Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss

Abstract

Blindness and deafness are the most frequent sensory disorders in humans. Whatever their cause - genetic, environmental, or due to toxic agents, or aging - the deterioration of these senses is often linked to irreversible damage to the light-sensing photoreceptor cells (blindness) and/or the mechanosensitive hair cells (deafness). Efforts are increasingly focused on preventing disease progression by correcting or replacing the blindness and deafness-causal pathogenic alleles. In recent years, gene replacement therapies for rare monogenic disorders of the retina have given positive results, leading to the marketing of the first gene therapy product for a form of childhood hereditary blindness. Promising results, with a partial restoration of auditory function, have also been reported in preclinical models of human deafness. Silencing approaches, including antisense oligonucleotides, adeno-associated virus (AAV)-mediated microRNA delivery, and genome-editing approaches have also been applied to various genetic forms of blindness and deafness The discovery of new DNA- and RNA-based CRISPR/Cas nucleases, and the new generations of base, prime, and RNA editors offers new possibilities for directly repairing point mutations and therapeutically restoring gene function. Thanks to easy access and immune-privilege status of self-contained compartments, the eye and the ear continue to be at the forefront of developing therapies for genetic diseases. Here, we review the ongoing applications and achievements of this new class of emerging therapeutics in the sensory organs of vision and hearing, highlighting the challenges ahead and the solutions to be overcome for their successful therapeutic application in vivo.

Keywords: CRISPR/Cas9; blindness; deafness (hearing loss); gene editing; gene therapy; hair cells; inherited retinal degeneration (IRD); retina.

FIGURE 1

Similarities between the sensory organs of vision and hearing. (A): Light signals are transduced by the outer segments of the photoreceptors, in the retina. (B): Hearing and balance are dependent on the processing of sound waves and/or movement within the hair bundles, which crown hair cells located in the cochlea and vestibule of the inner ear, respectively. Photoreceptors and hair cells have synaptic active zones different from those of brain conventional synapses in that they are associated with an electron-dense ribbon surrounded by tethered synaptic vesicles. The neurotransmitter released from all photoreceptor cells and hair cells is glutamate, which creates the electrical signal conveyed by afferent neurons through either the visual or auditory pathways to the brain.

FIGURE 2

(A,B) The retina, photoreceptor cells and light-sensitive outer segments. (A) The mammalian retina, located at the back of the eye is a laminated, multilayer sensory epithelium, made up of the retinal pigment epithelium (RPE) attached to the different neuronal layers that compose the neuroretina: the outer nuclear layer (ONL), the inner nuclear layer (INL) and the ganglion cell layer (GC), separated by the synaptic Outer plexiform (OPL) and inner plexiform (IPL) layers. The scanning electron micrograph illustrates the apical functional compartments of the rod (nocturnal vision) and cone (diurnal vision) photoreceptor cells, the outer (OS) and the inner (IS) segments, separated by the connecting cilium. (B) Schematic representation of a rod apical region, illustrating the outer segment composed of hundreds to thousands of specialized membrane discs that house the phototransduction cascade machinery. Briefly, in absence of light, there is a constant influx of sodium and calcium ions across the cGMP-gated channels (CNGα1/β1) at the outer segment plasma membrane. Incoming light directly activates the light-sensitive opsins, rhodopsin (Rho, rods) and cone opsin, triggering the phototransduction process via the G protein transducins and the effector enzyme, cGMP phosphodiesterase (PDE). The increase of cytosolic cGMP leads to the closure of the CNG channels, photoreceptor cell hyperpolarization, and membrane potential changes leading to graded modulation of glutamate release at the photoreceptor synaptic terminals. Photoresponse is halted upon phosphorylation of opsins by opsin kinases (e.g., GRK1, rods; GRK7, cones) and by arrestin’s binding inactivating transducin.

