CRISPR/Cas9 is an extremely powerful technology that allows scientists to easily rewrite human DNA to treat human disease. We envision that CRISPR/Cas9 technology can be applied in the therapy of all manner of disorders, from rare inherited genetic diseases such as Leber congenital amaurosis (link to https://rarediseases.info.nih.gov/diseases/634/leber-congenital-amaurosis), to common diseases such as high cholesterol and cancer.

So far, there are limited options available for patients with blinding diseases. The approval of Luxturna for RPE65 disease was a major breakthrough for gene therapy and ophthalmology. Now, a new generation of gene editing technologies, enabled by CRISPR/Cas9, is on the horizon to treat diseases untreatable by gene therapy. A new generation of CRISPR technologies, developed by the group of David Liu at the Broad Institute of MIT and Harvard (link to liugroup.us), named base and prime editors,  are even safer, more precise, more easily controlled, and more flexible in the mutations that are treatable by gene editing.

gene therapy

Base editing for inherited retinal diseases

We were one of the first to show that in vivo base editing rescues visual function in a mouse model of Leber congenital amaurosis, the rd12 model. The mice have a mutation that renders them unable to see, and by correcting the mutation by direct delivery of CRISPR/Cas9 to the eye, we restored vision, visual-guided behavior, and brain and visual cortex responses to visual input. We also showed protection of cone photoreceptors, which enable our ability to read and perceive color and is a critical unmet need for inherited retinal disease treatment.

base editing

Moving to the clinic

The first CRISPR/Cas9 clinical trials for a wide range of diseases has already begun. We believe that CRISPR/Cas9 will be shown to be safe for ex vivo and in vivo human use, and we are excited for the application for untreatable diseases. With the concurrent advances in genome sequencing, we are working toward a future where a patient can be sequenced, have their mutation corrected in a laboratory with patient derived stem cells, and then have the proper CRISPR/Cas9 treatment packaged into proper delivery vectors. We pioneered an even safer editing technique using eVLPs that will improve safety in humans.

clinic

Glaucoma is the second leading cause of irreversible blindness affecting over 70 million people worldwide. Glaucoma is characterized by loss of retinal ganglion axons, which send visual information to the brain. Primary Open Angle Glaucoma (POAG), a most common form of glaucoma, is often associated with increased eye pressure that can lead to axonal loss and irreversible blindness. This eye pressure is tightly maintained by specialized tissue called trabecular meshwork. In POAG, there is increased eye pressure due to the damage to trabecular meshwork. The exact mechanism of glaucomatous damage to trabecular meshwork is poorly understood. The current medical and surgical treatment to lower eye pressure have limited success since these treatments do not target main pathology within trabecular meshwork tissue. Vision loss continues to progress in some patients despite these treatments further emphasizing on the need of targeted treatments. Mutations in myocilin gene are the leading known genetic cause of POAG and it accounts for most cases of pediatric glaucoma. Myocilin-associated glaucoma is often less-responsive to current treatments leading to vision loss at early stage of life. Therefore, there is a critical need to develop novel treatment strategies to permanently cure this stage at early stage. While wild type myocilin is not required for maintenance of eye pressure, mutations in myocilin acquire toxic function in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and loss of trabecular meshwork cells. This results in build of eye pressure and vision loss. Therefore, knocking out myocilin at the DNA level makes an ideal strategy to permanently cure the disease in young patients.

New genome editing technologies allow investigators to directly alter the genes associated with disease pathology. Currently, CRISPR-Cas9 is the leading genome editing tool that allows knockout or genetic modification of specific genes. We have recently demonstrated that targeting MYOC by CRISPR-Cas9 system reduces mutant myocilin in TM and prevents glaucoma in transgenic mice. We utilized adenovirus (Ad) 5 to express Cas9 and guide RNA in TM. However, viral vectors have several limitations including ocular inflammation, safety concerns and off-target effects of Cas9 due to its expression for a longer duration of time. Recent improvements in mRNA technology and success of Covid-19 mRNA-based vaccine have opened new possibilities to utilize lipid nanoparticle (LPN)-mediated mRNA delivery approaches for ocular diseases. Here, we propose a novel and highly potent LPN-mediated CRISPR-Cas9 delivery system to express Cas9 mRNA and gRNA in the TM. This system consists of cationic polymer and other lipids encapsulated with Cas9 mRNA and guide RNA (gRNA) targeting MYOC. Due to phagocytic nature of TM, TM cells selectively uptake LPN and produce Cas9 protein forming ribonucleoprotein (RNP) complex with gRNA and editing MYOC in vitro and in vivo. We propose that LPN-mediated Cas9 mRNA delivery system will provide higher safety profile due to minimum ocular inflammation and significantly less off-target effects due to transient expression of Cas9.

Our approach will revolutionize glaucoma therapy by directly interfering with glaucomatous damage to trabecular meshwork and provide a one-time therapy to treat glaucoma.

gulab fig2

We are currently working toward treatments and cures for Leber congenital amaurosis, Stargardt disease, retinitis pigmenotsa, and age-related macular degeneration.

Selected Works

“Precision genome editing in the eye” (2022) https://www.pnas.org/doi/10.1073/pnas.2210104119

“In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration” (2022) https://doi.org/10.1038/s41467-022-29490-3

“Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing” (2020) https://doi.org/10.1038/s41551-020-00632-6

“Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins” (2022) https://doi.org/10.1016/j.cell.2021.12.021