Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation

Abstract

Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

FIG. 1

Myoblast differentiation increases transduction with rAAV-2cap5 but not rAAV-2 virus. Transduction of undifferentiated (A to D) and differentiated (E to H) C2C12 cells was evaluated for EGFP transgene expression following infection with 3,000 DNA particles/cell of either rAAV-2 (A, B, E, and F) or rAAV-2cap5 (C, D, G and H) virus for 24 h. EGFP expression was evaluated 72 h after infection by fluorescence microscopy. Nomarski and fluorescence photomicrographs are presented to the left and right of each panel, respectively. (I) Quantitative analysis of the percentage of EGFP-expressing cells. Values represent the means ± standard errors of the means (error bars) for more than 15 quantitated 10× magnification microscropic fields from three independent experiments.

FIG. 1

Myoblast differentiation increases transduction with rAAV-2cap5 but not rAAV-2 virus. Transduction of undifferentiated (A to D) and differentiated (E to H) C2C12 cells was evaluated for EGFP transgene expression following infection with 3,000 DNA particles/cell of either rAAV-2 (A, B, E, and F) or rAAV-2cap5 (C, D, G and H) virus for 24 h. EGFP expression was evaluated 72 h after infection by fluorescence microscopy. Nomarski and fluorescence photomicrographs are presented to the left and right of each panel, respectively. (I) Quantitative analysis of the percentage of EGFP-expressing cells. Values represent the means ± standard errors of the means (error bars) for more than 15 quantitated 10× magnification microscropic fields from three independent experiments.

FIG. 2

Quantitative analysis of RSV-luciferase expression from rAAV-2 and rAAV-2cap5 virus in differentiated and undifferentiated C2C12 cells. (A) Undifferentiated and differentiated C2C12 cells were infected with either rAAV-2 or rAAV-2cap5 virus for 24 h at an MOI of 3,000 DNA particles/cell. Mock-infected cells were used as a negative control for background enzyme activity. The luciferase activity was determined at 24, 48, and 72 h after infection. (B) Ratio of relative luciferase expression (rAAV-2cap5/rAAV-2) for the two vector types. Values in both panels represent the means ± standard errors of the means (error bars) for three independent data points.

FIG. 3

Examination of viral binding in C2C12 cells. (A) Viral binding was assessed by Southern blot analysis of viral DNA. C2C12 cells were precooled at 4°C for 10 min. After washing with serum-free DMEM, rAAV-2 (lanes 5, 6, 11, and 12) or rAAV-2cap5 (lanes 2, 3, 8, and 9) viruses (carrying the AAV-2 CMV-EGFP cassette) were applied to the cells at an MOI of 2,000 particles/cell for 60 min at 4°C. Mock-infected cells were included as negative controls (lanes 1, 4, 7, and 10). At the end of incubation, cells were either washed with PBS alone (lanes 1, 3, 4, 6, 7, 9, 10, and 12) or treated with 0.5% trypsin (lanes 2, 5, 8, and 11) before washing. Hirt DNA was then prepared and analyzed by Southern blotting with a transgene (EGFP)-specific ³²P-labeled probe. Abbreviations: Mock, mock-infected cells; Pseudo, rAAV-2cap5 virus; AAV-2, native rAAV-2 virus. (B) Viral binding from three independent experiments was quantified by densitometry. Values shown are means ± standard errors of the means (error bars). Lane numbers in panel B correspond to the labeling in panel A.

FIG. 4

Proteasome inhibitors differentially affect rAAV-2 and rAAV-2cap5 transduction in differentiated C2C12 cells. To analyze the effect of proteasome inhibitors on the intracellular processing of different AAV serotypes, fully differentiated C2C12 cells were infected with either rAAV-2 or rAAV-2cap5 luciferase vectors at an MOI of 600 DNA particles/cell for 4 h. Tripeptide proteasome inhibitors (40 μM LLnL or 4 μM Z-LLL) were also added to the media during the infection period. Luciferase expression was quantified at 24 h postinfection. The data represent the means ± standard errors of the means (error bars) for three independent samples for each experimental condition.

