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mRNA Mediated Expression of Novel Fusion Phage Tail Protein with Antimicrobial Peptides inside Macrophages for Targeted Clearance of Intracellular Mycobacterium tuberculosis
mRNA 介导的融合噬菌体尾部蛋白与抗菌肽在巨噬细胞中的表达，用于靶向清除细胞内结核分枝杆菌

Ziwei Chen

^{a #}

, Xueting Fan

^{c #}

, Lihui Zou

^{b #}

, Li Wan

^{a}

, Yayu Li

^{a}

, Chan

{Li}^{a}

, Yuan Chen

^{a}

, Lu Kuai

^{a}

, Jiahui Cai

^{a}

, Lili Zhang

^{a}

, Yifei Li

^{a}

, Hexin Li

^{a}

, Kanglin Wan

^{c}

, Haican Liu

^{c *}

, Hongtao Xu

^{d *}

, Fei Xiao

^{a, e *}

陈子维

^{a #}

, 樊雪婷

^{c #}

, 邹丽慧

^{b #}

, 倪婉

^{a}

, 李雅玉

^{a}

, 蔡晨

{Li}^{a}

, 陈远

^{a}

, 胡璐

^{a}

, 蔡佳慧

^{a}

, 张莉莉

^{a}

, 李逸飞

^{a}

, 李赫鑫

^{a}

, 关凌

^{c}

, 刘海灿

^{c *}

, 许鸿涛

^{d *}

, 肖飞

^{a, e *}

^{a}

Clinical Biobank, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China

^{a}

北京医院临床生物库，国家老年学中心，国家卫生健康委员会，老年医学研究所，中国医学科学院，北京，中国

^{b}

The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/ National Center of Gerontology of National Health Commission, P.R. China

^{b}

北京老年病研究所重点实验室，老年医学研究所，中国医学科学院，北京医院/国家卫生健康委员会国家老年学中心，中华人民共和国

^{c}

National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

^{c}

国家传染病智能追踪与预测重点实验室，国家传染病控制与预防研究所，中国疾病预防控制中心，北京 102206，中国

^{d}

Department of Laboratory Medicine, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China

^{d}

北京医院实验医学部，国家老龄化中心，国家卫生健康委员会，老年医学研究所，中国医学科学院，北京，中国

^{e}

Lead Contact

^{e}

主要联系人

^{#}

These authors have contributed equally to this work.

^{#}

这些作者对本工作贡献相同。

Corresponding authors. 通讯作者。

E-mail: xiaofei3965@bjhmoh.cn (Xiao F) & xuhongtao2911@bjhmoh.cn (Xu HT) & liuhaican@icdc.cn (Liu HC).
电子邮件：xiaofei3965@bjhmoh.cn (Xiao F) & xuhongtao2911@bjhmoh.cn (Xu HT) & liuhaican@icdc.cn (Liu HC)。

Abstract 摘要

Current anti-tuberculosis treatments primarily target extracellular Mycobacterium tuberculosis (Mtb), but exhibit limited efficacy against intracellular Mtb, leading to incomplete clearance of pathogens and an increased risk of recurrence. Antimicrobial peptides (AMPs) possess broad-spectrum antimicrobial activity and low potential for resistance development. Here we developed an in vivo mRNA expression platform which not only facilitates intracellular AMPs expression within macrophages, but also significantly enhances their bactericidal activity against $M t b$ post-infection. Notably, the combination of AMPs trimers demonstrated superior anti-Mtb activity compared to individual AMPs or other combinations. Furthermore, fusion of this AMP complex with either the minor tail protein Gp6 or lysin Gp10 from Mycobacterium phage L5 substantially improved macrophage-specific targeting and intracellular $M t b$ elimination. Thus, our current study provides novel insights and innovative strategies for the treatment of tuberculosis or other intracellular bacterial pathogens.
当前的抗结核治疗主要针对细胞外的结核分枝杆菌（Mtb），但对细胞内的 Mtb 效果有限，导致病原体无法完全清除，并增加复发的风险。抗菌肽（AMPs）具有广谱的抗菌活性和较低的耐药性发展潜力。在此，我们开发了一种体内 mRNA 表达平台，该平台不仅促进了巨噬细胞内抗菌肽的表达，还显著增强了其对 $M t b$ 的杀菌活性。值得注意的是，抗菌肽三聚体的组合表现出相比单一抗菌肽或其他组合更卓越的抗 Mtb 活性。此外，将该抗菌肽复合物与结核分枝杆菌噬菌体 L5 的次要尾蛋白 Gp6 或溶菌酶 Gp10 融合，显著改善了巨噬细胞特异性靶向和细胞内 $M t b$ 清除。因此，我们的研究为结核病或其他细胞内细菌病原体的治疗提供了新见解和创新策略。

Introduction 引言

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death among infectious diseases

^{1}

. Macrophages serve as the primary host cells for

M t b

during infection as they may facilitate the immune evasion of Mtb after the pathogen enters human body

^{2, 3}

. Currently, most existing drugs are only effective against extracellular

M t b

, and there are no effective strategies to disrupt

M t b

's immune evasion or eliminate the bacteria within macrophages, leading to a high rate of recurrence

^{2, 4}

. Therefore, novel approaches are urgently needed to target and eliminate intracellular Mtb to control TB more effectively.
肺结核（TB）是由分枝分隔分枝杆菌（Mtb）引起的，仍然是传染病中导致死亡的主要原因

^{1}

。巨噬细胞在感染过程中作为主要宿主细胞，可能促进 Mtb 在病原体进入人体后逃避免疫反应

^{2, 3}

。目前，大多数现有药物仅对细胞外

M t b

有效，并且没有有效的策略来破坏

M t b

的免疫逃逸或消除巨噬细胞内的细菌，这导致了高复发率

^{2, 4}

。因此，迫切需要新的方法来靶向并消除细胞内的 Mtb，以更有效地控制 TB。

Antimicrobial peptides (AMPs), also known as host defense peptides, are short peptides that are typically positively charged and exhibit antimicrobial properties

