AAV plasmid DNA simplifies liver‐directed in vivo gene therapy: comparison of expression levels after plasmid DNA‐, adeno‐associated virus‐ and adenovirus‐mediated liver transfection

Abstract

BackgroundSuccessful liver gene therapy depends on efficient gene transfer techniques and long‐lasting gene expression after successful transfer. Over the last decades, important progress has been made with the introduction of viral vectors using animal models, although their use is hampered by a complex and costly preparation compared to the simple and cost‐effective preparation of plasmid DNA. These problems become even more critical when considering the application of viral vectors in human gene therapy and gene therapy trials. In a previous study, we were able to show that the hydrodynamics‐based gene transfer of plasmid‐DNA, containing the adeno‐associated‐virus specific inverted terminal repeats (AAV‐ITR), prolongs gene expression in the liver, although it remained unclear whether plasmid gene transfer could achieve similar expression levels compared to viral‐vector gene transfer.MethodsRat livers were transfected in‐vivo with AAV‐ITR‐containing plasmid‐DNA using a modified hydrodynamics‐based procedure. Expression levels were monitored thereafter and compared with expression levels after viral‐vector gene transfer.ResultsA high and stable long‐term expression was achieved after in vivo transfection of rat livers with AAV‐ITR‐containing plasmids. The expression course resembled that after AAV‐mediated gene transfer, and the expression was at least as high, and lasted as long, compared to recombinant AAV‐mediated gene transfer.ConclusionsWe consider AAV‐ITR‐containing plasmids as a simple and cost‐effective alternative to recombinant viral vectors, especially for liver‐directed gene therapy in rodents. With ongoing progress in gene transfer methods for naked DNA, these plasmids may also become a successful alternative to recombinant viral vectors in human gene therapy. Copyright © 2010 John Wiley & Sons, Ltd.

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

“…Several options are available to deliver the transgene expression cassette to the target cell/organ Injection of naked DNA, either as plasmids (Doenecke et al 2010;Oishi et al 2016)o rmini-circles (Viecelli et al 2014;Hou et al 2016;Wuetal2016), is a simple mode of transgene delivery which lends itself to local delivery and is relatively non-immunogenic compared to some viral vector approaches (Wolff and Budker 2005). Mini-circles are devoid of plasmid backbone DNA; this may enhance transgene expression by overcoming heterochromatin formation and avoiding inflammation triggered by bacterial DNA (Mayrhofer et al 2009).…”

Chemoaffinity Material for Plasmid DNA Analysis by High-Performance Liquid Chromatography with Condition-Dependent Switching between Isoform and Topoisomer Selectivity

“…The key element bearing the correct gene is the plasmid DNA (or the derived RNA), which to date is most often packed into adeno-associated virus particles. 3 From three possible isoforms, the so-called covalently closed circular (ccc) form is considered to be most active for therapeutics. Pharmaceutical grade ccc pDNA is produced by fermentation in Escherichia coli and isolated subsequently from harvested cells by alkaline cell lysis and multistep purification of pDNA.…”

“…In November 2012, the first drug for gene therapy has been approved by the European Medicines Agency, which may mark a new therapeutic era, as there are numerous other genetic agents in clinical phases covering a broad range of health issues. The key element bearing the correct gene is the plasmid DNA (or the derived RNA), which to date is most often packed into adeno-associated virus particles . From three possible isoforms, the so-called covalently closed circular (ccc) form is considered to be most active for therapeutics.…”

Advances in Gene Delivery Systems

“…[168] Because of its simplicity, high efficiency and reproducibility, hydrodynamic gene delivery has been utilized for the delivery of DNA, small interfering RNA, proteins, small compounds and even viral vectors. [171][172][173][174][175][176][177][178][179] This technique has been widely used for gene therapy studies, gene knockdown, functional analysis of genetic elements and for establishing a disease model in animals. [171,176,180] In an attempt to apply this simple procedure of gene delivery to the clinic, efforts have been made to reduce the total injection volume.…”