Biology subjects

Tucker, B. A.

Publications and source records attributed to Tucker, B. A..

17 recordsLinked to original sources

bioRxiv2026

Validation and testing of an in vitro model to study medical treatments for anterior urethral stricture disease: assessing the potential efficacy of phosphodiesterase-4 (PDE4) inhibition and testosterone

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

pharmacology and toxicology↗

bioRxiv2026

Using Patient iPSC-derived Retinal Pigment Epithelial Cells to Evaluate Differential Susceptibility to MEK Inhibitor-Associated Retinopathy

PurposeCompare the effect of MEK inhibition on iPSC-derived retinal pigmental epithelial (RPE) cells generated from a patient who developed MEK inhibitor-Associated Retinopathy (MEKAR) versus a patient who did not develop retinopathy. DesignCase-control SubjectsTwo female patients with Neurofibromatosis Type 1 who were treated with MEK inhibitors. One patient developed MEKAR, the other did not. MethodsRPE were generated from human induced pluripotent stem cells (hiPSCs) from these two patients. These hiPSC-derived RPE were treated with selumetinib for 10 days. Main Outcome MeasuresPhagocytic activity and changes in gene expression ResultsAs previously reported, there was a significant increase in internalized rhodopsin in phagocytosis assays, yet this was only found in hiPSC-derived RPE from the patient who developed MEKAR. Selumetinib decreased expression of genes related to fluid transport and cell volume, including aquaporins and solute transporters. At baseline, cells from the patients without MEKAR had higher expression of these genes. Interestingly, selumetinib-induced changes in gene expression only reached statistical significance in cells from the patient who did not develop MEKAR, suggesting these changes may be a compensatory protective mechanism. Patients susceptible to forming MEKAR may have increased phagocytosis without a compensatory change in expression of genes related to fluid flux, thereby inhibiting their ability to transport fluid out of the subretinal space. ConclusionsMEK inhibitor-Associated Retinopathy may only affect susceptible patients whose retinal pigment epithelium cannot sufficiently regulate expression of genes related to fluid transport and cell volume, altering the ability of these cells to properly function.

pharmacology and toxicology↗

bioRxiv2026

Single-Cell Gene Expression and eQTL Analyses in the Human Retina, RPE, and Choroid in Macular Degeneration

Age-related macular degeneration (AMD) is a common, complex disease affecting older individuals that can lead to severe vision loss. It is characterized by early anatomical changes in the retina, retinal pigment epithelium (RPE), and choroid, especially in the central (macular) region. AMD can progress to severe atrophy and/or pathologic angiogenesis that leads to visual decline. Over 30 genetic loci have been identified as contributing to AMD risk; however, the mechanisms by which genetic variants affect pathology has not been thoroughly explored. In this report we examined single-nucleus gene expression in the retina, RPE and choroid of 88 individuals categorized by AMD stage, as well as 36 previously published samples. Genotyping was performed on 1.8 million SNPs, with additional SNPs imputed, on each donor to identify expression quantitative trait loci (eQTLs). We found that two AMD-risk loci (PILRB and ARMS2/HTRA1) affected the expression of PILRB and HTRA1, respectively. The risk allele of PILRB was associated with increased PILRB RNA in cones, fibroblasts, choroidal resident macrophages, and RPE, whereas the HTRA1 risk locus was associated with decreased HTRA1 RNA in the RPE. We also identified an age-related decrease in complement inhibitors in the choriocapillaris, a tissue susceptible to complement mediated damage in AMD. We have made the eQTL and gene expression data fully available on the web resource Spectacle for rapid and interactive data access.

cell biology↗

bioRxiv2026

Metabolic Analysis of Human Retinal Pigment Epithelium and Choroid Tissue in Aging and Macular Degeneration

