Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi

Abstract

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Keywords: COST; European Cooperation in Science and Technology; GenE-HumDi; MT: RNA/DNA Editing; base editors; delivery systems; genome editing; regulatory guidelines.

Graphical abstract

Figure 1

The map illustrates the global reach of GenE-HumDi COST Action, with 269 participants distributed across 38 countries Each participant is actively involved in at least one of the consortium’s working groups.

Figure 2

Gene editing technologies based on CRISPR-Cas systems The CRISPR-Cas toolbox contains multiple versions of Cas enzymes combined with other proteins to manipulate genomic DNA. For conventional genome editing, Cas9 nucleases are used to create DNA double-strand breaks (DSBs) that facilitate insertions or deletions (indels) of base pairs at the target site introduced by DSB correction via the non-homologous end-joining (NHEJ) pathway, leading to disruption of target DNA sequences. For precise edits, Cas9 nucleases are supplemented with a DNA template for its integration into the desired target locus by either homology-directed repair (HDR) or by homology-independent targeted integration (HITI). These approaches are accompanied by the simultaneous introduction of undesired indels, as such other approaches have fused different DNA modulatory proteins to Cas9 to alter the indel spectrum or to affect the HDR:indel ratio to favor HDR. The DNA break-free base and prime editors (BE and PE, respectively) display high product purity of the editing outcome and highly decrease the risks associated with DNA DSBs, including induction of gross chromosomal aberrations. BE mediates single-base substitutions, while PE can create small precise insertions, deletions, or base substitutions. To induce insertion of large DNA regions, some systems utilize Cas9 fused to transposases, serine integrases (PASTE) or CRISPR-associated transposases (CASTs) to insert large donor DNA templates. PASTE inserts an attB site into the desired genomic location by prime editing, followed by the integration of the donor DNA via the serine integrase (e.g., BxbI) acting on the flanking attP site. The CAST system uses CRISPR-associated transposases to insert transposon DNA engineered to carry the desired cargo. To manipulate the transcriptional status of a gene, a nuclease-deactivated version of Cas9 (dCas9) is employed that maintains the ability to bind a specific DNA target. When fused to transcriptional activators or repressors, target genes can be dialed up or down. By instead using fusion proteins that regulate the epigenetic status of a gene, inherited epigenetic marks can lead to permanent modulation of transcription.

Figure 3

Gene editing delivery systems Schematic illustration of the varieties of the tools to deliver genome editing components, classified into two categories based on the different constituents and cellular entry mechanisms: viral (A) and non-viral methods (B). In the first category, the most widely used viruses for delivery of GE tools are retroviruses, adeno-associated viruses, and adenoviruses, where entry mechanisms of the gene editing components into the target cell are virus specific. Viral methods can be used for both in vitro and in vivo applications. The non-viral delivery methods can be further split into three subgroups: physical methods utilized for in vitro gene editing (gene gun, electroporation and microinjection), and biological (extracellular vesicles, EVs) or chemical (lipid nanoparticles, LNPs; Poly lactic-co-glycolic acid nanoparticles, PLGA NPs; dendrimers and inorganic nanoparticles) methods for in vivo gene editing.

Figure 4

Sensitive assays and tools for prediction and detection of off-target sites Schematic overview of in-cellula off-target detection methods. Upper panel, from left to right: integrase-deficient lentivirus capture (IDLV Capture); genome-wide, unbiased identification of DSBs enabled by sequencing (Guide-Seq); direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS). Lower panel, from left to right: high-throughput, genome-wide translocation sequencing (HTGTS); chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-seq); discovery of in situ Cas off-targets and verification by sequencing (Discover-seq). DSB, double-stand break; NGS, next-generation sequencing; dsODN, double-strand oligo DNA; LTR, long terminal repeat. Schematic overview of biochemical off-target detection methods, from left to right Digenome-seq, Site-seq, Circle-seq and CHANGE-seq.

Figure 5

Applications of gene editing in the treatment of inherited rare diseases, cancer, and infectious diseases Summary diagram of conditions treatable by gene editing that are currently being investigated by GenE-HumDi COST Action members. Specific gene targets are listed in italics per each disease.