2022 Volume 45 Issue 11 Pages 1660-1668
Hereditary amyloidgenic transthyretin (ATTR) amyloidosis is caused by a genetic point-mutated transthyretin such as TTR Val30Met (TTR V30M), since it forms protein aggregates called amyloid resulting in the tissue accumulation and functional disorders. In particular, ATTR produced by retinal pigment epithelial cells often causes ATTR ocular amyloidosis, which elicits deterioration of ocular function and ultimately blindness. Therefore, development of novel therapeutic agents is urgently needed. Genome-editing technology using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas9) system is expected to be a therapeutic approach to treat genetic diseases, such as ATTR amyloidosis caused by a point mutation in TTR gene. Previously, we reported that glucuronylglucosyl-β-cyclodextrin conjugated with a polyamidoamine dendrimer (CDE) had excellent gene transfer ability and that underlying dendrimer inhibited TTR aggregation. Conversely, folate receptors are known to be highly expressed in retina; thus, folate has potential as a retinal target ligand. In this study, we prepared a novel folate-modified CDE (FP-CDE) and investigated its potential as a carrier for the retinal delivery of TTR-CRISPR plasmid DNA (pDNA). The results suggested that FP-CDE/TTR-CRISPR pDNA could be taken up by retinal pigment epithelial cells via folate receptors, exhibited TTR V30M amyloid inhibitory effect, and suppressed TTR production via the genome editing effect (knockout of TTR gene). Thus, FP-CDE may be useful as a novel therapeutic TTR-CRISPR pDNA carrier in the treatment of ATTR ocular amyloidosis.
Hereditary transthyretin (ATTR) amyloidosis is one of the intractable diseases caused by amyloidogenic TTR mutations (such as Val30Met (V30M)),1) and causes autonomic dysfunction, sensorimotor polyneuropathy, and gastrointestinal tract disorders.2) TTR is mainly produced from liver and exists as a tetramer in blood, dissociation of tetramer into a non-native monomer with low conformational stability can lead to amyloid formation, and subsequent insoluble amyloid deposition.3,4) In contrast to the systemic symptoms, typical ocular complications in ATTR amyloidosis include conjunctival vascular abnormalities, dry eye, pupillary abnormalities, vitreous opacity, and glaucoma, which are generally seen in both eyes.5–7) Vitreous opacity and glaucoma are the most common and most serious ocular complications, causing severe visual dysfunction. Because ATTR is also produced by retinal pigment epithelium (RPE) cells and becomes amyloid, the vitreous opacity due to amyloid deposition in the predilection sites, such as vitreous cavity as well as around ocular vessels, is often occurred. Amyloid deposition in the aqueous humor also causes elevated intraocular pressure, which eventually leads to glaucoma.8) Vitreous opacity caused by ATTR ocular amyloidosis can be cured by vitrectomy; nevertheless, the procedure may worsen glaucoma. In contrast, glaucoma is basically treated with drug therapy, however, in many cases, surgical treatment is used when it cannot be controlled. After surgical treatment, the intraocular pressure may be lowered for a while, but gradually, the filtering follicle becomes dysfunctional, leading to an increase in the intraocular pressure rising again. This is a poor outcome compared with other forms of glaucoma, and the continued production of ATTR may lead to amyloid deposition in the follicle and enhance the wound healing process.9)
In recent years, panretinal photocoagulation, in which laser beams are irradiated onto the retina to thermally coagulate the retina, has been performed in post-liver transplant patients to suppress intraocular amyloid production.10) This study showed that 30% reduction in RPE cells by photocoagulation significantly delayed the progression of vitreous opacity and glaucoma. However, panretinal photocoagulation may result in peripheral visual field loss and disruption of the blood–retinal barrier (BRB). Therefore, development of novel therapies for ATTR ocular amyloidosis is urgently needed.
An application of genome-editing technology to disease treatment has been expected in recent years. In particular, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas9) system is the latest tool for genome editing and has been applied to establishment of various genome-engineered animals and disease therapies, as it is faster and simpler than conventional technologies.11–13) Induction of the CRISPR-Cas9 system requires Cas9 (DNA-cleaving enzyme) and single-guide RNA (sgRNA; target DNA sequence recognition). Cas9 and sgRNA function as a complex and, after binding to a complementary sequence to sgRNA while moving to bump into genomic DNA, induce double-strand break (DSB) in the target sequence of genomic DNA.14) After induction of DSB, gene knockouts, point base substitutions and gene knock-ins can induce DNA repair mechanisms in the cell. There are three methods of introducing Cas9 and sgRNA into cells: 1) introduction of plasmid DNA encoding both, 2) co-transfection of two RNA molecules, Cas9 mRNA and sgRNA, and 3) direct introduction of a Cas9/sgRNA complex.15) In particular, plasmid DNA (pDNA) is the most widely used gene transfer technology because of its accessibility and economic efficiency.16,17) However, pDNA is not permeable to cell membranes and does not have nuclear transfer ability even if it is introduced into cells. Therefore, to apply pDNA encoding Cas9 and sgRNA for the treatment of ATTR ocular amyloidosis, it is essential to have a delivery carrier that can introduce pDNA into RPE cells, allow it to escape endosomes, and then transfer it to the nucleus to express encoded Cas9 and sgRNA.
