AAV-mediated gene therapies for glaucoma and uveitis: are we there yet?

Glaucoma and uveitis are non-vascular ocular diseases which are among the leading causes of blindness and visual loss. These conditions have distinct characteristics and mechanisms but share a multifactorial and complex nature, making their management challenging and burdensome for patients and clinicians. Furthermore, the lack of symptoms in the early stages of glaucoma and the diverse aetiology of uveitis hinder timely and accurate diagnoses, which are a cause of poor visual outcomes under both conditions. Although current treatment is effective in most cases, it is often associated with low patient adherence and adverse events, which directly impact the overall therapeutic success. Therefore, long-lasting alternatives with improved safety and efficacy are needed. Gene therapy, particularly utilising adeno-associated virus (AAV) vectors, has emerged as a promising approach to address unmet needs in these diseases. Engineered capsids with enhanced tropism and lower immunogenicity have been proposed, along with constructs designed for targeted and controlled expression. Additionally, several pathways implicated in the pathogenesis of these conditions have been targeted with single or multigene expression cassettes, gene editing and silencing approaches. This review discusses strategies employed in AAV-based gene therapies for glaucoma and non-infectious uveitis and provides an overview of current progress and future directions.

AAV-Based Strategies for Treatment of Retinal and Choroidal Vascular Diseases: Advances in Age-Related Macular Degeneration and Diabetic Retinopathy Therapies.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are vascular diseases with high prevalence, ranking among the leading causes of blindness and vision loss worldwide. Despite being effective, current treatments for AMD and DR are burdensome for patients and clinicians, resulting in suboptimal compliance and real risk of vision loss. Thus, there is an unmet need for long-lasting alternatives with improved safety and efficacy. Adeno-associated virus (AAV) is the leading vector for ocular gene delivery, given its ability to enable long-term expression while eliciting relatively mild immune responses. Progress has been made in AAV-based gene therapies for not only inherited retinal diseases but also acquired conditions with preclinical and clinical studies of AMD and DR showing promising results. These studies have explored several pathways involved in the disease pathogenesis, as well as different strategies to optimise gene delivery. These include engineered capsids with enhanced tropism to particular cell types, and expression cassettes incorporating elements for a targeted and controlled expression. Multiple-acting constructs have also been investigated, in addition to gene silencing and editing. Here, we provide an overview of strategies employing AAV-mediated gene delivery to treat AMD and DR. We discuss preclinical efficacy studies and present the latest data from clinical trials for both diseases.

MicroRNAs in cancer metastasis: biological and therapeutic implications.

Cancer metastasis is the primary cause of cancer-related deaths. The seeding of primary tumours at a secondary site is a highly inefficient process requiring substantial alterations in the genetic architecture of cancer cells. These alterations include significant changes in global gene expression patterns. MicroRNAs are small, non-protein coding RNAs which play a central role in regulating gene expression. Here, we focus on microRNA determinants of cancer metastasis and examine microRNA dysregulation in metastatic cancer cells. We dissect the metastatic process in a step-wise manner and summarise the involvement of microRNAs at each step. We also discuss the advantages and limitations of different microRNA-based strategies that have been used to target metastasis in pre-clinical models. Finally, we highlight current clinical trials that use microRNA-based therapies to target advanced or metastatic tumours.

Clinical and economic burden of benign and malignant skin lesions in renal transplant recipients.

BACKGROUND: Studies evaluating the economic burden of dermatological care in the transplant setting are currently not available in Australia. AIMS: To evaluate the clinical and economic burden of benign and malignant skin lesions in renal transplant recipients in Central Queensland. METHODS: A bottom-up approach was used to determine the clinical burden and direct costs from patient-level Medicare data obtained from Service Australia for skin lesions. RESULTS: Seventy-six percent of the renal transplant population in Central Queensland participated in this study. The median age was 57.0 years (standard deviation ± 13.6) and the majority (61.8%) of participants were men. The mean duration after transplant surgery was 99.9 months (interquartile range, 73.2-126.6 months). During a 2-year follow-up, 22 (40%) patients were diagnosed with benign skin lesions, 21 (38%) with nonmelanoma skin carcinoma (NMSC) and one (2%) with melanoma. There was a total of 231 visits to clinicians for diagnostic and therapeutic skin procedures and the direct costs to Medicare was $48 806 Australian Dollars (AUD) or $30 427 US Dollars (USD). Approximately 86% of the total direct costs was spent for nonNMSC and mean direct costs for NMSC was $763 AUD (or $476 USD). CONCLUSION: This Medicare data-based study provides further insight into the burgeoning clinical and economic burden of the care for benign and malignant skin lesions in the renal transplantation setting in Australia.

