Challenges in non-viral ocular gene transfer

2007 ◽  
Vol 35 (1) ◽  
pp. 47-49 ◽  
Author(s):  
L. Peeters ◽  
N.N. Sanders ◽  
J. Demeester ◽  
S.C. De Smedt
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Viral Gene ◽  
Target Tissue ◽  
Long Distance ◽  
Promising Alternative ◽  
Gene Complexes ◽  
Poly Ethylene ◽  
Review Reports ◽  
Subretinal Injection

Nowadays, there is no effective treatment for many retinal disorders. Knowledge of the genetic basis of many severe ocular diseases may allow for alternative treatments by gene therapy. Non-viral gene complexes, such as lipo- and poly-plexes, can be delivered to the posterior segment, most often the target tissue, by intravitreal or subretinal injection. Since subretinal injections are very invasive, intravitreal injection is a promising alternative route to deliver gene complexes into the eye. However, the drawback of this technique is the relative long distance the complexes have to travel through the vitreous gel before they reach the retina. This mini-review reports on how non-viral gene complexes behave in vitreous. It especially focuses on how the coating of lipoplexes with poly(ethylene glycol) influences their behaviour in vitreous and the transfection of retinal pigment epithelium.

The Human Fetal Retinal Pigment Epithelium: A Target Tissue for Thyroid Hormones

1999 ◽  
Vol 31 (6) ◽  
pp. 399-406 ◽  
Author(s):  
K.G. Duncan ◽  
K.R. Bailey ◽  
J.D. Baxter ◽  
D.M. Schwartz
Keyword(s):  
Retinal Pigment Epithelium ◽  
Thyroid Hormones ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Target Tissue

Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium

2010 ◽  
Vol 148 (1) ◽  
pp. e99-e100 ◽  
Author(s):  
D. Vercauteren ◽  
M. Piest ◽  
M. Al Soraj ◽  
A.T. Jones ◽  
J.F.J. Engbersen ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cellular Uptake ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Gene Complexes ◽  
In Cells

Semiquantitative Methods for GFP Immunohistochemistry and In Situ Hybridization to Evaluate AAV Transduction of Mouse Retinal Cells Following Subretinal Injection

2020 ◽  
pp. 019262332096480
Author(s):  
Gennadiy Bondarenko ◽  
Steven D. Sorden ◽  
Brian J. Christian ◽  
Sharron Webster ◽  
Alok K. Sharma
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Layer ◽  
Rna In Situ Hybridization ◽  
Semiquantitative Evaluation ◽  
Green Fluorescent ◽  
Subretinal Injection

The goal of this study was to develop methods for the evaluation of green fluorescent protein (GFP) and GFP transcript biodistribution in paraformaldehyde-fixed paraffin-embedded (PFPE) eye sections to assess the effectiveness of Adeno-associated virus (AAV) gene delivery in an experimental ocular toxicity study. Female C57BL/6NTac mice were administered AAV2-enhancedGFP vector once via subretinal injection. One group also received anti-inflammatory therapy (meloxicam). Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) for GFP were performed on PFPE serial eye sections and evaluated using semiquantitative methods. On day 43, GFP labeling in both IHC and ISH sections was greatest in the retinal pigment epithelium, compared with other retinal layers in which expression was negative to moderate. Despite the presence of IHC GFP labeling in the photoreceptor layer (PRL) in some animals, only low numbers of transduced cells were detected by ISH in the PRL. Simultaneous analysis of IHC and ISH may be needed for comprehensive assessment of gene transduction and protein biodistribution. This study demonstrates approaches for semiquantitative evaluation of IHC and ISH that allow interpretation and reporting of GFP expression in toxicity studies.

scholarly journals Advantages of robotic assistance over a manual approach in simulated subretinal injections and its relevance for gene therapy

Gene Therapy ◽  
2021 ◽  
Author(s):  
Reza Ladha ◽  
Thijs Meenink ◽  
Jorrit Smit ◽  
Marc D. de Smet
Keyword(s):  
Gene Therapy ◽  
Clinical Success ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Genetic Diseases ◽  
Cost Effective ◽  
Robotic Assistance ◽  
Injection Duration ◽  
Retinal Hole ◽  
Subretinal Injection

