DNA nanoparticle formulations for optimal ocular gene delivery

描述（由申请人提供）：该计划的目标是推进当前的DNA纳米颗粒（NP）递送和表达技术，以开发安全有效的治疗方法，用于治疗影响感光细胞（PR）和视网膜色素上皮（RPE）细胞的重要眼部疾病。 该计划将与分子生物工程，视觉科学，物理学和化学专家合并，以加速有效的眼部非病毒基因疗法的产生的重要步骤。 DNA NP由用赖氨酸-PEG聚阳离子压实的DNA单分子组成，并且具有8-11 nm的最小直径。 它们的小尺寸，加上有效地将NP运输到细胞核并绕过溶酶体降解系统的特异性摄取机制，可能是NP稳健地抑制有丝分裂后分化细胞的能力的原因。 配制时使用的抗衡离子决定了 NP形状以及杆状和椭圆形NP均显示对眼细胞（包括RPE、PR和视网膜神经节细胞（RGC））的稳健转染。 此外，鼠RDS NP已在RDS单倍不足的视网膜色素变性小鼠模型中证明了部分表型校正。 先前已经证明了这些NP在眼睛中的有效使用的原理证明，在本申请中，我们将采取必要的步骤来优化用于临床眼部使用的颗粒。 首先，我们将优化用于PR和RPE特异性基因转移的NP制剂（NP形状、大小和化学组成）（目标#1）。 第二，我们将设计临床适用的DNA载体（目标#2），以产生持续的高水平转基因表达。 第三，我们将测试这些NP在非人灵长类动物模型（狒狒）中靶向黄斑的能力（目标#3），包括评估NP的非侵入性玻璃体内递送是否可以抑制黄斑/中央凹视锥。 这是该技术临床应用的一个新的和关键的发展，因为许多视网膜变性靶向黄斑。 此外，由于啮齿动物视网膜中不存在黄斑，这是不能通过小鼠系统建模的步骤。 我们还将在狒狒中进行毒理学和DNA生物分布研究，包括对大脑视觉通路的详细评估。 基于现有NP制剂穿透视网膜深层的记录能力，我们假设这种效率可以通过目标#1中详述的NP制剂优化程序来提高，从而实现稳健的中央凹视锥基因转移。 总之，本申请的结果将促进用于眼部基因递送的DNA NP的临床前试验评价。 公共卫生相关性：该计划旨在推进我们的紧凑型DNA纳米技术，以促进其未来用作眼科疾病的临床基因治疗。 临床前研究计划为使用该技术作为眼部疾病如视网膜色素变性（RP），各种黄斑营养不良（MD）和糖尿病视网膜病变的临床可行的基因递送系统奠定基础。 我们的NP的最小直径（8-11 nm）比任何病毒载体小得多，并且可以允许独特有效和安全的玻璃体内眼部给药以将基因递送到RPE细胞和中央凹视锥中。

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery and expression technologies to develop safe and effective therapies for important ocular disorders affecting the photoreceptor (PR) and retinal pigment epithelial (RPE) cells. The program will merge experts with molecular bioengineering, vision science, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus and bypasses the lysosomal degradation system, likely accounts for the ability of the NPs to robustly transfect post-mitotic, differentiated cells. The counter-ion used at the time of formulation determines the NP shape and both rod-like and ellipsoidal NPs show robust transfection of ocular cells, including RPE, PRs and retinal ganglion cells (RGCs). Moreover, murine RDS NPs have demonstrated partial phenotypic correction in a retinitis pigmentosa mouse model of RDS haploinsufficiency. Having previously shown proof-of-principle for the effective use of these NPs in the eye, in this application we will take the necessary steps to optimize the particles for clinical ocular use. First, we will optimize the NP formulation (NP shape, size, and chemical composition) for PR- and RPE-specific gene transfer (Aim #1). Second, we will engineer clinically-appropriate DNA vectors (Aim #2) to generate persistent, high levels of transgene expression. Thirds, we will test the ability of these NPs to target the macula in a non-human primate model (baboon) (Aim #3) including an assessment of whether non-invasive intravitreal delivery of NPs can transfect macular/foveal cones. This is a novel and critical development for clinical application of this technology, as many retinal degenerations target the macula. Furthermore, it is a step that cannot be modeled by a mouse system due to the absence of a macula in the rodent retina. We will also conduct toxicology and DNA biodistribution studies in baboons, including a detailed evaluation of brain visual pathways. Building on the documented ability of the current NP formulation to penetrate deep retinal layers, we hypothesize that this efficiency can be improved by the NP formulation optimization program detailed in Aim #1, enabling robust foveal cone gene transfer. In summary, results from this application will facilitate preclinical trial evaluations of DNA NPs for ocular gene delivery. PUBLIC HEALTH RELEVANCE: This program is designed to advance our compacted DNA nanotechnology to facilitate its future use as a clinical gene therapy treatment for ocular diseases. Preclinical studies are planned to lay the foundation for use of this technology as a clinically viable gene delivery system for ocular diseases such as retinitis pigmentosa (RP), various macular dystrophies (MD), and diabetic retinopathy. The minimum diameter (8-11 nm) of our NPs is much smaller than any viral vector, and may permit uniquely effective and safe intravitreal ocular dosing to deliver genes into RPE cells and foveal cones.