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

使用腺病毒载体递送通过 CRISPR/Cas9 靶向整合因子 IX 基因进行 B 型血友病基因治疗

• 批准号: 9193681
• 负责人: Calvin J Stephens
• 金额: $ 3.02万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9193681
• 关键词: Address; Adenovirus Vector; Adenoviruses; Adverse effects; Affect; Animal Model; Antibody Formation; Blood Coagulation Disorders; CRISPR/Cas technology; Candidate Disease Gene; Clinical Treatment; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Cytomegalovirus; DNA; Disease; Dose; Embryo; Event; Factor IX; Frequencies; Gene Delivery; Gene Expression; Generations; Genes; Genomics; Guide RNA; Hemophilia B; Hemorrhage; Hepatocyte; Hepatotoxicity; Hereditary Disease; Human; Immune response; In Vitro; Individual; Injection of therapeutic agent; Interphase Cell; Joints; Knock-out; Knockout Mice; Lead; Life; Link; Liver; Mediating; Mendelian disorder; Modeling; Monitor; Mouse Strains; Mus; Mutation; Nature; Oncogenic; Patients; Phenotype; Plasmids; Positioning Attribute; Pre-Clinical Model; Production; Recurrence; Reporter; Reporter Genes; Serotyping; Severity of illness; Site; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Transgenic Animals; Treatment Cost; Tropism; Viral; Viral Vector; Virus; Virus Diseases; Wild Type Mouse; abstracting; adenoviral-mediated; arm; base; co-infection; design; endonuclease; experience; gene therapy; homologous recombination; human disease; improved; in vivo; inhibitor/antagonist; integration site; intravenous injection; male; mouse model; repaired; soft tissue; therapeutic gene; therapeutic transgene; vector

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.

抽象的 血友病B是一种X连锁的隐性单基因疾病，影响了25,000名男性中约1个， 以突变为特征，这些突变导致非功能或异常IX（固定）凝血活性。 这些突变会导致赞助商样出血成关节和软组织的表型，具体取决于 疾病的严重程度。血友病B的当前临床治疗包括注射寿命的治疗 修复。治疗费用既昂贵又具有侵入性，就像患者必须经常发生 静脉注射。这是基因治疗的核心对象之一，是建立的 凝血模型中凝血因子IX的长期治疗基因表达 不足。基因编辑技术的最新进步导致了目标的可能性 特定的位置，用于稳定整合和热转基因的长期表达。在此，我们假设 腺病毒介导的基因疗法可以建立长期治疗固定表达 同源性使用针对“安全港” ROSA26的基于CRISPR/CAS9的系统进行维修 鼠敲除和血友病的错义模型中的基因座B.人腺病毒C血清型5是一个井 特征的病毒矢量，由于其较大的克隆能力，它非常适合该概念项目证明 在复制不足的矢量，高滴度，能够感染分裂和非分裂细胞及其的能力中 天然对肝组织的向流，这是固定生产的自然部位。此外，腺病毒已被用作 CRISPR/CAS9策略的成功交付平台，已被证明是一个很好的向量 供体DNA。通过共同注射包含载体 Mfix，侧面是Rosa26的同源重组的特定序列，以及编码ROSA26的病毒 特定的guderna和cas9核酸内切酶。双重向量系统将首先在体外使用 然后，使用良好特征的固定kO小鼠菌株，用于体内基因递送的鼠肝细胞， 忠实地概括了人类疾病。将分析小鼠的固定表达水平， 与其他策略相比，治疗性修复活动，系统的效率以及改善的凝血 在监测载体介导的毒性和抑制剂抗体形成时表型。如果成功 研究将代表CRIS/CAS9系统的可行性，以靶向治疗基因的靶向整合 在胚胎躯体组织后，同时提供有关CRISPR/CAS9系统实用程序的进一步信息。