Nature biotech |无需提取回输！体内编程B细胞实现高效抗体产生
<div style="max-width:100%; overflow:hidden;"><p style="margin: 0px;padding: 0px;clear: both;min-height: 1em;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;orphans: 2;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;font-size: 16px;color: rgb(0, 0, 0);letter-spacing: 0.544px;caret-color: rgb(0, 0, 0);text-align: center;" data-mpa-powered-by="yiban.io"><strong style="color: rgb(51, 51, 51);font-size: 17px;letter-spacing: normal;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;"><span style="max-width: 100%;color: rgb(0, 122, 170);overflow-wrap: break-word !important;box-sizing: border-box !important;">前言</span></strong></p>
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<section style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;"><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">针对</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(123, 12, 0);overflow-wrap: break-word !important;box-sizing: border-box !important;"><strong>HIV的广义中和抗体</strong></span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;">（bNAbs）</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">可以<strong>有效抑制病毒血症</strong>。其中，bNAbs 3BNC117和10-1074的联合使用，可以在中断抗逆转录病毒治疗的情况下长期抑制对抗体敏感的病毒库。然而，bNAbs较短的半衰期与HIV对抗体的耐药性使治疗效果大打折扣，甚至引起病毒种群的反弹式增长</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;"></span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">。目前，<strong>解决此问题的方法主要分为两类</strong>：一是利用病毒载体转导肌肉细胞实现抗体的持续表达，但在肌肉细胞中成熟的抗体，其多样性与糖基化水平都大大降低，影响了针对病毒的中和能力</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;"></span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">。二是移植体外改造的B细胞以高效分泌抗体，这种方法的治疗效果良好，但需要专门的医疗中心和高技术要求的手术操作，并保证供体细胞和受体的生物相容性，在临床转化上仍有较高的难度。</span></section>
<p style="margin: 16px 0px 0px;padding: 0px;max-width: 100%;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;overflow-wrap: break-word !important;box-sizing: border-box !important;"><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">2022年6月9日，来自以色列特拉维夫大学的Adi Barzel团队在<strong><em>Nature Biotechnology</em></strong>上在线发表了题为</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(123, 12, 0);overflow-wrap: break-word !important;box-sizing: border-box !important;"><strong>In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice</strong></span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">的研究文章。研究人员将上述两种治疗方法的思路巧妙结合，设计腺相关病毒</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;">（AAV）</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;overflow-wrap: break-word !important;box-sizing: border-box !important;">载体搭载CRISPR/SaCas9系统，在小鼠体内B细胞的免疫球蛋白基因内<strong>敲入编码3BNC117的HIV抗体基因</strong>，并成功诱导记忆B细胞的产生与中和滴度为6.8 µg·ml<sup><span lang="EN-US" style="font-size:12.0pt;font-family:Times New Roman,serif;mso-fareast-font-family:宋体;mso-bidi-theme-font:
minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:ZH-CN;mso-bidi-language:
AR-SA;">−1</span></sup>的抗体表达。</span></p>
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<section style="line-height: 2em;text-align: center;margin-top: 8px;margin-bottom: 24px;"><span style="letter-spacing: 0.5px;font-size: 12px;color: rgb(136, 136, 136);">图1. 由两种AAV介导的体内B细胞基因编辑</span></section>
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<section style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;"><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">作者首先设计了</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(123, 12, 0);overflow-wrap: break-word !important;box-sizing: border-box !important;"><strong>两种重组AAV</strong></span><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">：一种编码<strong>来自金黄色葡萄球菌的Cas9蛋白</strong></span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;">（SaCas9）</span><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">及靶向鼠Ighj4基因内含子的单向导RNA</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;">（sgRNA）</span><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">，另一种<strong>编码所需抗体3BNC117的DNA重组模板</strong>。在用基于HIV包膜糖蛋白</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(136, 136, 136);overflow-wrap: break-word !important;box-sizing: border-box !important;">（Env）</span><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">的免疫原对小鼠进行免疫的6天后，作者向实验小鼠静脉注射上述两种重组AAV。经过几次相同的基于HIV-Env的加强免疫后，扩增活化B细胞的数量后，<strong>他们观察到小鼠的血清中开始表达bNAbs，并在3BNC117敏感的HIV菌株的中和实验中证明了该分泌抗体的效力</strong>。</span></section>
