Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo

Abstract

Adeno-associated virus (AAV) vectors are ideal for performing gene repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to gene repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation– based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable gene repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for repairing the mutated Fah (fumarylacetoacetate hydrolase) gene. Hepatic gene repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful gene repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10−3 were achieved and highly reproducible within typical dose ranges. In this model, repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion AAV-mediated gene repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates.

Induction of hepatocellular carcinoma by in vivo gene targeting

“…3). The frequency of Afp + foci was similar to that observed when targeting other genes in prior AAV experiments (20,21), suggesting that each targeting event produced a single Afp + hepatocyte that proliferated to form an HCC focus. If additional spontaneous oncogenic mutations were required they must have consistently occurred before each ∼1,000 cell malignancy developed.…”

“…Previous studies have shown that in addition to their potential for random, nonhomologous integration, AAV vectors can efficiently and accurately introduce mutations into homologous chromosomal target sequences (19). This process occurs in ∼1/10 4 hepatocytes after in vivo vector delivery to the liver (20,21). We reasoned that this gene-targeting frequency would be adequate to initiate multiple foci of HCC because of dysregulated gene expression after targeted promoter-enhancer insertion.…”

The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

“…This trend is also true in skeletal muscle (35, 60, 76), cardiac tissue (53, 68, 76), pancreas (72), and glioblastoma (21), and specialized cells in brain and retina are preferentially transduced by AAV8 (5, 9). In many animal models, AAV8 has already proven to provide an enormous therapeutic potential (7,18,38,47,54,55,62), although translation of the exceptional performance of AAV8 to higher primates has been challenging (25,74,75). Application of AAV8 vectors in a clinical trial has recently provided successful proof of concept for expression of the factor IX gene in human liver (48).…”

Gene Therapy in Liver Diseases: State-of-the-Art and Future Perspectives

“…Paulk et al, [103] demonstrated stable hepatic gene repair in both adult and neonatal mice with AAV-FAH serotypes 2 and 8, showing that AAV-mediated gene repair is feasible in vivo and can functionally correct enzymedeficiency consequences. In a study by Chen et al, [104] a murine model for hereditary tyrosinemia was used to evaluate in vivo gene therapy with AAV vectors expressing FAH.…”