Nanoparticle delivery of triplex-forming PNAs for thalassemia gene therapy

描述(申请人提供)：造血干细胞能够在一个人的一生中形成不同的血液成分。遗传性血液疾病，如地中海贫血和镰状细胞性贫血，有可能通过对造血干细胞和祖细胞(HSPC)的遗传操作来治疗或治愈。已有研究表明，三链形成肽核酸(PNA)可以协调50-60个碱基的“供体DNA”片段重组成基因组DNA，从而实现基因突变的定点纠正。然而，基于PNA的治疗方法在临床上的应用受到细胞内传递的挑战的限制，特别是在难以转化的HSPC中。我们的初步数据显示，聚乳酸-乙醇酸(PLGA)纳米颗粒可以运送PNA和供体DNA，用于在人类HSPC的地中海贫血部位进行定点重组。我们的中心假设是，可以设计出可生物降解的纳米颗粒，以高效、低毒和更高的细胞特异性靶向人类HSPC，提供PNA，用于位点特异性编辑地中海贫血基因。具体目标：我们将通过三个具体目标来检验我们的总体假设。我们的第一个目标将检验这样一个假设，即通过在颗粒表面添加特定的配体，可以增强纳米颗粒在HSPC中的吸收。我们将通过用细胞穿透性多肽对荧光纳米颗粒进行表面修饰，并在体外分析它们在人HSPC中的内化来实现这一目标。我们的第二个目标将检验这样一个假设，即通过在颗粒表面添加细胞特异性配体，可以增强纳米颗粒直接体内输送到HSPC的能力。我们将通过用针对HSPC的抗体对荧光纳米颗粒进行表面修饰来实现这一目标，并将其全身注射到由人类造血细胞重建的小鼠模型中，分析它们对HSPC的摄取。如果成功，在AIM 1中测试的配体也将被使用。我们的第三个目标将检验这样的假设，即纳米颗粒的优化将在体内和体外加强地中海贫血基因的编辑。这一目标将通过优化PNA和DNA在纳米颗粒中的载量以及使用AIMS 1和2中探索的表面修饰来实现。优化的PNA-DNA纳米颗粒将被评估在人类HSPC的体外基因修饰活性，以及在系统注射后在由人类造血细胞重建的小鼠模型中的基因修饰活性。由于我们的初步工作已经确定，我们的纳米粒子系统在HSPC中运行良好，并且每个目标都测试了建立在我们已证明的系统基础上的单独假设，所以这三个目标将同时实现。公共卫生意义：该项目将通过药物输送方面的进展，帮助将地中海贫血的基因疗法转化为临床实践，并开发高度通用的方法，将治疗药物输送到用于治疗血液疾病的造血干细胞。 与公共卫生相关：地中海贫血是一种破坏性的遗传性疾病，身体不能产生足够的血红蛋白来制造红细胞。对突变基因的特定纠正可以治愈地中海贫血和其他单基因血液疾病，如镰状细胞性贫血。该项目将利用药物输送方面的进展，将针对地中海贫血的靶向基因疗法转化为临床实践。

DESCRIPTION (provided by applicant): Hematopoietic stem cells are capable of forming the diverse components of an individual's blood throughout his/her lifetime. Inherited blood disorders such as ¿-thalassemia and sickle cell anemia can potentially be treated or cured through genetic manipulation of hematopoietic stem and progenitor cells (HSPCs). It has been shown that triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 bp "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations. However, translation of PNA based therapies to the clinic is limited by challenges in intracellular delivery, especially in difficult-to-transfect HSPCs. Our preliminary data shows that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can deliver PNA and donor DNA for site-specific recombination at a ¿-thalassemia site in human HSPCs. Our central hypothesis is that biodegradable nanoparticles can be engineered to deliver PNAs for site-specific editing of a ¿-thalassemia locus, with high efficiency, low toxicity, and increased cell-specific targeting to human HSPCs. SPECIFIC AIMS: We will test our overall hypothesis through three specific aims. Our first aim will test the hypothesis that uptake of nanoparticles in HSPCs can be enhanced by adding specific ligands to the particle surface. We will accomplish this aim by surface-modifying fluorescent nanoparticles with cell-penetrating peptides, and analyzing their internalization in human HSPCs in vitro. Our second aim will test the hypothesis that direct in vivo delivery of nanoparticles to HSPCs can be enhanced by adding cell-specific ligands to the particle surface. We will accomplish this aim by surface-modifying fluorescent nanoparticles with antibodies targeting HSPCs, and analyzing their uptake into HSPCs after systemic injection into a mouse model reconstituted with human hematopoietic cells. Ligands tested in Aim 1 will also be used if successful. Our third aim will test the hypothesis that nanoparticle optimization will enhance editing at the ¿-thalassemia locus both in vivo and in vitro. This aim will be accomplished by optimizing PNA and DNA loading in nanoparticles, and using surface modifications explored in Aims 1 and 2. Optimized PNA-DNA nanoparticles will be assessed for in vitro gene modifying activity in human HSPCs, and for in vivo gene modifying activity after systemic injection in a mouse model reconstituted with human hematopoietic cells. Since our preliminary work has established that our nanoparticle system works well in HSPCs, and each aim tests a separate hypothesis that builds on our proven system, all three aims will be pursued concurrently. PUBLIC HEALTH RELEVANCE: This project will help translate gene therapies for ¿-thalassemia to clinical practice through advances in drug delivery and develop highly versatile methods for delivery of therapeutics to hematopoietic stem cells for treatment of blood disorders. PUBLIC HEALTH RELEVANCE: ¿-thalassemia is a devastating inherited disorder in which the body cannot produce enough hemoglobin for red blood cells. Specific correction of mutated genes could provide a cure for ¿-thalassemia and other single-gene blood disorders such as sickle cell anemia. This project will use advances in drug delivery to translate targeted gene therapies for ¿-thalassemia to clinical practice.