Indian J Pediatr DOI 10.1007/s12098-017-2394-1
REVIEW ARTICLE
Leber’s Congenital Amaurosis and Gene Therapy Brijesh Takkar 1 & Pooja Bansal 1 & Pradeep Venkatesh 1
Received: 15 March 2017 / Accepted: 17 May 2017 # Dr. K C Chaudhuri Foundation 2017
Abstract Retinal blindness is an important cause of pediatric visual loss. Leber’s congenital amaurosis (LCA) is one of these causes, often wrongly included in the spectrum of retinitis pigmentosa. The disease has become the center of research after initial reports of success in management with gene therapy. This review discusses in brief the clinical presentation and investigative modalities used in LCA. Further, the road to gene discovery and details of currently applied gene therapy are presented. LCA is one of the first successfully managed human diseases and offers an entirely new dimension in ocular therapeutics. Keywords Leber's congenital amaurosis . Gene therapy . Pediatric blindness
Introduction Retinal blindness is a major cause of irreversible congenital visual dysfunction. A very large chunk of the causes of retinal blindness belongs to the umbrella diagnosis of pigmentary retinopathies [1, 2]. Though there is considerable genotypic and phenotypic variation, these diseases are broadly characterized by hyperplasia and hypertrophy of retinal pigment epithelial (RPE) cells and pigment migration into neuro-sensory retina. Several patterns of pigmentary retinopathies are recognized based on etiology, clinical appearance and systemic associations and these are enumerated in Table 1. * Pradeep Venkatesh
[email protected] 1
Retina and Uvea Services, Dr R P Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
Of all the causes of pediatric pigmentary retinopathies, retinitis pigmentosa (RP) is the most researched. RP is not a single disease, but a group of heterogenous, bilaterally symmetric disorders with a propensity to result in bilateral visual loss. The rate of disease progression is however very variable. The clinical signs are pigment deposition in the form of bony spicules, optic disc pallor and attenuation of the retinal vessels [2, 3]. Intrinsic degeneration of rod photoreceptor cells (responsible for scotopic vision) outweighs damage to cone photoreceptor cells (responsible for photopic vision). Functionally, this leads to loss of peripheral visual field with preservation of the central field (tunnel vision). In contrast to its adult variant, pediatric RP tends to be either X-linked, autosomal recessive and linked to several multi-organ syndromic associations [1, 2, 4]. The focus of this review is Leber’s congenital amaurosis (LCA), a disorder that has been linked to RP since its inception, and is sometimes erroneously included within its spectrum.
History The initial description of LCA was made by Dr. Theodore Leber in 1869 in his work titled BUeber Retinitis pigmentosa und angeborene Amaurose^ (BOn Retinitis pigmentosa and congenital amaurosis^) [2, 5]. He highlights the mirroring similarities between RP and LCA, and later alludes to the presence of Bmilder variants^ of the disease in whom preservation of vision extends into adulthood [2, 5, 6]. Several names have been ascribed to this variant, but it is now generally referred to as Severe Early Childhood Onset Retinal Dystrophy (SECORD). Today LCA and SECORD are generally discussed as similar retinopathies and share a large common genetic basis [2, 7, 8]. It is possible that variable genetics account for the
Indian J Pediatr Table 1
Pigmentary retinopathy in children
A. Retinitis Pigmentosa
Usually X-linked or autosomal recessive
B. Complicated Retinitis Pigmentosa 1. Usher syndrome
•Mental Retardation and deafness
2. Bardet-Biedl syndrome 3. MPS
•Hypogonadism, obesity, polydactyly •Corneal opacity, not associated with all MPS types
4. Kearns-Sayre syndrome
•Mitochondrial inheritance, ophthalmoplegia, conduction heart defects and mental retardation
5. Batten disease 6. Bassen-Kornzweig syndrome
•Neuronal ceroid lipofuscinoses, seizures •Defective fat absorption, failure to thrive, acanthocytosis
7. Refsums disease
•Phytanic acid oxidase deficiency, neurologic degeneration, peripheral neuropathy
C. Other dystrophy/degeneration 1. LCA/SECORD 2. Fundus albipunctatus 3. Wagner syndrome 4. Goldman-Favre syndrome D. Congenital E. Infective retinopathy 1. Rubella retinopathy 2. Syphilis retinopathy 3. Other infections
•See text for elaborate details •Flecked retina syndrome •Retinal detachment, optically empty vitreous •Night blindness, retinoschisis, vitreal degeneration •Grouped RPE hypertrophy, associated with Gardner syndrome (familial adenomatous polyposis), Bear-tracks are seen •Salt and pepper retinopathy •Features of congenital syphilis •Toxoplasmosis and Herpes/other viruses
MPS Mucopolysaccharidosis; LCA Leber’s congenital amaurosis; SECORD Severe early childhood onset retinal dystrophy; RPE Retinal pigment epithelial
heterogenic expression of LCA/SECORD and disease categorization based on gene defects has also been reported. Electrophysiological clues for diagnosis and some systemic associations were subsequently noted (discussed later) [7, 9–12].
