Recently developed engineered virus-like particles (eVLPs) have emerged as a promising delivery vehicle for ribonucleoprotein gene editing complexes. Variability in eVLP batch production may, however, hinder reproducibility and standardization across pre-clinical investigations designed to characterize and optimize this platform. Ultimately, stringent production, purification, and quantification processes will be required for this technology to reach the clinic. In this study, we developed two titration methods for base editor (BE)-eVLPs: a spacer-agnostic quantitative reverse-transcription PCR (RT-qPCR) assay to quantify the copy number of sgRNA molecules, and a NanoBiT luciferase-based approach to estimate vesicular stomatitis virus envelope glycoprotein (VSV-G) abundance, per μL of BE-eVLP preparation. We further engineered an LgBiT-expressing reporter cell line to monitor BE-eVLP transduction kinetics in real time. Our findings reveal that both RT-qPCR and HiBiT-based quantification enable effective batch-to-batch standardization of BE-eVLP preparations. Further, the LgBiT-expressing reporter cell line was effective in real-time monitoring of transduction kinetics. BE-eVLP transduction was proven dependent on endosomal acidification and was constrained by cellular endocytic capacity. Paired with accurate quantification of BE-eVLP preparations and using HiBiT-tagged virus-surface glycoproteins, the LgBiT reporter cell line can facilitate comparison of transduction and levels of editing achieved across emergent eVLP platforms and pseudotypes.

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

Recently developed engineered Virus-Like Particles (eVLPs) have emerged as a promising delivery vehicle for ribonucleoprotein gene editing complexes. Variability in eVLP batch production may, however, hinder reproducibility and standardization across pre-clinical investigations designed to characterize and optimize this platform. Ultimately, stringent production, purification and quantification processes will be required for this technology to reach the clinic. In this study, we developed two titration methods for base editor (BE)-eVLPs: a spacer-agnostic RT-qPCR assay to quantify the copy number of sgRNA molecules, and a NanoBiT® Luciferase-based approach to estimate VSV-G glycoprotein abundance, per μl of BE-eVLP preparation. We further engineered a LgBiT-expressing reporter cell line to monitor BE-eVLP transduction kinetics in real time. Our findings reveal that both RT-qPCR and HiBiT-based quantification enable effective batch-to-batch standardization of BE-eVLP preparations. Further, the LgBiT-expressing reporter cell line was effective in real-time monitoring of transduction kinetics. BE-eVLP transduction was proven dependent on endosomal acidification and was constrained by cellular endocytic capacity. Paired with accurate quantification of BE-eVLP preparations and using HiBiT-tagged virus-surface glycoproteins, the LgBiT-reporter cell line can facilitate comparison of transduction and levels of editing achieved across emergent eVLP platforms and pseudotypes.

Delivery strategies for gene editing applications can affect editing outcomes through distinct cellular responses. This work presents bulk RNA sequencing data from human bronchial epithelial CFF-16HBEge cells, following delivery of an adenine base editor and sgRNA either as plasmid DNA via lipofection or as ribonucleoprotein complexes via engineered virus-like particles (eVLPs). Differential gene expression analysis identified distinct transcriptional profiles between conditions, and gene ontology analysis highlighted enriched biological processes associated with each. This dataset provides a resource for researchers investigating cellular responses to gene editing delivery approaches in human airway epithelial cells in vitro.

Cystic fibrosis (CF) is a life-shortening autosomal recessive disease, caused by loss-of-function mutations that affect the CF transmembrane conductance regulator (CFTR) anion channel. G542X is the second-most common CF-causing variant, and it does not respond to current CFTR modulator drugs. Our study explores the use of adenine base editing to edit G542X to a non-CF-causing variant, G542R, and recover CFTR function. Using base editor engineered virus-like particles (BE-eVLPs) in patient-derived intestinal organoids, we achieved ∼2% G542X-to-G542R editing efficiency and restored CFTR-mediated chloride transport to ∼6.4% of wild-type levels, independent of modulator treatment, and with no bystander edits. This proof-of-principle study demonstrates the potential of base editing to rescue G542X and provides a foundation for future in - vivo applications.

Cystic fibrosis (CF) is a severe genetic disorder caused by loss-of-function mutations in the CFTR gene. Gene-editing approaches have the potential to correct such mutations. This systematic review outlines the mechanisms of the main CRISPR-based technologies, and, through cross-study comparisons, analyzes 27 research articles that applied them to target CF-causing variants. We report and discuss the strategy design, target cell selection, editing efficiency, prevalence of editing byproducts, and levels of CFTR functional restoration achieved in each work, with the aim of providing technical insights for further exploration of CRISPR-based gene-editing approaches. Our findings show that the F508del and W1282X mutations were the most extensively studied CF-causing variants, though over fifteen mutations were targeted overall. The majority of works under review explored the use of homology-directed repair or base editing, with a growing number of studies reporting efficient prime editing. Some studies tackled multiple individual mutations, compared different editors, or tested strategies across various models, while others focused on approaches that rescue CFTR function without directly correcting a mutation. Several works also proposed strategies that could address multiple variants with a single approach, while others highlighted technical difficulties in editing certain regions of the CFTR gene. This cross-study comparison also emphasizes the need for standardized reporting of editing efficiency and functional recovery, and stresses the importance of further single-cell RNA sequencing and in vivo studies to reach clinically relevant conclusions. As gene-editing techniques continue to evolve, and with over 60 ongoing CRISPR-based clinical trials, there is growing optimism for meaningful advancements in CF gene-editing therapeutics.

We are often confronted with a simple question, “which gene editing technique is the best?”; the simple answer is “there isn’t one”. In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, “classic” CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.

The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Background Labyrinthine haemorrhage is a rare vascular disorder often presenting with the triad of acute vertigo, sudden sensorineural hearing loss and tinnitus. There are minimal reports on imaging progression over the acute period.Index case A woman in her mid-40s presented with acute vertigo, sudden left-sided hearing loss and tinnitus, consistent with acute unilateral audiovestibular loss. Left peripheral vestibular hypofunction was confirmed acutely on video head impulse testing, and pure tone audiometry showed a profound left sensorineural hearing loss. An MRI brain including diffusion-weighted imaging within 24 hours was normal. Delayed MRI brain and internal acoustic canal after 7 days demonstrated increased 3D fluid-attenuated inversion recovery and T1 signal throughout the left cochlea and semicircular canals, without contrast enhancement. This was consistent with labyrinthine haemorrhage. She received early oral prednisone followed by three doses of intratympanic dexamethasone. At 12 months follow-up the patient remained profoundly deaf, however, balance and vestibular symptoms improved with early vestibular physical rehabilitation.Conclusion We report a case of acute labyrinthine haemorrhage missed on an early MRI brain sequence. This diagnosis should be considered in presentations of acute audiovestibular loss, and delayed MRI including internal auditory canal sequences may be important for diagnosis.

specific expression of the split-ABE to understand which cell types, and how many of them, must be corrected to restore CFTR function to therapeutic levels using our BCi W1282X-CFTR model.

Nephropathic cystinosis is a rare monogenetic kidney disease caused by mutations in the lysosomal transporter cystinosin (encoded by CTNS ) that, to date, has no cure. The hallmark of this disease is lysosomal accumulation of cystine and decline in proximal tubular function leading to kidney failure early in life. In this project, we developed a novel gene repair strategy using CRISPR/Cas9 Homology-Independent Targeted Integration (HITI) to restore CTNS . A novel, non-viral peptide-mediated approach was used to deliver the Cas9-guideRNA ribonucleoprotein (RNP) complex and repair templates to conditionally immortalized proximal tubule epithelial cell (ciPTEC) lines. The repair constructs contained either mCherry (1.7 kb), the CTNS Superexon (1.7 Kb) or both (3.2 Kb). The results demonstrated that the smaller mCherry construct achieved a higher repair efficiency (63%) compared to the CTNS -mCherry construct (16%). Clonal expansion of repaired cells showed restoration of lysosomal cystine levels in 70-75% of the clones, which was accompanied by improved mitochondrial bioenergetics. In conclusion, CRISPR/Cas9 HITI can be used to precisely insert repair templates into the genome, resulting in a functional cystinosin restoration, and a reversal of the cystinotic disease phenotype.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other mutations are pathogenic.

circuit, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot, and bulk RNA-seq.Methods: We used two model systems: primary nasal cells from individuals with CF who harbor nonsense variants and their family members and Flp-In stable cells carrying an expression minigene (EMGi21-i24) harboring nonsense variants.CFTR function and RNA studies were performed in NEs and CFBE Flp-In cells, and protein processing was assessed in Flp-In HEK293 stable cells.Twelve people with CF, 20 carrier parents, one unrelated carrier, and eight healthy controls were recruited.Each person with CF had at least one copy of nonsense variants L732X, R764X, R553X, E822X, R1162X, or W1282X.Cells were treated with combinations of antisense oligonucleotides (ASOs) to NMD-critical transcripts (SMG1, SMG6, Upf3b), small-molecule inhibitors of NMD (NMDi14, Emetine), readthrough compounds (G418, ELOX-02, CC-90009, PTC-124), and elexacaftor-tezacaftor-ivacaftor (ETI).CFTR function was measured in an Ussing chamber, and mRNA expression was assessed by qRT-PCR and RNA-seq.Results: Cells treated with a readthrough compound or ETI alone had minimal recovery of CFTR function, but treatment with a combination of an NMD-ASO, a readthrough compound, and ETI resulted in a remarkable recovery of CFTR function.For example, treating CFBE cells bearing W1282X CFTR EMGs with SMG6-ASO, ELOX-02, and ETI yielded ΔI sc = 32.5 ± 13.2 μA/cm 2 , corresponding to approximately 20% of those with wild-type CFTR EMGs.In NEs harboring W1282X, the same combination yielded a significant increase in CFTR function, with ΔI sc = 2.4 ± 0.1 μA/cm 2 , corresponding to approximately 18% of control.NMD-ASO, readthrough, and CFTR corrector combinations led to fulllength glycosylated CFTR in HEK293 cells stably transfected with the W1282X CFTR EMG.qRT-PCR and bulk RNA-seq results for CFBE cells and NEs support that CFTR RNA was 10 times as great under these treatment conditions, and RNA-seq confirmed that the ASOs drastically reduced NMD-associated targets.ASOs were the least toxic NMD inhibitors and ELOX-02 was the least toxic readthrough compound; a combination of the two resulted in highest recovery of CFTR expression and function.Of these variants, W1282X was most responsive, and R1162X was least.Conclusions: Our data suggest that therapeutic strategies for CF caused by nonsense variants resulting in NMD must consider RNA stability and compound toxicity.For some of these variants, including W1282X, CFTR function increases significantly with the addition of ETI after successful readthrough of stabilized RNA, but to treat those with nonsense variants that do not respond well to this protocol, such as R1162X, we must continue to pursue other innovative strategies such as tRNA suppression therapy and CRISPR-based approaches.