Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

Intelligent image-based deformation-assisted cell sorting with molecular specificity.

Although label-free cell sorting is desirable for providing pristine cells for further analysis or use, current approaches lack molecular specificity and speed. Here, we combine real-time fluorescence and deformability cytometry with sorting based on standing surface acoustic waves and transfer molecular specificity to image-based sorting using an efficient deep neural network. In addition to general performance, we demonstrate the utility of this method by sorting neutrophils from whole blood without labels.

A new hyperelastic lookup table for RT-DC.

Real-time deformability cytometry (RT-DC) is an established method that quantifies features like size, shape, and stiffness for whole cell populations on a single-cell level in real-time. A lookup table (LUT) disentangles the experimentally derived steady-state cell deformation and the projected area to extract the cell stiffness in the form of the Young's modulus. So far, two lookup tables exist but are limited to simple linear material models and cylindrical channel geometries. Here, we present two new lookup tables for RT-DC based on a neo-Hookean hyperelastic material numerically derived by simulations based on the finite element method in square and cylindrical channel geometries. At the same time, we quantify the influence of the shear-thinning behavior of the surrounding medium on the stationary deformation of cells in RT-DC and discuss the applicability and impact of the proposed LUTs regarding past and future RT-DC data analysis. Additionally, we provide insights about the cell strain and stresses, as well as the influence resulting from the rotational symmetric assumption on the cell deformation and volume estimation. The new lookup tables and the numerical cell shapes are made freely available.

Shear rheology of methyl cellulose based solutions for cell mechanical measurements at high shear rates.

Methyl cellulose (MC) is a widely used material in various microfluidic applications in biology. Due to its biocompatibility, it has become a popular crowding agent for microfluidic cell deformability measurements, which usually operate at high shear rates (>10 000 s-1). However, a full rheological characterization of methyl cellulose solutions under these conditions has not yet been reported. With this study, we provide a full shear-rheological description for solutions of up to 1% MC dissolved in phosphate-buffered saline (PBS) that are commonly used in real-time deformability cytometry (RT-DC). We characterized three different MC-PBS solutions used for cell mechanical measurements in RT-DC with three different shear rheometer setups to cover a range of shear rates from 0.1-150 000 s-1. We report viscosities and normal stress differences in this regime. Viscosity functions can be well described using a Carreau-Yasuda model. Furthermore, we present the temperature dependency of shear viscosity and first normal stress difference of these solutions. Our results show that methyl cellulose solutions behave like power-law liquids in viscosity and exhibit first normal stress difference at shear rates between 5000-150 000 s-1. We construct a general viscosity equation for each MC solution at a certain shear rate and temperature. Furthermore, we investigated how MC concentration influences the rheology of the solutions and found the entanglement concentration at around 0.64 w/w%. Our results help to better understand the viscoelastic behavior of MC solutions, which can now be considered when modelling stresses in microfluidic channels.

Importance of Nonhuman Primates as a Model System for Gene Therapy Development in Ophthalmology.

Gene therapy is a treatment concept that uses, in most cases, viral vectors to deliver a therapeutic transgene to target cells. Although the idea of gene therapy dates back over 50 years ago, due to the complexity of the treatment concept, it took until the last decade for the responsible agencies like FDA and EMA to recommend the first gene therapy products for clinical use. The development of these therapies relies on molecular engineering of specifically designed vectors and models to test the effectiveness and safety of the treatment. Despite an increasing effort to find effective surrogates, animal models are still irreplaceable in gene therapy development. Rodents are important for exploring pathways and disease mechanisms and identifying potential treatment targets. However, only the primate eye resembles the human eye to a degree where most structures are nearly identical. Some research questions can therefore only be answered using a nonhuman primate (NHP) model. In this review, we want to summarize these key features and highlight the importance of the NHP model for gene therapy development in ophthalmology.

CHANGES IN RETINAL SENSITIVITY AFTER GENE THERAPY IN CHOROIDEREMIA.

