Comparative Study of Adeno-associated Virus, Adenovirus, Bacu lovirus and Lentivirus Vectors for Gene Therapy of the Eyes

Kalesnykas, Giedrius; Kokki, Emmi; Alasaarela, Laura; Lesch, Hanna P.; Tuulos, Timo; Kinnunen, Kati; Uusitalo, Hannu; Airenne, Kari J.; Ylä‐Herttuala, Seppo

doi:10.2174/1566523217666171003170348

Cited by 40 publications

(27 citation statements)

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“…1 In this study we have taken advantage of long lasting transgene expression in RPE cells in vivo through Lentivirus transduction. 46 Also, AAV in our hand was unable to transduce iPSC-RPE cells as has been experienced by several other groups. This might be due to the higher efficacy of lentiviral vector to transduce post mitotic RPE cells as has been proved for nondividing cells.…”

Section: Discussionmentioning

confidence: 65%

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Shahi

Hermans

Sinha

et al. 2018

Preprint

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PurposeMutations in the KCNJ13 gene are known to cause Leber’s Congenital Amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 gene encodes the Kir7.1 subunit protein which acts as a tetrameric inwardly rectifying potassium ion channel in the retinal pigment epithelium to maintain ionic homeostasis thereby allowing photoreceptors to encode visual information. We sought to determine if genetic approaches might be effective in treating blindness due to mutations in KCNJ13.MethodsWe developed patient-derived hiPSC-RPE carrying an autosomal recessive nonsense mutation in the KCNJ13 gene (c.158G>A, p.Trp53*). We performed biochemical and electrophysiology assays of Kir7.1 function. Both small molecule read-through drug and gene-therapy approaches were tested using this disease-in-a-dish approach.ResultsWe found that the LCA16 hiPSC-RPE had normal morphology but did not express a functional Kir7.1 channel and was unable to demonstrate normal physiology. Following read-through drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV and Kir7.1 current was restored. Similarly, loss-of-function of Kir7.1 channel was circumvented by lentiviral gene delivery to the hiPSC-RPE cells. In either approach, Kir7.1 protein was expressing normally with restoration of membrane potential and Kir7.1 current.ConclusionLoss-of-function mutation in Kir7.1 is a cause of LCA. Using either read-through therapy or gene augmentation, we rescued Kir7.1 channel function in patient-derived iPSC-RPE cells via a precision medicine approach.

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Section: Discussionmentioning

confidence: 65%

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Shahi

Hermans

Sinha

et al. 2018

Preprint

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“…1 In this study, we have taken advantage of lentivirus transduction to mediate long-lasting transgene expression in RPE cells in vivo. 45 Also, we were unable to transduce hiPSC-RPE cells with adeno-associated virus (AAV), a difficulty reported by several other groups. .…”

Section: Discussionmentioning

confidence: 94%

Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness

Shahi

Hermans

Sinha

et al. 2019

The American Journal of Human Genetics

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Pathogenic variants of the KCNJ13 gene are known to cause Leber congenital amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 encodes the Kir7.1 subunit that acts as a tetrameric, inwardly rectifying potassium ion channel in the retinal pigment epithelium (RPE) to maintain ionic homeostasis and allow photoreceptors to encode visual information. We sought to determine whether genetic approaches might be effective in treating blindness arising from pathogenic variants in KCNJ13. We derived human induced pluripotent stem cell (hiPSC)-RPE cells from an individual carrying a homozygous c.158G>A (p.Trp53*) pathogenic variant of KCNJ13. We performed biochemical and electrophysiology assays to confirm Kir7.1 function. We tested both small-molecule readthrough drug and gene-therapy approaches for this ''disease-in-a-dish'' approach. We found that the LCA16 hiPSC-RPE cells had normal morphology but did not express a functional Kir7.1 channel and were unable to demonstrate normal physiology. After readthrough drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV, and the Kir7.1 current was restored. Similarly, we rescued Kir7.1 channel function after lentiviral gene delivery to the hiPSC-RPE cells. In both approaches, Kir7.1 was expressed normally, and there was restoration of membrane potential and the Kir7.1 current. Loss-of-function variants of Kir7.1 are one cause of LCA. Using either readthrough therapy or gene augmentation, we rescued Kir7.1 channel function in iPSC-RPE cells derived from an affected individual. This supports the development of precision-medicine approaches for the treatment of clinical LCA16.

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“…4), we also compared the production of adenoviral vectors in iCELLis bioreactor compared with scale-X bioreactor. Titers for AdGFP 11 produced in scale-X hydro system and iCELLis Nano bioreactors were analyzed using HPLC. 2 The titer for scale-X bioreactor run was 1.11 • 10 11 viral particles per milliliter and for the iCELLis Nano run 8.53 • 10 10 viral particles per milliliter after clarification.…”

Section: Lentiviral Vector Yields In Icellis Nano and Scale-x Bioreacmentioning

confidence: 99%

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

et al. 2020

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We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis Ò bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-XÔ hydro bioreactor (2.4 m 2) and iCELLis Nano system (2.67 m 2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.

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Comparative Study of Adeno-associated Virus, Adenovirus, Bacu lovirus and Lentivirus Vectors for Gene Therapy of the Eyes

Abstract: Our findings show that intravitreal gene delivery is safe and feasible with AAV, AdV and lentivirus vectors.

Cited by 40 publications

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Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

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