1: Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.
Science of the Month - May 2024
Nat Biotechnol.
Fabian P Suchy #, Daiki Karigane #, Yusuke Nakauchi, Maimi Higuchi, Jinyu Zhang, Katja Pekrun, Ian Hsu, Amy C Fan, Toshinobu Nishimur, Carsten T Charlesworth, Joydeep Bhadury, Toshiya Nishimura, Adam C Wilkinson, Mark A Kay, Ravindra Majeti #, Hiromitsu Nakauchi #
Gene editing using CRISPR/Cas9 and adeno-associated virus (AAV) vectors is being applied in clinical gene therapy for reasons such as 1) the high specificity of editing at the target locus and 2) high editing efficiency in cells relevant to gene therapy (e.g., hematopoietic stem cells and iPS cells). In this study, we discovered that incorrect gene editing occurs when multiple AAVs are inserted into the target locus in a concatenated manner. We also found that these concatemeric insertions lead to gene deletion rather than correction, occur at a high frequency of approximately 60% per cell, and present multiple challenges, including being undetectable by standard PCR. Therefore, we successfully reduced the frequency of concatemeric insertions to less than 5% by incorporating a sequence identical to the target locus cleaved by sgRNA into the AAV, thereby cleaving off the ITRs that cause concatenation. As this is a very simple solution, we expect this finding to be widely utilized in various fields, from basic research to clinical applications. This research was conducted jointly by the Majeti Lab, to which I belong, and the Nakauchi Lab, which focuses on organ regeneration research. I would like to take this opportunity to express my gratitude to everyone involved.
(Daiki Karigane, Class of '87, Stanford University (currently Department of Hematology, Tokyo Medical and Dental University))
2: Physician Estimates and Patient-Reported Health Status in Atrial Fibrillation
JAMA Netw Open.
Nobuhiro Ikemura, Shun Kohsaka, Takehiro Kimura, Philip G. Jones, Yoshinori Katsumata, Kojiro Tanimoto,Ikuko Ueda, Seiji Takatsuki, Masaki Ieda, Paul S. Chan, John A. Spertus
Atrial fibrillation is the most common arrhythmia in adults and is known to significantly impair patients' health status through symptoms such as palpitations and shortness of breath (compromising physical activity and quality of life, and causing anxiety about the disease and treatment). Understanding an individual's health status is considered crucial for selecting appropriate treatment methods, which is precisely why physicians conduct medical interviews. However, it has been pointed out that in actual clinical practice, physicians may not accurately perceive the patient's condition due to time constraints and patient reticence. Therefore, in collaboration with the Tokyo Medical Center (Dr. Tanimoto et al.) and the Mid-American Heart Institute (Dr. Spertus et al.), we conducted a subjective assessment of the health status of patients with atrial fibrillation using a disease-specific questionnaire (AFEQT) to examine whether there is a gap between physician perception and patient reality, and how this gap affects treatment choices. A survey of 330 new outpatients with atrial fibrillation revealed a significant discrepancy between patient reports and physician perceptions (Figure: agreement was found in only 34% of cases, with the remainder involving either overestimation or underestimation by physicians). Furthermore, we found that patients with this perception gap were less likely to receive appropriate interventions such as catheter ablation. Although these were challenging results for us as healthcare professionals, we believe that by incorporating patient-reported outcome tools like AFEQT into clinical practice and developing systems to more clearly convey the patient's voice to the physician, we can provide treatment that is more aligned with patient needs. We have already begun work toward this goal and will continue our efforts to report on its progress.
(Shun Kohsaka, Class of '76, and Nobuhiro Ikemura, equivalent to Class of '89, Department of Cardiology)
Other Published Papers
1: DMHPpp1r17 neurons regulate aging and lifespan in mice through hypothalamic-adipose inter-tissue communication.
Cell Metabolism.
Kyohei Tokizane, Cynthia S. Brace, Shin-ichiro Imai