RPE cells, a target cells for ATTR ocular amyloidosis therapy, highly express folate receptor-α (FR-α) on the basement membrane.18) Folic acid (FA), a ligand for FR-α, has the following advantages: 1) a water-soluble B vitamin with low antigenicity, 2) inexpensive, 3) high affinity to FR-α (dissociation constant; Kd = approx. 1 nM) and easy to internalize into RPE cells via FR-α-mediated endocytosis, and 4) small molecular weight, which may not affect intracellular kinetics of the FA-appended carrier.19–21)
In our laboratory, we prepared a novel conjugate of glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) with a polyamidoamine dendrimer (CDE) and reported its usefulness as a carrier for gene transfer.22) In addition, we also reported that dendrimer, a basis of CDE, inhibited amyloid formation and exhibited amyloid-breaking effects on ATTR.23) However, there are no previous studies applying CDE to delivery of TTR-CRISPR pDNA to retinal tissues. Therefore, modification of FA to CDE is expected to provide selective delivery to FR-α-expressing RPE cells. Indeed, FA-modified polyethyleneimine (PEI) efficiently delivered small interfering RNA (siRNA) to FR-α-expressing cancer cells.24) Based on these backgrounds, in this study, FA-modified CDE (FP-CDE) was newly synthesized by 1) polyethylene glycol (PEG)-mediated FA modification and 2) GUG-β-CyD modification via an amide bond to the dendrimer. Synthesized FP-CDE was used to prepare a complex with TTR-CRISPR pDNA. FP-CDE/TTR-CRISPR pDNA was evaluated for its usefulness in ATTR ocular amyloidosis treatment.
CDE was prepared according to a previously described method,23) using a polyamidoamine dendrimer (ethylenediamine core, generation 2.0) (Sigma-Aldrich Japan, Tokyo, Japan) and GUG-β-CyD (Ensuiko Sugar Refining, Tokyo, Japan). The number of GUG-β-CyD molecules attached to the CDE was determined by comparing the peak areas of the anomeric proton of GUG-β-CyD and the ethylene protons of the dendrimer in the 1H-NMR spectrum using an NMR spectrometer (JNM-α-500, JEOL, Tokyo, Japan). FP-COOH was prepared according to a previous method,25) using FA (Nacalai Tesque, Kyoto, Japan), N,N′-dicyclohexylcarbodiimide (NHS), N-hydroxysuccinimide, and SUNBRIGHT PA-020HC (Yuka Sangyo, Tokyo, Japan). FP-COOH (50 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (37.2 mg), and NHS (22.3 mg) were dissolved in borate buffer (0.2 M, pH 9, 1 mL) and stirred for 2 h. CDE (69.8 mg) was dissolved in borate buffer (0.2 M, pH 9, 1 mL) and stirred for 48 h at room temperature (r.t.) after addition of the solution containing activated FP-COOH. The product was dialyzed in H2O for 5 d (dialysis membrane; MWCO = 8000) and lyophilized to obtain FP-CDE.
Synthesis of TRITC-FP-CDETo prepare TRITC-modified FP-CDE, a NaCl solution (0.9% (w/v), 200 µL) containing FP-CDE (10 mg) and dimethyl sulfoxide (DMSO) (200 µL) containing tetramethylrhodamine (TRITC, 1 mg) (Thermo Fisher Scientific, Tokyo, Japan) were mixed, reacted at r.t. for 24 h, and purified by dialysis (dialysis membrane: MWCO = 1000).
Preparation of FP-CDE/pDNAVarious amounts of pDNA in Tris–ethylenediaminetetraacetic acid (EDTA) buffer were added to Hanks’ balanced salt solution (HBSS) buffer containing several amounts of FP-CDE. Subsequently, the sample solution was stirred using a vortex mixer for 10 s and incubated for 15 min before the experiments.
