Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells.

Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.

Benefits and Shortcomings of Laboratory Model Systems in the Development of Genetic Therapies.

Gene therapeutic approaches promise treatment or even a cure of diseases that were previously untreatable. Retinal gene therapies tested in clinical trials comprise a wide range of different strategies, including gene supplementation therapies, in vivo gene editing, modulation of splicing mechanisms, or the suppression of gene expression. To guarantee efficient transfer of genetic material into the respective target cells while avoiding major adverse effects, the development of genetic therapies requires appropriate in vitro model systems that allow tests of efficacy and safety of the gene therapeutic approach. In this review, we introduce various in vitro models of different levels of complexity used in the development of genetic therapies and discuss their respective benefits and shortcomings using the example of adeno-associated virus-based retinal gene therapy.

Kinetics of Heterogeneous Background in Stargardt's Disease over Time.

Stargardt's disease (STGD1) is caused by mutations in the ABCA4 gene. Different lesions characterised by decreased autofluorescence levels are found in fundus autofluorescence (FAF) from STGD1 patients and could be used as outcome indicators for disease progression. We investigated the fate of foci with reduced autofluorescence (FRA) within the heterogeneous background of STGD1 patients using FAF imaging. Genetically confirmed STGD1 patients presenting heterogeneous background autofluorescence on high-quality FAF images at a minimum of two visits at least 12 months apart were chosen. A grid centred on the fovea was used to define five different zones. Within each zone, five FRA were randomly selected for each eye. The eccentricity of foci was determined at different time points for each patient. Analysis of 175 randomly chosen FRA showed consistent centrifugal displacement over time, most notably in eyes showing areas with definitely decreased autofluorescence. Interestingly, FRA did not leave an area of hypo-autofluorescence on FAF in locations where they were previously located. These findings may help to better understand STGD1 progression, improve FAF interpretation, and shed light on the nature of heterogeneous background.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy.

BACKGROUND: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). METHODS: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. RESULTS: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. CONCLUSIONS: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.

Longitudinal Evaluation of Hyper-Reflective Foci in the Retina Following Subretinal Delivery of Adeno-Associated Virus in Non-Human Primates.

Purpose: The purpose of this study was to evaluate whether clinical grade recombinant adeno-associated virus serotype 8 (rAAV8) leads to increased appearance of hyper-reflective foci (HRF) in the retina of non-human primates (NHPs) following subretinal gene therapy injection. Methods: Different doses of rAAV8 vector (rAAV8. human phosphodiesterase 6A subunit (hPDE6A) at low dose: 1 × 1011 vector genomes (vg), medium dose: 5 × 1011 vg, or high dose: 1 × 1012 vg) were injected subretinally into the left eyes of NHPs in a formal toxicology study in preparation of a clinical trial. Right eyes received sham-injection. After 3 months of in vivo, follow-up retinal sections were obtained and analyzed. The number of HRF on spectral domain-optical coherence tomography (SD-OCT) volume scans were counted from both eyes at 30 and 90 days. Results: Animals from the high-dose group showed more HRF than in the low (P = 0.03) and medium (P = 0.01) dose groups at 90 days. There was a significant increase in the mean number of HRF in rAAV8-treated eyes compared with sham-treated eyes at 90 days (P = 0.02). Sham-treated eyes demonstrated a nonsignificant reduction of HRF numbers over time. In contrast, a significant increase over time was observed in the rAAV8-treated eyes of the high dose group (P = 0.001). The presence of infiltrating B- and T-cells and microglia activation were detected in rAAV8-treated eyes. Conclusions: Some HRF in the retina appear to be related to the surgical trauma of subretinal injection. Although HRF in sham-treated retina tends to become less frequent over time, they accumulate in the high-dose rAAV8-treated eyes. This may suggest a sustained immunogenicity when subretinal injections of higher doses of rAAV8 vectors are applied, but it has lower impact when using more clinically relevant doses (low and medium groups). Translational Relevance: An increase or persistence of HRFs following retinal gene therapy may indicate the need for immunomodulatory treatment.

ASYMETRIC GROWTH OF MACULAR ATROPHY ASSOCIATED WITH UNDERLYING MATURE, NONEXUDATIVE CHOROIDAL NEOVASCULARIZATION.

BACKGROUND/PURPOSE: To describe a case study that shows a possible association between the slow growth rate of macular atrophy and the presence of underlying mature, nonexudative choroidal neovascularization. METHODS: Case report. PATIENT: An 82-year-old woman with mixed age-related macular degeneration in both eyes was followed up for 6 years, with the last ranibizumab treatment given in the left eye more than 2 years previously. Evaluations included fluorescein angiography, spectral-domain optical coherence tomography, and optical coherence tomography angiography. RESULTS: During follow-up, there was a peculiar growth of macular atrophy, involving mainly the superior nasal sector. No signs of exudation on fluorescein angiography or spectral-domain optical coherence tomography were apparent throughout this period. However, optical coherence tomography angiography showed a mature, nonexudative choroidal neovascularization under the foveal sparing area and surrounding all the boundaries of atrophy except in the superonasal sector. Macular atrophy growth was observed mainly in the region devoid of blood vessels. CONCLUSION: Growth of macular atrophy was more prominent in the region where mature, nonexudative choroidal neovascularization was absent. Nutrients provided by vessels from choroidal neovascularization may provide support to adjacent retinal pigment epithelium cells, slowing down the progression of atrophy.

Immune responses to retinal gene therapy using adeno-associated viral vectors - Implications for treatment success and safety.