Attitudes of eye health practitioners towards diagnostic tests and therapies for dry eye disease in Australasia.

INTRODUCTION: The diagnosis of dry eye is challenging for eye health practitioners (EHP) and recently, a variety of new diagnostic tests have emerged. This study assesses the attitudes of EHP to dry eye and testing and compares these with attitudes in 2003. METHODS: An electronic questionnaire was disseminated to EHP in Australasia between December 2020 to March 2021. Participants rated the likelihood that presenting symptoms/signs were associated with dry eye, the utility of diagnostic tests, the value of test characteristics, and their satisfaction with dry eye diagnostics. Qualitative responses were categorised into positive, negative, or neutral themes. RESULTS: 144 responses were received, with 117 (81.3%) from Australia and 27 (18.7%) from New Zealand. Posterior blepharitis was significantly more likely to be associated with dry eye than other factors (p < 0.01). Clinical history, fluorescein staining and FBUT were judged significantly more useful in diagnosing dry eye compared to other tests (p < 0.01). Test validity was judged significantly more important in choosing a test than other qualities. Qualitative attitudes towards dry eye presentations and diagnostic tests were positive in 42.2% and 24.3%, negative in 32.4% and 41.9%, and neutral in 25.5% and 33.8% respectively. CONCLUSIONS: The opinions of EHP regarding dry eye tests were variable, but most favour history and corneal staining for diagnosis. Patterns of responses were similar to that reported by Turner et al 16-years ago, however, there is a higher satisfaction with available tests and therapeutic options. There is a need to develop a consensus amongst real-world clinicians regarding an optimum diagnostic pathway for dry eye, particularly in relation to newer diagnostic tests.

Differences in Uveal Melanoma Age-Standardized Incidence Rates in Two Eastern States of Australia Are Driven by Differences in Rurality and Ultraviolet Radiation.

Uveal melanoma (UM) is the second-most-common melanoma in humans and has a high age-standardized incidence rate (ASR) in Australia. Regional patterns of UM ASRs in Australia are unknown. The aim of this study was to determine and compare UM ASRs in two geographically disparate eastern states, Queensland (QLD) and Victoria (VIC), by using cancer registry data that was obtained from 2001 to 2013. World-standardized UM ASRs and incidence-rate ratios (IRRs) were calculated. Higher UM ASR was also observed in anterior UM compared to posterior UM ASR. UM ASR remained unchanged from 2001 to 2013 in QLD but decreased in VIC. A south-to-north latitude trend in UM ASR along the east of Australia is weakly evident, and rural populations have higher UM ASRs than major city populations in both states. Differences in ultraviolent radiation (UVR) susceptibility, indigenous populations, social behaviours, chemical exposure, and socioeconomic status could all be contributing to differences in UM rates between QLD and VIC and between rural compared to major city areas. It is possible that a minority of cases in QLD and VIC might be prevented by sun-protective behaviours. This is important, because these findings suggest that QLD, which is already known to have one of the highest cutaneous melanoma (CM) ASRs in the world, also has one of the highest UM ASRs.

Complement Factor H Loss in RPE Cells Causes Retinal Degeneration in a Human RPE-Porcine Retinal Explant Co-Culture Model.

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

The Impact of Ultraviolet Radiation on the Aetiology and Development of Uveal Melanoma.

Uveal melanoma (UM) is currently classified by the World Health Organisation as a melanoma caused by risk factors other than cumulative solar damage. However, factors relating to ultraviolet radiation (UVR) susceptibility such as light-coloured skin and eyes, propensity to burn, and proximity to the equator, frequently correlate with higher risk of UM. These risk factors echo those of the far more common cutaneous melanoma (CM), which is widely accepted to be caused by excessive UVR exposure, suggesting a role of UVR in the development and progression of a proportion of UM. Indeed, this could mean that countries, such as Australia, with high UVR exposure and the highest incidences of CM would represent a similarly high incidence of UM if UVR exposure is truly involved. Most cases of UM lack the typical genetic mutations that are related to UVR damage, although recent evidence in a small minority of cases has shown otherwise. This review therefore reassesses statistical, environmental, anatomical, and physiological evidence for and against the role of UVR in the aetiology of UM.

