Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

Bire, Solenne; Ley, Déborah; Casteret, Sophie; Mermod, Nicolas; Bigot, Yves; Rouleux‐Bonnin, Florence

doi:10.1371/journal.pone.0082559

Cited by 27 publications

(28 citation statements)

References 40 publications

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“…The low rate of conventional rAAV integration was strategically exploited to minimize persistent piggyBac transposase expression, which could cause multiple transposition cycles, leading to eventual loss of transposons and therefore therapeutic transgene expression, and additionally could increase damage to the genome through aberrant chromosomal break repair and recombination events . Thus, to add to the biosafety and efficacy of this hybrid vector system, a further advancement would be to deliver a more labile source of transposase expression, such as mRNA using lipid nanoparticles.…”

Section: Discussionmentioning

confidence: 99%

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV‐piggyBac Gene Therapy

et al. 2019

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Recombinant adeno‐associated viral (rAAV) vectors are highly promising vehicles for liver‐targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile‐onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV‐mediated liver‐targeted gene therapy. The Abcb4‐/‐ mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4‐/‐ mice with established liver disease, but not in disease‐free, wild‐type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase‐mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile‐onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life‐long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV‐piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.

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Section: Discussionmentioning

confidence: 99%

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV‐piggyBac Gene Therapy

et al. 2019

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“…However, we have some problems to be solved in this study: (i) we need to generate CAR T cells from leukemic patients treated with cytotoxic agents and/ or TKIs who are probably poor responders, (ii) we have no comparative data on the anti-leukemic potency of CAR T cells between retro/lentiviral and piggyBac gene modifications, (iii) we used an anti-CH2CH3 antibody, which is not compliant to cGMP, for selection of CAR-expressing T cells and culture plates inappropriate to cGMP, and (iv) we observed relatively high copy number integration of CD19.CAR-transposon plasmids (~5 copies per cell) with residual piggyBac -transposase plasmids (~2 copies per cell), which may be a potential risk of unexpected T-cell transformation, re-transposition of a transgene, or rejection of infused T cells in patients if expressing piggyBac protein. Regarding the safety concerns, co-delivery of a suicide gene or use of piggyBac -transposase mRNA may be helpful (11,13,31). All things considered, clinical-grade DNA plasmids are inexpensive and easy to manufacture relative to viral vectors; therefore, the piggyBac -transposon system in combination with our T-cell culture system would increase the cost-benefit and safety of CAR-modified T-cell therapy, thereby facilitating regulatory approval.…”

Section: Discussionmentioning

confidence: 99%

Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

et al. 2014

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Background aims To develop a treatment option for Philadelphia chromosome—positive acute lymphoblastic leukemia (Ph+ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR). Methods A CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15–containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph+ALL cell lines including three TKI-resistant (T315I–mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines. Results We obtained ~ 1.3 × 108 CART cells (CD4+, 25.4%; CD8+, 71.3%), co-expressing CD45RA and CCR7 up to ~80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CART cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor–related apoptosis-inducing ligand and interleukin-2. Conclusions We generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph+ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph+ALL.

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“…Thus, employment of a more labile (therefore, showing a short half-life) source of transposase, such as PB transposase mRNA or protein, is desirable. Indeed, efficient transposition using in vitro-transcribed PB transposase mRNAs has been documented in various species including mammals [89][90][91]. Notably, PB transposase mRNA (Improved Super piggyBac transposase mRNA) is now commercially available from Transposagen Biopharmaceuticals, Inc. (#SPB-100; Lexington, KY, USA).…”

Section: Pb Transposase Mrnamentioning

confidence: 99%

“…Expression of a GOI is guaranteed when it is placed between two insulators, which prevents propagation of a silencing chromatin structure over the GOI. Bire et al [89] demonstrated the effectiveness of insulators for increasing the expression rate of a GOI after PB-based gene delivery.…”

Section: Use Of Insulatorsmentioning

confidence: 99%

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

et al. 2020

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In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. piggyBac (PB), one of three transposons (PB, Sleeping Beauty (SB), and Tol2) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.

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Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

Cited by 27 publications

References 40 publications

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV‐piggyBac Gene Therapy

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV‐piggyBac Gene Therapy

Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

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