FIG. 5

The AAV-5 receptor is upregulated following differentiation of C2C12 cells. To correlate increased transduction of rAAV-2cap5 in differentiated C2C12 cells with AAV-5 receptors, cell surface α-2,3-linked sialic acid expression was determined using a MAL II lectin binding assay. MAL II lectin binding was visualized in undifferentiated (A and B) and differentiated (C and D) C2C12 cells using indirect avidin-FITC fluorescence microscopy (B and D). (A and C) Nomarski photomicrographs of panels B and D, respectively. Increased AAV-5 receptor expression in fully differentiated cells is clearly demonstrated in D.

FIG. 6

Factors affecting rAAV binding in C2C12 cells. (A) The effects of heparin competition or sialidase (NA III) treatment on rAAV-2 and rAAV-2cap5 virus infection in C2C12 cells were evaluated. rAAV-2 or rAAV-2cap5 infections (MOI of 1,000 DNA particles/cell) of undifferentiated (lanes 1 to 6) or differentiated (lanes 7 to 12) C2C12 cells were evaluated following no treatment (lanes 3, 6, 9, and 12), sialidase treatment (lanes 1, 4, 7, and 10), or heparin (final concentration, 20 μg/ml) competition (lanes 2, 5, 8, and 11). Hirt DNA was harvested after incubation at 4°C for 60 min and evaluated by Southern blotting against a ³²P-labeled EGFP probe. Pseudo, rAAV-2cap5 virus. (B) Results from densitometric quantification of DNA signals from three independent experiments. NAIII, type III neuraminidase (sialidase). Values are represented as percent inhibition (means ± standard errors of the means [error bars]; n = 3) in binding following sialidase treatment or heparin competition compared to untreated controls.

FIG. 7

Kinetic analysis of rAAV viral genome persistence in differentiated C2C12 cells. To better understand rAAV transduction in myotubes, differentiated C2C12 cells were infected with either rAAV-2cap5 (lanes 1, 2, and 3) or rAAV-2 (lanes 4, 5, and 6) at an MOI of 1,000 DNA particles/cell. Hirt DNA was harvested at 90 min (lanes 1 and 4), 24 h (lanes 2 and 4), and 48 h (lanes 3 and 6) postinfection. The left panel depicts a Southern blot hybridized with a ³²P-labeled EGFP probe. The right panel depicts the corresponding ethidium bromide-stained gel. The lane labels in both panels are identical with the exception of the DNA ladder. Pseudo, rAAV-2cap5 virus.

FIG. 8

Kinetic comparison of EGFP expression in normal and dystrophic muscles. The anterior tibialis muscles of 6-month-old normal or mdx mice were infected with 2 × 10¹⁰ particles of the indicated viruses as described in Materials and Methods. EGFP expression was determined at different time points by fluorescence microscopy. (A to H) Photographs of whole-mount tissue from freshly excised muscles 1 wk and 1 month after infection. Representative photographs from triplicate experiments are shown. Photomicrographs were taken at an 8-s (A, B, E, and F) or 1-s (C, D, G, and H) exposure. (I to N) EGFP expression 6 months after infection of mdx tibialis muscles was evaluated in paraformaldehyde-fixed, cryopreserved tissue sections (15 μm thick) following Evans blue perfusion to demarcate damaged myofibers. Photomicrographs in panels I to K (rAAV-2 infection) were taken from the right leg, and those in panels L and N (rAAV-2cap5 infection) were taken from the left leg of the same mouse. Photomicrographs were taken at 15-s (I and L) or 2-s (J, K, M, and N) exposures. FITC photomicrographs are represented (I, J, L, and M). Panels J and M (FITC channel) are identical to fields shown in panels K and N (Evans blue, rhodamine channel), respectively.

FIG. 9

Quantitative examination of luciferase activity following rAAV-2cap5 or rAAV-2 infection of tibialis muscles. rAAV luciferase expression vectors were used to evaluate transgene expression in normal and mdx anterior tibialis muscles at 1 week and 1 month postinfection with 2 × 10¹⁰ particles of rAAV-2 (AV2) or rAAV-2cap5 (AV2/5). The data represent the means ± standard errors of the means (error bars) for three independent muscle samples from each experimental group.