Recombinant adeno-associated virus (AAV) is the leading vector for gene therapy in the retina. As non-pathogenic, non-integrating, replication deficient vector, the recombinant virus efficiently transduces all key retinal cell populations. Successful testing of AAV vectors in clinical trials of inherited retinal diseases led to the recent approval of voretigene neparvovec (Luxturna) for the treatment of RPE65 mutation-associated retinal dystrophies. However, studies applying AAV-mediated retinal gene therapy independently reported intraocular inflammation and/or loss of efficacy after initial functional improvements. Both observations might be explained by targeted removal of transduced cells via anti-viral defence mechanisms. AAV has been shown to activate innate pattern recognition receptors (PRRs) such as toll-like receptor (TLR)-2 and TLR-9 resulting in the release of inflammatory cytokines and type I interferons. The vector can also induce capsid-specific and transgene-specific T cell responses and neutralizing anti-AAV antibodies which both limit the therapeutic effect. However, the target organ of retinal gene therapy, the eye, is known as an immune-privileged site. It is characterized by suppression of inflammation and promotion of immune tolerance which might prevent AAV-induced immune responses. This review evaluates AAV-related immune responses, toxicity and inflammation in studies of retinal gene therapy, identifies influencing variables of these responses and discusses potential strategies to modulate immune reactions to AAV vectors to increase the safety and efficacy of ocular gene therapy.

Generation and characterization of the human iPSC line CABi001-A from a patient with retinitis pigmentosa caused by a novel mutation in PRPF31 gene.

PRPF31 gene codes for a ubiquitously expressed splicing factor but mutations affect exclusively the retina, producing the progressive death of photoreceptor cells. We have identified a novel PRPF31 mutation in a patient with autosomal dominant retinitis pigmentosa. A blood sample was obtained and mononuclear cells were reprogrammed using the non-integrative Sendai virus to generate the cell line CABi001-A. The iPSC line has been characterized for pluripotency and differentiation capacity and will be differentiated toward photoreceptors and retinal pigment epithelium cells to study the molecular mechanism of the disease and test possible therapeutic strategies.

Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins.

BACKGROUND: Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. METHODS: In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. RESULTS: We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. CONCLUSIONS: Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.

Bone metabolism and clinical study of 44 patients with bisphosphonate-related osteonecrosis of the jaws

Osteonecrosis of the jaws is a clinical entity described and linked to treatment with bisphosphonates in 2003. Its real incidence is unknown and it could increase due to the large number of patients treated with these drugs, and its cumulative effect on the bone. State of the art knowledge regarding its etiopathogeny, clinical course and suitable treatments is limited. Objectives: To study the clinical characteristics of 44 patients with bisphosphonate-related osteonecrosis of the jaws and the state of their bone mineral metabolism: bone remodeling state, prevalence of fractures, bone mineral density study, and assessment of the different treatment strategies. Design of the Study: Observational. Information was gathered prospectively through interviews, clinical examinations, additional tests and review of medical records.Results: We studied 16 men and 28 women with a mean age of 64.7 years. Breast cancer was the most frequent underlying disease. Zoledronate was used in 82% of the cases and in the non-oncology group of patients; alendronate was the most frequently used bisphosphonate. The mean duration of the zoledronate and alendronate treatments was 25 months and 88 months respectively. The lower jaw was the most frequent location, and previous exodontias-among the triggering factors known-were the most closely linked to its onset. We found considerable osteoblastic activity in patients suffering from neoplasia, with artifacts present in their bone densitometry and a high percentage of vertebral fractures.Conclusions: According to our results, osteonecrosis of the jaws affects elderly patients. We found a direct relationship between the duration of exposure and the accumulated dose. Other relevant factors are: Poor oral and dental health, corticoids, diabetes and teeth extractions. In essence, it is a clinical diagnosis. Prevention is the best strategy to handle this clinical entity (AU)

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Bone metabolism and clinical study of 44 patients with bisphosphonate-related osteonecrosis of the jaws.

UNLABELLED: Osteonecrosis of the jaws is a clinical entity described and linked to treatment with bisphosphonates in 2003. Its real incidence is unknown and it could increase due to the large number of patients treated with these drugs, and its cumulative effect on the bone. State of the art knowledge regarding its etiopathogeny, clinical course and suitable treatments is limited. OBJECTIVES: To study the clinical characteristics of 44 patients with bisphosphonate-related osteonecrosis of the jaws and the state of their bone mineral metabolism: bone remodeling state, prevalence of fractures, bone mineral density study, and assessment of the different treatment strategies. DESIGN OF THE STUDY: Observational. Information was gathered prospectively through interviews, clinical examinations, additional tests and review of medical records. RESULTS: We studied 16 men and 28 women with a mean age of 64.7 years. Breast cancer was the most frequent underlying disease. Zoledronate was used in 82% of the cases and in the non-oncology group of patients; alendronate was the most frequently used bisphosphonate. The mean duration of the zoledronate and alendronate treatments was 25 months and 88 months respectively. The lower jaw was the most frequent location, and previous exodontias-among the triggering factors known-were the most closely linked to its onset. We found considerable osteoblastic activity in patients suffering from neoplasia, with artifacts present in their bone densitometry and a high percentage of vertebral fractures. CONCLUSIONS: According to our results, osteonecrosis of the jaws affects elderly patients. We found a direct relationship between the duration of exposure and the accumulated dose. Other relevant factors are: Poor oral and dental health, corticoids, diabetes and teeth extractions. In essence, it is a clinical diagnosis. Prevention is the best strategy to handle this clinical entity.