Eye on genome editing Du, Samuel W; Palczewski, Krzysztof
The Journal of experimental medicine,
05/2023, Volume:
220, Issue:
5
Journal Article
Peer reviewed
Open access
CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime ...editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.
MicroRNAs are short, evolutionarily conserved noncoding RNAs that are critical for the control of normal cellular physiology. In the retina, photoreceptors are highly specialized neurons that ...transduce light into electrical signals. Photoreceptors, however, are unable to process visual stimuli without the support of the retinal pigment epithelium (RPE). The RPE performs numerous functions to aid the retina, including the generation of visual chromophore and metabolic support. Recent work has underscored how microRNAs enable vision through their contributions to RPE functions. This review focuses on the biogenesis and control of microRNAs in rodents and humans, the roles microRNAs play in RPE function and degeneration, and how microRNAs could serve as potential therapeutics and biomarkers for visual diseases.
The retinal pigment epithelium (RPE) is crucial for maintaining photoreceptor and neural retinal health. Dysregulation of this postmitotic monolayer leads to retinal degeneration in various diseases such as age-related macular degeneration.MicroRNAs have been shown to regulate the gene expression of many vital RPE pathways. These include oxidative stress and metabolism, phagocytosis and endolysosomal function, exosomes and intercellular signaling, and vascular homeostasis.RPE development has been shown to be heavily dependent on the correct expression of microRNAs and their processing machinery, and altered microRNA levels can suppress differentiation or promote dedifferentiation of the RPE.Perturbations in the levels or composition of RPE microRNAs lead to RPE dysfunction and the progression of disease, revealing potential targets for therapeutic and biomarker development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent studies have identified critical roles for B cells in triggering autoimmune germinal centers (GCs) in systemic lupus erythematosus (SLE) and other disorders. The mechanisms whereby B cells ...facilitate loss of T cell tolerance, however, remain incompletely defined. Activated B cells produce interleukin 6 (IL-6), a proinflammatory cytokine that promotes T follicular helper (T
) cell differentiation. Although B cell IL-6 production correlates with disease severity in humoral autoimmunity, whether B cell-derived IL-6 is required to trigger autoimmune GCs has not, to our knowledge, been addressed. Here, we report the unexpected finding that a lack of B cell-derived IL-6 abrogates spontaneous GC formation in mouse SLE, resulting in loss of class-switched autoantibodies and protection from systemic autoimmunity. Mechanistically, B cell IL-6 production was enhanced by IFN-γ, consistent with the critical roles for B cell-intrinsic IFN-γ receptor signals in driving autoimmune GC formation. Together, these findings identify a key mechanism whereby B cells drive autoimmunity via local IL-6 production required for T
differentiation and autoimmune GC formation.
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•Limitations of current gene augmentation therapies need to be addressed.•Advances in CRISPR-Cas9 systems have potential to treat inherited retinal diseases.•Base editing and prime ...editing enable precise mutation corrections in the eye.•Advances in delivery methods have improved safe, effective precision gene editing.•Delivery of ribonucleoproteins or mRNA is ideal for therapeutic CRISPR treatments.
Gene augmentation and genome editing are promising strategies for the treatment of monogenic inherited retinal diseases. Although gene augmentation treatments are commercially available for inherited retinal diseases, there are many shortcomings that need to be addressed, like progressive retinal degeneration and diminishing efficacy over time. Innovative CRISPR-Cas9-based genome editing technologies have broadened the proportion of treatable genetic disorders and can greatly improve or complement treatment outcomes from gene augmentation. Progress in this relatively new field involves the development of therapeutics including gene disruption, ablate-and-replace strategies, and precision gene correction techniques, such as base editing and prime editing. By making direct edits to endogenous DNA, genome editing theoretically guarantees permanent gene correction and long-lasting treatment effects. Improvements to delivery modalities aimed at limiting persistent gene editor activity have displayed an improved safety profile and minimal off-target editing. Continued progress to advance precise gene correction and associated delivery strategies will establish genome editing as the preferred treatment for genetic retinal disorders. This commentary describes the applications, strengths, and drawbacks of conventional gene augmentation approaches, recent advances in precise genome editing in the retina, and promising preclinical strategies to facilitate the use of robust genome editing therapies in human patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, ...resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.
