Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Machine learning prediction of prime editing efficiency across diverse chromatin contexts.

The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates.

Enhancing prime editor activity by directed protein evolution in yeast.

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N4AH and N5HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N3VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

Mutant SF3B1 promotes malignancy in PDAC.

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1K700E alone is insufficient to induce malignant transformation of the murine pancreas, but that it increases aggressiveness of PDAC if it co-occurs with mutated KRAS and p53. We further show that Sf3b1K700E already plays a role during early stages of pancreatic tumor progression and reduces the expression of TGF-ß1-responsive epithelial-mesenchymal transition (EMT) genes. Moreover, we found that SF3B1K700E confers resistance to TGF-ß1-induced cell death in pancreatic organoids and cell lines, partly mediated through aberrant splicing of Map3k7. Overall, our findings demonstrate that SF3B1K700E acts as an oncogenic driver in PDAC, and suggest that it promotes the progression of early stage tumors by impeding the cellular response to tumor suppressive effects of TGF-ß.

Genetic ablation of ketohexokinase C isoform impairs pancreatic cancer development.

Although dietary fructose is associated with an elevated risk for pancreatic cancer, the underlying mechanisms remain elusive. Here, we report that ketohexokinase (KHK), the rate-limiting enzyme of fructose metabolism, is a driver of PDAC development. We demonstrate that fructose triggers KHK and induces fructolytic gene expression in mouse and human PDAC. Genetic inactivation of KhkC enhances the survival of KPC-driven PDAC even in the absence of high fructose diet. Furthermore, it decreases the viability, migratory capability, and growth of KPC cells in a cell autonomous manner. Mechanistically, we demonstrate that genetic ablation of KHKC strongly impairs the activation of KRAS-MAPK pathway and of rpS6, a downstream target of mTORC signaling. Moreover, overexpression of KHKC in KPC cells enhances the downstream KRAS pathway and cell viability. Our data provide new insights into the role of KHK in PDAC progression and imply that inhibiting KHK could have profound implications for pancreatic cancer therapy.

Predicting prime editing efficiency and product purity by deep learning.

Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications.

SF3B1 facilitates HIF1-signaling and promotes malignancy in pancreatic cancer.

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.

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 dissect PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identify Vps4b and Rnf31 as essential factors required for escaping CD8+ T cell killing. For Vps4b we find that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. For Rnf31 we demonstrate that it protects tumor cells from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction, a mechanism that is conserved in human PDA organoids. Orthotopic transplantation of Vps4b- or Rnf31 deficient pancreatic tumors into immune competent mice, moreover, reveals increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

In vivo targeting of a variant causing vanishing white matter using CRISPR/Cas9.

Vanishing white matter (VWM) is a leukodystrophy caused by recessive variants in subunits of eIF2B. At present, no curative treatment is available and patients often die at young age. Due to its monogenic nature, VWM is a promising candidate for the development of CRISPR/Cas9-mediated gene therapy. Here we tested a dual-AAV approach in VWM mice encoding CRISPR/Cas9 and a DNA donor template to correct a pathogenic variant in Eif2b5. We performed sequencing analysis to assess gene correction rates and examined effects on the VWM phenotype, including motor behavior. Sequence analysis demonstrated that over 90% of CRISPR/Cas9-induced edits at the targeted locus are insertion or deletion (indel) mutations, rather than precise corrections from the DNA donor template by homology-directed repair. Around half of the CRISPR/Cas9-treated animals died prematurely. VWM mice showed no improvement in motor skills, weight, or neurological scores at 7 months of age, and CRISPR/Cas9-treated controls displayed an induced VWM phenotype. In conclusion, CRISPR/Cas9-induced DNA double-strand breaks (DSBs) at the Eif2b5 locus did not lead to sufficient correction of the VWM variant. Moreover, indel formation in Eif2b5 induced an exacerbated VWM phenotype. Therefore, DSB-independent strategies like base- or prime editing might better suited for VWM correction.

CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-ß Pathway Regulators.

The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-ß signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-ß pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.

In vivo prime editing of a metabolic liver disease in mice.

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

miR-802 Suppresses Acinar-to-Ductal Reprogramming During Early Pancreatitis and Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood. METHODS: The Ptf1aCre/+LSL-KrasG12D/+ and Ptf1aCre/+LSL-KrasG12D/+LSL-p53R172H/+ and caerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM. RESULTS: We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic KrasG12D-induced transformation. Genetic ablation of mir-802 cooperates with KrasG12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels of ductal and attenuated expression of acinar identity genes. Consistently with these findings, we show that this miR-802-RhoA-F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival. CONCLUSIONS: We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802-RhoA-F-actin network may be a new strategy to interfere with pancreatic carcinogenesis.

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 DNA (ssDNA) 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 a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) 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.

Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens.

Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.

miR-802 regulates Paneth cell function and enterocyte differentiation in the mouse small intestine.

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.