Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn.To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background.We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections.The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue.in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.We successfully developed a novel PLA method that can, in the future, be used on  Synucleinopathies are pathologically characterized by the presence of α-synuclein (asyn)-positive protein inclusions in the brain called Lewy bodies (LBs) and Lewy neurites . Prior w review, ).Given the strong ties between pS129 asyn and the pathology of synucleinopathies, it is critical to have tools that specifically detect physiological pS129 asyn. Elucidating the physiologically relevant function of this post-translational modification could allow us to further understand its role in asyn aggregation. Previously developed monoclonal antibodies raised against the pS129 asyn epitope preferentially detect pS129 asyn over non-phosphorylated asyn and readily detect aggregated pS129 asyn but not the soluble form. It has been challenging to limit the cross-reactivity of developed pS129 asyn monoclonal antibodies with other proteins that have similar phosphorylated epitopes, such as neurofilament light chain . The sigProximity ligation assay (PLA) is a staining method that creates a robust signal when two primary antibodies are within an estimated range of <40 nm . This ra∧6 cells/ml. 1.0×10∧6 cells were added to each well in a 24-well plate with poly-D-lysine - Laminin coated coverslips. The 24-well plate was then placed in a 37°C and 5% CO2 incubator. After 24 h, 80% of BME + 5% FBS was removed and replaced with fresh BME containing 2.5μM Cytosine Arabinoside. Day In Vitro (DIV) 6 primary neurons were treated with 1μl adeno-associated virus (AAV6) expressing human asyn or mouse asyn under the synapsin 1 promoter (7×10∧13 vg/ml) for 4 h. Cultures were not kept past DIV 9. Primary neurons were fixed by addition of 4 % PFA for 30 min, washed and stored in 1x PBS at 4°C.Postnatal day 0 WT or SNCA KO cortices were dissected in ice cold HBSS. The cortices were then incubated in papain for 30 min at 37°C. After incubation, cortices were washed twice with BME  with centrifugations at 1000 rpm for 3 min. Tissue was triturated in BME containing 50μg/ml of DNAse and then washed twice in BME with centrifugations at 1000 rpm for 6 min. Cells were diluted with BME + 5% fetal bovine serum (FBS) to a concentration of 1.0×10HeLa cells were grown in DMEM (Gibco) + 10% FBS (Gibco) and plated on matrigel (Corning) coated glass coverslips at 30,000 cells/well of a 24 well plate. Cells were treated with polo-like kinase (PLK) inhibitor BI2536 at a final concentration of 1μM. DMSO was used as a control. After 4 h, cells were fixed by addition of 4 % PFA for 30 min, washed and stored in 1x PBS at 4°C.tm1nbm (SNCA KO) . A Next, we evaluated the specificity of the pS129 asyn monoclonal antibodies MJF-R13, pSyn#64, 81A, and EP1536Y (abcam) using immunohistochemistry (IHC) . IHC wasWe next aimed to develop an assay to detect pS129 asyn that would work equally well in rodent and human tissues. Many asyn antibodies that are commercially available recognize the C-terminus of asyn, and typically these antibodies do not recognize both human and rodent asyn with the same affinity due to sequence differences in this region . The synWe then evaluated the brightfield version of the PLA (BF-PLA) utilizing DAB to stain for physiological pS129 asyn on PFA fixed brain tissue . pS129 aNext, we evaluated the ability of the BF-PLA to stain for physiological pS129 asyn in additional major brain areas in the WT mouse brain . A punctate staining was noted throughout the stained mouse brain sections. In the prefrontal cortex (PFC), similar to the neocortex, the staining was not only seen throughout  but was particularly strong in layer IV, likely due to staining of neuronal cell bodies . As expected, the pS129 asyn staining was homogenous throughout the striatum and there was no obvious staining of cell bodies . The hippocampus was mainly stained in the CA3 region (stratum lucidum) with possible intense labeling of cell bodies . Additional somal staining was visible especially in the paraventricular nucleus of the thalamus , as well as in the amygdala . Even though asyn is well known to be highly expressed in dopaminergic neurons in the substantia nigra and total staining can be visible in their cell bodies , we did Since pS129 asyn is often used as a marker of asyn pathology, we also wanted to validate our BF-PLA staining of pS129 asyn in relevant animal models . DetectiNext, we evaluated the use of BF-PLA to detect non-aggregated and aggregated pS129 asyn of LBs in human brain tissue. Brain tissues from neurologically unimpaired controls and patients with DLB were acquired from the Alzheimer Disease Research Center (ADRC) at the University of California, San Diego (UCSD). In both the control and DLB human brain tissue, BF-PLA showed extensive pS129 asyn staining in frontal cortical areas . InteresHere, we describe a PLA protocol that specifically and sensitively detects endogenous and physiologically relevant pS129 asyn in cell cultures, mouse, and human brain tissue. For the first time, we show here the localization and distribution of non-aggregated pS129 asyn in brains that have no pathological attributes present. We additionally show that this PLA can be used to detect increased levels of pS129 asyn in primary cell cultures and mouse models overexpressing asyn, as well as in PFF injected mice. Moreover, we show that a reduction in normal pS129 asyn levels by means of kinase inhibitor can be readily picked up which is further evidence that our assay produces a specific pS129 asyn signal. Therefore, we believe that this PLA method is a great tool to answer some of the major questions still surrounding pS129 asyn biology. Understanding the physiological function of pS129 asyn could help us discern its role in aggregation and pathology. For example, this PLA staining method could be used in combination with other cell organelle markers, such as TGN38 (trans-Golgi network) or Lamp1 (lysosomes), to identify the localization of pS129 asyn, which could suggest a possible function. Furthermore, several co-expression experiments or drug screens could be performed in combination with this method to understand when and where asyn is phosphorylated. Lastly, one could also investigate how the location of pS129 asyn may change over time, such as changes with age, which is a fundamental risk factor for synucleinopathies.® PLA kit, we noticed that staining variations were seen between different batches of kits. We believe this may be in part due to the low-temperature activated Ligase and Polymerase enzymes used during the protocol, which might make them more vulnerable at room temperature. Therefore, special care needs to be taken when using these enzymes. Vials should always be kept in cryo containers and only as short as possible at room temperature. Additional staining differences were seen depending on the experimental setup. When staining 30μm thick brain sections with the BF-PLA, only a thin upper layer (≈5μm) of the section was successfully stained, especially if the section was already mounted before staining and not free floating. Sections were mostly stained already mounted to glass slides to reduce the amount of reagents needed. Better penetration and staining was seen for 6μm thick ethanol-fixed paraffin embedded sections. These penetration issues could come from the antibodies or enzymes not being able to successfully penetrate deep into the sections. Further optimization could be done by using varying amounts of detergent, different fixative, or antigen retrieval to improve staining thickness. While the protocol for our pS129 asyn PLA is more time intensive and expensive compared to traditional ICC or IHC and uses two antibodies produced in mouse and rabbit, it is still possible to co-stain other antigens on the same tissue. In brain sections stained with BF-PLA, immuno-alkaline phosphatases and fast blue can be used as long as the primary antibody is not produced in mice or rabbits. Likewise, F-PLA stainings on sections or cultured cells only require one fluorophore and thus other antigens can be stained if the antibodies are not hosted in mouse or rabbit. Additionally, both F-PLA and BF-PLA are easily interchangeable as the protocols only differ by an additional 2 steps at the end for the BF-PLA (see Method section). The 1st step adds a peroxidase instead of a fluorophore to the amplicon while the second uses DAB to visualize the staining. Therefore, brain sections can easily be stained either with DAB or a fluorophore, making this assay a very versatile method.While using the Sigma-Aldrich Duolinkin vivo without major cross-reactivity of the monoclonal antibodies. Interestingly, in the mouse brain, pS129 asyn can be located as a general signal in most parts of the brain but also as more dense staining specifically in some, but not all, cell bodies. This could either suggest a different function of pS129 asyn in distinct neurons or that phosphorylation of asyn is more prominent in cells that highly express the relevant kinases. It is worth noting that the localization of these somal stainings of pS129 asyn in the various subregions do not necessarily overlap with the localization of pathology seen in synucleinopathies [Using this novel PLA method, we show here the distribution of pS129 asyn throughout the mouse brain and its cellular localization opathies . In the opathies . We alsoOur PLA using R13 / syn-1 was not able to detect pathologically aggregated pS129 asyn in human brain tissue from DLB patients. As the assay does stain accumulated pS129 asyn in the PFF animal model, we hypothesize that this may be due to the chronically aggregated, dense LBs having a different structure or different post-translational modifications when compared to the short-term aggregated asyn in animal models. The syn-1 antibody binds near the 90th amino acid on asyn, which is part of the motif involved in building the β-sheet structure in asyn aggregates  and inclOverall, our pS129 asyn PLA with the R13 and syn-1 monoclonal antibodies shows higher specificity than previous stainings with pS129 asyn antibodies alone and is also capable of detecting the low physiological levels of pS129 asyn in cell culture, mouse, and human tissue.in vitro and in vivo, our PLA can be utilized to answer major questions still surrounding pS129 asyn biology and its role in the pathology of synucleinopathies.The PLA described here addresses the issue of cross-reactivity of pS129 asyn monoclonal antibodies by establishing a specific signal for pS129 asyn with minimal background signal. We show how this PLA protocol can be used to specifically detect physiological, non-aggregated pS129 asyn in cell culture, wild-type mouse brain tissue, and human brain tissue. Furthermore, we demonstrate its use in cell cultures and mouse models to detect increased levels of pS129 asyn. While soluble pS129 asyn was readily detected in human brain tissue, LBs were not stained by our PLA protocol in brain tissue from DLB patients. Due to its diverse utility in specifically detecting endogenous and physiologically relevant pS129 asyn Click here for additional data file.


