Less is more: Self-amplifying mRNA becomes self-killing upon dose escalation in immune-competent retinal cells.

In the last few years, mRNA therapeutics experienced a new wave of interest as therapy for retinal diseases. Nevertheless, despite the widespread use of mRNA vaccines in the COVID-19 pandemic, mRNA delivery to the eye is still in its infancy. Recently, our research group has demonstrated that after subretinal and intravitreal delivery of modified mRNA, the number of transfected retinal cells and protein expression per cell remains limited. In this study, we aimed to tackle this limitation by using self-amplifying mRNA (saRNA), which in theory will increase the duration and level of protein expression when only a few mRNA molecules reach their target cells. A one-on-one comparison between modified mRNA and saRNA in two immune-competent human retinal cell types, including Müller cells and retinal pigment epithelial cells, and in immune-deficient BHK-21 cells revealed that saRNA delivery induced an innate immune response blocking its own translation above a certain dose threshold. Removal of double-stranded (ds)RNA byproducts by cellulose-based purification and addition of the innate immune inhibitor B18R remarkably improved translation from saRNA through a reduction in innate immune response. Taken together, when saRNA is applied for retinal disease, the dose should be controlled and measures should be taken to limit immunogenicity.

Every nano-step counts: a critical reflection on do's and don'ts in researching nanomedicines for retinal gene therapy.

INTRODUCTION: Retinal disease affects millions of people worldwide, generating a massive social and economic burden. Current clinical trials for retinal diseases are dominated by gene augmentation therapies delivered with recombinant viruses as key players. As an alternative, nanoparticles hold great promise for the delivery of nucleic acid therapeutics as well. Nevertheless, despite numerous attempts, 'nano' is in practice not as successful as aspired and major breakthroughs in retinal gene therapy applying nanomaterials are yet to be seen. AREAS COVERED: In this review, we summarize the advantages of nanomaterials and give an overview of nanoparticles designed for retinal nucleic acid delivery up to now. We furthermore critically reflect on the predominant issues that currently limit nano to progress to the clinic, where faulty study design and the absence of representative models play key roles. EXPERT OPINION: Since the current approach of in vitro - in vivo experimentation is highly inefficient and creates misinformation, we advocate for a more prominent role for ex vivo testing early on in nanoparticle research. In addition, we elaborate on several concepts, including systematic studies and open science, which could aid in pushing the field of nanomedicine beyond the preclinical stage.

Photodisruption of the Inner Limiting Membrane: Exploring ICG Loaded Nanoparticles as Photosensitizers.

The inner limiting membrane (ILM) represents a major bottleneck hampering efficient drug delivery to the retina after intravitreal injection. To overcome this barrier, we intend to perforate the ILM by use of a light-based approach which relies on the creation of vapor nanobubbles (VNBs) when irradiating photosensitizers with high intensity laser pulses. Upon collapse of these VNBs, mechanical effects can disrupt biological structures. As a photosensitizer, we explore indocyanine green (ICG) loaded nanoparticles (NPs) specifically designed for our application. In light of this, ICG liposomes and PLGA ICG NPs were characterized in terms of physicochemical properties, ICG incorporation and VNB formation. ICG liposomes were found to encapsulate significantly higher amounts of ICG compared to PLGA ICG NPs which is reflected in their VNB creating capacity. Since only ICG liposomes were able to induce VNB generation, this class of NPs was further investigated on retinal explants. Here, application of ICG liposomes followed by laser treatment resulted in subtle disruption effects at the ILM where zones of fully ablated ILM were alternated by intact regions. As the interaction between the ICG liposomes and ILM might be insufficient, active targeting strategies or other NP designs might improve the concept to a further extent.

ICG-mediated photodisruption of the inner limiting membrane enhances retinal drug delivery.

