DNA nanoparticles are safe and nontoxic in non-human primate eyes

Abstract

Introduction: DNA nanoparticles (NPs) comprising polylysine conjugated to polyethylene glycol efficiently target murine photoreceptors and the retinal pigment epithelium (RPE) and lead to long-term phenotypic improvement in models of retinal degeneration. Advancing this technology requires testing in a large animal model, particularly with regard to safety. So, herein we evaluate NPs in non-human primates (baboon).

Methods and results: NPs with plasmids carrying GFP and a ubiquitous, RPE-specific, or photoreceptor-specific promoter were delivered by either subretinal or intravitreal injection. We detected GFP message and protein in the retina/RPE from eyes dosed with NPs carrying ubiquitously expressed and RPE-specific vectors, and GFP message in eyes injected with NPs carrying photoreceptor-specific vectors. Importantly, we observed NP DNA in the retina/RPE following intravitreal injection, indicating the inner limiting membrane does not prevent NP diffusion into the outer retina. We did not observe any adverse events in any baboon, and there were no NP-associated changes in retinal function. Furthermore, no systemic or local inflammatory reaction to the vectors/injections was observed, and no NP DNA was found outside the eye.

Conclusion: Taken together with the well-established rodent safety and efficacy data, these findings suggest that DNA NPs may be a safe and potentially clinically viable nonviral ocular therapy platform for retinal diseases.

Keywords: DNA nanoparticles; baboon; gene therapy; non-human primate; nonviral gene transfer; ocular gene transfer; safety.

Figure 1

GFP expression after a single subretinal or intravitreal injection of NP-CBA-GFP in baboon eyes. Notes: Tissues were harvested from animals subretinally or intravitreally injected with either naked CBA-GFP, NP CBA-GFP, or vehicle (saline). (A) Expression of GFP message by qRT-PCR in the anterior segment (cornea and lens, left) and posterior segment (retina+PECS and optic nerve, right). GFP values were normalized to actin. Each eye is shown as an individual symbol with NPs in red, naked DNA in blue, saline in yellow, and subretinal/intravitreal in circles/squares, respectively. Legend indicates animal #/eye. (B) Western blot analysis for GFP protein (or actin as a loading control) in lysates (50 µg/lane) from retina+PECS. Retinal lysates from GPI-GFP transgenic mice were used as a positive control. Abbreviations: IV, intravitreal injection; Nak, naked plasmid; NP, nanoparticle; PECS, pigment epithelium, choroid, and sclera; qRT-PCR, quantitative reverse transcription polymerase chain reaction; Sal, saline; SR, subretinal injection.

Figure 2

GFP expression after a single subretinal injection of VMD2-GFP-S/MAR in baboon eyes. Notes: Tissues were harvested at the times indicated in Table 1 from animals subretinally injected with naked VMD2-GFP or NP VMD2-GFP (or vehicle). (A) Expression of GFP message was assessed by qRT-PCRCR in the RPE and retina from animal #5 (naked DNA, blue circles), animal #1 (uninjected, black triangles), and animal #2 (saline, yellow circles). (B) In subsequent animals, qRT-PCR for GFP was performed on RPE tissue isolated from various quadrants after a single subretinal injection. GFP values were normalized to β-actin. Each eye is shown as an individual symbol. Symbols are consistently colored from quadrant to quadrant; legend indicates animal #/eye. Black lines indicate mean. Abbreviations: Nak, naked plasmid; NP, nanoparticle; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RPE, retinal pigment epithelium; Sal, saline.

