Comparing a Head-Mounted Smartphone Visual Field Analyzer to Standard Automated Perimetry in Glaucoma: A Prospective Study.

PRECIS: Wang et al compare an FDA-registered head-mounted smartphone device (PalmScan VF2000) with standard automated perimetry (SAP) in glaucoma patients and find that the head-mounted device may not fully recapitulate SAP testing. PURPOSE: This study prospectively compared visual field testing using the PalmScan VF2000 Visual Field Analyzer, a head-mounted smartphone device, with standard automated perimetry (SAP). METHODS: Patients with glaucoma undergoing Humphrey Field Analyzer SAP testing were asked to complete in-office PalmScan testing using a Samsung S5 smartphone in a virtual reality-style headset. Glaucoma severity was defined as SAP mean deviation (MD) >-6 dB for mild, between -6 and -12 dB for moderate, and <-12 dB for severe. Global parameters MD and pattern standard deviation (PSD) from PalmScan and SAP were compared using t-tests and Bland-Altman analyses. Bland-Altmann analyses of PalmScan and SAP MD were conducted for the superonasal, superotemporal, inferonasal, and inferotemporal visual field quadrants. The repeatability of PalmScan was assessed using Spearman's correlations and intraclass correlation coefficients (ICCs). RESULTS: Fifty-one patients (51 eyes) completed both SAP and PalmScan testing and met criteria for analysis. Compared to SAP, global MD and PSD measurements from PalmScan differed by an average of +0.62±0.26 dB (range: -3.25 to +4.60 dB) and -1.00±0.24 dB (range: -6.03 to +2.77 dB), respectively, while MD scores from individual visual field quadrants differed by as much as -6.58 to +11.43 dB. Agreement of PalmScan and SAP in classifying glaucoma severity was 86.3% across all eyes. PalmScan and SAP identified the same quadrant as having the worst visual field defect in 66.7% of eyes. CONCLUSIONS: Despite advantages in cost and accessibility, the PalmScan head-mounted perimetry device may not be able to fully recapitulate SAP testing.

Organ- and Cell-Selective Delivery of mRNA In Vivo Using Guanidinylated Serinol Charge-Altering Releasable Transporters.

Selective RNA delivery is required for the broad implementation of RNA clinical applications, including prophylactic and therapeutic vaccinations, immunotherapies for cancer, and genome editing. Current polyanion delivery relies heavily on cationic amines, while cationic guanidinium systems have received limited attention due in part to their strong polyanion association, which impedes intracellular polyanion release. Here, we disclose a general solution to this problem in which cationic guanidinium groups are used to form stable RNA complexes upon formulation but at physiological pH undergo a novel charge-neutralization process, resulting in RNA release. This new delivery system consists of guanidinylated serinol moieties incorporated into a charge-altering releasable transporter (GSer-CARTs). Significantly, systematic variations in structure and formulation resulted in GSer-CARTs that exhibit highly selective mRNA delivery to the lung (∼97%) and spleen (∼98%) without targeting ligands. Illustrative of their breadth and translational potential, GSer-CARTs deliver circRNA, providing the basis for a cancer vaccination strategy, which in a murine model resulted in antigen-specific immune responses and effective suppression of established tumors.

VISUAL OUTCOMES AFTER INTERNAL LIMITING MEMBRANE PEELING VERSUS FLAP IN THE CLOSURE OF FULL-THICKNESS MACULAR HOLES.

PURPOSE: To examine postoperative outcomes of internal limiting membrane peeling (ILMP) versus flap (ILMF) in the closure of full-thickness macular holes. METHODS: Retrospective chart review of patients who underwent pars plana vitrectomy and gas tamponade with ILMP or ILMF to close full-thickness macular hole at the Atrium Health Wake Forest Baptist from January 2012 to October 2022 with at least 3 months follow-up. Main outcome measures were type 1 primary full-thickness macular hole closure and postoperative best-corrected visual acuity in mean logMAR. RESULTS: One hundred thirty and 30 eyes underwent ILMP and ILMF, respectively. There were no significant differences in baseline characteristics between the groups. Ninety-six percent of ILMP eyes and 90% of ILMF eyes achieved primary hole closure ( P = 0.29). Among all eyes with primary hole closure, best-corrected visual acuity at 1 year was not different between the groups, but when stratified by lens status, it was superior in the ILMP versus ILMF group in pseudophakic eyes: the estimated least-squares mean best-corrected visual acuity (Snellen equivalent) (95% confidence interval) was 0.42 (20/50) (0.34, 0.49) in the ILMP group and 0.71 (20/100) (0.50, 0.92) in the ILMF group. CONCLUSION: Internal limiting membrane peeling and ILMF techniques yielded similarly high full-thickness macular hole closure rates. In pseudophakic eyes with primary hole closure, ILMF eyes had worse best-corrected visual acuity at 1 year.

