Therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity. Molecular engineering was recently used to identify key immunogenic amino acid residues of the viral capsid and generate mutants with decreased antibody recognition. Here we explored the importance of finely tuning amino acid identity at immunogenic sites to optimize vector phenotype. A capsid library was generated by codon randomization at five positions where substitutions were shown to yield antibody evading phenotypes. Screening this library to isolate immune-escaping mutants allowed an exhaustive scan of combinations of the 20 natural amino acids at each position and yielded variants that remained infectious when incubated with serum or IVIG concentrations that completely neutralize AAV-2. Clones obtained replacing different residues at the same positions displayed strikingly different phenotypes, demonstrating that a precise choice of amino acid substitutions is fundamental to optimize immune-escaping, packaging ability, infectivity and tropism.
Recent research into periodontal disease pathology focuses on the role of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in periodontal bone destruction processes. RANKL regulates the differentiation of osteoclast by binding to its specific receptor RANK, while OPG inhibits the differentiation of osteoclasts by binding RANKL and therefore preventing RANKL to bind RANK. The aim of the present study was to investigate the influence of Porphyromonas gingivalis lipopolysaccharide (LPS) and interleukin-6 (IL-6) on RANKL and OPG expression and release in periodontal ligament (PDL) cells. Human PDL cells were stimulated for 48 h with purified P. gingivalis LPS and IL-6. OPG and sRANKL release were assessed by using enzyme-linked immunosorbent assay technique. OPG and RANKL expression was quantitatively measured by using the real-time PCR technique. Whereas P. gingivalis LPS induced sRANKL release, expression was only slightly increased, IL-6 did not show an effect on RANKL expression or release. In conclusion the data demonstrate that stimulation of PDL cells with P. gingivalis LPS leads to an increased release of sRANKL, rather than increased RANKL expression. Through this action, P. gingivalis LPS may exert its biological effect on osteoclast formation and bone resorption.
Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.
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