Age-related macular degeneration is a common ocular disease that causes vision loss in the elderly, with a complex set of risk factors and proposed mechanisms of pathogenesis. A powerful method for investigating changes in disease is metabolomics, by which small molecules can be identified and quantified simultaneously. We report here the metabolic analysis of human RPE-choroid tissue in aging and macular degeneration (AMD), as well as comparisons of human macular and extramacular RPE-choroid and neural retina. Levels of 215 metabolites were determined in young donors, AMD donors (early/intermediate, geographic atrophy, and neovascularization) and age-matched controls. The largest number of metabolite differences were observed between young and healthy aged controls, as opposed to between aged controls and any stage of AMD. Two notable metabolites found to be increased in aging choroids are trimethylamine N-oxide and uric acid, both of which were significant after Bonferroni correction. A mouse endothelial cell line treated with a high concentration of uric acid exhibited reduced migration in a wound closure assay. This study provides initial insights into the metabolome of human choroids in varying states of age and macular degeneration, as well as functional implications of these changes in the aging choroid.

cell biology↗

bioRxiv2025

Acriflavine delivery via Polyurethane nanocapsules to treat neovascular age-related macular degeneration

Choroidal neovascularization is a complication associated with retinal diseases such as age-related macular degeneration (AMD), a leading cause of vision loss in the developed world. Choroidal neovascular membrane (CNVM) refers to the abnormal growth of blood vessels in the retina which results in exudation and/or hemorrhage, leading to photoreceptor damage and vision loss. Currently first-line treatment for CNVM include intravitreal injections of vascular endothelial growth factor (VEGF)-binding antibodies that prevent the growth of these leaky blood vessels. Unfortunately, anti-VEGF drugs often require frequent injections, and prolonged VEGF inhibition has been associated with retinal atrophy and decreased long term effectiveness in some patients. This study presents the use of Acriflavine, a small molecule HIF1 inhibitor loaded polyurethane nanocapsules to treat CNVM in a rat model. Fourteen days following laser injury and intravitreal drug administration, CMVM size was significantly reduced in acriflavine nanocapsule and free acriflavine treated animals as compared to drug free controls. Moreover, acriflavine nanocapsules reduce CNVM incidence compared to drug free controls by approximately 25%. Among the different delivery routes tested, intravitreal delivery of acriflavine nanocapsules was found to be superior to subretinal and suprachoroidal delivery for reducing CNVM area without causing significant damage to the neural retina. This paper presents the synthesis, characterization and the effectiveness of the polyurethane based acriflavine delivery system in treating choroidal neovascularization.

pharmacology and toxicology↗

bioRxiv2025

Modeling MEK-inhibitor Associated Retinopathy in vitro using human induced-1 pluripotent stem cell-derived retinal pigment epithelial cells

Pharmacologic inhibitors of MEK are important anti-cancer drugs but can result in MEK inhibitor-Associated Retinopathy (MEKAR) in which vision is lost due to serous retinal detachments that form via an unknown mechanism. We hypothesized that the cause of this side effect is drug-induced dysfunction of retinal pigment epithelial (RPE) cells. To test this hypothesis, we used human induced pluripotent stem cell-derived RPE cells. We treated mature, hiPSC-derived RPE cells with selumetinib and measured impacts on RPE-specific function, structure, and gene expression. Selumetinib increases the ability of hiPSC-derived RPE to internalize bovine rod outer segments (1.9 vs 3.0, p=0.0024). It also decreases expression of aquaporin 1 during the first 10 days of treatment (2.7 vs 1.1, p=0.0015). It has no effect on the ability of hiPSC-derived RPE to maintain membrane integrity. Selumetinib alters gene expression of hiPSC-derived RPE, with significant changes in genes involved in transport of ions and small molecules regulating cell volume and lysosomal acidification. Selumetinib may lead to subretinal fluid accumulation by both increasing secretions into this space and decreasing outflow.

molecular biology↗

bioRxiv2025

Development of a Targeted Choroidal Injury Model for the Study of Retinal Degenerations and Therapeutic Cell Replacement

