Direct Gene Transfer with Compacted DNA Nanoparticles in Retinal Pigment Epithelial Cells: Expression, Repeat Delivery and Lack of Toxicity

Abstract

Aim To evaluate the safety of compacted DNA nanoparticles (NPs) in retinal pigment epithelial (RPE) cells. Materials & Methods Enhanced GFP expression cassettes controlled by the RPE-specific vitelloform macular dystrophy promoter were constructed with and without a bacterial backbone and compacted into NPs formulated with polyethylene glycol-substituted lysine 30-mers. Single or double subretinal injections were administered in adult BALB/c mice. Expression levels of enhanced GFP, proinflammatory cytokines and neutrophil/macrophage mediators, and retinal function by electroretinogram were evaluated at different time-points postinjection. Results Immunohistochemistry and real-time PCR demonstrated that NPs specifically transfect RPE cells at a higher efficiency than naked DNA and similar results were observed after the second injection. At 6 h postinjections, a transient inflammatory response was observed in all cohorts, including saline, indicating an adverse effect to the injection procedure. Subsequently, no inflammation was detected in all experimental groups. Conclusion This study demonstrates the safety and efficacy of NP-mediated RPE gene transfer therapy following multiple subretinal administrations.

Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy

“…Although viral vector based gene delivery has been proven to be robust in gene delivery, it has several inherent concerns [17,18]. We have previously shown the effectiveness of CK30PEG10K compacted DNA NPs in efficiently transfect retinal tissues [18–21]. The expression of the therapeutic gene can result in long-term expression, rescue retina cells, and improve phenotypes in different neurodegenerative models (ABCA4 −/− , RPE65 −/− , Rds +/− , Rho −/− mice) [20,22–26].…”

“…Earlier research on these DNA NPs determined that an acetate counterion to lysine amines could compact plasmid DNA into a rod shape with an 8–11 nm diameter and a length proportional to the plasmid size [27,28]. These NPs have a tolerable safety profile with a lack of immunogenicity in the retina after delivery through intravitreal and subretinal routes, including multiple dosing [21,25,29]. Our strategy here is to intravitreally deliver CK30PEG10k compacted therapeutic miR200-b plasmid DNA to 6 month old diabetic Ins2 Akita mice and evaluate its therapeutic role against VEGF-mediated angiogenesis at 3 months post injection (PI-3 m).…”

DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice

“…Compacted DNA nanoparticles (NPs) (8-10 nm in diameter) formulated with polyethylene glycol-substituted polylysine (CK30PEG) are highly efficient in transfecting postmitotic cells (1)(2)(3)(4)(5), are biodegradable (3,4), and exhibit minimal toxicity, even after repeated dosing, to the eye, lung, and brain (1,2,(5)(6)(7). In contrast with many other nonviral approaches, these NPs drive long-term gene expression (1,2) after subretinal delivery to the mouse eye, making them an excellent choice for chronic retinal degenerations such as Stargardt.…”

Nanoparticle-based technologies for retinal gene therapy

“…Using the same strategy, subretinal injection of CK30-PEG NPs carrying the RDS gene induced sustained gene expression and partial rescue of photoreceptor degeneration in Rds +/− mice, as indicated by elevated ERG a- and b-wave amplitudes and improved retinal morphology compared to control Rds +/− retinas (injected with naked DNA) [85, 86]. Importantly, CK30-PEG NPs were found to be non-toxic to the retina [194], even when a second subretinal injection of the NPs was delivered 30 days after the first injection [195]. Considering the effectiveness of nucleolin-dependent endocytosis in gene transfer to photoreceptor cells, other forms of DNA-NPs can be formulated to potentially exploit this mechanism for cell internalization.…”