Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

Analysis of copy number alterations reveals the lncRNA ALAL-1 as a regulator of lung cancer immune evasion.

Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.

CTCF is dispensable for immune cell transdifferentiation but facilitates an acute inflammatory response.

Three-dimensional organization of the genome is important for transcriptional regulation1-7. In mammals, CTCF and the cohesin complex create submegabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs)8-12. Although TADs can contribute to transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex causes modest gene expression changes13-16. In contrast, CTCF is required for cell cycle regulation17, embryonic development and formation of various adult cell types18. To uncouple the role of CTCF in cell-state transitions and cell proliferation, we studied the effect of CTCF depletion during the conversion of human leukemic B cells into macrophages with minimal cell division. CTCF depletion disrupts TAD organization but not cell transdifferentiation. In contrast, CTCF depletion in induced macrophages impairs the full-blown upregulation of inflammatory genes after exposure to endotoxin. Our results demonstrate that CTCF-dependent genome topology is not strictly required for a functional cell-fate conversion but facilitates a rapid and efficient response to an external stimulus.

Design of modified U1i molecules against HIV-1 RNA.

Several gene therapeutic approaches have been proposed to add to current antiretroviral therapy against HIV-1. U1 interference (U1i) is a promising new gene therapy tool that targets mRNAs with modified U1 snRNAs. For efficient inhibition, the 3'-terminal exon of pre-mRNAs must be recognized by the modified U1 snRNA. Subsequent interaction between the U1-associated 70K protein and poly(A) polymerase leads to inhibition of polyadenylation and consequently degradation of the pre-mRNA. We designed 14 new U1i inhibitors against HIV-1 mRNA regions that are 100% complementary to at least 70% of HIV-1 sequences listed in the HIV database. All U1i inhibitors were tested transiently in HIV-1 production assays as well as luciferase reporter experiments and three candidates were examined further in stably lentivirus-transduced T cell lines. We identified U1i-J that targets the region encoding the NF-κB binding sites as the most effective inhibitor that substantially reduced viral protein expression. The potency of J is determined in part by the presence of a duplicated target within the HIV-1 mRNA. The stably transduced SupT1 T cells were challenged with HIV-1 but no antiviral effect was detected. U1i inhibitors can be potent suppressors of HIV-1 production in transient assays but further optimization of this antiviral approach is needed.