Therapeutic homology-independent targeted integration in retina and liver

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs

Deciphering multiple sclerosis disability with deep learning attention maps on clinical MRI

Deep learning; Disability; Structural MRIAprendizaje profundo; Discapacidad; Resonancia magnética estructuralAprenentatge profund; Discapacitat; Ressonància magnètica estructuralThe application of convolutional neural networks (CNNs) to MRI data has emerged as a promising approach to achieving unprecedented levels of accuracy when predicting the course of neurological conditions, including multiple sclerosis, by means of extracting image features not detectable through conventional methods. Additionally, the study of CNN-derived attention maps, which indicate the most relevant anatomical features for CNN-based decisions, has the potential to uncover key disease mechanisms leading to disability accumulation. From a cohort of patients prospectively followed up after a first demyelinating attack, we selected those with T1-weighted and T2-FLAIR brain MRI sequences available for image analysis and a clinical assessment performed within the following six months (N = 319). Patients were divided into two groups according to expanded disability status scale (EDSS) score: ≥3.0 and < 3.0. A 3D-CNN model predicted the class using whole-brain MRI scans as input. A comparison with a logistic regression (LR) model using volumetric measurements as explanatory variables and a validation of the CNN model on an independent dataset with similar characteristics (N = 440) were also performed. The layer-wise relevance propagation method was used to obtain individual attention maps. The CNN model achieved a mean accuracy of 79% and proved to be superior to the equivalent LR-model (77%). Additionally, the model was successfully validated in the independent external cohort without any re-training (accuracy = 71%). Attention-map analyses revealed the predominant role of frontotemporal cortex and cerebellum for CNN decisions, suggesting that the mechanisms leading to disability accrual exceed the mere presence of brain lesions or atrophy and probably involve how damage is distributed in the central nervous system.MS PATHS is funded by Biogen. This study has been possible thanks to a Junior Leader La Caixa Fellowship awarded to C. Tur (fellowship code is LCF/BQ/PI20/11760008) by “la Caixa” Foundation (ID 100010434). The salaries of C. Tur and Ll. Coll are covered by this award

CRISPR/Cas9-mediated specific knock-down of dominant mutations in Rhodopsin gene

Rhodopsin (RHO) mutations represent a common cause of blindness, accounting for 25% of autosomal dominant Retinitis Pigmentosa (RP) and 8-10% of all RP. Although gene therapy has been successfully applied to retinal degeneration caused by recessive mutations, therapeutic intervention for dominant mutations are still lagging behind. In this study, we explored the efficacy of newly described CRISPR/Cas9 variants with altered PAM specificity and nearly completely reduced off-target effects, to specifically inactivate two highly frequent dominant mutations, P23H and P347S, mapped in the N-terminal and the C-terminal region of the RHO gene, respectively. We designed gRNAs on the mutations to compare allele-specific targeting of the high fidelity SpCas9 (SpCas9-HF1), the respective VQR variant (SpCas9-VQR-HF1) or the SaCas9, and we tested gRNAs in vitro on HeLa clones stably expressing P23H, P347S or wild-type RHO. Analysis of insertions or deletions (Indels) in the genomic DNA specifically in the RHO gene, by Cel-I assay and sequencing, identified the most efficient and mutation-specific system able to induce Indels in the P23H or P347S RHO mutated allele, with almost undetectable editing of the wild-type allele. We are going to package the selected CRISPR/Cas9-gRNA in AAV2/8 particles to test this approach in P23H or P347S RHO transgenic mice, to evaluate retina functionality and vision recovery upon CRISPR/Cas9-mediated editing. Our results will provide clear evidences about the employment of CRISPR/Cas9 system to selectively target dominant mutations and the preclinical application of this strategy for patients affected by RP due to mutations in the RHO gene

Therapeutic homology-independent targeted integration in retina and liver

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs