CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

Abstract

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Novel Diagnostic Tool for p47 -Deficient Chronic Granulomatous Disease Patient and Carrier Detection

“…This 1-day method determines the NCF1 gene CNV by quantification of GTGT content in the NCF1 gene and pseudogene loci, and thus it detects the presence or absence of the ΔGT mutation within NCF1 gene and pseudogene alleles. It can be established in any molecular biology laboratory, and it allows for the robust discrimination of homozygous ΔGT p47 phox CGD patients from heterozygous carriers and healthy individuals for rapid diagnostic purposes, as well as for the monitoring of NCF1 genome-editing-based gene therapy 8 …”

“…Calculation of the GTGT content (Figure 1G) allowed for the differentiation between NCF1 ΔGT mutation carriers and healthy individuals. Representative PCR-RFLP samples developed by PAGE and agarose electrophoresis (Figures 2A–2C) compare controls (healthy individuals with GTGT in two NCF1 alleles and different 20-nt intronic repeat numbers), X-CGD and autosomal recessive CGD NCF2 (gp91 phox and p67 phox deficiency, respectively), two NCF1 ΔGT carriers (Carrier ΔGT #1 and Carrier ΔGT #2), an induced pluripotent stem cell (iPSC) NCF1 ΔGT cell line, 9 a human acute myeloid leukemia cell line PLB-985 (wild-type), and a PLB-985 NCF1 ΔGT cell line 8 …”

“…PCR products were purified using the QIAquick Gel Purification Kit (QIAGEN). 20 ng gel-purified barcoded PCR products of individual subjects were pooled and analyzed with SMRT sequencing 11 by Functional Genomics Center Zurich, ETH/University of Zurich, Switzerland, as described 8 …”

Recent advances in understanding and treating chronic granulomatous disease

“…Targeted insertion of an exon 2–13 minigene into exon 2 of CYBB resulted in normal regulation of gp91 phox expression by the endogenous CYBB promoter and restored gp91 phox and NOX activity, demonstrating the necessity of retention of intronic elements for expression of the minigene 66 . CRISPR-Cas9 correction of CYBB 67 and NCF1 68 has also been demonstrated recently. Finally, a zinc finger nuclease has been used to correct the GT deletion at the start of exon 2 in an NCF1 pseudogene and demonstrate restoration of function 69 .…”

Novel Translational Research Methodology and the Prospect to a Better Understanding of Neurodegenerative Disease

“…Her own and her team’s research has resulted in the hypothesis that expansion mutations as observable in HD may also have an etiological role in more frequent psychiatric diseases [16, 17], allowing for novel therapeutic approaches [18]. Gene therapy aimed at single pathological targets, as performed by Janine Reichenbach and her team at the University of Zurich [19-21], might thus attain therapeutic relevance beyond the initially targeted monogenetic disorder. Rodent models play a fundamental role to test novel mechanistic hypotheses as well as the resulting therapeutic interventions.…”