Hojun Li

In vivo genome editing restores haemostasis in a mouse model of haemophilia

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.

Assessing the potential for AAV vector genotoxicity in a murine model

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cytosine-rich regions. [corrected] Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation.

Robust ZFN-mediated genome editing in adult hemophilic mice

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Diverse IgG Subclass Responses to Adeno-associated Virus Infection and Vector Administration

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. J. Med. Virol. 81:65–74, 2009.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.