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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. Limits of AAV-mediated gene therapy include targeting dominant mutations and inducing long-term transgene expression. Here, the authors show that AAV-HITI results in efficient allele-independent integration of a donor DNA in both retina and liver providing therapeutic benefit in mouse models of either a genetic form of blindness or a lysosomal storage disease, respectively.

Mesenchymal stromal cells (MSCs) are multipotent cells found in different tissues: bone marrow, peripheral blood, adipose tissues, skeletal muscle, perinatal tissues, and dental pulp. MSCs are able to self-renew and to differentiate into multiple lineages, and they have been extensively used for cell therapy mostly owing to their anti-fibrotic and immunoregulatory properties that have been suggested to be at the basis for their regenerative capability. MSCs exert their effects by releasing a variety of biologically active molecules such as growth factors, chemokines, and cytokines, either as soluble proteins or enclosed in extracellular vesicles (EVs). Analyses of MSC-derived secretome and in particular studies on EVs are attracting great attention from a medical point of view due to their ability to mimic all the therapeutic effects produced by the MSCs (i.e., endogenous tissue repair and regulation of the immune system). MSC-EVs could be advantageous compared with the parental cells because of their specific cargo containing mRNAs, miRNAs, and proteins that can be biologically transferred to recipient cells. MSC-EV storage, transfer, and production are easier; and their administration is also safer than MSC therapy. The skeletal muscle is a very adaptive tissue, but its regenerative potential is altered during acute and chronic conditions. Recent works demonstrate that both MSCs and their secretome are able to help myofiber regeneration enhancing myogenesis and, interestingly, can be manipulated as a novel strategy for therapeutic interventions in muscular diseases like muscular dystrophies or atrophy. In particular, MSC-EVs represent promising candidates for cell free-based muscle regeneration. In this review, we aim to give a complete picture of the therapeutic properties and advantages of MSCs and their products (MSC-derived EVs and secreted factors) relevant for skeletal muscle regeneration in main muscular diseases.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety ( n  = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox. Cas9 nucleases hold clinical significance for genome editing therapies. Here the authors characterize CoCas9, a compact, efficient and precise Cas9 from the human microbiome, and show that delivery via AAV vectors enables efficient editing in the mouse retina, expanding the genome editing toolbox.

[...]it is still unclear whether neurodegeneration is a direct consequence of inflammatory CNS injury or whether it represents a primitive independent process, and a better understanding of the interplay between these two aspects of the disease is warranted. Noteworthy, the prioritized effects were enriched in genes involved in lymphocyte functions and in particular in T cell activation and proliferation. [...]genes linked to Vitamin D metabolism, such as CYP27B1, CYP24A1, and genes coding for targets of MS immune-modulatory therapies, including VCAM1 for natalizumab and IL2RA for daclizumab, were also highlighted. [...]the main role played by the immune system is also reflected in the significant proportion of MS risk variants that is shared with other autoimmune diseases (Richard-Miceli and Criswell, 2012), such as type 1 diabetes, rheumatoid arthritis and intestinal bowel diseases, even though sometimes with opposite effect. First of all, candidate gene studies have assessed the role of genes implicated in MS susceptibility in determining disease course, failing to identify any significant association (Jensen et al., 2010) except for the HLA-DRB1*1501 allele (Hauser et al., 2000; Barcellos et al., 2003).

Background/Objectives: Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of immune pathways and may hold diagnostic and therapeutic relevance in autoimmune diseases such as Multiple Sclerosis (MS). However, research on lncRNAs in MS remains fragmented and geographically clustered. This systematic review aimed to collate and critically evaluate studies of lncRNA expression in MS, assess consistency of findings across studies, and synthesize proposed functional implications of the most frequently studied lncRNAs. Methods: This PROSPERO-registered review (CRD420250575938), conducted in accordance with PRISMA, searched PubMed, Scopus, Embase, and Web of Science (2010–2024) for studies evaluating lncRNA expression in adult MS (≥18 years of age). Eligible studies included ≥20 participants and assessed lncRNAs in blood, PBMCs, serum, plasma, or CSF using qRT-PCR, RNA-seq, or microarrays. Pediatric, review, animal, and in vitro studies were excluded. Two reviewers independently screened and extracted data, with risk of bias evaluated using QUADAS-2. Results: Narrative synthesis of 51 studies identified 77 unique lncRNAs. A limited set (MALAT1, GAS5, MEG3, H19) demonstrated consistent dysregulation in MS, whereas others (THRIL, IFNG-AS1, HOTAIR, TUG1) exhibited context-dependent expression influenced by treatment, relapse status, or demographics. Functional annotations converged on immune pathways, including NF-κB, STAT3, IFN-γ/Th1, and glucocorticoid signaling. Conclusions: This review identifies reproducible and context-specific lncRNA dysregulation in MS, emphasizing the need for transcriptome-wide approaches, standardized methods, and multi-center validation. Current evidence is constrained by geographic clustering, preselection bias, and methodological heterogeneity.

Inefficient targeting of muscle stem cells (MuSCs), also called satellite cells, represents a major bottleneck of current therapeutic strategies for muscular dystrophies, as it precludes the possibility of promoting compensatory regeneration. Here we describe a muscle-targeting delivery platform, based on gold nanoparticles, that enables the release of therapeutic oligonucleotides into MuSCs. We demonstrate that AuNPs conjugation to an aptamer against α7/β1 integrin dimers directs either local or systemic delivery of microRNA-206 to MuSCs, thereby promoting muscle regeneration and improving muscle functionality, in a mouse model of Duchenne Muscular Dystrophy. We show here that this platform is biocompatible, non-toxic, and non-immunogenic, and it can be easily adapted for the release of a wide range of therapeutic oligonucleotides into diseased muscles. Lack of muscle stem cell targeting limits the treatment of muscular dystrophies. Here, the authors describe a platform based on functionalized gold nanoparticles that allows selective delivery of therapeutic oligonucleotides and promotes skeletal muscle regeneration.

Genome-wide association studies (GWAS) have identified more than 50,000 unique associations with common human traits. While this represents a substantial step forward, establishing the biology underlying these associations has proven extremely difficult. Even determining which cell types and which particular gene(s) are relevant continues to be a challenge. Here, we conduct a cell-specific pathway analysis of the latest GWAS in multiple sclerosis (MS), which had analyzed a total of 47,351 cases and 68,284 healthy controls and found more than 200 non-MHC genome-wide associations. Our analysis identifies pan immune cell as well as cell-specific susceptibility genes in T cells, B cells and monocytes. Finally, genotype-level data from 2,370 patients and 412 controls is used to compute intra-individual and cell-specific susceptibility pathways that offer a biological interpretation of the individual genetic risk to MS. This approach could be adopted in any other complex trait for which genome-wide data is available. Genome-wide association studies (GWAS) have so far uncovered more than 200 loci for multiple sclerosis (MS). Here, the authors integrate data from various sources for a cell type-specific pathway analysis of MS GWAS results that specifically highlights the involvement of the immune system in disease pathogenesis.

The presence of α-synuclein aggregates in the retina of Parkinson’s disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.

Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes ( CHD1L , PRKAB2 ) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis. This study identifies distinct genetic-epigenetictranscriptional interplay at the 1q21.1 locus in relation to neuronal deficits in primary progressive multiple sclerosis (PPMS).

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.