Reto Eggenschwiler

The lysosomal storage disorder Fabry disease results from α‐galactosidase A deficiency, leading to excessive glycosphingolipid substrate accumulation, primarily globotriaosylceramide (Gb3). While the underlying molecular mechanisms remain elusive, multi‐systemic complications ultimately culminate in premature death, with heart failure being the leading cause of death. Current treatment options fail to treat Fabry disease adequately and only delay its progression. Preclinical studies on an alternative approach, systemic delivery of nucleoside‐modified GLA mRNA (modGLA), suggest improved effectiveness over existing therapies in reducing glycosphingolipid levels in the heart. It remains unclear whether modGLA can rescue Fabry cardiomyopathy phenotypes at the cellular level, which are not faithfully recapitulated in current animal models. To address this, we investigated characteristic phenotypes in two new models of Fabry cardiomyopathy utilizing human iPSC‐derived cardiomyocytes in transcriptomic and functional analyses. These human Fabry disease cardiomyocytes displayed broad transcriptional dysregulation, apoptosis, mitochondrial dysfunction, impaired reactive oxygen species handling, altered contractility, and enhanced calcium transient decay parameters. Mechanistically, phospholamban hyperphosphorylation may contribute to this calcium dysregulation. Consistently, modGLA therapy restored α‐galactosidase A activity, reduced glycosphingolipid deposition, and normalized molecular alterations, including phospholamban hyperphosphorylation and calcium decay parameters, supporting modGLA as a promising therapeutic strategy for Fabry disease.

The lysosomal storage disorder Fabry disease results from α-galactosidase A deficiency, leading to excessive glycosphingolipid substrate accumulation, primarily globotriaosylceramide (Gb3). While the underlying molecular mechanisms remain elusive, multi-systemic complications ultimately culminate in premature death, with heart failure being the leading cause of death. Current treatment options fail to treat Fabry disease adequately and only delay its progression. Preclinical studies on an alternative approach, systemic delivery of nucleoside-modified GLA mRNA (modGLA), suggest improved effectiveness over existing therapies in reducing glycosphingolipid levels in the heart. It remains unclear whether modGLA can rescue Fabry cardiomyopathy phenotypes at the cellular level, which are not faithfully recapitulated in current animal models. To address this, we investigated characteristic phenotypes in two new models of Fabry cardiomyopathy utilizing human iPSC-derived cardiomyocytes in transcriptomic and functional analyses. These human Fabry disease cardiomyocytes displayed broad transcriptional dysregulation, apoptosis, mitochondrial dysfunction, impaired reactive oxygen species handling, as well as enhanced decay parameters of calcium transients. Mechanistically, we identified hyperphosphorylated phospholamban as a major player in this calcium dysregulation. Strikingly, modGLA therapy of Fabry cardiomyocytes restored α-galactosidase A enzyme activity, reduced glycosphingolipid deposition, and normalized the observed molecular alterations, supporting modGLA therapy as a promising strategy for the treatment of Fabry disease.

Background: Hereditary intrahepatic cholestasis is caused by variants of various genes involved in enterohepatic bile circulation, metabolization, and conjugation. Originally classified into 3 groups, the number of contributing genes is still increasing, underlining the need for a deeper understanding of the molecular interaction during intrahepatic cholestasis. Methods: In the present study, we investigate the interplay of heterozygous variants in 3 cholestasis-associated genes (ABCB11, ABCB4, and MYO5B) by exploiting iPSC-based hepatic organoids from a patient suffering from recurrent intrahepatic cholestasis. Results: Functional characterization of MRP2-mediated cholyl-lysyl-fluorescein (CLF) and BSEP-mediated Tauro-nor-THCA-24-DBD transport demonstrated a marked reduction of transport in MYO5B-deficient organoids, in comparison to unaffected control organoids. Moreover, iPSC-based organoids derived from the patient carrying 3 heterozygous variants in ABCB11, ABCB4, and MYO5B also exhibited absence of BSEP-mediated Tauro-nor-THCA-24-DBD transport, but functional MRP2-mediated CLF-transport. Interestingly, CRISPR/Cas9-mediated correction of the mutated ABCB11 allele could not restore the impaired BSEP function, suggesting the heterozygous MYO5B variant as the main driver of the transport deficiency. In fact, CRISPR/Cas-mediated correction of the MYO5B variant finally resulted in a restoration of the BSEP-mediated Tauro-nor-THCA-24-DBD transport. Conclusions: iPSC-based organoids serve as an authentic model for functional assessment of the hepatobiliary transport with fluorescent substrates. This allows the characterization of variants of uncertain significance and other variants in cholestasis-associated genes and revealed that a heterozygous MYO5B variant increases the susceptibility to defective hepatobiliary BSEP-mediated transport.