Many groundbreaking therapies for the treatment of blindness require delivery of biologics or cells to the inner retina by intravitreal injection. Unfortunately, the advancement of these therapies is greatly hampered by delivery difficulties where obstruction of the therapeutics at the inner limiting membrane (ILM) represents the dominant bottleneck. In this proof-of-principle study, we explore an innovative light-based approach to locally ablate the ILM in a minimally invasive and highly controlled manner, thus making the ILM more permeable for therapeutics. More specifically, we demonstrate that pulsed laser irradiation of ILM-bound indocyanine green (ICG), a clinically applied ILM dye, results in the formation of vapor nanobubbles which can disrupt the bovine ILM as well as the extraordinary thick human ILM. We have observed that this photodisruption allows for highly successful retinal delivery of model nanoparticles which are otherwise blocked by the intact ILM. Strikingly, this treatment is furthermore able of enhancing the efficacy of mRNA-loaded lipid nanoparticles within the bovine retina by a factor of 5. In conclusion, this study provides evidence for a light-based approach to overcome the ILM which has the potential to improve the efficacy of all retinal therapies hampered by this delivery barrier.

Laser-induced nanobubbles safely ablate vitreous opacities in vivo.

In myopia, diabetes and ageing, fibrous vitreous liquefaction and degeneration is associated with the formation of opacities inside the vitreous body that cast shadows on the retina, appearing as 'floaters' to the patient. Vitreous opacities degrade contrast sensitivity function and can cause notable impairment in vision-related quality of life. Here we introduce 'nanobubble ablation' for safe destruction of vitreous opacities. Following intravitreal injection, hyaluronic acid-coated gold nanoparticles and indocyanine green, which is widely used as a dye in vitreoretinal surgery, spontaneously accumulate on collagenous vitreous opacities in the eyes of rabbits. Applying nanosecond laser pulses generates vapour nanobubbles that mechanically destroy the opacities in rabbit eyes and in patient specimens. Nanobubble ablation might offer a safe and efficient treatment to millions of patients suffering from debilitating vitreous opacities and paves the way for a highly safe use of pulsed lasers in the posterior segment of the eye.

Vaccinia Virus Protein B18R: Influence on mRNA Immunogenicity and Translation upon Non-Viral Delivery in Different Ocular Cell Types.

In the last few years, interest has grown in the use of nucleic acids as an ocular therapy for retinal genetic diseases. Recently, our research group has demonstrated that mRNA delivery could result in effective protein expression in ocular cells following subretinal injection. Yet, although mRNA therapy comes with many advantages, its immunogenicity resulting in hampered mRNA translation delays development to the clinic. Therefore, several research groups investigate possible strategies to reduce this innate immunity. In this study, we focus on B18R, an immune inhibitor to suppress the mRNA-induced innate immune responses in two ocular cell types. We made use of retinal pigment epithelial (RPE) cells and Müller cells both as immortalized cell lines and primary bovine cells. When cells were co-incubated with both B18R and mRNA-MessengerMAX lipoplexes we observed an increase in transfection efficiency accompanied by a decrease in interferon-ß production, except for the Müller cells. Moreover, uptake efficiency and cell viability were not hampered. Taken together, we showed that the effect of B18R is cell type-dependent but remains a possible strategy to improve mRNA translation in RPE cells.

Ocular barriers to retinal delivery of intravitreal liposomes: Impact of vitreoretinal interface.

Drug delivery to the posterior segment of the eye is challenging due to several anatomical and physiological barriers. Thus, there is a need for prolonged action and targeted drug delivery to treat retinal diseases. Intravitreal injections avoid anterior eye barriers, but the vitreoretinal interface and inner limiting membrane (ILM) may prevent access of drug delivery systems to the retina. Existing data on retinal permeation of intravitreal nanoparticles are sparse and probably misleading due to the inter-species differences of retinal structures in rodents and humans. To bridge this gap, retinal permeation of light-activated liposomes was studied in an ex vivo bovine explant system that simulates the structure of vitreoretinal interface and intact ILM. Our findings indicate that the particle size plays a significant role in determining the retinal penetration as the liposomes of >100 nm sized failed to overcome the ILM and could not permeate into the retina. In addition, our results demonstrate the impact of surface charge and PEG-coating on retinal penetration. Small (≈ 50 nm) anionic liposomes with PEG coating showed the most extensive distribution and cellular localization in the retina. In summary, this study extends understanding of ocular barriers, and provides valuable information to augment design of retinal drug delivery systems.