N = 50) and liver . In Study 1, subjects were first divided equally using a mental rotations test (MRT) into low and high spatial ability groups. Participants memorised a 2D-labeled heart valve diagram and sketched it rotated 180°, before self-reporting their intrinsic cognitive load (ICL). For Study 2, participants studied a liver PR3DM or its corresponding A3DM with texture-homogenisation, followed by a liver anatomy post-test, and reported extraneous cognitive load (ECL). All participants reported no prior anatomy experience. Participants with low spatial ability (N = 25) had significantly lower heart drawing scores (p = 0.001) than those with high spatial ability (N = 25), despite no significant differences in reported ICL (p = 0.110). Males had significantly higher MRT scores than females (p = 0.011). Participants who studied the liver A3DM (N = 22) had significantly higher post-test scores than those who studied the liver PR3DM (N = 24) (p = 0.042), despite no significant differences in reported ECL (p = 0.720). This investigation demonstrated that increased spatial ability and colour-coding of 3D models are associated with improved anatomy performance without significant increase in cognitive load. The findings are important and provide useful insight into the influence of spatial ability and photorealistic and artistic 3D models on anatomy education, and their applicability to instructional and assessment design in anatomy.Photorealistic 3D models (PR3DM) have great potential to supplement anatomy education; however, there is evidence that realism can increase cognitive load and negatively impact anatomy learning, particularly in students with decreased spatial ability. These differing viewpoints have resulted in difficulty in incorporating PR3DM when designing anatomy courses. To determine the effects of spatial ability on anatomy learning and reported intrinsic cognitive load using a drawing assessment, and of PR3DM versus an Artistic colour-coded 3D model (A3DM) on extraneous cognitive load and learning performance. First-year medical students participated in a cross-sectional (Study 1) and a double-blind randomised control trial (Study 2). Pre-tests analysed participants' knowledge of anatomy of the heart (Study 1,  However, there is an increasing demand for alternatives teaching methods due to financial, emotional, safety, and practical challenges associated with the use of cadavers4. While technology has given rise to new methods, these must be evaluated for their effectiveness as realistic supplements to anatomy teaching and learning.Anatomy has traditionally been delivered and conducted through cadaveric dissections5. Their increased accessibility and improved user-interaction6 has allowed for increased performance in tasks involving spatial ability (SA) and structural recognition9. However, cost and decreased authenticity remain key disadvantages in terms of applicability and reproducibility6.One such method is using three-dimensional (3D) models, which includes both digital and physical models, which can be manipulated spatially to enhance visualisation of structures and their relationships11. Hence, photorealistic 3D models can be defined as digital replicas of real-world objects that look the same as their physical counterparts from a human's point of view10. Photogrammetry’s advantages over other 3D models are that they can be rotated, altered digitally, and viewed from different perspectives6.An example of a 3D anatomy model is a photorealistic one produced via photogrammetry. Photorealistic 3D models (PR3DMs) are produced by taking a series of pictures from different angles to gather spatial information about an object13. Perception of realistic objects has been explained through geon theory, where primitive 3D shapes are geons, perceptual units used in segmenting and processing visualisations15. Thus, the perception of 3D objects may be hindered by complex, realistic surfaces that have more geons. Several studies have reported higher cognitive load and worse learning performances in realism due to higher perceptual complexity16, as extraneous details impair both learning and transfer of information17. However, there was a report of the paradoxical effect of realism, explained through the disfluency theory that realism better captures learners’ attention and aids information retrieval, improving learning performance19.There is conflicting evidence about the effects of PR3DMs in anatomy20. The overall cognitive load of a visualisation is defined as the summation of ICL and ECL21. Visual signalling can minimise ECL of photorealistic visualisations24, assisting students by modelling more effective search strategies and directing learners to crucial elements27. These cues may be additive  or non-additive 28. Colour-coding is an established signalling strategy that has been repeatedly shown to positively-affect learning, including in information retention, transfer and matching31, while minimising ECL33. Lokka and Çöltekin had shown the benefits of combining realism with abstraction on recall accuracy and learning performance34, and a similar method was suggested to be applied to anatomy education14. However, there is limited information regarding effects of colour coding PR3DMs on anatomy learning performance and ECL. To our knowledge, there is only one study reporting the effect of adding the signalling effect of colour-coding to PR3DMs on learning performance, but it was not based on a structure that exists in human anatomy14.The inherent difficulty of a subject contributes to intrinsic cognitive load (ICL) while the nature of content presentation that is designed counterproductively imposes extraneous cognitive load (ECL)35, and its effects on anatomy learning have been widely studied. Several studies have shown positive correlations between SA and anatomy knowledge, hypothesised to be because anatomy requires firm comprehension of 3D relationships between anatomical structures, especially when viewed from different perspectives38. Spatial ability is also important for one to produce mental 3D images based on the assimilation and synthesis of two-dimensional imagery39. However, very few studies involved drawing as a measure of post-intervention knowledge, a method that provides comprehensive insight into learners’ mental 3D-representation of structures40, of which only one was about human anatomy41 and another related to cross-sectional anatomy of the dog42. Thus, there remains lack of guidance and consensus for the design of anatomy courses based on SA.Spatial ability (SA), the ability to retain, generate, retrieve and transform well-structured visualisationsThis study aimed to investigate how spatial ability affects anatomy learning using drawing assessment, and the effects of colour coded PR3DMs on extraneous cognitive load and learning performance. We hypothesised that (1) learners with high spatial ability would have better anatomy performance in drawing assessment, (2) colour coded PR3DM  of the liver will decrease ECL, and (3) learning with A3DM models would results in better anatomy performance. Gaining a deeper understanding about these will hopefully aid in the design of tools and curriculum for anatomy teaching and learning, including possible customisation based on individual needs and abilities.A cross-sectional study Study 1) followed 3 days later by a double-blinded randomised control trial (Study 2) with pre- and post-testing was designed to compare student’s learning performance and ICL/ECL. Experimental design and flow of activities of studies one and two is detailed in Fig.  followedEthical approval was obtained from the Nanyang Technological University Institute Review Board (IRB-2022-476). Study methods were performed in accordance with approved guidelines and regulations. Participation in the study was voluntary and would neither replace the existing cardiac and liver anatomy curriculum nor impact on formal assessment for the participants. Signed informed consent was obtained from participants prior to enrolment into the study. Confidentiality was ensured by de-identification of participants’ names during data collection. Singapore Dollar $10 vouchers were provided as tokens of appreciation.13.The study preceded formal teaching on cardiac and liver anatomy. The study was only publicised in the week leading up to the study through class announcement and email. Study 1 was conducted on 15 August 2022 and Study 2 was conducted on 18 August 2022.Participants were recruited from the first-year undergraduate medical student cohort of the Lee Kong Chian School of Medicine in Singapore. Fifty participants were recruited based on a similar power calculation to that used in the study by Skulmowski and Rey43. Responses were scored out of 48. Participants were sorted into equal groups of high or low SA by a median split depending on their relative scores upon completion of the MRT45.Participants completed the redrawn Vandenberg & Kuse (1978) mental rotations test (MRT), following protocols outlined by Peters et al.Pre-tests Appendix  were desStudy 1: Participants had three minutes to memorise a 2D-labelled diagram of the superior view of heart valves . The resources were developed in-house via photogrammetry of plastinated liver specimen. The photographs of plastinated liver specimens  were taken at every 5 degrees of rotation using an automated turntable. This was performed 4–5 times at different angles to capture all the specimen's details. The best 100 photos  were stitched together using Autodesk Recap Photo  to generate a 3D model. In Autodesk Maya , the 3D model was refined, and a texture map was applied to generate PR3DM. In A3DM, textures were homogenised and structures colour-coded using Blender 13. Since the model in this study required rotation and touch to reveal labels, three minutes (180 s) were given to study the model. Participants then answered a 10-question  post-test, with diagrams from their respective models  on a 7-point Likert scale, and ECL questions for study 2Appendix . This cop < 0.05 was statistically significant.Means and standard deviations were reported for normally distributed continuous variables, median and interquartile range for non-normal variables, and frequencies and percentages for categorical variables. Student’s t-test was used to compare normally distributed continuous variables, Mann–Whitney U test for non-normal variables, and chi-squared/Fisher’s exact test for categorical variables as appropriate. Pearson correlation was conducted for the relationship between MRT scores and drawing assessment scores. Cronbach Fifty participants were recruited, and demographics are detailed in Table p = 0.110) and difficulty of heart drawing (p = 0.187) between low and high SA groups. However, low SA participants reported significantly higher difficulty in remembering names of valves and cusps (p = 0.038). The low SA group scored significantly lower for overall heart drawing , and for individual valves. MRT scores were moderately correlated with heart drawing scores .The results from Study 1 are detailed in Table p = 0.720) between PR3DM and A3DM groups. The A3DM group had significantly higher liver post-test scores than the PR3DM group .The results from Study 2 are detailed in Table A stratified random sample of 19 males was obtained for comparison between sexes, to account for the large difference in numbers of female and male participants. There were significantly more males in the high SA group in both the original population and stratified sample, detailed in Table p = 0.023). Males also had significantly higher MRT scores than females . Otherwise, there were no significant differences in heart drawing scores (p = 0.254) and ICL (p = 0.065) for Study 1, and ECL (p = 0.099) and liver post-test scores (p = 0.263) for Study 2 between sexes.Details of comparison between females and stratified sample of males are indicated in Table Through this study, it was found that (1) participants with low SA had significantly lower heart drawing scores than those with high SA despite no significant differences in reported ICL, (2) males had significantly higher SA than females, (3) there were no significant differences in reported ECL when signalling in the form of colour-coding was added to the PR3DM, and (4) participants who studied the liver A3DM had significantly higher post-test scores than those who studied the liver PR3DM. The findings are important and provide useful insights on the impact that spatial ability and photorealistic and artistic 3D models have on anatomical education and their application to the research of instructional and assessment design in anatomy.50. From our study, it was found that males had significantly higher SA than females (p = 0.011), and there were significantly more males in the high SA group (p = 0.023). As modalities like drawing rely more on spatial reasoning, they can exacerbate effects favouring higher SA students, such as males. This emphasizes that anatomy assessment should be designed from both spatial and non-spatial perspectives, especially for anatomy beginners where SA has a greater effect on anatomy performance52. In several studies, SA improved with repetition and practice55, and mentored sketching (in engineering fields)56. For example, Provo et al. (2002) found that males had superior spatial ability, but that at 8 months, spatial abilities were comparable42. Our findings suggest that drawing can be encouraged during anatomy teaching and learning sessions to promote SA, increasing experiences of knowledge construction. However, this should be further investigated.The finding that participants with a low SA had significantly lower heart drawing scores supported our first hypothesis that learners with a high SA have superior anatomy performance in drawing assessment. The correlation between MRT and heart drawing scores was moderately positive. This corroborates with the meta-analysis by Roach et al. reporting significant