a percentage of the titer of unmodified AAV vector particles of the same serotype. Infectious titers were determined by gene transduction assay on HeLa C12 cells. Adenovirus-infected HeLa C12 cells (3 IU/cell) were exposed for 2 h at 37°C to BAP-modified AAV vectors or standard AAV vector (unmodified) containing eGFP transgenes at the same multiplicity of infection (DRP/cell), which varied depending on the serotype being evaluated. Fresh medium was added and the cells were analyzed by FACS for eGFP expression after 48 h. Infectious titer of each vector with BAP-modified capsid, expressed as a ratio of gene transducing units (TU) per DRP, is presented as a percentage of the infectious titer (TU/DRP) of unmodified AAV vector of the same serotype. All data are shown as the means of triplicate determinations from three to five different vector preparations. Bars indicate the standard error of the mean. (A) AAV2-based vectors with BAP-modified capsids and (B) AAV1-, AAV3-, AAV4-, and AAV5-based vectors with BAP-modified capsids. Vectors are designated by capsid protein, and all contained the same eGFP transgene flanked by AAV2 ITRs.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Biotinylation of BAP-modified AAV capsid proteins. BAP-modified AAV helper plasmids, or unmodified AAV helper plasmids, were used for the production of recombinant AAV by triple transfection in HEK 293 cells as previously described [8]. In some instances pEGFP-BirA, containing the E. coli biotin ligase gene, was also transfected into these cells to mediate biotinylation of the engineered BAP motif. Cell extracts were fractionated on iodixanol gradients after 48 h to select assembled AAV particles and then analyzed by Western blot using mAb b1, to detect AAV capsid protein, or SAvHRP, to detect biotinylated proteins. Vector preparations are designated by serotype and capsid protein modification. Preparations that received the BirA plasmid are designated by the “biotin” prefix (e.g., biotin.AAV1.D590_P591insBAP). (A) Detection of BAP-modified and unmodified AAV capsid proteins. Proteins were detected with anti-AAV2 capsid mAb b1, which recognizes a shared motif common to the other AAV serotype, with the exception of AAV4. (B) Detection of biotinylated capsid proteins with SAvHRP.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Solid-phase binding of AAV vectors with biotinylated BAP-modified capsids to streptavidin. Iodixonal-fractionated AAV vector preparations with unmodified capsid, BAP-modified capsid, or biotinylated BAP-modified capsid were adsorbed nonspecifically to assay plates. The plates were blocked and the accessibility of biotin on the virion surface was assessed by detecting the ability of immobilized AAV particles to capture SAvHRP (SAv). The assay was performed in triplicate using several different vector preparations. To assess differences in adsorption caused by capsid modification, AAV2 and BAP-modified AAV2 particles were also detected by standard ELISA using anti-AAV2 virion mAb (A20) as previously described [8].
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Purification of biotinylated BAP-modified AAV vectors on immobilized monomeric avidin-affinity resin. Biotinylated AAV1-based eGFP vector was produced in HEK 293 cells by transfection of pXR1-Cap1.D590_P591insBAP, pEGFP-BirA, and standard recombinant AAVeGFP and adenovirus helper plasmids [8]. Packaging cell lysate was fractionated on an iodixanol step gradient and applied to the avidin-affinity column by gravity flow. Flowthrough (FT) was collected, the matrix was washed three times with PBS, and each wash fraction was collected (W1, W2, and W3). Virus was eluted with PBS, pH 8.0, containing 150 mM NaCl, 1.0% DOC, and 5 mM biotin (E). The indicated fractions were analyzed for: (A) yield of purified AAV particles by DRP titer, expressed as a percentage of DRP applied to the column, and (B) AAV capsid protein, biotinylated protein, and total protein, by Western blot with anti-AAV capsid antibody (B1 mAb), streptavidin-HRP (SAv), and Sypro tangerine staining (Sypro tangerine), respectively.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Biotinylated AAV vectors purified on immobilized monomeric avidin-affinity resin maintain endogenous tissue tropism and effectiveness in vivo. (A) The transduction efficiency of the biotin elution fraction following monomeric avidin-affinity purification of biotinylated AAV1eGFP vector (AAV1.D590_P591insBAP capsid) was assessed by injection of 3 × 1010 DRP into the tibialis anterior muscles of immunocompetent BALB/c mice. (B) Standard AAV1eGFP vector purified by ion-exchange chromatography [25] was used as a control. Muscles were harvested 28 days after injection and frozen, cut, and stained for eGFP expression using a rabbit anti-GFP–Alexa 488-conjugated antibody. Representative sections are shown. (C) Quantification of eGFP levels by ELISA-based assay. The mean eGFP levels (pg/μg total cellular protein) are shown for mice injected with AAV1eGFP (n = 3) and biotinylated AAV1eGFP (biotin.AAV1eGFP) (n = 3). Results demonstrate equivalent in vivo gene transduction efficiency.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Targeting of biotinylated BAP-modified AAV vectors to receptor-bearing cells. For assessment of targeted gene transduction, rat glioma BT4C cells expressing the artificial biotin receptor Scavidin [28] were transduced with purified AAVeGFP vector preparations (100 DRP/cell) and analyzed for eGFP expression after 72 h. (A) Gene transduction mediated by AAV1eGFP. (B) Gene transduction mediated by AAVeGFP vector with biotinylated AAV1.D590_P591insBAP capsid.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions