Novel caprine adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties

Abstract

Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8 administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

FIG. 1.

Comparison of the amino acid sequence of AAV-5 and caprine AAV (AAV-Go.1) VP1. Amino acid differences are shown. The dots in the alignment represent the amino acids that are identical in the two sequences. The insertion of two amino acids (SS) in AAV-Go.1at positions 481 and 482 of VP1 is indicated by dashes in the AAV-5 sequence.

FIG. 2.

Predicted location of differences between AAV-5 and caprine AAV VP3, modeled on the crystal structure of AAV-2 VP3. (A) External surface amino acids are color coded by amino acid type using the RasMol software. (B) Magnified top view of an asymmetric structural unit (1/60 of the full virus). External amino acids that are different between AAV-5 and caprine AAV VP3 are indicated in black (29 of the 42 changes identified). The spike is located in the lower left and lower right corners. (C) Lateral view of a representative trimer showing all 29 external differences (in black), 10 buried differences (in yellow), and two internal differences (in cyan). Because the spikes in AAV-5 and caprine AAV are predicted to be shorter than those in AAV-2, the amino acids 315 to 320 in AAV-2 were deleted in this figure to simulate this difference in sequence and structure.

FIG. 3.

Comparative expression of AAV vectors in primary neuronal cultures. Transduction efficiency of the reporter gene lacZ in the rat striatal neurons was highest in AAV-6 lacZ (A) followed by AAV-8 lacZ (B), AAV-2 lacZ (C), AAV-5 lacZ (D), AAV-Go.1 lacZ (E), and AAV-4 lacZ (F). AAV-6 lacZ transduced neurons exclusively, whereas AAV-5 lacZ-mediated gene transfer was inefficient in neurons but significant in glial cells. All other vectors transduced both neurons and glial cells.

FIG. 4.

Transduction of muscle in SCID mice. Male mice were injected intramuscularly with 2 × 10¹¹ vector genomes of rAAV-1 hFIX9, rAAV-8 hFIX9, and rAAV-Go.1 hFIX9 with (empty symbols) and without (filled symbols) previous administration of IVIG. Human factor IX concentration was measured by ELISA. Each data point corresponds to the mean of five animals. The human factor IX concentration in the control animals was considered a blank and was subtracted from human factor IX levels in the experimental animals. The amount of IVIG present in the plasma of the animal at the time of injection with the vector was calculated to be 10 mg/ml based on the AAV-2 neutralizing titer of 1:300. Squares, AAV-1; diamonds, AAV-8; circles, AAV-Go.1.

FIG. 5.

Transduction of liver in SCID mice. (A) Male SCID mice were injected via the tail vein with 5 × 10¹¹ vector genomes of rAAV-Go.1 hFIX16 (circles) or rAAV-8 hFIX16 (diamonds) (n = 5). Retro-orbital blood was collected 1, 2, and 4 (n = 5) and 8 (n = 3) weeks after vector injection. Transduction was in the presence (empty symbols) or absence (filled symbols) of IVIG. Mice tested with IVIG were injected via the tail vein (250 μl at 100 mg/ml), 24 h before injection with the vector. In the case of AAV-Go.1 and AAV-8 the neutralizing titer was 1:120. Human factor IX was measured by ELISA. (B) In a separate study SCID mice were injected with rAAV-2 hFIX16. Empty and filled symbols, presence and absence of IVIG, respectively. Mice tested with IVIG were injected with the same total volume of 250 μl used for the injections above but now containing 9 μl at 100 mg/ml; the neutralizing titer when the virus was injected was 1:10.

FIG. 6.

Biodistribution of rAAV-Go.1 hFIX16 after intravenous administration in mice. Human factor IX double-stranded DNA was quantitated in eight tissues 4 weeks after AAV administration. Bars represent the mean numbers of copies and standard deviations after testing of two different animals. Black bars, AAV-Go.1 hFIX16; gray bars, AAV-8 hFIX16. Liver, lung, and spleen were also analyzed when vector delivery was performed in the presence of IVIG (crosshatched bars). ds, double stranded.