positive pooled correlation between SA and anatomy performance when drawing tasks were used to assess anatomical knowledge57. This indicates that a deficiency in drawing abilities should not be a problem in recalling the image. Therefore, using drawing in this study as an assessment tool to evaluate spatial knowledge is reasonable. Interestingly, ICL was not significantly different between low and high SA. The low SA group’s self-reported scores on the difficulty of drawing in different orientation and remembering valve and cusp positions were comparable to the high SA group. This phenomenon could possibly be explained by the Dunning-Kruger effect, where students who perform poorly overestimate their performance in self-ratings58, which has been observed in the medical field60. However, low SA participants reported much increased difficulty remembering cardiac valve and their cusp names. Engaging in active learning techniques such as sketching and labelling of the cardiac valves and cusps can help students’ memory and retention of information.A possible explanation for the low SA group's performance in heart drawing is that they were unable to mentally imagine, orient, and map the heart valves with their components from an unfamiliar perspective. This impacted their ability to recall and present the image correctly. Previous research found that participants who could not draw objects from an imagined viewpoint could do it from their actual viewpoint10 but can increase ECL due to their anatomical and textural details that may not be essential to learning61. Significant ECL reduction through colour coding has also been reported14. In this study, non-additive signalling—colour-coding—was added, highlighting structural differentiation28. However, the current study observed no significant difference in ECL when PR3DM was colour coded. The reason for this could be because the distinct colours for each structure in the liver A3DM coupled with outlines of structures that were in close proximity to each other reduced ambiguity in determining the borders of anatomical structures, mitigating the increased cognitive load from photorealistic geometric and structural details even though textural and colour realism had to be sacrificed. Another possible reason for the similar ECL is that other factors contributing to ECL were kept constant between groups in the current study. For example, the transient information effect, describing provision of free-control of dynamic visual aids, also interferes with learning spatially-complex information62. This effect was kept constant as participants studying both models could independently control the rotation, size, and revelation of labels. Another example would be the redundancy effect, elicited as both 3D models were viewable from all directions, where presenting extra materials like multiple spatially-challenging views increases ECL and decreases working memory63, affecting anatomy learning. It was also reported that non-essential information should be discarded from visualisations, with only key structural views presented, especially in initial learning phases65. Further studies can be conducted to investigate how presenting a series of static key views with sacrificed user-interactivity compares with freely rotatable PR3DMs with good interactivity but presents all views .Our second hypothesis was that colour-coded 3D model would decrease ECL. Previously, literature showed that PR3DMs can provide accurate details10. Our findings substantiate that PR3DM enhancement through signalling may increase the learner’s attention and interest on the 3D anatomy models. This may have implications for the creation of learning tools and instructional design, in that realistic and color-coded 3D anatomy models can be displayed in tandem when guiding students in learning human anatomical regions that are more difficult and complicated to understand. However, our findings must be taken with the caveat that in the post-test, question diagrams corresponded to the model the participants studied. The question thus arises whether visual aids inadvertently increased the reliance of learners on these cues, potentially lowering their performance when tested without cues.Perhaps what is most important is the impact of instructional design on learning performance, as per our third hypothesis. Our results demonstrated that participants who studied the A3DM performed significantly better in post-testing than the PR3DM group, suggesting that colour-coding translated to better recognition, distinction of structures and information retention. Photorealism is useful because it preserves true geometry, giving accurate representations of subject materialWe acknowledge several study limitations. Firstly, Study 1 was limited to the heart and Study 2 was limited to the liver. Future larger studies should evaluate if similar effects can be replicated with other human anatomical regions and organs. Secondly, learning strategies between the high and low SA groups could not be delineated and may have been different. Given the short learning phase, low SA participants may simply lack time to generate effective visualisation strategies to retain knowledge. Eye motion analyses would be helpful in identifying differences in viewing patterns, providing clearer explanations on how the high SA group performed better. Thirdly, self-selection bias may be another limitation in that students interested to learn anatomy before the formal course may have participated compared to those uninterested. Lastly, this investigation was undertaken prior to the formal anatomy course, thus limiting its validity to untrained individuals. It is possible that experienced learners may develop mechanisms to compensate for decreased spatial ability, and further studies in this cohort may be useful.This investigation demonstrated the positive effects of SA and colour-coding on anatomy performance. The participants in the photorealistic and artistic 3D groups indicated similar ECL. These may have significance for the instructional and evaluation design based on PR3DMs and techniques of drawing anatomy.Supplementary Information.


Active surveillance has been proposed as a therapeutic option in selected intermediate risk patients with biopsy grade group 2 prostate cancer. However, its oncologic safety in this setting is debated. Therefore, we conducted a non-systematic literature research of contemporary surveillance protocols including patients with grade group 2 disease to collect the most recent evidence in this setting. Although no randomized controlled trial compared curative-intent treatments, namely radical prostatectomy and radiotherapy vs. active surveillance in patients with grade group 2 disease, surgery is associated with a benefit in terms of disease control and survival when compared to expectant management in the intermediate risk setting. Patients with grade group 2 on active surveillance were at higher risk of disease progression and treatment compared to their grade group 1 counterparts. Up to 50% of those patients were eventually treated at 5 years, and the metastases-free survival rate was as low as 85% at 15-years. When considering low- and intermediate risk patients treated with radical prostatectomy, grade group 2 was one of the strongest predictors of grade upgrading and adverse features. Available data is insufficient to support the oncologic safety of active surveillance in all men with grade group 2 prostate cancer. Therefore, those patients should be counselled regarding the oncologic efficacy of upfront active treatment modalities and the lack of robust long-term data supporting the safety of active surveillance in this setting. Active surveillance (AS) is the standard of care for patients with low-risk localized prostate cancer (PCa) to postpone or avoid treatment-related side effects without losing the window of curability . A closeThree randomized trials assessed the role of surgery in the management of PCa patients with localized disease –15. AlthThe ProtecT study randomized individuals with screening-detected clinically localized PCa to active monitoring vs. RP vs. radiotherapy, approximately 25% of participants had features of intermediate risk disease according to contemporary risk-stratification tools . This stA summary of relevant studies reporting the oncologic outcomes of ISUP biopsy GG 2 PCa patients managed with AS is depicted in Table The Sunnybrook cohort included 213 (22.5%) intermediate risk patients, 60% of which had biopsy Gleason 7 PCa (102 had Gleason score 3 + 4) and the remaining had PSA between 10 and 20 ng/ml or Gleason score ≥ 4 + 3. Patients with intermediate risk PCa had 15-year metastases-free survival rates of 84% and lower cancer-specific free survival compared to the low-risk setting  . Of noteThe Royal Marsden Hospital protocol included only 33 men (7%) with GG 2 PCa and failed to show an increased risk of being treated with curative-intent treatments at a median follow-up of 5.7 years. However, Gleason 7 was the strongest predictor of adverse pathology at RP . The relApproximately 10% of patients managed with AS within the UCSF had Gleason 3 + 4 disease. In their landmark publication, Cooperberg et al. reported that patients with intermediate risk disease were not at increased risk of progression  or active treatment (30 vs. 35%) . An updaIn the multicentre Canary PASS cohort, 9% of men included had intermediate risk PCa, and although the five-year reclassification rates were similar between GG 2 and 1 disease (30% vs. 37%), a higher proportion of men with GG 2 were treated at 5 years . Moreover, among men without grade reclassification, those with initial GG 2 switched to treatment more often than those with GG 1. Both adverse pathology and risk of BCR were similar between men who received delayed RP after initial surveillance .A retrospective study reported the outcomes of more than 200 patients with GG 2 disease managed with AS at the Memorial Sloan Kettering Cancer Center (MSKCC). The authors report a 5-year treatment-free survival rate of 61% , which iFinally, the University College London Hospital (UCLH) recently reported the outcomes of an AS program where biopsies were omitted in favour of MRI monitoring . In partBaboudjian et al. recently summarized findings of AS in intermediate risk PCa in a systematic review and meta-analysis, demonstrating higher risk of metastases and PCa-related mortality for patients with intermediate risk features . HoweverTaken together, these findings highlight that AS in men with intermediate risk features carries a non-negligible risk of disease progression and curative-intent treatment. Moreover, the 15-year metastases-free survival rate can be as low as 85% in intermediate risk patients managed with AS. Therefore, patients with GG 2 PCa, and those with intermediate risk features, who are interested in expectant management should be counselled regarding the increased risk of disease progression and adverse outcomes after curative-intent therapies.per se an adverse prognostic factor for disease recurrence after treatment. However, studies testing this association in the AS setting are lacking, therefore, these notions cannot be directly applied for patients with GG 2 disease managed on AS.When considering men treated with RP, who were otherwise eligible for AS, adverse pathology should be defined as the presence of non-organ confined disease, or GG ≥ 3, or lymph node invasion . As suchSeveral retrospective studies which included patients with low- and intermediate risk disease undergoing RP evaluated predictors of favourable pathology to propose novel AS criteria based on individual characteristics. Up to 25% of patients with low-volume intermediate risk PCa defined as the presence of ≤ 2 cores of GG 2 PCa, PSA < 20 ng/ml harbour adverse pathologic features at RP , and theThe advent of novel imaging modalities such as mpMRI for diagnosis and staging might alter this scenario. Lantz et al. recently reported that, although GG 2 PCa is still associated with misclassification at RP, a higher nomogram-calculated threshold can be used to select AS candidates among men diagnosed with mpMRI . MoreoveThe use of AS has been proposed in selected intermediate risk PCa patients with GG 2 disease to reduce the risk of treatment-related side effects without compromising oncologic outcomes , 6. OverBesides prospective AS cohorts, other authors reported that a GG 2 disease roughly doubles the risk of adverse pathology if patients with low- or intermediate risk PCa were treated with RP –32. WhenNevertheless, physicians shall also carefully weight benefits and harms of aggressive treatment modalities, considering age and comorbidities in adjunction to clinical and pathologic features of intermediate risk PCa . Some maIn the next future we can expect that the widespread availability of mpMRI and molecular imaging techniques such as Prostate Specific Membrane Antigen Positron Emission Tomography (PSMA PET), with consequent improved ability to correctly stratify patients will allow for a better classification of patients with GG 2 disease . PSMA PEAS is the preferred management for patients with low-risk localized PCa and some authors advocated the use of this approach in men with favourable GG 2 disease. However, available data is insufficient to support the oncologic safety of AS in this setting. At the time being, patients with GG 2 PCa at prostate biopsy should be counselled regarding the oncologic efficacy of upfront treatment and the lack of robust long-term data supporting the safety of AS in this setting. Most importantly, these men should be counselled on the almost 3-fold increase in the risk of developing metastases if managed expectantly. Until optimal risk stratification tools are available, such as novel imaging techniques and contemporary metrics to quantify the amount of pattern 4 in biopsies, AS shall not be routinely implemented in all patients with GG 2 disease.


We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using  While MUnderstanding the genetic bases of naturally occurring, highly divergent adaptive phenotypes in novel systems that parallel human clinical variation, such as ‘evolutionary mutant’ models –18, provEmerging fish model systems include the rapidly evolving East African and Cameroon cichlid radiations, in which a small number of genetic changes underlie immense morphological disparity –32 and tCyprinodon pupfishes for discovering and validating the craniofacial function of a new gene associated with jaw evolution via QTL and GWAS analyses. Pupfishes offer some advantages over other evolutionary fish systems because they (1) rapidly evolved highly divergent and unique craniofacial phenotypes (Cyprinodon variegatus (a), which is broadly distributed across the Caribbean and North American Atlantic coast and occurs in sympatry with two microendemic trophic specialist species found only in the hypersaline lakes of San Salvador Island (SSI), Bahamas. Each trophic specialist displays highly divergent behaviour, pigmentation and craniofacial morphology: the molluscivore C. brontotheroides has a novel nasal protrusion that is a skeletal extension of the maxilla and foreshortened robust oral jaws (b); and the scale-eater, C. desquamator, exhibits two-fold larger oral jaws and overall brachycephalic features (c) ..galr2a egalr2a in the maxilla of only molluscivores at hatching (a–c), absent in the scale-eaters and generalists, consistent with the uniquely enlarged and anteriorly protruding head of the maxilla in this species [galr2a expression in the intermandibular muscles of the scale-eaters at hatching time (c) suggests that galr2a expression can also modulate the development of the observed hypertrophic musculature of the adductor mandibulae in the adult scale-eating pupfish [galr2a in musculoskeletal development and may contribute to the divergent craniofacial morphology observed in SSI pupfishes.We noted expression of hatching a–c, abse species ,50,59,98ing time c suggest pupfish . Altoget (c)c). Importantly, M871 exhibits a higher binding constant for Galr2 receptors than the Galr1 + 2 antagonist M35 [galanin and Galr1 mRNA transcript abundance does not vary among the three SSI species .Interestingly, the response of Meckel's cartilage length and chondrocyte density to the inhibition of Galr-receptor pathways was highly dependent on the species' genetic background. Scale-eaters responded only to the Galr2-specific antagonist M871 with substantially reduced Meckel's cartilage length and increased chondrocyte density but were unaffected by the Galr1 + 2 antagonist M35 c. Importnist M35 ,99. Prevnist M35  also indgalr2a transcripts and presumably contain overall fewer Galr2 receptors in their craniofacial tissue, M871's higher binding affinity [galr2a at 2 dpf, suggesting that, as in scale-eaters treated with M35, fewer Galr2 receptors may be present for inhibition in these species’ background. However, the response to M871 appears to be specialist-specific, with little effect on the generalist Meckel's cartilage length, and thus, we cannot rule out more complex species-specific factors interacting with Galr regulatory pathways.Therefore, we conclude that even though scale-eaters expressed the lowest amount of affinity  was suffaffinity . We alsoGalr2 activation inhibits cell proliferation in neuronal cell lines [galr2a abundance and, consequently, have low Galr2 activity during craniofacial development resulting in increased chondrocyte density and Meckel's cartilage length. Thus, the increased chondrocyte density observed in the M871-treated scale-eater larvae likely results from the strong and continued inhibition of the fewer Galr2 receptors available during development due to their genetic background. Lastly, due to the variable expression of galr2a between species at stage 24 but similar expression in chondrocytes of the Meckel's and palatoquadrate cartilages of different species at stage 33, we speculate that galr2a may have a species-specific morphogenetic role during pharyngeal arch differentiation and early jaw development and an osteogenic role at later stages.Our work is consistent with previous work showing that ll lines ,100, suggalr2a during early development may result in their reduced oral jaw lengths as adults through increased opportunities for endogenous agonistic binding interactions . We also acknowledge the polygenic nature of jaw development and note that our previous genetic mapping experiments place an upper bound of 15% of oral jaw variation that may be due to differences in galr2a regulation among the myriad craniofacial genes driving the evolution of divergent craniofacial traits in this system.Our results support a novel role of the second receptor for galanin, . 5Full details on pupfish husbandry, HCR FISH staining, microscopy, Galr2-inhibition, sequencing, TFBS prediction, gene expression and statistical analyses are available in the supplemental material.


