VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy

Alsing, Sidsel; Doktor, Thomas Koed; Askou, Anne Louise; Jensen, Emilie Grarup; Ahmadov, Ulvi; Kristensen, Lasse Sommer; Andresen, Brage Storstein; Aagaard, Lars; Corydon, Thomas J.

doi:10.1016/j.omtn.2022.02.019

Cited by 10 publications

(32 citation statements)

References 64 publications

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“…Furthermore, the vector contains an expression cassette with eGFP expressed from the ubiquitously active phosphoglycerate kinase 1 (PGK) promoter. The back-toback orientation of the two expression cassettes as well as the full cloning strategy can be found in (Alsing et al, 2022). VSV-Gpseudotyped LV particles were produced, purified, and the infectious titer determined by RT-qPCR as described in (Alsing et al, 2022).…”

Section: Vectors Primers and Antibodiesmentioning

confidence: 99%

“…The back-toback orientation of the two expression cassettes as well as the full cloning strategy can be found in (Alsing et al, 2022). VSV-Gpseudotyped LV particles were produced, purified, and the infectious titer determined by RT-qPCR as described in (Alsing et al, 2022). In this paper, LV/shRNA VEGF -eGFP corresponds to LV/VMD2-miR451-12, and LV/shRNA Irr -eGFP corresponds to LV/VMD2-miR451-S1.…”

Section: Vectors Primers and Antibodiesmentioning

confidence: 99%

“…This protocol describes delivery of LVs encoding anti-angiogenic RNAi molecules and eGFP to murine RPE cells (Figures 1A,B) as described in (Alsing et al, 2022). Steps involving gene therapy and eGFP co-delivery to murine RPE, in vivo and ex vivo assessment of expression and localization in the retina, and isolation of RPE cells and autofluorescence-restrictive cell sorting (Figure 1C), are outlined in Figure 1C.…”

Section: Protocolmentioning

confidence: 99%

“…However, since efficacy is strongly correlated with delivery, assessment of efficacy in the rodent eye can be precluded by sub-optimal delivery. Optimal delivery of gene-coding, virus-based vectors to the RPE cells can be achieved by a subretinal injection (Askou et al, 2012;Askou et al, 2015), a micro-precise process with inherent variability between injections (Alsing et al, 2022). In mice, this leads to differences in the size of the transduced area, and thus a variation among mice in the same treatment group.…”

Section: Introductionmentioning

confidence: 99%

“…When assessing the efficacy of a novel gene therapy tool, isolation of a specific cell subpopulation is desired; the target cells that received the gene therapy or gene editing tool. By co-delivering genes expressing fluorescent proteins, such as the enhanced green fluorescent protein (eGFP) (Askou and Corydon, 2018), isolation of gene therapy target cells is possible by FACS methods (Holmgaard et al, 2017;Alsing et al, 2022). In this context, isolation of targeted RPE cells can be a hurdle.…”

Section: Introductionmentioning

confidence: 99%

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Simple Autofluorescence-Restrictive Sorting of eGFP+ RPE Cells Allows Reliable Assessment of Targeted Retinal Gene Therapy

Alsing

Lindholm

Haldrup

et al. 2022

Front. Drug. Deliv.

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Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.

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Section: Vectors Primers and Antibodiesmentioning

confidence: 99%

Section: Vectors Primers and Antibodiesmentioning

confidence: 99%

Section: Protocolmentioning

confidence: 99%