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1.
Am J Hum Genet ; 111(4): 714-728, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38579669

ABSTRACT

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.


Subject(s)
Argininosuccinate Lyase , Argininosuccinic Aciduria , Humans , Argininosuccinate Lyase/genetics , Argininosuccinic Aciduria/genetics , Argininosuccinic Aciduria/therapy , Clustered Regularly Interspaced Short Palindromic Repeats , RNA, Guide, CRISPR-Cas Systems , Urea , Gene Editing/methods
2.
Mol Genet Metab Rep ; 31: 100863, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35782600

ABSTRACT

Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, causing muscular degeneration and retinochoroidal atrophy that progresses to blindness. HOGA arises from mutations in the ornithine aminotransferase (OAT) gene, and nearly one-third of the known patients worldwide are homozygous for the Finnish founder mutation OAT c.1205 T > C p.(Leu402Pro). We have corrected this loss-of-function OAT mutation in patient-derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9. The correction restored OAT expression in stem cells and normalized the elevated ornithine levels in cell lysates and cell media. These results show an efficient recovery of OAT function in iPSC, encouraging the possibility of autologous cell therapy for the HOGA disease.

3.
Stem Cell Reports ; 16(12): 3064-3075, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34822772

ABSTRACT

Human induced pluripotent stem cells (hiPSCs) allow in vitro study of genetic diseases and hold potential for personalized stem cell therapy. Gene editing, precisely modifying specifically targeted loci, represents a valuable tool for different hiPSC applications. This is especially useful in monogenic diseases to dissect the function of unknown mutations or to create genetically corrected, patient-derived hiPSCs. Here we describe a highly efficient method for simultaneous base editing and reprogramming of fibroblasts employing a CRISPR-Cas9 adenine base editor. As a proof of concept, we apply this approach to generate gene-edited hiPSCs from skin biopsies of four patients carrying a Finnish-founder pathogenic point mutation in either NOTCH3 or LDLR genes. We also show LDLR activity restoration after the gene correction. Overall, this method yields tens of gene-edited hiPSC monoclonal lines with unprecedented efficiency and robustness while considerably reducing the cell culture time and thus the risk for in vitro alterations.


Subject(s)
Cellular Reprogramming/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Editing , Base Sequence , Cells, Cultured , Endoderm/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mutation/genetics , Phenotype , RNA/genetics , Receptor, Notch3/genetics , Receptors, LDL/genetics , Transgenes
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