Can we improve time to treatment using circulating tumor DNA (ctDNA) genotyping before tissue diagnosis among patients with suspected advanced lung cancer?In this nonrandomized clinical trial, 150 patients with suspected advanced lung cancer underwent ctDNA testing during initial diagnostic workup; 90 patients had tissue confirmation of advanced nonsquamous non–small cell lung cancer. Median time to treatment was 39 days vs 62 days for a reference cohort, with faster turnaround time for genotyping results.The use of plasma ctDNA testing before tissue diagnosis among patients with suspected advanced lung cancer may expedite biomarker testing and accelerate time to treatment. Liquid biopsy has emerged as a complement to tumor tissue profiling for advanced non–small cell lung cancer (NSCLC). The optimal way to integrate liquid biopsy into the diagnostic algorithm for patients with newly diagnosed advanced NSCLC remains unclear.To evaluate the use of circulating tumor DNA (ctDNA) genotyping before tissue diagnosis among patients with suspected advanced NSCLC and its association with time to treatment.This single-group nonrandomized clinical trial was conducted among 150 patients at the Princess Margaret Cancer Centre–University Health Network  between July 1, 2021, and November 30, 2022. Patients referred for investigation and diagnosis of lung cancer were eligible if they had radiologic evidence of advanced lung cancer prior to a tissue diagnosis.Patients underwent plasma ctDNA testing with a next-generation sequencing (NGS) assay before lung cancer diagnosis. Diagnostic biopsy and tissue NGS were performed per standard of care.The primary end point was time from referral to treatment initiation among patients with advanced nonsquamous NSCLC using ctDNA testing before diagnosis (ACCELERATE [Accelerating Lung Cancer Diagnosis Through Liquid Biopsy] cohort). This cohort was compared with a reference cohort using standard tissue genotyping after tissue diagnosis.P < .001). Among the ACCELERATE cohort, the median turnaround time from sample collection to genotyping results was 7 days  for plasma and 23 days  for tissue NGS (P < .001). Of the 90 patients with advanced nonsquamous NSCLC, 21 (23%) started targeted therapy before tissue NGS results were available, and 11 (12%) had actionable alterations identified only through plasma testing.Of the 150 patients  enrolled, 90 (60%) had advanced nonsquamous NSCLC. The median time to treatment was 39 days  for the ACCELERATE cohort vs 62 days  for the reference cohort (This nonrandomized clinical trial found that the use of plasma ctDNA genotyping before tissue diagnosis among patients with suspected advanced NSCLC was associated with accelerated time to treatment compared with a reference cohort undergoing standard tissue testing.NCT04863924ClinicalTrials.gov Identifier:  This nonrandomized clinical trial evaluates the use of circulating tumor DNA genotyping before tissue diagnosis among patients with suspected advanced non–small cell lung cancer and its association with time to treatment. Herein we report the clinical utility of the use of plasma ctDNA genotyping prior to a pathologic diagnosis of lung cancer using InVisionFirst-Lung (Inivata Ltd), a plasma ctDNA comprehensive NGS assay including variants, fusions, and copy number alterations relevant in lung cancer, as part of a multidisciplinary centralized referral program to expedite diagnosis and treatment of lung cancer.Liquid biopsies are minimally invasive blood tests that identify circulating tumor DNA (ctDNA) in patients’ plasma.21 All participants provided written informed consent prior to study enrollment. This study adhered to the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline. The trial protocol is available in The ACCELERATE (Accelerating Lung Cancer Diagnosis Through Liquid Biopsy) study is a prospective, single-group, minimally invasive nonrandomized clinical trial conducted at the Princess Margaret Cancer Centre–University Health Network (UHN), Toronto, Ontario, Canada. Conduct of this study was approved by the institutional research ethics board (UHN Board C) and was carried out in accordance with the principles of the Declaration of Helsinki.22Between July 1, 2021, and November 30, 2022, 150 patients were enrolled in the ACCELERATE cohort. Patients referred to the UHN Lung Rapid Assessment and Management Program (LungRAMP) were eligible to participate if they had (1) radiologic evidence of unresectable stage III or IV lung cancer; (2) measurable disease with 1 cm or more of disease detected by computed tomography; or (3) a diagnostic tissue biopsy planned or performed without a diagnosis of NSCLC yet. Eligibility was confirmed by the weekly Lung RAMP multidisciplinary case conference . Patients with pleural effusions but no measurable disease were eligible if the MCC favored a diagnosis of malignant disease based on imaging. Patients were excluded if they had concurrent cancer, had a cancer diagnosis other than lung cancer in the past 2 years, or were pregnant due to potentially confounding ctDNA results. Patient self-reported race and/or ethnicity were obtained from the electronic medical record to further characterize the study population, as the frequency of molecular alterations in lung cancer can vary across different ancestral populations.After eligibility confirmation, patients were invited to participate in the study . Consent23 Blood samples were processed to plasma by centrifugation and stored at −80 °C according to validated specifications until analysis in batch. Plasma circulating free DNA (cfDNA) was extracted with the QIAamp Circulating Nucleic Acid kit (QIAGEN). After quality control, sequencing libraries were prepared using a 2-step amplification process, and libraries were sequenced by the NextSeq500 system (Illumina Inc). Sequencing data were analyzed using the Inivata analytical pipeline to identify genomic alterations. Results were returned to the study team and treating clinician within a median of 7 calendar days from blood collection.Plasma ctDNA testing was performed using InVisionFirst-Lung, a highly sensitive and specific NGS assay targeting 37 genes that has been prospectively validated and demonstrates high concordance with tissue results and high sensitivity and specificity for variants, single-nucleotide variants, fusions, and copy number alterations  and IHC for ALK (5A4 antibody) and ROS-1  were performed.27Reflex molecular testing of tumor tissue was performed per institutional standard of care, including comprehensive NGS   and ROS1  and PD-L1 tumor expression, were used. Further details on institutional testing methods are listed in eTable 1 in For patients in the reference cohort, reflex testing using an NGS assay targeting 15 genes  .The LungRAMP program maintains a prospective database of all patients referred for investigation of suspected lung cancer. A reference cohort of 89 patients with advanced NSCLC diagnosed through LungRAMP and treated at the Princess Margaret Cancer Centre prior to the COVID-19 pandemic (2018-2019) was identified. Demographic characteristics, molecular data, result turnaround time, and time to treatment were abstracted.P values were from 2-sided tests, and results were deemed statistically significant at P < .05.Patient characteristics and molecular data are summarized using median, IQR, frequency, and percentage. The nonparametric Wilcoxon-Mann-Whitney test was used to compare time to treatment for patients with advanced nonsquamous NSCLC enrolled in the ACCELERATE cohort and the reference cohort. The Wilcoxon signed rank test was used to compare the turnaround time of plasma ctDNA testing vs tissue testing in the ACCELERATE cohort. All analyses were performed using SAS, version 9.4 (SAS Institute Inc). All Between July 1, 2021, and November 30, 2022, 150 patients  were enrolled in the ACCELERATE cohort . Most paBiopsy-proven NSCLC was demonstrated in 107 patients (71%), although 3 patients were later found to have early-stage disease and underwent surgical resection . Of 104 patients with advanced NSCLC, 90 patients had nonsquamous histologic characteristics, 2 had atypical carcinoid tumors, and 12 had squamous carcinoma . AnotherTP53 (n = 10), KRASG12V (n = 6), KRASG12D (n = 1), STK11 (n = 2), and PIK3CA (n = 1). Comparison of tissue vs ctDNA results for the guideline-recommended biomarkers in NSCLC is shown in eTable 2 in All participants underwent plasma testing; 115 (77%) had tumor-associated variants detected . Of the Among the 18 patients with other cancer diagnoses, 7 did not have detectable cfDNA in plasma, but 3 had potentially informative results based on cancer type  had actionable alterations identified by either method: 35 of 53 (66%) in both, 11 of 53 (21%) in plasma only, and 7 of 53 (13%) in tissue only  (Most patients (72 of 90 [80%]) started systemic therapy for advanced nonsquamous NSCLC. Median time to treatment was 39 days  for the ACCELERATE cohort compared with 62 days  for the reference cohort ( < .001) .P < .001). Furthermore, 21 of 90 patients (23%) with advanced nonsquamous NSCLC started targeted therapy based on ctDNA findings before tissue genotyping results were available. For this subgroup, the median time to treatment was 27 days . Patients who received both plasma and tissue NGS results before starting therapy had a median time to treatment of 41 days .Three-fourths (40 of 53 [75%]) of patients with actionable alterations started targeted therapy, with a median time to treatment of 33 days  in the ACCELERATE cohort vs 61 days in the reference cohort  . One patient with EGFR L858R–variant lung cancer received chemoradiotherapy after multidisciplinary team review. Another patient with MET ex14 skip–variant lung cancer experienced rapid deterioration and died before starting treatment.Reasons for not starting targeted therapy among patients with actionable alterations included no approved or available first-line targeted therapy in Canada at that time (7 P < .001)  vs 23 days  for tissue NGS, measured from tissue biopsy to reporting ( < .001) . The timIn the reference cohort, referred before the COVID-19 pandemic , the medIn our study, plasma ctDNA testing before diagnosis in the initial diagnostic workup of patients with suspected advanced NSCLC was associated with shorter time to molecular results than tissue testing and shorter median time to treatment compared with a reference cohort using tissue molecular testing only (39 vs 62 days). Plasma testing was associated with greater detection of ESCAT tier 1 actionable alterations compared with tissue testing alone. Nearly one-fourth of patients started treatment based on plasma results after tissue diagnosis and before tissue NGS results were available, with a median time to treatment of 27 days. These results suggest the clinical utility of adding plasma ctDNA testing before tissue biopsy or confirmed cancer diagnosis to the standard diagnostic workup of patients with suspected advanced lung cancer. Compared with a reference cohort using tissue molecular testing only, the use of liquid biopsy was associated with faster return of molecular results and accelerated time to treatment for all patients, both with and without targetable alterations identified.31 This testing leads to increased delivery of targeted therapy for patients with advanced NSCLC. To improve access and timely treatment initiation, liquid biopsy could be performed even earlier, as in our study, as part of the initial diagnosis workup for patients with suspected advanced lung cancer before tissue biopsy or diagnosis. Several recent small studies have demonstrated that plasma testing for patients with suspected advanced lung cancer at the time of or before tumor biopsy is associated with decreased time to treatment.34Preliminary evidence suggests that concurrent plasma ctDNA and tissue NGS testing after lung cancer diagnosis increases the proportion of patients receiving comprehensive molecular testing vs a tissue-only approach.32 allowing for prompt treatment initiation when needed for symptomatic or deteriorating patients. As many as 70% of patients with advanced nonsquamous NSCLC in the ACCELERATE cohort could have started treatment based on ctDNA results, tissue diagnosis, and PD-L1 tumor assessment. These patients include those with actionable alterations but also those with nonactionable driver alterations, such as KRAS non-G12C variants. The detection of these molecular alterations in plasma is informative and can also help guide treatment decisions.To our knowledge, this is the largest study exploring this prediagnostic approach for suspected advanced lung cancer. Our study demonstrated that plasma ctDNA testing before lung cancer diagnosis was associated with faster time to molecular results, more frequent identification of actionable targets than tissue testing alone, and accelerated time to treatment for all patients with nonsquamous NSCLC participating in the study. Nearly one-fourth of patients (23%) started treatment before tissue molecular testing was available, similar to the pilot study by Cui et al,19 Plasma ctDNA-based NGS has lower sensitivity than tissue-based NGS, with low levels of ctDNA available for analysis, limited sensitivity for detection of fusions, and challenges in assessment of copy number variation. Cell-free RNA assays may yield higher sensitivity.35 We believe our results may also have been subject to the Hawthorne effect and affected by the COVID-19 pandemic. Time from referral to tissue biopsy was shorter among the ACCELERATE cohort compared with the reference group (12 days vs 24 days), which suggests a potential effect of the COVID-19 pandemic on access to care when patients with suspected metastatic disease were prioritized for diagnostic tests. ACCELERATE participants were also prioritized for investigation and assessment, including expedited biopsies and rapid medical oncology assessment after ctDNA results. The effect of multidisciplinary diagnostic assessment programs in shortening wait times for diagnosis and treatment of lung cancer, rather than sequential assessment by clinicians, is also highlighted in our study.Our study has limitations, including its single-group prospective design and the fact that it was conducted at a single institution. Despite expert selection of patients with radiologic evidence of advanced lung cancer by a multidisciplinary team, only 70% of patients had biopsy-proven advanced NSCLC, while 30% had other diagnoses or no malignant neoplasms. These results reinforce the need for tissue biopsy for lung cancer diagnosis and pathologic subtyping: plasma first does not mean plasma only.36 the addition of complementary liquid biopsy to tissue testing in patients with newly diagnosed advanced nonsquamous NSCLC did not increase overall treatment costs and led to more patients receiving appropriate targeted therapy. In our study, we provide a possible strategy to optimize molecular testing in NSCLC and time to treatment using plasma ctDNA prior to diagnosis. This approach may permit diagnostic cancer tissue sparing in some scenarios and decrease treatment delays (Finally, despite the known benefits of liquid biopsy, cost is a potential barrier limiting its implementation. Although 71% of patients in our study had advanced NSCLC, this percentage will vary across geographic regions. Justifying the cost of liquid biopsy for patients without NSCLC may be challenging, although results were potentially informative for some patients with a diagnosis of other cancers. In a cost-effectiveness analysis by Ezeife et al,t delays .In this nonrandomized clinical trial, the use of liquid biopsy before lung cancer diagnosis in the initial diagnostic workup of patients with suspected advanced NSCLC was associated with shortened time to treatment and molecular results and yielded a higher rate of detection of actionable alterations. Complementing standard tissue testing with plasma testing before diagnosis could increase access to precision medicine and may improve patient outcomes. Although we believe this is a promising strategy to improve the diagnostic journey and treatment decision-making for patients with advanced NSCLC, the effect of this approach on clinically meaningful outcomes, such as quality of life, survival, and cost-effectiveness, still needs to be demonstrated.


