Hemophilia B Gene Therapy via CRISPR/Cas9-Targeted Integration of the Factor IX Gene using Adenovirus Vector Delivery

摘要 血友病B是一种X连锁隐性单基因疾病，大约每25,000名男性中就有1人患病， 以导致凝血因子IX(FIX)无功能或异常活性的突变为特征。 这些突变可能导致自发性出血进入关节和软组织的表型，这取决于 疾病的严重程度。目前血友病B的临床治疗包括终生注射治疗 当然是FIX。治疗费用昂贵，而且具有身体侵入性，因为患者必须反复接受治疗。 静脉注射。因此，血友病B基因治疗的核心目标之一是建立 凝血因子IX在临床前凝血模型中长期治疗性基因表达的研究 缺乏症。最近基因编辑技术的进步使靶向成为可能 治疗性转基因稳定整合和长期表达的特异性基因座。在此，我们假设 腺病毒介导的基因治疗可以建立长期的治疗性FIX表达 使用以CRISPR/CAS9为基础的系统针对ROSA26的同源定向修复 小鼠血友病B基因敲除和错义模型中的基因座人腺病毒C5型血清是一个很好的 由于其巨大的克隆能力，非常适合此概念验证项目的特征化病毒载体 在复制缺陷载体中，高滴度，感染分裂细胞和非分裂细胞的能力，以及其 对肝组织的天然取向，FIX生产的自然场所。此外，腺病毒已被用作一种 CRISPR/CAS9战略的成功交付平台，并已被证明是 捐献者DNA。在rosa26基因座的固定整合将通过共注射包含以下内容的载体来实现 MFIX，两侧是用于同源重组的rosa26特定序列，以及编码rosa26的病毒 特异性指南RNA和Cas9内切酶。双载体系统将首先在体外进行验证， 然后利用特征良好的FIX KO小鼠品系在体内利用小鼠肝细胞进行基因传递， 它忠实地概括了人类疾病。将对小鼠进行修复表达水平的分析， 治疗FIX活性、系统与其他策略比较的有效性以及改善凝血功能 表型，同时监测媒介介导的毒性和抑制抗体的形成。如果成功，请执行此操作 研究表明CRISPR/Cas9系统用于靶向整合治疗基因的可行性。 并就CRISPR/CAS9系统的用途提供了进一步的信息。

Abstract Hemophilia B is an X-linked recessive monogenic disorder affecting approximately 1 in 25,000 males, characterized by mutations which result in non functional or aberrant Factor IX (FIX) coagulation activity. These mutations can lead to a phenotype of spontaneous bleeding into joints and soft tissue, depending on the severity of the disease. Current clinical treatment of hemophilia B consists of life-long therapy with injections of FIX. The cost of treatment is expensive as well as physically invasive as patients must undergo recurrent intravenous injections. Thus, one of the core objectives of gene therapy for Hemophilia B is the establishment of long term therapeutic gene expression of coagulation factor IX in preclinical models of coagulation deficiency. Recent advancements in gene editing technology has given rise to the possibility of targeting specific loci for stable integration and long term expression of therapeutic transgenes. Herein, we hypothesize adenoviral-mediated gene therapy can establish long term therapeutic FIX expression following homology directed repair using a CRISPR/Cas9-based system targeted to the ‘safe harbor’ ROSA26 locus in murine knockout and missense models of Hemophilia B. Human adenovirus C serotype 5 is a well characterized viral vector that is ideally suited for this proof of concept project due to its large cloning capacity in replication deficient vectors, high titers, ability to infect both dividing and non dividing cells, as well as its native tropism to liver tissue, the natural site of FIX production. In addition, adenovirus has been utilized as a successful delivery platform for CRISPR/Cas9 strategies and has been shown to be an excellent vector for donor DNA. FIX integration at the ROSA26 locus will be achieved through co-injection of a vector containing mFIX, flanked by ROSA26 specific sequences for homologous recombination, and a virus encoding a ROSA26 specific guideRNA and the Cas9 endonuclease. The dual vector system will first be validated in vitro using murine hepatocytes then utilized for gene delivery in vivo using the well characterized FIX KO mouse strain, which faithfully recapitulates the human disease. The mice will be analyzed for levels of FIX expression, therapeutic FIX activity, efficacy of the system compared to other strategies, and improved coagulation phenotypes while monitoring for vector mediated toxicity and inhibitor antibody formation. If successful this study will represent the feasibleness of CRISPR/Cas9 system for the targeted integration of therapeutic genes in post embryonic somatic tissue while providing further information on the utility of CRISPR/Cas9 system.