Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Abstract

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOC^Y437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Keywords: ER stress; Gene therapy; Genome editing for glaucoma; Intraocular pressure; Lentiviral particles; Myocilin-associated Glaucoma; Trabecular meshwork; Viral vectors.

Figure 1. AAV2-mediated GFP transduction in ocular tissues of C57BL/6J mice.

ssAAV2, scAAV2 and scAAV2^Trp-Mut expressing GFP (2 × 10¹⁰ GC/eye) were injected in mouse eyes via IVT or IC bolus injections or slow IC infusion (n = 3 eyes each). GFP expression was examined by confocal imaging 2 weeks post-injections in anterior segment (A) and retina (B). Non-injected eyes serve as control for background fluorescent intensity. TM—trabecular meshwork; SC—Schlemm’s canal; CB—ciliary body; C—cornea; I—iris. White arrows show TM.

Figure 2. LV-mediated GFP transduction in ocular tissues of C57BL/6J mice.

LV particles expressing GFP (2.5 × 10⁶ TU/eyes) were injected in mouse eyes via IVT or IC bolus injections or slow IC infusion (n = 3 eyes each). GFP expression was examined by confocal imaging 2 weeks post-injections in anterior segment (A) and retina (B). Non-injected eyes served as control for background fluorescent intensity. TM—trabecular meshwork; SC—Schlemm’s canal; CB—ciliary body; C—cornea; I—iris. White arrows show TM.

Figure 3. Comparison of AAV2- and LV-mediated MYOCediting in TM cells.

(A) Representative images showing AAV2_crMYOCor LV_crMYOC treatment of TM3 cells stably expressing DsRed tagged mutant MYOC. AAV2_crMYOC or LV_crMYOCreduces intracellular MYOC and ER stress marker GRP78 (cyan) (scale bar = 50 mm; n=3). (B) Quantitative analysis of fluorescent intensities demonstrates a significant reduction of MYOC fluorescence in both LV and AAV2-CrMYOC treated TM cells. For GRP78 immunostaining, only LV_crMYOC cells showed significant decrease. Unpaired (two-tailed) student t test, with *p < 0.05, **p < 0.01, ***p< 0.001 and ****p < 0.0001. Quantitative data represented as mean ± SEM.

Figure 4. Effect of AAV2- and LV-mediated MYOCediting on ER stress markers in TM cells.

A) Representative Western blot showing decreased protein levels of MYOC, GRP78, GRP94, and CHOP predominantly in LV_crMYOC treated cells compared to AAV2_crMYOC(n = 3). B) The densitometric analysis confirms significant decrease in MYOC and associated ER stress markers with LV_crMYOC treatment only, with no significant effect observed in AAV2_crMYOC treated cells. Unpaired (two-tailed) student t test, with *p < 0.05, **p< 0.01, ***p < 0.001 and ****p < 0.0001. Quantitative data represented as mean ± SEM.

Figure 5. LV-CrMYOC knockout of human MYOC reduces mutant myocilin in TM and lowers elevated IOP in Tg-MYOC^Y437H mice.

(A) IOP measurements in Tg- MYOC^Y437H mice injected with LV_CrMYOC. Ocular hypertensive Tg- MYOC^Y437H mice were injected with LV_Cas9-Null or LV_CrMYOC (2.5 × 10⁶ TU/eyes) and IOPs were measured weekly (n = 6 mice each; >9 months old). LV_CrMYOC reduced elevated IOP significantly compared to ocular hypertensive LV_CrNull injected Tg- MYOC^Y437H mice. (B) Representative images showing decreased MYOC in TM of Tg-MYOC^Y437H mice transduced with LV_crMYOC (n=2). (C) Representative slit-lamp images revealed no ocular inflammation in Tg-MYOC^Y437H mice injected intravitreally with LV_crMYOC (2.5 × 10⁶ TU/eyes) compared to eyes transduced with Ad5_crMYOC (2 × 10⁶ pfu/eyes; n = 3 each). Data represented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Two-way ANOVA with repeated measures and Bonferroni post-hoc analysis.