PURPOSE: Choroideremia (CHM) is a rare inherited retinal degeneration resulting from mutation of the CHM gene, which results in absence of functional Rab escort protein 1 (REP1). We evaluated retinal gene therapy with an adeno-associated virus vector that used to deliver a functional version of the CHM gene (AAV2-REP1). METHODS: THOR (NCT02671539) is a Phase 2, open-label, single-center, randomized study. Six male patients (51-60 years) with CHM received AAV2-REP1, by a single 0.1-mL subretinal injection of 10 genome particles during vitrectomy. Twelve-month data are reported. RESULTS: In study eyes, 4 patients experienced minor changes in best-corrected visual acuity (-4 to +1 Early Treatment Diabetic Retinopathy Study [ETDRS] letters); one gained 17 letters and another lost 14 letters. Control eyes had changes of -2 to +4 letters. In 5/6 patients, improvements in mean (95% confidence intervals) retinal sensitivity (2.3 [4.0] dB), peak retinal sensitivity (2.8 [3.5] dB), and gaze fixation area (-36.1 [66.9] deg) were recorded. Changes in anatomical endpoints were similar between study and control eyes. Adverse events were consistent with the surgical procedure. CONCLUSION: Gene therapy with AAV2-REP1 can maintain, and in some cases, improve, visual acuity in CHM. Longer term follow-up is required to establish whether these benefits are maintained.

High-Throughput Microfluidic Characterization of Erythrocyte Shapes and Mechanical Variability.

The motion of red blood cells (RBCs) in microchannels is important for microvascular blood flow and biomedical applications such as blood analysis in microfluidics. The current understanding of the complexity of RBC shapes and dynamics in microchannels is mainly based on several simulation studies, but there are a few systematic experimental investigations. Here, we present a combined study that systematically characterizes RBC behavior for a wide range of flow rates and channel sizes. Even though simulations and experiments generally show good agreement, experimental observations demonstrate that there is no single well-defined RBC state for fixed flow conditions but rather a broad distribution of states. This result can be attributed to the inherent variability in RBC mechanical properties, which is confirmed by a model that takes the variation in RBC shear elasticity into account. This represents a significant step toward a quantitative connection between RBC behavior in microfluidic devices and their mechanical properties, which is essential for a high-throughput characterization of diseased cells.

AAV8 Can Induce Innate and Adaptive Immune Response in the Primate Eye.

Ocular gene therapy has evolved rapidly into the clinical realm due to promising pre-clinical proof-of-concept studies, recognition of the high unmet medical need of blinding disorders, and the excellent safety profile of the most commonly used vector system, the adeno-associated virus (AAV). With several trials exposing subjects to AAV, investigators independently report about cases with clinically evident inflammation in treated eyes despite the concept of ocular immune privilege. Here, we provide a detailed analysis of innate and adaptive immune response to clinical-grade AAV8 in non-human primates and compare this to preliminary clinical data from a retinal gene therapy trial for CNGA3-based achromatopsia (ClinicalTrials.gov: 02610582).

High-throughput viscoelastic characterization of cells in hyperbolic microchannels.

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress-strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress-strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, shows strain-thickening following a power law up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation times of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid-liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Young's moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with latrunculin B and nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.

Occlusive retinal vasculitis associated with intravitreal Faricimab injections.

PURPOSE: We describe a case of occlusive vasculitis associated with intravitreal Faricimab (Vabysmo) injections. METHODS: A retrospective case report. RESULTS: A 52-year old man treated with monthly Faricimab injections for diabetic macula oedema presented with sudden reduced vision, new retinal hemorrhages, significant retinal vascular occlusions and ischemia. After screening for differential diagnoses was unremarkable, the patient was treated with oral and intravitreal steroid therapy under which the occlusive vasculitis was stabilized. CONCLUSION: Occlusive vasculitis, though rare, is a potential complication of Faricimab therapy. Comprehensive reporting and large-scale analyses are essential to better understand and manage this adverse event.

Dose-Dependent Progression of Chorioretinal Atrophy at the Injection Site After Subretinal Injection of rAAV2/8 in Nonhuman Primates.

