Age-Related Seroprevalence of Antibodies Against AAV-LK03 in a UK Population Cohort

Abstract

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapy. The presence of neutralizing antibodies (Nab) against AAV capsids decreases cell transduction efficiency and is a common exclusion criterion for participation in clinical trials. Novel engineered capsids are being generated to improve gene delivery to the target cells and facilitate success of clinical trials; however, the prevalence of antibodies against such capsids remains largely unknown. We therefore assessed the seroprevalence of antibodies against a novel synthetic liver-tropic capsid AAV-LK03. We measured seroprevalence of immunoglobulin (Ig)G (i.e., neutralizing and nonneutralizing) antibodies and Nab to AAV-LK03 in a cohort of 323 UK patients (including 260 pediatric) and 52 juvenile rhesus macaques. We also performed comparative analysis of seroprevalence of Nab against wild-type AAV8 and AAV3B capsids. Overall IgG seroprevalence for AAV-LK03 was 39% in human samples. The titer increased with age. Prevalence of Nab was 23%, 35%, and 18% for AAV-LK03, AAV3B, and AAV8, respectively, with the lowest seroprevalence between 3 and 17 years of age for all serotypes. Presence of Nab against AAV-LK03 decreased from 36% in the youngest cohort (birth to 6 months) to 7% in older primary school-age children (9-11 years) and then progressively increased to 54% in late adulthood. Cross-reactivity between serotypes was >60%. Nab seroprevalence in macaques was 62%, 85%, and 40% for AAV-LK03, AAV3B, and AAV8, respectively. When planning for AAV gene therapy clinical trials, knowing the seropositivity of the target population is critical. In the population studied, AAV seroprevalence for AAV serotypes tested was low. However, high cross-reactivity between AAV serotypes remains a barrier for re-injection. Shifts in Nab seroprevalence during the first decade need to be confirmed by longitudinal studies. This possibility suggests that pediatric patients could respond differently to AAV therapy according to age. If late childhood is an ideal age window, intervention at an early age when maternal Nab levels are high may be challenging. Nab-positive children excluded from trials could be rescreened for eligibility at regular intervals because this status may change.

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

“…Using the luciferase-based neutralizing antibody detection assay, and a cutoff of 1:5, the seroprevalence to WT AAV3B in our cohort of 100 individual serum samples was 37%. This was very similar to the 35% previously reported for a UK patient population 54 and 36% for a North American population 55 respectively. In a separate study, we analyzed a similar number of North American patient samples and found a seroprevalence to AAV3B of 45% (I.Z., unpublished data).…”

Human Immune Responses to Adeno-Associated Virus (AAV) Vectors

“…Although AAV seroprevalence varies geographically, neutralizing antibodies (NAbs) that recognize virtually all AAV serotypes can be found in a large proportion of the human population (ranging from 30 to 60%). Successive infections may explain the high prevalence of anti-AAV NAbs in humans (37)(38)(39), resulting in broad cross-reactivity across AAV serotypes of different origins, including human, mammalian or engineered. Regardless of the geographic region, the most prevalent NAbs are directed against AAV2, followed by AAV1 (38).…”

Suprachoroidal and Subretinal Injections of AAV Using Transscleral Microneedles for Retinal Gene Delivery in Nonhuman Primates

“…We screened 24 rhesus macaques (4-10 years of age) for absence of pre-existing serum neutralizing antibodies (NAbs) to AAV8 using an in vitro transduction inhibition assay, 47 and identified 6 animals (mean age, 8.3 ± 2.4 years; 3 males/3 females) with no detectable NAbs (<1:2,560), consistent with the high prevalence of anti-AAV8 NAbs reported in rhesus macaques. 48 We injected both eyes of three of these animals with nonhuman primate-grade AAV8 expressing enhanced green fluorescent protein (GFP) under a ubiquitous cytomegalovirus (CMV) promoter at two different doses (7 Â 10 11 or 7 Â 10 12 vector genomes [vg]/eye) using a 700-mm-long 30-gauge microneedle ( Figure 1B, Clearside Biomedical, Alpharetta, GA, USA) inserted through the conjunctiva and sclera into the suprachoroidal space in the superotemporal quadrant (single 100-mL injection) of left eyes, and both superotemporal and inferonasal quadrants (two 50-mL injections) of right eyes ( Figure 1C). No surgical microscope or vitrectomy surgery was required.…”