Torella, L. (Laura)
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- CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I(2018) Vilas, A. (Amaia); González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); Betancor, I. (Isabel); Rodriguez-Madoz, J.R. (Juan Roberto); Vales, A. (África); Rodriguez, S. (Saray); Lara-Astiaso, D. (David); Martínez-Turrillas, R. (Rebeca); Castro-Labrador, L. (Laura); Olagüe, M. (María); Torella, L. (Laura); Salido, E. (Eduardo); Barberia, M. (Miren); Prosper-Cardoso, F. (Felipe); Martin-Higueras, C. (Cristina); Zapata-Linares, N.M. (Natalia María)CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
- mRNA and gene editing: Late breaking therapies in liver diseases(Wiley, 2022) González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); Weber, N.D. (Nicholas D.); Torella, L. (Laura)The efficient delivery of RNA molecules to restore the expression of a missing or inadequately functioning protein in a target cell and the intentional spe-cific modification of the host genome using engineered nucleases represent therapeutic concepts that are revolutionizing modern medicine. The initia-tion of several clinical trials using these approaches to treat metabolic liver disorders as well as the recently reported remarkable results obtained by patients with transthyretin amyloidosis highlight the advances in this field and show the potential of these therapies to treat these diseases safely and ef-ficaciously. These advances have been possible due, firstly, to significant improvements made in RNA chemistry that increase its stability and prevent activation of the innate immune response and, secondly, to the development of very efficient liver-targeted RNA delivery systems. In parallel, the breakout of CRISPR/CRISPR-associated 9–based technology in the gene editing field has marked a turning point in in vivo modification of the cellular genome with therapeutic purposes, which can be based on gene supplementation, correc-tion, or silencing. In the coming years we are likely to witness the therapeutic potential of these two strategies both separately and in combination. In this review we summarize the preclinical data obtained in animal models treated with mRNA as a therapeutic agent and discuss the different gene editing strategies applied to the treatment of liver diseases, highlighting both their therapeutic efficacy as well as safety concerns.
- In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9(2020) González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); López-Manzaneda, S. (Sergio); Alberquilla, O. (Omaira); García-Torralba, A. (Aída); Olivier, E. (Emmanuel); Ramírez, J.C. (Juan C.); Ojeda-Pérez, I. (Isabel); Mountford, J. (Joanne); Torella, L. (Laura); Sánchez-Domínguez, R. (Rebeca); Torres, R. (Raúl); Bueren, J.A. (Juan A.); Segovia, J.C. (Jose-Carlos)The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.
- Advances in precision and safety of CRISPR-based gene targeting for Primary Hyperoxaluria Type 1(Universidad de Navarra, 2024-02-22) Torella, L. (Laura); González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea)Primary hyperoxaluria type 1 (PH1) is a rare metabolic disorder caused by pathogenic mutations in the AGXT gene. It is characterized by excessive oxalate production in the liver, resulting in its toxic accumulation in the kidneys. CRISPR-Cas9 targeting the Hao1 gene, encoding the glycolate oxidase (GO) protein, has proven effective as a substrate reduction therapy (SRT) in a preclinical PH1 model. This thesis aims to refine this gene targeting strategy to enhance the predictability, precision, and overall safety of this therapeutic approach. First, we compared the editing outcomes of single Cas9 nuclease and paired Cas9 nucleases guided by two gRNAs, 64 nucleotides apart and targeting opposite strands, aiming for a more precise disruption of Hao1. Utilizing adeno-associated viral vectors serotype 8 (AAV8) to deliver editing tools into mouse liver, and confining Cas9 expression to hepatocytes via a liver-specific promoter, we demonstrated that paired Cas9 nucleases yielded more predictable modifications, primarily resulting in a precise deletion between the two cuts. Subsequently, we explored the use of paired Cas9 nickases to minimize off-target effects and achieve a more accurate gene disruption. Our findings revealed that paired Cas9 nickases, delivered by two independent AAV vectors, exhibited efficacy comparable to individual and paired Cas9 nucleases in reducing the target enzyme level in vivo. Notably, we observed that paired nick-induced double-strand breaks were likely repaired through the microhomology-mediated end-joining pathway, resulting in heterogeneous modifications of variable sizes. Conversely and as expected, single nicks failed to disrupt the target gene. Importantly, the use of paired Cas9 nickases significantly reduced AAV integration at the target site compared to Cas9 nucleases, potentially due to differences in repair mechanisms. To facilitate clinical translation, we developed an all-in-one AAV vector for nickase-mediated targeted cleavage, maintaining therapeutic efficacy with a reduced AAV dose. Our comprehensive off-target analysis confirmed the specificity of selected gRNAs with no off-target activity or chromosomal translocations. Finally, paired gRNAs targeting the HAO1 human orthologous gene were tested in human cells demonstrating efficiency and laying the groundwork for future studies. In parallel, we explored the use of CRISPR-CasRx, a programmable RNA editor, for a safer SRT avoiding permanent DNA modifications. The successful reduction of GO expression in vitro paved the way for in vivo testing. However, AAV8 vectors delivering CasRx with single or multiple gRNAs did not result in significant GO protein reduction in the livers of mice, despite indications of collateral effects. This emphasizes the need for further investigation to improve the efficiency of this technology. In conclusion, this thesis addresses the refinement of CRISPR-Cas9-mediated gene targeting strategies for treating PH1. It demonstrates the efficacy of paired Cas9 nucleases in achieving more predictable modifications. Additionally, it underscores the promise of paired Cas9 nickases for in vivo gene disruption in a therapeutic context, with minimized on-target AAV integration and off-target effects. Finally, it highlights the need for further optimization of CRISPR-CasRx application for therapeutic purposes.