Influence of pathogenic stimuli on Müller cell transfection by lipoplexes.

Neuroprotection is a mutation-independent therapeutic strategy that seeks to enhance the survival of neuronal cell types through delivery of neuroprotective factors. The Müller cell, a retinal glial cell type appreciated for its unique morphology and neuroprotective functions, could be regarded as an ideal target for this strategy by functioning as a secretion platform within the retina following uptake of a transgene of our choice. In this in vitro study we aimed to investigate the capability of Müller cells to take up a standard liposomal vector (i.e. Lipofectamine 2000) and process its pDNA or mRNA cargo into the reporter GFP protein. By doing so, we found that mRNA outperformed pDNA in Müller cell transfection efficiency. Since neuroprotection is explored as a therapy for diabetic retinopathy and glaucoma, we furthermore examined the Müller cell's lipoplex-induced transfection efficiency and cytotoxicity in stressful conditions linked to these diseases - i.e. hypoxia, hyperglycemia and oxidative stress. Interestingly, Müller cells were able of maintaining high GFP expression regardless of these noxious stimuli. In terms of lipoplex-induced toxicity, hyperglycemia seemed to have a protective effect while hypoxia and oxidative stress led to a slightly higher toxicity. In conclusion, our study indicates that mRNA-lipoplexes have potential in transfecting Müller cells in healthy as well as diseased conditions.

Photoablation of Human Vitreous Opacities by Light-Induced Vapor Nanobubbles.

Myopia, diabetes, and aging are the main causes of progressive vitreous collagen aggregation, resulting in vitreous opacities, which can significantly disturb vision. As vitreous opacities, which induce the visual phenomenon of "floaters", are accessible with nanomaterials and light, we propose a nanotechnology-based approach to locally ablate them with highly reduced light energy compared to the more traditional YAG laser therapy. Our strategy relies on the plasmon properties of gold nanoparticles that generate vapor nanobubbles upon pulsed-laser illumination whose mechanical force can ablate vitreous opacities. We designed gold nanoparticles coated with hyaluronic acid (HA), which have excellent diffusional mobility in human vitreous, an essential requirement to reach the vitreous opacities. In addition, we found that HA-coated gold nanoparticles can accumulate extensively on human vitreous opacities that were obtained by vitrectomy from patients with vision-degrading myodesopsia. When subsequently applying nanosecond laser pulses, the collagen aggregates were efficiently destroyed with ∼1000 times less light energy than typically used in YAG laser therapy. This low-energy "floater-specific destruction", which is due to the accumulation of the small gold nanoparticles on the opacities, is attractive, as it may be safer to the surrounding ocular tissues while at the same time being easier and faster to apply compared to YAG laser therapy, where the opacities need to be ablated piece by piece by a tightly focused laser beam. Gold nanoparticle-assisted photoablation may therefore provide a safer, faster, and more reliable destruction of vitreous opacities in the treatment of ophthalmologic diseases.

Non-viral delivery of chemically modified mRNA to the retina: Subretinal versus intravitreal administration.

mRNA therapeutics have recently experienced a new wave of interest, mainly due to the discovery that chemical modifications to mRNA's molecular structure could drastically reduce its inherent immunogenicity and perceived instability. On this basis, we aimed to explore the potential of chemically stabilized mRNA for ocular applications. More specifically, we investigated the behavior of mRNA-loaded lipid-based carriers in human retinal cells (in vitro), in bovine retinal explants (ex vivo) and in mouse retinas (in vivo). We demonstrate a clear superiority of mRNA over pDNA to induce protein expression in different retinal cell types, which was further enhanced by chemical modification of the mRNA, providing up to ~1800-fold higher reporter gene expression compared to pDNA. Moreover, transgene expression could be detected for at least 20 days after a single administration of chemically modified mRNA in vitro. We furthermore determined the localization and extent of mRNA expression depending on the administration route. After subretinal (SR) administration, mRNA expression was observed in vivo and ex vivo. By contrast, intravitreal (IVT) administration resulted in limited expression in vivo. Using ex vivo bovine explants with an intact vitreoretinal (VR) interface we could attribute this to the inner limiting membrane (ILM), which presents a large barrier for non-viral delivery of mRNA, trapping mRNA complexes at the vitreal side. When the vitreous was removed, which compromises the ILM, mRNA expression was apparent and seemed to colocalize with Müller cells or photoreceptors after respectively IVT or SR administration. Taken together, this study represents a first step towards mRNA-mediated therapy for retinal diseases.

