Enhancing transduction of the liver by adeno-associated viral vectors

Abstract

A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 x 10(7) vg per mouse (4 x 10(8) vg kg(-1)) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4+/-0.6 to 9+/-2 microg ml(-1) in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10-20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.

Figure 1. In-vivo expression of hFIX from AAV pseudotyped vectors

Plasma human FIX (hFIX) levels (mean ± SEM) measured by ELISA between days 1 and 57 in mice after tail vein injection of scAAV-LP1-hFIX packaged with capsid derived from serotype -rh.10 (Panel A), -9, (Panel B), -8 (Panel C) and 7 (Panel D) and at different particle doses consisting of 2×10¹¹ (triangle), 4×10¹⁰ (circle), 4×10⁹ (diamond) and 4×10⁸ (square) vg/kg. Panel E shows the relationship between vector dose and transgene expression on day 57 for scAAV-LP1-hFIX packaged with capsid derived from serotype -rh.10 (◇), -9, (△), -8 (×) and 7 (○).

Figure 2. Biodistribution of scAAV vector

Genomic DNA was isolated from three individual mice in each of the four serotype cohorts (serotype -rh.10 [Black filled bar], -9, [Grey filled bar], -8 [Bar with criss-crosses] and 7 [Clear bar]) following tail vein administration of 4×10¹⁰vg/kg from the liver (see Table 1) and nonhepatic tissues (panel B). Transgene copy number in each tissue was quantified by a vector specific qPCR assay. Shown is transgene copy number per diploid genome corrected for variation in loading and amplification efficiency using GAPDH primers.

Figure 3

A. Anti-AAV 8 (left panel) and rh.10 (right panel) antibody titers in mice 57 days after tail vein administration of 4×10¹⁰vg/kg of scAAV-LP1-hFIX packaged with capsid from AAV serotype 8 or rh.10 compared to naïve untransduced animals. Shown are relative anti-AAV antibody titers from 3 animals. B. Re-administration of rAAV vector pseudotyped with serotype 8 (left panel) or rh.10 (right panel) capsids encoding human FVII. Shown are plasma levels (mean ± SEM) of human FVII protein measured by ELISA 42 days after tail vein administration of LP1-hFVII.

Figure 4. Influence of gender and proteasome inhibition on scAAV mediated transgene expression

Human FIX concentration (hFIX) in murine plasma after tail vein administration of 4 × 10⁹ vg/kg of scAAV2/8-LP1-hFIXco in cohorts (n=6) of male (Panel A) and female (Panel B) C57Bl/6 mice. A proportion of these animals were treated with a single dose of bortezomib 24 hours (Grey bar) before or with two doses of bortezomib given 24 hours before and again 24 hours (Clear Bar) after scAAV administration and their hFIX expression compared with mice that received excipient (Crisscross bar) alone. All transgene expression values are depicted as average together with the standard error of the mean.

Figure 5

A. Human FIX expression (mean ± SEM) following tail vein administration of 2 × 10¹² vg/kg rAAV2 CAGG-FIX vector particles in C57Bl/6 mice (n=6/cohort). 4 × 10⁹vg/kg of AV5LacZ, an E1/E3 deleted adenoviral vector encoding the β galactosidase gene was administered into one cohort (△) at 10 weeks after rAAV2 CAGG-FIX mediated gene transfer. The second cohort received an equal volume of PBS (◇). B. Genomic DNA (top panel) and total cellular RNA (bottom panel) were extracted from the liver of mice that had been sequentially transduced with rAAV2 CAGG-FIX and AV5LacZ adenoviral particles (as described above). rAAV transgene copy number was determined by digesting 10mg of DNA with NheI followed by Southern blot transfer and hybridization with a hFIX specific probe. Northern blot transfer of 10mg of total hepatocellular RNA onto nylon membrane followed by hybridization with a hFIX specific probe. N = samples from naïve mouse, - = samples from mice exposed to rAAV2 CAGG-FIX only and + = mice sequentially transduced with rAAV2 and adenovirus. C. Truncated soluble vascular endothelial growth factor receptor (tsFlk-1) levels (mean ± SEM) in mice transduced with rAAV2 CAGG-tsFlk-1 alone (filled bar, N=14) or sequentially with rAAV2 CAGG-tsFlk-1 followed 15 weeks later by 4 × 10⁹ vg/kg of AV5LacZ (clear bar, N=10). D. Plasma levels of hFIX as determined by ELISA 4 weeks after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=4) or PBS (filled bar, N=4) into BalB/c mice, that were 20 weeks previously transduced with 4 × 10¹² vg/kg of rAAV2 LSP-FIX. E. Plasma levels of hFIX as determined by ELISA 10 days after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=3) or PBS (filled bar, N=3) into C56Bl/6 mice, that were 8 weeks previously transduced with 4×10⁸ vg/kg of scAAV2/8-LP1-hFIXco.