^{5, 6}

. As key effector molecules of the innate immune response, AMPs play a critical role in controlling infections and latent tuberculosis infection

^{5}

. Studies have demonstrated that AMPs, such as human neutrophil peptides (HNPs)

^{7}

, neurotoxin-X (NZX)

^{8}

, human cathelicidin LL-37 (LL-37)

^{9, 10}

, ubiquitin-derived peptide 2 (Ub2)

^{11}

, Proline-Arginine
抗菌肽（AMPs），也称为宿主防御肽，是通常带正电荷并表现出抗菌特性的短肽

^{5, 6}

。作为先天免疫反应的关键效应分子，AMPs 在控制感染和潜伏性肺结核感染中发挥着关键作用

^{5}

。研究表明，AMPs，如人类中性粒细胞肽（HNPs）

^{7}

、神经毒素-X（NZX）

^{8}

、人类猫白细胞素 LL-37（LL-37）

^{9, 10}

、源自泛素的肽 2（Ub2）

^{11}

、脯氨酸-精氨酸。

Rich Peptide 39 (PR-39)

^{12}

, enterocin E6 (E6)

^{10}

, and hepcidin

^{13}

, have direct antimicrobial activity against

M t b

. However, high manufacturing costs and degradation risks limit the clinical application of AMPs

^{5}

.
富集肽 39 (PR-39)

^{12}

、肠肽 E6 (E6)

^{10}

和肝调素

^{13}

对

M t b

具有直接的抗微生物活性。然而，高制造成本和降解风险限制了 AMPs 的临床应用

^{5}

。

In vitro transcribed mRNA (IVT mRNA) technology is characterized by its high safety, broad target range, and ease of manufacturing

^{14}

. The research & development of COVID-19 mRNA vaccines, along with ongoing technological advancements, has markedly increased mRNA expression and stability, ensuring efficient and sustained protein synthesis

^{15}

. These improvements render IVT mRNA in tumors, chronic diseases, and infectious diseases as an increasingly feasible and promising technology

^{14}

. In our present study, we developed an in vitro mRNA expression platform for AMPs that enabled macrophages to express AMPs intracellularly, thereby enhancing the intracellular bactericidal activity of macrophages following Mtb infection. In addition, we found a combination of AMPs fused with phage protein showing high specificity and stronger antibacterial activity.
体外转录 mRNA (IVT mRNA) 技术以其高安全性、广泛的靶标范围和易于制造为特征

^{14}

。随着 COVID-19 mRNA 疫苗的研发以及持续的技术进步，mRNA 的表达和稳定性显著提高，确保了高效而持续的蛋白质合成

^{15}

。这些改进使得 IVT mRNA 在肿瘤、慢性疾病和传染病中的应用越来越可行且前景广阔

^{14}

。在我们目前的研究中，我们开发了一个用于 AMPs 的体外 mRNA 表达平台，使巨噬细胞能够在细胞内表达 AMPs，从而增强巨噬细胞在 Mtb 感染后的细胞内杀菌活性。此外，我们发现与噬菌体蛋白融合的 AMPs 组合显示出高特异性和更强的抗菌活性。

Results 结果

To enhance the intracellular anti-

M t b

activity of macrophages, we selected three antiMtb AMPs -LL-37, UB2, and E50-52. We designed three IVT-mRNAs (LL-37 mRNA, UB2 mRNA, and E50-52 mRNA), each consisting of a cap structure,

5^{'}

and

3^{'}

untranslated regions (UTRs), an AMP coding sequence, and a poly(A) tail for expression in macrophages (Figure 1A). Additionally, we constructed an GFPexpressing mRNA using the same system as a positive control to confirm the successful construction and expression of the mRNAs. After the IVT mRNAs were transfected into macrophages for 6 hours, green fluorescence was observed in the GFP mRNA group, and the expression of AMPs did not induce any cytotoxic effects on the macrophages (Supplementary Figure 1A), confirming both the successful transfection and safe expression of the AMPs in macrophages. We also added the chemically synthesized peptides LL-37, UB2, and E50-52 separately to the macrophages as control groups. Next, macrophages were infected with Mtb H37Rv for 2 hours. After 5 days of incubation,

M t b

viability in the Ctrl group (without mRNA transfection) and the GFP
为了增强巨噬细胞内的抗-

M t b

活性，我们选择了三种抗 Mtb 的 AMPs -LL-37、UB2 和 E50-52。我们设计了三种 IVT-mRNA（LL-37 mRNA、UB2 mRNA 和 E50-52 mRNA），每种都由一个帽结构、

5^{'}

和

3^{'}

非翻译区（UTR）、一个 AMP 编码序列和一个 poly(A)尾组成，以便在巨噬细胞中表达（图 1A）。此外，我们还使用相同的系统构建了一个表达 GFP 的 mRNA，作为阳性对照，以确认 mRNA 的成功构建和表达。在将 IVT mRNA 转染到巨噬细胞 6 小时后，在 GFP mRNA 组中观察到绿色荧光，AMP 的表达未对巨噬细胞产生任何细胞毒性效应（补充图 1A），确认了 AMP 在巨噬细胞中成功转染和安全表达。我们还分别向巨噬细胞添加了化学合成的肽 LL-37、UB2 和 E50-52 作为对照组。接下来，将巨噬细胞感染 Mtb H37Rv 2 小时。经过 5 天的培养，

M t b

在对照组（未转染 mRNA）和 GFP 组中的存活率