Nanoparticles for retinal gene therapy

Abstract

Ocular gene therapy is becoming a well-established field. Viral gene therapies for the treatment of Leber's congentinal amaurosis (LCA) are in clinical trials, and many other gene therapy approaches are being rapidly developed for application to diverse ophthalmic pathologies. Of late, development of non-viral gene therapies has been an area of intense focus and one technology, polymer-compacted DNA nanoparticles, is especially promising. However, development of pharmaceutically and clinically viable therapeutics depends not only on having an effective and safe vector but also on a practical treatment strategy. Inherited retinal pathologies are caused by mutations in over 220 genes, some of which contain over 200 individual disease-causing mutations, which are individually very rare. This review will focus on both the progress and future of nanoparticles and also on what will be required to make them relevant ocular pharmaceutics.

Figure 1. Relative sizes of various nanoparticle-based delivery vectors

Shown are scale depictions of the relative sizes of different non-viral vectors. Sizes are either hydrodynamic diameters (HD) or measured by electron microscopy (EM) or dynamic light scattering (DLS). (A) Plasmid DNA [DLS ~1200 nm] (Parker Read, S. et al. 2010). (B) Toroidal, spermine compacted phage DNA [HD ~50 nm] (Wilson, R.W. et al. 1979). (C) CK30-PEG trifluoroacetate ellipsoid-shaped nanoparticle [EM ~22 × 50 nm] (Farjo, R. et al. 2006). (D) CK30-PEG acetate rod-shaped nanoparticle [EM ~8–11 × 200 nm] (Farjo, R. et al. 2006). (E) PEG-POD spherical nanoparticle [DLS ~130 nm] (Parker Read, S. et al. 2010). (F,G) Untargeted and RGD targeted PLGA nanoparticles [HD ~220–420 nm] (Singh, S.R. et al. 2009).

Figure 2. Nucleolin is not expressed on the plasma membrane of cultured ocular cells

Transformed cone-derived 661W cells (A) and transformed human RPE ARPE-19 cells (B) were fixed in 4% paraformaldehyde and stained with either mouse monoclonal C-23 MS-3 anti-nucleolin (red) or the plasma membrane marker rabbit polyclonal anti-GLUT1 (green) as indicated. 661W and ARPE-19 cells only expressed nucleolin within the nucleus. Co-labeling with GLUT1 did not reveal any expression of nucleolin on the plasma membrane. Scale bar, 10 µm.

Figure 3. Nucleolin is expressed in the murine retina

Paraffin-embedded (A) or frozen (B) retinal sections from postnatal day (P) 30 wild-type (WT) mice were fixed in 4% paraformaldehyde. (A) Sections were stained with H&E, no primary antibody, or immunoreacted with C-23 MS-3 monoclonal anti-nucleolin antibody (1:5). The secondary antibody was gold-conjugated goat-anti-mouse IgG (5 nm gold particles, 1:1000). Nucleolin is expressed in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). (B) Sections were stained with rabbit polyclonal anti-syntaxin-3 (−1:500, green) to label photoreceptor plasma membrane and anti-nucleolin C-23 F-18 goat polyclonal antibody (1:5, red). Shown is a single plane from a confocal micrograph. Punctate co-labeling (yellow-arrows) suggests that nucleolin is expressed on the plasma membrane of photoreceptors. RPE, retinal pigment epithelium; OS/IS, outer/inner segments; OPL, outer plexiform layer; IPL, inner plexiform layer. Scale Bar, 20 µm.

Figure 4. CK30-PEG nanoparticles drive GFP expression after subretinal injection at postnatal day 5

Wild-type (WT) mice underwent subretinal injection on postnatal day (P)5 with CK30-PEG nanoparticles containing the pZEO-GFP vector which incorporates the CMV promoter. Retinal sections were stained with DAPI and imaged. (A) On post-injection day (PI)-2, GFP expression was detected throughout the retina and in the cornea and lens. (B) On PI-7 no significant expression was detected, likely due to CMV promoter down-regulation. Scale bar, 25 µm.