2023 Gene Therapy Mini-Series.

Since the discovery of the structure of DNA and its transcription, it has been a scientific dream to treat monogenic disorders by rewriting and correcting the genetic code. Development of recombinant DNA technology and DNA sequencing in the 1970s laid the foundation for gene-insertion therapies, with the goal to deliver a healthy functioning copy of the mutated gene in patients. Development of retrovirus and adenovirus delivery vectors in the 1980s led to the first gene-therapy treatment of a patient with adenosine deaminase–deficient severe combined immune-deficiency in 1990. The field received a major setback in 1999 as the result of the death of a patient from a severe immune reaction in the liver related to adenovirus delivery. Development of safer vectors (lentivirus, herpes simplex and adeno-associated viruses, or AAVs), improved transduction procedures and increased safety parameters in clinical trials led to a resurgence in the field in 2000s. This decade also saw the development of gene-editing technologies like zinc finger nucleases and transcription activator-like effector nucleases and the emergence of rapid gene-sequencing methods. Discovery of clustered regularly interspaced short palindromic repeats (Crispr)-Cas9 as a simple, elegant tool for creating precise double-stranded breaks in DNA in 2012 re-energized the field and has led to major applications of the platform for gene-editing and correction of monogenic disorders. Improvement in delivery platforms using lipid nanoparticles, oligonucleotides and AAV to deliver DNA or Crispr guides-Cas9 RNAs at high frequency in the cells of different tissues has ushered in the era of “in vivo” gene-editing therapies. This has led to multiple approvals of gene therapy–based treatments by the Food and Drug Administration, for example, voretigene neparvovec-rzyl (Luxturna) for bi-allelic RPE65 mutation-associated retinal dystrophy, etranacogene dezaparvovec-drlb (Hemgenix) for hemophilia B, onasemnogene abeparvovec (Zolgensma) for spinal muscular atrophy and betibeglogene autotemcel (Zynteglo) for transfusion-dependent thalassemia, to name a few. The rapid advancements have also created major challenges, especially related to assay development for safety and potency assessments, manufacturing bottlenecks for delivery to patients and regulatory and ethical issues, specifically related to germline editing. The mini-series on gene-therapy was planned to describe the state-of-the-art, recent innovations, applications and current challenges. A major goal of this mini-series is to provide the readers with an overview of these novel therapies that are already approved or are in clinical testing. Although Crispr-Cas9 is a precise DNA-editing tool, the double-stranded break created by Crispr-Cas9 can lead to large deletions and chromosomal aberrations. Hence, base-editing, which leads to single-strand breaks and leads to single-base edits in the DNA by deamination, is considered a safer methodology. Walker Lahr, Christopher Sipe, Joseph Skeat and others describe the development of cytosine and adenosine base editors and applications of this technology as a refinement of Crispr-Cas9 editing. AAV remains the major delivery tool for the guides or DNA for gene modification. Recent advances in AAV technology, specifically related to rapid creation and analyses of AAV capsid libraries, are highlighted in the paper by Joanna Szumska and Dirk Grimm. The two manuscripts, by Laura Ugalde, Sara Fananas, Raul Torres and others and Senthil Bhoopalan, Jonathan Yen, Rachel Levine and others, provide an overview of the basics of Crispr-Cas9 technology, applications and challenges of gene therapy for treating blood disorders. The potential of these one-time, functionally curative treatments is expected to grow exponentially in the near future, as the manufacturing, safety, durability, regulatory and reimbursement challenges are addressed. Recent endeavors by Food and Drug Administration in this direction, for instance, creation of Initial Targeted Engagement for Regulatory Advice on CBER products (INTERACT) and Gene Therapy Pilot Program are also steps in the right direction. We hope this series will prove of interest to the readers from pre-clinical, translational research and clinical scientists alike.