Heparin-binding correlates with increased efficiency of AAV1- and AAV6-mediated transduction of striated muscle, but negatively impacts CNS transduction

Abstract

Gene delivery vectors derived from adeno-associated virus (AAV) have great potential as therapeutic agents. rAAV1 and rAAV6, efficiently target striated muscle, but the mechanisms that determine their tropism remain unclear. It is known that AAV6, but not AAV1, interacts with heparin-sulfate proteoglycans (HSPG). HSPGs are not primary receptors for AAV6, but heparin interactions may affect tissue tropism and transduction. To investigate these possibilities, we generated rAAV1 and rAAV6 capsids that do or do not bind heparin. We evaluated the transduction profile of these vectors in vivo across multiple routes of administration, and found that heparin-binding capability influences tissue transduction in striated muscle and neuronal tissues. Heparin-binding capsids transduce striated muscle more efficiently than non-binding capsids, via both intramuscular and intravenous injection. However, rAAV6 achieved greater muscle transduction than the heparin-binding rAAV1 variant, suggesting that there are additional factors that influence differences in transduction efficiency between AAV1 and AAV6. Interestingly, the opposite trend was found when vectors were delivered via intracranial injection. Non-binding vectors achieved robust and widespread gene expression, whereas transduction via heparin-binding serotypes was substantially reduced. These data indicate that heparin-binding capability is an important determinant of transduction that should be considered in the design of rAAV-mediated gene therapies.

Figure 1

Salt elution profile of AAV2, AAV6, and AAV1 chimeric capsids. Capsids were passed through a heparin column and eluted with increasing concentrations of NaCl. Focus forming units (ffu) per ml were determined using various dilutions of fractions added to HT1080 cells seeded at 9 ×104 cells per well of a 12 well plate 24 hours pre-transduction. At 72 hours post-transduction cells were fixed and stained for hPLAP activity.

Figure 2

IM injection of rAAV vectors into tibialis anterior. Images are representative fields from cross-sections of tibialis anterior stained for alkaline phosphatase activity in weight-matched, adult mice. Injected serotype is indicated horizontally. Total vector genome dose is indicated in the left vertical column. vg, vector genomes. Scale bar: 50 μm.

Figure 3

Systemic injection: hPLAP protein expression profile and vector genome quantification. (a) Representative cross-sections of hearts collected from injected animals and stained for alkaline phosphatase activity. Injected serotype is indicated above each image. The levels of hPLAP activity [in relative light units (RLU) per μg protein] (b) and persistence of vector genomes [vg per μg total DNA] (c) were measured in select tissues following intravenous injection of 2 x1012 vector genomes into adult mice. Data are presented as mean values ±SEM. (n=3) Scale bar: 1 mm. *, p<0.05 **, p<0.01

Figure 4

Intracranial injection: hPLAP protein expression profile and vector genome quantification. Animals received 1 ×109 vector genomes in 0.75 μl, administered bilaterally within the dorsal striatum via stereotaxic injection. hPLAP activity [in relative light units (RLU) per μg protein] (a) and quantification of vector genomes [vg per μg total DNA] (b) are reported. Data are presented as mean values ±SEM. (n=5) Scale bar: 50 μm. * indicates statistically significant difference compared to AAV6 (p<0.05).