Development of AAV-based liver gene therapies for stable correction of Ornithine Transcarbamylase Deficiency

Vicidomini, Antonio (2024). Development of AAV-based liver gene therapies for stable correction of Ornithine Transcarbamylase Deficiency. PhD thesis The Open University.


Ornithine transcarbamylase deficiency (OTCD) is a rare X-linked liver disorder resulting from mutations in the OTC gene, which produces a key mitochondrial enzyme involved in the urea cycle. The type of mutation defines the severity and age of onset. Current treatments focus on managing symptoms through diet and medications. Still, the only cure is an invasive organ transplantation, which increases the demand for non-invasive, more effective, and long-lasting treatments.

Liver-directed episomal gene therapy has demonstrated efficacy in adults; however, in neonatal and juvenile patients the long-term efficacy and re-administration are challenged by the loss of episomal DNA during liver growth or pathological processes and immune susceptibility. Re-administration of the same vector later in time is hindered by the presence of anti-AAV neutralising antibodies generated during the first administration.

During my PhD, I focused on developing AAV-mediated gene therapies tailored for neonatal and juvenile needs, using a widely used non-lethal mouse model of OTCD, the OTCSpf-Ash strain.

In the case of juvenile patients, to allow re-administration of the AAV therapy we used the Rapamycin-based immunomodulator (ImmTOR®) to generate a drug-specific tolerogenic environment without broadly suppressing the immune system. Our research showed that treating juvenile OTCSpf-Ash mice with a single AAV infusion and with ImmTOR® every 28 day preserved the therapeutic efficacy and reduced the formation of drug-specific neutralizing antibodies to a titre (<1:10) that would allow efficient AAV-vector re-administration.

For neonatal patients, we addressed the limitations of episomal gene therapy in rapidly growing livers by permanently modifying the mouse genome, resulting in the transmission of the therapeutic gene to daughter cells during liver growth. We employed the GeneRide technology coupled with CRISPR/Cas9., which consist in the targeting of the therapeutic cDNA into the albumin locus, without inactivating the targeted albumin allele, and enabling expression of the OTC cDNA by the robust albumin gene promoter. Neonatal OTCSpf-Ash mice were treated shortly after birth (P2) with an AAV8 donor vector containing the optimized human OTC cDNA and a second vector expressing the SaCas9 and the sgRNA. We observed a significant increase in OTC enzyme activity and normalization of the phenotype.

We also compared the long-term efficacy of the integrative approach with the episomal, non-integrative, gene therapy in a model of liver regeneration. After the induction of liver regeneration by resection of 2/3 of the liver, mice treated with the integrative approach maintained stable OTC enzyme levels, whereas those treated with episomal therapy showed a significant reduction in the therapeutic efficacy due to DNA loss.

The results obtained with the non-lethal OTCD mice paved the way for treating a more severe OTCD mouse model, the OTCKO mice which presents early mortality within 24-hour after birth, preventing either episomal or integrating approaches. To extend the life span and enable integrating gene therapy, we are developing a strategy based on the delivery of mRNA-loaded lipid nanoparticles (LNPs) to hepatocytes, that results in the rapid translation into the OTC protein and a fast therapeutic effect. In vitro experiments in liver-derived tissue culture cells showed protein expression as early as 6 hours post-transfection in vitro. We are developing lipid nanoparticle carriers for mRNA using a microfluidic device, which still requires further optimization for in vivo delivery.

With these strategies, we aim to propose impactful treatments for OTCD in juvenile and neonatal patients and set the basis for the development of more effective therapies in short- and long-term settings despite the severity of the disease.

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