Safety of liver gene transfer following peripheral intravascular delivery of adeno-associated virus (AAV)-5 and AAV-6 in a large animal model

Abstract

Intravascular delivery of adeno-associated virus (AAV) vector is commonly used for liver-directed gene therapy. In humans, the high prevalence of neutralizing antibodies to AAV-2 capsid and the wide cross-reactivity with other serotypes hamper vector transduction efficacy. Moreover, the safety of gene-based approaches depends on vector biodistribution, vector dose, and route of administration. Here we sought to characterize the safety of AAV-5 and AAV-6 for liver-mediated human factor IX (hFIX) expression in rabbits at doses of 1 × 10(12) or 1 × 10(13) viral genomes/kg. Circulating therapeutic levels of FIX were observed in both cohorts of AAV-6-hFIX, whereas for AAV-5-hFIX only the high dose was effective. Long-lasting inhibitory antibodies to hFIX were detected in three of the 10 AAV-6-injected animals but were absent in the AAV-5 group. Overall, vector shedding in the semen was transient and vector dose-dependent. However, the kinetics of clearance were remarkably faster for AAV-5 (3-5 weeks) compared with AAV-6 (10-13 weeks). AAV-6 vector sequences outside the liver were minimal at 20-30 weeks post-injection. In contrast, AAV-5 exhibited relatively high amounts of vector DNA in tissues other than the liver. Together these data are useful to further define the safety and potential for clinical translation of these AAV vectors.

FIG. 1.

Time course of expression of hFIX antigen and formation of antibodies to hFIX in rabbits following delivery of AAV-6 vectors. Rabbits were injected via the peripheral vein with AAV-6 at doses of 1 × 10¹³ vg/kg (high-dose, A and B) or 1 × 10¹² vg/kg (low-dose, C and D). Data are shown for each individual animal. Note the differences in the scales from (A) and (C). Color images available online at www.liebertonline.com/hum.

FIG. 2.

Circulating FIX antigen levels and anti-hFIX IgG titers in rabbits injected with AAV-5 vectors. Rabbits were injected via the peripheral vein with AAV-5 at 1 × 10¹³ vg/kg (high-dose, A and B) or 1 × 10¹² vg/kg (low-dose, C and D). Data are shown for each individual animal. Color images available online at www.liebertonline.com/hum.

FIG. 3.

Humoral responses to AAV capsid protein. Rabbits were injected with AAV-5 or AAV-6 at doses of 1 × 10¹² vg/kg (low-dose) or 1 × 10¹³ vg/kg (high-dose). Titers of specific IgG to each dose cohort (n = 5 animals per dose cohort) are shown. Values of p < 0.05 (by analysis of variance) reflect comparison between groups of the high-dose cohorts.

FIG. 4.

Kinetics of appearance of vector DNA sequences in total semen from the low-dose cohort rabbits: (top panel) animals injected with AAV-6 and (bottom panel) animals injected with AAV-5 at doses of 1 × 10¹² vg/kg. The numbers of triplicate PCR signals from each individual animal are indicated. *Denotes animals followed up to week 20 after vector injection. Color images available online at www.liebertonline.com/hum.

FIG. 5.

Kinetics of appearance of vector DNA sequences in total semen from the high-dose cohort rabbits: (top panel) animals injected with AAV-6 and (bottom panel) animals injected with AAV-5 at doses of 1 × 10¹³ vg/kg. The numbers of triplicate PCR signals from each individual animal are indicated. *Denotes animals followed up to week 20 after vector injection. Color images available online at www.liebertonline.com/hum.

FIG. 6.

Biodistribution of AAV DNA in rabbits following intravenous injection of AAV vectors. (A) Rabbits received a low dose (1 × 10¹² vg/kg) of AAV-5 (n = 3) or AAV-6 (n = 4) vectors, and the indicated tissues were harvested at week 30. Three animals were sacrificed at week 20 (two for AAV-5 and one for AAV-6). (B) Rabbits were injected with a high dose (1 × 10¹³ vg/kg) of AAV-5 (n = 5) or AAV-6 (n = 5) vectors, and the indicated tissues were harvested at week 30. Three animals (AAV-5, n = 2; AAV-6, n = 1) were sacrificed at week 20. Vector DNA content was obtained by real-time quantitative PCR assay. Combined data from the week 20 or 30 time points are shown as mean ± SD values. *p < 0.05 between serotype groups. AC Gland, accessory gland; L. and R. Testes, left and right testes, respectively.

FIG. 7.

Relative concentration of vector DNA in tissues compared with the liver. Data show the difference in the mean concentration of vector DNA in low-dose (top panel) and high-dose (bottom panel) cohorts. AC Gland, accessory gland; L. and R. Testes, left and right testes, respectively.