Caution: Plasmid DNA topology affects luciferase assay reproducibility and outcomes

Tudini, Emma; Burke, Les J.; Whiley, Phillip J.; Ševčı́k, Jan; Spurdle, Amanda B.; Brown, Melissa A.

doi:10.2144/btn-2019-0060

Cited by 5 publications

(3 citation statements)

References 7 publications

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“…The mechanism underlying the influence of the DNA topology on the ultimate transfection efficiency is, however, still unknown. Nevertheless, it was found that sc-pDNA displays a very tiny hydrodynamic size that, in turn, may be responsible for its great intracellular mobility through entangled and cross-linked composite networks of actin and microtubules [78][79][80][81][82][83].…”

Section: Plasmid Dnamentioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

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Section: Plasmid Dnamentioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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“…Supercoiled pDNA (sc-pDNA) is superior to its open-circular or linearized counterparts in gene expression due to a reduced susceptibility to intracellular degradation. Although how DNA topology influences transfection efficiency remains unclear, sc-pDNA was found to display a small hydrodynamic size, which in turn may provide greater mobility through cross-linked and composite networks of actin and microtubules inside the cytoplasm of targeted cells (Bono et al, 2020;Cherng et al, 1999;Dhanoya et al, 2011Dhanoya et al, , 2012Tudini et al, 2019;Weintraub et al, 1986).…”

Section: Barriers To Nucleic Acid Deliverymentioning

confidence: 99%

Lipid‐based nucleic acid therapeutics with in vivo efficacy

Sufian

Ilies

2022

WIREs Nanomed Nanobiotechnol

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Synthetic vectors for therapeutic nucleic acid delivery are currently competing significantly with their viral counter parts due to their reduced immunogenicity, large payload capacity, and ease of manufacture under GMP‐compliant norms. The approval of Onpattro, a lipid‐based siRNA therapeutic, and the proven clinical success of two lipid‐based COVID‐19 vaccines from Pfizer‐BioNTech, and Moderna heralded the specific advantages of lipid‐based systems among all other synthetic nucleic acid carriers. Lipid‐based systems with diverse payloads—plasmid DNA (pDNA), antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), and messenger RNA (mRNA)—are now becoming a mature technology, with growing impact in the clinic. Research over four decades identified the key factors determining the therapeutic success of these multi‐component systems. Here, we discuss the main nucleic acid‐based technologies, presenting their mechanism of action, delivery barriers facing them, the structural properties of the payload as well as the component lipids that regulate physicochemical properties, pharmacokinetics and biodistribution, efficacy, and toxicity of the resultant nanoparticles. We further detail on the formulation parameters, evolution of the manufacturing techniques that generate reproducible and scalable outputs, and key manufacturing aspects that enable control over physicochemical properties of the resultant particles. Preclinical applications of some of these formulations that were successfully translated from in vitro studies to animal models are subsequently discussed. Finally, clinical success and failure of these systems starting from 1993 to present are highlighted, in a holistic literature review focused on lipid‐based nucleic acid delivery systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

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“…I was interested to see Tudini et al's letter 'Caution: Plasmid DNA topology affects luciferase assay reproducibility and outcomes' that, I believe, incorrectly identified plasmid supercoiling as a variable that affects reporter plasmid output [1]. It is important for all authors to consider issues that may affect experimental reproducibility, especially in a systemic manner.…”

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confidence: 99%

Plasmid Multimer Status and not DNA Topology Likely Affects Luciferase Assay Reproducibility

Jobling

2020

BioTechniques

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Caution: Plasmid DNA topology affects luciferase assay reproducibility and outcomes

Cited by 5 publications

References 7 publications

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

Lipid‐based nucleic acid therapeutics with in vivo efficacy

Plasmid Multimer Status and not DNA Topology Likely Affects Luciferase Assay Reproducibility

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