DD) fused to an RNA binding protein. While it has been shown that endogenous ADARs can be recruited to a defined target site with the sole help of an ADAR-recruiting guide RNA, thus freeing up packaging space, decreasing the chance of an immune response against a foreign protein, and decreasing transcriptome-wide off-target effects, this approach has been limited by a low editing efficiency. Through the recent development of novel circular ADAR-recruiting guide RNAs as well as the optimisation of ADAR-recruiting antisense oligonucleotides, RNA editing with endogenous ADAR is now showing promising target editing efficiency in vitro and in vivo. A target editing efficiency comparable to RNA editing with exogenous ADAR was shown both in wild-type and disease mouse models as well as in wild-type non-human primates (NHP) immediately following and up to 6 weeks after application. With these encouraging results, RNA editing with endogenous ADAR has the potential to present an attractive option for the treatment of inherited retinal diseases (IRDs), a field where gene replacement therapy has been established as safe and efficacious, but where an unmet need still exists for genes that exceed the packaging capacity of an adeno associated virus (AAV) or are expressed in more than one retinal isoform. This review aims to give an overview of the recent developments in the field of RNA editing with endogenous ADAR and assess its applicability for the field of treatment of IRD.RNA editing holds great promise for the therapeutic correction of pathogenic, single nucleotide variants (SNV) in the human transcriptome since it does not risk creating permanent off-targets edits in the genome and has the potential for innovative delivery options. Adenine deaminases acting on RNA (ADAR) enzymes catalyse the most widespread form of posttranscriptional RNA editing in humans and their ability to hydrolytically deaminate adenosine to inosine in double stranded RNA (dsRNA) has been harnessed to change pathogenic single nucleotide variants (SNVs) in the human genome on a transcriptional level. Until now, the most promising target editing rates have been achieved by exogenous delivery of the catalytically active ADAR deaminase domain (ADAR RPE-65-associated IRD now available and others being evaluated in clinical trial (ABCA4 or USH2A), the gold standard delivery vehicle for most gene replacement therapies, or genes that are expressed as multiple isoforms in the human retina   in particular have been at the forefront of diseases for which gene therapies are currently being developed. There is an imperative need for therapies to cure IRDs: they have a world-wide prevalence of about 1 in 2000, affect more than two million people and cause significant economic as well as and psychological burden . While gal trial , an unme., CRB1) .CEP290- and USH2A-associated IRDs (DD) and have multiple double stranded RNA binding domains (dsRBD), with which they bind to double stranded RNA (dsRNA) and edit tens of thousands to millions of sites in the human transcriptome  . The secTo unlock the utility of ADARs as an RNA editing toolset, editing must be achieved with a high on-target efficiency and specificity which precludes off-target editing in the form of bystander editing and transcriptome-wide off-target editing. For editing with endogenous ADAR, the central challenges are improving on-target editing efficiency while limiting bystander editing in the gRNA:target RNA duplex, while the central challenge for exogenous ADAR editing is restricting promiscuous transcriptome-wide off target editing and achieving target specificity while retaining high on target editing rates.This review aims to give an outline of the history and recent developments in the field of RNA editing with endogenous ADARs and discuss the potential for treatment of IRDs. Since an understanding of the structure, function and role of ADAR enzymes is essential to understanding the design, challenges and strategies in the RNA editing field, an overview of ADAR enzymes will proceed the discussion on RNA editing with endogenous ADAR.Three members of the ADAR family are encoded in the mammalian genome and are highly conserved across species : ADAR1, The presence of ADAR2 and ADAR1 protein expression levels in the neuronal cell types of the retina has not yet been comprehensively analysed, but immunohistochemical evidence points toward ADAR2 expression in the retinal ganglion cell (RGC) layer . It is aA receptor ] and long interspersed elements LINEs . ADAR1 iic cells . In a huic cells . In humaic cells . ADAR1 hIt has yet to be fully understood which characteristics determine whether an adenosine within dsRNA is deaminated, but it is thought that ADAR exhibits a sequence preference for particular RNA motifs  while thADAR preferentially deaminates adenosines that occur in an A-C mismatch over those that occur as A-A or A-G mismatches or in an A-U pairing . These cAlthough ADARs do not exhibit strict sequence specificity, the enzymes do exhibit a preference for certain neighbouring nucleotides. The most pronounced preference is seen for the base flanking the 5′ end of the target adenosine. Here, both ADAR1 and ADAR2 favour Uridine (U) as a neighbouring base, followed by A, C and G (U > A > C > G). While the nature of the 3′ neighbouring base is thought to be less important, both ADARs prefer guanosine (G) at this position. More specifically, ADAR1 prefers G > C ≈ A > U and ADAR2 exhibits a slightly different order of preference: G > C > U ≈ A . These pBeyond sequence-specific preferences, ADAR-dsRNA interactions rely on the structure of the RNA substrate to confer target editing specificity. Generally, ADAR acts on both inter- and intramolecular dsRNA of >20 bp in length . Long >. In geneDD alone also has targeting capacities  an antisense sequence complementary to the target RNA substrate located at the 3′ end and (2) the R/G GluR2 ADAR-recruiting domain at the 5′ end. The antisense region contained a C mismatch opposite the target A, and the optimal length was determined to be 18–20 nt. Lengthening of the antisense region beyond this point led to declining editing rates as well as bystander edits.In an eGFP – reporter plasmid with a premature stop codon, on-target editing rates up to 50% were observed, which increased to 65% when the ADAR2 expression was integrated in the host genome of HEK293T cells under the CMV promotor. When editing components as well as ADAR2 were given at high concentrations, massive off target effects were seen and a gRNA lacking the R/G motif was able to elicit editing. These effects were decreased when lowering the concentrations of all transfected components, indicating that dosing is a central question when trying to achieve desired editing while lowering bystander edits. Furthermore, the authors showed editing of several endogenous housekeeping gene transcripts with editing rates ranging from 10 to 35% in 12 out of 13 target sites. Interestingly enough, one site in beta actin consistently achieved 0% editing, indicating the target variability in editing rates. Apart from showing editing of several endogenous housekeeping genes, a 10% correction of the PINK W437X nonsense mutation, a functional rescue of cellular phenotype was also shown. In a longitudinal analysis of editing, peak editing was observed at 48 h after transfection which remained constant until 72 h and then started to decline at 96 h.In a follow-up study by Using a very similar gRNA design to RABA7 transcript in HEK293T cells through the recruitment of endogenous ADAR. Even when the GluR2 recruitment domains were removed from the adRNA, the adRNA elicited editing. An A-C mismatch placed at the centre of the antisense region exhibited the most effective editing. While the RNA editing through adRNA recruitment of endogenous ADAR exhibited a target editing efficiency lower than the RNA editing achieved with overexpressed wild type and hyperactive ADAR2 (E488Q), this was the first indication that endogenous ADAR was able to be recruited by an ADAR-recruiting gRNA, with and without the ADAR-recruiting domain R/G GluR2. These results held true in vivo, when different adRNA designs were tested on a G > A point mutation in a sparse fur ash (ashspf) mouse model of ornithine transcarbamylase (OTC) deficiency. One month after retroorbital injection adRNA alone resulted in low but significant RNA editing yields in the liver. Nevertheless, the highest edited fraction came from adRNA delivery together with the hyperactive ADAR2 mutations (E488Q) (4.6–33.8% RNA editing).In 2019, Katrekar et al. used the design of the ADAR-recruiting gRNA (named adRNA after ADAR-recruiting gRNA) described in PPIB gene with editing rates >80%. When targeting this same transcript with lentiviral transduction in HEK293T cells, the editing rates dropped to 6% at 6 days post transduction. This efficiency increased to 20% when chemical modifications were introduced in the form of 2-O-methyl and phosphorothioate backbone modifications at either end of the arRNA. This highlights the wide range of editing rates in human primary cell lines, that are dependent on target sequence, delivery mechanism and endogenous ADAR expression in the target tissue.Building on this work, Qu et al. developed LEAPER (leveraging endogenous ADAR for programmable editing of RNA), an endogenous ADAR-recruiting gRNA (arRNA), consisting of a long, single arRNA without ADAR-recruiting sequences or chemical modifications. During the optimisation of LEAPER, several design principles were established: similar to Katrekar et al. which required an adRNA of at least 60 bp for endogenous ADAR recruitment, a minimum length of at least 71 nt was required for efficient endogenous ADAR recruitment and subsequent editing. The length of the arRNA correlated positively with the target editing efficiency, with arRNA counting 111 and 151 nt being used in experiments in standard and primary cell lines. A mismatch position placed in the middle of the arRNA lead to the highest editing efficiency and the preferred 5′ neighbouring base was 5’U. The standard cell lines all lacked ADAR2 expression, indicating that ADAR1 was being recruited for editing. But in a knockout cell line, editing was rescued by the addition of ADAR2 as well as ADAR1 isoforms, indicating that LEAPER can