2020: Gene Therapy Enters Its Fourth Decade

HUMAN GENE THERAPY BURST ONTO THE SCENE IN 1990, with the first therapeutic gene transfer attempted for patients with severe combined immune deficiency due to adenosine deaminase deficiency. The pioneering steps of the 1990s led to early evidence of clinical efficacy in the 2000s and a number of gene therapy products approved by the Food and Drug Administration and European Medicines Agency in the 2010s. As clinical gene therapy enters its fourth decade, the 2020s, it is appropriate to take stock of gains that the field has enjoyed and to discuss continued limitations of this crucial set of platform technologies. Gene therapy originated as a concept when Theodore Friedmann and Richard Roblin published a seminal paper in Science in 1972, indicating clearly that all of the elements were present to deliver genes into human cells with the necessary transcriptional promoter and polyadenylation signals required for an mRNA to be expressed and subsequently translated into a therapeutic protein. Friedmann anticipated that for monogenic disorders, particularly autosomal recessive Mendelian disorders, this could prevent or reverse the phenotypic manifestations of a disease. In the decades that followed, the concept of hijacking the mechanisms that viruses have evolved to deliver genes into human cells became a reality, first in eukaryotic cells, then in animals, and finally in patients. This pathway was reproduced with several viral vector classes: gammaretroviruses (RV), adenoviruses (Ad), adeno-associated viruses (AAV), herpes simplex viruses (HSV), lentiviruses (LV), and a few others. Additional advances in understanding the basic biology of life, such as the discovery of RNA interference as a mechanism to downregulate gene expression and the CRISPR-Cas system as a means of editing genes, have opened new avenues for the design of gene therapeutics. Meanwhile, more breakthroughs in studying stem-cell biology, particularly with regard to hematopoietic stem cells and in cancer immunotherapy, have provided highly effective platforms for ex vivo gene therapy. OPPORTUNITIES AND CHALLENGES As we enter a new decade, the field of gene therapy is faced with unprecedented opportunities, along with certain persistent challenges and even some potential serious risks. The opportunities that are nearest at hand include those in gene silencing, gene editing, RNA editing, base editing, and prime editing. The last two of these, both developed in David Liu’s lab, are cutting-edge adaptations of CRISPR-mediated RNA guidance, in which the Cas-sgRNA complex directs the genome sequence specificity and is combined with additional enzymatic functions either to change nucleotide bases by deamination or to rewrite them by reverse transcription. In the next few years, each of these new molecular mechanisms will be tested in various applications in molecular medicine, including cells, animals, and patients. Those efforts will help to create a more complete picture of how well each modality works in terms of efficiency and safety. Limitations of current and future gene therapy technologies are bound to linger for several years. One of the most acute limitations is still the availability of largescale, low-cost manufacturing capacity for gene therapy vectors. The specifics of this limitation include the necessity for further understanding viral vector replication and packaging biology and producer cell biology, and the complexity of the upstream cell-based manufacturing platform, which often begins with transfection of adherent cells in culture. While stable packaging lines exist for some vector types, this is not true of all right now, and the adaptation of manufacturing cell lines to suspension to microcarriers or higher complexity substrates requires ongoing engineering efforts. Parallel issues associated with downstream effective separation of empty from full viral particles and purification of vectors are also a major focus of current efforts. While each of these issues is addressable, they will require significant time, effort, and energy. Another intrinsic limitation of current systems is that of delivery to large or difficult to access target cell populations within the body. For gene therapy to the central