In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

BackgroundKallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur.Methodology/Principal FindingsUsing murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice.Conclusion/SignificanceKLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis.

Bacterial superantigens bind directly to human leukocyte antigen (HLA) class II molecules and vigorously activate T cells expressing certain T-cell receptor variable region families. As interaction with HLA class II molecules is the primary step in this process, polymorphic variations in HLA class II can determine the extent of superantigen binding to HLA class II molecules, govern the magnitude of immune activation induced by given superantigens and determine the outcome of superantigen-mediated diseases. As direct assessment of the influence of HLA class II polymorphism in humans is impossible because of expression of more than one HLA class II alleles in a given individual and toxicity of superantigens, transgenic mice expressing HLA-DQ6 (HLA-DQA1*0103 and HLA-DQB1*0601) and HLA-DQ8 (HLA-DQA1*0301 and HLA-DQB1*0302) were used to achieve this goal. HLA-DQ6 and HLA-DQ8 elicited comparable in vitro and in vivo immune response to staphylococcal enterotoxins (SE) A, SEB, SEH and SEK, toxic shock syndrome toxin-1, streptococcal pyrogenic exotoxin (SPE) A and SPEC and streptococcal mitogenic exotoxin Z (SMEZ). However, each superantigen had a unique T-cell receptor activation profile. In vivo challenge with Streptococcus pyogenes, H305, capable of elaborating SPEA and SMEZ, yielded a similar clinical outcome in HLA-DQ6 and HLA-DQ8 transgenic mice. In conclusion, HLA-DQ6 and HLA-DQ8 elicited comparable response to certain bacterial superantigens. Our report highlights the advantages of HLA class II transgenic mice in such studies.

With the recent advent of several generations of targeted DNA nucleases, most recently CRISPR/Cas9, genome editing has become broadly accessible across the biomedical community. Importantly, the capacity of these nucleases to modify specific genomic loci associated with human disease could render new classes of genetic disease, including autosomal dominant or even idiopathic disease, accessible to gene therapy. In parallel, the emergence of adeno-associated virus (AAV) as a clinically important vector raises the clear possibility of integrating these two technologies towards the development of gene editing therapies. Though clear challenges exist, numerous proof of concept studies in preclinical models offer exciting promise for the future of gene therapy.