Figure 5

A. Human FIX expression (mean ± SEM) following tail vein administration of 2 × 10¹² vg/kg rAAV2 CAGG-FIX vector particles in C57Bl/6 mice (n=6/cohort). 4 × 10⁹vg/kg of AV5LacZ, an E1/E3 deleted adenoviral vector encoding the β galactosidase gene was administered into one cohort (△) at 10 weeks after rAAV2 CAGG-FIX mediated gene transfer. The second cohort received an equal volume of PBS (◇). B. Genomic DNA (top panel) and total cellular RNA (bottom panel) were extracted from the liver of mice that had been sequentially transduced with rAAV2 CAGG-FIX and AV5LacZ adenoviral particles (as described above). rAAV transgene copy number was determined by digesting 10mg of DNA with NheI followed by Southern blot transfer and hybridization with a hFIX specific probe. Northern blot transfer of 10mg of total hepatocellular RNA onto nylon membrane followed by hybridization with a hFIX specific probe. N = samples from naïve mouse, - = samples from mice exposed to rAAV2 CAGG-FIX only and + = mice sequentially transduced with rAAV2 and adenovirus. C. Truncated soluble vascular endothelial growth factor receptor (tsFlk-1) levels (mean ± SEM) in mice transduced with rAAV2 CAGG-tsFlk-1 alone (filled bar, N=14) or sequentially with rAAV2 CAGG-tsFlk-1 followed 15 weeks later by 4 × 10⁹ vg/kg of AV5LacZ (clear bar, N=10). D. Plasma levels of hFIX as determined by ELISA 4 weeks after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=4) or PBS (filled bar, N=4) into BalB/c mice, that were 20 weeks previously transduced with 4 × 10¹² vg/kg of rAAV2 LSP-FIX. E. Plasma levels of hFIX as determined by ELISA 10 days after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=3) or PBS (filled bar, N=3) into C56Bl/6 mice, that were 8 weeks previously transduced with 4×10⁸ vg/kg of scAAV2/8-LP1-hFIXco.

Figure 5

A. Human FIX expression (mean ± SEM) following tail vein administration of 2 × 10¹² vg/kg rAAV2 CAGG-FIX vector particles in C57Bl/6 mice (n=6/cohort). 4 × 10⁹vg/kg of AV5LacZ, an E1/E3 deleted adenoviral vector encoding the β galactosidase gene was administered into one cohort (△) at 10 weeks after rAAV2 CAGG-FIX mediated gene transfer. The second cohort received an equal volume of PBS (◇). B. Genomic DNA (top panel) and total cellular RNA (bottom panel) were extracted from the liver of mice that had been sequentially transduced with rAAV2 CAGG-FIX and AV5LacZ adenoviral particles (as described above). rAAV transgene copy number was determined by digesting 10mg of DNA with NheI followed by Southern blot transfer and hybridization with a hFIX specific probe. Northern blot transfer of 10mg of total hepatocellular RNA onto nylon membrane followed by hybridization with a hFIX specific probe. N = samples from naïve mouse, - = samples from mice exposed to rAAV2 CAGG-FIX only and + = mice sequentially transduced with rAAV2 and adenovirus. C. Truncated soluble vascular endothelial growth factor receptor (tsFlk-1) levels (mean ± SEM) in mice transduced with rAAV2 CAGG-tsFlk-1 alone (filled bar, N=14) or sequentially with rAAV2 CAGG-tsFlk-1 followed 15 weeks later by 4 × 10⁹ vg/kg of AV5LacZ (clear bar, N=10). D. Plasma levels of hFIX as determined by ELISA 4 weeks after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=4) or PBS (filled bar, N=4) into BalB/c mice, that were 20 weeks previously transduced with 4 × 10¹² vg/kg of rAAV2 LSP-FIX. E. Plasma levels of hFIX as determined by ELISA 10 days after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=3) or PBS (filled bar, N=3) into C56Bl/6 mice, that were 8 weeks previously transduced with 4×10⁸ vg/kg of scAAV2/8-LP1-hFIXco.

