The vectors went in two-by-two: Transduction efficiency and tolerability of dual and triple rAAV vector delivery following intravitreal injection for genome-editing applications.

Genome or prime editing has become a promising tool for the treatment of hereditary disorders affecting the inner retina, such as dominant optic neuropathies. In vivo delivery of gene editors, such as Cas9, is typically achieved using recombinant adeno-associated virus (rAAV) vectors, which have a broad range of cellular tropisms and are well tolerated following intravitreal administration. Owing to the large size of gene editing constructs and the limited carrying capacity of rAAV (<5.

Characterization of drusen formation in a primary porcine tissue culture model of dry AMD.

Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled In this study, we further characterize a "drusen-in-a-dish" primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes.

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies.

Evolution of CRISPR/Cas Systems for Precise Genome Editing.

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects.

Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage.

Purpose: ARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry.

A Comprehensive Look at the -13910 C>T LCT Gene Polymorphism as a Molecular Marker for Vitamin D and Calcium Levels in Young Adults in Central and Eastern Europe: A Preliminary Study.

Intolerance to dairy products resulting from the abnormal digestion of milk sugar (lactose) is a common cause of human gastrointestinal disorders. The aim of this study was to show that the -13910 C>T LCT gene polymorphism, together with genotypes of selected VDR gene polymorphisms and diet and nutritional status parameters, can impact the prevalence of vitamin D and calcium deficiency in young adults. This study was conducted on a group of 63 people, which comprised 21 individuals with primary adult lactase deficiency, and a control group of 42 individuals with no hypolactasia.

Tolerability and tropism of recombinant adeno-associated virus vectors in the African green monkey (Chlorocebus sabaeus) anterior chamber.

While many studies have investigated the use of recombinant adeno-associated vectors (rAAV) in the posterior chamber for treatment of inherited retinal diseases, fewer studies have looked at rAAV's ability to transduce cells within the anterior chamber. This study focuses on evaluating the tropism and tolerability of three rAAV serotypes-rAAV2/6, rAAV2/9, and rAAV2/2[MAX]-expressing a green fluorescent protein (GFP) reporter following intracameral injection in the non-human primate (NHP) African green monkey (Chlorocebus sabaeus) model. Injection of high dose (1 × 10 vg/eye) rAAV vector resulted in transient inflammation characterized by aqueous flare and cellular infiltrate that resolved without intervention in all serotypes.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC.

Prostaglandin-based rAAV-mediated glaucoma gene therapy in Brown Norway rats.

Prostaglandin analogs are first-line treatments for open angle glaucoma and while effective at lowering intraocular pressure, they are undermined by patient non-compliance, causing atrophy of the optic nerve and severe visual impairment. Herein, we evaluate the safety and efficacy of a recombinant adeno-associated viral vector-mediated gene therapy aimed at permanently lowering intraocular pressure through de novo biosynthesis of prostaglandin F2α within the anterior chamber. This study demonstrated a dose dependent reduction in intraocular pressure in normotensive Brown Norway rats maintained over 12-months.

Isolation, culture, and cryosectioning of primary porcine retinal pigment epithelium on transwell cell culture inserts.

Primary culture and long-term maintenance of primary retinal pigment epithelium (RPE) is a useful model system for the study of ocular pathologies such as age-related macular degeneration. Here, we detail the steps for the isolation and long-term culture of primary porcine RPE. We also describe steps for cryoprotecting, cryosectioning, and interrogating with immunofluorescence and histochemistry RPE cells grown on transwell membranes.

The Relative Abundances of Human Leukocyte Antigen-E, α-Galactosidase A and α-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells.

The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from h×h× triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from h, h and transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants.

Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution.

Purpose: Retinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge.

Methods: We introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes.

rAAV-mediated over-expression of acid ceramidase prevents retinopathy in a mouse model of Farber lipogranulomatosis.

Farber disease (FD) is a rare monogenic lysosomal storage disorder caused by mutations in ASAH1 that results in a deficiency of acid ceramidase (ACDase) activity and the abnormal systemic accumulation of ceramide species, leading to multi-system organ failure involving neurological decline and retinopathy. Here we describe the effects of rAAV-mediated ASAH1 over-expression on the progression of retinopathy in a mouse model of FD (Asah1) and its littermate controls (Asah1 and Asah1). Using a combination of non-invasive multimodal imaging, electrophysiology, post-mortem histology and mass spectrometry we demonstrate that ASAH1 over-expression significantly reduces central retinal thickening, ceramide accumulation, macrophage activation and limits fundus hyper-reflectivity and auto-fluorescence in FD mice, indicating rAAV-mediated over-expression of biologically active ACDase protein is able to rescue the anatomical retinal phenotype of Farber disease.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency.