Figure 3

GFP is expressed in the RPE after delivery of VMD2-GFP-S/MAR. Notes: (A–E) Immunohistochemistry was performed on retinal sections with antibodies against GFP (A, B, E), RPE65 as a control for RPE labeling (C), or with secondary antibodies alone (D). The purple coloring indicates GFP/RPE65 antibody labeling with nuclei counterstained with methyl green (teal). White arrowheads indicate expression in the RPE. (E) Images at 20× from a region not expressing GFP (left) and a region expressing GFP (right). Animal/eye numbers are as follows: (A, C, D, E) 7R, (B) 7L. Scale bar: 25 µm. Abbreviations: INL, inner nuclear layer; IS, inner segment; Nak, naked plasmid; NP, nanoparticle; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium; Sal, saline; UN, uninjected.

Figure 4

RK-GFP DNA was detected in the retina and RPE. Notes: Retinas and RPE were harvested from animals subretinally (two-site or one-site) or intravitreally injected with NP RK-GFP or saline. (A) Expression of GFP message as measured by qRT-PCR in the four quadrants of eyes receiving two-site subretinal injections (triangle), one-site subretinal injections (circle), or intravitreal injections (square). Colors correspond to individual eyes and are the same in (B). Black lines indicate mean. (B) Relative levels of RK-GFP DNA were measured in injected eyes by qPCR in the retina (closed symbols) and RPE (open symbols). Black bar indicates mean. Legend indicates animal #/eye. Abbreviations: IV, intravitreal injection; SR, subretinal injection; NP, nanoparticle; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RPE, retinal pigment epithelium.

Figure 5

Intraocular delivery of nanoparticle DNA caused a modest decrease in retinal function. Notes: Animals from group 2 (Tables 1 and S2) underwent electroretinography immediately before injection and 45 days after injection. (A, C) Shown are representative (A) scotopic and (C) photopic wave forms before (blue) and after (black) injection. Arrows indicate the places where the A-wave was measured, while the arrowheads indicate the places where the B-wave was measured. (B, D) Graphical representation of the percent change in maximum (B) scotopic and (D) photopic amplitudes. Black lines indicate mean. Colors correspond to individual eyes; legend lists animal #/eye. *P<0.05, ns = nonsignificant in one-way Student’s t-test testing the hypothesis that the mean % difference is different from 0 (ie, that there is significant change from baseline). Abbreviations: IV, intravitreal injection; SR, subretinal injection.

Figure 6

Inflammatory cytokines are not upregulated by NP injection in the baboon eye. Notes: Aqueous humor and serum were collected before the injections (baseline, filled symbols) and at the endpoint (postinjection, open symbols; time as indicated in Table 1). Samples were assayed for levels of IL-6, IFN-γ, IL-1β, or TNF-α protein using a non-human primate cytokine magnetic bead panel kit. Each eye is shown as an individual symbol with NPs in red, naked DNA in blue, saline in green, uninjected/positive control in black, and subretinal/intravitreal injection in circles/squares, respectively. Serum from a dengue fever virus–infected baboon was used as a positive control (diamond) and an uninjected animal was used as a negative control (triangle). (A) Graphical representation of data obtained from the aqueous humor of animals that received subretinal injections. (B) Graphical representation of data obtained from the aqueous humor of animals that received intravitreal injections. (C, D) Graphical representation of data obtained from the serum of animals that received subretinal injections in groups 1 and 2, respectively. Abbreviations: IFN, interferon; IL, interleukin; IV, intravitreal injection; Nak, naked plasmid; NP, nanoparticle; SR, subretinal injection; TNF, tumor necrosis factor.

Figure 7

No RK-GFP DNA is detected in the brain. Notes: The optic nerve, lateral geniculate nucleus, and visual cortex were harvested from animals subretinally (two-site or one-site) or intravitreally injected with NP RK-GFP or saline at PI-45 days. Relative levels of GFP DNA were assessed by qPCR. Colors correspond to individual eyes; legend lists animal #/eye. The positive control is optic nerve DNA spiked with naked RK-GFP (black diamond). Abbreviations: IV, intravitreal injection; NP, nanoparticle; PI, postinjection; qPCR, quantitative polymerase chain reaction; RPE, retinal pigment epithelium; SR, subretinal injection.