The Effects of Continual Consumption of Origanum vulgare on Liver Transcriptomics.

Pathogen control is re-emerging as a significant challenge to the health of both humans and animals. The livestock industry is in the process of massively replacing in-feed antibiotics with organic production friendly plant-based products. Nutrigenomics as a science of the effects of food constituents on gene expression is shedding more light on both benefits and detrimental side-effects of feed additive prolonged consumption on the host, indicating the need to understand the feed-host interactions and their influence on the host disease profile. In this study, we investigated the effects of 2% oregano powder supplementation on the liver gene expression in healthy male broilers from the hatch to 6 weeks of age. Deep RNAseq was performed on average 113.3 million paired and quality trimmed sequences per sample and four samples for the control and treatment each. The results demonstrate the severity of oregano effect on liver gene expression with substantial modifications in steroid hormone regulation, fat and carbohydrate metabolism alterations and strong influence on the host disease and function profile. Oregano supplementation was able to interfere with the transcriptional effects of a range of registered drugs and to significantly transcriptionally inhibit a range of cancer disease categories including liver cancer, and to modify fat and carbohydrate metabolism.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice.

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages.

Prolonged continual consumption of oregano herb interferes with the action of steroid hormones and several drugs, and effects signaling across the brain-gut axis.

Herbs and spices have been used throughout human history for their medicinal qualities. The advent of cheap and readily available medicines have lessened the need for herbs and spices as traditional medicines, however, they are rapidly regaining popularity with rising interest of the general population in health, natural products and nutrition. The need for alternative medicines with antimicrobial properties, such as herbs and spices, has also come to the forefront in light of the recent bans of antibiotic use in the livestock industry, including the poultry industry. This large scale use presents an opportunity to observe nutrigenomic effects of prolonged use of herbs on a substantial number of birds fed high concentrations of these products throughout the production cycle. In this manuscript, we investigated the transcriptional effect of continual prolonged oregano supplementation on chicken ileum gene expression. Based on ileum transcriptomics, we report that continual supplementation with 2% oregano altered microbiota-gut-brain axis signalling, rearranged cancer susceptibility towards reduced steroid hormone-related cancers and altered expression of genes targeted by many registered drugs, thus likely affecting their efficiency and side effects. Transcriptional toxicology analysis indicated significant activation of Ventricular Septal Defect and Congenital Heart Disease categories. Our results, counter the notion that natural products such as oregano have the potential for little to no side-effects as they are "natural". The nutrigenomic approach of understanding benefits and side effects of the food we eat, can revolutionize disease management and therapy and have special significance in designing the diets for individuals or livestock with known disease predispositions.

Natural product-derived phytochemicals as potential agents against coronaviruses: A review.

Coronaviruses are responsible for a growing economic, social and mortality burden, as the causative agent of diseases such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), avian infectious bronchitis virus (IBV) and COVID-19. However, there is a lack of effective antiviral agents for many coronavirus strains. Naturally existing compounds provide a wealth of chemical diversity, including antiviral activity, and thus may have utility as therapeutic agents against coronaviral infections. The PubMed database was searched for papers including the keywords coronavirus, SARS or MERS, as well as traditional medicine, herbal, remedy or plants, with 55 primary research articles identified. The overwhelming majority of publications focussed on polar compounds. Compounds that show promise for the inhibition of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin B2, quercetin, myricetin, caffeic acid, psoralidin, isobavachalcone, and lectins such as griffithsin. Other compounds such as lycorine may be suitable if a therapeutic level of antiviral activity can be achieved without exceeding toxic plasma concentrations. It was noted that the most promising small molecules identified as coronavirus inhibitors contained a conjugated fused ring structure with the majority being classified as being polyphenols.

Characterisation and validation of the 8-fold quadrant dissected human retinal explant culture model for pre-clinical toxicology investigation.

