Blood. primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in Rabbit Polyclonal to KLF10/11 >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression Ionomycin in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients. Introduction Systemic administration of adeno-associated virus (AAV) vectors has been used to transduce the liver for the subsequent production of a therapeutic protein. This approach has shown robust efficacy in mouse models for several lysosomal storage diseases (LSDs).1,2,3,4 For example, an AAV8 vector bearing -galactosidase A (gal) was used to transduce the liver of a mouse model for Fabry disease, resulting in the correction of both biochemical and functional deficits.1 This same strategy has been used successfully to generate factor IX (FIX) in mice,5,6,7,8,9 dogs,10,11,12 nonhuman primates (NHPs),8,13,14,15 and hemophilia B patients.16 Although host immune responses have been the major concern in patients, there have also been anecdotal reports that the expression levels produced from AAV transduction of mouse liver exceed those that can be obtained from primates.7,15,17 Thus, for a well-secreted protein like FIX, expression levels attained in patients are generally less than those seen in mouse models.9,16 Compared to FIX, the Ionomycin secretion efficiency of LSD proteins is significantly lower, and the target blood levels for therapy are significantly higher. For example, FIX levels of 200?ng/ml are considered sufficient, while for gal, serum levels approaching 1,000?ng/ml are likely to be required1 because gal must be taken up from the circulation into the lysosomes of the target endothelial cells. Thus, generating necessary serum levels of an LSD protein such as gal in primates using a liver-directed approach may represent a higher hurdle than an analogous approach for a well-secreted protein like FIX. Primates, both monkeys18 and humans,19,20 are known to have prior exposure to AAV, although Ionomycin the Ionomycin fraction of the population with identified exposure may vary by viral serotype and assay used to characterize that exposure. By any measure, a significant fraction of NHPs have been exposed to AAV, and in those with high neutralizing anti-AAV titers, attempts to transduce the liver are largely blocked. Indeed, recent studies have pointed out that very low levels of neutralizing antibodies are sufficient to prevent liver transduction by AAV.7,15,17 However, neither the relationships between viral dose, preexisting anti-AAV antibody level and liver transduction, nor between total and neutralizing anti-AAV antibodies are well characterized. Prior exposure of the primate liver to AAV also has the potential to alter viral trafficking and transgene expression. For example, latent AAV in mammalian hepatocytes is likely maintained by low levels of viral expression.21 How this might impact a subsequent transduction of the same hepatocyte by a gene therapy vector is largely unknown. By quantifying the role played by preexisting anti-AAV antibodies in expression from the primate liver, we reasoned that any remaining differences between mouse and primate expression from the same vector would be attributable to either fundamental differences between Ionomycin vector fate in mouse and primate hepatocytes, or would be related to the prior exposure of the primate liver to AAV. To address possible translational issues related to the prior exposure of primates to AAV, we have used identical dosing [in DNase-resistant particles (drp)/kg] of a single preparation of an AAV2/8-DC190-h-gal (AAV8-gal) vector in mice and NHPs. Here, the use of one preparation is valuable as differences between preparations may impact vector expression levels. In our study, at equivalent vector dose the resulting expression levels in NHPs averaged 1.5-logs lower than those seen in mice. Through experiments in mouse and primate primary hepatocytes, we show that these differences in expression are unlikely to reflect species-specific differences in relative vector or promoter efficiency or in the efficiency of transgene translation or secretion. The potential role of preexisting antivector antibodies was characterized using the passive transfer.