Alexander Georgia M, He Bo, Leikvoll Austin, Jones Stephanie, Wine Rob, Kara Prakash, Martin Negin, Dudek Serena M
Neurobiology Laboratory, National Institute of Environmental Health Sciences, Division of Intramural Research, National Institute of Health, Research Triangle Park, North Carolina 27713, USA.
Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA.
bioRxiv. 2024 Nov 28:2024.11.27.625768. doi: 10.1101/2024.11.27.625768.
Hippocampal area CA2 is unique in many ways, largely based on the complement of genes expressed there. We and others have observed that CA2 neurons exhibit a uniquely robust tropism for adeno-associated viruses (AAVs) of multiple serotypes and variants. In this study, we aimed to systematically investigate the propensity for AAV tropism toward CA2 across a wide range of AAV serotypes and variants, injected either intrahippocampally or systemically, including AAV1, 2, 5, 6, 8, 9, DJ, PHP.B, PHP.eB, and CAP-B10. We found that most serotypes and variants produced disproportionally high expression of AAV-delivered genetic material in hippocampal area CA2, although two serotypes (AAV6 and DJ) did not. In an effort to understand the mechanism(s) behind this observation, we considered perineuronal nets (PNNs) that ensheathe CA2 pyramidal cells and, among other functions, buffer diffusion of ions and molecules. We hypothesized that PNNs might attract AAV particles and maintain them in close proximity to CA2 neurons, thereby increasing exposure to AAV particles. However, genetic deletion of PNNs from CA2 had no effect on AAV transduction. Next, we next considered the AAV binding factors and receptors known to contribute to AAV transduction. We found that the AAV receptor (AAVR), which is critical to transduction, is abundantly expressed in CA2, and knockout of AAVR nearly abolished expression of AAV-delivered material by all serotypes tested. Additionally, we found CA2 enrichment of several cell-surface glycan receptors that AAV particles attach to before interacting with AAVR, including heparan sulfate proteoglycans, N-linked sialic acid and N-linked galactose. Indeed, CA2 showed the highest expression of AAVR and the investigated glycan receptors within the hippocampus. We conclude that CA2 neurons are endowed with multiple factors that make it highly susceptible to AAV transduction, particularly to the systemically available PHP variants, including CAP-B10. Given the curved structure of hippocampus and the relatively small size of CA2, systemic delivery of engineered PHP or CAP variants could all but eliminate the need for intrahippocampal AAV injections, particularly when injecting recombinase-dependent AAVs into animals that express recombinases in CA2.
海马体CA2区在许多方面都很独特，这在很大程度上基于该区域所表达的基因互补情况。我们和其他研究人员观察到，CA2神经元对多种血清型和变体的腺相关病毒（AAV）表现出独特的强大嗜性。在本研究中，我们旨在系统地研究在广泛的AAV血清型和变体中，经海马内或全身注射后，AAV对CA2的嗜性倾向，这些血清型和变体包括AAV1、2、5、6、8、9、DJ、PHP.B、PHP.eB和CAP-B10。我们发现，尽管两种血清型（AAV6和DJ）并非如此，但大多数血清型和变体在海马体CA2区产生了不成比例的高AAV传递遗传物质表达。为了理解这一观察结果背后的机制，我们考虑了包裹CA2锥体细胞的神经周网（PNN），它具有多种功能，包括缓冲离子和分子的扩散。我们假设PNN可能会吸引AAV颗粒并使其保持在靠近CA2神经元的位置，从而增加对AAV颗粒的暴露。然而，从CA2中基因删除PNN对AAV转导没有影响。接下来，我们考虑了已知有助于AAV转导的AAV结合因子和受体。我们发现，对转导至关重要的AAV受体（AAVR）在CA2中大量表达，敲除AAVR几乎消除了所有测试血清型传递的物质的表达。此外，我们发现CA2中几种细胞表面聚糖受体富集，这些受体是AAV颗粒在与AAVR相互作用之前附着的对象，包括硫酸乙酰肝素蛋白聚糖、N-连接唾液酸和N-连接半乳糖。事实上，CA2在海马体内显示出AAVR和所研究的聚糖受体的最高表达。我们得出结论，CA2神经元具有多种使其对AAV转导高度敏感的因素，特别是对全身可用的PHP变体，包括CAP-B10。鉴于海马体的弯曲结构和CA2相对较小的尺寸，工程化PHP或CAP变体的全身递送几乎可以消除海马内注射AAV的需要，特别是当将依赖重组酶的AAV注射到在CA2中表达重组酶的动物体内时。
