Biology subjects

Rothgangl, T.

Publications and source records attributed to Rothgangl, T..

8 recordsLinked to original sources

bioRxiv2025

Cell-stereotyped DNA repair outcomes are widespread during genome editing

Genome editing outcomes are governed by DNA repair pathways that vary with cell type and state. We developed scOUT-seq (single-cell Outcomes Using Transcript sequencing), a scalable approach that jointly profiles transcriptomes and matched multi-allelic editing outcomes ranging from homology directed repair (HDR) to inter-chromosomal translocations. We mapped editing outcomes in human CD34 hematopoietic stem and progenitor cells (HSPCs), mouse LSK HSPC equivalents, human upper airway organoids, and mouse multi-organ in vivo editing. Profiling 500,000 alleles across 74 cell types, scOUT-seq revealed that outcomes in most cell subtypes differ markedly from the bulk average. Various cell types shifted major repair classes, preferred different molecular sequences, and even enriched large structural variants, with distinctive patterns of allelic co-occurrence. Surprisingly, rare stem subtypes diverged from prevalent progenitors, and inhibitory neuron subtypes efficiently incorporated HDR alleles. These data suggest the potential for tailored therapeutic editing that may have been missed by bulk measurements.

genomics↗

bioRxiv2024

Therapeutic liver cell transplantation to treat a genetic liver defect

For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human of phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.

molecular biology↗

bioRxiv2024

Treatment of a genetic liver disease in mice through transient prime editor expression

Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 {micro}mol/l to below the therapeutic threshold of 600 {micro}mol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

bioengineering↗

bioRxiv2023

Prime editing of the beta-1 adrenoceptor in the brain reprograms mouse behavior

Prime editing is a highly versatile genome editing technology that holds great potential for treating genetic diseases1, 2. While in vivo prime editing has recently been conducted in the brain, liver, heart, and retina3-6, application of this technology to modulate neural circuits in the brain has not been reported yet. Here, we employ adeno-associated viral vectors to deliver optimized intein-split prime editors into the brain of mice. Delivery into newborn pups via intracerebroventricular injection resulted in up to 44.0% editing at the Dnmt1 locus in the cortex (on average 34.8{+/-}9.8% after 6 months). In addition, we obtained up to 28.1% editing at the Adrb1 locus in the cortex (on average 14.7{+/-}11.6% after 6 months). The introduced Adrb1A187V mutation is a naturally occurring variant of the {beta}1-adrenergic receptor, which has previously been linked to increased activity and natural short sleep7. Similarly, we observed an increase in the activity and exploratory behavior of treated animals. This study demonstrates the potential of prime editing for treating genetic diseases in the central nervous system and for reprogramming molecular pathways that modulate animal behavior.

genetics↗

bioRxiv2023

Safe delivery of AAV vectors to the liver of small weaned pigs by ultrasound guided percutaneous transhepatic portal vein injection

One challenge for liver-directed gene therapy is sufficient vector delivery to the target tissue while minimizing loss of the applied vector dose to other tissues. Infusion via peripheral veins is the least invasive approach; however, it results in systemic diffusion and substantial vector dilution. Here, we describe a safe and minimally invasive method to deliver adeno-associated virus (AAV) vectors to the liver of small weaned pigs by ultrasound-guided percutaneous trans-hepatic portal vein injection. 4-week-old piglets were infused with [~]2.5x1014 vector genomes comprising a dual-rAAV2/9 vector system with a split adenine base editor for in vivo inactivation of PCSK9 to reduce LDL-cholesterol levels. Animals had no signs of discomfort and tolerated the procedure well. However, despite 45% editing of the target site with the applied adenine base editor system in cultivated porcine cells, we only found low amounts of AAV vector genomes and neither detectable transgene-expression nor successful editing in the treated pig livers. We hypothesize that rapid proliferation of pig hepatocytes caused AAV vector dilution, leading to a loss of the vectors from the nucleus, and hence insufficient base editor protein expression for achieving detectable editing rates. Nonetheless, ultrasound-guided percutaneous transhepatic injection to the portal vein is well-tolerated in piglets and has potential for human (neonate) application.

molecular biology↗

bioRxiv2021

Treatment of a metabolic liver disease by in vivo prime editing in mice

Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of pathogenic mutations (1). However, correction of a disease phenotype in vivo in somatic tissues has not been demonstrated thus far. Here, we establish proof-of-concept for in vivo prime editing and repair the metabolic liver disease phenylketonuria (PKU) in mice. We first developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (PE2{Delta}RnH), and a linker- and NLS-optimized intein-split PE construct (PE2 p.1153) for delivery by adeno-associated virus (AAV) vectors. Systemic dual AAV-mediated delivery of this variant into the liver of neonatal mice enabled installation of a transversion mutation at the Dnmt1 locus with an average efficiency of 15%, and delivery of unsplit PE2{Delta}RnH using human adenoviral vector 5 (AdV5) further increased editing rates to 58%. PE2{Delta}RnH-encoding AdV5 was also used to correct the disease-causing mutation of the phenylalanine hydroxylase (Pah)enu2 allele in phenylketonuria (PKU) mice with an average efficiency of 8% (up to 17.3%), leading to therapeutic reduction of blood phenylalanine (L-Phe) levels. Our study demonstrates in vivo prime editing in the liver with high precision and editing rates sufficient to treat a number of metabolic liver diseases, emphasizing the potential of prime editing for future therapeutic applications. One Sentence SummaryIn vivo prime editing corrects phenylketonuria in mice.

genetics↗

bioRxiv2021

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. Using murine PDA cells and human PDA organoids, we demonstrate that Rnf31 protects from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction. For Vps4b we found that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Orthotopic transplantation of Rnf31- or Vps4b deficient pancreatic tumors, moreover, revealed increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth in mice. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

cancer biology↗

bioRxiv2020

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors, and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

bioengineering↗