High temporal frequency light response in mouse retina is mediated by ON and OFF bipolar cells and requires FAT3 signaling.

Vision is initiated by the reception of light by photoreceptors and subsequent processing via parallel retinal circuits. Proper circuit organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. Indeed, similar deficits were observed in mice lacking the bipolar cell glutamate receptors GRIK1 (OFF-bipolar cells) and GRM6 (ON-bipolar cells). Mechanistically, FAT3 binds to the synaptic protein PTPσ and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. How FAT3 impacts ON-cone bipolar cell function at high temporal frequency remains to be uncovered. These findings expand the repertoire of FAT3's functions and reveal the importance of both ON- and OFF-bipolar cells for high frequency light response.

Charge-altering releasable transporters enhance mRNA delivery in vitro and exhibit in vivo tropism.

The introduction of more effective and selective mRNA delivery systems is required for the advancement of many emerging biomedical technologies including the development of prophylactic and therapeutic vaccines, immunotherapies for cancer and strategies for genome editing. While polymers and oligomers have served as promising mRNA delivery systems, their efficacy in hard-to-transfect cells such as primary T lymphocytes is often limited as is their cell and organ tropism. To address these problems, considerable attention has been placed on structural screening of various lipid and cation components of mRNA delivery systems. Here, we disclose a class of charge-altering releasable transporters (CARTs) that differ from previous CARTs based on their beta-amido carbonate backbone (bAC) and side chain spacing. These bAC-CARTs exhibit enhanced mRNA transfection in primary T lymphocytes in vitro and enhanced protein expression in vivo with highly selective spleen tropism, supporting their broader therapeutic use as effective polyanionic delivery systems.

Telemedicine retinopathy of prematurity severity score (TeleROP-SS) versus modified activity score (mROP-ActS) retrospective comparison in SUNDROP cohort.

Identifying and planning treatment for retinopathy of prematurity (ROP) using telemedicine is becoming increasingly ubiquitous, necessitating a grading system to help caretakers of at-risk infants gauge disease severity. The modified ROP Activity Scale (mROP-ActS) factors zone, stage, and plus disease into its scoring system, addressing the need for assessing ROP's totality of binocular burden via indirect ophthalmoscopy. However, there is an unmet need for an alternative score which could facilitate ROP identification and gauge disease improvement or deterioration specifically on photographic telemedicine exams. Here, we propose such a system (Telemedicine ROP Severity Score [TeleROP-SS]), which we have compared against the mROP-ActS. In our statistical analysis of 1568 exams, we saw that TeleROP-SS was able to return a score in all instances based on the gradings available from the retrospective SUNDROP cohort, while mROP-ActS obtained a score of 80.8% in right eyes and 81.1% in left eyes. For treatment-warranted ROP (TW-ROP), TeleROP-SS obtained a score of 100% and 95% in the right and left eyes respectively, while mROP-ActS obtained a score of 70% and 63% respectively. The TeleROP-SS score can identify disease improvement or deterioration on telemedicine exams, distinguish timepoints at which treatments can be given, and it has the adaptability to be modified as needed.

High-Yield, Case-Based, Interactive Workshop on Telehealth and Teleneurology With Pediatric Resident Physicians.