PurposeChoroidal loss is an important pathophysiological step in many retinal diseases, but few reliable translational models of choroidal injury exist. Here, we report a new targeted choroidal injury model using bioconjugated saporins and compare it models of systemic sodium iodate administration. MethodsWild-type Sprague-Dawley rats were given suprachoroidal injections of anti-CD38 or anti-CD105 antibodies conjugated to saporin immunotoxin (10 {micro}l at 0.05 {micro}g/{micro}L) to induce selective choroidal endothelial cell injury. These animals were compared to wild-type rats given sodium iodate (75 mg/kg) via tail vein injections, with a dose escalation study (25, 50, and 75 mg/kg) in immunocompromised (Sprague-Dawley Rag2/Il2g double-knockout) rats. Animals were examined at 1-, 2-, and 3-weeks post-treatment, and the degree of choroidal injury compared using fundus photography, optical coherence tomography, and immunohistochemistry. ResultsSuprachoroidal administration of anti-CD38 or anti-CD105 saporins resulted in severe choroidal vascular injury localized to the injection site, without damage to adjacent choroidal vasculature, progressive injury over time, or development of choroidal neovascularization. By contrast, sodium iodate treated animals had rapid, diffuse choroidal loss which progressed throughout the study time points, with fatal systemic side effects at the highest (75 mg/kg) dose. ConclusionsSuprachoroidal injection of anti-CD38 and anti-CD105 saporins results in targeted, localized, non-progressive choroidal injury in rats. These models offer alternatives to systemic sodium iodate administration, which causes diffuse, progressive choroidal injury. Translational RelevanceImmunotoxin-based models of targeted choroidal injury may be useful for understanding pathways of retinal degeneration and facilitating development of therapies for diseases involving choroidal cell loss.

neuroscience↗

bioRxiv2025

Sialoglycoconjugate Profiling of Human Choroid, Retinal Pigment Epithelium, and Macular Degeneration Related Lesions

Age-related macular degeneration is a leading cause of central vision loss in the elderly. Early hallmarks of the disease include basal laminar deposit and choriocapillaris degeneration. The location and composition of sialoglycoconjugates in healthy and diseased choroid and disease-related lesions have not been thoroughly examined. This study utilized lectins to examine sialoglycoconjugates in human tissue, specifically Sambucus nigra/Elderberry Bark Lectin (EBL) and Maackia amurensis lectin II (MAL-II), to examine -2,6 and -2,3 sialic acids, respectively. EBL and MAL-II both label the choroid and basal laminar deposit, with slightly different patterns. Whereas MAL-II predominantly labels the choriocapillaris endothelium, EBL also labels Bruchs membrane and extracellular domains surrounding the vasculature (intercapillary pillars). EBL labeling overlaps with the distribution of complement factor H to a greater extent than MAL-II. After treatment with neuraminidase to remove terminal sialic acids, a battery of lectins was applied to sections of choroids. Lectins that recognize {beta}-galactose, N-acetyllactosamine, galactose ({beta}-1,3) N-acetylgalactosamine, and - or {beta}-N-acetylgalactosamine showed increased reactivity, including increased labeling of glycans in basal laminar deposits. This study provides insight into the location and partial identities of sialoglycoconjugates in the human choroid, with possible implications for the pathogenesis of macular degeneration.

cell biology↗

bioRxiv2025

Production of clinical grade patient iPSC-derived 3D retinal organoids containing transplantable photoreceptor cells

