Effective gene therapy of Stargardt disease with PEG-ECO/ pGRK1-ABCA4-S/MAR nanoparticles

Abstract

Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 ^-/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 ^-/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).

Keywords: ABCA4; ECO; MT: Delivery strategies; Stargardt disease; inherited retinal disorders; nanoparticles; non-viral gene therapy.

Graphical abstract

Figure 1

Formulation and characterization of ECO plasmid DNA nanoparticles (A) Schematic of self-assembly formulation of ECO/plasmid DNA nanoparticles. (B and C) Flow cytometry histograms of GFP expression in ARPE-19 cells 48 h after transfection with EM-PEG-ECO/pCMV-GFP and AAV2-CMV-GFP (B) and fluorescence images of retinal flat mounts of GFP expression in BALB/c mice 4 months after a subretinal injection of EM-PEG-ECO/pCMV-GFP and AAV2-CMV-GFP (C). (D) Diagram of the therapeutic pGRK1-ABCA4-S/MAR plasmid construct and confirmation of the structure by restriction enzyme digestion agarose gel electrophoresis. (E and F) Characterization of different ECO/pGRK1-ABCA4-S/MAR nanoparticle formulations by agarose gel electrophoresis (E) and DLS of size and zeta potential distribution (F). (G) qRT-PCR of ABCA4 mRNA expression in Abca4^−/− mice 1 week after subretinal treatment with (1) ECO/pGRK1-ABCA4-S/MAR, (2) ECO/pGRK1-ABCA4-S/MAR (10% sucrose), (3) PEG-ECO/pGRK1-ABCA4-S/MAR, and (4) PEG-ECO/pGRK1-ABCA4-S/MAR (10% sucrose). The mRNA expressions were normalized to that by PEG-ECO/pGRK1-ABCA4-S/MAR. (H–K) Characterization of in vivo gene expression of PEG-ECO/pGRK1-ABCA4-S/MAR in Abca4^−/− mice with different injection routes (intravitreal versus subretinal) (H), injection volume (0.5 or 1 μL, expression normalized to 0.5 μL) (I), injection dose (25, 50, or 100 ng/eye, expression normalized to 50 ng/eye) (J), and age at treatment (K). Analyses were done by qRT-PCR of ABCA4 mRNA expression 1 week or 1 month, as indicated, after single treatment of nanoparticles (100 ng/eye, 0.5 μL). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005.

Figure 2

Treatment efficacy after a single dose of 50 ng or 100 ng/eye with PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles in Abca4^−/− mice (A) Timeline of treatment with PEG-ECO/pGRK1-ABCA4-S/MAR in Abca4^−/− mice. (B and C) ABCA4 expression distribution in the retina of Abca4^−/− mice after subretinal treatment with ECO/pGRK1-ABCA4-S/MAR nanoparticles (200 ng/μL, 1 μL) (B) and PBS (C) for 7 days. Analysis was conducted on confocal images of immunohistochemistry (IHC) staining with an ABCA4 antibody (ABCA4 expression is shown in white). (D) Fluorescence IHC staining of ABCA4 in the retina (red, RPE65; green, ABCA4; blue, DAPI) of Abca4^−/− mice 4 months after a single treatment of PBS and PEG-ECO/pGRK1-ABCA4-S/MAR (50 ng or 100 ng/eye). (E) qRT-PCR of ABCA4 mRNA expression in Abca4^−/− mice 4 months and 1 year after treatment. (F and G) HPLC chromatograms (F) and UV spectra (G) of the A2E standard and A2E from eye samples of 1-year old Abca4^−/− mice, where the A2E peak was identified with the same elution time as the A2E standard. (H and I) Representative HPLC chromatograms of A2E (H) and the A2E levels quantified from HPLC relative to the control (I) 4, 8, and 12 months after a single treatment with PEG-ECO/pGRK1-ABCA4-S/MAR (50 ng or100 ng/eye) in Abca4^−/− mice (∗p < 0.05, ∗∗p < 0.01).

Figure 3

Gene expression and therapeutic efficacy of repeated dosing of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles in Abca4^−/− mice (A) Timeline of repeated injections of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles. (B) Time course of ABCA4 mRNA expression, analyzed by qRT-PCR after 1 injection of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles (100 ng/eye) in Abca4^−/− mice. (C and D) Fluorescence IHC staining of ABCA4 (green) and RPE65 (red) of retina sections from Abca4^−/− mice that received 1, 2, and 3 subretinal injections of PEG-ECO/pGRK1-ABCA4-S/MAR (100 ng/eye; blue, DAPI) (C) and of the whole retina after 3 injections (D). (E) qRT-PCR of ABCA4 mRNA expression in Abca4^−/− mice that received 1, 2, and 3 injections of PEG-ECO/pGRK1-ABCA4-S/MAR. (F) Representative HPLC chromatograms of A2E in the eyes of Abca4^−/− mice treated with PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles in comparison with that in control mice. (G) Quantitative comparison of A2E levels in treated Abca4^−/− mice relative to control mice. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001.

Figure 4

Safety of subretinal injection of PEG-ECO/DNA nanoparticles (A and B) Scotopic electroretinogram (ERG) responses of BALB/c mice 1 month after subretinal injection of ECO/pGRK1-ABCA4-S/MAR nanoparticles (A) and of Abca4^−/− mice 10–10.5 months after subretinal injection of a single dose (100 ng/eye) or 3 doses (every 3 months, 100 ng/eye) of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles (B) at a test light intensity of 1.6 log(cd × s × m⁻²). (C) Eye morphology, demonstrated by scanning laser ophthalmoscopy (SLO), of Abca4^−/− mice that received multiple treatments of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles 7 months after the initial treatment (control, untreated). (D) Eye morphology (SLO) of BALB/c mice treated with EM-PEG-HZ-ECO/pCMV-GFP nanoparticles and AAV2-CMV-GFP (dose, 5 × 10⁹ gene copies; control, untreated) 1, 2, and 3 months after treatment (large white area, potential inflammation). (E and F) PEG antibody analysis of anti-PEG IgM (E) and anti-PEG IgG (F) levels from the blood of Abca4^−/− mice that received multiple treatments of PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.0001.