Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus

Abstract

Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs.

Figure 1

Maps of scAAV and ssAAV. (A) A schematic representation of the single-stranded (ss) AAV genome containing a full-length actin intron as well as a Neomycin resistance cassette. This construct contains the CBA promoter driving the expression of GFP. (B) A schematic representation of the self-complementary (sc) AAV genome containing a mutated 5' inverted terminal repeat (ITR), an actin intron from which 722 nucleotides have been deleted, and no Neomycin resistance cassette. This construct also contains the CBA promoter driving the expression of GFP. Both of these constructs contain the AAV2 ITRs and were packaged into AAV1 capsids.

Figure 2

Rapid onset of gene expression using scAAV in the murine heart. CD-1 mice (Charles Rivers) were injected 4 days after birth with 1.85 × 10¹¹ vector genomes (vg) of scAAV using previously established methods [14]. Animals were returned to their dams for 4 days (A) or 6 days (B) at which time their hearts were harvested, cut into thirds representing the apical region (apex), the mid-region (mid), or base of the heart (base). The tissues were fixed in 4% paraformaldehyde for 12 hours, rinsed in PBS and allowed to equilibrate in 20% sucrose for 12–24 hours. The next day, the hearts were frozen in cryomolds containing OCT compound (Tissue-Tek) to prepare for cryostat sectioning into 5 μm sections. Slides were mounted using Vectashield mounting media containing DAPI (Vector Labs) to counter stain the nuclei. A Leica TCS SP2 AOBS Spectral Confocal Microscope with a 10× objective was used to obtain fluorescent images. Staining was documented using the Leica Confocal Software (LCS) Version 2.61. Sections with GFP expression can be seen in the top panels while merged images containing GFP and DAPI signals can be seen in the lower panels. The bar represents 150 μm in the 6 day apex and 300 μm in all other panels.

Figure 3

Increased gene expression with scAAV after longer intervals. CD-1 mice (Charles Rivers) injected 4 days after birth with 1.85 × 10¹¹ vector genomes (vg) of either scAAV or ssAAV using sub-xiphoid injections [14]. The animals were returned to their dams for 11 days (A) or 21 days (B) and then processed as described in Figure 2. Representative sections with GFP fluorescence are pictured. The bar represents 150 μm in the 21 day ssAAV-apex and 300 μm in all other panels.

Figure 4

Similar copy numbers of ssAAV and scAAV in infected hearts. Serial sections of tissues used for microscopy were collected for real-time PCR analysis of AAV vector genomes. Genomic DNA (gDNA) was extracted from the tissues using a Qiagen DNeasy tissue kit according to the manufacturer's protocol. One microgram of extracted gDNA was used in all quantitative PCR reactions. The PCR conditions were 50 cycles of 94.8°C for 40 s, 37.8°C for 2 min, 55.8°C for 4 min, and 68.8°C for 30 sec. DNA samples were assayed in triplicate. The third replicate was spiked with CBA DNA at a ratio of 100 copies/μg of gDNA. If at least 40 copies of the spike-in DNA were detected, the DNA sample was considered acceptable for reporting vector DNA copies. Data were averaged by group and plotted with standard deviation for comparisons. No statistically significant differences were measured between any of the groups of scAAV and ssAAV treated animals.