Recent evidence shows that climate change is leading to irreversible and existential impacts on vulnerable communities and countries across the globe. Among other effects, this has given rise to public debate and engagement around notions of climate crisis and emergency. The Loss and Damage (L&D) policy debate has emphasized these aspects over the last three decades. Yet, despite institutionalization through an article on L&D by the United Nations Framework Convention on Climate Change (UNFCCC) in the Paris Agreement, the debate has remained vague, particularly with reference to its remit and relationship to adaptation policy and practice. Research has recently made important strides forward in terms of developing a science perspective on L&D. This article reviews insights derived from recent publications by the Intergovernmental Panel on Climate Change (IPCC) and others, and presents the implications for science and policy. Emerging evidence on hard and soft adaptation limits in certain systems, sectors and regions holds the potential to further build momentum for climate policy to live up to the Paris ambition of stringent emission reductions and to increase efforts to support the most vulnerable. L&D policy may want to consider actions to extend soft adaptation limits and spur transformational, that is, nonstandard risk management and adaptation, so that limits are not breached. Financial, technical, and legal support would be appropriate for instances where hard limits are transgressed. Research is well positioned to further develop robust evidence on critical and relevant risks at scale in the most vulnerable countries and communities, as well as options to reduce barriers and limits to adaptation.

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1–68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1–68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2–4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.