Too stressed to de-stress? The experience of work stress and recovery among attorneys during the COVID-19 pandemic.

It is well-established that legal professionals experience high stress and often high rates of associated health problems. Much less is known about attitudes and behaviours around stress prevention in this occupation. Our study examined views of stress and recovery among 131 U.S. attorneys. In open-ended data, attorneys commonly expressed that their job is very demanding, and it impacts their health. Many respondents felt it was important to manage their stress but had difficulty doing so. Quantitative analyses showed that attitudes about stress (stress-related comparisons, viewing stress as achievement, stress-related impression management, and stigma around stress concerns) demonstrated several significant relationships with perceived stress, recovery experiences, remorse for relaxation, and work-family conflict. Our findings suggest that practical interventions to support the health and well-being of legal professionals may need to target the workload norms, as well as attitudes and beliefs about the normalness of high stress and insufficient recovery.

CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

The Degree of Adeno-Associated Virus-Induced Retinal Inflammation Varies Based on Serotype and Route of Delivery: Intravitreal, Subretinal, or Suprachoroidal.

Adeno-associated virus (AAV)-mediated gene therapy has great potential for treating a wide range of retinal degenerative diseases. However, some initial enthusiasm for gene therapy has been tempered by emerging evidence of AAV-associated inflammation, which in several instances has contributed to clinical trial discontinuation. Currently, there is a paucity of data describing the variable immune responses to different AAV serotypes, and similarly, little is known regarding how these responses differ depending on route of ocular delivery, including in animal models of disease. In this study, we characterize the severity and retinal distribution of AAV-associated inflammation in rats triggered by delivery of five different AAV vectors (AAV1, AAV2, AAV6, AAV8, and AAV9), each of which contained enhanced green fluorescent protein (eGFP) driven under control of the constitutively active cytomegalovirus promoter. We further compare the inflammation across three different potential routes (intravitreal, subretinal, and suprachoroidal) of ocular delivery. Compared to buffer-injected controls for each route of delivery, AAV2 and AAV6 induced the most inflammation across all routes of delivery of vectors tested, with AAV6 inducing the highest levels of inflammation when delivered suprachoroidally. AAV1-induced inflammation was highest when delivered suprachoroidally, whereas minimal inflammation was seen with intravitreal delivery. In addition, AAV1, AAV2, and AAV6 each induce infiltration of adaptive immune cells like T cells and B cells into the neural retina, suggesting an innate adaptive response to a single dose of virus. AAV8 and AAV9 induced minimal inflammation across all routes of delivery. Importantly, the degree of inflammation was not correlated with vector-mediated transduction and expression of eGFP. These data emphasize the importance of considering ocular inflammation when selecting AAV serotypes and ocular delivery routes for the development of gene therapy strategies.

Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy.

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

Biocompatibility of Human Induced Pluripotent Stem Cell-Derived Retinal Progenitor Cell Grafts in Immunocompromised Rats.

Loss of photoreceptor cells is a primary feature of inherited retinal degenerative disorders including age-related macular degeneration and retinitis pigmentosa. To restore vision in affected patients, photoreceptor cell replacement will be required. The ideal donor cells for this application are induced pluripotent stem cells (iPSCs) because they can be derived from and transplanted into the same patient obviating the need for long-term immunosuppression. A major limitation for retinal cell replacement therapy is donor cell loss associated with simple methods of cell delivery such as subretinal injections of bolus cell suspensions. Transplantation with supportive biomaterials can help maintain cellular integrity, increase cell survival, and encourage proper cellular alignment and improve integration with the host retina. Using a pig model of retinal degeneration, we recently demonstrated that polycaprolactone (PCL) scaffolds fabricated with two photon lithography have excellent local and systemic tolerability. In this study, we describe rapid photopolymerization-mediated production of PCL-based bioabsorbable scaffolds, a technique for loading iPSC-derived retinal progenitor cells onto the scaffold, methods of surgical transplantation in an immunocompromised rat model and tolerability of the subretinal grafts at 1, 3, and 6 months of follow-up (n = 150). We observed no local or systemic toxicity, nor did we observe any tumor formation despite extensive clinical evaluation, clinical chemistry, hematology, gross tissue examination and detailed histopathology. Demonstrating the local and systemic compatibility of biodegradable scaffolds carrying human iPSC-derived retinal progenitor cells is an important step toward clinical safety trials of this approach in humans.

Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Development and biological characterization of a clinical gene transfer vector for the treatment of MAK-associated retinitis pigmentosa.

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP.

AUTOIMMUNE RETINOPATHY MIMICKING HERITABLE RETINAL DEGENERATION IN A PATIENT WITH COMMON VARIABLE IMMUNE DEFICIENCY.