Agarose Gel ElectrophoresisTTR-CRISPR pDNA (0.5 µg) complexes with FP-CDE were prepared at various charge ratios (0–5) in HBSS. FP-CDE/TTR-CRISPR pDNA solution (10 µL) and 6x loading dye (2 µL) (Thermo Fisher Scientific) were mixed and the mixed solution was applied to 2% (w/v) agarose S (Nippon Gene, Tokyo, Japan). Agarose gel electrophoresis was performed in Tris-borate–EDTA (TBE) buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) at 100 V for 30 min. The gel was stained with TBE buffer containing ethidium bromide (r.t., 30 min), and an Amersham Typhoon scanner (FLA-9000; FUJIFILM, Tokyo, Japan) was used for TTR-CRISPR pDNA band visualization.
Serum Tolerance of FP-CDE/pDNAFP-CDE/TTR-CRISPR pDNA (1 µg pDNA) was incubated for 15 min at 37 °C in reaction buffer containing fetal bovine serum (FBS) (1 unit/µL). Agarose gel electrophoresis was performed as described previously.
Mean Diameter and ζ-PotentialsAfter preparation of the TTR-CRISPR pDNA (5 µg) complexes with FP-CDE (charge ratio = 5, 10, and 100) in aqueous solvent (1 mL), the particle sizes and ζ-potentials of FP-CDE/TTR-CRISPR pDNA were measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). Samples showing polydispersity of less than 0.5 were used in the analysis.
Cytotoxicity AssayHuman retinal pigment epithelial ARPE-19 cells were seeded in 96 well plates at 1 × 104 cells/well and cultured in 10% (v/v) FBS contained Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium for 24 h. After washing with serum-free medium, 20 µL solutions containing FP-CDE/TTR-CRISPR pDNA (0.5 g pDNA) complex (charge ratio = 0, 10, 50, and 100) and 80 µL serum-free medium were added and incubated at 37 °C for 6 h under 5% CO2. After washing with serum-free medium, cells were incubated in 10% (v/v) FBS contained DMEM/F12 medium for 18 h. After washing with 200 µL of HBSS, cellular viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) in accordance with the manufacturer’s protocol. Cell viability was calculated as 100% with non-FP-CDE treatment.
Flow Cytometric AssayFP-CDE/fluoresceion-pDNA (charge ratio = 100) was transfected to ARPE-19 cells with FA (1 or 2 mM) and incubated for 2 h. The fluorescence intensity of fluorescein-pDNA in the cells was determined by flow cytometry (Guava® easyCyte 6HT/2L, Millipore, Massachusetts). The mean fluorescence intensity was calculated.
Real-Time PCRARPE-19 cells were seeded in a 24-well plate at a density of 1 × 105 cells/well in DMEM/F12 with FBS (10%) for 24 h. After removing the medium, fresh medium containing TTR-CRISPR pDNA (2 µg) complex with FP-CDE (charge ratio = 100) was added for 6 h. The medium was then removed and the ARPE-19 cells were incubated for 2 or 3 d in a CO2 incubator. After collecting the culture supernatant for 2 or 3 d to measure the TTR protein level, total RNA in ARPE-19 cells was extracted using TRIzol® reagent (Invitrogen, Tokyo, Japan) in accordance with the manufacturer’s protocol. Reverse transcription (RT) and real-time PCR were carried out using a PrimeScript™ RT reagent kit (TaKaRa Bio, Shiga, Japan), SYBR® Premix Dimer Eraser™ (TaKaRa Bio), primers (Hokkaido System Science Co., Ltd., Hokkaido, Japan), and a Light Cycler 480® (Roche, Pennsburg, Germany). The comparative Ct method (2−ΔΔCt) was used to calculate the relative mRNA expression of human TTR/β-actin. The sequences of the forward and reverse primers for human TTR and β-actin are as follows:
To measure TTR protein levels in the culture supernatant of ARPE cells treated with FP-CDE/TTR-CRISPR pDNA by ELISA, the cell culture supernatant treated with the TTR-CRISPR pDNA (2 µg) complex with FP-CDE (charge ratio = 100) was collected as described above. The cell culture supernatant (15 µL) and coating buffer (1.59 g/L Na2CO3 and 2.93 g/L NaHCO3) (100 µL) were added to a 96 well plate, then incubated overnight at 4 °C. After washing three times with phosphate-buffered saline containing Tween 20 (PBS-T, 0.05% Tween, 100 µL), blocking buffer (0.5% gelatin in coating buffer) (100 µL) was added. After incubation (r.t., 1 h) and washing three times with PBS-T, polyclonal rabbit anti-human prealbumin (Dako, Tokyo, Japan) (1 : 1000 in PBS-T), a primary antibody against TTR, was added and incubated at 37 °C for 1 h. The cells were washed with PBS-T and polyclonal goat anti-rabbit immunoglobulins/horseradish peroxidase (HRP) (Dako, Tokyo, Japan) (1 : 5000 in PBS-T), as a secondary antibody against immunoglobulin G (IgG) and incubated at 37 °C for 1 h. After washing with PBS-T, SureBlue TMB Reserve (Funakoshi, Tokyo, Japan) was used to detect the HRP substrate in accordance with the manufacturer’s protocol, and the absorbance was measured (450 nm) using a microplate reader (Epoch, Wakenyaku Co., Kyoto, Japan). Calibration curves were prepared using recombinant WT TTR protein (Structural Biology Laboratory at Toyama University, Toyama, Japan).