Der Begriff „Gentherapie“ ist nicht mehr passgenau verwendbar, wenn beispielsweise neben der additiven Ergänzung defekter Gene auch gezielte DNA- und Genexpression-Änderungen mittels RNA und CRISPR/Cas-basierter Verfahren adressiert werden. Solche neuen somatischen genomischen Therapien stellen aber die Medizin vor neue Herausforderungen. Insbesondere die Individualisierung solcher Therapien bricht mit herkömmlichen Paradigmen der Arzneimittelentwicklung und bedingt neue Konzepte für eine kosteneffiziente Herstellung und angepasste Regulierung.

Cas9–SpRY is an engineered variant of the Streptococcus pyogenes Cas9 with relaxed PAM recognition, which can technically be utilized at any target in the genome but some targets are addressed with low efficiency. Here, we show that a previously unexplored motif at the turn and beginning of α-helix 51 (TH51) can be engineered to improve both nuclease and prime-editing activity of Cas9–SpRY. Interaction of the lysine-rich PID loop 2 (PL2) with the target DNA downstream of the PAM (post-PAM) mediates initiation of R-loop formation and subsequent cleavage yet it was unclear if other regions of the PID engage with post-PAM as well. To this end, the NAAN–PAM-targeting iSpyMac hybrid nuclease, which lacks all lysine residues in PL2, was compared with Cas9–SpRY at identical targets using molecular dynamics simulation and in cell culture models, uncovering four crucial post-PAM-interacting lysines in TH51 and TH53 of iSpyMac. Ectopic insertion of a lysine-rich PL2 into iSpyMac boosted its nuclease and prime-editing activities and, in turn, Cas9–SpRY benefited from certain lysine-rich TH51 motifs. Specifically, TH51 from an uncultured Abiotrophia Cas9 species boosted overall Cas9–SpRY activity. Together, this study demonstrates that engineering of post-PAM interacting motifs opens new avenues for the design of advanced CRISPR enzymes.

The advent of innovative techniques, such as the CRISPR/Cas system, has opened up a new range of possibilities for modifying the genome, with the potential to address previously unmet therapeutic needs of patients with genetic diseases. These new possibilities have not only raised ethical concerns but also challenged existing classifications of genome modification techniques. While the legal status of some of these new therapies remains uncertain, there is an ongoing debate within philosophy of biology about the information-related metaphors adopted by scientists to describe and classify the genome and its therapeutic modification. Given the continuing advance of new genomic therapies, we show, employing an interdisciplinary approach, that a comprehensive framework for the classification of these technologies is needed to resolve legal and philosophical issues. The first section provides an analysis of the current state of novel genome-modifying techniques in medical genetics. In the second section, we assess the regulatory status of these techniques within the European regulatory framework for advanced therapy medicinal products (ATMPs). Drawing on these results, we argue in the third section from a philosophical perspective that metaphors, such as ‘editing’ the genome, which are based on a conception of the genome as linear information, cannot adequately capture the breadth of advanced genomic technologies. To accurately categorise these techniques in a manner that meets their diverse applications, we propose introducing the umbrella term ‘somatic genomic therapies’ (SGTs). Urging an integrative approach to defining and classifying new technologies in medical genetics, we advocate for the development of an integrative concept of SGTs.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%–45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.

Certain point-mutations in the human SERPINA1-gene can cause severe α1-antitrypsin-deficiency (A1AT-D). Affected individuals can suffer from loss-of-function lung-disease and from gain-of-function liver-disease phenotypes. However, age of onset and severity of clinical appearance is heterogeneous amongst carriers, suggesting involvement of additional genetic and environmental factors. The generation of authentic A1AT-D mouse-models has been hampered by the complexity of the mouse Serpina1-gene locus and a model with concurrent lung and liver-disease is still missing. Here, we investigate point-mutations in the mouse Serpina1a antitrypsin-orthologue, which are homolog-equivalent to ones known to cause severe A1AT-D in human. We combine in silico and in vitro methods and we find that analyzed mutations do introduce potential disease-causing properties into Serpina1a. Finally, we show that introduction of the King’s-mutation causes inactivation of neutrophil elastase inhibitory-function in both, mouse and human antitrypsin, while the mouse Z-mutant retains activity. This work paves the path to generation of better A1AT-D mouse-models.

The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.