Treating Blindness with Combined CRISPR-Cas9/RecA
Photoreceptor cell death was successfully halted in mice–results are promising for preventing vision loss in humans.
Retinitis Pigmentosa (RP) is an inherited form of retinal degeneration that causes the death of light-sensitive cells called rods and cones. It is the leading cause of visual disability and blindness in people under 60 years old, with 1 million cases estimated worldwide.¹𝄒² While there is currently only one FDA-approved treatment available, suitable for those with a mutation in the RPE65 gene (about 2% of patients), the CRISPR-Cas9 gene-editing tool provides a promising new treatment option. However, replacing mutated genes in neurons is significantly more difficult since they rarely undergo homology-directed repair (HDR) under normal conditions. Cai et al.³ explore a novel method of increasing the frequency of HDR using the Escherichia coli protein recombinase A (RecA) and reducing vision loss in mice with a mutated RP gene.
The human retina (Fig 1a) has three types of photoreceptor cells: rods, cones, and intrinsically photosensitive retinal ganglion cells (Fig 1b). Rods are responsible for low-light and peripheral vision and cones are for color vision and visual acuity.¹ Cone cells depend on rods for survival because rods secrete a protein that enables glucose transport.⁴ Therefore, any mutation causing rod degeneration will also affect cones.
Figure 1. Anatomy of the human eye. (a) The human retina, highlighted in yellow, is located at the back of the eye. (b) Three types of photoreceptor cells: rods and cones (consisting of a synapse, nucleus, inner and outer segment) and intrinsically photosensitive retinal ganglion cells.
There are at least 45 known genes implicated in RP, and the mode of inheritance is variable.¹ Autosomal recessive RP (arRP) is the most common, occurring in 50-60% of cases, and has the most severe phenotype. Patients with arRP tend to experience symptoms before their teenage years. A mutation in the PDE6B gene interrupts visual processing by inhibiting the activity of phosphodiesterases and is responsible for 4-5% of cases of arRP. Phosphodiesterases are enzymes responsible for hydrolyzing cGMP into GMP. Decreased levels of cGMP prompt the closure of Ca2+ and Na+ channels, so failure of this mechanism causes an increased concentration of Na+ in cells, eventually leading to cell death.⁵
Cai et al.³ targeted the PDE6B locus of retinal degeneration model 1 (rd1) mice using bacterial recombinase A (RecA), a protein isolated from E. Coli to promote the frequency of HDR in photoreceptor cells. HDR is often preferable in gene editing because it does not cause indels that contribute to a loss of genetic information. However, HDR only occurs during the S/G2 phase of the cell cycle, when cells are replicating. Most neurons are terminally differentiated–they can no longer undergo mitosis. They do not perform HDR under normal conditions. RecA is known to increase HDR by mediating the homology search and exchange of homologous DNA sequences, but it has not been used in combination with the CRISPR-Cas9 gene-editing system.⁶
Figure 2. In Vitro Testing of the Cas9/RecA System. Three groups (Cas9/RecA, Cas9, and Control) were applied to HEK293FT cells, and fluorescence was measured to determine which type of DNA repair occurred.
Cai et al.³ transfected their Cas9/RecA, Cas9, and control elements (Fig 2) into human embryonic kidney 293FT cells expressing a blue fluorescent protein. When the cells have a C allele at position 196, they express a blue fluorescent protein, but a point mutation changing it to a T allele can cause the expression of enhanced green fluorescent protein. Loss of fluorescence indicates that non-homologous end joining occurred because an indel was introduced at position 196, while expression of enhanced green fluorescent protein indicates HDR occurred and the T allele is present. They saw a 1.7-fold increase in green fluorescent cells in the Cas9/RecA group compared to Cas9 only. Next, they tried their technique in vivo by electroporating rd1 mice with the Cas9/RecA and Cas9 plasmids. The Cas9/Rec- treated mice had a fivefold increase in surviving rods and a fourfold increase in surviving cones compared to Cas9 treated mice. Electroretinogram data showed that the greatest functional difference occurred in brighter light. However, the protein expressed in Cas9/RecA-treated mice was only about 2% of what was expressed in wild-type mice.
Cai et al.³ chose the rd1 mouse model for their experiment, which has an aggressive phenotype. The rd10 mouse, also with a PDE6B mutation, has been suggested as an alternative for modeling RP in humans.⁷ Since they found more success treating postnatal day 0 mice than postnatal day 3 mice, allowing a longer treatment window would likely provide better results and be more representative of the human disease progression. Additionally, mice have a 35:1 rod-to-cone ratio, while humans have a ratio of 20:1. Experimentation in an animal with a more similar retina would better predict the effect on humans. The Sloughi Dog, or Arabian Greyhound, is affected by retinal atrophy due to PDE6B mutations, but people are generally hesitant to test potentially dangerous treatments on animals that are common pets.
Expression of 2% of the wild-type amount of protein seems trivial. However, patients can lose a significant amount of rods and cones (up to 90%) before noticing a difference in the clarity of their vision. Restoration to 20/20 vision is not necessarily the goal. About 90% of rods must be lost before cone degeneration, so maintaining beneath that threshold can prevent the most deleterious effects. Overall, the combined CRISPR-Cas9/RecA therapy shows promise for editing terminally differentiated cells, and in tandem with other tools for promoting HDR like vSLENDR, a technique using an adeno-associated virus, could provide significant benefits to patients with neurological disease.
Bibliography
- Dyonne T Hartong MD, Prof Eliot L Berson MD, & Prof Thaddeus P Dyrja MD. Retinitis pigmentosa. Lancet 368, 1795–1809 (2006).
- Cross, N., van Steen, C., Zegaoui, Y., Satherley, A. & Angelillo, L. Retinitis Pigmentosa: Burden of Disease and Current Unmet Needs. Clin Ophthalmol 16, 1993–2010 (2022).
- Cai, Y. et al. In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway. Sci Adv 5, eaav3335 (2019).
- Krol, J. & Roska, B. Rods Feed Cones to Keep them Alive. Cell 161, 706–708 (2015).
- Cote, R. H., Gupta, R., Irwin, M. J. & Wang, X. Photoreceptor Phosphodiesterase (PDE6): Structure, Regulatory Mechanisms, and Implications for Treatment of Retinal Diseases. in Protein Reviews: Volume 22 (ed. Atassi, M. Z.) 33–59 (Springer International Publishing, Cham, 2022). doi:10.1007/5584_2021_649.
- Shcherbakova, O. G., Lanzov, V. A., Ogawa, H. & Filatov, M. V. Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells. Mutation Research/DNA Repair 459, 65–71 (2000).
- Han, J. et al. Review: The history and role of naturally occurring mouse models with Pde6b mutations. Mol Vis19, 2579–2589 (2013).