FIGURE 3

The inner ear, the hair cells and the sound-sensitive hair bundles. (A) The mammalian coiled, snail-shaped cochlea houses the auditory sensory organ, also called organ of Corti. Our hearing relies on two types of hair cells: a single row of inner hair cells (IHCs), the genuine sensory cells that transmit sound-induced electrical signals to the brain, and three rows of outer hair cells (OHCs), responsible for sound amplification. The scanning electron micrograph illustrate the highly organized structure of an OHC hair bundle, with three rows of stereocilia arranged in a staircase pattern. (B) Schematic representation of the mechanosensitive hair bundle, composed of 50–100 F-actin-filled microvillus structures, the stereocilia, arranged in staircase pattern at the apical hair cell surface. Incoming sound waves to the cochlea ultimately lead to the deflection of the hair bundle. Positive deflection, in the direction of the longest stereocilium, triggers the opening of the mechano-electrical transduction (MET) channels; located at the lower end of an extracellular fibrous link, the tip link. The ensuing influx of Ca²⁺ and K⁺ ions leads to hair-cell depolarization, resulting in membrane potential changes leading to graded modulation of glutamate release at the IHC synaptic active zones, which convey signal information to the brain through the auditory primary neurons (adapted from Dulon et al., 2018; Delmaghani and El-Amraoui, 2020).

FIGURE 4

(A) Viral and non-viral delivery, and gene editing therapies in the eye and the inner ear. (A) Current therapeutic strategies with promising success in the eye and the inner ear involve gene replacement using adeno-associated viruses (AAVs), and gene editing targeting specific mutations causing vision and/or hearing loss. (B) An important challenge in eye and ear therapeutics is the route of administration. In the retina, the subretinal injection may be favored to ensure high transduction rates of photoreceptor and RPE cells. The intravitreal route is recommended for targeting the ganglion and inner nuclear cell layers, and particularly, the central retina. In the inner ear, main administration routes include local injections into the perilymph through the round-window membrane (RWM), or through the oval-window (trans-stapedial injection). Injections into the endolymph, in the cochlear scala media (cochleostomy) or through a semicircular canal (canalostomy), remain challenging, with high risk of sensory damage. Thanks to smaller size cas9 enzymes, it’s possible to use AAVs to transfer the CRISPR-Cas9/gRNA complex into target cells. Alternatives exist for larger nucleases such as base or prime editors, which include the use of dual AAVs, or non-viral vectors (e.g., liposomes), which additionally can be used for delivering Cas protein instead of DNA.

FIGURE 5

DNA- and RNA-based genome editing to manipulate disease gene-of-interest. Different mechanisms occur, depending on the type of CRISPR/Cas nuclease and supplied editing tools. (A1) The DNA CRISPR/Cas nucleases such as Cas9 or Cas12 can bind and cleave the genomic DNA. The double-stranded DNA breaks can then undergo repair by either the nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is a random and highly error prone mechanism that incorporates insertion/deletion mutations, called indels, causing gene disruption (or silencing). In HDR, the DSB can be repaired by externally adding a donor DNA template that is homologous to the target sequence. The donor template is copied into the targeted site, resulting in a directed precise repair of the defective DNA sequence. (A2) Base editing relies on inactivated Cas nucleases (dcas) with preserved DNA targeting but without DNA cleavage, and could thus be used in non-dividing cells. The dCas nuclease (for instance Cas9 nickase, nCas9) is coupled to a deaminase, cytidine (CBE) or adenosine (ABE), which make it possible to edit specific nucleotide without DNA double break: C-to-T transitions by CBE editors or A-to-G transitions by ABE editors. (A3) Prime editing is suitable to edit both point mutations and larger deletions or insertions. Here, dCas9 is fused with an engineered reverse transcriptase enzyme (RT), combined with a prime editing guide RNA (pegRNA) that serve to both position the enzyme at target site and provide the template sequence necessary to correct or replace the defective DNA region. (B) CRISPR‐Cas RNA systems, such as Cas13, can be used to manipulate cellular RNA both for basic research and therapeutics. While catalytically active Cas13 variants can cleave and disrupt the targeted RNA, the RNase-defective dCas13 (B1) and dCas13-effector fusion (B2) variants further expand possible RNA manipulations. These include regulation of RNA stability, splicing, intracellular localization, epitranscriptome modulation, translational activation/repression, RNA imaging, labeling of RNA‐interacting proteins, or site-directed nucleobase editing (possible by ADAR effector domains), Abbreviations: ADAR, adenosine deaminase acting on RNA; PFS, protospacer flanking sequences; A, adenosine; T, thymidine; C, cytidine; G, guanosine; I, inosine; and U, uracile.