The obstacle course to the inner retina: Hyaluronic acid-coated lipoplexes cross the vitreous but fail to overcome the inner limiting membrane.

Considerable research over the last few years has revealed dysregulation of growth factors in various retinal diseases, such as glaucoma, diabetic retinopathy and photoreceptor degenerations. The use of messengerRNA (mRNA) to transiently overexpress a specific factor could compensate for this imbalance. However, a critical challenge of this approach lies in the ability to efficiently deliver mRNA molecules to the retinal target cells. In this study we found that intravitreal (IVT) injection is an attractive approach to deliver mRNA to the retina, providing two critical barriers can be overcome: the vitreous and the inner limiting membrane (ILM). We demonstrated that the vitreous is indeed a major hurdle in the delivery of the cationic mRNA-complexes to retinal cells, both in terms of vitreal mobility and cellular uptake. To improve their intravitreal mobility and avoid unwanted extracellular interactions, we evaluated the use of hyaluronic acid (HA) as an electrostatic coating strategy. This HA-coating provided the complexes with a negative surface charge, markedly enhancing their mobility in the vitreous humor, without reducing their cellular internalization and transfection efficiency. However, although this coating strategy allows the mRNA-complexes to successfully overcome the vitreal barrier, the majority of the particles accumulated at the ILM. This study therefore underscores the crucial barrier function of the ILM toward non-viral retinal gene delivery and the need to smartly design mRNA-carriers able to surmount the vitreous as well as the ILM.

Müller cells as a target for retinal therapy.

Müller cells are specialized glial cells that span the entire retina from the vitreous cavity to the subretinal space. Their functional diversity and unique radial morphology render them particularly interesting targets for new therapeutic approaches. In this review, we reflect on various possibilities for selective Müller cell targeting and describe how some of their cellular mechanisms can be used for retinal neuroprotection. Intriguingly, cross-species investigation of their properties has revealed that Müller cells also have an essential role in retinal regeneration. Although many questions regarding this subject remain, it is clear that Müller cells have unique characteristics that make them suitable targets for the prevention and treatment of numerous retinal diseases.

Morphology and Composition of the Inner Limiting Membrane: Species-Specific Variations and Relevance toward Drug Delivery Research.

The inner limiting membrane (ILM) represents the structural boundary between the vitreous and the retina, and is suggested to act as a barrier for a wide range of retinal therapies. While it is widely acknowledged that the morphology of the human ILM exhibits regional variations and undergoes age-related changes, insight into its structure in laboratory animals is very limited. Besides presenting a detailed overview of the morphology and composition of the human ILM, this review specifically reflects on the species-specific differences in ILM structure. With these differences in mind, we furthermore summarize the most relevant reports on the barrier role of the ILM with regard to viral vectors, nanoparticles, anti-VEGF medication and stem cells. Overall, this review aims to deliberate on the impact of species-specific ILM variations on drug delivery research as well as to pinpoint knowledge gaps which future basic research should resolve.

In vitro and ex vivo models to study drug delivery barriers in the posterior segment of the eye.

Many ocular disorders leading to blindness could benefit from efficient delivery of therapeutics to the retina. However, despite extensive research into drug delivery vehicles and administration techniques, efficacy remains limited because of the many static and dynamic barriers present in the eye. Comprehension of the various barriers and especially how to overcome them can improve our ability to estimate the potential of existent drug delivery vectors and support the design of new ones. To this end, this review gives an overview of the most important ocular barriers for each administration route to the back of the eye. For each barrier, its biological composition and its role as an obstacle towards macromolecules, nanoparticles and viral vectors will be discussed; special attention will be paid to the influence of size, charge and lipophilicity of drug(s) (carrier) on their ability to overcome each barrier. Finally, the most significant available in vitro and ex vivo methods and models to test the potential of a therapeutic to cross each barrier are listed.

