Durability of transgene expression after rAAV gene therapy

Abstract

Recombinant adeno-associated virus (rAAV) gene therapy has the potential to transform the lives of patients with certain genetic disorders by increasing or restoring function to affected tissues. Following the initial establishment of transgene expression, it is unknown how long the therapeutic effect will last, although animal and emerging human data show that expression can be maintained for more than 10 years. The durability of therapeutic response is key to long-term treatment success, especially since immune responses to rAAV vectors may prevent re-dosing with the same therapy. This review explores the non-immunological and immunological processes that may limit or improve durability and the strategies that can be used to increase the duration of the therapeutic effect.

Keywords: animals; gene expression; gene expression regulation; gene therapy; gene transfer techniques; genetic therapy/methods; genetic vectors; human genetics; transgenes.

Figure 1

Factors that may reduce durability Non-immunological (orange) and immunological factors (green) that may affect durability over weeks, months, and years post-gene therapy administration. CpG, 5′-cytosine-phosphate-guanine-3′; dsRNA, double-stranded RNA.

Figure 2

From rAAV administration to long-term transgene expression The rAAV vector binds to receptors on the target cell surface (1) and is taken into the cell by endocytosis (2). rAAV traffics from early to late endosomes (3) and is delivered to the nucleus. Once inside the nucleus, the capsid is removed (uncoating) (4), releasing the genetic material. The transgene, if single-stranded, undergoes second-strand synthesis before forming a double-stranded DNA episome (5), which is maintained in the nucleus of the cell where it can be expressed (6), resulting in the production of the protein of interest (7).

Figure 3

Immune processes affecting the durability of rAAV gene therapy (A) Immune responses can occur against the rAAV capsid, transgene, or transgene protein. (B) In the case of recipients with prior exposure to rAAV or a similar serotype, NAbs may prevent initial transduction, resulting in low or absent expression of the transgene. Innate immune responses could result in no expression or a rapid loss of expression, but they are more likely to provide important signals to the adaptive immune system, with the potential to reduce or eliminate expression at a later stage after transgene expression has been established. On subsequent exposure, memory cells of the adaptive immune system can respond rapidly, with the release of NAbs against the rAAV capsid. dsRNA, double-stranded RNA; NAbs, neutralizing antibodies; rAAV, recombinant adeno-associated virus.

Figure 4

Mechanisms of rAAV detection by the innate immune response (1) TLRs are membrane-bound pattern-recognition receptors responsible for detecting pathogen-associated molecular patterns, for example, in the genomes of bacteria and viruses. The rAAV genome, particularly unmethylated CpG motifs, can be detected by TLR9 on the endosomal membranes of dendritic cells following endocytosis and degradation by the endosome. In addition, TLR2, which recognizes microbial protein and glycolipid structures on the dendritic cell surface and within endosomes, has been shown to detect the AAV capsid in circulation.^, TLR2 and TLR9, with the downstream mediator MyD88 are believed to be the key pattern-recognition receptors responsible for initiating an innate immune response against rAAV-mediated gene therapy, triggering the expression of pro-inflammatory cytokines and type I IFNs via downstream signaling pathways involving the transcription factors nuclear factor κB (NF-κB) and interferon (IFN) regulatory factor (IRF), respectively.^, (2) Following successful transduction, dsRNA can form and be detected by cytosolic RNA sensors, RIG-1 and MDA5, leading to the expression of IFN-β via downstream signaling pathways. In addition to triggering the destruction of the cell, these mechanisms led to an antiviral state in the surrounding tissue and the recruitment of innate immune cells, as well as providing activation signals for an adaptive immune response.^,

Figure 5

Mechanisms of the adaptive immune system affecting durable transgene expression Following rAAV vector administration and successful transduction, transduced cells present capsid-derived epitopes via MHC class I on the cell surface. This triggers the potential clearance of transduced cells by CTLs and recruitment of APCs, which are responsible for presenting antigens on their cell surfaces via MHC classes I and II. This, in turn, recruits additional CD8⁺ CTLs and CD4⁺ T helper cells and facilitates the release of antibodies by B cells, and establishment of a population of antigen-specific memory T and B cells.^,,, APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex; rAAV, recombinant adeno-associated virus.