Successful Repeated Hepatic Gene Delivery in Mice and Non-human Primates Achieved by Sequential Administration of AAV5ch and AAV1

Abstract

In the gene therapy field, re-administration of adeno-associated virus (AAV) is an important topic because a decrease in therapeutic protein expression might occur over time. However, an efficient re-administration with the same AAV serotype is impossible due to serotype-specific, anti-AAV neutralizing antibodies (NABs) that are produced after initial AAV treatment. To address this issue, we explored the feasibility of using chimeric AAV serotype 5 (AAV5^ch) and AAV1 for repeated liver-targeted gene delivery. To develop a relevant model, we immunized animals with a high dose of AAV5^ch-human secreted embryonic alkaline phosphatase (hSEAP) that generates high levels of anti-AAV5^ch NAB. Secondary liver transduction with the same dose of AAV1-human factor IX (hFIX) in the presence of high levels of anti-AAV5^ch NAB proved to be successful because expression/activity of both reporter transgenes was observed. This is the first time that two different transgenes are shown to be produced by non-human primate (NHP) liver after sequential administration of clinically relevant doses of both AAV5^ch and AAV1. The levels of transgene proteins achieved after delivery with AAV5^ch and AAV1 illustrate the possibility of both serotypes for liver targeting. Furthermore, transgene DNA and RNA biodistribution patterns provided insight into the potential cause of decrease or loss of transgene protein expression over time in NHPs.

Keywords: AAV; gene therapy; re-administration of gene therapy.

Figure 1

Experimental Setup and Humoral Immune Response against AAV Vectors in Mice (A–E) Mice receiving intravenous injections of PBS or AAV5^ch-hSEAP at week 0 and second intravenous injection of PBS, AAV1-hFIX, or AAV5^ch-hFIX at week 3 (A) develop total anti-AAV5^ch antibodies (B) and total anti-AAV1 antibodies (C) in plasma over time, which correlates with anti-AAV5^ch NAB (D) and anti-AAV1 NAB (E) levels at week 6. Data are presented as means ± SEM of all mice (n = 6).

Figure 2

Transgene Activity or Presence in Mouse Plasma and Liver Tissue (A–D) Mice intravenously cross-administered with AAV5^ch followed by AAV1 present with stable reporter gene hSEAP activity (A and C) and hFIX presence (B) in mouse plasma over time and expression of GFP in representative mouse liver sections (D). Mice received intravenous injections of PBS or AAV5^ch-hSEAP at week 0 and a second i.v. injection of PBS, AAV1-hFIX, or AAV5^ch-hFIX at week 3 (n = 6/group) (A and B), or a first i.v. injection of PBS or AAV5^ch-hSEAP at week 0 and a subsequent one of PBS, AAV1-GFP at week 3 (C and D). (D) PBS, PBS group (I); PBS, AAV1-GFP group (II); AAV5^ch-hSEAP, AAV1-GFP group (III). RLU, relative luminescence units. Data in graphs (A)–(C) are presented as means ± SEM of all mice (n = 6).

Figure 3

Experimental Setup and Presence of hSEAP and hFIX Vector DNA and mRNA in the Liver (A) Monkeys were injected i.v. at day 0 with an AAV5^ch-based vector (AAV5^ch-hSEAP) or PBS and at day 30 with either an AAV1- or AAV5^ch-based vector (AAV1-hFIX or AAV5^ch-hFIX). (B–E) Reporter gene hSEAP DNA (B), mRNA (C), and hFIX DNA (D) and mRNA (E) in the monkey liver tissue at sacrifice. Mean ± SEM of eight different liver regions was plotted for every monkey. Monkeys were injected i.v. at day 0 with an AAV5^ch-hSEAP (HNPs 10–15) or nothing (n. 1, n. 2) and at day 30 with AAV1-hFIX (HNPs 4–6 and 10–13) or AAV5^ch-hFIX (HNPs 14 and 15) and sacrificed at day 364.

Figure 4

Short-Term Transgene Activity or Presence in the Monkey Plasma (A and B) Stable reporter gene hSEAP activity (A) and hFIX expression (B) in monkey plasma over time in animals cross-administered with AAV5^ch-hSEAP followed by AAV1-hFX and in animals injected with AAV5^ch-hFIX or AAV1-hFIX only. Monkeys were injected i.v. at day 0 with an AAV5^ch-based vector (AAV5^ch-hSEAP) or PBS and on day 30 with either an AAV1- or AAV5^ch-based vector (AAV1-hFIX or AAV5^ch-hFIX) or PBS. RLU, relative luminescence units. Data are presented as means ± SEM of all monkeys in each experimental group.

Figure 5

Long-Term Transgene Activity or Presence in the Monkey Plasma (A–E) Long-term reporter gene hSEAP activity (A), hFIX expression (grouped, B, means ± SEM), and hFIX expression in individual monkeys in PBS followed by AAV1-hFIX-injected group (C), PBS followed by AAV5^ch-hFIX-injected group (D), and AAV5^ch-hSEAP followed by AAV1-hFIX-injected group (E) measured in monkey plasma over time. Monkeys were injected i.v. at day 0 with an AAV5^ch-based vector (AAV5^ch-hSEAP) or PBS and on day 30 with either an AAV1- or AAV5^ch-based vector (AAV1-hFIX or AAV5^ch-hFIX) or PBS. RLU, relative luminescence units.

Figure 6

Humoral Immune Response against Transgene Products (A and B) Fold change of anti-hSEAP (A) and anti-hFIX (B) IgG in monkey plasma over time. Monkeys were injected i.v. on day 0 with an AAV5^ch-based vector (AAV5^ch-hSEAP) or PBS and on day 30 with either an AAV1- or AAV5^ch-based vector (AAV1-hFIX or AAV5^ch-hFIX).

Figure 7

Neutralizing Humoral Immune Response against AAV Vectors in Monkeys (A and B) Monkeys injected i.v. at day 0 with an AAV5^ch-based vector (AAV5^ch-hSEAP) or PBS and at day 30 with either an AAV1- or AAV5^ch-based vector (AAV1-hFIX or AAV5^ch-hFIX) develop anti-AAV5^ch (A) and anti-AAV1 NABs (B).