Emerging Issues in AAV-Mediated In Vivo Gene Therapy

Abstract

In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective "therapeutic products" is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.

Keywords: AAV; AAV capsid; AAV genome; gene therapy; genotoxicity; inherited diseases; liver immunogenicity; persistence.

Figure 1

Schematic Representation of Oversized and Dual AAV Vector Strategies for Large Transgene Expression Transgene expression cassettes larger than 4.7 kb can be packaged in oversized AAV vectors (A) or in regular-size dual AAV vectors that undergo genome re-assembly after cell co-transduction (B–D). (B–D) The dual AAV genome re-assembly is driven by homologous recombination between homology regions (HR) within the transgene sequence (B), inverted terminal repeats (ITRs)-mediated genome concatemerization (C), or homologous recombination between highly recombinogenic heterologous homology regions (HHR) (D). CDS, coding sequence; polyA, poly-adenylation signal; SA, splicing acceptor signal; SD, splicing donor signal.

Figure 2

Current Issues in AAV Liver Gene Transfer AAV liver gene transfer provided evidence of safety and efficacy in recent clinical trials. However, several issues related to the AAV vector platform and to the vector-host interaction are emerging (white box). These issues will need to be addressed in the future to expand the application of AAV in vivo gene therapy. (1) Vector immunogenicity: neutralizing antibodies (NAbs) against the AAV capsid prevent/limit cell transduction, whereas cytotoxic CD8⁺ T cell responses eliminate AAV-transduced cells that present AAV capsid antigens loaded on major histocompatibility complex class I molecule (MHC-I) complexes. Innate immune responses contribute to the overall vector immunogenicity. (2) Potency and efficacy: the efficiency of AAV vectors at infecting and transducing the desired target cells impacts on therapeutic doses and therapeutic efficacy. (3) Genotoxicity: integration of the AAV vector DNA in the genome of the infected cell, despite being a rare event, may have genotoxic effects. (4) Persistence: because the AAV genome mainly persists in an episomal form in the nucleus of the infected cells, it can be lost in conditions of cell proliferation (such as liver growth), limiting therapeutic efficacy. ER, endoplasmic reticulum.

Figure 3

Schematic Path for the Optimization of AAV-Based Approaches for Human Gene Therapy Preclinical studies performed in animal models are key to address the current limitations of AAV gene therapy approaches (white boxes) related to the vector genome and the transgene expression cassette (upper boxes) or the vector capsid (bottom boxes). Examples of various available strategies aimed at overcoming these limitations are depicted (gray boxes). pts., patients; CTL, cytotoxic T lymphocyte; IgG, immunoglobulin G.