Adeno-associated virus vector as a platform for gene therapy delivery

Abstract

Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. Recent advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Preclinical and clinical successes in AAV-mediated gene replacement, gene silencing and gene editing have helped AAV gain popularity as the ideal therapeutic vector, with two AAV-based therapeutics gaining regulatory approval in Europe or the United States. Continued study of AAV biology and increased understanding of the associated therapeutic challenges and limitations will build the foundation for future clinical success.

Fig. 1 |. 50 years of AAv.

A timeline is pictured showing selected key milestones in adeno-associated virus (AAV) gene therapy development. Following the first reports on the discovery of AAV in 1965 and 1966 (REFS^,), the next 15–20 years of basic biology research culminated in the cloning and sequencing of the AAV2 genome^–. AAV was vectorized in 1984 for in vitro gene delivery^,, and the development of a trans-complementing system to produce high-quality recombinant AAV (rAAV) in the late 1980s greatly facilitated the use of rAAV as a gene delivery vehicle^–, leading to the first in vivo application. Persistent gene expression by rAAVs in mammalian tissues was first reported in 1994 (REF.), followed by seminal publications in 1996 (REFS^–). An AAV vector was first used in a human patient in 1995 for the treatment of cystic fibrosis. A new family of primate AAV serotypes was reported in the early 2000s, greatly expanding the AAV toolbox for in vivo gene delivery^–. The efficacy of AAV gene therapy was convincingly demonstrated in 2008 for the treatment of Leber congenital amaurosis^–. The first AAV-based gene therapy drug, Glybera, was approved by the European Medicines Agency (EMA) in 2012, with Luxturna becoming the first AAV gene therapy product to receive US Food and Drug Administration (FDA) approval 5 years later. We emphasize that early studies of the basic biology of AAV laid the foundation for vector development and therapeutic application. During the translation process, the application of AAV vectors for gene therapy delivery further stimulated interest in studying AAV biology.

Fig. 2 |. Diagram of rAAv transduction pathway.

Adeno-associated virus (AAV) is recognized by glycosylated cell surface receptors of the host. This triggers internalization of the virus via clathrin-mediated endocytosis. AAV then traffics through the cytosol mediated by the cytoskeletal network. Owing to the somewhat low pH environment of the endosome, the VP1/VP2 region undergoes a conformational change. Following endosomal escape, AAV undergoes transport into the nucleus and uncoating. AAV can also undergo proteolysis by the proteasome. There are currently two classes of recombinant AAVs (rAAVs) in use: single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). ssAAVs are packaged as either sense (plus-stranded) or anti-sense (minus-stranded) genomes. These single-stranded forms are still transcriptionally inert when they reach the nucleus and must be converted to double-stranded DNA as a prerequisite for transcription. This conversion can be achieved by second strand synthesis via host cell DNA polymerases or by strand annealing of the plus and minus strands that may coexist in the nucleus. Because scAAVs are already double-stranded by design, they can immediately undergo transcription. The viral inverted terminal repeats (ITRs) present in the rAAV genome can drive inter-molecular or intra-molecular recombination to form circularized episomal genomes that can persist in the nucleus. Vector genomes can also undergo integration into the host genome at very low frequencies, depicted by the dashed line (BOX 2).

Fig. 3 |. infographic of the four primary methods for capsid discovery and engineering.

a | Directed evolution via methods such as capsid shuffling or error-prone PCR can create numerous unique capsid combinations that may harbour distinct and favourable vector properties. b | Discovery of naturally occurring adeno-associated virus (AAV) surveys proviral sequences present in host tissues that may have been infected with wild-type AAVs. c | Rational design utilizes pre-existing knowledge of capsid biology and host cell targets to engineer capsids that specifically recognize tissue-specific or cell-specific extracellular markers or to evade immune surveillance. d | In silico design, a somewhat new method for capsid discovery, utilizes computational approaches to predict novel capsid designs that are not seen in nature. Reconstruction of ancestral AAVs from contemporary capsids is one form of in silico design. The inner circle depicts the primary methods for candidate capsid screening: in vitro (for example, cell culture) and in vivo using small animal models (for example, mice) and large animal models (for example, non-human primates) that serve as proxies for the human patient.

Fig. 4 |. overview of rAAv interventional gene therapy clinical trials.

The data set is from ClinicalTrials.gov, accessed on 13 November 2018. The 145 registered trials are categorized on the basis of adeno-associated virus (AAV) capsid serotype (part a), primary tissue target for gene delivery (part b) and clinical trial phase (part c). rAAV, recombinant AAV.

Fig. 5 |. immunological barriers to successful rAAv gene delivery.

The recombinant adeno-associated virus (rAAV) may encounter neutralizing antibodies (NAbs) that are widely found in the human population, which greatly compromises gene delivery , especially following intravascular administration. Furthermore, administration of rAAV can induce capsid-specific NAb generation (not shown). The rAAV capsid and genome may trigger innate immunity via activation of Toll-like receptor 2 (TLR2) and TLR9, respectively. Activation of innate immunity can further promote adaptive immune responses. The capsid undergoes proteasomal degradation, and the resulting peptides are presented by major histocompatibility complex (MHC) class I molecules to CD8⁺ T cells. The CD8⁺ T cell can exert destructive cytotoxic effects to eliminate rAAV-transduced cells, resulting in the loss of transgene expression. The transgene product can elicit a humoral immune response to generate transgene product-specific antibodies that can compromise therapeutic efficacy. AAV, adeno-associated virus.