In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses

Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA 94305, USA.
Journal of Virology (Impact Factor: 4.44). 07/2008; 82(12):5887-911. DOI: 10.1128/JVI.00254-08
Source: PubMed


Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.

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Available from: Mark A Kay, Aug 14, 2014
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    • "Chimeric capsids based on in vitro evolution strategy was first described by Grimm et al.,, in 2008 [63]. In this study, cap sequences from 8 AAV serotyopes (AAV2,4,5,8,9, avian, caprine and bovine AAV) were randomly shuffled and reassembled and selected first for its ability to transduce hepatoma cell lines in the presence of IVIG. "
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    ABSTRACT: Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose.
    Full-text · Article · Nov 2014 · Journal of Biomedical Science
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    • "The capsid is the primary interface between virus and host cell, mediating vector binding to cell surface receptors (Wu et al., 2006). Moreover, the capsid but not the ITR sequence influences cell and tissue tropism (Grimm et al., 2008; Vandenberghe et al., 2008). The propensity of certain AAVs serotypes to bypass anatomical barriers is directly related to their capsid composition since only several serotypes with identical genome are able to efficiently cross the BBB (Zhang et al., 2011). "
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    ABSTRACT: Adeno-associated virus (AAV)-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.
    Full-text · Article · Jun 2014 · Frontiers in Molecular Neuroscience
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    • "This class of viruses also exhibits stable and long lasting transgene expression and a wide range of infectivity [17,21]. Numerous AAV serotypes have been isolated from adenoviral stocks [22,23], from humans [24-26] and from primate tissues [24-26]; some AAV serotypes have also been engineered by directed evolution [27-29]. These AAV serotypes possess three different capsid proteins: Virion Proteins 1, 2, 3 (VP1, VP2 and VP3). "
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    ABSTRACT: In recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype’s ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (alphaCaMKII). We determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5[prime]s decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined. For laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.
    Full-text · Article · Feb 2014 · BMC Neuroscience
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