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，称为第八因子(FVIII)，其功能是促进血液凝固。血友病A的最新治疗包括频繁静脉输注含有FVIII的产品。目前治疗血友病的局限性是：1)FVIII产品的成本，3)对FVIII的免疫反应阻碍了治疗效果，3)由于个别关节反复出血导致的关节疾病的发病率，以及4)治疗限于世界人口的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，已经进行了三个阶段的临床试验。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的非治疗性水平极低。我们最近发现了一种名为BDDpfVIII的猪FVIII转基因基因，可以促进非常高水平的蛋白质表达，并且我们在转基因造血干细胞(HSCs)移植后的血友病A小鼠模型中证明了这种转基因基因非常有效地发挥作用。具体地说，我们已经证明了BDDpfVIII的表达优于其他生物工程化的人类FVIII表达结构，并且基因修饰和移植HSCs可以产生治愈的FVIII水平。此外，移植BDDpfVIII转导的HSCs后，即使在先前存在的抗人FVIII抑制剂的情况下，也可以在低毒的移植前用靶向免疫抑制进行调节后达到治愈的FVIII活性水平。因此，我们已经克服了低水平表达的主要障碍，使用了编码已成功用于血友病A患者的蛋白质的转基因。在当前的应用中，我们建议更全面地描述高表达构建体的使用，并进一步了解与这一新的基因治疗策略相关的关键参数，并研究BDDpfVIII在造血细胞(血液)中非生理性表达的生物学。为了推动我们的研究走向临床意义，我们建议1)测试临床相关的HSC移植条件方案，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)测试已被证明与肿瘤逆转录病毒相比显示出插入突变减少的重组慢病毒载体。最后，编码BDDpfVIII的优化慢病毒载体(S)将被测试对人类HSCs进行基因修饰的能力。 公共卫生相关性：血友病A是一种由凝血因子(指定为因子VIII)不足引起的出血性疾病，基因疗法为其提供了一种潜在的治疗方法。然而，临床前研究和临床试验表明，成功的基因治疗的一个主要限制因素是人类FVIII转基因的FVIII表达极低，最近我们通过引入猪FVIII转基因克服了这一问题。我们证明了使用猪转基因比使用人类转基因产生的FVIII高达100倍，最近，我们证明了使用重组逆转录病毒将基因转移到血液干细胞后也观察到了类似的差异。我们目前的研究重点是利用血友病A的小鼠模型更好地了解使用这一程序产生治愈的FVIII水平所涉及的关键药理学参数，目的是了解这种应用如何最好地转化为临床成功。

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.