One of the major challenges in studying ocular toxicology is a lack of clinically-relevant retinal experimental models. In this study we describe the use of an in vitro human retinal explant strategy to generate a reproducible experimental model with utility in neuro-toxicity retinal studies. A retinal dissection strategy, referred to as the 8 fold quadrant dissection, was developed by dissecting human donor retinas into 4 fragments through the fovea in order to obtain 8 experimentally reproducible retinal explants from a single donor. This quadrant dissection gave rise to equivalent proportions of CD73+ photoreceptors and CD90+ ganglion cells in 8 fragments from a single donor and this remained stable for up to 3 days in culture. Major retinal cell types continued to be observed after 8 weeks in culture, despite breakdown of the retinal layers, suggesting the potential to use this model in long-term studies where observation of individual cell types is possible. The utility of this system was examined in a proof of principle neuro-toxicology study. We showed reproducible induction of toxicity in photoreceptors and retinal ganglion cells by glutamate, cobalt chloride and hydrogen peroxide insults, and observed the therapeutic positive effects of the administration of memantine, formononetin and trolox. The quadrant dissected human retinal explants have the potential to be used in toxicology studies in human ocular diseases.

Adeno-associated virus neutralising antibodies in type 1 diabetes mellitus.

Recombinant Adeno-associated viruses (AAVs) are an attractive vector for gene therapy delivery which may be blocked by AAV neutralising antibodies (NAbs). As Type 1 Diabetes (T1DM) is an endocrine disease of immunological origin, it is likely that NAb profiles are altered in the disease. In this study NAb to AAV2, AAV5, AAV6, and AAV8 in 72 subjects with T1DM and 45 non-diabetic patients were measured over a 4-year follow-up period. AAV2 NAb titres were significantly lower in non-diabetic subjects (P = 0.036). The T1DM group had more AAV8 NAb activity at baseline (P = 0.019), whilst after 4 years follow-up the T1DM group displayed developed increased AAV 5 (P = 0.03), 6 (P = 0.03) and 8 (P = 0.002) activity relative to the control group, however, overall AAV5 and 8 NAb levels were very low in patients <40. AAV NAb titre activity and prevalence generally appears higher in T1DM, however, low levels of AAV 5 and 8, particular in younger adult age groups at which T1DM can be targeted, could make these attractive vectors to target the disease.

Retinal explant culture: A platform to investigate human neuro-retina.

The retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex inter-cellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture.

Spotlight on Bortezomib: potential in the treatment of hepatocellular carcinoma.

INTRODUCTION: This study reviews the evidence for the use of Bortezomib (BZB), a first-in-class proteasome inhibitor in advanced Hepatocellular carcinoma (HCC). This review aims to delineate the role of BZB within the management of non-surgical and metastatic HCC, either as an alternative or as an adjunct to the current treatment paradigm. AREAS COVERED: In addition to BZB pharmacology and mechanism of action, safety and tolerance profiles of the drug obtained from clinical trials are explored. The utility of BZB as a therapeutic agent either alone or in combination with other therapies against HCC, including its application in both preclinical and clinical settings has been reviewed. In particular, we highlight the importance of preclinical evaluation of BZB as a combinatorial agent in synergism with other therapies for the use in the management of HCC. EXPERT OPINION: There has been much interest surrounding the use of BZB, a first-in-class proteasome inhibitor for HCC therapy. The discernment of outcomes of BZB clinical trials for HCC need to take into consideration the disease-specific factors that can affect survival outcomes including patient selection and aetiological differences. Further preclinical testing of BZB in combination with other therapeutic modalities can be important for eliciting enhanced anti-HCC effects.

Gene therapy and the adeno-associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations.

Voretigene neparvovec-rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno-associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age-related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future.

Synergistic and independent action of endogenous microRNAs 122a and 199a for post-transcriptional liver detargeting of gene vectors.

In hepatocellular carcinoma (HCC), which usually develops in a cirrhotic liver, treatments preserving normal liver function and viability are vitally important. Here, we utilise the differential expression of miRNAs 122a and 199a between normal hepatocytes and HCC to generate vectors harbouring their binding sites for hepatocyte detargeting. Using a reporter gene, we observed a synergistic detargeting of cells expressing both miRNAs as well as cells expressing either of the miRNAs; while expression was retained in HCC cells negative for both miRNA122a and miRNA199a. Mimics and inhibitors for individual miRNAs were used to confirm these results. Furthermore, suicide gene therapy with cytosine deaminase (CD)/5-fluorocytosine system resulted in limited killing of cells expressing either of the two miRNAs. Finally, we report feasibility of using adeno associated virus (AAV) based vectors for delivery of this dual regulated gene delivery system. These results present a novel dual targeted system whereby miRNA122a and miRNA199a act either synergistically or independently in regulating transgene expression with vectors harbouring binding sites of both miRNAs and have implications in detargeting vectors from multiple cell types in the liver.

MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma.

A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy.