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 Willer 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 Willer 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 Willer cell transfection efficiency. Since neuroprotection is explored as a therapy for diabetic retinopathy and glaucoma, we furthermore examined the Willer cell's lipoplex-induced transfection efficiency and cytotoxicity in stressful conditions linked to these diseases - i.e. hypoxia, hyperglycemia and oxidative stress. Interestingly, Willer 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 Willer cells in healthy as well as diseased conditions

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-β 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

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the “first come – first serve” principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells

Evading innate immunity in nonviral mRNA delivery : don't shoot the messenger

In de field of non-viral gene therapy, in vitro transcribed (IVT) mRNA has emerged as a promising tool for the delivery of genetic information. Over the past few years it has become widely known the introduction of IVT mRNA into mammalian cells elicits an innate immune response which has favored mRNA use towards immunotherapeutic vaccination strategies. However, for non-immunotherapy related applications this intrinsic immune-stimulatory activity directly interferes with the aimed therapeutic outcome, as it can seriously compromise the expression of the desired protein. This review presents an overview of the immune-related obstacles that limit mRNA advance for non-immunotherapy related applications

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

Müller cells as a target for retinal therapy

Muller 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 Willer 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 Muller cells also have an essential role in retinal regeneration. Although many questions regarding this subject remain, it is clear that Muller 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