Retinal Degeneration Secondary to MERTK Mutations: Potential Candidate for Gene Therapy

INTRODUCTION

Inherited retinal diseases (IRD) are a clinically and genetically heterogeneous group of disorders that all share in common progressive photoreceptor degeneration.¹ To date, around 300 genes have been associated with IRD, which can be inherited by Mendelian, mitochondrial, and rarely, digenic inheritance.^2,3 MERTK (MER proto-oncogene, tyrosine kinase) is an uncommon cause of autosomal recessive IRD, but the mechanism of photoreceptor degeneration in MERTK-related IRD has been studied extensively. The protein coded by MERTK in the eye plays a key role in facilitating photoreceptor outer segment phagocytosis by the retinal pigment epithelium (RPE) cells, and disruption in the MERTK gene results in impaired phagocytosis with eventual photoreceptor dysfunction and death.⁴ Studies in rodent models with MERTK-related IRD, including the Royal College of Surgeons (RCS) rat, have demonstrated the primary role that RPE cells play in MERTK-related IRD, where photoreceptor degeneration was rescued by introduction or transplantation of healthy RPE cells.^5,6 Since the RPE is a primary site of degeneration, MERTK-related IRD is an attractive candidate for gene augmentation therapy, because it is likely to be easier to target RPE cells than photoreceptor cells undergoing degeneration.

ROYAL COLLEGE OF SURGEONS (RCS) RATS

The RPE cells interact with photoreceptor cells to facilitate vision, participating in the visual cycle and degrading photoreceptor outer segments on a daily basis.⁴ Photoreceptor outer segments are synthesized and shed continuously, such that the outer segments turn over completely every 7–10 days.^7,8 The RCS rat is one of the earliest reported rodent models of IRD, in which RPE dysfunction results in defective outer segment phagocytosis with subsequent progressive, postnatal loss of photoreceptor cells.⁹ D’Cruz et al.¹⁰ used a positional cloning approach to study the rdy (retinal dystrophy) locus of the RCS rat. They identified a small deletion in the gene that encodes the receptor tyrosine kinase MerTK. The deletion included the splice acceptor site upstream of the second coding exon of MERTK; the resulting transcript was shortened by skipping the second exon and joining the first and third coding exons, which led to a frameshift and a translation termination signal 20 codons after the AUG. The findings conclusively demonstrated that mutation of the MERTK gene is responsible for retinal degeneration in RCS rats.¹⁰

To further investigate the connection between MERTK and retinal dystrophy, Duncan et al.¹¹ characterized the retinal phenotype in mice with disruption in the MERTK gene. They utilized the mer^kd mice, which had both copies of MERTK knocked out and found that the retinal phenotype of mer^kd mice was similar to RCS rats.¹¹ The similarity in phenotypes between the two rodent models suggests that defective RPE phagocytosis is a feature of retinal degeneration caused by loss of Mer tyrosine kinase (MerTK) function, likely including mutations in human MERTK.

HUMAN MERTK MECHANISM

Gal et al.¹² screened the human orthologue, MERTK, located at 2q14.1, in 328 DNA samples from individuals with various retinal dystrophies and found 3 mutations in 3 unrelated individuals with autosomal recessive retinitis pigmentosa (RP). These findings established that RPE phagocytosis is implicated in human retinal degeneration.¹² MERTK-related retinal degeneration accounts for a small percentage of IRD patients; by documenting 79 MERTK variants in IRD patients including early onset autosomal recessive rod-cone and cone-rod degeneration with macular involvement, Audo et al.² estimated that about 2% of IRD cases are attributable to pathogenic variants in MERTK.

The MERTK gene encodes the tyrosine‐protein kinase MerTK, a transmembrane receptor kinase protein and a member of the TAM (TYRO3/AXL/MER) receptor kinases that regulate phagocytosis of apoptotic cells in many tissues throughout the body.¹⁴ In conjunction with αvβ5 integrin, which facilitates photoreceptor outer segment binding to RPE cells, MerTK controls the outer segment binding to and engulfment by RPE cells.^{15, 16} Furthermore, the regulation of phagocytosis is achieved by the soluble form and the transmembrane location of the MerTK protein, which limits outer segment binding to the apical surface of RPE cells.^{16, 17}