<p style="margin: 16px 0px 0px;padding: 0px;max-width: 100%;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;overflow-wrap: break-word !important;box-sizing: border-box !important;"><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">为进一步证明这种方法在体的有效性和安全性，作者首先通过下一代测序对B细胞中的编辑效率和3BNC117 VH基因的体细胞超突变进行分析，<strong>发现脾脏中出现了一组富集的突变，表明表达3BNC117的B细胞正在产生与扩增</strong>。之后，作者还使用特异性抗体染色在脾脏中定位了生发中心内的被编辑B细胞，以及骨髓中分泌抗体的浆细胞，证明了该方法的有效性。</span></p>
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<section style="line-height: 2em;text-align: center;margin-top: 8px;margin-bottom: 24px;"><span style="letter-spacing: 0.5px;font-size: 12px;color: rgb(136, 136, 136);">图2. 在淋巴组织中追踪体内工程B细胞的产生</span></section>
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<section style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;"><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">作者接着评估了<strong>在体编辑的脱靶效应与AAV的组织分布</strong>，发现Cas9诱导的双链切割中，95%是在目标位点上，大部分脱靶位点处于内含子中，不影响正常基因表达。在肝脏和血液中发现AAV基因组的强烈富集，随着时间推移，肝脏中的AAV拷贝数逐渐减少，而在免疫小鼠的淋巴结和骨髓中的拷贝数增加，这表明在</span><span style="margin: 0px;padding: 0px;max-width: 100%;font-size: 14px;letter-spacing: 0.5px;color: rgb(123, 12, 0);overflow-wrap: break-word !important;box-sizing: border-box !important;"><strong>这些组织中被编辑的B细胞正在扩增</strong></span><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">。</span></section>
<section style="margin: 16px 0px 0px;padding: 0px;max-width: 100%;color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 17px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: normal;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;white-space: normal;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;line-height: 2em;overflow-wrap: break-word !important;box-sizing: border-box !important;"><span style="margin: 0px;padding: 0px;max-width: 100%;overflow-wrap: break-word !important;box-sizing: border-box !important;font-size: 14px;letter-spacing: 0.5px;">为了进一步提高该方法的安全性，作者<strong>进</strong><strong>一步修改了他们的AAV设计</strong>，将驱动Cas9表达的通用启动子换成了B细胞特异性CD19启动子，并把sgRNA转移到含有抗体重组模板的AAV上，这样只有两种AAV同时转导的B细胞才能被编辑。<strong>改进的方法实现了与原方法相似的基因敲入和血清抗体水平，同时Cas9在肝脏中的表达和编辑明显减少</strong>。</span></section>
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<p style="clear: both;min-height: 1em;"><span style="color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 14px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: 0.5px;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;display: inline !important;float: none;">综上，该研究</span><span style="font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 14px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: 0.5px;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;float: none;color: rgb(123, 12, 0);display: inline !important;"><strong>成功建立了一种利用基因编辑在体永久性产生工程化B细胞的方法</strong></span><span style="color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 14px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: 0.5px;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;display: inline !important;float: none;">，与之前在体产生治疗性CAR-T细胞的研究</span><span style="font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 14px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: 0.5px;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;float: none;color: rgb(136, 136, 136);display: inline !important;"></span><span style="color: rgb(51, 51, 51);font-family: mp-quote, -apple-system-font, BlinkMacSystemFont, Helvetica Neue, PingFang SC, Hiragino Sans GB, Microsoft YaHei UI, Microsoft YaHei, Arial, sans-serif;font-size: 14px;font-style: normal;font-variant-ligatures: normal;font-variant-caps: normal;font-weight: 400;letter-spacing: 0.5px;orphans: 2;text-align: justify;text-indent: 0px;text-transform: none;widows: 2;word-spacing: 0px;-webkit-text-stroke-width: 0px;text-decoration-thickness: initial;text-decoration-style: initial;text-decoration-color: initial;display: inline !important;float: none;">遥相呼应，<strong>共</strong><strong>同开辟了在体工程化免疫细胞的治疗思路</strong>。但在走上临床之前，仍有许多的问题需要继续探索，比如能否在体工程化B细胞以产生多种抗体，以实现对HIV更为有效的治疗；或是减少HIV-Env的加强免疫次数，仍<strong>保持诱导抗体的水平和持续时间</strong>，<strong>提高患者对该种治疗的接受度</strong>。</span></p>
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<p style="line-height: normal;"><span style="letter-spacing: 0.5px;font-size: 12px;color: rgb(136, 136, 136);">参考文献</span></p>
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<p style="line-height: normal;"><span style="letter-spacing: 0.5px;font-size: 12px;color: rgb(136, 136, 136);"> Priddy, F. H. et al.
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