Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids

Völkner, Manuela; Pavlou, Marina; Büning, Hildegard; Michalakis, Stylianos; Karl, Mike O.

doi:10.1089/hum.2020.321

Cited by 25 publications

(47 citation statements)

References 49 publications

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“…This did not affect karyotype (Fig S1B ,C). Human mCar-GFP retinal organoids were produced using a modified version of a previously published protocol which has been shown to generate robust numbers of cone photoreceptors (Fig S1A) (15)(16)(17). The mCar-driven GFP signal was predominantly located in the outer neuroepithelial layer as to be expected for cones (Fig 1A).…”

Section: Resultsmentioning

confidence: 99%

“…The Personal Genome Project hiPS cell line PGP1 (47) was a gift from George Church The mCar-GFP and Crx-mCherry iPSC lines (kind gift from Olivier Goureau -see ( 23)) were maintained in mTeSR1 (Stem cell technologies) on matrigel coated plates and split using ReleSR at room temperature (Stem cell technologies). Stem cells were differentiated to retinal organoids using an optimized protocol as previously described see also supplementary methods (17).…”

Section: Generation Of a Hipsc Cone Reporter Linementioning

confidence: 99%

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Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

Gasparini

Tessmer

Reh

et al. 2021

Preprint

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Once human photoreceptors die, they do not regenerate, thus photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation and synaptic connectivity to the host will be critical in advancing this technology to clinical practice. Unlike the unstructured grafts of prior cell suspension transplantations into end-stage degeneration models, we describe extensive incorporation of iPSC retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extend throughout the graft, even forming a common outer limiting membrane. Donor-host interaction appears to promote polarisation as well as development of morphological features critical for light detection, namely formation of inner and well stacked outer segments oriented towards the RPE. Putative synapse formation and graft function is evident both at a structural and electrophysiological level. Overall, these results show that human photoreceptors interact readily with a partially degenerated retina. Moreover, incorporation into the host retina appears to be beneficial to graft maturation, polarisation and function.

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Section: Resultsmentioning

confidence: 99%

Section: Generation Of a Hipsc Cone Reporter Linementioning

confidence: 99%

Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

Gasparini

Tessmer

Reh

et al. 2021

Preprint

Self Cite

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“…For analysis of human photoreceptor cells, human retinal organoids (HRO) derived from the Crx-mCherry iPSC line (kindly provided by O. Goureau, Paris) and generated as described in ref. [52] were dissociated as follows: organoids were washed 3x in 37 °C PBS, transferred to 37 °C Papain solution and incubated for 2 h at 37 °C on a horizontal shaker shaking at 90 rpm [53] . Then, DNase I stock solution was added to 5% v/v (> 400 KU/ml) and HRO triturated carefully using a glass pipette.…”

Section: Methodsmentioning

confidence: 99%

Label-free imaging flow cytometry: analysis and sorting of enzymatically dissociated tissues

Herbig

Tessmer

Nötzel

et al. 2021

Preprint

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Biomedical research often relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells' properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on their physical properties. Sorting real-time deformability and fluorescence cytometry (soRT-FDC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with additional challenges including survival, changes in morphology, or presence of aggregates. Here, we introduce methods for robust analysis and sorting of single cells from mammalian nervous tissue and provide DNNs which are capable of distinguishing visually similar cells. Exemplarily, we employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye. Results provide evidence that the combination of machine learning and soRT-FDC allows label-free enrichment of target cells from dissociated tissues.

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“…Cloning, production, purification, and titration of AAV vectors were performed as described in detail previously (Börner et al, 2020; Strobel et al, 2019; Völkner et al, 2021; Weinmann et al, 2020). Expression of the enhanced green fluorescent protein (GFP) reporter was driven by the ubiquitously active cytomegalovirus promotor.…”

Section: Methodsmentioning

confidence: 99%

“…Expression of the enhanced green fluorescent protein (GFP) reporter was driven by the ubiquitously active cytomegalovirus promotor. We selected two second generation to efficiently transduce various cell types in human retinal explants and human retinal organoids (Pavlou et al, 2021;Völkner et al, 2021). Capsid variant AAV9-NVVRSSS was selected based on a high transduction efficiency in AAV library screens in human cells focusing on the NXXRXXX peptide motif (Börner et al, 2020) (Weinmann et al, unpublished data).…”

Section: Aav Production and Transduction Of Cell Culturesmentioning

confidence: 99%

Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

Fischer

Weinmann

Gillardon

2021

Preprint

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Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates. In the present study, we tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes. AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1x10E5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1x10E4 viral genomes per cell) lead to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker, which may indicate functional alterations induced by viral transduction and/or transgene expression.

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Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids

Cited by 25 publications

References 49 publications

Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

Label-free imaging flow cytometry: analysis and sorting of enzymatically dissociated tissues

Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

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