recruit ADAR1 and ADAR2. In addition to a testing in a reporter plasmid assay, where arRNA achieved a target efficiency of approximately 13%, LEAPER was tested on an array of standard and primary cell lines, showing a wide range of target editing rates. The highest editing rates were shown in the primary bronchial epithelial cells targeting transcripts of the endogenous KRAS gene transcript, where one-third of As were subject to bystander editing. In an attempt to curb this bystander editing, A to G mismatches were placed across from the potential target As. Clinical relevancy of LEAPER was shown by editing the tumour suppressor gene transcript TP53 as well as a nonsense mutation in the IUDA gene transcript in a primary cell line isolated from a Hurler syndrome patient. Editing rates were modest but nevertheless significant. Expression levels of targeted transcripts were monitored to rule out possible RNA interference (RNAi) effect of the arRNA.Due to the length of the antisense domain, As occurring in the target sequence were promiscuously deaminated, although the extent of this varied between targets. The highest bystander editing was seen in the in vitro, the E342K missense mutation (PiZZ mutation) in the SERPINA gene, the most common cause for alpha1 antitrypsin deficiency, was targeted. In a HeLa cell line expressing mutated SERPINA, cDNA editing rates of 10% were found without IFN-alpha, which increased to 20% with IFN-alpha treatment. When targeting the Tyr701 site in the 5′ UAU codon of the endogenous STAT1 in HeLa and primary cells, the highest editing yields were achieved with IFN-alpha induction and ranged from 20 to 30%. Neither LEAPER nor RESTORE were tested in vivo.In the same year, Merkle et al. chemically modified their previously described  ADAR2-reBMPR target transcript, but CLUSTER yielded higher on target efficiencies for the mIDUA target transcript. In contrast to LEAPER, which showed high bystander editing rates up to 50%, CLUSTER did not elicit any detectable bystander edits. Coding and non-coding regions of endogenous transcripts with varying levels of expression were targeted in HEK293Ft cells and yielded on target rates of 19–44% without bystander editing. Encouragingly, editing efficiencies were similar for targets in the ORF and the 3’UTR. Fibroblasts taken from a Hurler patient were treated with CLUSTER gRNA in the form of chemically stabilised ASO. Editing rates of 24% were seen as well as an increase in IDUA enzyme activity. Off-target effects were investigated and found to be mainly located in repetitive Alu sequences with three novel exonic off target effects also observed. Of note, Adenovirus (AV) not AAV was used. Finally, two different CLUSTER designs were tested in wild-type C57/BL6 mice by co-delivering the dual luciferase reporter plasmid with a CLUSTER gRNA into the liver by hydrodynamic tail vein injection. Both gRNAs had a similar design with a 20 nt target specificity domain, an invariant GluR2 ADAR-recruiting sequence and 3 × RS. The length of the RS varied between the two constructs with the first having 15-, 13-, and 11 nt and the second gRNA having 20-, 15-, and 15 nt. 72 h post-injection, the first CLUSTER gRNA demonstrated 5% editing rate both in luminescence detection and Sanger sequencing while the second gRNA demonstrated a 10% editing rate.In 2022, the Stafforst group further modified their original gRNA design and created CLUSTER, which retained the basic design principle of RESTORE, but achieved recruitment of endogenous ADAR without chemical modification. While the short, 20 nt antisense target specific domain as well as the invariant GluR2 recruitment domain remained, a series of single stranded RNA recruitment sequences (RS), a “cluster,” were added to the gRNA design. These RS bind to the target mRNA in various regions distal to the target site and distal to each other. These RS were 7–20 nt in length and could be flexibly chosen to exclude adenines that would otherwise be prone to bystander editing. The spacer regions between the RS binding sites were generally between 10 and 460 nt. The Stafforst group provides an open-source bioinformatics tool named recruitment cluster finder (RCF) to enable users a customised CLUSTER gRNA design. This strategy allowed for an increased binding affinity of the gRNA to the target sequence by adding RS while retaining the minimised chance of bystander editing that occurs when using a short specificity domain. Another important difference to RESTORE was the type of ADAR recruited. RESTORE preferentially recruited ADARp150 for editing and thus required IFN-alpha induction, but CLUSTER preferentially recruited the ubiquitously expressed ADARp110 and was also able to show recruitment of ADAR2. In contrast to LEAPER, CLUSTER was shown to benefit from the GluR2 ADAR-recruiting domain, but not from an extension of the specificity domain, so the specificity domain was kept at 20 nt to avoid extraneous bystander editing. At least 2 RS were needed to enable significant editing through the recruitment of endogenous ADAR. A luciferase reporter assay in HeLa cells was used for CLUSTER gRNA optimisation and testing of ADAR-specific recruitment. In HeLa cells using transfection of disease relevant transcripts and CLUSTER gRNA, editing yields between 3 and 61% without bystander edits were observed. When comparing CLUSTER to LEAPER, LEAPER outperformed CLUSTER by 20% on the In 2022, two groups utilised the elegant method of RNA circularisation, developed by MALAT1 and KRAS), a 50 nt, flexible polyAC linker (AC50) was included to increase the structural flexibility of the circ-arRNA. This circ-arRNA_AC50 showed increased editing efficiencies across all target sites. Editing efficiency was tested for 20 sites across 9 endogenous target genes in HEK293T cells. On average, editing efficiency increased by 2.3 for circ-arRNA and by 3.1 for cir-arRNA_AC50 across all target sites when compared to the linear arRNA LEAPER design. Sustained editing >13 days was shown when targeting the PPIA transcript in HEK293T cells via transfection. When transduced with AAV containing cir-arRNA, HEK293T, primary human hepatocyte cell line and cerebral organoids showed long term, sustained editing over 9 days, whereas the linear counterparts did not elicit significant editing. KRAS was one of two sites that exhibited initial editing efficiencies below that of linear arRNA. Yet over 7 days, editing rates with linear arRNA fell precipitously, while circ-arRNA exhibited continuous, stable editing, resulting in more efficient cumulative editing by circ-arRNA. Similarly, when targeting a reporter plasmid, the expression of arRNA was still detectable at high levels on day 21, whereas the linear arRNA counterpart was not detectable at day 21. While circ-arRNAs produced sustained, high editing efficiencies, they also exhibited pronounced bystander editing along the length of their antisense region. To reduce these bystander edits, the base flipping mechanism required to access ADARs catalytic centre was found to be hindered by deleting the Us opposite the adenosines prone to bystander editing. Different numbers of base deletions were strategically placed across the antisense regions. The cir-arRNA with 14 deletions in the antisense region as well as a flexible AC50 linker was shown to eliminate all bystander edits while retaining a 60% on-target editing efficiency across 8 target sites of the endogenous PPIA transcript. A cir-arRNA with a AC50 linker and 4 deletions opposite unwanted adenosines showed a 70% editing rate in a clinically relevant p53 nonsense mutation as well a restoration of full-length protein expression in p53−/− HEK293T cells. Of note, even though the editing rate of cir-arRNA_AC50 (not carrying deletions) was much higher than that of the construct with the deletions, the functional recovery was higher despite the lower editing rate, likely due to the low rate of bystander editing.Yi et al. based their circularised ADAR-recruiting gRNA (circ-arRNA) on their previously described arRNA, LEAPER. Like in the original design, the target specific antisense region of LEPAER2.0 was 151 nt long, with the A-C mismatch placed at the centre of the sequence and no chemical modifications . To incrRABA7 transcript as a target. Once again, a positive correlation was seen between gRNA length and on-target editing efficiency, with a linear arRNA of 200 bp resulting in a 1.6-fold increase in on-target editing efficiency compared to the linear 100 bp adRNA counterpart. While addition of ADAR recruitment domains resulted in an only marginal increase in editing efficiency compared to the simple 100 bp linear guide RNA, addition of the stabilising U6 + 27 cassette resulted in a 2-fold increase in target editing and circularisation of the adRNA resulted in a 3.5-fold on-target editing improvement over the linear adRNA. ADAR1 recruitment was confirmed by knockdown of ADAR1 and concomitant abrogation of editing efficiency. To decrease bystander editing, Katrekar et al. employed two strategies  was shown when K562 cells were electroporated with genetically encoded cadRNA targeting the 3’UTR of the RABA7 transcript. These editing rates were similarly high (70% editing) when electroporating the cell line with in vitro transcribed RNA (IVT RNA).To optimise their previously described adRNA, Katrekar et al. undertook a screening of linear adRNA of 100 and 200 nt with and without different ADAR-recruiting domains (GluR2 and Alu recruiting domains as well as the stabilising U6 + 27 cassette) as well as circular adRNA (cadRNA) in HEK293T cells using the 3’ UTR of the rategies : (1) A-GBoth Yi et al. and Katrekar et al. investigated transcriptome-wide off-target editing of cadRNA/circ-arRNA and compared these with transcriptome- wide off-target editing of exogenously delivered ADARs. In Yi et al. 17 off-target transcriptome wide edits were seen using circ-arRNA, whereas overexpression of exogenous ADAR led to >16,000 transcriptome-wide off-target effects. Katrekar et al. found similar results: With enzyme overexpression 10^3–10^4 off targets edits were routinely observed, this decreased by 2–3-fold when using cadRNAs. Both groups noted cadRNA and circ-arRNA did not lead to a change in mRNA expression levels, indicating the there was no RNA interference mechanism at play.in vivo