Abstract
Purpose :
Non-viral vectors present several advantages for retinal gene therapy, in particular by carrying genes above 4.5 kb; however, these still need to be optimized to achieve higher transfection efficiencies. Previous results from our group revealed that entering the nucleus was the bottleneck for our nonviral vectors (Bitoque, et al. Materials Science and Engineering: C, 2018; Bitoque, et al. Bioscience Reports, 2021). Therefore, we aimed to increase the efficiency of a polymeric nonviral vector by conjugating nuclear localization signals (NLSs), which are short amino acid sequences capable of directing molecules to the nucleus.
Methods :
To test our hypothesis, we have produced and characterized a nonviral vector by combining a plasmid encoding a reference gene with our NLS-conjugated PAEMA (poly(2-aminoethyl methacrylate polymer). We further tested it in vitro using a human pigment epithelial cell line (D407) to assess its effect on cell viability and transfection efficiency and in vivo in the rd10 mouse. All experimental procedures were carried out according to the Portuguese, European Union, FELASA, and the Association for Research in Vision and Ophthalmology (ARVO) regulations for the use of animals in ophthalmic and vision research.
Results :
We have found that PAEMA can be efficiently modified to incorporate NLSs, and this modified polymer efficiently complexes plasmid-based expression systems, with size and charge adequate for cell entry. We have further confirmed this polymer to be capable of protecting its load from degradation but adequately releasing it 24 hours after cell entry. Furthermore, in RPE cells, the qualitative (fluorescence microscopy) and quantitative (flow cytometry) analysis of the transfection efficiency show that PAEMA conjugated with NLSs is able to increase the expression of the gene of interest by 63%. Lastly, intravitreal injection of PAEMA-NLSs polyplexes in the retina of rd10 mice has shown in vivo compatibility, and negligible, fast-resolving, inflammatory response. Further studies are focused on the capacity of the optimized polymer to deliver the β subunit of PDE6 gene to the nucleus of retinal cells.
Conclusions :
Overall, these results demonstrate the potential of PAEMA nonviral vectors for retinal gene therapy, in particular for overcoming the nuclear bottleneck for an efficient delivery.
This is a 2021 ARVO Annual Meeting abstract.