Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNA^hCFTRin vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNA^hCFTRin vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNA^hCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNA^hCFTR was quantified by hCFTR ELISA, and cmRNA^hCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV_0.1, suggesting NP-cmRNA^hCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.

β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control. We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBBIVS1–110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs). Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1–110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells—HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs. Our approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines’ cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.

Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even among patients with similar CFTR mutations due to the possible effect of ‘modifier genes’. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype. Global gene expression and enrichment analyses revealed that genes of the type I interferon response and ribosomal stalk proteins are potential modifiers of CF related lung dysfunction. The results provide a new set of CF modifier genes with possible implications as new therapeutic targets for the treatment of CF.

Mutations of the IL2RG gene, which encodes for the interleukin-2 receptor common gamma chain (γ_C, CD132), can lead to X-linked severe combined immunodeficiency (X-SCID) associated with a T⁻B⁺NK⁻ phenotype as a result of dysfunctional γ_C-JAK3-STAT5 signaling. Lately, hypomorphic mutations of the IL2RG gene have been described causing atypical SCID with a milder phenotype. Here, we report three brothers with low-normal lymphocyte counts and susceptibility to recurrent respiratory infections and cutaneous warts. The clinical presentation combined with dysgammaglobulinemia suspected an inherited immunity disorder, which has been proven by Next Generation Sequencing as a novel c.458T > C; p.Ile153Thr IL2RG missense-mutation. Subsequent functional characterization revealed impaired T-cell proliferation, low TREC levels and a skewed TCR Vβ repertoire in all three patients. Interestingly, investigation of various subpopulations showed normal expression of CD132 but with partially impaired STAT5 phosphorylation compared to healthy controls. Additionally, we performed precise genetic analysis of subpopulations revealing spontaneous somatic reversion, predominately in lymphoid derived CD3⁺, CD4⁺ and CD8⁺ T cells. Our data demonstrate that the atypical SCID phenotype noticed in these three brothers is due to the combination of hypomorphic IL-2RG function and somatic reversion.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.