Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized “nanoblades,” a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30–40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

Autosomal dominant Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in an inborn error of immunity characterized by chronic mucocutaneous candidiasis, recurrent viral and bacterial infections, and diverse autoimmune manifestations. Current treatment consists of chronic antifungal therapy, antibiotics for concomitant infections, and immunosuppressive therapy in case of autoimmune diseases. More recently, treatment with Janus kinases 1 and 2 (JAK1/2) inhibitors have shown promising yet variable results. We describe a STAT1 GOF patient with an incidental finding of elevated cardiac troponins, leading to a diagnosis of a longstanding, slowly progressive idiopathic myocarditis, attributed to STAT1 GOF. Treatment with a JAK-inhibitor (baricitinib) mitigated cardiac inflammation on MRI but was unable to alter fibrosis, possibly due to the diagnostic and therapeutic delay, which finally led to fatal arrhythmia. Our case illustrates that myocarditis could be part of the heterogeneous disease spectrum of STAT1 GOF. Given the insidious presentation in our case, a low threshold for cardiac evaluation in STAT1 GOF patients seems warranted.

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