Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity

Abstract

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

FIG. 1.

Construction and characterization of serotype clones. (A) Each serotype capsid domain, generated by PCR, was cloned into the pBS+ AAV2rep plasmid. The serotype-specific capsid insertions (shaded rectangles) were inserted into pBS+ AAV2rep and are listed in order from type 1 to 5. Restriction sites are shown in the AAV2 diagram. Additionally, modifications containing the coding region of the carboxy termini of each serotype’s rep coding domain (gray hatched) were cloned into the constructs as needed. (B) Acrylamide gel of AAV serotypes 1 through 5 digested with BstNI. White arrowheads point to common bands in the backbone and replication gene. The 50-bp ladder (Amersham Pharmacia) flanks the serotype lanes.

FIG. 2.

Western blot of cell lysates from serotype-specific transfections. At 24 h after triple transfection, 5 μg of total protein was loaded into each well. After transfer the blots were incubated with the anti-Rep monoclonal antibody 1F11, with the sizes of the proteins listed on the right side (A), or the anticapsid monoclonal antibody B1, with the capsid subunits listed on the right side (B). The serotype-specific helpers used in the transfection are listed above each blot. (C) B1 recognition site as determined by Wobus et al. (44) is shown at the bottom of the figure. The amino acid sequence from this region for all five serotyes is shown. Asterisks indicate amino acids identical to those of type 2.

FIG. 3.

Hirt assay. Low-molecular-weight DNA was isolated from 293 cells, at 24 h after triple transfected with serotype-specific plasmids. Then, 2.5 μg of undigested (lanes 1 to 5) and DpnI-digested DNA (lanes 7 to 11) from each serotype sample was loaded onto a 1% agarose gel (lane 6, DNA ladder). After transfer the blot was probed with a 735-bp fragment of the GFP gene. Input DNA digested with DpnI reveals the replicating monomer and dimer transgene (arrowed). The lower bands in lanes 7 to 11 are DpnI digestion products of input plasmids.

FIG. 4.

Transduction efficiency of fractions off a heparin-Sepharose affinity column for rAAV serotypes 1 through 5. The elution conditions were optimized originally for AAV2 (50). Numbered fractions were collected in 0.5-ml volumes, waste (W) was collected in a single 10-ml fraction, and the control (C) was a 1-ml aliquot of virus applied to the column. Infections were done in reference cell lines, with between 1/100 to 20 μl of each fraction. Salt elution began with fraction 26. Each bar represents the average of three separate infections, with the standard deviation indicated by an error bar.

FIG. 5.

Transduction efficiency in CHO mutant and reference cell lines. Cells (cell types are specified in the legend on the right) were transduced with ca. 0.3 TU/cell, as determined by the reference cell lines (HeLa cells for AAV serotypes 1, 2, 3, and 5 and Cos1 cells for AAV4). The transducing titers are given for each serotype in each cell line. The particle numbers used in this experiment (per microliter) were as follows: rAAV1, 1.2 × 10⁸; rAAV2, 5.6 × 10⁸; rAAV3, 9.1 × 10⁸; rAAV4, 2.3 × 10⁸; and rAAV5, 5.4 × 10⁸. Each bar represents the average of three separate infections, with the standard deviation indicated by an error bar.

FIG. 6.

Subretinal injection and in vivo fluorescence imaging. Subretinal injections were performed via a transcleral transchoroidal approach on wild-type Wistar rats, as previously described (33). Briefly, the sclera and the choroid were punctured, and a 33-gauge needle was then inserted in a tangential direction under an operating microscope. Three microliters of each of the five rAAV serotypes (5 × 10¹⁰ particles/ml) was delivered into the subretinal space of rats (n = 3). A new method using fundus photography has been developed and was performed here in order to control the accuracy and reproducibility of subretinal injections (Rolling et al., unpublished). GFP protein expression in live rats was monitored by fluorescent retinal imaging with a Canon UVI retinal camera connected to a digital imaging system (Lhedioph Win Software). Retinas were examined at 12, 26, and 46 days postinjection.