Particle-Mediated Gene Transfection and Organotypic Culture of Postmortem Human Retina

Masri, Rania A.; Lee, Sammy; Madigan, Michele C.

doi:10.1167/tvst.8.2.7

Cited by 7 publications

(7 citation statements)

References 64 publications

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“…Shooting the nucleic acids with the gene gun method leads to the transfection of the sole GCL. 33 Instead, viral vectors transduce all retinal layers but only if the retina is isolated from embryos (from E15) or early postnatal days (P0−P2). 34,35 Electroporation does not transfect the retinal explants efficiently if the nucleic acids are made approaching the tissue from the GCL side, but it works when the explant is treated from the RPE side.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Shooting the nucleic acids with a biolistic method transfects isolated groups of cells in the GCL but not deeper. 33 Electroporation 16 and viral vectors 34,35 appear somewhat to be the best method to drive genetic modifications in retinal explants, mainly when organs are isolated from a young age. Efficiency decreases in explants from adult animals.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Nonviral vectors applied either in the culture media (RPE side) or directly on the explants (ganglion cell layer (GCL) side) can transfect only the photoreceptors or the GCL, respectively. Shooting the nucleic acids with a biolistic method transfects isolated groups of cells in the GCL but not deeper . Electroporation and viral vectors , appear somewhat to be the best method to drive genetic modifications in retinal explants, mainly when organs are isolated from a young age.…”

Section: Introductionmentioning

confidence: 99%

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New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection

et al. 2021

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The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed “Reverse Magnetofection” provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.

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Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection

et al. 2021

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“…Donor eyes were obtained and prepared as retinal explant cultures as described above, with trephines taken from areas of interest (macular, midperiphery, periphery). During Round 2 selection (described below) the "Interphase Retinal Explant Culture System" was used (Retinal ganglion cell side up, photoreceptors down), to simulate intravitreal injection and with the aim to select for photoreceptors (adapted from [20]). Four retinal tissue pieces, each of approximately 1 x 2 cm 2 in size, were dissected out and placed onto the prepared tissue culture insert for second round AAV library selection.…”

Section: Methodsmentioning

confidence: 99%

AAV capsid bioengineering in primary human retina

Westhaus,

Eamegdool,

Fernando

et al. 2023

Preprint

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Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To this end, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

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“…These two RGC types receive a lot of attention due to their reliable occurrence across non-human primates (Goodchild et al 1996;) and humans (Dacey and Petersen 1992;Kolb et al 1992;Rodieck et al 1985;Soto et al 2020), where they make up around 80% of ganglion cells (Dacey , 2004Dacey and Petersen 1992;Masri et al 2019b;Yan et al 2020b). They were first described by Polyak, who extended the efforts of Cajal (1893) with Golgi staining in the primate retina (Poljak 1935;Polyak 1941).…”

Section: P R I M At E Modelmentioning

confidence: 99%

Functional Decomposition of Retinal Ganglion Cell Receptive Fields

Zapp¹

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I reviewed the literature about past and current methods of subunit inference and about the involvement of subunits in direction selectivity, I contributed to writing the manuscript, and prepared the figures Figure 1, Figure 2, and Figure 3. SN reviewed the literature about subunit function and adaptation, contributed to writing the manuscript, and prepared the figures Figure 4, Figure 5, and Figure 6. TG supervised the literature research and prepared the initial draft. All authors helped revise the manuscript.

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Particle-Mediated Gene Transfection and Organotypic Culture of Postmortem Human Retina

Cited by 7 publications

References 64 publications

New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection

New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection

AAV capsid bioengineering in primary human retina

Functional Decomposition of Retinal Ganglion Cell Receptive Fields

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