Thioflavin-T (Th-T) AssayTo detect the inhibitory effects on TTR V30M amyloid formation, TTR V30M protein (20 µM) dissolved in glycine–HCl buffer (20 µL) was incubated for 6 h at 37 °C with or without TTR-CRISPR pDNA (0.1 µg), FP-CDE (0.1 mM), CDE (0.1 mM)/TTR-CRISPR pDNA (0.1 µg) (charge ratio = 100), and diflunisal (50 µM). The solutions (3 µL) were mixed with Th-T (10 µM) (Wako, Osaka, Japan) in glycine buffer (50 mM, 600 µL) and the fluorescence intensity at 489 nm (excitation wavelength 442 nm) was determined using a fluorescence spectrophotometer (F-4500, Hitachi High Technologies, Tokyo, Japan).
To break the TTR V30M amyloid, glycine–HCl buffer containing TTR V30M protein (20 µM) was incubated for 6 h at 37 °C. After incubation, The TTR V30M solution was added to TTR-CRISPR pDNA (0.1 µg), FP-CDE (0.1 mM), FP-CDE (0.1 mM)/TTR-CRISPR pDNA (0.1 µg) (charge ratio = 100), and TUDCA (50 µM)/doxycycline (50 µM). Th-T assay was performed as described above. In addition, to compare the disruption inhibition efficiency of CDE/FP-CDE and already known drug candidates, TTR V30M solution (20 µM) incubated for 6 h was treated with or without diflunisal (50 µM) and FP-CDE (0.1 mM)/TTR-CRISPR pDNA (0.1 µg) (charge ratio = 100), and a Th-T assay was performed.
In Vivo Retinal Migration of FP-CDE/pDNAAll animal experiments were performed with the approval of the Kumamoto University ethical committee (Approval ID: A2019-081). Under isoflurane anesthesia, BALB/c mice (male, 4 weeks old) were treated with HBSS (3 µL) containing TRITC-carrier/fluorescein-pDNA (0.2 µg pDNA, charge ratio = 100). After 30 min, the collected eyes were fixed in a 4% neutral buffered formaldehyde solution and immersed in a 20% sucrose solution for 48 h. After embedding, the eye was cut horizontally in a cryostat, and frozen sections were prepared. The retina was imaged using a fluorescence microscope (Biorevo BZ-9000, Keyence, Osaka, Japan).
In Vivo Gene Transfer Ability of FP-CDE/pDNABALB/c mice (male, 4 weeks old) were treated with HBSS (3 µL) containing pDNA encoding GL3 luciferase (pGL3) (0.2 µg pDNA) complexes with CDE or FP-CDE (charge ratio = 100). After 24 h, the collected eyes were added to 400 µL of reporter assay buffer (Promega, Madison, WIUA), and homogenized using a homogenizer (T25 basic from IKA Works). The samples were frozen and thawed three times, and then centrifuged at 10000 rpm for 10 min. The supernatant was used as a cell extract to determine luciferase activity using a luminometer (Lumat LB9507, EG&G BERHTOLD, Wildbad, Germany), and the relative light unit (RLU) was calculated.
Histological Observation of the RetinaBALB/c mice (male, 4 weeks old) were treated with TTR-CRISPR pDNA (0.2 µg pDNA) complexed with CDE or FP-CDE (charge ratio = 100). After 24 h, frozen sections of the collected eyes were prepared, as described above. Retina was imaged using a fluorescence microscope.
Data AnalysisData are presented as the mean ± standard error (S.E.). The significance test was performed using a Scheffe’s test with p-value of <0.05 and unpaired t-test p-value of <0.05.
