血友病 A（Hemophilia A）是一种由凝血因子 VIII（F8）缺乏或功能异常引起的严重 X 连锁隐性遗传性出血性疾病。患者通常表现为自发性出血，严重影响生活质量。目前，血友病 A 的主要治疗策略包括凝血因子替代疗法、非因子替代疗法和腺相关病毒（AAV）介导的基因治疗。然而，这些方法仍面临多种挑战：凝血因子替代疗法需要终身频繁输注，费用昂贵，且可能引发抑制因子的产生； 非因子替代疗法只能在一定程度上减少出血事件的发生，并不能从根本上治愈血友病； AAV 介导的基因治疗尽管在部分临床研究中展现了疗效，但由于 AAV episome 的逐渐丢失、宿主免疫应答以及载量限制，其长期稳定性和有效性仍有待提升，特别是在儿童患者中，由于肝细胞的增殖， AAV 携带的 F8 基因可能逐步丢失。此外， AAV 递送CRISPR 存在长期表达 Cas9 蛋白的风险，可能引发持续基因切割、脱靶效应以及针对 AAV 衣壳和 Cas9 蛋白的免疫反应，限制了其临床应用。 综上，开发一种安全、高效、长期稳定的基因治疗策略至关重要。

针对上述问题， 我们提出了一种脂质体纳米颗粒（LNP）递送 CRISPR 基因编辑系统联合 AAV8-BDDF8 供体模板的协同治疗策略，旨在提高治疗效果、降低免疫风险及基因毒性。 我们首先对 LNP 配方进行了系统优化，以提高 CRISPR 成分的递送效率和安全性。研究结果表明，阳离子脂质 ALC-0315 在所有测试配方中展现出最优的 mRNA 递送效率，同时优化胆固醇比例（30%-50%）能够显著提升基因编辑效率。此外，为了进一步优化 LNP 的体内分布和生物相容性， 我们引入了生物膜来源的辅助脂质（如 DOPE 和鞘磷脂），以增强 LNP 的稳定性，并结合肝靶向性脂质（如 C12-GalCer、 C18-GalCer）来提升肝细胞的摄取效率。实验表明，这些改进能够有效提高 LNP 在肝脏中的靶向递送效率， 降低潜在的系统性免疫激活风险。

在血友病 A 小鼠模型中， 我们进一步验证了 LNP-CRISPR 联合 AAV8-BDDF8策略的体内治疗效果。研究结果表明，该策略能够显著提高基因编辑效率，在肝脏中实现稳定的 F8 基因表达，并有效恢复凝血功能。通过长期随访观察发现，接受治疗的小鼠能够长期维持正常的 F8 凝血因子活性，并且未出现明显的脱靶效应、免疫毒性和器官毒性。此外，生物化学指标分析显示， LNP 递送 CRISPR 系统未导致显著的肝损伤或炎症反应，进一步验证了其安全性。

为了精准评估 CRISPR 介导的大片段基因插入事件， 我们开发了一种长片段PCR（long-range PCR）联合纳米孔测序（Nanopore sequencing）的检测方法。传统的短读长测序技术（如 Illumina）在检测大片段基因插入事件时存在较大的局限性，难以准确解析整合位点的序列信息。 我们提出的优化的长片段 PCR 联合纳米孔测序的方法能够全面解析大片段基因整合事件，为后续的安全性和有效性评估提供了精准的分子检测手段。

综上所述， 我们通过优化 LNP 递送系统、 创新地使用 LNP-CRISPR 及 AAV8- BDDF8协同治疗血友病A的策略，成功提高了血友病A基因治疗的疗效和安全性。此外，结合长片段 PCR 与纳米孔测序的精准检测方法，我们全面解析了大片段基因整合事件的复杂性。 本研究的创新性成果为血友病 A 的基因治疗提供了新的技术路径，并为未来的临床转化奠定了坚实的理论基础和技术支撑。

Hemophilia A is a severe X-linked recessive hereditary bleeding disorder caused by the deficiency or dysfunction of coagulation factor VIII (F8). Patients usually present with spontaneous bleeding, which seriously affects their quality of life. Currently, the main treatment strategies for hemophilia A include coagulation factor replacement therapy and adeno-associated virus (AAV)-mediated gene therapy. However, these methods still face multiple challenges: coagulation factor replacement therapy requires lifelong and frequent infusions, which are costly and may lead to the development of inhibitory antibodies. Nonfactor replacement therapies can only partially reduce bleeding episodes and do not offer a curative solution for hemophilia. Although AAV-mediated gene therapy has shown efficacy in some clinical studies, its long-term stability and effectiveness still need to be improved due to the gradual loss of AAV episomes, host immune responses, and load limitations, especially in pediatric patients, where the F8 gene carried by AAV may be gradually lost due to the proliferation of hepatocytes. In addition, there is a risk of longterm expression of Cas9 in the delivery of CRISPR by AAV, which may induce continuous gene cutting, off-target effects, and immune responses to AAV capsid and Cas9 proteins, limiting its clinical application. In summary, it is crucial to develop a safe, efficient, longterm and stable gene therapy strategy.

To address these challenges, we proposed a synergistic therapeutic strategy of liposome nanoparticle (LNP) delivery of CRISPR gene editing system combined with AAV8-BDDF8 donor template, aiming to improve the therapeutic effect, reduce immune risks and genotoxicity. This study first systematically optimized the LNP formulation to improve the delivery efficiency and safety of CRISPR components. The results showed that cationic lipid ALC-0315 showed the best mRNA delivery efficiency among all tested formulations, and optimizing the cholesterol ratio (30%-50%) can significantly improve the gene editing efficiency. In addition, in order to further optimize the in vivo distribution and biocompatibility of LNP, this study introduced auxiliary lipids derived from biomembranes (such as DOPE and sphingomyelin) to enhance the stability of LNP, and combined with liver-targeted lipids (such as C12-GalCer, C18-GalCer) to improve the uptake efficiency of hepatocytes. Experiments show that these improvements can effectively improve the targeted delivery efficiency of LNP in the liver and reduce the potential risk of systemic immune activation.

In the hemophilia A mouse model, we further verified the in vivo therapeutic effect of the LNP-CRISPR combined with AAV8-BDDF8 strategy. The results showed that this strategy can significantly improve the efficiency of gene editing, achieve stable F8 gene expression in the liver, and effectively restore coagulation function. Through long-term follow-up observations, it was found that the treated mice were able to maintain normal coagulation factor activity for a long time, and no obvious off-target effects, immunotoxicity or organ toxicity occurred. In addition, biochemical index analysis showed that the LNP delivery CRISPR system did not cause significant liver damage or inflammatory response, further verifying its safety.

To accurately evaluate CRISPR-mediated large-fragment gene insertion events, we developed a long-range PCR combined with nanopore sequencing detection method. Traditional short-read sequencing technologies (such as Illumina) have great limitations in detecting large-fragment gene insertion events, and it is difficult to accurately analyze the sequence information of the integration site. The optimized long-range PCR combined with nanopore sequencing method we proposed can comprehensively analyze the events of large-fragment gene integration, providing an accurate molecular detection method for subsequent safety and efficacy evaluation.

In summary, we have successfully improved the efficacy and safety of gene therapy for hemophilia A by optimizing the LNP delivery system and innovatively using LNPCRISPR and AAV8-BDDF8 to synergistically treat hemophilia A. In addition, by combining the precise detection methods of long-fragment PCR and nanopore sequencing, we have comprehensively analyzed the complexity of large-fragment gene integration events. The innovative results of this study provide a new technical path for gene therapy of hemophilia A and lay a solid theoretical foundation and technical support for future clinical transformation.