May 8, 2024 | ASGCT, ORAL SESSION, ABSTRACT #87
Development of an In Vivo Non-Viral Delivery Platform for Ocular Editing and Application as a Potential Treatment for Glaucoma
Mary-Lee Dequéant, Ph.D. | Senior Director, CRISPR Therapeutics
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CRISPR THERAPEUTICS | 2 |
Disclosures
Mary-Lee Dequéant, Ph.D.
Senior Director, CRISPR Therapeutics
Disclosure Information
I have the following relevant financial relationships to disclose: Employee of: CRISPR Therapeutics
Stockholder in: CRISPR Therapeutics
CRISPR THERAPEUTICS | 3 |
Non-Viral Delivery - A Promising Approach
for Ocular Gene Editing
● Potential to use CRISPR/Cas9 technology to address
genetic diseases of the eye once considered untreatable | Lipid nanoparticle (LNP) formulated with |
Cas9 mRNA and guide RNA (gRNA) |
- Advantages of localized delivery to the eye:
- Accessible for localized delivery
○ Small volume
- Enclosed structure / limited systemic exposure
- Immune-privilegedorgan
- Non-viraldelivery of Cas9 mRNA offers several advantages:
- Transient expression of Cas9 enzyme, no potential for vector integration
- Ability to deliver larger cargoes
- Simplified manufacturing
- Repeat dosing feasible
CRISPR THERAPEUTICS | 4 |
MYOC Mutations are the Most Common
Genetic Cause of Glaucoma
- Glaucoma is the second leading cause of blindness worldwide1
- Myocilin (MYOC) mutations are the most common genetic cause of glaucoma.2 MYOC-associated glaucoma affects ~150k people in the United States alone1,3
- Myocilin mutations are autosomal dominant4 and associated with earlier onset and more rapid progression of the disease5
- Mutations cause impaired outflow of aqueous humor through the trabecular meshwork (TM), leading to elevated intraocular pressure (IOP)6
Abnormal aqueous humor outflow
through the TM Increased intraocular pressure (IOP)
Damage to optic nerve and ganglion cells
1 Allingham R.R. et al. 2009, 2 Fingert J.H. et al., 2011, 3 Zhang N. et al., 2021, 4 Sheffield V.C. et al. 1993, 5 Fingert J.H. et al., 1999, | CRISPR THERAPEUTICS | 5 |
6 Alward W.L.M. et al. 1998.
Myocilin Gene is a Promising Target for the Treatment of Glaucoma
- Myocilin is a glycoprotein highly expressed in the trabecular meshwork (TM)1
- Gain-of-functionMYOC mutations cause misfolded protein aggregates in the endoplasmic reticulum (ER), inducing cellular stress that can lead to TM cell death/dysfunction2
- Knocking out MYOC expression in the TM using CRISPR/Cas9 was shown to reverse the ER stress, restore TM function, and reduce IOP3,4
- WT MYOC expression is not required for normal ocular function5 and homozygous MYOC loss of function in humans is not associated with any disease6
Adapted from Kasetti et al., 2021
1 Pang C.P. et al. 2002, 2 Kasetti R.B. et al. 2016, 3 Jain A. et al., 2017, 4 Patil S.V. et al., 2024, 5 Kim B.S. 2001, | CRISPR THERAPEUTICS | 6 |
6 Lam D.S. 2000. |
In Vivo Screen Identified LNPs that Deliver Efficiently to Mouse TM
- ~200 LNPs formulated with EGFP1 mRNA were tested in vitro in primary TM cells
● ~40 LNP formulations were selected for in vivo testing | EGFP Detected in TM by IHC3 |
24h After LNP Dosing | |
EGFP Expression in Mouse TM 24h After IVT2 Injection | Trabecular | |
3 | Meshwork | |
Score | ||
2 | ||
GFP 1 | Ciliary body | Iris |
0 | 100μm | |
LNP Formulations |
1 EGFP - Enhanced Green Fluorescent Protein; 2 IVT - Intravitreal; 3 IHC - Immunohistochemistry | CRISPR THERAPEUTICS 7 |
LNPs Selected for Targeted Delivery to Non-
Human Primate TM
Intracameral Injection | EGFP Detected in TM by IHC |
in Cynomolgus Monkey | 24h After Dosing with LNP |
Iris | |
TM |
Ciliary
Body
Cornea
Injection in the anterior segment of LNP formulated with EGFP mRNA
CRISPR THERAPEUTICS | 8 |
LNP Formulation Also Successfully
Delivered to Human TM in Ex Vivo Model
Human Eye Anterior Segment | EGFP Detected in Human TM by IHC |
Organ Culture (ASOC)1 | 24h After LNP Infusion |
TM
200μm
CRISPR THERAPEUTICS | 9 |
1 LNP formulated with EGFP mRNA was infused through syringe/pump. Flow: 2.5 μl/min (method adapted from Boice and Snider, JOVE, 2021)
Prioritized MYOC gRNA Shows Efficient
Editing in Primary Human TM cells
>90% MYOC Gene Editing In Vitro | MYOC Protein Reduction Up to 85% |
with Prioritized gRNA1 | with Prioritized gRNA |
Editing (%)
100
80
60
40
20
0
No LNP | Low | Medium | High |
LNP Dose
Protein Expression | (norm.) |
MYOC |
1.0
0.8
0.6
0.4
0.2
0.0
No LNP | Low | Medium | High |
LNP Dose
Cross-reactive human/NHP MYOC gRNA selected based on in vitro screen
Comprehensive off- target assessment completed with no off-target editing detected for prioritized gRNAs
1 gRNA - guide RNAs | CRISPR THERAPEUTICS | 10 |
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CRISPR Therapeutics AG published this content on 08 May 2024 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 08 May 2024 19:24:01 UTC.