The RPE phagocytosis of outer segments involves two ligand-receptor pairs.¹⁸ Initially, phagocytosis is triggered when phosphatidylserine is exposed extracellularly at the surface of outer segments.⁴ Recognition of phosphatidylserine by the ligand-receptor pair formed by the milk fat globule‐EGF‐factor 8 (MFG‐E8) and αvβ5 integrin activates tyrosine kinase 2 at the apical membrane of the RPE, which subsequently autophosphorylates.¹⁹ This process is important for the activation of the second ligand-receptor pair, since the loss of αvβ5 integrin receptors and MFG-E8 is linked to reduced phagocytosis synchronicity with the circadian rhythm.²⁰ Acting as the receptor in the second ligand-receptor pair in the process, MerTK uses Gas6/Protein S as ligands to interact with phosphatidylserine, then MerTK subsequently autophosphorylates.¹⁸ Once the binding of the two ligand-receptor pairs is complete, outer segment membranes are internalized and degraded.⁴ Levels of Tyro3, another member of the TAM family, modulate the severity of photoreceptor degeneration due to mertk deletion, and Tyro3 acts as a genetic modifier for MERTK-related retinal degeneration.²¹ Therefore, mutations in MERTK interrupt the recycling of light-sensitive photoreceptor outer segment membranes by RPE cells, and result in photoreceptor degeneration and loss.^{11, 21}

EPIDEMIOLOGY

The prevalence of IRD is estimated to be between 1 in 3,500 and 1 in 5,000 worldwide with variable age of onset and severity.¹ Large-scale sequencing approaches have shown that approximately 2–3% of retinal dystrophies may result from mutations in MERTK in an autosomal recessive pattern. However, the prevalence of MERTK-related IRD is higher in some populations, such as in North Africa where it accounts for 18% of rod-cone dystrophy, and in the Faroe Islands where a 91 kb founder deletion is responsible for up to 30% of IRD.^{2, 22, 23, 24} Bi-allelic mutations of MERTK have been associated with early onset retinitis pigmentosa,²⁵ with a phenotype characterized by childhood onset rod and cone dysfunction and macular atrophy,^{2, 26} while some studies reported MERTK variants in almost 4% of patients with cone-rod dystrophy.²

GENE THERAPY

MERTK-related IRDs represent an attractive candidate for gene therapy for several reasons.²⁷ Since RPE phagocytosis is the primary defect in MERTK-related IRD,⁵ the target of gene augmentation therapy is RPE cells which may be more amenable to gene augmentation than photoreceptors that are undergoing retinal degeneration. Based on results in animal models, photoreceptor structure in eyes with MERTK-related retinal degeneration should be rescued by restoration of RPE phagocytosis,⁵ which should result after successful delivery of the MERTK gene to RPE cells.^{21, 28} In addition, due to the early onset nature of IRD associated with MERTK mutations,²⁶ the lifelong reduction of disease burden provided by an effective gene therapy could be substantial if it were delivered to young patients at early stages of degeneration.

With a firm understanding of the degenerative mechanism, several groups demonstrated proof-of-concept for gene augmentation treatment of MERTK-related IRD. Gene replacement using recombinant adeno-associated virus (AAV) vectors to express the murine MERTK gene, controlled by either a ubiquitous promoter (CMV)²¹ or RPE-specific promoter (RPE65),²⁹ significantly improved photoreceptor function and cell survival compared to contralateral non-injected eyes in RCS rats. By using a BEST1 promoter to target RPE cells in RCS rats, Deng et al.²⁸ delivered human MERTK cDNA with an AAV8-vector, which in turn preserved retinal function and structure, rescued RPE phagocytosis and prevented remodeling in the treated eyes for at least 8 months.

The preclinical studies described above provided evidence to support the initiation of a first-in-human clinical trial of gene augmentation for MERTK-related IRD. Ghazi et al.³⁰ reported results of a Phase I treatment trial in 6 patients with early onset retinitis pigmentosa secondary to MERTK mutations. Visual acuity at baseline for eligible patients was 20/50 to light perception. After 2 years, patients treated with subretinal rAAV2-VMD2-hMERTK experienced acceptable ocular and systemic safety; visual acuity improved in 3 of the 6 patients following treatment, but declined over 2 years. Patients with better vision at baseline were more likely to demonstrate improved visual acuity after treatment. Future studies involving patients with higher visual function at baseline will be needed to assess more fully the efficacy of this potential therapeutic approach.

CONCLUSION

IRD due to MERTK mutations represents an exciting opportunity for development of gene therapy. The mechanism of MERTK-related retinal degeneration has been well-characterized in pre-clinical models, permitting proof-of-concept studies. The primary site of disease in the RPE, evidence of photoreceptor rescue in rodent models treated with MERTK gene augmentation, early onset of disease and potentially long-lasting treatment effects all make MERTK-related IRD an attractive candidate for gene augmentation therapy. Results from a Phase I clinical treatment trial provide support for future clinical studies that may lead to an efficacious treatment option for patients affected by this early onset, visually debilitating disease.