Objective: Progressive retinal atrophy has been described after subretinal gene therapy utilizing the adeno-associated virus (AAV) vector platform. To elucidate whether this atrophy is a consequence of inherent properties of AAV, or if it is related to the surgical trauma of subretinal delivery, we analyzed data from an Investigational New Drug-enabling study for PDE6A gene therapy in nonhuman primates. Design: Animal study (nonhuman primates), retrospective data analysis. Subjects: Forty eyes of 30 healthy nonhuman primates (macaca fascicularis) were included in the analysis. Two AAV dose levels (low: 1x10E11, high: 1x10E12) were compared with sham injection (balanced saline solution; BSS). Twenty untreated eyes were not analyzed. Methods: Animals were treated with a sutureless 23G vitrectomy and single subretinal injections of AAV.PDE6A and/or BSS. The follow-up period was 12 weeks. Atrophy development was followed using fundus autofluorescence (AF), OCT, fluorescence angiography, and indocyanine green angiography. Main Outcome Measures: Area [mm2] of retinal pigment epithelium atrophy on AF. Presence of outer retinal atrophy on optical coherence tomography. Area [mm2] of hyperfluorescence in fluorescence angiography and hypofluorescence in indocyanine green angiography. Results: Progressive atrophy at the injection site developed in 54% of high-dose-treated, 27% of low-dose-treated, and 0% of sham-treated eyes. At the end of observation, the mean ± SD area of atrophy in AF was 1.19 ± 1.75 mm2, 0.25 ± 0.50 mm2, and 0.0 ± 0.0 mm2, respectively (sham × high dose: P = 0.01). Atrophic lesions in AF (P = 0.01) and fluorescence angiography (P = 0.02) were significantly larger in high-dose-treated eyes, compared with sham-treated eyes. Rate of progression in high-dose-treated eyes was 4.1× higher compared with low-dose-treated eyes. Conclusion: Subretinal injection of AAV.PDE6A induced dose-dependent, progressive retinal atrophy at the site of injection. Findings from multimodal imaging were in line with focal, transient inflammation within the retina and choroid and secondary atrophy. Atrophic changes after gene therapy with AAV-based vector systems are not primarily due to surgical trauma and increase with the dose given. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Peripapillary retinal nerve fibre layer thinning in patients with X-linked retinoschisis.

AIMS: To assess peripapillary retinal nerve fibre layer (pRNFL) thickness in patients with X-linked retinoschisis (XLRS), as pRNFL thinning may limit functional improvements in gene therapy trials. METHODS: This retrospective multicentre study included 49 eyes from 25 patients diagnosed with XLRS. Data collected with multimodal imaging at baseline and last follow-up (when available) included age, best-recorded visual acuity (BRVA), central retinal thickness, macular volume (MV), presence and location of peripheral retinoschisis and pRNFL thickness in the global (G), superotemporal (TS), superonasal (NS), inferotemporal (TI), inferonasal (NI), nasal (N) and temporal (T) sectors. Retinal sensitivity, assessed by microperimetry, was also recorded for seven patients at baseline. RESULTS: pRNFL was thinner (below the fifth percentile) in at least one sector in 72% of right eyes and 79% of left eyes, with thinning across three or more sectors in 20% of right and 17% of left eyes. In 44% of cases, thinning occurred in the temporal sectors of both eyes, with no nasal sectoral thinning. Number of peripheral retinoschisis quadrants matched thinned pRNFL sectors. A strong positive correlation was found between MV and temporal pRNFL thickness (r=0.71, p<0.01), while weak negative correlation trends were noted with age (p=0.05) and BRVA (logMAR; p=0.12) related to temporal thickness of pRNFL sectors. CONCLUSION: pRNFL thinning, predominantly sectoral and linked to macular or peripheral retinoschisis, occurs in about three-quarters of patients with XLRS, while diffuse thinning occurs in one-fifth. Temporal pRNFL thinning might occur only after the collapse of intraretinal cystoid cavities in the macula.

Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec.

