AAV Variant Characterization for Liver Gene Therapy
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Solution Overview
Problem
The understanding of wild-type adeno-associated virus (AAV) infection in the liver is limited, particularly regarding the specific AAV genotypes and their persistence, which hampers the therapeutic potential of AAV variants in gene therapy.
Innovation Solution
Characterization of natural AAV variants and their molecular forms in liver tissues through quantitative analysis and in silico analysis of viral capture data, identifying new clonal insertions and their relationship with helper viruses, to elucidate the biology of wild-type AAV infection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant AAV vectors are used for gene therapy, then transduction efficiency and long-term transgene expression are improved, but understanding of wild-type AAV infection biology remains limited
Solution Approach 1:
Instead of studying wild-type AAV infection directly (which is difficult due to latent infection and integration), the inventors used recombinant AAV vectors as a model system to investigate wild-type infection biology. By inverting the approach and using the well-characterized rAAV system to gain insights into natural infection, they were able to identify viral genotypes and molecular forms in liver tissues that reflect wild-type infection patterns.
2Adaptability or versatility
If AAV vectors are used for gene therapy, then tissue-specific transduction is improved, but specificity of viral tropism needs further characterization
Solution Approach 1:
The inventors applied local quality by characterizing specific AAV genotypes (AAV2-like and AAV13-like) and their distribution in different liver tissue compartments. They identified that different viral genotypes and molecular forms (episomal vs. integrated) are present in specific tissue locations, providing detailed spatial and genotypic resolution of viral tropism in the liver.
3Measurement precision
If quantitative analysis of viral DNA is performed, then detection sensitivity is improved, but differentiation between episomal and integrated forms requires precise measurement
Solution Approach 1:
The inventors segmented the viral DNA analysis into distinct molecular forms by using specific molecular biology techniques. They separated and quantified episomal AAV DNA from integrated AAV DNA using DNAse treatment and TaqMan-based assays, allowing precise differentiation and quantification of each molecular form independently.
Data Source
AI summary
Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. The aimed of the inventors was to characterize the natural history of AAV infection in the liver. Viral DNA was thus quantified in tumor and non-tumor liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analyzed using a DNAse/TaqMan based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. AAV DNA was detected in 21% of the patients equally distributed in 2 major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Thus the inventors provided an integrated analysis of the wild type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. These findings are important to understand wild type AAV biology and particularly relevant considering the large usage of AAV vector in liver-targeted gene therapy. Thus, the present invention relates to new adeno-associated virus (AAV) variants and uses thereof for gene therapy.