Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.

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.

Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Hepsin promotes breast tumor growth signaling via the TGFß-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFß) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFß and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFß signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFß and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFß and EGFR pathways in cancer.

CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy.

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.

Simultaneous high-efficiency base editing and reprogramming of patient fibroblasts.

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.

Curative gene therapies for rare diseases.

Diseases caused by alterations in the DNA can be overcome by providing the cells or tissues with a functional copy of the mutated gene. The most common form of gene therapy implies adding an extra genetic unit into the cell. However, new genome engineering techniques also allow the modification or correction of the existing allele, providing new possibilities, especially for dominant diseases. Gene therapies have been tested for 30 years in thousands of clinical trials, but presently, we have only three authorised gene therapy products for the treatment of inherited diseases in European Union. Here, we describe the gene therapy alternatives already on the market in the European Union and expand the scope to some clinical trials. Additionally, we discuss the ethical and regulatory issues raised by the development of these new kinds of therapies.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.