Electrically Induced Angular Momentum Flow between Separated Ferromagnets.

Converting angular momentum between different degrees of freedom within a magnetic material results from a dynamic interplay between electrons, magnons, and phonons. This interplay is pivotal to implementing spintronic device concepts that rely on spin angular momentum transport. We establish a new concept for long-range angular momentum transport that further allows us to address and isolate the magnonic contribution to angular momentum transport in a nanostructured metallic ferromagnet. To this end, we electrically excite and detect spin transport between two parallel and electrically insulated ferromagnetic metal strips on top of a diamagnetic substrate. Charge-to-spin current conversion within the ferromagnetic strip generates electronic spin angular momentum that is transferred to magnons via electron-magnon coupling. We observe a finite angular momentum flow to the second ferromagnetic strip across a diamagnetic substrate over micron distances, which is electrically detected in the second strip by the inverse charge-to-spin current conversion process. We discuss phononic and dipolar interactions as the likely cause to transfer angular momentum between the two strips. Moreover, our Letter provides the experimental basis to separate the electronic and magnonic spin transport and thereby paves the way towards magnonic device concepts that do not rely on magnetic insulators.

Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a BRET-Based Sensor.

Stargardt disease (STGD) leads to blindness in children and young adults. So far, no curative therapy is available and gene augmentation therapies have not yet advanced to the clinics, in part, due to the limited packaging capacity of adeno-associated viruses used to transfer genes into photoreceptor cells. Prime editing offers a new perspective to treat mutations on the genomic level. A nicking variant of Cas9 fused to a reverse transcriptase complex with an elongated guideRNA force intracellular mismatch repair to correct the targeted mutation even in postmitotic cells such as photoreceptors in the eye. Using a custom-made bioluminescence resonance energy transfer (BRET)-based editing sensor in HEK293 cells, we tested 27 different prime editing guide RNAs (pegRNAs) and additional 4 nicking guide RNAs (ngRNAs) with regard to their efficiency to induce sequences changes in exon 43 of the porcine ATP binding cassette subfamily A member 4 (ABCA4) gene that eliminate a mutagenic adenine frameshift insertion, which has been associated with STGD in humans. We identified nine working pegRNAs, and in combination with ngRNAs, we achieved a correction rate of up to ≈92% measured with the BRET-based reporter system. Our data prove the high efficiency of prime editors to correct mutations and highlight the importance of optimal ngRNA design, thus offering a promising editing tool to correct ABCA4 mutations in the disease context.

Characterization of Double-Strand Break Repair Protein Ku80 Location Within the Murine Retina.

Purpose: To characterize the spatial distribution of the DNA-double strand break-repair protein Ku80 in the murine retina. Even though robust data exist on the complexity of DNA repair mechanisms in dividing cells in vitro, almost nothing is known about it in post-mitotic neurons or photoreceptors (PRs). This knowledge is an important prerequisite for in vivo therapeutic approaches by genome editing in retina and PRs. Recently, it was shown that mouse rod PRs are incapable of repairing double-strand breaks induced by radiation. Material and Methods: Retinae from wild-type, rd10, and RPGR-KI mouse lines were obtained and stained with antibodies against Ku80, and cellular markers CtBP2, beta-Dystropglycan, Lamin B, and peanut agglutinin. Organotypic explant cultures were generated and maintained for up to 10 days. Laser microdissection was performed to obtain photoreceptor nuclei, and Ku80 expression was compared to whole retina by real-time PCR (RT-PCR). Results: Strong Ku80 immunoreactivity was observed in rod but not cone photoreceptor terminals localized in the outer plexiform layer of the retina in all three mouse lines. During retinal explant culture, we observed that Ku80-positive globules translocate into the heterochromatin region of nuclei in the outer nuclear layer (ONL). By quantitative PCR, we showed upregulation of relative Ku80 expression in the ONL during wild-type retinal explant culture. Discussion: The unexpected localization of Ku80 to murine rod terminals indicates another tissue-specific modification to the canonical DNA repair mechanisms and warrants further investigation.

A Bioluminescence Resonance Energy Transfer-Based Reporter System: Characterization and Applications.

Genome editing strategies and DNA repair research need powerful analytical tools. We generated a bioluminescence resonance energy transfer (BRET)-based reporter for the quantification of indel frequencies induced by DNA repair. The BRET reporter, expressed as a single molecule, consists of a mutated Renilla reniformis luciferase domain and a GFP2 domain separated by a shuttle-cloning box for the integration of any given endonuclease target sequence. The luciferase activity acts both as energy donor and as the internal standard, while the loss of GFP2 fluorescence acts as a reporter for the out-of-frame sequence alterations that result from the DNA repair via the non-homologous end joining/microhomology-mediated end joining DNA repair pathways of the endonuclease-mediated DNA double-strand break. This results in a decrease of the fluorescence/luminescence ratio. Employing this reporter in different experimental scenarios, using different cell lines and diseases targeted, we quantified the influence of both protein knockdown of DNA repair pathways as well as guide RNA mismatches on CRISPR-mediated nuclease activity and subsequent repair based on mutagenic repair on the reporter. In conclusion, we demonstrated this BRET-based reporter to be a robust and sensitive analytical tool for assessment of variety of different genome editing-based approaches.

