Improved Induction of Immune Tolerance to Factor IX by Hepatic AAV-8 Gene Transfer

Abstract

Gene therapy for hemophilia B has been shown to result in long-term expression and immune tolerance to factor IX (F.IX) after in vivo transduction of hepatocytes with adeno-associated viral (AAV-2) vectors in experimental animals. An optimized protocol was effective in several strains of mice with a factor 9 gene deletion (F9 À=À ). However, immune responses against F.IX were repeatedly observed in C3H=HeJ F9 À=À mice. We sought to establish a gene transfer protocol that results in sustained expression without a requirement for additional manipulation of the immune system. Compared with AAV-2, AAV-8 was more efficient in transgene expression and induction of tolerance to F.IX in three different strains of wild-type mice. At equal vector doses, AAV-8 induced transgene product-specific regulatory CD4 + CD25 + FoxP3 + T cells at significantly higher frequency. Moreover, sustained correction of hemophilia B in C3H=HeJ F9 À=À mice without antibody formation was documented in all animals treated with !4Â10 11 vector genomes (VG)=kg and in 80% of mice treated with 8Â10 10 VG=kg. Therefore, it is possible to develop a gene transfer protocol that reliably induces tolerance to F.IX largely independent of genetic factors. A comparison with other studies suggests that additional parameters besides plateau levels of F.IX expression contributed to the improved success rate of tolerance induction.

Adeno-Associated Virus-Mediated Gene Transfer Leads to Persistent Hepatitis B Virus Replication in Mice Expressing HLA-A2 and HLA-DR1 Molecules

“…To ensure the targeting of the HBV genome to the liver, we used an AAV serotype 8 vector that displays liver-specific transduction (22). We cloned 1.2 copies of the HBV genome between the two ITRs of AAV serotype 2 in a carrier plasmid, which was then used to produce the hybrid AAV2/8-HBV vector.…”

“…This involved the use of the hepatotropic hybrid serotype 2 and 8 AAV vector to deliver the entire HBV genome into mouse hepatocytes. AAV2/8 efficiently transduces hepatocytes (22). Mouse liver is permissive for HBV replication (24), so the use of AAV2/8 vectors allows the entry step of HBV infection to be bypassed.…”

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

“…15 Nonetheless, AAV8 and AAV2(Y444/500/ 730F) vectors were able to tolerize this strain to hF.IX on gene transfer to the liver, prompting us to speculate that innate responses to these capsids may differ. 7,10 Thus, we compared the innate immune profile of several ssAAV vectors in the murine liver, but we found an identical mild and highly transient response regardless of capsid sequence. However, changing the genome to scAAV substantially increased innate immunity in a TLR9-dependent manner.…”

“…49 In addition, the level of gene transfer to professional antigen presenting cells such as DCs and the transduction pattern and distribution of the antigen in the tissue impact the response. 7,50 Implications for gene therapy…”

“…For example, AAV8 shows substantially higher transduction efficiency in mouse liver and reduced activation of capsid-specific T cells, and it facilitates tolerance induction to transgenes. 6,7 Furthermore, prevalence for neutralizing antibodies in humans is markedly lower to AAV8 than to AAV2. 8 In another set of investigations, replacing surfaceexposed tyrosine residues to phenylalanine has been shown to improve gene transfer for several serotypes.…”