Understanding AAV vector immunogenicity: from particle to patient

Abstract

Gene therapy holds promise for patients with inherited monogenic disorders, cancer, and rare genetic diseases. Naturally occurring adeno-associated virus (AAV) offers a well-suited vehicle for clinical gene transfer due to its lack of significant clinical pathogenicity and amenability to be engineered to deliver therapeutic transgenes in a variety of cell types for long-term sustained expression. AAV has been bioengineered to produce recombinant AAV (rAAV) vectors for many gene therapies that are approved or in late-stage development. However, ongoing challenges hamper wider use of rAAV vector-mediated therapies. These include immunity against rAAV vectors, limited transgene packaging capacity, sub-optimal tissue transduction, potential risks of insertional mutagenesis and vector shedding. This review focuses on aspects of immunity against rAAV, mediated by anti-AAV neutralizing antibodies (NAbs) arising after natural exposure to AAVs or after rAAV vector administration. We provide an in-depth analysis of factors determining AAV seroprevalence and examine clinical approaches to managing anti-AAV NAbs pre- and post-vector administration. Methodologies used to quantify anti-AAV NAb levels and strategies to overcome pre-existing AAV immunity are also discussed. The broad adoption of rAAV vector-mediated gene therapies will require wider clinical appreciation of their current limitations and further research to mitigate their impact.

Keywords: humoral immunity; neutralizing antibodies; patient exclusion; redosing; seroprevalence.

Figure 1

Schematic representation of AAV genome, the AAV life cycle and the engineering of a generic rAAV expression cassette . (A) AAVs consist of a single-stranded DNA genome of ~4.7 Kb enclosed by a capsid ~26 nm in diameter. The AAV genome contains three open reading frames flanked by two T-shaped inverted terminal repeats (ITRs). The cap, rep, MAAP and AAP of the AAV genome encode three capsid proteins, four replication proteins, the membrane-associated accessory protein (MAAP) that facilitates AAV egress and encapsulation and the assembly-activating protein (AAP) that facilitates capsid assembly in some serotypes. (B) In rAAV, the AAV genome is replaced by a transgene expression cassette, including a specialized promoter, enhancer, transgene of interest, and terminator, flanked with ITRs. (C) rAAV vectors transduce cells through (1) binding to cell surface receptors and/or co-receptors), followed by (2) internalization by endocytosis. AAV then (3) traffics through the endosomal and Golgi compartment and after endosomal escape, undergoes (4) nuclear transport and (5) uncoats releasing the genome, which is then (6) expressed. Relative size representations are not to scale.

Figure 2

Exemplar AAV serotypes and their preferential tissue tropism , , . Differences in the capsid sequence and the presence of specific host cell receptor(s) determine cell/tissue tropism of exemplar AAV serotypes. AAV, adeno-associated virus. CNS, central nervous system.

Figure 3

Canonical mechanism of action of NAbs , . In the absence of pre-existing NAbs, robust transgene expression is possible. In the presence of NAbs, developed following previous exposure to AAV, NAbs bind to the AAV vector and may prevent binding to the target cell receptor which can block vector transduction and transgene expression. The NAb may also inhibit the interaction of viral envelope protein and cell-surface receptors after the vector has bound to the cell. Other mechanisms by which NAbs impair transgene expression not shown include NAb-coated vector binds to the cellular receptor and is internalized and subsequently destroyed intracellularly; the NAbs bind to the cell receptor and block the rAAV binding and subsequent internalization. NAb, neutralizing antibody; rAAV, recombinant adeno-associated virus.

Figure 4

Assays used to detect immunity to AAVs. (A) An in vitro cell-based transduction inhibition assay, using a “reporter” gene such as GFP, β-galactosidase, or luciferase, which provides convenient and sensitive detection of transduction , , . The degree of inhibition of reporter gene expression is plotted against the dilutions of the serum sample. The anti-AAV NAb titer is defined as the highest serum dilution that inhibits vector transduction by a specified amount (eg, ≥50%) in comparison with the negative control sample. (B) An in vitro cell-based neutralization assay measures the binding of rAAV to target cells. Increased levels of NAbs are associated with a proportionate decrease in rAAV cell binding , , . (C) ELISAs can be used to measure antibody binding to the AAV capsid or other serotype-specific proteins or peptides. This method includes coating of the assay plate with AAV capsids (full or empty) or peptides, the addition of patient sample, and finally detection of signal , , . AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; NAb, neutralizing antibody; qRT-PCR, quantitative reverse transcription polymerase chain reaction; rAAV, recombinant adeno-associated virus; IC50, half maximal inhibitory concentration.