Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors

Abstract

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine

“…To overcome these obstacles, doggybone DNA vectors containing the same expression cassettes without both OriP/EBNA1 and shp53 and lacking bacterial sequences were developed, which may reduce the immune response. 18 Whereas reprogramming using cell-penetrating proteins suffered from extremely low iPSC induction efficiency and requirement of proteins in large quantities, 19 , 20 use of synthetic modified mRNAs, which can be cost-effectively produced on a large scale, provides the clearest solution to generate the most unambiguously footprint-free iPSCs suitable for the clinic application. 21 However, reprogramming using synthetic modified mRNAs requires serial transgene delivery, and blood cells seem to be difficult for reprogramming using the modified mRNA technology.…”

SARS-CoV-2 Doggybone DNA Vaccine Produces Cross-Variant Neutralizing Antibodies and Is Protective in a COVID-19 Animal Model

“…The synthesized spike expression cassette for the stabilized dbDNA construct was identical to the wild-type version except for 2956–2961 nucleotide bases of spike, resulting in amino acid changes K986P, V987P [ 18 , 19 , 20 , 21 , 22 ]. The dbDNA with the spike expression cassette was manufactured as previously described [ 14 , 23 , 24 ].…”

CLN7 mutation causes aberrant redistribution of protein isoforms and contributes to Batten disease pathobiology

“…iPSC lines were previously generated as described using episomal doggybone DNA vectors ( 45 ) using dermal fibroblasts held by the UCL NCL database and cell repository (https://www.ucl.ac.uk/ncl-disease/), donated by two CLN7 patients (Pa380; homozygous for c.881C>A, p.(Thr294Lys) and Pa474; homozygous for c.1393C>T, p.(Arg465Trp)) and two age-matched controls (PromoCell). All iPSC were maintained on inactivated MEFs (iMEFs) feeder layer and transitioned to feeder-free in preparation for neural specification.…”