BACKGROUND/AIMS: Voretigene neparvovec (VN) is the first and only subretinal gene therapy approved by the Food and Drug Administration and European Medicines Agency. Real-world application has started in 2018 in patients with vision impairment due to biallelic retinal pigment epithelium (RPE) 65 mutation-associated inherited retinal degenerations. Herein, we evaluated the development of retinal atrophy within in a single-centre patient cohort treated with VN. METHODS: 13 eyes of eight patients treated with VN were retrospectively analysed for areas of retinal atrophy over a period of 6-24 months following surgery. Ultrawide field images were used to measure the area of atrophy. Fundus autofluorescence imaging is presented as an instrument for early detection of signs of retinal atrophy in these patients. RESULTS: Atrophic changes beyond the retinotomy site were observed in all eyes. Areas of atrophy developed within the area of detachment (bleb) in all eight patients and outside the bleb in three patients. Changes in autofluorescence preceded the development of retinal atrophy and were already evident 2 weeks after surgery in the majority of patients. The areas of atrophy increase with time and progression continued over year 1. Functional outcomes remained stable (VA, FST, visual field). CONCLUSION: Subretinal injection of VN can lead to RPE atrophy with consequent photoreceptor loss in and outside of the bleb area. Fundus autofluorescence is an important tool to monitor atrophic changes in patients after gene therapy. Interestingly, while areas of atrophy also included central areas, the functional benefits of the treatment did not appear to be affected and remained stable.

Evaluation of a liquid membrane-based tunable lens and a solid-state LIDAR camera feedback system for presbyopia.

Presbyopia is an age-related loss of accommodation ability of the eye which affects individuals in their late 40s or early 50s. Presbyopia reduces the ability of a person to focus on closer objects at will. In this study, we assessed electronically tunable lenses for their aberration properties as well as for their use as correction lenses. The tunable lenses were evaluated in healthy subjects with cycloplegia by measuring visual acuity and contrast sensitivity for their use in presbyopia correction. Furthermore, we have developed and demonstrated the feasibility of a feedback mechanism for the operation of tunable lenses using a portable solid-state LIDAR camera with a processing time of 40 ± 5 ms.

Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium.

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability-including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

Spatial and temporal resolution of the photoreceptors rescue dynamics after treatment with voretigene neparvovec.

BACKGROUND: Voretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue. METHODS: Seven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months. RESULTS: All except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R2>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R2=0.5). CONCLUSIONS: The presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.

Three-year results of phase I retinal gene therapy trial for CNGA3-mutated achromatopsia: results of a non randomised controlled trial.

AIMS: To determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582). METHODS: Details of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy. RESULTS: No adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye. CONCLUSION: The results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

An Optimized Treatment Protocol for Subretinal Injections Limits Intravitreal Vector Distribution.

Purpose: Subretinal injections (SRis) are commonly used in retinal gene therapy procedures to deliver adeno-associated virus (AAV) to photoreceptors and retinal pigment epithelial cells. We present an optimized surgical protocol to minimize off-target application of AAV in the vitreous, which in turn reduces the risk of extensive biodistribution and inflammation, ultimately leading to enhanced safety of the therapy. Design: Experimental animal research study. Participants: Eight cynomolgus monkeys (Macaca fascicularis). Methods: Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10ˆ 11 and 5×10ˆ 11 viral genomes [vg]). Samples of intravitreal fluid were taken at the end of the SRi procedure and again after a 3-minute lavage (wash-out) with balanced salt solution (BSS). Main Outcome Measures: Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups. Results: Even uneventful SRi leads to dissemination of millions of AAV particles (0.1-0.7% of viral vector loading dose) into the vitreous cavity. Three minutes of lavage led to a substantial decrease (on average 96%) of intravitreal vector load. Conclusions: Multiple studies have shown that the intravitreal space is not as immune privileged as the subretinal space. Intravitreal AAV particles disseminate into the bloodstream, lead to increased biodistribution into lymphatic tissue, and help to stage an immune response with implications for both safety and efficacy. Therefore, minimizing off-target vector application after reflux of vector from the subretinal space is of significant interest. We show that a simple lavage of intravitreal fluid efficiently decreases the intravitreal vector load. Such a step should be considered when performing subretinal gene therapy.

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.