Nanoparticle delivery of triplex-forming PNAs for thalassemia gene therapy

描述（由申请人提供）：造血干细胞能够在其一生中形成个体血液的各种成分。遗传性血液疾病，如地中海贫血和镰状细胞贫血，可以通过造血干细胞和祖细胞（HSPC）的遗传操作来治疗或治愈。已经显示，三链体形成肽核酸（PNA）可用于协调短的50-60 bp“供体DNA”片段重组到基因组DNA中，导致基因突变的位点特异性校正。然而，基于PNA的疗法向临床的转化受到细胞内递送的挑战的限制，特别是在难以阻断的HSPC中。我们的初步数据表明，聚（乳酸-羟基乙酸）（PLGA）纳米粒子可以提供PNA和供体DNA的位点特异性重组在一个地中海贫血位点在人类HSPCs。我们的中心假设是，可生物降解的纳米颗粒可以被工程化以递送PNA用于对地中海贫血基因座的位点特异性编辑，具有高效率、低毒性和增加的对人类HSPC的细胞特异性靶向。具体目标：我们将通过三个具体目标来测试我们的总体假设。我们的第一个目标是测试HSPC中纳米颗粒的摄取可以通过向颗粒表面添加特定配体来增强的假设。我们将通过用细胞穿透肽对荧光纳米颗粒进行表面修饰，并在体外分析它们在人HSPCs中的内化来实现这一目标。我们的第二个目标将测试的假设，直接在体内交付的纳米粒子HSPCs可以通过添加细胞特异性配体的颗粒表面增强。我们将通过用靶向HSPC的抗体对荧光纳米颗粒进行表面修饰，并在全身注射到用人类造血细胞重建的小鼠模型中后分析其吸收到HSPC中来实现这一目标。如果成功，也将使用目标1中测试的配体。我们的第三个目标将测试纳米颗粒优化将在体内和体外增强对地中海贫血基因座的编辑的假设。这一目标将通过优化纳米颗粒中的PNA和DNA负载，并使用目标1和2中探索的表面修饰来实现。将评估优化的PNA-DNA纳米颗粒在人HSPC中的体外基因修饰活性，以及在用人造血细胞重建的小鼠模型中全身注射后的体内基因修饰活性。由于我们的初步工作已经确定我们的纳米颗粒系统在HSPC中工作良好，并且每个目标都测试了建立在我们已验证的系统基础上的单独假设，因此所有三个目标都将同时进行。

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.