In class II, much like class I, sera from AAV2 immunization suppressed AAV2 and mutant transductions with related efficiencies. DNA computer virus that requires a helper computer virus, such as adenovirus (Ad) or herpes simplex virus, for efficient replication. Despite the lack of swelling or additional AAV-associated complications following administration of AAV2 vectors in several organs, neutralizing antibody (NAb) titers against the AAV2 capsid were found to be significantly increased following vector administration, particularly in the lung, muscle, and liver (7, 8, 43C45, 49, 54, 63). NAbs in blood circulation are able to block AAV transduction after systemic administration. Recently, Manno et al. (44) performed a phase I study of AAV-mediated FIX transgene delivery in individuals with hemophilia B, in which one patient with a higher preexisting NAb titer shown lower YIL 781 levels of FIX in the serum than another patient with lower preexisting NAbs against AAV2. These results suggest that preexisting NAbs in the human population can attenuate vector transduction effectiveness and inhibit transgene manifestation upon systemic software (44). In the general human population, over 95% of individuals have been infected by AAV2, with 50% of them having NAbs (3, 4, 6, 10, 18, 21, 24, 27, 35, 48, 62, 65). The prevalence of NAb in children is lower, ranging from 13 to 25% (9, 35). Although 11 additional types of AAV have been isolated for gene therapy purposes, little to no cross-reactivity of NAbs is definitely demonstrated among these types in animals (27, 30, 38, 60, 65). In humans, recent studies have shown that different examples of NAb cross-reactivity exist between AAV2 and other types (6, 10, 27, 45). There is a lower prevalence of NAbs against AAV1, -5, -6, -7, and -8 than against AAV2 (6, 10). These findings present a concern to the gene therapy community as to how we can avoid antagonistic NAb activity during systemic delivery of AAV vectors. To conquer this concern, several approaches have been exploited in PIK3CB our lab and by additional organizations, including (i) the utilization of polymers to protect the AAV capsid and block NAb acknowledgement (11, 32, 33), (ii) the development of NAb YIL 781 escape mutants by error-prone PCR (31, 41, 56), (iii) the application of other types of AAV vectors (27, 30, 38, 60, 65), and (iv) the generation of chimeric types via AAV shuffling (2, 36, 37). In this study, we have systematically explored the possibility of using different types and AAV mutants as option vectors for intramuscular gene delivery in mice preimmunized against different AAV types. MATERIALS AND METHODS Plasmids and viruses. We first generated a series of AAV type vectors (AAV1 to -6) YIL 781 as explained previously (57). The plasmid pRep6cap6 was a kind gift from David Russell (University or college of Washington) (61). All constructs were generated in the pXR2 backbone (57). Site-directed mutagenesis (Stratagene QuikChange site-directed mutagenesis kit) was used to place nucleotides or alternative point mutations. The pXR2.5 plasmid was constructed by replacement of amino acids at positions 263, 265, 709, 712, and 720 of AAV2 from the corresponding amino acids in AAV1. Primer 5-CCAGCCAATCANNKGGAGCCTCGAACG-3 was used to generate clones with insertion of an amino acid(s) at residue 265 of the AAV2 capsid. AAV was produced in 293 cells using a triple-transfection protocol (68). Computer virus was concentrated by double cesium chloride gradient centrifugation. YIL 781 The titer was quantitated by Southern dot blotting, and the contamination of preparations with vacant particle was less than 5%. Dedication of immunoglobulin titers for different AAV types. For assessment of the humoral immune response to AAV types 1 to 6, 1 1010 particles of AAV/green fluorescent protein (GFP) (100 l) were intraperitoneally injected into 6- to 10-week-old mice (BALB/c; Jackson Laboratory, ME) at day time 0, and mice were boosted with the same computer virus as for the primary immunization at day time 14. Blood sera were collected via the retro-orbital plexus in the indicated time points for NAb assays. Serially diluted serum samples from immunized mice were analyzed for AAV-specific immunoglobulins (total Ig, IgG, IgM, IgG1, IgG2a, IgG2b,.