CRISPR-ABE Mediated Correction of ASLD via Lipid Nanoparticle Delivery: A Novel Therapeutic Approach


Argininosuccinate lyase deficiency (ASLD) is a rare inherited disorder characterized by the body’s inability to properly break down argininosuccinate due to mutations in the ASL gene. This deficiency disrupts the urea cycle, leading to the accumulation of ammonia in the bloodstream, which can cause severe neurological damage and even death if not properly managed. The traditional treatment modalities for ASLD, which include dietary restrictions, supplementation, and in severe cases, liver transplantation, offer only symptomatic relief without addressing the root cause of the disorder. Thus, the development of gene therapy approaches represents a significant advancement in the potential treatment of ASLD and similar genetic disorders. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and gene therapy represent groundbreaking approaches in the treatment of genetic disorders. These technologies offer the potential to correct the underlying genetic errors that cause such conditions, providing a more definitive solution than traditional treatments.  For instance CRISPR-Cas9 is a gene-editing tool that allows scientists to cut DNA at a specific location and either remove, add, or replace the DNA in that location. In argininosuccinate lyase deficiency, CRISPR-Cas9 system could be designed to target the specific mutations in the gene that codes for argininosuccinate lyase. By correcting these mutations, the normal function of the enzyme could be restored, allowing the urea cycle to proceed properly and prevent the accumulation of ammonia. Alternatively, a new, functional copy of the gene could be inserted into the genome of liver cells, compensating for the defective gene and restoring enzyme function.

One of the major challenges of using CRISPR and gene therapy is ensuring that the gene editing or gene insertion does not unintentionally alter other parts of the genome, which could potentially lead to unwanted side effects or new health issues. Moreover, the body’s immune system might recognize the viral vectors used in gene therapy as foreign invaders, leading to an immune response that could reduce the therapy’s effectiveness or cause other complications. Furthermore, the long-term effects and stability of gene edits or inserted genes need further study. Continuous monitoring and research are essential to ensure their safety and effectiveness over time. Indeed, CRISPR and gene therapy hold promise for treating argininosuccinate lyase deficiency and other similar genetic disorders by addressing the root cause of the disease.  To this account, a new study published in the American Journal of Human Genetics and conducted by Sami Jalil, Timo Keskinen, Juhana Juutila, Rocio Sartori Maldonado, Liliya Euro, Anu Suomalainen, Risto Lapatto, Emilia Kuuluvainen, Ville Hietakangas, Timo Otonkoski, Mervi Hyvönen, and Kirmo Wartiovaara from Helsinki University in Finland, the team developed an innovative approach aimed at addressing ASLD through a novel gene therapy approach using CRISPR technology.

They generated human-induced pluripotent stem cells (hiPSCs) from ASLD Patients where skin biopsies from two individuals homozygous for the Finnish founder ASL variant (c.1153C>T [p.Arg385Cys]) were reprogrammed to generate hiPSCs. This variant is known for its severe impact on the ASL enzyme’s function, critical in the urea cycle. This way they reported the successful generation and verification of hiPSCs provided a patient-specific model for studying ASLD in vitro, allowing for the examination of disease mechanisms and therapeutic interventions. The researchers used adenine base editors (ABEs) to specifically target and correct the c.1153C>T mutation in the hiPSCs. Edited cells were then differentiated into hepatocyte-like cells to assess functional recovery. They found a significant reduction in argininosuccinate levels was observed in edited cells compared to non-edited controls, indicating the restoration of ASL function and, by extension, the urea cycle. The team tested three different FDA-approved LNP formulations for their efficiency in delivering the ABE-encoding RNA and the sgRNA targeting the ASL variant into fibroblasts derived from ASLD patients. They found all three LNP formulations successfully delivered the CRISPR components and achieved efficient editing of the ASL variant with no apparent cell toxicity. The best performing formulations significantly reduced argininosuccinate levels to those seen in healthy donors, showcasing a potential route for clinical application.

Afterward they conducted comprehensive analyses to evaluate the potential off-target effects of the CRISPR editing and the cytotoxicity of the LNP formulations. The team observed minimal off-target effects, and the LNP formulations demonstrated a favorable safety profile, supporting the potential of this approach for safe clinical use. Moreover, the researchers analyzed the metabolic impact of correcting the ASLD mutation in hepatocyte-like cells derived from the edited hiPSCs. This included measuring the levels of argininosuccinate and other relevant metabolites. Edited hepatocyte-like cells showed a significant normalization of metabolic profiles, with a drastic reduction in argininosuccinate levels, further validating the functional correction of the urea cycle.

The authors effectively used human-induced pluripotent stem cells (hiPSCs) derived from individuals with ASLD to model the disorder in vitro. They also employed CRISPR-Cas9 technology, specifically ABEs, to target the disease-causing genetic variant in ASL, achieving a remarkable correction of the genetic defect. The differentiation of these edited hiPSCs into hepatocyte-like cells and the subsequent demonstration of normalized argininosuccinate levels validate the efficacy of this gene editing approach. Importantly, the researchers addressed a critical aspect of therapeutic application by testing various FDA-approved lipid nanoparticle formulations for delivering the gene editing components, thereby highlighting a viable pathway for in vivo application of this therapy.

The study’s focus on minimizing off-target effects and ensuring the specificity of the gene editing process underscores the careful consideration of the potential clinical implications of this therapy. The use of CRISPR technology in this manner has the potential to offer a permanent cure for ASLD by directly addressing the genetic cause of the disease, as opposed to merely managing its symptoms. Additionally, this approach could be applicable to a wide range of genetic disorders beyond ASLD, where specific genetic mutations have been identified as the cause of the disease.

In conclusion, the research conducted by Helsinki University scientists is an important step forward in the treatment of ASLD and potentially other genetic disorders. By combining cutting-edge CRISPR technology with an innovative delivery mechanism, it paves the way for future clinical applications that could offer hope to patients with ASLD and other similar genetic conditions. The implications of this research extend beyond ASLD, offering insights into the broader field of genetic medicine and the ongoing quest to develop more effective, targeted treatments for genetic diseases.

CRISPR-ABE Mediated Correction of ASLD via Lipid Nanoparticle Delivery: A Novel Therapeutic Approach - Medicine Innovates

About the author

Timo Otonkoski
Principal investigator, M.D., Ph.D. Professor
University of Helsinki

Otonkoski lab is interested in nuclear reprogramming and genome editing, and how these can be applied to understand mechanisms behind pancreatic beta-cell failure leading to diabetes.


Sami Jalil, Timo Keskinen, Juhana Juutila, Rocio Sartori Maldonado, Liliya Euro, Anu Suomalainen, Risto Lapatto, Emilia Kuuluvainen, Ville Hietakangas, Timo Otonkoski, Mervi E. Hyvönen, Kirmo Wartiovaara. Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors. The American Journal of Human Genetics, 2024; 111 (4): 714 DOI: 10.1016/j.ajhg.2024.03.004 .

Go To The American Journal of Human Genetics