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

描述（由申请人提供）：环磷酸鸟苷（cGMP）是一种关键信使分子，其将脊椎动物光感受器中的光子吸收与神经信号传导联系起来。视杆细胞中PDE基因的<$-亚基（PDE <$）的自然突变导致rd小鼠的感光细胞迅速退化。一种新的PDE突变小鼠模型，rd 10小鼠，其中视杆细胞变性在约P16开始，仅在P30保留视锥细胞体，将用于测试突变基因的及时校正是否可以导致长期稳定的视网膜拯救。这需要治疗基因的非常快速的启动，这对常规载体是一个挑战。在并行实验中，我们建议使用AAV载体和DNA纳米颗粒（LPEI）方法单独和组合进行视网膜基因递送，以测试rd 10小鼠中的治疗。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，作为在单个系统中联合收割机结合载体和纳米颗粒技术的优点的一种方式。具体目标1。测试AAV递送的PDE在rdlO小鼠中的治疗功效。具体目标2。在rd 10小鼠中测试DNA/LPEI纳米颗粒递送的PDE â的治疗功效。具体目标3。在rdlO小鼠中测试组合的AAV + DNA/LPEI纳米颗粒递送的PDE的治疗功效。具体目标4。在rd 10小鼠中测试睡美人转座子DNA/LPEI纳米颗粒递送PDE的治疗功效。我们的总体目标是通过比较这四种用于视网膜基因递送的替代方案来确定在这种具有挑战性的、快速发病的隐性RP模型中实现最有效和最安全的治疗。我们的最终目标是建立一个快速、持续时间长并能容纳大量治疗性cDNA的视网膜基因递送系统。我们的总体目标是确定PDE基因的视网膜基因递送的四种替代方案中的哪一种在这种具有挑战性的隐性RP的快速发病模型（rd 10小鼠）中实现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、与DNA/LPEI纳米颗粒组合的AAV和睡美人转座子DNA/LPEI纳米颗粒。我们的最终目标是建立一个快速、持续时间长并能容纳大量治疗性cDNA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.