AbstractSubretinal injection is a method for gene delivery to treat genetic diseases of the photoreceptors and retinal pigment epithelium. A reflux-free subretinal injection is important to allow effective, safe, and cost-effective gene therapy to the retina. We report on a comparison between manual and robotic assistance in simulated subretinal injections using an artificial retina model. Nine surgeons carried out the procedure with and without the Preceyes Surgical System, using an OPMI Lumera 700 Zeiss surgical microscope equipped with intra-operative optical coherence tomography. Success in creating a bleb without reflux, injection duration, drift, tremor, and increase in the diameter of the puncture hole were analyzed. Robotic assistance improved drift (median 16 vs 212 µm), tremor (median 1 vs 18 µm), enlargement of the retinal hole, and allowed for prolonged injection times (median 52 vs 29 sec). Robotic assistance allowed higher rate of bleb formation (8/9 vs 4/9 attempts) with a moderate reduction in reflux (7/9 vs 8/9 attempts) in this artificial model. Robotic assistance can significantly contribute to subretinal injections and provide quantifiable parameters in assessing surgical and clinical success of novel retinal gene therapies.

scholarly journals Novel non-sense mutation in RP2 (c.843_844insT/p.Arg282fs) is associated with a severe phenotype of retinitis pigmentosa without evidence of primary retinal pigment epithelium involvement

2019 ◽  
Vol 12 (5) ◽  
pp. e224451 ◽  
Author(s):  
Faye Horner ◽  
James Wawrzynski ◽  
Robert MacLaren
Keyword(s):  
Retinal Pigment Epithelium ◽  
Retinitis Pigmentosa ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Gene Replacement ◽  
Viral Gene ◽  
Retinal Layer ◽  
Viral Gene Therapy ◽  
Gene Therapy Vectors ◽  
Route Of Delivery

Retinitis pigmentosa (RP) relates to a heterogeneous group of rod-cone dystrophies of varying genetic aetiology. There is currently great interest in gene replacement therapy. Phenotyping is of particular importance because some RP genes are expressed ubiquitously and it is critically important to understand which retinal layer is primarily affected. RP2 is increasingly diagnosed in patients suffering from X-linked RP, which causes outer retinal degeneration. We present a case of a previously unreported null mutation in RP2 associated with severe RP. Loss of the retinal pigment epithelium (RPE) was noted in the central macula but not around the disc or peripherally. There was therefore no evidence of independent degeneration of the RPE. Hence despite expression in all retinal cells, RP2 deficiency does not appear to be pathogenic to the RPE. This observation may be helpful in considering the promoter and route of delivery of adeno-associated viral gene therapy vectors encoding RP2.

scholarly journals The learning curve of murine subretinal injection among clinically trained ophthalmic surgeons

2021 ◽  
Author(s):  
Peirong Huang ◽  
Siddharth Narendran ◽  
Felipe Pereira ◽  
Shinichi Fukuda ◽  
Yosuke Nagasaka ◽  
...  
Keyword(s):  
Learning Curve ◽  
Success Rate ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Training System ◽  
Training Data ◽  
Surgeon Experience ◽  
Systematic Training ◽  
Cusum Analysis ◽  
Subretinal Injection

PURPOSE: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. METHODS: To evaluate the LC for SRI in a murine model, we analyzed training data from 3 clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffer saline injection and the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. A Cumulative Sum (CUSUM) analysis was performed and plotted individually to monitor each surgeon's simultaneous performance. RESULTS: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364-training case as a "cutoff" point, the predicted probability of success before and after the 364th case was 65.38% and 99.32%, respectively (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. CONCLUSIONS: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time.

scholarly journals Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

2012 ◽  
Vol 29 (2) ◽  
pp. 83-93 ◽  
Author(s):  
MAREN ENGELHARDT ◽  
CHINATSU TOSHA ◽  
VANDA S. LOPES ◽  
BRYAN CHEN ◽  
LISA NGUYEN ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Host Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Host Cells ◽  
Apical Surface ◽  
Noninvasive Methods ◽  
Rpe Cells ◽  
Subretinal Injection

AbstractReplacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch’s membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host’s cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

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