Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4⁺ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

中文翻译：

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雷帕霉素治疗和 AAV6 皮下递送后腺相关病毒血清阳性恒河猴的基因转移，但未重新靶向 AAV6 载体

腺相关病毒 (AAV) 载体，如 AAV6，在体外显示出对原代人 CD4 ⁺ T 细胞的趋向性，正在探索用于体内递送抗 HIV 治疗方式. 然而，非靶器官中预先存在的免疫和隔离会显着阻碍它们的表现。为了克服这些挑战，我们研究了免疫抑制是否允许 AAV6 或靶向 AAV6 衍生物在血清反应阳性的恒河猴中传递基因。在静脉内 (IV) 或皮下 (SC) 递送 AAV 之前，用雷帕霉素对动物进行免疫抑制，我们监测了载体的生物分布、基因转移和安全性。猕猴接受磷酸盐缓冲盐水、单独的 AAV6 或等剂量的 AAV6 和通过直接锚蛋白重复蛋白 (DARPin) 重新靶向 CD4 的 AAV6-55.2 载体。AAV6 和 AAV6-55.2 载体基因组在给药后 28 天内在外周血单个核细胞和大多数器官中发现，在肝脏、脾脏、淋巴结 (LN) 和肌肉中发现的水平最高，表明重新定位并不能阻止载体隔离。尽管进行了载体基因组检测，但在任何组织中均未检测到来自 AAV6-55.2 的基因表达。SC 注射 AAV6 促进了注射部位附近肌肉中的有效基因表达，以及脾脏、LNs 和肝脏中的低水平基因表达，而 IV 注射 AAV6 后的基因表达主要在脾脏中观察到。尽管在雷帕霉素停药 14 天后，在四只 AAV 治疗的动物中的三只中检测到肝酶升高，但 AAV 载体的耐受性良好。一只注射 SC 的动物在注射部位附近出现肌肉炎症，在研究结束时，可检测到针对转基因和 AAV6 衣壳的 T 细胞反应。全面的，我们的数据表明，雷帕霉素治疗可能提供一种可能的策略来表达抗 HIV 疗法，例如广泛中和肌肉中的抗体。这项研究提供了重要的安全性和有效性数据，将有助于未来抗 HIV 基因疗法的研究设计。