Introduction: Increasing prevalence of neurologic disorders with an aging global population and limited availability of neurologists may lead to worse patient outcomes. As a result of the COVID-19 pandemic, telehealth services surged, and despite easing public health measures, the demand has remained. Telehealth technology has the potential to close the physical gaps in expanding the reach of care. This academic half-day workshop sought to provide a learning opportunity in response to these concerns. Methods: The workshop consisted of small- and large-group case discussions among pediatric resident physicians (PGY 1-PGY 3) moderated by two child neurology faculty physicians over Zoom. Participants received a learner document with prereading articles and questions for each case. PowerPoint presentations with video demonstrations were used to introduce the cases and guide discussions. Results: Of the 25 attendees, 14 (56% response rate) answered a nonmandatory postsession survey. Eighty-six percent of the respondents were very or extremely satisfied with the content covered and were similarly satisfied with the effectiveness of content delivery. Seventy-nine percent of the respondents found the content helpful or very helpful in preparation for the board, and 93% anticipated applying the content covered occasionally or frequently in their clinical practice. Discussion: Small-group discussions with video demonstrations are helpful in increasing proficiency with telehealth technology and in examining board-relevant cases on pediatric patients. There is strong interest in subsequent telehealth half-day workshops that incorporate teaching through group discussions on relevant patient case scenarios.

Chromophore supply modulates cone function and survival in retinitis pigmentosa mouse models.

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.

Engineering circular RNA for enhanced protein production.

Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.

Effective field of view of wide-field fundus photography in the Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP).

Five-field 130° wide-angle imaging is the standard of care for retinopathy of prematurity (ROP) screening with an ideal hypothetical composite field-of-view (FOV) of 180°. We hypothesized that in many real-world scenarios the effective composite FOV is considerably less than ideal. This observational retrospective study analyzed the effective FOV of fundus photos of patients screened for ROP as part of the Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP) initiative. Five fundus photos were selected from each eye per image session. Effective FOV was defined as the largest circular area centered on the optic disc that encompassed retina in each of the four cardinal views. Seventy-three subjects were analyzed, 35 without ROP and 34 with ROP. Mean effective FOV was 144.55 ± 6.62° ranging from 130.00 to 153.71°. Effective FOV was not correlated with the presence or absence of ROP, gestational age, birth weight, or postmenstrual age. Mean effective FOV was wider in males compared to females. Standard five-field 130° fundus photos yielded an average effective FOV of 144.54° in the SUNDROP cohort. This implies that an imaging FOV during ROP screening considerably less than the hypothetical ideal of 180° is sufficient for detecting treatment warranted ROP.

Single-cell multiome of the human retina and deep learning nominate causal variants in complex eye diseases.

Genome-wide association studies (GWASs) of eye disorders have identified hundreds of genetic variants associated with ocular disease. However, the vast majority of these variants are noncoding, making it challenging to interpret their function. Here we present a joint single-cell atlas of gene expression and chromatin accessibility of the adult human retina with more than 50,000 cells, which we used to analyze single-nucleotide polymorphisms (SNPs) implicated by GWASs of age-related macular degeneration, glaucoma, diabetic retinopathy, myopia, and type 2 macular telangiectasia. We integrate this atlas with a HiChIP enhancer connectome, expression quantitative trait loci (eQTL) data, and base-resolution deep learning models to predict noncoding SNPs with causal roles in eye disease, assess SNP impact on transcription factor binding, and define their known and novel target genes. Our efforts nominate pathogenic SNP-target gene interactions for multiple vision disorders and provide a potentially powerful resource for interpreting noncoding variation in the eye.

Targeting Microglia to Treat Degenerative Eye Diseases.

Microglia have been implicated in many degenerative eye disorders, including retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment. While the exact roles of microglia in these conditions are still being discovered, evidence from animal models suggests that they can modulate the course of disease. In this review, we highlight current strategies to target microglia in the eye and their potential as treatments for both rare and common ocular disorders. These approaches include depleting microglia with chemicals or radiation, reprogramming microglia using homeostatic signals or other small molecules, and inhibiting the downstream effects of microglia such as by blocking cytokine activity or phagocytosis. Finally, we describe areas of future research needed to fully exploit the therapeutic value of microglia in eye diseases.

Modeling absolute zone size in retinopathy of prematurity in relation to axial length.

