Gene therapy with genetically modified human CD34+ hematopoietic stem cells (HSCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno associated virus (AAV) 6 delivery of donor constructs in human HSCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus-positive HSCs with 6–16% human cell marking were observed following engraftment into mice. In HSCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 in resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.
X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the
CYBB
gene, resulting in loss of gp91
phox
protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of
CYBB
exon 1–13 or 2–13 cDNAs from adeno-associated virus donors at endogenous
CYBB
exon 1 or exon 2 sites. Targeted insertion of exon 1–13 cDNA did not restore physiologic gp91
phox
levels, consistent with a requirement for intron 1 in
CYBB
expression. However, insertion of exon 2–13 cDNA fully restored gp91
phox
and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91
phox
expression in exon 2–13 corrected cells, indicating that retention of intron 1 was sufficient for optimal
CYBB
expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit non-homologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91
phox
and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.
The evolution of resistance to insecticides is well known to be closely associated with the overexpression of detoxifying enzymes. Although the role of glutathione S-transferase (GST) genes in insecticide resistance has been widely reported, the underlying regulatory mechanisms are poorly understood. Here, one GST gene (GSTu1) and its antisense transcript (lnc-GSTu1-AS) were identified and cloned, and both of them were upregulated in several chlorantraniliprole-resistant Plutella xylostella populations. GSTu1 was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Furthermore, we demonstrated that lnc-GSTu1-AS interacted with GSTu1 by forming an RNA duplex, which masked the binding site of miR-8525-5p at the GSTu1-3′UTR. In summary, we revealed that lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation that could have been induced by miR-8525-5p and thus increased the resistance of P. xylostella to chlorantraniliprole. Our findings reveal a new noncoding RNA-mediated pathway that regulates the expression of detoxifying enzymes in insecticide-resistant insects and offer opportunities for the further understanding of the mechanisms of insecticide and drug resistance.
Kidney renal clear cell carcinoma (KIRC) is the most general subtype of renal cell carcinoma, which composes about 1/20 of adult malignancies. The anomaly of long noncoding RNAs (lncRNAs) expression is proved to mediate cancer progression of various types. The function and mediation mechanism of MSC‐AS1 has rarely been detected in KIRC before. This study started with the mediation of MSC‐AS1 on cell function. In this study, MSC‐AS1 was dramatically upregulated in KIRC and correlated with dismal prognosis of KIRC patients. Knockdown of MSC‐AS1 would suppress the proliferative and migratory properties of KIRC cells. MSC‐AS1 was found to directly downregulate miR‐3924 expression while miR‐3924 directly downregulated WNT5A expression. Meanwhile, MSC‐AS1 could promote the expression of WNT5A, indicating the existence of MSC‐AS1/miR‐3924/WNT5A. Further assays indicated that MSC‐AS1 could enhance Wnt/β‐catenin pathway. By means of rescue assays, the mediation of MSC‐AS1/miR‐3924/WNT5A/β‐catenin axis on KIRC cell proliferation, migration and migration was verified. This study revealed that MSC‐AS1 regulates KIRC cell proliferation and migration via miR‐3924/WNT5A/β‐catenin axis. MSC‐AS1 might contribute to new strategies for KIRC treatment.
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