Advancing lentiviral gene therapy for cystic fibrosis.

项目摘要 囊性纤维化（CF）是一种常见的，缩短寿命的遗传性疾病。囊性纤维化传导中的突变 调节子（CFTR）基因导致CFTR缺陷或缺失，其不能有效地在许多细胞中转运阴离子。 但肺部并发症是最常见的死亡原因。CF治疗的最新进展 包括能够挽救蛋白质功能药物。然而，这些治疗是突变类别特异性的，并且是有效的。 对某些突变无效。因此，持续需要开发突变不可知论治疗， 使所有CF患者受益，例如基因添加。 慢病毒载体非常适合于CF基因治疗，因为它们具有足够的携带能力，并且它们的 整合到宿主基因组中的能力确保了长期利益。然而，目前可用的载体， 产生低的载体滴度或不能有效地从顶端表面除去气道上皮细胞。尽管有这些 局限性，用携带CFTR的功能性拷贝的慢病毒载体治疗新生CF猪部分地降低了CFTR的表达。 挽救了CF表型，表明实现治疗性CFTR表达是可以实现的。 我的长期目标是设计一种适合于有效治疗人类肺CF的慢病毒载体。 他的建议的总体目标是评估两个独立但互补的战略， CFTR向人气道上皮细胞的慢病毒递送。慢病毒载体可以容纳来自 其他病毒，称为假型化，这可以导致它们优先选择特定的细胞类型。为 第一个策略，我筛选了七个病毒包膜，并确定了两个来自狒狒内源性逆转录病毒 （BaEV），其产生高载体滴度并有效地将原代人气道上皮细胞从 顶面第二种策略是增加转导细胞中的CFTR表达。因为航空公司 上皮细胞通过缝隙连接电化学偶联，少数细胞表达高水平的CFTR， 可以进行足够的CFTR介导的阴离子转运，以纠正CF表型。增加的基因表达 可以通过密码子优化来实现。我比较了三个密码子优化的CFTR（coCFTR）序列， 并鉴定了一种显著增加CFTR介导的跨上皮Cl-转运的酶。我的核心假设是 增加转导效率和转基因表达将足以达到野生型水平 CFTR介导的阴离子转运。为了验证我的假设，我提出了以下具体目标：1）确定 如果BaEV假型慢病毒载体可以有效地将CFTR递送至原代人气道上皮细胞， 2)确定coCFTR递送是否减少实现野生型所需的补充细胞的比例 CFTR介导的阴离子转运水平。这些策略将在体外使用人CF供体来源的 气道上皮，并在体内使用CF猪模型。这些实验将提供有价值的临床前数据 这将指导载体设计，并为未来使用慢病毒载体治疗CF的临床试验提供信息。

Project Summary Cystic fibrosis (CF) is a common, life-shortening genetic disease. Mutations in the cystic fibrosis conductance regulator (CFTR) gene lead to defective or absent CFTR that is unable to effectively transport anions in many tissues, but pulmonary complications are the most common cause of death. Recent advances in CF treatment include drugs able to rescue protein function. However, these treatments are mutation-class specific and are ineffective for some mutations. Thus, there is a persistent need to develop mutation agnostic treatments that can benefit all people with CF, such as gene addition. Lentiviral vectors are well suited for CF gene therapy because they have sufficient carrying capacity, and their ability to integrate into the host’s genome ensures long-term benefits. Currently available vectors, however, produce low vector titers or inefficiently transduce airway epithelial cells from the apical surface. Despite these limitations, treatment of newborn CF pigs with a lentiviral vector carrying a functional copy of CFTR partially rescued CF phenotypes, suggesting that achieving therapeutic CFTR expression is within reach. My long-term goal is to design a lentiviral vector suitable for the effective treatment of pulmonary CF in humans. The overall objective of his proposal is to evaluate two independent but complementary strategies, to improve lentiviral delivery of CFTR to human airway epithelial cells. Lentiviral vectors can accommodate envelopes from other viruses, termed pseudotyping, which can lead them to preferentially transduce specific cell types. For the first strategy, I screened seven viral envelopes and identified two derived from baboon endogenous retrovirus (BaEV) that produce high vector titers and efficiently transduce primary human airway epithelial cells from the apical surface. The second strategy is to increase CFTR expression in transduced cells. Because airway epithelial cells are electrochemically coupled through gap junctions, a few cells expressing high levels of CFTR may perform sufficient CFTR-mediated anion transport to correct CF phenotypes. Increased gene expression can be achieved through codon optimization. I compared three codon optimized CFTR (coCFTR) sequences and identified one that significantly increases CFTR-mediated transepithelial Cl- transport. My central hypothesis is that increasing transduction efficiency and transgene expression will be sufficient to achieve wild type levels of CFTR-mediated anion transport. To test my hypothesis, I propose the following Specific Aims: 1) Determine if BaEV pseudotyped lentiviral vectors can efficiently deliver CFTR to primary human airway epithelial cells, and 2) Determine if coCFTR delivery reduces the proportion of complemented cells needed to achieve wild type levels of CFTR-mediated anion transport. These strategies will be tested in vitro using human CF donor-derived airway epithelia, and in vivo using the CF pig model. These experiments will provide valuable preclinical data that will guide vector design and inform future clinical trials using lentiviral vectors to treat CF.