Treatment outcomes in retinopathy of prematurity (ROP) are closely correlated with the location (i.e. zone) of disease, with more posterior zones having poorer outcomes. The most posterior zone, Zone I, is defined as a circle centered on the optic nerve with radius twice the distance from nerve to fovea, or subtending an angle of 30 degrees. Because the eye enlarges and undergoes refractive changes during the period of ROP screening, the absolute area of Zone I according to these definitions may likewise change. It is possible that these differences may confound accurate assessment of risk in patients with ROP. In this study, we estimated the area of Zone I in relation to different ocular parameters to determine how variability in the size and refractive power of the eye may affect zoning. Using Gaussian optics, a model was constructed to calculate the absolute area of Zone I as a function of corneal power, anterior chamber depth, lens power, lens thickness, and axial length (AL), with Zone I defined as a circle with radius set by a 30-degree visual angle. Our model predicted Zone I area to be most sensitive to changes in AL; for example, an increase of AL from 14.20 to 16.58 mm at postmenstrual age 32 weeks was calculated to expand the area of Zone I by up to 72%. These findings motivate several hypotheses which upon future testing may help optimize treatment decisions for ROP.

Augmentation of CD47/SIRPα signaling protects cones in genetic models of retinal degeneration.

Inherited retinal diseases, such as retinitis pigmentosa (RP), can be caused by thousands of different mutations, a small number of which have been successfully treated with gene replacement. However, this approach has yet to scale and may not be feasible in many cases, highlighting the need for interventions that could benefit more patients. Here, we found that microglial phagocytosis is upregulated during cone degeneration in RP, suggesting that expression of "don't-eat-me" signals such as CD47 might confer protection to cones. To test this, we delivered an adeno-associated viral (AAV) vector expressing CD47 on cones, which promoted cone survival in 3 mouse models of RP and preserved visual function. Cone rescue with CD47 required a known interacting protein, signal regulatory protein α (SIRPα), but not an alternative interacting protein, thrombospondin-1 (TSP1). Despite the correlation between increased microglial phagocytosis and cone death, microglia were dispensable for the prosurvival activity of CD47, suggesting that CD47 interacts with SIRPα on nonmicroglial cells to alleviate degeneration. These findings establish augmentation of CD47/SIRPα signaling as a potential treatment strategy for RP and possibly other forms of neurodegeneration.

A Cross-Sectional Cohort Study of the Effects of FGF23 Deficiency and Hyperphosphatemia on Dental Structures in Hyperphosphatemic Familial Tumoral Calcinosis.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder caused by mutations in FGF23, GALNT3, KLOTHO, or FGF23 autoantibodies. Prominent features include high blood phosphate and calcific masses, usually adjacent to large joints. Dental defects have been reported, but not systematically described. Seventeen patients with HFTC followed at the National Institutes of Health underwent detailed clinical, biochemical, molecular, and dental analyses. Studies of teeth included intraoral photos and radiographs, high-resolution µCT, histology, and scanning electron microscopy (SEM). A scoring system was developed to assess the severity of tooth phenotype. Pulp calcification was found in 13 of 14 evaluable patients. Short roots and midroot bulges with apical thinning were present in 12 of 13 patients. Premolars were most severely affected. µCT analyses of five HFTC teeth revealed that pulp density increased sevenfold, whereas the pulp volume decreased sevenfold in permanent HFTC teeth compared with age- and tooth-matched control teeth. Histology revealed loss of the polarized odontoblast cell layer and an obliterated pulp cavity that was filled with calcified material. The SEM showed altered pulp and cementum structures, without differences in enamel or dentin structures, when compared with control teeth. This study defines the spectrum and confirms the high penetrance of dental features in HFTC. The phenotypes appear to be independent of genetic/molecular etiology, suggesting hyperphosphatemia or FGF23 deficiency may be the pathomechanistic driver, with prominent effects on root and pulp structures, consistent with a role of phosphate and/or FGF23 in tooth development. Given the early appearance and high penetrance, cognizance of HFTC-related features may allow for earlier diagnosis and treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disease affecting >20 million people worldwide. Loss of daylight vision typically occurs due to the dysfunction/loss of cone photoreceptors, the cell type that initiates our color and high-acuity vision. Currently, there is no effective treatment for RP, other than gene therapy for a limited number of specific disease genes. To develop a disease gene-agnostic therapy, we screened 20 genes for their ability to prolong cone photoreceptor survival in vivo. Here, we report an adeno-associated virus vector expressing Txnip, which prolongs the survival of cone photoreceptors and improves visual acuity in RP mouse models. A Txnip allele, C247S, which blocks the association of Txnip with thioredoxin, provides an even greater benefit. Additionally, the rescue effect of Txnip depends on lactate dehydrogenase b (Ldhb) and correlates with the presence of healthier mitochondria, suggesting that Txnip saves RP cones by enhancing their lactate catabolism.