Age‐associated B cells (ABC), a novel subset of activated B cells defined by CD11b and CD11c expression, have been linked with both protective anti‐viral responses and the pathogenesis of systemic ...autoimmunity. Expression of the TH1 lineage transcription factor T‐bet has been identified as a defining feature of ABC biology, with B cell‐intrinsic expression of this transcription factor proposed to be required for ABC formation. In contrast to this model, we report that Tbx21 (encoding T‐bet)‐deficient B cells upregulate CD11b and CD11c surface expression in vitro in response to integrated TLR and cytokine signals. Moreover, B cell‐intrinsic T‐bet deletion in a murine lupus model exerted no impact of ABC generation in vivo, with Tbx21−/− ABCs exhibiting an identical surface phenotype to wild‐type (WT) ABCs. Importantly, WT and Tbx21−/− ABCs sorted from autoimmune mice produced equivalent amounts of IgM and IgG ex vivo following TLR stimulation, indicating that T‐bet‐deficient ABCs are likely functional in vivo. In summary, our data contradict the established literature by demonstrating that T‐bet expression is not uniformly required for ABC generation.
Integrated B cell receptor, Toll‐like receptor, co‐stimulatory and cytokine signals promote “age‐associated B cells” (ABCs) generation during autoimmunity. Surprisingly, ABCs can form without B cell T‐bet (Tbx21). Functionally, both Tbx21+/+ and Tbx21−/− ABCs produced autoantibodies ex vivo after TLR7 stimulation, with the impact of T‐bet limited to IgG2c class‐switched recombination.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The B cell survival cytokine BAFF has been linked with the pathogenesis of systemic lupus erythematosus (SLE). BAFF binds distinct BAFF-family surface receptors, including the BAFF-R and ...transmembrane activator and CAML interactor (TACI). Although originally characterized as a negative regulator of B cell activation, TACI signals are critical for class-switched autoantibody (autoAb) production in BAFF transgenic mice. Consistent with this finding, a subset of transitional splenic B cells upregulate surface TACI expression and contribute to BAFF-driven autoAb. In the current study, we interrogated the B cell signals required for transitional B cell TACI expression and Ab production. Surprisingly, despite established roles for dual BCR and TLR signals in autoAb production in SLE, signals downstream of these receptors exerted distinct impacts on transitional B cell TACI expression and autoAb titers. Whereas loss of BCR signals prevented transitional B cell TACI expression and resulted in loss of serum autoAb across all Ig isotypes, lack of TLR signals exerted a more limited impact restricted to autoAb class-switch recombination without altering transitional B cell TACI expression. Finally, in parallel with the protective effect of TACI deletion, loss of BAFF-R signaling also protected against BAFF-driven autoimmunity. Together, these findings highlight how multiple signaling pathways integrate to promote class-switched autoAb production by transitional B cells, events that likely impact the pathogenesis of SLE and other BAFF-dependent autoimmune diseases.
MicroRNAs (miRs) are short, evolutionarily conserved noncoding RNAs that canonically downregulate expression of target genes. The miR family composed of miR-204 and miR-211 is among the most highly ...expressed miRs in the retinal pigment epithelium (RPE) in both mouse and human and also retains high sequence identity. To assess the role of this miR family in the developed mouse eye, we generated two floxed conditional KO mouse lines crossed to the RPE65-ERT2-Cre driver mouse line to perform an RPE-specific conditional KO of this miR family in adult mice. After Cre-mediated deletion, we observed retinal structural changes by optical coherence tomography; dysfunction and loss of photoreceptors by retinal imaging; and retinal inflammation marked by subretinal infiltration of immune cells by imaging and immunostaining. Single-cell RNA sequencing of diseased RPE and retinas showed potential miR-regulated target genes, as well as changes in noncoding RNAs in the RPE, rod photoreceptors, and Müller glia. This work thus highlights the role of miR-204 and miR-211 in maintaining RPE function and how the loss of miRs in the RPE exerts effects on the neural retina, leading to inflammation and retinal degeneration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Compact ellipticals (cEs) are outliers from the scaling relations of early-type galaxies, particularly the mass-metallicity relation, which is an important outcome of feedback. The formation of such ...low-mass, but metal-rich and compact, objects is a long-standing puzzle. Using a pair of high-resolution N-body+gas simulations, we investigate the evolution of a gas-rich low-mass galaxy on a highly radial orbit around a massive host galaxy. As the infalling low-mass galaxy passes through the host's corona at supersonic speeds, its diffuse gas outskirts are stripped by ram pressure, as expected. However, the compactness increases rapidly because of bursty star formation in the gas tidally driven to the center. The metal-rich gas produced by supernovae and stellar winds is confined by the ram pressure from the surrounding environment, leading to subsequent generations of stars being more metal-rich. After the gas is depleted, tidal interactions enhance the metallicity further via the stripping of weakly bound, old, and metal-poor stars, while the size of the satellite is changed only modestly. The outcome is a metal-rich cE that is consistent with observations. These results argue that classical cEs are neither the stripped remnants of much more massive galaxies nor the merger remnants of normal dwarfs. We present observable predictions that can be used to test our model.
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