Figure 5

A. Human FIX expression (mean ± SEM) following tail vein administration of 2 × 10¹² vg/kg rAAV2 CAGG-FIX vector particles in C57Bl/6 mice (n=6/cohort). 4 × 10⁹vg/kg of AV5LacZ, an E1/E3 deleted adenoviral vector encoding the β galactosidase gene was administered into one cohort (△) at 10 weeks after rAAV2 CAGG-FIX mediated gene transfer. The second cohort received an equal volume of PBS (◇). B. Genomic DNA (top panel) and total cellular RNA (bottom panel) were extracted from the liver of mice that had been sequentially transduced with rAAV2 CAGG-FIX and AV5LacZ adenoviral particles (as described above). rAAV transgene copy number was determined by digesting 10mg of DNA with NheI followed by Southern blot transfer and hybridization with a hFIX specific probe. Northern blot transfer of 10mg of total hepatocellular RNA onto nylon membrane followed by hybridization with a hFIX specific probe. N = samples from naïve mouse, - = samples from mice exposed to rAAV2 CAGG-FIX only and + = mice sequentially transduced with rAAV2 and adenovirus. C. Truncated soluble vascular endothelial growth factor receptor (tsFlk-1) levels (mean ± SEM) in mice transduced with rAAV2 CAGG-tsFlk-1 alone (filled bar, N=14) or sequentially with rAAV2 CAGG-tsFlk-1 followed 15 weeks later by 4 × 10⁹ vg/kg of AV5LacZ (clear bar, N=10). D. Plasma levels of hFIX as determined by ELISA 4 weeks after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=4) or PBS (filled bar, N=4) into BalB/c mice, that were 20 weeks previously transduced with 4 × 10¹² vg/kg of rAAV2 LSP-FIX. E. Plasma levels of hFIX as determined by ELISA 10 days after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=3) or PBS (filled bar, N=3) into C56Bl/6 mice, that were 8 weeks previously transduced with 4×10⁸ vg/kg of scAAV2/8-LP1-hFIXco.

Figure 5

A. Human FIX expression (mean ± SEM) following tail vein administration of 2 × 10¹² vg/kg rAAV2 CAGG-FIX vector particles in C57Bl/6 mice (n=6/cohort). 4 × 10⁹vg/kg of AV5LacZ, an E1/E3 deleted adenoviral vector encoding the β galactosidase gene was administered into one cohort (△) at 10 weeks after rAAV2 CAGG-FIX mediated gene transfer. The second cohort received an equal volume of PBS (◇). B. Genomic DNA (top panel) and total cellular RNA (bottom panel) were extracted from the liver of mice that had been sequentially transduced with rAAV2 CAGG-FIX and AV5LacZ adenoviral particles (as described above). rAAV transgene copy number was determined by digesting 10mg of DNA with NheI followed by Southern blot transfer and hybridization with a hFIX specific probe. Northern blot transfer of 10mg of total hepatocellular RNA onto nylon membrane followed by hybridization with a hFIX specific probe. N = samples from naïve mouse, - = samples from mice exposed to rAAV2 CAGG-FIX only and + = mice sequentially transduced with rAAV2 and adenovirus. C. Truncated soluble vascular endothelial growth factor receptor (tsFlk-1) levels (mean ± SEM) in mice transduced with rAAV2 CAGG-tsFlk-1 alone (filled bar, N=14) or sequentially with rAAV2 CAGG-tsFlk-1 followed 15 weeks later by 4 × 10⁹ vg/kg of AV5LacZ (clear bar, N=10). D. Plasma levels of hFIX as determined by ELISA 4 weeks after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=4) or PBS (filled bar, N=4) into BalB/c mice, that were 20 weeks previously transduced with 4 × 10¹² vg/kg of rAAV2 LSP-FIX. E. Plasma levels of hFIX as determined by ELISA 10 days after a bolus tail vein injection of 4 × 10⁹ vg/kg AV5GFP (clear bar, N=3) or PBS (filled bar, N=3) into C56Bl/6 mice, that were 8 weeks previously transduced with 4×10⁸ vg/kg of scAAV2/8-LP1-hFIXco.