The pleiotropic effects of natural AAV infections on liver-directed gene transfer in macaques

Abstract

Adeno-associated viral (AAV) vectors hold great potential for liver-directed gene therapy. Stable and high levels of transgene expression have been achieved in many murine models. Systemic delivery of AAV vectors in nonhuman primates (NHPs) that are natural hosts of AAVs appear to be challenging due to the high prevalence of pre-existing neutralizing antibodies (NAbs). This study evaluates the performance of AAV8, hu.37, and rh.8 vectors expressing green fluorescent protein (GFP) from a liver-specific promoter in rhesus macaques. Two of the animals that received AAV8 showed transduction of 24 and 40% of hepatocytes 7 days after systemic vector delivery. Importantly, expression was detected in several animals after 35 days despite the elevation of liver enzymes and development of transgene-specific T cells in liver. Pre-existing low levels of NAbs profoundly impacted the outcome of gene transfer and redirected vector DNA to spleen. We developed a sensitive in vivo passive transfer assay to detect low levels of NAbs to these novel AAV serotypes. Other strategies need to be developed to reduce immune response to the transgene in order to maintain long-term gene expression.

Figure 1

GFP expression in rhesus macaque livers after intravenous infusion of 3 × 10¹² GC/kg of AAV.TBG.EGFP pseudotyped with AAV8, hu.37, and rh.8 capsids. (a) Livers are harvested at day 7. (b) Livers are harvested at day 35. Representative images of each animal are shown. Bar = 100 µm.

Figure 2

In vitro neutralizing antibody assay on preinjected monkey sera. Serial twofold dilutions of monkey and naive mouse serum were incubated with 1 × 10⁹ genome copies of (a) AAVrh.8.CMV.LacZ or (b) AAV8.CMV.LacZ. Transduction efficiency at each serum dilution was measured by β-galactosidase levels (relative light unit per second, RLU/second) using a luminometer 24 hours after infection. AAV neutralization titer for each sample was determined by the highest serum dilution that inhibited AAV.CMV.LacZ transduction by ≥50%, compared with the mouse serum control. Pre-samples for 608137, 606145, and 607177 were obtained 14 days before gene transfer, and 12 days before gene transfer for 605103 and 605067. AAV, adeno-associated virus.

Figure 3

Detection of neutralizing AAV antibodies in preinjected monkey sera by in vivo passive transfer experiment. C57BL/6 mice were infused with of 200 µl of serum sample from an individual NHP or naive mouse serum. Two hours after the passive transfer, mice received an intravenous injection of 1 × 10⁹ genome copies of AAV.LSP.cFIX-W vector packaged with the respective capsids (AAV8, hu.37, or rh.8). cFIX expression levels in the plasma at days 7, 21, and 28 after vector administration are shown. Data are presented as mean ± SD (n = 3). AAV, adeno-associated virus; cFIX, canine factor IX; NHP, nonhuman primate.

Figure 4

Time course of liver enzyme levels (ALT and AST) in monkeys before and after intravenous infusion of 3 × 10¹² genome copies/kg of AAV.TBG.EGFP pseudotyped with AAV8, hu.37, and rh.8. Pre1 samples were taken 71, 79, and 87 days before vector administration of AAV8, hu.37, and rh.8, respectively. Pre2 samples were taken 12, 20, and 35 days before vector administration of AAV8, hu.37, and rh.8, respectively. AAV, adeno-associated virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LFT, liver function test.

Figure 5

Time course of T-cell responses to AAV capsid and GFP in the PBMCs of monkeys after vector administration. PBMCs, isolated before and weekly after vector injection, were stimulated with AAV8 capsid and GFP-peptide libraries and assayed by IFNγ ELISPOT. Background from unstimulated control (<30 SFU/10⁶ lymphocytes) was subtracted from each sample. Asterisk indicates a positive response, which is defined as more than threefold of background and >55 SFU/10⁶ lymphocytes. AAV, adeno-associated virus; ELISPOT, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; IFNγ, interferon-γ PBMC, peripheral blood mononuclear cell; SFU, spot forming units.

Figure 6

T-cell response to GFP in different tissues at day 35 after vector administration. PBMCs and lymphocytes isolated from spleen, bone marrow (BM), and liver were stimulated with GFP-peptide library and assayed by IFNγ ELISPOT. Background from unstimulated control (<30 SFU/10⁶ PBMCs, <65 SFU/10⁶ splenocytes, <70 SFU/10⁶ bone marrow lymphocytes, and <50 SFU/10⁶ liver lymphocytes) was subtracted from each sample. Asterisk indicates a positive response, which is defined as more than threefold of background and >55 SFU/10⁶ lymphocytes. AAV, adeno-associated virus; ELISPOT, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; IFNγ, interferon-γ PBMC, peripheral blood mononuclear cell; SFU, spot forming units.