Development of DREADD-based gene therapy targeting HCN4 in spinal dorsal horn

2022 Fiscal Year Final Research Report

• Project/Area Number: 18K16465
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 55050:Anesthesiology-related
• Research Institution: Kurume University
• Principal Investigator: Oshita Kensuke 久留米大学, 医学部, 助教 (70529510)
• Project Period (FY): 2021-03-01 – 2023-03-31
• Keywords: 慢性疼痛 / DREADD / HCN / 脊髄後角

Outline of Final Research Achievements

We generated transgenic mouse (HCN4+/luc), in which luciferase and stop codon was knocked-in at the upstream of HCN4 translation initiation site to visualize HCN4-expressing cells. We found that HCN4 was expressed in spinal dorsal horn by assessing the chemiluminescence in HCN4+/luc.In addition, we generated another line knock-in mouse which in HCN4 expression level could be reversibly suppressed by oral doxycycline (DOX) administration using tetracycline trans activator and the responsive element (HCN4+/tTA-TRE). We crossed these two lines and generated a double knock-in mouse to knockdown HCN4 completely with DOX and clarified that the locus of HCN4 is specifically in spinal dorsal horn. HCN4 positive cells in spinal dorsal horn was co-expressed with parvalbumin and protein kinase Cγ, which indicated that the most of HCN4 positive cells was included in excitatory interneuron. We will perform further investigation of contribution of HCN4 positive cells on development of allodynia.

Free Research Field

麻酔学

Academic Significance and Societal Importance of the Research Achievements

我々はHCN4遺伝子の翻訳開始点にテトラサイクリントランスアクチベータ(tTA) とその応答配列(TRE) をノックインしたマウス(HCN4+/tTA-TRE)とHCN4+/lucを交配したマウス(HCN4luc/tTA-TRE)を作成した。このマウスを用いて、HCN4 陽性細胞の細胞体が主にII 層深部、III 層に局在していることが明らかとした。さらに脊髄後角においてHCN4陽性細胞はパルブアルブミンもしくはプロテインキナーゼCγ（PKCγ）と共発現しており、興奮性介在ニューロンであることが判明した。PKCγ陽性の興奮性介在ニューロンはアロディニア形成に関与している可能性がある。