The type of DNA damage response after decitabine treatment depends on the level of DNMT activity.

Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.

Deep Learning-Based Retinal Layer Segmentation in Optical Coherence Tomography Scans of Patients with Inherited Retinal Diseases.

BACKGROUND: In optical coherence tomography (OCT) scans of patients with inherited retinal diseases (IRDs), the measurement of the thickness of the outer nuclear layer (ONL) has been well established as a surrogate marker for photoreceptor preservation. Current automatic segmentation tools fail in OCT segmentation in IRDs, and manual segmentation is time-consuming. METHODS AND MATERIAL: Patients with IRD and an available OCT scan were screened for the present study. Additionally, OCT scans of patients without retinal disease were included to provide training data for artificial intelligence (AI). We trained a U-net-based model on healthy patients and applied a domain adaption technique to the IRD patients' scans. RESULTS: We established an AI-based image segmentation algorithm that reliably segments the ONL in OCT scans of IRD patients. In a test dataset, the dice score of the algorithm was 98.7%. Furthermore, we generated thickness maps of the full retinal thickness and the ONL layer for each patient. CONCLUSION: Accurate segmentation of anatomical layers on OCT scans plays a crucial role for predictive models linking retinal structure to visual function. Our algorithm for segmentation of OCT images could provide the basis for further studies on IRDs.

Genome length determination in adeno-associated virus vectors with mass photometry.

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the adeno-associated virus (AAV) capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption, and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (coefficient of variation [%CV] < 2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV < 2.9%) considering calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with precision of %CV < 0.7% and accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their calibration materials and efforts.

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

[Gene therapy in ophthalmology]. / Gentherapie in der Augenheilkunde.

In 2017 the gene therapy medication voretigene neparvovec-rzyl was approved by the U.S. Food and Drug Administration (FDA) for retinal gene therapy of hereditary retinal dystrophies caused by mutations in the RPE65 gene. Voretigene neparvovec-rzyl is a gene augmentation therapy using an  adeno-associated virus-based vector to express a healthy copy of the human RPE65 gene in the patient's retinal pigment epithelial (RPE) cells. The success of gene augmentation therapy in RPE65-linked retinal dystrophy encouraged research activities on the concept of gene supplementation to be extended to nongenetic diseases, such as age-related macular degeneration; however, it also showed that the principle of success cannot be easily extended to other retinal dystrophies. This review article presents the most commonly used principles and technologies of gene therapy and provides an overview of the current challenges and limitations. Furthermore, practice-relevant aspects of the indications and the treatment procedure are discussed. Particular attention is paid to the consideration of disease stages, especially with respect to patient's expectations and the evaluation of treatment success.

Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

CNG channel-related retinitis pigmentosa.

The genes CNGA1 and CNGB1 encode the alpha and beta subunits of the rod CNG channel, a ligand-gated cation channel whose activity is controlled by cyclic guanosine monophosphate (cGMP). Autosomal inherited mutations in either of the genes lead to a progressive rod-cone retinopathy known as retinitis pigmentosa (RP). The rod CNG channel is expressed in the plasma membrane of the outer segment and functions as a molecular switch that converts light-mediated changes in cGMP into a voltage and Ca2+ signal. Here, we will first review the molecular properties and physiological role of the rod CNG channel and then discuss the characteristics of CNG-related RP. Finally, we will summarize recent activities in the field of gene therapy aimed at developing therapies for CNG-related RP.

Biology, Pathobiology and Gene Therapy of CNG Channel-Related Retinopathies.

The visual process begins with the absorption of photons by photopigments of cone and rod photoreceptors in the retina. In this process, the signal is first amplified by a cyclic guanosine monophosphate (cGMP)-based signaling cascade and then converted into an electrical signal by cyclic nucleotide-gated (CNG) channels. CNG channels are purely ligand-gated channels whose activity can be controlled by cGMP, which induces a depolarizing Na+/Ca2+ current upon binding to the channel. Structurally, CNG channels belong to the superfamily of pore-loop cation channels and share structural similarities with hyperpolarization-activated cyclic nucleotide (HCN) and voltage-gated potassium (KCN) channels. Cone and rod photoreceptors express distinct CNG channels encoded by homologous genes. Mutations in the genes encoding the rod CNG channel (CNGA1 and CNGB1) result in retinitis-pigmentosa-type blindness. Mutations in the genes encoding the cone CNG channel (CNGA3 and CNGB3) lead to achromatopsia. Here, we review the molecular properties of CNG channels and describe their physiological and pathophysiological roles in the retina. Moreover, we summarize recent activities in the field of gene therapy aimed at developing the first gene therapies for CNG channelopathies.

More than meets the eye: The role of microglia in healthy and diseased retina.

