The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

Abstract

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

“…Importantly, alongside the elevated PEDF level, a decrease of MMP-2 expression is according to the observed antiangiogenic environment following tIPE and tRPE cell therapy. These results are consistent with our previous pilot studies showing safety, effectiveness at a higher single dose, and survival of PEDF-expressing cells transplanted in the rat subretinal space 31 , 49 , 53, 54, 55, 56, 57, 58…”

“…As a consequence, there is a great demand for developing a treatment that allows for the maintenance of high PEDF level for an extended period of time. In Europe, a phase Ib/IIa ex vivo gene therapy clinical trial for long-term expression of PEDF in IPE cells by combining pFAR4 miniplasmid and SB transposon technologies is planned and has been submitted for approval to Swiss regulatory authorities Swissmedic 31 , 49 , 55 (https://www.targetamd.eu/), with all preclinical tests completed 82 . Our approach is based on the use of RPE and IPE cells because they show similar characteristics and many functions in common.…”

“…Construction of the PEDF transposon plasmid, whose expression cassette contains the hPEDF gene flanked by SB transposon ITRs, and stable gene delivery using the hyperactive SB100X transposase system have been previously reported 31 , 49 , 54 , 55 . The schematic diagram of the plasmids and constructs used in the study were shown in Figure 7A.

Figure 7Diagram of the Plasmids Used in the Study and Timeline(A) Schematic diagram of the various constructs used in the study: (i) pFAR4 CMV SB100X SV40 plasmid, (ii) pFAR4-ITRs CMV PEDF BGH plasmid, (iii) pFAR4-ITRs CMV PEDF-His BGH plasmid, and (iv) pFAR4-ITRs CAGGS Venus SV40 plasmid.

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F‐box/WD Repeat‐Containing Protein 5 Mediates the Ubiquitination of Apoptosis Signal‐Regulating Kinase 1 and Exacerbates Nonalcoholic Steatohepatitis in Mice

“…The amino acid sequences of the FBXW5 (S1) and FBXW5 (S3) are highly conserved between mouse and human on the basis of sequences alignment. After 8 weeks of HFHC diet feeding, we delivered a hemagglutinin (HA)‐tagged pT3 plasmid carrying human FBXW5 (S1) or FBXW5 (S3) (pT3‐FBXW5(S1 or S3)‐HA) and the SB100X transposase plasmid into mice liver followed by another 8 weeks of HFHC diet feeding (Supporting Fig. A).…”

Preliminary findings from the Implementation of a wet macular degeneration patient-reported outcome and experience measure

“…Age-related macular degeneration (AMD) can result in significantly decreased vision, subsequently impacting a patient's independence with activities of daily living, social relationships, financial security, and general mental well-being. [1][2][3][4] A utility value study of MD found that patients with minor visual impairment would trade 11% of their remaining lifetime to restore their vision, while patients with severe impairment would trade 60%; highlighting the significant impact AMD can have on the individual. [5] An understanding of the concerns, experiences, and perspectives of patients with wet MD is critical in delivering patient-centered care and improving life quality over a long period.…”