results generated in both these studies were the first to show sustained editing of a disease-causing mutation using endogenous ADAR  Type I (Hurler syndrome) with the pathogenic nonsense variant W392X in the Idua gene to show transcript editing efficiency of cadRNA/circ-arRNA. Both studies used a genetically encoded cadRNA/circ-gRNA packaged in scAAV or AAV8, respectively, to target the liver. Yi et al. showed a 10% targeted editing rate at 4 weeks post injection while Katrekar et al. showed a 7–17% correction of the premature stop codon 2 weeks post-injection. On a protein level endpoint both studies detected a decrease in GAG accumulation  as an indirect marker for partial restoration of alpha-L-iduronidase activity  animal model, Monian et al. used fully chemically modified, short, 30 nt antisense oligonucleotides (AIMers) for endogenous ADAR recruitment. Optimisation of the chemical modifications of the AIMers showed that in addition to the selectively placed 2′ ribose modifications 2’deoxy-, 2’fluoro and 2’-O-methyl, a phosphorothioate (PS) backbone increased target editing activity. PS backbone chirality also influenced editing activity with left-handed (Sp) PS modifications increasing editing efficiency, while right-handed (Rp) PS chirality decreased editing efficiency. Modifications that preferentially enhanced ADAR1 rather than ADAR2 were selected since ADAR1 is expressed ubiquitously at higher levels than ADAR2 and thus may be considered a more therapeutically relevant recruitment target than ADAR2. In addition to optimising chemical modifications for editing activity, an N-acetylgalactosamine  modification was included to improve AIMer delivery to hepatocytes via asialoglycoproteins receptor . The use5 to 39% . While tThe feasibility of targeting pre-mRNA with RNA editing has been investigated in several studies. Apart from determining which guide RNA is most effective, the ability to target the pre-mRNA would broaden the RNA editing scope to intronic and splice site mutations, which currently present important therapeutic targets in DNA editing and ASO therapy . FurtherGUSB target transcript, there was no difference in editing rates between the two gRNAs. For the other two targets, GPI and NUP43, the pre-mRNA gRNA was found to be one-third and one half as effective as a gRNA targeting mature mRNA. The Wei group investigated pre-mRNA and mRNA gRNA targeting the coding sequence of in the IDUA transcript both in vitro and in vivo  was seen.Localisation of the components required for editing (endogenous ADAR and gRNA) is an important indication of the feasibility of pre-mRNA targeting. Both Yi et al. and Reautschnig et al. demonstrated gRNA localisation to both the cytoplasm and the nucleus, albeit in different proportions. While Yi et al. found the circ-arRNA to localise to both cellular compartments in equal proportions, Reautschnig et al. found the CLUSTER 16p8 gRNA predominantly localised to the nucleus when transfected. These findings suggest that RNA editing takes place at least partially in the nucleus. The nuclear localisation of ADAR1p110 (and ADAR2) underscores the probability of this assumption. Reautschnig et al. compared the editing yields of a pre-mRNA gRNA to those of a gRNA targeting the mRNA in the coding sequence close to an exon/intron border of three endogenously expressed genes in HEK293FT cell lines. For the  in vivo . In a prnor site . A 4.6–8Ush2a gene was detected in photoreceptor cells up to 259 days post-injection, which was associated with desired exon 12 skipping  to the retina . InitialAn alternative strategy may be to use nanoparticles to carry DNA that enables transcription of the required RNA structures. Whilst various nanoparticle forms provide efficient delivery to the RPE, the polyethylene glycol (PEG)-substituted polylysine (CK30PEG) nanoparticles appear to be a promising form for delivery of therapeutic DNA to photoreceptor cells , 2015. HRNA editing by recruitment of endogenous ADAR is a promising method of harnessing the endogenous posttranscriptional A-to-I editing ADAR enzyme to correct pathogenic SNVs on a transcriptional level without the safety risks of inducing permanent genomic off-target effects. When compared to RNA editing with exogenous ADAR delivery, RNA editing with native ADAR shows three main advantages: (1) Since it requires only the delivery of a nucleic acid sequence and does not require co-delivery of a protein, it harbours potential to decrease immune response to foreign proteins of bacterial origin. (2) It frees up packaging capacity in delivery vehicles such as an AAV, which is limited to 4.7 kB. (3) Lastly, endogenous ADARs achieves target editing with significantly less transcriptome-wide off-target editing compared with strategies relying on ADAR overexpression. Generally, there is a trade-off between achieving high on-target editing with ADAR overexpression and transcriptome wide off-target effects, even when a system is used that only overexpresses the deaminase domain of ADAR (and does not include the dsRBDs). Creating a gRNA that can recruit endogenous ADAR to engage in site-selective RNA editing at high enough levels to show a response at a functional protein level is the goal of gRNA optimisation. Three main caveats should be considered before applying this strategy to a tissue such as the retina. (1) Target tissues and cell types within those target tissues must express ADAR enzymes for editing to occur. Since many of the more recently described ADARs have been optimised for recruitment of the ubiquitously expressed ADAR1p110, the inquiry into ADAR expression needs to establish not only if ADAR expression is present but if it is present at sufficient levels for editing to occur since the gRNA:targetRNA duplex is in competition with natural substrates of endogenous ADARs. (2) Apart from the self-evident sequence limitation that RNA editing with endogenous ADAR can only target G > A SNVs, the target is also limited by the sequence preference of native ADAR, which makes editing of 5’-GAN-3′ motifs particularly inefficient. (3) Thirdly, a feasible delivery to target cells must be considered. Chemically modified gRNA cannot be genetically encoded and packaged into a viral vector and delivery of a naked chemically modified gRNA is only feasible if the gRNA is short. If ADAR expression is low in the target tissue, if the target sequence has a 5′-G and if the target base requires cytidine rather than adenine deamination, RNA editing with exogenous ADAR should be considered the more viable option.in vivo, where an antisense region was able to elicit significant, but low editing. Overall, this study showed that recruitment of endogenous ADAR was possible with an antisense domain of at least 60 bp, but the approach was limited due to low target efficiency and was far outperformed by approaches utilising ADAR overexpression, in particular overexpression of the hyperactive ADAR (E488Q) mutant, with full length ADAR or just the deaminase domain. In the same year, two methods were established that enabled efficient gRNA recruitment in vitro, LEAPER and RESTORE. LEAPER was able to show editing rates up to 80% in vitro by lengthening the antisense domain to 150 bp and beyond, but was unsurprisingly hampered by significant bystander effects, given ADARs propensity for deamination of perfectly paired dsRNA. RESTORE showed encouraging editing rates in vitro using the original GluR2 design not by lengthening the antisense domain, but by fully chemically modifying the invariant and the antisense domain of the gRNA and thus increasing the binding affinity of the gRNA  for chemically modified mRNA have not yet been established in this setting. In an exciting new development, circularisation of the LEAPER arRNA mark another increase in on-target editing efficiency for RNA editing with endogenous ADAR. It should be noted that throughout both Yi et al. and Katrekar et al. while by and large circularisation dramatically improved editing efficiencies, the target efficiencies varied greatly between targets and cell lines. Within the same target gene transcript, editing results varied greatly, which perhaps points to the sequence preference and structural selectivity that native ADAR exhibits. However, even with low initial target efficiencies, it could be argued that due to the increased stability and longer half-life, a circularised gRNA could still outperform an equivalent linear version due to sustained editing over time, an effect essential for RNA based therapies. Circularised gRNA also showed robust and sustained editing rates in both wild-type and disease mouse models. From a delivery perspective, both groups used genetically encoded gRNA packaged into an AAV, a highly relevant mode of delivery for IRDs. Future modifications of circular gRNA might include further stabilisation by chemical modification or addition of recruitment segments. A further exciting development was shown in Monian’s et al. proof-of-concept study demonstrated very promising, high target efficiency both 2 days post AIMer delivery (up to 50%) and over 1 month time period (up to 40%) without bystander and with minimal off-target effects. It remains to be seen if these results can be replicated on a protein and phenotype level targeting a coding sequence in a disease-causing mutation. Katrekar et al. experienced a drop in efficacy when moving from a wild type non-coding sequence to disease causing transcript target. For retinal application, these short, 30 nt AIMers are of particular interest since they hold promise of a straightforward intravitreal delivery, whereas the length of previous chemically modified gRNA such as RESTORE would require delivery in a non-viral packaging vehicle such as a lipid nanoparticle. While these are promising, their primary target has to date been the liver, an organ that by its nature is designed for optimal uptake (similarly to the RPE). It should be noted that all reviewed trials looking for therapeutic efficacy of RNA editing with endogenous ADAR in vivo have been designed to target the liver, and therapeutic efficiacy in other organs has yet to be established. While there have been reports of using Mini-dCas13X for Duchenne muscular dystrophy  and the NIHR Oxford Biomedical Research Centre . 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