Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles

Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352, USA.
Molecular Therapy (Impact Factor: 6.43). 08/2008; 16(7):1252-60. DOI: 10.1038/mt.2008.100
Source: PubMed

ABSTRACT We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

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Available from: Stefan Leichtle, Feb 09, 2014
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    • "The GP3 is one of the most variable structure proteins of PRRSV with 86–98% aa sequence identity among type 2 PRRSV strains (Kapur et al., 1996; Mardassi et al., 1995; Meng et al., 1995; Morozov et al., 1995), and only 54–60% identity between type 1 and type 2 strains (Mardassi et al., 1995; Murtaugh et al., 1995). DNA shuffling mimics the natural recombination process at a much accelerated rate and has been used for directing the evolution of genes (Apt et al., 2006; Callison et al., 2005; Li et al., 2008; Locher et al., 2005; Pekrun et al., 2002; Soong et al., 2000; Yang et al., 2009). By using DNA shuffling, the recombinants with new patterns of phenotypes can be rapidly screened and identified (Soong et al., 2000). "
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    ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen. Here we applied the DNA shuffling approaches to molecularly breed the PRRSV GP3 gene, a neutralizing antibodies inducer, in an attempt to improve its heterologous cross-neutralizing ability. The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling. Additionally, synthetic DNA shuffling of the GP3 gene was also performed using degenerate oligonucleotides. The shuffled-GP3-libraries were cloned into the backbone of a DNA-launched PRRSV infectious clone pIR-VR2385-CA. Four traditional-shuffled chimeras each representing all 6 parental strains and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication both in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain FL-12.
    Virology 10/2012; 434(1). DOI:10.1016/j.virol.2012.09.005 · 3.28 Impact Factor
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    • "Despite some clinical success (Maguire et al., 2008, 2009), AAV2 is limited by the relatively low efficiency of gene transfer, slower onset of gene expression, and higher prevalence of preexisting immunity in human populations (Gao et al., 2005). To overcome such drawbacks, many investigators have successfully explored either naturally occurring (Gao et al., 2002) or genetically engineered and modified (Schaffer and Maheshri, 2004; Muzyczka and Warrington, 2005; Li et al., 2008; Yang et al., 2009) novel AAV capsids, which are the major determinants of tropism and transduction efficiency. To date more than 11 AAV serotypes (Wu et al., 2006) and more than 100 naturally occurring primate AAV variants have been reported (Gao et al., 2005). "
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    ABSTRACT: Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or β-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart.
    Human Gene Therapy Methods 02/2012; 23(1):29-37. DOI:10.1089/hgtb.2011.229 · 1.64 Impact Factor
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    • "'Dynamic' regulators acting in an immune response phase-related manner Multiple new inducers of cell migration have been identified, but counterbalancing mechanisms (Friedl and Weigelin, 2008; Steevels and Meyaard, 2011; Steevels et al., 2011 "
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    ABSTRACT: Development of novel cell migration modulators for anti-inflammatory and cardiovascular therapy is a complex task since any modulator will necessarily interfere with a balanced system of physiological regulators directing proper positioning of diverse immune cell types within the body. Whereas this shall serve efficient pathogen elimination, lack of proper control over these processes may result in counterproductive chronic inflammation and progressive tissue injury instead of healing. Prediction of the therapeutic potential or side effects of any migration modulator is not possible based on theoretical considerations alone but needs to be experimentally evaluated in preclinical disease models and by clinical studies. Here, we briefly summarize basic mechanism of cell migration, and groups of synthetic drugs currently in use for migration modulation. We then discuss one fundamental problem encountered with single-target approaches that arises from the complexity of any inflammation, with multiple interacting and often redundant factors being involved. This issue is likely to arise for any class of therapeutic agent (small molecules, peptides, antibodies, regulatory RNAs) addressing a single gene or protein. Against this background of studies on synthetic migration modulators addressing single targets, we then discuss the potential of endogenous proteins as therapeutic migration modulators, or as parent compounds for the development of mimetic drugs. Regulatory proteins of this type commonly address multiple receptors and signalling pathways and act upon the immune response in a phase-specific manner. Based on recent evidence, we suggest investigation of such endogenous migration modulators as novel starting points for anti-inflammatory and cardiovascular drug development.
    British Journal of Pharmacology 10/2011; 165(7):2044-58. DOI:10.1111/j.1476-5381.2011.01762.x · 4.99 Impact Factor
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