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route.

Development of a Preclinical Laser Speckle Contrast Imaging Instrument for Assessing Systemic and Retinal Vascular Function in Small Rodents.

Purpose: To develop and test a non-contact, contrast-free, retinal laser speckle contrast imaging (LSCI) instrument for use in small rodents to assess vascular anatomy, quantify hemodynamics, and measure physiological changes in response to retinal vascular dysfunction over a wide field of view (FOV).

Methods: A custom LSCI instrument capable of wide-field and non-contact imaging in small rodents was constructed. The effect of camera gain, laser power, and exposure duration on speckle contrast variance was standardized before the repeatability of LSCI measurements was determined in vivo.

CRISPR/Cas Technology in Pig-to-Human Xenotransplantation Research.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats linked to Cas nuclease) technology has revolutionized many aspects of genetic engineering research. Thanks to it, it became possible to study the functions and mechanisms of biology with greater precision, as well as to obtain genetically modified organisms, both prokaryotic and eukaryotic. The changes introduced by the CRISPR/Cas system are based on the repair paths of the single or double strand DNA breaks that cause insertions, deletions, or precise integrations of donor DNA.

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells.

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from h×h bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence.

Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation.

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation.

Application of Genetically Engineered Pigs in Biomedical Research.

Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation.

Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells.

Validating a low-cost laser speckle contrast imaging system as a quantitative tool for assessing retinal vascular function.

The ability to monitor progression of retinal vascular diseases like diabetic retinopathy in small animal models is often complicated by their failure to develop the end-stage complications which characterize the human phenotypes in disease. Interestingly, as micro-vascular dysfunction typically precedes the onset of retinal vascular and even some neurodegenerative diseases, the ability to visualize and quantify hemodynamic changes (e.g.

A Comparison of Inducible Gene Expression Platforms: Implications for Recombinant Adeno-Associated Virus (rAAV) Vector-Mediated Ocular Gene Therapy.

The ability to temporally control levels of a therapeutic protein in vivo is vital for the development of safe and efficacious gene therapy treatments for autosomal dominant or complex retinal diseases, where uncontrolled transgene overexpression may lead to deleterious off-target effects and accelerated disease progression. While numerous platforms exist that allow for modulation of gene expression levels - ranging from inducible promoters to destabilizing domains - many have drawbacks that make them less than ideal for use in recombinant adeno-associated virus (rAAV) vectors, which over the past two decades have become the mainstay technology for mediating gene delivery to the retina. Herein, we discuss the advantages and disadvantages of three major gene expression platforms with regard to their suitability for ocular gene therapy applications.

Transgenic Plant Detection Using an AuNPs Based SPR Biosensor.

The intensive development and commercialization of genetically modified plants observed over the last decade has led to the development of transgenic detection methods that are rapid and sensitive. Among the strategies used for the detection/monitoring of genetically modified organisms (GMOs), surface plasmon resonance (SPR) meets the necessary criteria. This optical technique measures the changes in the refractive index in the vicinity of thin metal layers (i.

Expression of pluripotency-related genes is highly dependent on trichostatin A-assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos.

The present study sought to examine whether trichostatin A (TSA)-assisted epigenetic transformation of porcine bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) affects the transcriptional activities of pluripotency-related genes (Oct4, Nanog, c-Myc, Sox2 and Rex1), multipotent stemness-related gene (Nestin) and anti-apoptotic/anti-senescence-related gene (Survivin). Epigenetically transformed or non-transformed BM-MSCs that had been transcriptionally profiled by qRT-PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA-mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti-ageing properties of porcine BM-MSCs.

Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1.

Throughout the CNS, interactions between pre- and postsynaptic adhesion molecules establish normal synaptic structure and function. Leucine-rich repeat (LRR) domain-containing proteins are a large family that has a diversity of ligands, and their absence can cause disease. At the first retinal synapse, the absence of LRIT3 expression leads to the disassembly of the postsynaptic glutamate signaling complex (signalplex) expressed on depolarizing bipolar cell (DBC) dendrites.