CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Keywords: AAV; CRISPR; gene therapy; glycolysis; hypoxia inducible factor; metabolic reprogramming; prolyl hydroxylase; rejuvenation; retinal degeneration; therapeutic editing.

Graphical abstract

Figure 1

PHD inhibitor enhances photoreceptor survival and preserves retinal function by increasing retina glycolysis Pde6β^H620Q/H620Q mice were fed the PHD inhibitor, FG-4592, at one dose every 2 days from P5. (A) Metabolites were extracted from the retinas of mice at P23. ¹³C-labeled glycolytic intermediates increased in retinas from mice in the treatment group compared to those in the control group. Minimal effects were detected in TCA-cycle intermediates and mitochondrial oxidation metabolites, shown here as treated vs. control. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01; n = 5–6 per group. (B) H&E staining was performed to analyze retinas from the Pde6β^H620Q/H620Q mouse model at P35, with a representative section comparing the treatment group and control group at 500 μm from the optic nerve head. The green bar indicates the outer nuclear layer (ONL). Scale bar, 25 μm. (C) Spider plot analysis of ONL thickness. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n = 6 per group. (D) Pde6β^H620Q/H620Q mice were subjected to three types of serial ERG recordings—scotopic (rod-specific), maximal (rod and cone), and photopic (cone-specific)—at P28. Single tracings of the three-step ERG are shown. Blue represents the ERG tracing from a mouse with FG4592, while the gray tracing is from sham-fed control. (E) Analysis of the amplitude of b wave as shown in (D). Each dot or diamond represents the average ERG amplitude of both eyes per mouse. Error bars indicate SEM. ∗p ≤ 0.05; n = 6 per group.

Figure 2

PHD deficiency up-regulates key regulators of glycolytic metabolism in photoreceptors Experimental (PHD^−/−;Pde6β^H620Q/H620Q;Pde6γ^CreERT2/+) and control (PHD^FL/FL;Pde6β^H620Q/H620Q;Pde6γ^CreERT2/+) mice were treated with tamoxifen or sham solution, respectively, for three consecutive days (P8, P9, and P10). Mice were sacrificed at postnatal 3 weeks, and retinas were collected and snap-frozen in liquid nitrogen until further processing. (A) mRNA expression of Hif1a and Hif2a and the downstream glycolytic targets (Glut1, Glut2, Glut3, Glut5, Hk1, Hk2, Pfkp, Pfkm, Pfkl, Aldoa, Pgk1, Eno1, Pkm1, 2, Ldha, Ldhb, and Pdk1) were quantified to assess changes before and after PHD was ablated. β-Actin was used as the internal control. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001; n = 4–5 per group. (B) Representative immunoblots of glycolytic metabolism enzymes and regulators in the retinas of treated and untreated mice at P21 (before the onset of degeneration) to detect HIF1A, HIF2A, GLUT1, HK2, and LDHA protein levels. β-Actin was used as a loading control. Membrane was stripped and reprobed for all targets. (C) Quantitative analysis of protein levels shown in (B). Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01; n = 3 per group.

Figure 3

Loss of PHD enhances glycolysis and diminishes mitochondrial oxidation in PDE6-deficient retinas Eyes from control PHD^fl/fl and PHD^−/− (homozygous for Pde6β^H620Q/^H620Q) mice were dissected at P21 before retinal degeneration. Retinas were isolated under ambient illumination, incubated with 5 mM [U-¹³C]glucose, and harvested at 30-s and 90-s time points. Metabolites were extracted with 80% methanol, derivatized, and quantified by GC-MS. Flux of carbons from [U-¹³C]glucose through glycolysis is faster in the PHD-deficient retinas, and TCA-cycle activity is slower than controls. The bar graphs in the upper-right section show that the average rates over 40 min at which glucose is consumed and at which lactate is exported are not significantly different between experimental and control retinas. Axis units are indicated in the center field, with the y axis representing picomoles or micrograms of protein and the x axis representing time (seconds). Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01; n = 6–9 per group.

Figure 4

Upregulation of glycolysis through ablation of PHD enhances cone survival in a preclinical Pde6β^H620Q/H620Q RP model PHD deficiency improves rod and cone survival in the Pde6β^H620Q/H620Q mutant background. Experimental (PHD^−/−;Pde6β^H620Q/H620Q;Pde6γ^CreERT2/+) and control (PHD^FL/FL;Pde6β^H620Q/H620Q;Pde6γ^CreERT2/+) mice were treated with tamoxifen or sham solution for three consecutive days (P9, P10, and P11). (A–C) Analysis of amplitudes of electroretinogram tracings comparing experimental and control mice. Mice were subjected to three types of serial ERG recordings: scotopic rod (A), maximal rod and cone (B), and photopic cone (C) at 6, 8, and 10 weeks postnatally. Experimental mice with precise PHD1,2,3 ablation in rod photoreceptors are shown in red, while the signal from control mice is in gray. The average ERG amplitude of both eyes per mouse was used for analysis. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001; n = 5–6 in each group per time point. (D) Representative H&E-stained central retinal sections from experimental and control mice at 4 and 6 weeks at a distance of 500 μm from the optic nerve head. Yellow bars indicate ONL thickness. Scale bars, 25 μm. (E) Spider plot analysis of ONL thickness at 4 weeks. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n = 6 per group. (F) Spider plot analysis of ONL thickness at 6 weeks. Error bars indicate SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n = 6 per group. (G) Peanut agglutinin (PNA) staining of cones in 12-week-old PHD^fl/fl control and PHD^−/− (deficient) mice. The inset shows representative central retinal cones. (H) Bar chart of cone cell counts, quantified by percent area of the total area measured in both groups in (G). Error bars indicate SEM. ∗∗p ≤ 0.01; n = 3 in each group.