Neurodegenerative conditions that affect the retina are currently the leading cause of incurable blindness in the developed world. Although gene and drug therapies are being developed to slow disease progression in some cases, restorative cell replacement approaches are needed for patients with significant vision impairment due to retinal degeneration. While a variety of different cell types have been evaluated in the context of retinal cell replacement, induced pluripotent stem cells (iPSCs), which can be generated and delivered as an autologous therapeutic, are in many ways the most attractive donor cell source currently available. Like embryonic stem cells, iPSCs must be differentiated into the target therapeutic cell type prior to transplantation. For instance, for patients with retinitis pigmentosa who have primary photoreceptor cell disease, photoreceptor cell derivation and enrichment are required prior to transplantation. Although other effective retinal differentiation protocols exist, they are often not fully compatible with clinical manufacturing. In this study, we report development of a xeno-free 3D retinal differentiation protocol based on the most robust adherent/non-adherent 3D differentiation strategies published to date. In addition, we demonstrate that while iPSC reprogramming efficiency is enhanced under reduced oxygen tension (i.e., 5%), efficient embryoid body and subsequent retinal organoid production require standard oxygen levels (i.e., 21%). Finally, we show that photoreceptor precursor cells obtained from 3D retinal organoids derived using the developed protocol under current good manufacturing practices (cGMP) survive in the subretinal space of dystrophic Pde6b-null rats for 1-month post-transplantation and form new synaptic connections with host bipolar neurons.

neuroscience↗

bioRxiv2024

Development of self-healing hydrogels to support choroidal endothelial cell transplantation for the treatment of early age related macular degeneration

In retinal diseases such as age-related macular degeneration (AMD) and choroideremia, a key pathophysiologic step is loss of endothelial cells of the choriocapillaris, the dense vascular bed required for maintaining health and function of the retina. As such, repopulation of choroidal vasculature early in the disease process may halt disease progression. Prior studies have shown that injection of donor cells in suspension results in significant cellular efflux and poor cell survival. As such, the goal of this study was to develop a hydrogel system designed to support CEC transplantation. A library of hydrogels was synthesized using laminin (i.e., LN111, LN121, and LN421), carboxy methyl chitosan, and oxidized dextran via reversible Schiff base chemistry. Each of the developed hydrogels was readily injectable into the suprachoroidal space, with excellent gelation, mechanical, and degradation properties. Laminin-based hydrogels were compatible with immortalized CEC survival in vitro, and suprachoroidal injection of LN111 and LN121 containing gels were well-tolerated in an in vivo rat model, whereas LN421 containing gels caused significant chorioretinal inflammation. Hydrogels were detected in the suprachoroidal space of immunosuppressed rats at 1-week post-injection and were completely resorbed by 1-month post-injection. There were significantly more CECs retained in immunosuppressed rats that received cell laden hydrogels than those that received unsupported cell suspensions (i.e., CECs only). These findings pave the way for future CEC replacement studies in animal models of choroidal cell loss toward the development of future therapies. Statement of significanceAge related macular degeneration (AMD) is a leading cause of untreatable blindness in the industrial world. A key pathologic step in AMD is loss of the choriocapillaris endothelial cells, which provide vascular support to the overlying retina, including the light-sensing photoreceptors. We believe that choroidal cell replacement early in disease may prevent retinal cell death and subsequent vision loss. In this study, we present a strategy for repopulating the choriocapillaris using choroidal endothelial cell laden hydrogels. Specifically, we demonstrate the synthesis and characterization of 3 different laminin-based hydrogel systems. LN111 and LN121 hydrogels were found to increase retention of choroidal endothelial cells following suprachoroidal transplantation. These findings pave the way for future cell replacement studies in animal models of choroidal cell dropout.

bioengineering↗

bioRxiv2024

Exonic splice variant discovery using in vitro models of inherited retinal disease

Correct identification of the molecular consequences of pathogenic genetic variants is essential to the development of allele-specific therapies. However, in some cases, such molecular effects may be ambiguous following genetic sequence analysis alone. One such case is exonic, codon-altering variants that are also predicted to disrupt normal RNA splicing. Here, we identify such cases in the context of inherited retinal disease. NR2E3 c.932G>A (p.Arg311Gln) is a variant commonly associated with Enhanced S Cone Syndrome (ESCS). Previous studies using mutagenized cDNA constructs have shown that the arginine to glutamine substitution at position 311 of NR2E3 does not meaningfully diminish function of the rod-specific transcription factor. Using retinal organoids, we explored the molecular consequences of NR2E3 c.932G>A when expressed endogenously during human rod photoreceptor cell development. Retinal organoids carrying the NR2E3 c.932G>A allele expressed a transcript containing a 186-nucleotide deletion of exon 6 within the ligand binding domain. This short transcript was not detected in control organoids or control human donor retina samples. A minigene containing exons 5 and 6 of NR2E3 showed sufficiency of the c.932G>A variant to cause the observed splicing defect. These results support the hypothesis that the pathogenic NR2E3 c.932G>A variant leads to photoreceptor disease by causing a splice defect and not through an amino acid substitution as previously supposed. They also explain the relatively mild effect of Arg311Gln on NR2E3 function in vitro. We also used in silico prediction tools to show that similar changes are likely to affect other inherited retinal disease variants in genes such as CEP290, ABCA4, and BEST1.