Microglia are the main resident immune cells of the nervous system and as such they are involved in multiple roles ranging from tissue homeostasis to response to insults and circuit refinement. While most knowledge about microglia comes from brain studies, some mechanisms have been confirmed for microglia cells in the retina, the light-sensing compartment of the eye responsible for initial processing of visual information. However, several key pieces of this puzzle are still unaccounted for, as the characterization of retinal microglia has long been hindered by the reduced population size within the retina as well as the previous lack of technologies enabling single-cell analyses. Accumulating evidence indicates that the same cell type may harbor a high degree of transcriptional, morphological and functional differences depending on its location within the central nervous system. Thus, studying the roles and signatures adopted specifically by microglia in the retina has become increasingly important. Here, we review the current understanding of retinal microglia cells in physiology and in disease, with particular emphasis on newly discovered mechanisms and future research directions.

Understanding opalescence measurements of biologics - A comparison study of methods, standards, and molecules.

Opalescence measurements are broadly applied to assess the quality and stability of biopharmaceutical products at all stages of development and manufacturing. They appear to be simple and straight forward but detect complex light scattering phenomena. Despite a routine calibration step, opalescence values obtained with the same biopharmaceutical sample but on different instruments and/or with different methods may vary significantly. Since the reasons for this high variability are generally not well understood, comparison of opalescence results from different biopharmaceutical laboratories is difficult. Here, we characterized a comprehensive set of biopharmaceutically relevant samples with five opalescence methods to illustrate fundamental differences in method performance and explore the reasons for poor comparability. In addition, we developed a high-throughput method for measuring opalescence in a conventional light scattering plate reader that yields opalescence values in the same range as compendial methods. The presented results underline the impact of sample properties, instrument type, and calibration standards on the determined opalescence value. Based on our findings we provide recommendations for the appropriate application of each method during biopharmaceutical drug product development. Overall, our study contributes to an improved understanding of opalescence measurements in the biopharmaceutical field.

Mutations within the cGMP-binding domain of CNGA1 causing autosomal recessive retinitis pigmentosa in human and animal model.

Retinitis pigmentosa is a group of progressive inherited retinal dystrophies that may present clinically as part of a syndromic entity or as an isolated (nonsyndromic) manifestation. In an Indian family suffering from retinitis pigmentosa, we identified a missense variation in CNGA1 affecting the cyclic nucleotide binding domain (CNBD) and characterized a mouse model developed with mutated CNBD. A gene panel analysis comprising 105 known RP genes was used to analyze a family with autosomal-recessive retinitis pigmentosa (arRP) and revealed that CNGA1 was affected. From sperm samples of ENU mutagenesis derived F1 mice, we re-derived a mutant with a Cnga1 mutation. Homozygous mutant mice, developing retinal degeneration, were examined for morphological and functional consequences of the mutation. In the family, we identified a rare CNGA1 variant (NM_001379270.1) c.1525 G > A; (p.Gly509Arg), which co-segregated among the affected family members. Homozygous Cnga1 mice harboring a (ENSMUST00000087213.12) c.1526 A > G (p.Tyr509Cys) mutation showed progressive degeneration in the retinal photoreceptors from 8 weeks on. This study supports a role for CNGA1 as a disease gene for arRP and provides new insights on the pathobiology of cGMP-binding domain mutations in CNGA1-RP.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

The protein biosynthesis inhibitor vioprolide A evokes anti-angiogenic and pro-survival actions by targeting NOP14 and decreasing VEGF receptor 2- and TAZ-signaling.

Angiogenesis contributes to the progression of several diseases including cancer or age-related macular degeneration and is crucially driven by pathologically hyperactive endothelial cells (ECs). Targeting angiogenic processes in ECs thus represents a promising strategy to treat these conditions. Vioprolide A (vioA) is a myxobacterial cyclic depsipeptide that targets the nucleolar protein 14 (NOP14) and possesses strong anti-cancer and anti-inflammatory actions. Here, we present evidence that vioA promotes anti-angiogenic actions in vivo and in ECs in vitro. VioA reduced the choroidal neovascularization after laser-induced photocoagulation in mice in vivo, the sprouting of choroidal explant cultures ex vivo and key angiogenic features of ECs in vitro. Mechanistically, vioA decreased VEGFR2 protein levels and phosphorylation leading to impaired downstream pro-angiogenic signaling. Concurrently, vioA influenced TAZ signaling by diminishing its nuclear translocation and protein level, resulting in a reduced expression of pro-angiogenic target genes and dynamic cytoskeletal remodeling. Surprisingly, vioA induced pro-survival signaling in ECs by activating Akt and inhibiting p53-dependent apoptosis. Knockdown of the cellular target NOP14 further revealed a partial involvement in the anti-angiogenic and pro-survival actions of vioA. Taken together, our study introduces vioA as an interesting anti-angiogenic compound that warrants further investigations in preclinical studies.