Figure 5

Ablation of PHD2 with gRNAs is sufficient for Hif1a augmentation and target engagement in vitro (A) Schematic summary of the outcomes produced by gRNA therapeutic deletion. The gRNAs used target exon 1 in human PHD2 sequence. (B and C) Ninety-five percent of non-homologous end-joining insertions and deletions (indels) resulted in a frameshift mutation in both mouse N2A (B) and human HEK293 (C) cells. Indels that are indivisible by 3 represent a successful insertion of a frameshift-mediated stop codon, truncating gene function. Unsuccessful edits are indicated with an asterisk and account for approximately 5% of the cases in both N2A (B) and HEK293 (C) cells. (D) Immunoblotting was performed, and the level of PHD2 from mouse N2A cells with control (PX459) or plasmids containing gRNAs targeting PHD2 was determined. β-Actin was used as a loading control. (E) Immunoblot was performed, and the level of PHD2 from human HEK293 cells with control (PX459) or plasmids containing gRNAs targeting PHD2 was quantified. β-Actin was used as a loading control. After transfection with scramble gRNAs or gRNAs targeting PHD2, HEK293 cells were cultured in standard media. Immunoblots revealed lower levels of PHD2 after gRNA therapeutic gene deletion of PHD2 compared to scramble gRNA controls. (F) Increased expression of LDHA, PDK1, GLUT1, and GLUT3 (glycolytic markers of the Warburg effect) after ablation of PHD2 in normoxic conditions. Control transduced with plasmids containing scramble gRNAs. Error bars indicate SEM. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n = 3 in each group.

Figure 6

Approximately 35% of CD73⁺ rod photoreceptors were transduced by AAV8::hGRK1-GFP (A–C) Mouse retinas receiving a subretinal injection (A–C) of AAV8:hGRK1-GFP for 1 month were dissociated, labeled with the rod photoreceptor marker anti-CD73, and analyzed by flow cytometry. BL6 wild-type mice were used here as a control. (A) Representative results in control mice with no injection and (B and C) in two injected mice. The GFP⁺/CD73⁺ cells are gated with the visible blue square and labeled as “P3”; the GFP⁻/CD73⁺ cells were gated with the red square and labeled as “P4.” x axis, CD73; y axis, GFP.

Figure 7

AAV8::U6-gRNAs_PHD2 CRISPR-Cas9 therapy improves photoreceptor function and structure in Pde6β^H620Q/Cas9 arRP and Rho^C110R/+ adRP mouse models Experiments were conducted using Pde6β^H620Q/Cas9 arRP (A–C) and Rho^C110R/+ adRP (D–H) mouse models. (A) Injection with a single dual-vector, AAV8:U6-gRNAs_PHD2;hGRK1-GFP, allowed marking of ventral subretinal transduction sites. Cas9 expression was transgenic in this line. (B) Representative global electroretinography (ERG) traces of AAV-treated right eyes (red) and untreated left fellow eyes (black) from the experimental groups. There were improvements in the scotopic ERG b-wave, mixed rod-cone ERG a- and b-wave, and photopic ERG b-wave recordings (μV) from the AAV2/8(Y733F)-gRNA transduced eyes compared with uninjected fellow eyes at 8 weeks of age. The subretinal injection was performed on one eye of each mouse at P14. ERG was performed 8 weeks post injection. (C) Gray bars represent untreated fellow eyes, red bars represent vector-transduced eyes, and black dots represent each mouse after virus treatment. AAV8-transduced and uninjected fellow eyes were compared at each time point. Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01; n = 4 for all groups. (D) Co-injection of two vectors AAV8:U6-gRNAs_PHD2;hGRK1-GFP and AAV8:hGRK1-Cas9 allowed marking of subretinal transduction sites in dominant Rho^C110R/+ at 1 month post injection. (E) Representative global ERG traces of AAV-treated right eyes (red) and untreated left fellow eyes (black) from the experimental groups. Subretinal injection was performed on one eye of each mouse at P28. There were statistically significant improvements in the scotopic ERG b-wave, mixed rod-cone ERG a- and b-wave, and photopic ERG b-wave recordings (μV) from the AAV2/8(Y733F)-gRNA transduced eyes compared with the uninjected fellow eyes at 5 months of age. Gray bars represent untreated fellow eyes, and red bars represent vector-injected eyes (black dots represent each mouse after virus treatment). (F) AAV8-transduced and uninjected fellow eyes were compared at each time point. Error bars indicate SEM. ∗∗p ≤ 0.01, ∗∗∗p ≤0.001; n = 4 for all groups. (G) H&E-stained retinal sections taken from peripheral Rho^C110R/+ retina transduced ventrally with AAV8:U6-gRNAs_PHD2 in the right eye. Yellow bar denotes ONL thickness. Scale bar (black, top left), 25 μm. (H) Quantification of ONL thickness in Rho^C110R/+ mice injected with the dual AAV8 compared to the non-injected fellow eye as seen in (G). Error bars indicate SEM. ∗p ≤ 0.05; n = 4 in each group.