genetics↗

bioRxiv2024

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

Autologous photoreceptor cell replacement therapy shows great promise for treating patients with multiple forms of inherited retinal degenerative blindness. Specifically, in disorders such as retinitis pigmentosa and Stargardts disease, selective death of photoreceptor cells results in irreversible blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of photoreceptor cells required for these therapies. However, in addition to photoreceptor cells, retinal organoids also contain many other retinal cell types. Therefore, approaches for isolating fate committed photoreceptors from dissociated retinal organoids are desirable to produce photoreceptor cell replacement therapies. In this work, we present a partial dissociation strategy, which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. Given that this technique can be performed with only standard plasticware and cGMP compliant reagents, it is an ideal candidate for use in the preparation of clinical cell therapies.

bioengineering↗

bioRxiv2023

Loss of NR2E3 disrupts rod photoreceptor cell maturation causing a fate switch late in human retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease due to mutations in NR2E3, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation, up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than those observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation.

developmental biology↗

bioRxiv2023

GENE EXPRESSION WITHIN A HUMAN CHOROIDAL NEOVASCULAR MEMBRANE USING SPATIAL TRANSCRIPTOMICS

Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process. In this study, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We identified genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes. Within the area of neovascularization, endothelial cells were predicted to increase expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we also investigated spatial gene expression patterns within the macular neural retina and between the macular and peripheral choroid. We recapitulated previously described regional-specific gene expression patterns across both tissues. Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

genomics↗

bioRxiv2022

Non-random distribution of mitochondrial m.3243A>G heteroplasmy in human retina and its impact on cellular phenotype

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non- random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm- derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm- derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non- random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/496449v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@10b1c41org.highwire.dtl.DTLVardef@6c2d7corg.highwire.dtl.DTLVardef@1e4d09org.highwire.dtl.DTLVardef@153a5a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Genetic modifiers of Cep290-mediated retinal degeneration

Mutations in CEP290 cause up to 30% of cases of Leber congenital amaurosis (LCA), a severe childhood blindness resulting from abnormalities in the photoreceptor connecting cilia that lead to rapid retinal degeneration. Like many genetic diseases, CEP290-LCA has considerable variable expressivity, indicating the presence of other factors that influence phenotypic outcome. Here, we have undertaken a phenotype-driven approach in mice to identify genetic modifiers of CEP290-mediated retinal degeneration through backcrosses and intercrosses between BXD24-Cep290rd16 mice and the genetically distinct inbred CAST strain to introduce genetic variation. Optical coherence tomography was used to quantitatively measure retinal thickness as a surrogate indication of photoreceptor degeneration in the resulting rd16-mutant mice. It was readily apparent that CEP290-mediated retinal degeneration in the resulting mice is sensitive to genetic background, with some mice exhibiting relatively thick laminated retinas and others having thin retinas with advanced disease. Quantitative trait locus (QTL) analysis identified multiple genomic loci capable of influencing the retinal degeneration phenotype in Cep290-mutant mice that together account for 71.7% of the phenotypic variation in retinal thickness observed in our population. Following the QTL analysis, two suppressor loci were studied in detail through a combination of physical and molecular approaches to narrow the critical region for each QTL and identify the probable causative genetic variations.

genetics↗

bioRxiv2019

Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

genetics↗