Immunogenicity of Novel AAV Capsids for Retinal Gene Therapy.

OBJECTIVES: AAV vectors are widely used in gene therapy, but the prevalence of neutralizing antibodies raised against AAV serotypes in the course of a natural infection, as well as innate and adaptive immune responses induced upon vector administration, is still considered an important limitation. In ocular gene therapy, vectors applied subretinally bear the risk of retinal detachment or vascular leakage. Therefore, new AAV vectors that are suitable for intravitreal administration for photoreceptor transduction were developed. METHODS: Here, we compared human immune responses from donors with suspected previous AAV2 infections to the new vectors AAV2.GL and AAV2.NN-two capsid peptide display variants with an enhanced tropism for photoreceptors-with the parental serotype AAV2 (AAV2 WT). We investigated total and neutralizing antibodies, adaptive and innate cellular immunogenicity determined by immunofluorescence staining and flow cytometry, and cytokine secretion analyzed with multiplex beads. RESULTS: While we did not observe obvious differences in overall antibody binding, variants-particularly AAV2.GL-were less sensitive to neutralizing antibodies than the AAV2 WT. The novel variants did not differ from AAV2 WT in cellular immune responses and cytokine production in vitro. CONCLUSION: Due to their enhanced retinal tropism, which allows for dose reduction, the new vector variants are likely to be less immunogenic for gene therapy than the parental AAV2 vector.

A Bioengineered In Vitro Model to Assess AAV-Based Gene Therapies for Cyclic GMP-Related Disorders.

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy CNGA3 gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function.

Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1-/- double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1-/- double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Systematic Studies on Stabilization of AAV Vector Formulations by Lyophilization.

Adeno-associated virus (AAV) vectors have evolved as one of the most promising delivery systems for gene therapy. The current standard for AAV vector storage is deep-freezing below -60 °C. While this allows for long-term vector storage without loss of activity, it is inconvenient and involves high costs and logistical challenges. Therefore, there is a need for AAV formulations, such as freeze-dried formulations, that allow for long-term storage at 2-8 °C. A major challenge in developing a lyophilization process for complex biological structures like an AAV vector is to minimize the stress on the capsid during the lyophilization cycle. Here, we evaluated different conditions for freeze-drying of AAV8 vectors and found that undesirable instability can be significantly reduced if secondary drying is performed at lower temperatures, kept as short as possible, and the residual moisture is kept between 1.5 and 2%. In a next step, we explored formulations with different salt concentration or excipient compositions and found that a combination of 10 mM phosphate buffer, 5.67% (150 mM) trehalose, 5% hydroxyectoine and 0.1% poloxamer with a residual moisture of approx. 1.5% provided stable long-term storage at 2-8 °C and for at least 4 weeks at 25 °C. These results pave the way for future optimizations of freeze-drying processes for AAV vector-based gene therapy products.

dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy.

Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent. Here, we describe a protocol for the design, execution and evaluation of such gene therapies using dCas9-VPR. We offer guidelines on how to identify functionally equivalent genes, design and clone single guide RNAs and evaluate transcriptional activation in vitro. Moreover, focusing on inherited retinal diseases, we provide a detailed protocol on how to apply this strategy in mice using dual recombinant adeno-associated virus vectors and how to evaluate its functionality and off-target effects in the target tissue. This strategy is in principle applicable to all organisms that possess functionally equivalent genes suitable for transcriptional activation and addresses pivotal unmet needs in gene therapy with high translational potential. The protocol can be completed in 15-20 weeks.

Achromatopsia: Genetics and Gene Therapy.

Achromatopsia (ACHM), also known as rod monochromatism or total color blindness, is an autosomal recessively inherited retinal disorder that affects the cones of the retina, the type of photoreceptors responsible for high-acuity daylight vision. ACHM is caused by pathogenic variants in one of six cone photoreceptor-expressed genes. These mutations result in a functional loss and a slow progressive degeneration of cone photoreceptors. The loss of cone photoreceptor function manifests at birth or early in childhood and results in decreased visual acuity, lack of color discrimination, abnormal intolerance to light (photophobia), and rapid involuntary eye movement (nystagmus). Up to 90% of patients with ACHM carry mutations in CNGA3 or CNGB3, which are the genes encoding the alpha and beta subunits of the cone cyclic nucleotide-gated (CNG) channel, respectively. No authorized therapy for ACHM exists, but research activities have intensified over the past decade and have led to several preclinical gene therapy studies that have shown functional and morphological improvements in animal models of ACHM. These encouraging preclinical data helped advance multiple gene therapy programs for CNGA3- and CNGB3-linked ACHM into the clinical phase. Here, we provide an overview of the genetic and molecular basis of ACHM, summarize the gene therapy-related research activities, and provide an outlook for their clinical application.