Toward smart design of retinal drug carriers: a novel bovine retinal explant model to study the barrier role of the vitreoretinal interface.

Retinal gene delivery via intravitreal injection is hampered by various physiological barriers present in the eye of which the vitreoretinal (VR) interface represents the most serious hurdle. In this study, we present a retinal explant model especially designed to study the role of this interface as a barrier for the penetration of vectors into the retina. In contrast to all existing explant models, the developed model is bovine-derived and more importantly, keeps the vitreous attached to the retina at all times to guarantee an intact VR interface. After ex vivo intravitreal injection into the living retinal explant, the route of fluorescent carriers across the VR interface can be tracked. By applying two different imaging methods on this model, we discovered that the transfer through the VR barrier is size-dependent since 40 nm polystyrene particles are more easily taken up in the retina than 100 and 200 nm sized particles. In addition, we found that removing the vitreous, as commonly done for culture of conventional explants, leads to an overestimation of particle uptake, and conclude that the ultimate barrier to overcome for retinal uptake is undoubtedly the inner limiting membrane. Damaging this matrix resulted in a massive increase in particle transfer into the retina. In conclusion, we have developed a highly relevant ex vivo model that maximally mimics the human in vivo physiology which can be applied as a representative test set-up to assess the potential of promising drug delivery carriers to cross the VR interface.

Ovarian tissue cryopreservation in female-to-male transgender people: insights into ovarian histology and physiology after prolonged androgen treatment.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

Effect of hyaluronic acid-binding to lipoplexes on intravitreal drug delivery for retinal gene therapy.

Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the uncoated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration.

Coating of Quantum Dots strongly defines their effect on lysosomal health and autophagy.

In the last decade the interest in autophagy got an incredible boost and the phenomenon quickly turned into an extensive research field. Interestingly, dysfunction of this cytoplasmic clearance system has been proposed to lie at the root of multiple diseases including cancer. We therefore consider it crucial from a toxicological point of view to investigate if nanomaterials that are developed for biomedical applications interfere with this cellular process. Here, we study the highly promising 'gradient alloyed' Quantum Dots (QDs) that differ from conventional ones by their gradient core composition which allows for better fluorescent properties. We carefully examined the toxicity of two identical gradient alloyed QDs, differing only in their surface coatings, namely 3-mercaptopropionic (MPA) acid and polyethylene glycol (PEG). Next to more conventional toxicological endpoints like cytotoxicity and oxidative stress, we examined the influence of these QDs on the autophagy pathway. Our study shows that the cellular effects induced by QDs on HeLa cells were strongly dictated by the surface coat of the otherwise identical particles. MPA-coated QDs proved to be highly biocompatible as a result of lysosomal activation and ROS reduction, two cellular responses that help the cell to cope with nanomaterial-induced stress. In contrast, PEGylated QDs were significantly more toxic due to increased ROS production and lysosomal impairment. This impairment next results in autophagy dysfunction which likely adds to their toxic effects. Taken together, our study shows that coating QDs with MPA is a better strategy than PEGylation for long term cell tracking with minimal cytotoxicity. STATEMENT OF SIGNIFICANCE: Gradient alloyed Quantum Dots (GA-QDs) are highly promising nanomaterials for biomedical imaging seeing they exhibit supremely fluorescent properties over conventional QDs. The translation of these novel QDs to the clinic requires a detailed toxicological examination, though the data on this is very limited. We therefore applied a systematic approach to examine the toxicity of GA-QDs coated with two commonly applied surface ligands, this while focusing on the autophagy pathway. The impact of QDs on this pathway is of importance since it has been connected with various diseases, including cancer. Our data accentuates that the coating defines the impact on autophagy and therefore the toxicity induced by QDs on cells: while MPA coated QDs were highly biocompatible, PEGylated QDs were toxic.

Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells.

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.