Adeno-associated viral vectors at the frontier between tolerance and immunity

International audienceIn recent years, the field of in vivo gene transfer with adeno-associated virus (AAV) vectors has seen an extraordinary expansion of applications and investments. Results emerging from clinical trials (1) and the recent market approval of a gene therapy drug for lipoprotein lipase deficiency (2) contributed to the hype around this vector system (3). Indeed, AAV vectors have several features that make them an ideal tool for gene transfer, for example, parental virions are replication deficient and non-pathogenic (4), and vectors can drive expression of a transgene for several years (5, 6) despite the fact that they do not integrate efficiently into the host genome. In recent years, a portfolio of natural AAV isolates (AAV serotypes) differing in tissue tropism has been developed as vectors. This toolbox has been further expanded with engineered AAV capsids developed to enhance efficiency and specificity of gene delivery, and to escape antibody neutralization (7). At the vector genome level, availability of potent promoter/enhancer sequences, codon-optimization of transgenes, and development of self-complementary AAV vectors (8) further enhanced efficacy of gene transfer. Finally, the availability of scalable processes to produce AAV vectors in GMP contributed significantly to the expansion of the field

Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities

Adeno-associated viral (AAV) vectors have emerged as one of the most popular gene transfer systems in both research and clinical gene therapy. As AAV vectors are derived from a stealth, nonpathogenic virus and lack active integrase activity, these vectors are frequently applied for in vivo gene therapy of liver, muscle, and other postmitotic tissues. Although long-term transgene expression from AAV vector episomes is reported from these tissues, the episomal nature of AAV-once regarded as disadvantage-has become an attractive feature for gene-editing approaches targeting proliferating cells. In response to the high demand, AAV vector production is receiving special attention. Besides particle yields and biological activity, the most important concern is improving vector purity. The most difficult task in this regard is removal of defective particles, that is, capsids that are either empty or contain DNA other than the full-length vector genomes. Herein, we characterize and discuss these so-called product-related impurities, methods for their detection, as well as strategies to avoid or reduce their formation

Small But Increasingly Mighty: Latest Advances in AAV Vector Research, Design, and Evolution

Recombinant gene delivery vectors derived from naturally occurring or genetically engineered adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion, and cell specificity in order to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology, including virus-host interactions, and/or that expand our arsenal of technologies for AAV capsid design and evolution. This review highlights a collection of latest advances in these areas, which, in the authors' opinion, hold particular promise to propel the AAV vector field forward in the near future, especially when applied in combination. These include fundamental novel insights into the AAV life cycle, from an unexpected role of autophagy and interactions with other viruses to the (re-)discovery of a universal AAV receptor and the function of AAV-AAP for capsid assembly. Concurrently, recent successes in the rational design of next-generation synthetic AAV capsids are pointed out, exemplified by the structure-guided derivation of AAV mutants displaying robust in vivo immune evasion. Finally, a variety of new and innovative strategies for high-throughput generation and screening of AAV capsid libraries are briefly reviewed, including Cre recombinase-based selection, ancestral AAV capsid reconstruction, and DNA barcoding of AAV genomes. All of these examples showcase the present momentum in the AAV field and, together with work by many other academic or industrial entities, raise substantial optimism that the remaining hurdles for human gene therapy with AAV vectors will (soon) be overcome

Surface-Engineered Viral Vectors for Selective and Cell Type-Specific Gene Delivery

Recent progress in gene transfer technology enables the delivery of genes precisely to the application-relevant cell type ex vivo on cultivated primary cells or in vivo on local or systemic administration. Gene vectors based on lentiviruses or adeno-associated viruses can be engineered such that they use a cell surface marker of choice for cell entry instead of their natural receptors. Binding to the surface marker is mediated by a targeting ligand displayed on the vector particle surface, which can be a peptide, single-chain antibody, or designed ankyrin repeat protein. Examples include vectors that deliver genes to specialized endothelial cells or lymphocytes, tumor cells, or particular cells of the nervous system with potential applications in gene function studies and molecular medicine

Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy

Introduction: Gene therapy clinical trials with adeno-associated virus (AAV) vectors report impressive clinical efficacy data. Nevertheless, challenges have become apparent, such as the need for high vector doses and the induction of anti-AAV immune responses that cause the loss of vector-transduced hepatocytes. This fostered research focusing on development of next-generation AAV vectors capable of dealing with these hurdles. Areas Covered: While both the viral vector genome and the capsid are subjects to engineering, this review focuses on the latter. Specifically, we summarize the principles of capsid engineering strategies, and describe developments and applications of engineered capsid variants for liver-directed gene therapy. Expert Opinion: Capsid engineering is a promising strategy to significantly improve efficacy of the AAV vector system in clinical application. Reduction in vector dose will further improve vector safety, lower the risk of host immune responses and the cost of manufacturing. Capsid engineering is also expected to result in AAV vectors applicable to patients with preexisting immunity toward natural AAV serotypes

A Novel Directed Evolution Method to Enhance Cell-Type Specificity of Adeno-Associated Virus Vectors

Clinical application of viral vectors is often hampered by the lack of selectivity of viral particles for the targeted tissue. This drawback decreases the efficiency of gene delivery and raises safety concerns. We successfully established a novel in vitro evolution protocol to engineer adeno-associated virus vectors with increased selectivity for designated target cells. Subjecting a peptide-display library of AAV capsids to negative selection cycles on human primary fibroblasts and to positive selection cycles on a human melanoma cell line, we isolated several variants with up to 3.7-fold increased specificity for malignant cells in comparison to fibroblasts and other cell types. These mutants can be used to achieve high levels of gene transfer to target cells reducing undesired transduction of neighbouring tissues

Engineering the AAV capsid to optimize vector-host-interactions

Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of preexisting neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAV's tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAV's host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously non-permissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes. These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine