Hematopoietic gene therapy for hemophilia A

描述（由申请人提供）：血友病A是一种先天性出血性疾病，由影响血浆蛋白的基因突变引起，称为因子VIII (fVIII)，其功能是促进血液凝固。目前最先进的A型血友病治疗包括频繁静脉输注含fVIII的产品。目前治疗血友病的限制是1)fVIII产品的成本，3)针对fVIII的免疫反应的发展阻碍了治疗效果，3)单个关节反复出血导致的关节疾病的发病率，4)治疗限制在世界人口的30%。由于为患者提供临床益处所需的fVIII数量有限，血友病A是一个有吸引力的基因治疗疾病靶点，已经进行了三个一期临床试验。这些试验的结果令人失望，因为在每种基因治疗策略中产生的fVIII极低，非治疗水平。我们最近发现，一种被命名为BDDpfVIII的改良猪fVIII转基因促进了非常高水平的蛋白表达，并且我们证明了这种转基因在移植转基因造血干细胞（hsc）后的血友病a小鼠模型中非常有效地发挥作用。具体来说，我们已经证明BDDpfVIII的表达优于其他生物工程人类fVIII表达构建体，并且遗传修饰和hsc移植可导致治疗性fVIII水平。此外，在移植bddpfviii转导的造血干细胞后，经过靶向免疫抑制的低毒性移植前调节，即使在预先存在抗人fVIII抑制剂的情况下，也能达到治疗性fVIII活性水平。因此，我们已经克服了低水平表达的主要障碍，使用转基因编码一种已成功用于a型血友病患者的蛋白质。在目前的应用中，我们建议更充分地表征高表达构建体的使用，并进一步了解这种新型基因治疗策略所涉及的关键参数，并研究BDDpfVIII在造血细胞中非生生性表达的生物学。为了将我们的研究推向临床意义，我们建议1)测试临床相关的HSC移植调节方案，这些方案更接近于人类骨髓移植方案中常规使用的方案；2)测试重组慢病毒载体，该载体已被证明与肿瘤逆转录病毒相比，可以减少插入突变。最后，优化的慢病毒载体编码BDDpfVIII将测试其基因修饰人类造血干细胞的能力。

DESCRIPTION (provided by applicant): Hemophilia A is a congenital bleeding disorder caused by genetic mutations affecting a plasma protein, termed factor VIII (fVIII), whose function is to facilitate blood clotting. State of the art treatment for hemophilia A consists of frequent intravenous infusions of fVIII containing products. The current limitations to treating hemophilia are 1) the cost of fVIII products, 3) the development of immune responses against fVIII that block treatment efficacy, 3) morbidity due to joint disease resulting from repeated bleeding into individual joints and 4) the limitation of treatment to 30% of the world population. Due to the limited amount of fVIII needed to provide clinical benefit to the patient, hemophilia A is an attractive disease target for gene therapy, and three phase 1 clinical trials have been conducted. The outcome of these trials has been disappointing due to the extremely low, non-therapeutic levels of fVIII produced in each of the gene therapy strategies. We recently showed that a modified porcine fVIII transgene, designated BDDpfVIII, facilitates very high-level protein expression, and we demonstrated proof-of-concept that this transgene functions extremely efficiently in a mouse model of hemophilia A following transplantation of genetically-modified hematopoietic stem cells (HSCs). Specifically, we have shown that the expression of BDDpfVIII is superior to other bioengineered human fVIII expression constructs and that genetic modification and transplantation of HSCs results in curative fVIII levels. Additionally, curative fVIII activity levels are achieved after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pre-transplantation conditioning with targeted immunosuppression, even in the context of pre-existing anti-human fVIII inhibitors. Therefore, we have overcome the major hurdle of low-level expression using a transgene that encodes a protein that has been used successfully in patients with hemophilia A. In the current application, we propose to more fully characterize the use of the high-expression construct and further our understanding of the critical parameters involved with this novel gene therapy strategy and study the biology of non-physiological BDDpfVIII expression in hematopoietic (blood) cells. To advance our studies toward clinical significance, we propose to 1) test clinically relevant HSC transplant conditioning regimens that more closely resemble those used routinely in human bone marrow transplant protocols and 2) test recombinant lentiviral vectors that have been demonstrated to display a reduction of insertional mutagenesis compared to oncoretroviruses. Finally, the optimized lentiviral vector(s) encoding BDDpfVIII will be tested for the ability to genetically modify human HSCs. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder caused by insufficiency of a blood clotting factor, designated factor VIII, for which gene therapy offers a potential cure. However, pre-clinical studies and clinical trials showed that a major limitation to a successful gene therapy treatment is the extremely poor expression of fVIII from the human fVIII transgene, which we recently overcame by introducing a porcine fVIII transgene. We demonstrated that use of the porcine transgene results in up to 100-fold greater fVIII production than the human version, and more recently, we demonstrated that a similar differential is observed following gene transfer into blood stem cells using recombinant retroviruses. Our current studies focus on better understanding the critical pharmacologic parameters involved in generating curative fVIII levels with this procedure using a mouse model of hemophilia A with the goal of understanding how this application can best be translated to clinical successes.