Retinal tissue develops an inflammatory reaction to tobacco smoke and electronic cigarette vapor in mice.

Cigarette smoke has been identified as a major risk factor for the development of age-related macular degeneration (AMD). As an alternative to conventional cigarettes (C-cigarette), electronic cigarettes (E-cigarette) have been globally promoted and are currently widely used. The increasing usage of E-cigarettes raises concerns with regard to short- (2 weeks), medium- (3 months), and long- (8 months) term consequences related to retinal tissue. In this report, a controlled study in mouse models was conducted to probe the comprehensive effects of E-cigarette vapor on retina, retinal pigmented epithelium (RPE), and choroidal tissues by (1) comparing the effects of C-cigarette smoke and E-cigarette vapor on retina separately and (2) determining the effects of E-cigarette vapor on the RPE and analyzing the changes with regard to inflammatory (IL-1ß, TNFα, iNOS) and angiogenic (VEGF, PEDF) mediators in retina/RPE/choroid by ELISA assays. The data showed that C-cigarette smoke exposure promoted an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor developed inflammatory and angiogenic reactions more pronounced in RPE and choroid as compared to retinal tissue, while nicotine-containing E-cigarette vapor caused even a more serious reaction. Both inflammatory and pro-angiogenic reactions increased with the extension of exposure time. These results demonstrate that exposure to C-cigarette smoke is harmful to the retina. Likewise, the exposure to E-cigarette vapor (with or without nicotine) increases the occurrence and progression of inflammatory and angiogenic stimuli in the retina, which might also be related to the onset of wet AMD in humans. KEY MESSAGES: C-cigarette smoke exposure promotes an inflammatory reaction in the retina in vivo. Mice exposed to E-cigarette (nicotine-free) vapor develop inflammatory and angiogenic reactions more pronounced in RPE and choroid compared to retinal tissue, while nicotine-containing E-cigarette vapor causes even a more serious reaction. Both inflammatory and pro-angiogenic reactions increase with the extension of E-cigarette vapor exposure time.

Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces.

PURPOSE: To study the efficacy of a novel needle for intravitreal injection (IVI) in comparison to the conventional needle under experimental conditions. METHODS: The newly designed 30-gauge (G) needle (NDN) (EP 18158 542.3, patent pending) with occluded outer orifice and a side port for drug delivery was compared to the conventional standard hypodermic 30 G needle for IVI (SHN). An animal study to obtain needle tip aspirates was performed on 10 albino rat eyes. During IVIs, cellular content, which was cut by the needle tip, was aspirated. Cellular material was studied in regard to cell types and their quantity. The injection stream was studied using trypan blue dye in vitro and pig cadaver eyes. The penetration force was tested on polyurethane Testing Foil Strips PU 04 (Melab, Leonberg, Germany) by applying a velocity of 100 mm/min. The results were analyzed using descriptive statistics, correlation matrices and t-test methods with p<0.05 as statistically significant. RESULTS: Cytological analysis of the needle aspirates showed the presence of cellular content in each case. The amount of conjunctival, ciliary body epithelial cells and granulated basophilic protein sediments (sign of cellular damage) in the case of the NDN tips was significantly lower compared to the SHN. The average penetration force of the NDN was 0.791 N, and in the case of the SHN was 0.566 N. The injection stream study revealed a difference in the initial injection phase between the two needle types, although the diffuse filling of the vitreous area which surrounded the needle tip appeared to be similar. DISCUSSION: The NDN demonstrated superior performance with regard to a significantly reduced number of cells being captured by the needle tip. Delivery of the injected fluid into the vitreous cavity was comparable. In order to investigate superior properties of the NDN needle design, further studies with improved prototypes would be necessary.

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF.

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in vivo Genome Editing.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Detection of the Vascular Endothelial Growth Factor with a Novel Bioluminescence Resonance Energy Transfer Pair Using a Two-Component System.

In this paper we describe a two-component BRET (bioluminescence resonance energy transfer)-based method to detect vascular endothelial growth factor (VEGF) molecules in unknown samples as the basis for subsequent in vivo use. A luminescent VEGF binding molecule, which binds in the receptor binding motif of VEGF, is used as the energy donor, transferred to a fluorophore-coupled VEGF binding molecule (acceptor), which binds to the neuropilin binding motif of VEGF, thus enabling energy transfer from the donor to the acceptor molecule. This leads to the emission of light at a longer wavelength and thus the generation of an increased BRET signal only when VEGF is bound to both the donor and acceptor molecules. We further describe a novel BRET pair that uses the Renilla reniformis mutant luciferase RLuc8 and the chemically engineered fluorophore PerCP-Cy5.5®, which exhibits superior peak separation of approximately 300 nm. The implantation of capsules consisting of the two BRET components in solution, permeable for VEGF for its in vivo detection, would provide a new and improved method for monitoring VEGF-induced pathologies and thus an adjustment of therapy to patient needs.

Functional Characterization of AAV-Expressed Recombinant Anti-VEGF Single-Chain Variable Fragments In Vitro.

PURPOSE: Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy. METHODS: The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02. RESULTS: Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02). CONCLUSION: Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.