The physicochemical properties of the FP-CDE/TTR-CRISPR pDNA complex are important factors that contribute to the efficiency of TTR-CRISPR pDNA transfection into cells. Therefore, we investigated the physicochemical properties of FP-CDE/TTR-CRISPR pDNA complex. Agarose gel electrophoresis was performed by adding FP-CDE to TTR-CRISPR pDNA at a charge ratio of 0–5 (Fig. 1A). The bands derived from TTR-CRISPR pDNA faded in a charge-ratio-dependent manner and disappeared at a charge ratio of 5, suggesting that FP-CDE forms a complex with TTR-CRISPR pDNA at charge ratios higher than 5. Next, we examined the effect of charge ratio on the stability of pDNA in serum (Fig. 1B). The pDNA-derived bands were attenuated by FBS treatment. In contrast, the bands were maintained in FP-CDE/TTR-CRISPR pDNA complex at all charge ratios, suggesting that FP-CDE/TTR-CRISPR pDNA is enzymatically stable. Next, the particle size and ζ-potential of FP-CDE/TTR-CRISPR pDNA complex were examined (Figs. 1C, D). FP-CDE/TTR-CRISPR pDNA exhibited particle sizes of approximately 160 and 170 nm and ζ-potentials of approximately 2 and 7 mV at charge ratios of 10 and 100, respectively. These results suggested that FP-CDE/TTR-CRISPR pDNA is a submicron-sized particle under physiological conditions.
(A) Agarose gel electrophoretic analysis of TTR-CRISPR pDNA complex with FP-CDE. These figures show the representative image for 3 experiments. (B) Effects of FP-CDE on electrophoretic mobility of TTR-CRISPR pDNA treated with FBS. These figures show the representative image for 3 experiments. (C) Particle size and (D) ζ-Potentials of FP-CDE/TTR-CRISPR pDNA. Each value represents the mean ± standard error (S.E.) of 3 experiments.
Carriers for gene delivery should exhibit as low cytotoxicity as possible. However, Aramaki et al. reported that cationic liposomes induced apoptosis in B cells and macrophages, and Fischer et al. also reported that cationic polymers such as polyethyleneimine (PEI) and dendrimers induced necrosis in mouse fibroblasts.26,27) Therefore, we investigated the cytotoxicity of FP-CDE on ARPE-19 cells using WST method. Figure 2A showed the cytotoxicity of FP-CDE/TTR-CRISPR pDNA complex in ARPE-19 cells after 24 h treatment with carrier/pDNA. Because the FP-CDE complex showed no cytotoxicity up to a charge ratio of 100, further experiments were performed at this ratio of 100. Next, to determine whether FP-CDE/CRISPR pDNA could express the protein encoded by pDNA, we transfected FP-CDE/CRISPR pDNA into ARPE-19 cells and investigated the intracellular expression of GFP encoded by CRISPR pDNA. While no intracellular expression of GFP was observed in the transfection of pDNA alone, GFP-derived fluorescence was observed in ARPE-19 cells transfected with FP-CDE/CRISPR pDNA (Fig. 2B). Next, we investigated the effect of FA, a FR-α competitive inhibitor, on cellular uptake of the FP-CDE complex in ARPE-19 cells using fluorescein-pDNA (Fig. 2C). The fluorescence intensity derived from fluorescein-pDNA was evaluated by flow cytometry after transfection with FP-CDE/fluorescein-pDNA in the presence of various concentrations of FA. As shown in Fig. 2D, the cellular uptake of FP-CDE/fluorescein-pDNA complex decreased in a folate-concentration-dependent manner. These results suggest that FP-CDE introduced pDNA into ARPE-19 cells via FR-α-mediated intracellular migration to induce the expression of protein encoded by pDNA.
(A) Cytotoxicity of FP-CDE/TTR-CRISPR pDNA in ARPE-19 Cells. (B) Observation of intracellular expression GFP encoded TTR-CRISPR pDNA in ARPE19 cells. These figures show the representative data for 3 experiments. (C, D) Effects of FA on cellular association of FP-CDE/Fluorescein-pDNA with ARPE19 Cells. (C) The figure shows the representative data for 9 experiments. (D) Each value represents the mean ± S.E. of 9 experiments. * p < 0.05, compared with without FA.
In this section, the suppressive effect of FP-CDE/TTR-CRISPR pDNA complex on TTR mRNA production in ARPE-19 cells was examined by real-time PCR. Figure 3A shows the inhibitory effect of FP-CDE/TTR-CRISPR pDNA on TTR mRNA production after transfection with FP-CDE/TTR-CRISPR pDNA. The CRISPR pDNA complex reduced TTR mRNA production compared with that in the control group. In addition, TTR levels in the culture medium were measured by ELISA. In FR-CDE/TTR-CRISPR pDNA treated cells, the concentration of secreted TTR protein in culture medium were decreased as well as TTR mRNA levels (Fig. 3B).
