Nanoparticle & AAV Approaches to Rapid Onset, Stable Retinal Gene Therapy

描述(申请人提供)：环鸟苷一磷酸(CGMP)是脊椎动物光感受器中一个关键的信使分子，它将光子的吸收与神经信号联系起来。视杆细胞中PDE(PDE)基因亚基的自然突变会导致RD小鼠快速的光感受器退化。一种新的具有PDE基因突变的小鼠模型--RD10小鼠，在大约P16开始杆状感光细胞退化，在P30只剩下锥体细胞体，将被用来测试突变基因的及时纠正是否可以导致长期稳定的视网膜挽救。这需要非常迅速的治疗性基因，这对传统载体来说是一个挑战。在并列实验中，我们建议分别和联合使用AAV载体和DNA纳米颗粒(LPEI)方法在RD10小鼠身上测试视网膜基因传递的治疗效果。此外，我们建议通过DNA/LPEI纳米颗粒方法测试睡美人(SB)转座子系统，作为一种将载体技术和纳米颗粒技术的优势结合在一个系统中的方法。具体目的1.检测AAV介导的PDE对RD10小鼠的治疗效果。具体目的2.检测DNA/LPEI纳米粒子对rd10小鼠的治疗效果。具体目的3.检测联合应用AAV+DNA/LPEI纳米粒给药对rd10小鼠的治疗效果。具体目的4.检测睡美人转座子DNA/LPEI纳米粒对rd10小鼠的治疗效果。我们的总体目标是通过比较这四种视网膜基因输送方案来确定在这一具有挑战性的、快速发病的隐性RP模型中实现最有效和最安全的治疗方案。我们的最终目标是建立一种快速、持续时间长、能够容纳大量治疗性DNA的视网膜基因递送系统。我们的总体目标是确定在这一具有挑战性的、快速发病的隐性RP模型rd10小鼠中，PDE基因的四种视网膜基因输送方案中的哪一种实现了最有效和最安全的治疗。这些替代方案包括单独的AAV，单独的DNA/LPEI纳米颗粒，AAV与DNA/LPEI纳米颗粒的组合，以及睡美人转座子DNA/LPEI纳米颗粒。我们的最终目标是建立一种快速、持续时间长、能够容纳大量治疗性DNA的视网膜基因递送系统。

DESCRIPTION (provided by applicant): Cyclic guanosine monophosphate (cGMP) is a key messenger molecule that links absorption of photons to neural signaling in the vertebrate photoreceptor. Naturally occurring mutations of the ¿-subunit of PDE (PDE¿) gene in rods leads to rapid photoreceptor degeneration in rd mice. A new mouse model with PDE¿ mutation, rd10 mouse, in which rod photoreceptor degeneration initiates at about P16, with only cone cell bodies remaining at P30, will be employed to test if in time correction of mutant gene can lead to long-term stable retinal rescue. This requires a very rapid onset of therapeutic genes, which is a challenge for conventional vectors. In side-by-side experiments we propose to test for therapy in the rd10 mouse using AAV vector and DNA nanoparticle (LPEI) approaches to retinal gene delivery, both separately and in combination. In addition we propose to test the Sleeping Beauty (SB) transposon system via the DNA/LPEI nanoparticle approach as a way to combine the advantages of both the vector and nanoparticle techniques in a single system. Specific Aim 1. Test the therapeutic efficacy of AAV delivered PDE¿ in the rd10 mouse. Specific Aim 2. Test the therapeutic efficacy of DNA/LPEI nanoparticle delivered PDE¿ in the rd10 mouse. Specific Aim 3. Test the therapeutic efficacy of combined AAV + DNA/LPEI nanoparticle delivered PDE¿ in the rd10 mouse. Specific Aim 4. Test the therapeutic efficacy of Sleeping Beauty transposon DNA/LPEI nanoparticle delivered PDE¿ in the rd10 mouse. Our overall aim is to determine by comparison of these four alternatives for retinal gene delivery which achieves the most effective and safest therapy in this challenging, rapid onset model of recessive RP. Our ultimate goal is to establish a retinal gene delivery system that is fast, of long duration and can accommodate large therapeutic cDNAs. Our overall aim is to determine which of four alternatives for retinal gene delivery of the PDE¿ gene achieves the most effective and safest therapy in this challenging, rapid onset model of recessive RP, the rd10 mouse. Theses alternatives include AAV alone, DNA/LPEI nanoparticle alone, AAV combined with DNA/LPEI nanoparticle, and the Sleeping Beauty transposon DNA/LPEI nanoparticle. Our ultimate goal is to establish a retinal gene delivery system that is fast, of long duration and can accommodate large therapeutic cDNAs.