PURPOSE: 1) To describe a case of autoimmune retinopathy mimicking heritable photoreceptor degeneration in a patient with common variable immune deficiency and 2) to investigate the humoral and cell-mediated branches of the immune system in this patient to better understand the mechanism of immune-mediated photoreceptor damage in this disease. METHODS: Retrospective chart review with evaluation of multimodal imaging, genotype analysis, and investigation of circulating autoantibodies and T-cell response to retinal antigens. RESULTS: A 40-year-old woman with bilateral, progressive vision loss was referred for evaluation of a possible inherited retinal degeneration. She was found to have asymmetric peripheral visual field constriction, cystoid macular edema, vitreous cells, and bone spicule-like pigmentary changes in both eyes. An extensive workup for underlying infectious or inflammatory causes was unrevealing, and molecular analysis for heritable retinal degeneration failed to identify a plausible disease-causing genotype. Screening for antiretinal antibodies showed the presence of multiple antiretinal antibodies, consistent with a diagnosis of autoimmune retinopathy. Immunologic workup demonstrated markedly decreased levels of serum IgA and IgG, consistent with common variable immune deficiency. T-cells isolated from the patient showed increased proliferation when stimulated with human retinal proteins, supporting a role for both cell- and humoral-mediated autoimmunity. Treatment with mycophenolate mofetil and intravenous immunoglobin therapy slowed the progression of disease and resulted in preservation of her central vision. CONCLUSION: Autoimmune retinopathy can be seen in common variable immune deficiency and has clinical findings similar to heritable photoreceptor degeneration. Both the humoral and cellular immune responses are involved in the pathophysiology. Immune modulatory therapy has stabilized the disease course in this patient and may play an important role in the management of autoimmune retinopathy.

Chimeric Helper-Dependent Adenoviruses Transduce Retinal Ganglion Cells and Müller Cells in Human Retinal Explants.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 µL (1 × 106 vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

The effect of retinal scaffold modulus on performance during surgical handling.

Emerging treatment strategies for retinal degeneration involve replacing lost photoreceptors using supportive scaffolds to ensure cells survive the implantation process. While many design aspects of these scaffolds, including material chemistry and microstructural cues, have been studied in depth, a full set of design constraints has yet to be established. For example, while known to be important in other tissues and systems, the influence of mechanical properties on surgical handling has not been quantified. In this study, photocrosslinked poly(ethylene glycol) dimethacrylate (PEGDMA) was used as a model polymer to study the effects of scaffold modulus (stiffness) on surgical handling, independent of material chemistry. This was achieved by modulating the molecular weight and concentrations of the PEGDMA in various prepolymer solutions. Scaffold modulus of each formulation was measured using photo-rheology, which enabled the collection of real-time polymerization data. In addition to measuring scaffold mechanical properties, this approach gave insight on polymerization kinetics, which were used to determine the polymerization time required for each sample. Scaffold handling characteristics were qualitatively evaluated using both in vitro and ex vivo trials that mimicked the surgical procedure. In these trials, scaffolds with shear moduli above 35 kPa performed satisfactorily, while those below this limit performed poorly. In other words, scaffolds below this modulus were too fragile for reliable transplantation. To better compare these results with literature values, the compressive modulus was measured for select samples, with the lower shear modulus limit corresponding to roughly 115 kPa compressive modulus. While an upper mechanical property limit was not readily apparent from these results, there was increased variability in surgical handling performance in samples with shear moduli above 800 kPa. Overall, the knowledge presented here provides important groundwork for future studies designed to examine additional retinal scaffold considerations, including the effect of scaffold mechanical properties on retinal progenitor cell fate.

Retinal Tropism and Transduction of Adeno-Associated Virus Varies by Serotype and Route of Delivery (Intravitreal, Subretinal, or Suprachoroidal) in Rats.

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) vary significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, and AAV2/9) expressing enhanced green fluorescent protein driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina through subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared with data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

Development of High-Resolution Three-Dimensional-Printed Extracellular Matrix Scaffolds and Their Compatibility with Pluripotent Stem Cells and Early Retinal Cells.

Purpose: Widely used approaches for retinal disease modeling and in vitro therapeutic testing can be augmented by using tissue-engineered scaffolds with a precise 3-dimensional structure. However, the materials currently used for these scaffolds are poorly matched to the biochemical and mechanical properties of the in vivo retina. Here, we create biopolymer-based scaffolds with a structure that is amenable to retinal tissue engineering and modeling. Methods: Optimal two-photon polymerization (TPP) settings, including laser power and scanning speed, are identified for 4 methacrylated biopolymer formulations: collagen, gelatin, hyaluronic acid (HA), and a 50/50 mixture of gelatin/HA, each with methylene blue as a photoinitiator. For select formulations, fabrication accuracy and swelling are determined and biocompatibility is evaluated by using human induced pluripotent stem cells and rat postnatal retinal cells. Results: TPP is feasible for each biopolymer formulation, but it is the most reliable for mixtures containing gelatin and the least reliable for HA alone. The mean size of microscaffold pores is within several microns of the intended value but the overall structure size is several times greater than the modeled volume. The addition of HA to gelatin scaffolds increases cell viability and promotes neuronal phenotype, including Tuj-1 expression and characteristic morphology. Conclusion: We successfully determined a useful range of TPP settings for 4 methacrylated biopolymer formulations. When crosslinked, these extracellular matrix-derived molecules support the growth and attachment of retinal cells. We anticipate that when combined with existing patient-specific approaches, this technique will enable more efficient and accurate retinal disease modeling and therapeutic testing in vitro than current techniques allow.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility.

Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797&809.

Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose in situ has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and three-dimensional printing via stereolithography. PCL prepolymers with different molecular weights and functionalities were studied to understand the role of molecular structure in reaction kinetics, mechanical properties, and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High-molecular weight, low-functionality PCLDA prepolymers exhibited a lower modulus and a higher strain at break, while low-molecular weight, high-functionality PCLTA prepolymers exhibited a lower strain at break and a higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than those of more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both in vitro and in vivo, causing no observed detrimental effects on seeded murine-induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create three-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low-molecular weight, high-functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 µm. This work lays the foundation for future work in fabricating microscale PCL structures for a wide range of tissue regeneration applications.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.