(A) Human TTR mRNA expression levels in ARPE19 cells and (B) concentration of secreted TTR protein in culture medium determined after transfection with FP-CDE/TTR-CRISPR pDNA. Each value represents the mean ± S.E. of 5–7 experiments. * p < 0.05, compared with FP-CDE/Control-CRISPR pDNA. (C, D) Human TTR protein levels in the culture medium (C) before and (D) after passaged ARPE19 cells. Each value represents the mean ± S.E. of 3 experiments. * p < 0.05, compared with carrier/control complexes.
Next, we investigated the genome-editing effects of FP-CDE/TTR-CRISPR pDNA complex on TTR suppression in ARPE-19 cells (Figs. 3C, D). Figure 3C shows the effects of TTR production after transfection with the FP-CDE/TTR-CRISPR pDNA complex. After 72 h of transfection, the culture supernatant (before passage) was collected, the cells were passaged, and after 72 h, the culture supernatant (after passage) was collected again. The amount of TTR protein in the recovered medium was evaluated using ELISA. Before passage, both TTR-siRNA and TTR-CRISPR pDNA inhibited the production of TTR protein (Fig. 3C). In contrast, after passaging, TTR-CRISPR pDNA treatment sustained suppressing TTR protein production, while the effect of siRNA disappeared (Fig. 3D). These results suggest that transfection with the FP-CDE/TTR-CRISPR pDNA complex may result in TTR gene knockout.
In Vitro Inhibitory and Breaking Effects of the FP-CDE/TTR-CRISPR pDNA Complex on TTRWe previously reported that dendrimer exhibits a two-step therapeutic effect on the TTR amyloidosis cascade. Dendrimer, a three dimensional (3D)-structural nanomaterial, which has a branched cationic polymer repeating poly(amidoamine) (PAMAM) units, showed inhibitory and breaking effects of TTR amyloid via electrostatic interaction and hydrogen bonding with TTR protein. In addition, we also reported that GUG-β-CyD inhibits TTR amyloid formation by hydrophobic interaction with aromatic amino acid residues of TTR protein. However, it is unclear whether FP-CDE alone and complexes have the therapeutic effects described above. Therefore, we next examined the effects of FP-CDE/TTR-CRISPR pDNA on both inhibiting TTR amyloid formation and amyloid breaking effect were investigated using a thioflavin-T method. Hereafter we used diflunisal as a positive control for inhibiting TTR amyloid formation since it has a nonsteroidal anti-inflammatory drug with inhibitory activity in TTR amyloidosis.28) In addition, Tauroursodeoxycholic acid (TUDCA)/Doxycycline is also utilized as a positive control in amyloid breaking experiments, since it has an amyloid break function and is in the clinical trial phase.29) FP-CDE alone significantly decreased the fluorescence intensity of the thioflavin-T dye, suggesting that it significantly inhibited amyloid formation (Fig. 4A). Furthermore, the FP-CDE/TTR-CRISPR pDNA complex also showed amyloid breaking effect (Fig. 4B). These results suggest that FP-CDE/pDNA inhibits TTR amyloid formation and breakage.
(A, B) TTR V30M alone shows untreated control with sample and its fluorescence intensity is set at 100%. Each value represents the mean ±S.E. of 6 experiments. * p < 0.05, compared with control. † p < 0.05, compared with TTR-CRISPR pDNA. ‡ p < 0.05, compared with FP-CDE.
In this study, we evaluated the usefulness of FP-CDE as a carrier for CRISPR/Cas9 pDNA delivery to the retina for ATTR ocular amyloidosis treatment. FP-CDE complex with CRISPR/Cas9 expression vector targeting TTR (TTR-CRISPR pDNA) was prepared, and its gene transfer ability and inhibitory effect on TTR production in ARPE cells were examined in vitro.
First, we confirmed the ability of FP-CDE to form a complex with TTR-CRISPR pDNA, and the physicochemical properties of the FP-CDE/CRISPR pDNA complex (Fig. 1). It is generally known that gene–polymer complexes are formed by spontaneous condensation via electrostatic interactions between negatively charged genes and positively charged polymers.30) FP-CDE forms a complex with TTR-CRISPR pDNA via electrostatic interactions because the core dendrimer molecule has a high-density primary amine group on its surface (Fig. 1A). FP-CDE/TTR-CRISPR pDNA showed a charge-ratio-dependent decrease in particle size and increase in ζ-potential (Figs. 1C, D). This phenomenon is similar to that observed in previous studies, suggesting that the condensation of the pDNA results in a decrease in size and an increase in ζ-potential due to the dense assembly of the CDE.23)
In the no cytotoxicity concentration of FP-CDE/TTR-CRISPR pDNA up to a charge ratio of 100 (Fig. 2A), we examined the effect of FA on the intracellular uptake of FP-CDE/TTR-CRISPR pDNA, as FR-α expression has been reported on the basement membrane side of ARPE-19 cells (Figs. 2C, D). Cellular uptake of this complex was significantly decreased by the addition of FA, suggesting that this complex is taken up by ARPE-19 cells through FR-α-mediated endocytosis. Reddy et al. reported that folate-modified carrier/gene complexes of approximately 150 nm or less were efficiently taken up into the cell by FR-mediated endocytosis.31) The complex used in this study exhibited the particle size of approximately 170 nm (Fig. 1C). However, the high polydispersity (PDI) of approximately 0.22 (data not shown) suggests that there are complexes with a particle size of 150 nm or less, and that these particles are incorporated into the cells by FR-mediated intracellur uptake.
