Refining strategies to translate genome editing to the clinic

Journal name:
Nature Medicine
Year published:
Published online


Recent progress in developing programmable nucleases, such as zinc-finger nucleases, transcription activator–like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas nucleases, have paved the way for gene editing to enter clinical practice. This translation is a result of combining high nuclease activity with high specificity and successfully applying this technology in various preclinical disease models, including infectious disease, primary immunodeficiencies, hemoglobinopathies, hemophilia and muscular dystrophy. Several clinical gene-editing trials, both ex vivo and in vivo, have been initiated in the past 2 years, including studies that aim to knockout genes as well as to add therapeutic transgenes. Here we discuss the advances made in the gene-editing field in recent years, and specify priorities that need to be addressed to expand therapeutic genome editing to further disease entities.

At a glance


  1. Designer-nuclease-induced genome editing.
    Figure 1: Designer-nuclease-induced genome editing.

    For ex vivo applications, TALENs and ZFNs are typically delivered in the form of mRNA (in red), and the CRISPR–Cas complex as mRNA/gRNA (in red) or as a ribonucleoprotein (RNP, a gRNA complexed with a Cas9 protein; in red and green). The resulting nuclease-induced DNA double-strand break will then be repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). NHEJ is error-prone and will introduce insertion or deletion (indel) mutations of variable length at the break site. Depending on nuclease activity, indel mutations are inserted either on one allele or both target alleles (orange cells), the latter of which might potentially result in a complete functional-gene knockout. For precise HDR, donor DNA must be co-delivered with the customized nuclease. The donor contains homologous sequences (purple boxes) to the target locus. Classically, nonintegrating vectors based on adeno-associated virus (AAV) or integrase-deficient lentiviral vector (IDLV), which contain viral repeat sequences at their termini (ITR, inverted terminal repeat; LTR, long terminal repeat), are employed for this purpose. The donor DNA serves as the HDR template and can be used to correct a mutation (red asterisks) in a gene or to target the integration of either an autonomous therapeutic expression cassette (yellow box) with its own promoter (blue arrow), or a corrective exon (yellow box), which is spliced to the endogenous upstream exons by use of the splice acceptor (red box). HDR events may result in mono- or biallelic gene editing (green cells). Alternatively, a monoallelic HDR event can occur in combination with an NHEJ-mediated indel mutation on the second allele (green–orange cells).

  2. Improving the specificity of engineered nucleases.
    Figure 2: Improving the specificity of engineered nucleases.

    (a) For TALENs, improved specificity can be achieved by optimizing the number or the nature of the TALE modules that compose the DNA-binding domain. Each module recognizes a single nucleotide of the target sequence. The specificity of a module is depicted by the color code: e.g., green oval represents a TALE module that binds to 'A'. DNA-binding specificity of this module is determined by the di-residue asparagine isoleucine (NI). The overall activity and specificity of a TALEN is further determined by the length and/or the composition of the C-terminus of the TALE domain, the so-called linker, which connects the DNA-binding domain with the FokI cleavage domain6, 53, 54, 55, 83, 84, 85. TALENs cleave their cognate target site only upon dimerization of the FokI domains of the two subunits. To minimize cleavage at off-target sites by homodimers, i.e., two identical TALEN subunits, obligate heterodimeric FokI variants, illustrated as (+) and (−), have been developed47, 48, 49, 50. (b) The specificity of CRISPR–Cas nucleases can be enhanced by various means, such as by truncating the gRNA or by exchanging amino acids in the Cas9 protein that alter the protein–DNA interface, resulting in a so-called high-fidelity Cas9 (ref. 63). Alternatively, orthologous CRISPR–Cas9 systems with more restrictive requirements for binding the protospacer-adjacent motif (PAM) reduce the number of potential off-target binding sites66, 67, 68. Specificity can be further improved by using dimeric CRISPR–Cas systems, in which a catalytic-dead Cas9 (dCas9) protein is fused to the FokI cleavage domain in an architecture similar to that of TALENs72, 86, 87. Sp, Streptococcus pyogenes; St, Streptococcus thermophilus; Nm, Neisseria meningitidis; Sa, Streptococcus aureus. HNH and RuvC, cleavage domains.

  3. Production of an off-the-shelf CAR T cell product.
    Figure 3: Production of an off-the-shelf CAR T cell product.

    A CAR is an artificial construct typically based on an extracellular monoclonal-antibody domain directed against a tumor (or tumor-associated) antigen, which is fused to a signaling domain on the inside of the cell that promotes cell proliferation when the engineered T cell encounters its target tumor cell. A notable advancement in the CAR T cell field is the effort to manufacture these immune cell therapies ahead of need, so that they can be transfused on demand to a wide range of patients. Such universal, nonalloreactive CAR T cells can be produced by knocking out the TRAC locus that encodes the T cell receptor (TCR) α chain19, 20. This approach can be combined with a knockout of the CD52 locus that renders the CAR T cells resistant to alemtuzumab, a monoclonal antibody used in the treatment of various T cell malignancies. To manufacture such an off-the-shelf product, T cells are collected from a healthy donor through apheresis. After the addition of the CAR by lentiviral gene transfer, TALEN-encoding mRNA are transferred to the cells to disrupt the TRAC and CD52 loci. After depletion of TCR-positive cells, the fully modified CAR T cells are expanded and stored until needed.

  4. Roadmap: translating targeted genome editing into the clinic.
    Figure 4: Roadmap: translating targeted genome editing into the clinic.

    POC, proof of concept; CMC, Chemistry, Manufacturing, and Controls; IND, Investigational New Drug; FDA, Food and Drug Administration; IMPD, Investigational Medicinal Product Dossier; EMA, European Medicines Agency.


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  1. Institute for Cell and Gene Therapy, Medical Center—University of Freiburg, Freiburg, Germany.

    • Tatjana I Cornu,
    • Claudio Mussolino &
    • Toni Cathomen
  2. Center for Chronic Immunodeficiency, Medical Center—University of Freiburg, Freiburg, Germany.

    • Tatjana I Cornu,
    • Claudio Mussolino &
    • Toni Cathomen
  3. Faculty of Medicine, University of Freiburg, Freiburg, Germany.

    • Toni Cathomen

Competing financial interests

T.C. is a consultant for TRACR Hematology.

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