Alejandra Elena Arbetman

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-8administration 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.

89. Isolation of a Close AAV5 Relative from Goat Tissues: Evidence of Host Promiscuity

Top of pageAbstract Preexisting immunity to AAV vectors can potentially limit their clinical usefulness by blocking tissue transduction and/or by causing the elimination of vector-transduced cells. We previously demonstrated that the AAV2-neutralizing capacities of human plasmas (as measured by in vitro assay) are frequently substantial relative to proposed doses of AAV vectors. Out of a group of 50 hemophiliacs, approximately 40% had AAV2-neutralizing capacities exceeding 1e13 viral particles/ml or about 6e16 viral particles/total blood volume. Furthermore, the majority of the group with high anti-AAV2 titers also had significant titers against other AAV serotypes (AAV1, and 3–6). To create improved gene therapy vectors, we are currently screening tissues and adenovirus preparations from a wide variety of non-primate species to isolate AAV capsids that have less preexisting immunity in humans. One of the more surprising results from this screen was the PCR amplification of a close AAV5 relative from a crude goat adenovirus preparation. AAV5 is a human parvovirus, originally isolated from a penile flat condylomatous lesion, with a high sero-prevalence in the post-pubertal population. The goat adenovirus preparation used as the PCR template was produced during routine diagnostic adenovirus screening of a tissue sample from a goat that had died of enteritis. The presence of parvovirus-like particles in the initial stock was demonstrated by electron microscopy and no primate-derived products were used at any point in the viral culture. The 2,805 bp goat AAV PCR fragment, which encodes 603 bp of rep, the central intron, and all of cap, is 94 % homologous to AAV5. The DNA and protein homologies to AAV5 for the partial rep region are 98% and 99%, respectively, and are 93% and 94% for the cap region, respectively. With respect to the linear amino acid sequence, the distribution of the amino acid differences between the AAV5 and goat AAV capsids is highly polar. All of the 44 amino acid differences occur exclusively in the C-terminal hypervariable region of the capsid protein in a scattered fashion. It has been proposed that this region comprises the surface loops of AAV5 by analogy to AAV2. Because of its high homology to AAV5, it is likely that the goat AAV arose from a recent species jump of AAV5 from humans to goats. The fact that all of the amino acid differences between the AAV5 and the goat capsids occur in regions that are probably surface exposed implies that capsid evolution is being driven primarily by the humoral immune system of the new host or by adaptation to ruminant receptors. The lack of amino acid changes in non-surface exposed regions of cap may imply a lack of pressure from the cellular immune response. Interspecies infection, particularly in the case of domestic animals, may be a previously unappreciated but significant mechanism for diversity generation and virus evolution for AAVs. This novel goat AAV capsid has been successfully used to pseudo-type AAV2 vector genomes and is currently being evaluated for tissue tropism and resistance to human preexisting humoral immunity.