CRISPR genome surgery in a novel humanized model for autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

Mutation-Independent Gene Therapies for Rod-Cone Dystrophies.

The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.

From the Sea for the Sight: Marine Derived Products for Human Vision.

Visual impairment, at different degrees, produce a reduction of patient wellness which negatively impact in many aspects of working and social activities. Eye diseases can have common cellular damages or dysfunctions (e.g., inflammation, oxidative stress, neuronal degeneration), and can target several eye compartments, primarily cornea and retina. Marine organisms exhibit high chemical diversity due to the wide range of marine ecosystems where they live; thus, molecules of marine origin are gaining increasing attention for the development of new mutation-independent therapeutic strategies, to reduce the progression of retina pathologies having a multifactorial nature and characterized by high genetic heterogeneity. This review aims to describe marine natural products reported in the recent literature that showed promising therapeutic potential for the development of new drugs to be used to contrast the progression of eye pathologies. These natural compounds exhibited beneficial and protective properties on different in vitro cell systems and on in vivo models, through different mechanisms of action, including anti-inflammatory, antioxidant, antiangiogenic/vasoprotective or cytoprotective effects. We report compounds produced by several marine source (e.g., sponges, algae, shrimps) that can be administrated as food or with target-specific strategies. In addition, we describe and discuss the uses of opsin family proteins from marine organisms for the optimization of new optogenetic therapeutic strategies.

Neoantigen-based cancer vaccination using chimeric RNA-loaded dendritic cell-derived extracellular vesicles.

Cancer vaccines critically rely on the availability of targetable immunogenic cancer-specific neoepitopes. However, mutation-based immunogenic neoantigens are rare or even non-existent in subgroups of cancer types. To address this issue, we exploited a cancer-specific aberrant transcription-induced chimeric RNA, designated A-Pas chiRNA, as a possible source of clinically relevant and targetable neoantigens. A-Pas chiRNA encodes a recently discovered cancer-specific chimeric protein that comprises full-length astrotactin-2 (ASTN2) C-terminally fused in-frame to the antisense sequence of the 18th intron of pregnancy-associated plasma protein-A (PAPPA). We used extracellular vesicles (EVs) from A-Pas chiRNA-transfected dendritic cells (DCs) to produce the cell-free anticancer vaccine DEXA-P . Treatment of immunocompetent cancer-bearing mice with DEXA-P inhibited tumour growth and prolonged animal survival. In summary, we demonstrate for the first time that cancer-specific transcription-induced chimeric RNAs can be exploited to produce a cell-free cancer vaccine that induces potent CD8+ T cell-mediated anticancer immunity. Our novel approach may be particularly useful for developing cancer vaccines to treat malignancies with low mutational burden or without mutation-based antigens. Moreover, this cell-free anticancer vaccine approach may offer several practical advantages over cell-based vaccines, such as ease of scalability and genetic modifiability as well as enhanced shelf life.

Myriocin Effect on Tvrm4 Retina, an Autosomal Dominant Pattern of Retinitis Pigmentosa.

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP.

Mutation-Independent Therapies for Retinal Diseases: Focus on Gene-Based Approaches.

Gene therapy is proving to be an effective approach to treat or prevent ocular diseases ensuring a targeted, stable, and regulated introduction of exogenous genetic material with therapeutic action. Retinal diseases can be broadly categorized into two groups, namely monogenic and complex (multifactorial) forms. The high genetic heterogeneity of monogenic forms represents a significant limitation to the application of gene-specific therapeutic strategies for a significant fraction of patients. Therefore, mutation-independent therapeutic strategies, acting on common pathways that underly retinal damage, are gaining interest as complementary/alternative approaches for retinal diseases. This review will provide an overview of mutation-independent strategies that rely on the modulation in the retina of key genes regulating such crucial degenerative pathways. In particular, we will describe how gene-based approaches explore the use of neurotrophic factors, microRNAs (miRNAs), genome editing and optogenetics in order to restore/prolong visual function in both outer and inner retinal diseases. We predict that the exploitation of gene delivery procedures applied to mutation/gene independent approaches may provide the answer to the unmet therapeutic need of a large fraction of patients with genetically heterogeneous and complex retinal diseases.