Linear Polyethylenimine-Based Nanoparticle DNA Complexed With AAV Vector Rescues Retinal Degeneration in the Rd1 Mouse

Abstract

Purpose: : The purpose of this study is to test whether a complex of Linear Polyethylenimine-based (LPEI) DNA nanoparticle and an AAV vector can preserve retinal function and structure in rd1 mice. Our aim was to develop a rapid onset but long-lasting gene therapy technique to treat retinal degeneration.

Methods: : : An AAV5-CBA- mPDE_β vector was prepared according to standard procedures in our lab. The plasmid pTR-CBA- mPDE_β was precipitated into an LPEI nanoparticle at an N/P ratio of 6. It was then mixed with vector at a ~ 20,000 LPEI molecules per vector particle and delivered to the subretinal space of PN 3-11 rd1 mice. Rod derived ERGs were recorded and retinal histology then analyzed at PN21, 28 and 35. The effect of treatment on photoreceptor survival was analyzed by quantifying the number of nuclei in the ONL and the level of biochemical preservation by rhodopsin immunohistochemistry. Untreated partner left-eyes served as controls. Additionally, photoreceptor apoptotic cell death was quantified by TUNEL analysis. To examine the structure of nanoparticle-vector complexes, EM images were taken of vector, nanoparticle and the vector-nanoparticle complexes.

Results: : Rod ERG data showed that there was a 15~30% of normal b-wave preservation in treated eyes compared to nearly unrecordable ERGs in partner control eyes at PN21, 28, and 35. Histological analysis showed 2~4 layers of ONL nuclei in treated eyes compared to an almost total loss of ONL nuclei in control eyes at PN35. Histochemically, rhodopsin was evident above the inner segment layer of treated retinas and nearly absent in controls at PN35. TUNEL assays revealed a significant decrease in apoptotic photoreceptors in treated versus control eyes. By EM analysis, nanoparticles were 30 to 50nm spheroids while the AAV5 vector was 20nm to 25nm in diameter. The nanoparticle-vector complex appeared as an unstructured aggregate of apparently loosely coiled DNA/nanoparticle associated with multiple vectors.

Conclusions: : Linear polyethylenimine-based nanoparticle DNA complexed with AAV vector was demonstrated to preserve photoreceptor function and structure in spite of the rapid onset retinal degeneration seen in the rd1 mouse. Thus far preservation is maintained for 35 days.

Keywords: gene transfer/gene therapy • immunohistochemistry • retinal degenerations: hereditary