Next, we examined the suppressive effect of FP-CDE/TTR-CRISPR pDNA on TTR expression in ARPE-19 cells and found that this complex had significantly higher suppressive effect on TTR production than the control-CRISPR pDNA complex (Figs. 3A, B). We previously reported that the gene transfer efficiency of α-CDE increased in a charge ratio-dependent manner and that free α-CDE, not complexed with pDNA, was responsible for gene transfer efficiency.32,33) Therefore, it is possible that free carriers contribute to the gene transfer effect of FP-CDE.
We examined whether the suppressive effect of FP-CDE/TTR-CRISPR pDNA on TTR production was due to DSB-induced knockout of the target sequence of the TTR gene. The FP-CDE complex with TTR-siRNA or TTR-CRISPR pDNA complex suppressed TTR expression in the culture supernatant before passaging (Fig. 3C). Furthermore, after passaging, the TTR-CRISPR pDNA complex still had inhibitory effect on TTR production, whereas the TTR-siRNA complex lost its transient effect (Fig. 3D). Thus, cells treated with the TTR-CRISPR pDNA complex retained this trait after passaging. These results suggested that the TTR-CRISPR pDNA complex might suppress TTR production by knocking out the TTR target gene (Figs. 3C, D). Currently, it has been reported that photocoagulation to thermally coagulate the retina in patients with TTR ocular amyloidosis delays the progression of vitreous opacity and glaucoma when RPE cells are reduced by 30%. Therefore, the 30% inhibition of TTR production by FP-CDE/TTR CRISPR pDNA may have a potential as a clinical use.
Local siRNA therapy for ATTR ocular amyloidosis is currently being intensively investigated. The RNAi effect of siRNAs has a rapid medicinal effect. However, because the effect is transient, continuous siRNA administration is required. In addition, because of the various barrier functions in the eye (e.g., blood–ocular barrier, blood–vasal barrier, and tear fluid barrier), topical ocular siRNA therapy has been investigated using vitreous injections. However, vitreous injection is considered invasive and may cause several side effects, such as increased intraocular pressure and cataracts. However, it is necessary to examine the effect of genome editing on TTR target gene knockout by DNA sequencing and T7E assay.
FP-CDE/TTR-CRISPR pDNA exhibited inhibitory effect on TTR V30M amyloid formation and breaking effect on the TTR amyloid. In addition, the therapeutic effect of FP-CDE/TTR-CRISPR pDNA was significantly higher than that of FP-CDE (Fig. 4). We reported that dendrimers not only inhibited TTR amyloid by interacting with TTR through hydrogen bonds but also reduced TTR amyloid by reduction of β-sheet structure in TTR amyloid.24) We also reported that simply mixing cationic PEG and protein prevented protein aggregation.34) Therefore, it is possible that FP-CDE alone and the TTR-CRISPR pDNA complex may also have inhibited TTR aggregation and reduced the β-sheet structure in TTR via the interaction of TTR with dendrimers and PEG. Although the factors that contributed to the higher TTR amyloid inhibitory and breaking effect of the FP-CDE/TTR-CRISPR pDNA complex compared to the FP-CDE alone are unknown, it is possible that the complexation of FP-CDE increased the steric hindrance to TTR and clustered FP-CDE molecules to enhance their electrostatic and hydrophobic interactions. Further studies using Biacore and quartz crystal microbalances are needed to elucidate the interaction between the FP-CDE/TTR-CRISPR pDNA complex and TTR in more detail.
