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IntroductionLiver-directed gene transfer using adeno-associated virus (AAV) vectors has the potential to serve as therapy for several inherited hematologic diseases. One such disease is the bleeding disorder hemophilia B, caused by a deficiency in coagulation factor IX (FIX). Currently, there are 2 clinical trials for hemophilia B that use liver-directed AAV-mediated gene transfer of the F9 gene (www. clinicaltrials.gov; identifiers NCT00515710 and NCT00979238). One of these trials reported transient efficacious circulating FIX levels (ϳ 10%) with the use of the vector AAV2-hFIX16. 1 Although AAV vectors are predominantly nonintegrating, with most of the transgene expression from stable episomes, 2 it has been shown through direct sequencing that integration can occur. 3,4 When integration takes place, there is a preference for integrating in regions where DNA breaks occur. These can be regions of endonuclease cleavage, 5 active transcription, 6-8 cytosine-phosphateguanosine (CpG) islands, 7,8 and palindromes. 9 All of these studies describing AAV vector genome integration identified vector integration sites through plasmid rescue of vectors containing bacterial origins of replication (ori). 4 Amplification of these plasmids in bacterial culture allows for sequencing of the integration junction between vector and host genome. Because of the bacterial selection involved in this method, bias may occur against recovering integrants whose size or sequence negatively affect bacterial growth, resulting in incomplete mapping of the full spectrum of integrants.Vector genome integration has been associated with adverse events; integrating ␥-retroviral vectors were implicated in the clonal expansion of transduced cells in 3 clinical studies, 2 for X-linked severe combined immunodeficiency 10,11 and the other for chronic granulomatous disease. 12,13 Although AAV vectors integrate at a much lower frequency than retroviral vectors, low-level AAV vector integration in transduced cells may still be a concern. A compelling argument supporting low genotoxic risk of AAV vectors comes from long-term follow-up of liver-directed AAVmediated gene transfer in canine and murine models. Of 77 dogs receiving AAV vector at doses up to 3.4 ϫ 10 12 vector genomes(vg)/kg and followed for Յ 10 years, none developed liver tumors as assayed by ultrasound, computed tomographic (CT) scan, and magnetic resonance imaging (MRI) 14,15 (K.A.H., V.R.A., and Timothy C. Nichols, unpublished data, October 15, 2010). Similarly, Ͼ 300 mice receiving AAV vectors with a therapeutic transgene at doses up to 4 ϫ 10 13 vg/kg and followed Յ 14 months have not shown a difference in tumor incidence compared with control mice. 16,17 However, a study by one group reported an increase in tumor incidence that was attributed to AAV vectors. 18 These investigators reported that administration of an AAV serotype 2 (AAV2) vector encoding ␤-glucuronidase in neonatal mice resulted in a significant increase in incidence of hepatocellular carcinoma (HCC), a tumor commonly fou...

Key Points AAV delivery of ZFNs and corrective Donor vectors to adult mouse liver results in stable human factor IX levels, normalizing hemophilic clotting times.

Significance Immune-mediated diseases are prevalent, debilitating, and costly. Unfortunately, current treatments rely on nonspecific immunosuppression, which also shuts down a protective immune response. To circumvent this, we exploited the noninflammatory natural means of clearance of red blood cells (RBCs), in combination with sortase-mediated RBC surface modification to display disease-associated autoantigens as RBCs’ own antigens. We found that this strategy holds promise for prophylaxis and therapy, as shown in a mouse model of multiple sclerosis and of type 1 diabetes.

Humoral immune responses occur following exposure to Adeno-associated virus (AAV) or AAV vectors. Many studies characterized antibody responses to AAV, but human IgG subclass responses to AAV have not been previously described. In this study, IgG subclass responses were examined in serum samples of normal human subjects exposed to wild-type AAV, subjects injected intramuscularly with AAV vectors and subjects injected intravascularly with AAV vectors. A diversity of IgG subclass responses to AAV capsid were found in different subjects. IgG1 was found to be the dominant response. IgG2, IgG3, and IgG4 responses were also observed in most normal human subjects; IgG2 and IgG3 each represented the major fraction of total anti-AAV capsid IgG in a subset of normal donors. Subjects exposed to AAV vectors showed IgG responses to AAV capsid of all four IgG subclasses. IgG responses to AAV capsid in clinical trial subjects were inversely proportional to the level of pre-existing anti-AAV antibody and independent of the vector dose. The high levels of anti-AAV capsid IgG1 can mask differences in IgG2, IgG3, and IgG4 responses that were observed in this study. Analysis of IgG subclass distribution of anti-AAV capsid antibodies indicates a complex, non-uniform pattern of responses to this viral antigen.