Illustration of AAV-HITI for sustained transgene expression in liver gene therapyIntroduction

Gene therapy has long been a beacon of hope for curing genetic disorders. Among various methods, adeno-associated viral (AAV) vectors have emerged as a prominent tool due to their safety and efficacy in delivering therapeutic genes. Recently, a research team led by researchers at the Telethon Institute of Genetics and Medicine (TIGEM) in Italy has developed an innovative approach called AAV-mediated homology-independent targeted integration (AAV-HITI). This method, published in the journal Cell Reports Medicine, has shown remarkable potential in treating inherited liver diseases.

What is AAV-HITI?

AAV-HITI leverages the non-homologous end-joining (NHEJ) repair pathway, which is a cell cycle-independent mechanism, to achieve targeted gene integration. The AAV-HITI system employs CRISPR-Cas9 to introduce double-strand breaks (DSBs) at specific genomic loci, facilitating the integration of therapeutic genes. This approach stands out because it does not rely on homologous recombination, making it effective even in non-dividing cells such as hepatocytes.

Mechanism of Action

The mechanism of action of AAV-HITI combines CRISPR-Cas9 genome editing with AAV vector delivery to achieve stable gene integration in hepatocytes. Two AAV8 vectors are used: one carrying SpCas9 and another containing the HITI donor DNA with the therapeutic transgene. Upon administration, the vectors transduce hepatocytes. SpCas9, guided by gRNA, creates double-strand breaks in the albumin locus and the donor DNA extremities. The cell’s non-homologous end joining (NHEJ) repair mechanism then integrates the donor DNA into the albumin locus, facilitated by inverted gRNA sequences on the donor DNA. Once integrated, the therapeutic transgene is expressed under the control of the endogenous albumin promoter, resulting in a fusion mRNA that produces both modified albumin and the therapeutic protein. This integration ensures stable long-term transgene expression even in dividing hepatocytes, making it particularly effective for treating rapidly growing tissues like the liver.

Key Findings

Robust and Sustained Gene Expression

The study demonstrated that AAV-HITI achieves stable and prolonged transgene expression. In newborn mice, approximately 16% of hepatocytes exhibited successful transgene integration, leading to therapeutic levels of protein production. Notably, this expression was sustained for up to a year post-treatment, highlighting the potential of AAV-HITI for long-term therapeutic applications.

Targeted Integration at the Albumin Locus

The researchers designed their AAV-HITI system to target the 3′ end of the highly expressed albumin gene in mouse hepatocytes. By inserting the therapeutic transgene at this location, they aimed to hijack the strong endogenous promoter activity to drive long-term expression.

Efficient Integration in Neonatal Liver

When administered to newborn mice, the AAV-HITI system achieved productive integration in approximately 15% of hepatocytes. This relatively high efficiency is crucial for reaching therapeutic thresholds in many genetic disorders.

Therapeutic Efficacy in Disease Models

The researchers tested their approach in mouse models of two inherited diseases:

  • Mucopolysaccharidosis type VI (MPS VI): AAV-HITI delivery of the ARSB gene resulted in supraphysiological levels of the enzyme in the blood, leading to normalization of glycosaminoglycan levels in urine and various tissues.
  • Hemophilia A: Integration of a B-domain deleted factor VIII transgene achieved therapeutic levels of clotting factor activity, correcting the bleeding phenotype.

Preferential Integration of Full-Length Donor DNA

Detailed molecular analysis revealed that the majority of integration events involved the full-length HITI donor DNA as intended by the system’s design. This is important for ensuring the integrity of the therapeutic transgene.

Safety Profile

Several key findings support the safety of the AAV-HITI approach:

  • No evidence of gross chromosomal rearrangements or off-target insertions/deletions was detected using CAST-Seq analysis.
  • Histopathological examination showed no signs of hepatocellular carcinoma up to 1 year post-treatment.
  • The system was effective at vector doses that would be considered potentially safe for human translation.

Efficacy in Adult Liver

Importantly, the researchers demonstrated that AAV-HITI could also achieve therapeutic transgene expression when administered to adult mice, expanding its potential applications.

Implications for Human Therapy

The promising results in mouse models pave the way for translating AAV-HITI to human therapies. The ability to achieve stable gene integration in the liver without adverse effects is a significant advancement. Future studies will focus on optimizing the vectors and assessing the long-term safety and efficacy in larger animal models before moving to clinical trials.


The research findings mark a significant milestone in gene therapy, showcasing AAV-HITI as a safe and effective method for treating inherited liver diseases. With sustained gene expression, minimal off-target effects, and robust therapeutic outcomes, AAV-HITI holds great promise for revolutionizing the treatment of genetic disorders. As research progresses, we move closer to realizing the full potential of gene therapy in curing previously untreatable conditions.

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