In order to show the efficacy of FP-CDE/TTR-CRISPR in vivo, the administered FP-CDE/TTR-CRISPR must reach the retinal tissue of mice. In addition, to accurately evaluate the in vivo therapeutic effect of FP-CDE/TTR-CRISPR, it is necessary to administer FP-CDE/TTR-CRISPR to the TTR ocular amyloidosis pathological model and evaluate the drug effect. In order to investigate the in vivo genome editing effects of FP-CDE/TTR-CRISPR pDNA in the future, we evaluated the retinal migration of FP-CDE/pDNA administered by eye drop administration as a preliminary study. In order to easily and visually observe the retinal migration of CDE and pDNA, we used the tetramethylrhodamine (TRITC), a fluorescence probe, modified FP-CDE complex with fluorescein-pDNA (charge ratio = 100) was eye drop administration to BALB/c mouse eyes, and the retinal migration of TRITC-FP-CDE/fluorescein-pDNA complexes was examined. Supplementary Fig. 1A shows the intraocular behavior of TRITC-CDE or TRITC-FP-CDE complexed with fluorescein-pDNA by eye drop administration in mice. For retinal migration evaluation, 30 min after eye drop administration of TRITC-FP-CDE/fluorescein-pDNA, frozen sections of collected eyes were observed under a fluorescence microscope. Although the red fluorescence derived from TRITC-CDE was weak, that derived from FP-CDE was clear in the retina. In addition, the images of fluorescein-pDNA were comparable to those of TRITC carriers, and the merged image of FP-CDE/pDNA was clearly observed. On the other hand, some part of fluorescein-pDNA was observed in photoreceptor cells (Supplementary Fig. 1A). Therefore, further carrier optimization to deliver pDNA to RPE cells with high cell selectivity is necessary. Next, to evaluate the gene transfer activity of FP-CDE/TTR-CRISPR pDNA in healthy mice, a luciferase assay was performed using the pDNA encoding luciferase as a model gene. The luciferase gene expression levels in the whole eye were measured 24 h after eye drop administration of the carrier/pDNA solution (Supplementary Fig. 1B). FP-CDE showed approximately 5-fold higher gene transfer effect in the whole eye than CDE. These results suggest that FP-CDE is more useful than CDE as a carrier for gene transfer in the eye. Several reports show that FR-α is expressed in RPE cells and photoreceptor cells,35,36) but TTR is only expressed in RPE, so it is assumed to be selective in its therapeutic effect. In the future, a larger model such as the rabbit, which is commonly used in ocular therapy, should be used to isolate each layer for a detailed study of gene transfer sites. In addition, FR-α is highly expressed on the basement membrane side of RPE cells.31) Therefore, it is inferred that the FP-CDE/pDNA complex is taken up by RPE cells via the choroid and through FR-α-mediated intracellular migration from the basement membrane side of RPE cells, resulting in efficient gene transfer. Luong et al. demonstrated that folate-modified dendrimer nanoparticles (particle size; 160 nm, ζ-potential; 10 mV) are taken up via FR-α-mediated endocytosis.37) Importantly, FP-CDE/TTR-CRISPR pDNA has similar physicochemical properties (particle size; 170 nm, ζ-potential; 8 mV). Therefore, FP-CDE/TTR-CRISPR pDNA is considered to be introduced into the cells via FR-α-mediated endocytosis. As a result, The FP-CDE could migrate to the retina significantly more than folate-unmodified CDE/pDNA, and has a significantly higher gene transfer ability than CDE. In the future, based on the FP-CDE/TTR-CRISPR profiles obtained by these in vivo experiments, the in vivo therapeutic effects of FP-CDE/TTR-CRISPR will be evaluated using the animal model of TTR ocular amyloidosis. Next, pDNA polyplexes with CDE and FP-CDE were treated with eye drop administration to healthy mice, and 24 h later, the eyes were collected for histological evaluation using hematoxylin–eosin (H&E) staining (Supplementary Fig. 2). Histological observations of the retina showed that the pDNA complex with CDEs had little effect on the histological structure of the retina, suggesting that FP-CDE/pDNA may not induce retinal damage in healthy mice 24 h after administration. To evaluate further safety, long term safety study should be necessary.
We are currently preparing to investigate the in vivo genome editing effects of FP-CDE/TTR-CRISPR pDNA using the TTR ocular amyloidosis model. The TTR profile must be observed over a long period of time to determine whether genome editing continues to decrease the production of TTR. In addition, a longer period of time may be required to confirm the safety of genome editing. Therefore, we are proceeding with the most up-to-date attention on the in vivo genome editing effects of FP-CDE/TTR-CRISPR pDNA.
In conclusion, FP-CDE/TTR-CRISPR pDNA was taken up by RPE cells via FR-α-mediated endocytosis, suggesting that it may have a suppressive effect on TTR production by knocking out TTR. Based on the in vivo experiments showing the excellent safety of FP-CDE/TTR-CRISPR pDNA, FP-CDE is expected to be applied as the TTR-CRISPR pDNA retinal delivery carrier for treatment of ATTR ocular amyloidosis. The findings obtained in this study provide useful basic data for the development of novel ATTR ocular amyloidosis therapies using genome editing technology.
The authors declare no conflict of interest.
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