Gene delivery technologies have been developed for various biotechnology applications. In gene therapy, they are promising for the treatment of several inherited and acquired human diseases. When therapies require the transfection of a transgene, the vector integration is one of the solutions that is used for maintaining and sustaining expression. On the basis of their origin, vectorisation technologies are currently divided in two fields, gathering on one hand viral vectors and, on the other hand, non-viral approaches. In the case of the non-viral therapies, three main sub-fields are in progress to integrate transgenes. The first uses oligonucleotides to stimulate targeted gene repair by homologous recombination. The second is based on site-specific endonucleases for which the cleavage activity is used to stimulate the host recombination mechanisms in the presence of a DNA vector. The third one is developed from phage and transposon enzymatic systems. The two lasts sub-fields use non-viral enzymes and are the scope of this review. Here, our objective was to overview the main non-viral enzymatic systems able to integrate DNA cassettes. Their molecular and functional characteristics are summarized, and their properties and limits in the current state of the art highlighted. An overview of the safety and quality issues is also presented and discussed, taking into account the solutions that might circumvent problems, intellectual property and economic status for each system. As a conclusion, we propose projections of the future technological developments in the context of the different interests for public and private bodies.
DNA transposons are considered to be good candidates for developing tools for genome engineering, insertional mutagenesis and gene delivery for therapeutic purposes, as illustrated by the recent first clinical trial of a transposon. In this article we set out to highlight the interest of patent information, and to develop a strategy for the technological development of transposon tools, similar to what has been done in many other fields. We propose a patent landscape for transposon tools, including the changes in international patent applications, and review the leading inventors and applicants. We also provide an overview of the potential patent portfolio for the prokaryotic and eukaryotic transposons that are exploited by spin-off companies. Finally, we discuss the difficulties involved in tracing relevant state-of-the-art of articles and patent documents, based on the example of one of the most promising transposon systems, including all the impacts on the technological development of transposon tools.
Despite the strong presence of Chinese scientists in genome-editing research, little attention has been paid to the legal, economic, and scientific development of patented CRISPR technologies in China. In this study, we focus on CRISPR patent documents from academic and industrial Chinese players to assess their positioning on this breakthrough technology. We review the fields of application and the CRISPR components claimed in the relevant patent documents. Our results show different profiles observed for academic or industrial assignees. Most of the patent families in our data set cover applications in genome editing and nucleic-acid detection for human therapeutic and diagnostic purposes. Trends in the patent data since 2014 confirm that China' R&D has rapidly developed a significant CRISPR patent landscape of its own, covering a diverse range of systems and applications. These recent developments deserve closer scrutiny from the international CRISPR community.
Proofs of concept have shown that chromosomal gene clusters encoding ribosomal RNA (rRNA) constitute gene delivery integration loci that are optimal for transgene expression. However, because homologous recombination is efficient to integrate DNA segments into these genes in animals, new molecular tools are required to construct systems able to target molecules in the immediate vicinity of the rRNA genes.We investigated the properties of several DNA binding domains (DBDs) able to recognize specifically a motif within a 100-bp region of the rRNA genes that is 99-100% conserved among eukaryotes. Our findings demonstrate that two Myb-like DBDs originating from the endonucleases encoded by R2 non-LTR retrotransposons are promising candidates since they i) specifically recognize, with high affinity, a 20-bp binding site located within the expected genomic rDNA target, ii) act as monomers, iii) contain a nuclear localization signal, iv) remain functional when fused to another domain and, v) do not alter the functionality of the protein to which they are fused. However, results obtained in vivo with several R2DBD fusions reveal that two properties remain to be engineered before these DBDs can be integrated into a molecular targeting system directed into rRNA genes. The first concerns the ability of R2DBD to locate within the nucleolus, the organelle in which the rRNA genes reside. The second is the tendency of R2DBD to accumulate in certain parts of the nuclei, which limits its diffusion within nuclei. Solutions are discussed to circumvent these current limitations.Our results supply important information concerning the R2DBD properties and the targeting of plasmid DNA within nuclei. They will need to be further analyzed from three aspects; the unexploited advantages of the R2DBDs, the possibilities and limitations of fusion peptides for targeting integrations of non-viral vector, and the alternatives to fusion peptides for targeting vectors.
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