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      Engineering adeno-associated viruses for clinical gene therapy.

      Nature reviews. Genetics

      Capsid, chemistry, metabolism, Clinical Trials as Topic, DNA, Viral, genetics, Dependovirus, Directed Molecular Evolution, Genetic Engineering, Genetic Therapy, methods, Genetic Vectors, Genome, Viral, Hemophilia A, pathology, therapy, Humans, Metabolic Diseases, Muscular Dystrophies, Viral Proteins

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          Abstract

          Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

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          Most cited references 44

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          In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.

          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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            Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure.

            Adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase was assessed in a randomized, double-blind, placebo-controlled, phase 2 study in patients with advanced heart failure. Thirty-nine patients received intracoronary adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase or placebo. Seven efficacy parameters were assessed in 4 domains: symptoms (New York Heart Association class, Minnesota Living With Heart Failure Questionnaire), functional status (6-minute walk test, peak maximum oxygen consumption), biomarker (N-terminal prohormone brain natriuretic peptide), and left ventricular function/remodeling (left ventricular ejection fraction, left ventricular end-systolic volume), plus clinical outcomes. The primary end point success criteria were prospectively defined as achieving efficacy at 6 months in the group-level (concordant improvement in 7 efficacy parameters and no clinically significant worsening in any parameter), individual-level (total score for predefined clinically meaningful changes in 7 efficacy parameters), or outcome end points (cardiovascular hospitalizations and time to terminal events). Efficacy in 1 analysis had to be associated with at least a positive trend in the other 2 analyses. This combination of requirements resulted in a probability of success by chance alone of 2.7%. The high-dose group versus placebo met the prespecified criteria for success at the group-level, individual-level, and outcome analyses (cardiovascular hospitalizations) at 6 months (confirmed at 12 months) and demonstrated improvement or stabilization in New York Heart Association class, Minnesota Living With Heart Failure Questionnaire, 6-minute walk test, peak maximum oxygen consumption, N-terminal prohormone brain natriuretic peptide levels, and left ventricular end-systolic volume. Significant increases in time to clinical events and decreased frequency of cardiovascular events were observed at 12 months (hazard ratio=0.12; P=0.003), and mean duration of cardiovascular hospitalizations over 12 months was substantially decreased (0.4 versus 4.5 days; P=0.05) on high-dose treatment versus placebo. There were no untoward safety findings. The Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) study demonstrated safety and suggested benefit of adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase in advanced heart failure, supporting larger confirmatory trials. http://www.clinicaltrials.gov. Unique identifier: NCT00454818.
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              Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.

              Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.
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                Author and article information

                Journal
                24840552
                4393649
                10.1038/nrg3742

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