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.
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.
...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:
Hand-motor impairment affects a large proportion of multiple sclerosis (MS) patients; however, its substrates are still poorly understood.
Objectives:
To investigate the association ...between global disability, hand-motor impairment, and alterations in motor-relevant structural and functional magnetic resonance imaging (MRI) networks in MS patients with different clinical phenotypes.
Methods:
One hundred thirty-four healthy controls (HC) and 364 MS patients (250 relapsing-remitting MS (RRMS) and 114 progressive MS (PMS)) underwent Expanded Disability Status Scale (EDSS) rating, nine-hole peg test (9HPT), and electronic finger tapping rate (EFTR). Structural and resting state (RS) functional MRI scans were used to perform a source-based morphometry on gray matter (GM) components, to analyze white matter (WM) tract diffusivity indices and to perform a RS seed-based approach from the primary motor cortex involved in hand movement (hand-motor cortex). Random forest analyses identified the predictors of clinical impairment.
Result:
In RRMS, global measures of atrophy and lesions together with measures of structural damage of motor-related regions predicted EDSS (out-of-bag (OOB)-R2 = 0.19, p-range = <0.001–0.04), z9HPT (right: OOB-R2 = 0.14; left: OOB-R2 = 0.24, p-range = <0.001–0.03). No RS functional connectivity (FC) abnormalities were identified in RRMS models. In PMS, cerebellar and sensorimotor regions atrophy, cerebellar peduncles integrity and increased RS FC between left hand-motor cortex and right inferior frontal gyrus predicted EDSS (OBB-R2 = 0.16, p-range = 0.02–0.04).
Conclusion:
In RRMS, only measures of structural damage contribute to explain motor impairment, whereas both structural and functional MRI measures predict clinical disability in PMS. A multiparametric MRI approach could be relevant to investigate hand-motor impairment in different MS phenotypes.
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.
Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in ...a reduced number of self-renewing muscle stem cells (MuSCs) and an impairment of their activation and differentiation, which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study, we focused on an unexplored strategy to improve MuSC function and to preserve their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), that are multipotent cells recognized to have a role in tissue repair in different disease models. We demonstrate that the hAMSC secretome (CM hAMSC) and extracellular vesicles (EVs) isolated thereof directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Furthermore, we demonstrate that hAMSC secreted factors modulate the muscle stem cell niche in dystrophic-
-mice. Interestingly, local injection of EV hAMSC in
muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs and in new fiber formation. EV hAMSCs also significantly reduced muscle collagen deposition, thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Herein for the first time we demonstrate that CM hAMSC and EVs derived thereof promote muscle regeneration by supporting proliferation and differentiation of resident muscle stem cells. These results pave the way for the development of a novel treatment to counteract DMD progression by reducing fibrosis and enhancing myogenesis in dystrophic muscles.
Background:
Fingolimod (FTY) is an effective second-line drug for relapsing-remitting multiple sclerosis, with ~50% patients showing no evidence of disease activity (NEDA) after 2 years. Nonetheless, ...the early identification of non-responders is extremely important, to promptly address them to more aggressive drugs.
Objectives:
This cohort study evaluates FTY medium-term effectiveness, searching for early markers of treatment failure.
Patients and methods:
Three hundred eighty patients starting FTY were enrolled and classified according to NEDA and time to first relapse criteria at 4-year follow-up. Logistic and Cox regression analyses were applied to identify early predictors of non-response.
Results:
At 4 years, 65.6% of patients were free from relapses and 35.4% had NEDA. Female gender was associated with a higher risk of non-response. Moreover, evidence of clinical and/or magnetic resonance imaging (MRI) activity during the first year of treatment was highly predictive of disease activity in the follow-up: the positive predictive value for non-response was 0.74 for the presence of ⩾1 relapse, 0.73 for the presence of ⩾1 active MRI lesion, and 0.83 for the presence of both clinical and MRI activity.
Conclusions:
FTY effectiveness persists at medium-term follow-up; a close monitoring during the first year of treatment is warranted to early identify non-responders requiring treatment optimization.
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.
Background:
Hand motor impairment has considerable effects on daily-life activities of patients with multiple sclerosis (pwMS). Understanding its anatomo-functional substrates is relevant to provide ...more specific therapeutic interventions.
Objectives:
To investigate the association between hand motor performance and anatomo-functional magnetic resonance imaging (MRI) abnormalities in pwMS.
Methods:
A total of 134 healthy controls (HC) and 366 pwMS underwent the Nine-Hole-Peg-Test (9HPT), structural and resting state (RS) functional MRI. Multivariate analyses identified the independent predictors of hand motor performance.
Results:
PwMS versus HC showed widespread gray matter atrophy, microstructural white matter abnormalities, and decreased RS functional connectivity in motor and cognitive networks. Predictors of worse right-9HPT (R2 = 0.52) were decreased right superior cerebellar peduncle and right lemniscus fractional anisotropy (FA) (p ⩽ 0.02), left angular gyrus atrophy (p < 0.003), decreased RS connectivity in left superior frontal gyrus, and left posterior cerebellum (p < 0.001). Worse left 9HPT (R2 = 0.56) was predicted by decreased right corticospinal FA (p = 0.003), atrophy of left anterior cingulum and left cerebellum (p ⩽ 0.02), decreased RS connectivity of left lingual gyrus and right posterior cerebellum in cerebellar and executive networks (p ⩽ 0.02).
Conclusion:
Structural and functional abnormalities of regions involved in motor functions contribute to explain motor disability in pwMS. The integration of clinical and advanced MRI measures contributes to improve our understanding of multiple sclerosis clinical manifestations.
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.
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