DNA nanoparticles deliver genes into the retina

Biologists from the University of Oklahoma Health Science Center, in collaboration with researchers from Copernicus Therapeutics Inc., have developed DNA nanoparticles which they used to transfer genes into the mouse retina.

According to the Copernicus Therapeutics website:

[We have] discovered how to compact DNA into complexes containing only a single molecule of DNA, resulting in a volume 30-1000 times smaller than other complexes. This simple difference makes Copernicus’ “DNA nanoparticles” highly stable and uniquely able to deliver DNA to the nuclei of non-dividing human cells – which includes most cells in the body.

Two different types of complexes were formulated – those made in the presence of trifluoroacetate formed short rods, while those made in the presence of acetate formed long rods (left and right, respectively, in the image below). Both types of particle had a diameter of less than 25 nanometres (billionths of a meter), which is the size of the pores in the membrane surrounding the nucleus.

Both types of complex complexes contained the gene encoding enhanced green fluorescent protein (EGFP), which is of no therapeutic value, but acted as a ‘reporter’ which enabled the research team to determine the efficiency of gene transfer. Solutions of the nanoparticles were injected directly into the eyes of mice. Retinal cells exposed to the solution took up the particles readily. The small size of the particles facilitated their entry through the nuclear membrane pores, so that the reporter gene was incorporated into the cells’ chromosomes. Its expression produced a green fluorescence within the cell, which was then visualized using fluorescence microscopy.

The mammalian retina is a highly organized tissue consisting of 6 layers, each containing different cell types. By varying the injection site, the nanoparticles were targeted to different layers in the retina, where they transferred the reporter gene only to cells in that layer. Injection of the solution into the space behind the retina led to EGFP expression in nearly all of the photoreceptors, the cells at the back of the retina which are responsive to light.

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Electron micrographs of the compacted DNA nanoparticles of varying sizes used in the study (from Farjo, et al, 2007).

Efforts to develop gene therapies usually use modified viral particles as vectors to deliver genes into target tissues. The use of viral vectors can eb problematic for a number of reasons. For example, the viral particles can be toxic, and may elicit immune and inflammatory responses. Furthermore, although viral vectors are engineered specifically to shuttle genes into target cells, there is always a possibility that they may regain their pathogenic (disease-causing) properties.

To overcome these problems, researchers have developed a number of non-viral vectors for delivering genes into the body. DNA molecules can be inserted into gold nanoparticles and into spherical vesicles called liposomes, which are made from the same materials as cell membranes. Alternatively, ‘naked’ DNA can be injected directly into tissue, but this results in low levels of expression of the tranferred gene.

The DNA nanoparticles used in this study delivered the EGFP reporter gene highly efficiently – subretinal injection of the solution led to the reporter being expressed at similar levels to opsin, which is the most highly-expressed gene in the retina. The particles did not elicit an immune response in the animals, and no deleterious effects on retinal function were observed.

This study shows that compacted DNA nanoparticles could potentially be very useful in developing gene therapies for a number of eye diseases. The highly efficient transfer of the reporter gene into photoreceptor cells could lead to effective treatments for conditions such as retinitis pigmentosa, in which there is progressive degeneration of these cells. By modifying the size and surface properties of the nanoparticles, they could be made to target specific types of cells in other organs, including the skin and liver.

Reference:

Farjo R, et al (2006). Efficient Non-Viral Ocular Gene Transfer with Compacted DNA Nanoparticles. PLoS ONE 1: e38. doi:10.1371/journal.pone.0000038