Retinitis pigmentosa is an inherited eye disease affecting around one in every 4,000 people. It results from genetic defects in light sensitive cells of the retina, called photoreceptor cells, which line the back of the eye. Though vision loss can occur from birth, retinitis pigmentosa usually involves a gradual loss of vision, sometimes leading to blindness. Rod photoreceptors, which are responsible for vision in low light, are impacted first. The disease then affects cone photoreceptors, the cells that detect light during the day, providing both color and sharp vision. Around 100 mutated genes associated with retinitis pigmentosa have been identified, but only a handful of families with one of these mutant genes have been treated with a gene therapy specific for their mutated gene. There are currently no therapies available to treat the vast number of people with this disease. The mutations that cause retinitis pigmentosa directly affect the rod cells that detect dim light, leading to loss of night vision. There is also an indirect effect that causes cone photoreceptors to stop working and die. One theory to explain this two-step disease process relates to the fact that cone photoreceptors are very active cells, requiring a high level of energy, nutrients and oxygen. If surrounding rod cells die, cone photoreceptors may be deprived of some essential supplies, leading to cone cell death and daylight vision loss. To examine this theory, Xue et al. tested a new gene therapy designed to alleviate the potential shortfall in nutrients. The experiments used three different strains of mice that had the same genetic mutations as humans with retinitis pigmentosa. The gene therapy used a virus, called adeno-associated virus (AAV), to deliver 20 different genes to cone cells. Each of the 20 genes tested plays a different role in cells' processing of nutrients to provide energy. After administering the treatment, Xue et al. monitored the mice to see whether or not their vision was affected, and how cone cells responded. Only one of the 20 genes, Txnip, delivered using gene therapy, had a beneficial effect, prolonging cone cell survival in all three mouse strains. The mice that received Txnip also retained their ability to discern moving stripes on vision tests. Further investigations demonstrated that activating Txnip forced the cones to start using a molecule called lactate as an energy source, which could be more available to them than glucose, their usual fuel. These cells also had healthier mitochondria ­ the compartments inside cells that produce and manage energy supplies. This dual effect on fuel use and mitochondrial health is thought to be the basis for the extended cone survival and function. These experiments by Xue et al. have identified a good gene therapy candidate for treating retinitis pigmentosa independently of which genes are causing the disease. Further research will be required to test the safety of the gene therapy, and whether its beneficial effects translate to humans with retinitis pigmentosa, and potentially other diseases with unhealthy photoreceptors.

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa.

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).

In Situ Detection of Adeno-associated Viral Vector Genomes with SABER-FISH.

Gene therapy with recombinant adeno-associated viral (AAV) vectors is a promising modality for the treatment of a variety of human diseases. Nonetheless, there remain significant gaps in our understanding of AAV vector biology, due in part to the lack of robust methods to track AAV capsids and genomes. In this study, we describe a novel application of signal amplification by exchange reaction fluorescence in situ hybridization (SABER-FISH) that enabled the visualization and quantification of individual AAV genomes after vector administration in mice. These genomes could be seen in retinal cells within 3 h of subretinal AAV delivery, were roughly full length, and correlated with vector expression in both photoreceptors and the retinal pigment epithelium. SABER-FISH readily detected AAV genomes in the liver and muscle following retro-orbital and intramuscular AAV injections, respectively, demonstrating its utility in different tissues. Using SABER-FISH, we also found that retinal microglia, a cell type deemed refractory to AAV transduction, are in fact efficiently infected by multiple AAV serotypes, but appear to degrade AAV genomes prior to nuclear localization. Our findings show that SABER-FISH can be used to visualize AAV genomes in situ, allowing for studies of AAV vector biology and the tracking of transduced cells following vector administration.