Etiology and Pathogenesis Twenty three causative genotypes have so far been identified and these defects account for more than half of the cases, while some remain elusive [13]. The commonly implicated genes include CEP290 (15%), GUCY2D (12%), CRB1(10%) and RPE65 (8%) [10, 11, 13]. Of these, RPE65 is the most highly reported because of the success achieved with gene therapy using this variant. Most of these genes are inherited as autosomal recessive traits, though some dominantly inherited genes (CRX) are also known [14]. Pathogenesis of LCA involves aplasia, dysplasia (dysfunction) and degeneration [10]. The initial description by Leber may have been of the degenerative types. The heterogeneity in pathogenesis arises from the underlying genetic variations. For example defective RPE65 gene, located on chromosome 1 and involved in the retinoid cycle (responsible for generation of 11 cis-retinal, an intermediary
that is important for regeneration of visual pigments) produces slow degeneration of photoreceptors in mouse models [2, 15]. In contrast, the defect in GUCY2D gene located on chromosome 17 and which encodes enzyme retinal guanylate cyclase (important in the process of phototransduction – conversion of light signals to neuronal impulses), results in severe retinal dysfunction [2, 16]. On the other hand, CRX gene on chromosome 19 is a cone-rod homeobox gene and is responsible for transcription factors needed for retinal genesis. In a mouse model, deficiency of this gene has been shown to result in aplasia of the outer segments of the photoreceptors [2, 17].
Clinical Features The estimated occurrence of LCA is 2–3 in a million. Historically, close family history may not be present due to recessive inheritance. Usually poor visual development is noted by the parents. Oculo-digital sign is a characteristic feature and thought to be an attempt to stimulate dysfunctional photoreceptors by production of phosphenes [11]. Because of the constant pressure on the orbit due to digital compression, enophthalmos (sunken eyeball) may ensue due to orbital fat atrophy [18]. Corneal ectasia may also be a sequela [19]. Pupils may be amaurotic with accompanying nystagmus.
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Nyctalopia or photophobia may also be present. Characteristically, there is high hyperopia. The retina appears strikingly normal initially, followed in later years by RP like pigmentary disturbance. These findings perhaps depend upon the gene defects discussed earlier. Gradually a Bmarbled fundus^, Coats like reaction and a pseudo-macular coloboma representing extensive tissue loss at fovea may also be seen [11]. There is severe vision loss in majority of cases (characterized by irregular eye movements, absence of light perception and fixation). In majority of the cases the visual status remains stable, in few it deteriorates, while in extremely rare circumstances spontaneous marginal improvement may occur. The term complicated LCA has been used for LCA associated with other systemic/ocular anomalies. Non-ocular associations include central nervous depression, olfactory disturbance, deafness, renal anomalies, cardio-myopathy, hepatic dysfunction and skeletal anomalies [2, 20]. Syndromic association and/or differential diagnosis have been presented in Table 2. Mental retardation was believed to be the most frequent association but recent literature refutes this claim. With regards to SECORD, it should be remembered that it presents later than LCA, but within the first decade of life. The symptoms are initially mild, like night blindness, but progress later. As compared to LCA, some vision is preserved till later in life. The rate of RPE65 mutation in SECORD is much less than LCA. Overall, SECORD may be considered as a milder, slowly progressive form of LCA. Disease progression is however faster than adult onset RP [8].
Table 2 Syndromic associations and differential diagnosis of LCA
Bardet-Biedl syndrome Alstrom syndrome Neuronal ceroid lipofuscinosis Senior-Loken syndrome Saldino–Mainzer syndrome Bassen-Kornzweig syndrome Joubert syndrome
Investigations and Workup A major breakthrough in understanding of LCA, was discovery of extinguished or non-recordable electroretinography (ERG) [9]. In essence, normal ERG rules out LCA altogether and should alert the physician to look for cortical or neuronal causes of visual loss. This test is of utmost value in the initial stages of LCA when fundus findings are absent. In later stages, the Belectric atrophy^ converts to Banatomical atrophy^ and fundus findings become more important for diagnosis. Photoreceptors have been shown to be preserved even in later stages of the disease on imaging with Optical Coherence Tomography (OCT) in LCA patients with RPE65 deficiency [11]. Genetic workup is crucial. Mutations may be detected in nearly half of the cases. Knowledge of genetic change is crucial as it indicates prognosis, risk of disease transmission and possible systemic associations and forms the basis of gene therapy [10, 11].
Therapeutic Options There is no cure for LCA. Reduced exposure to light has been suggested but has no proven benefits. There is suggestion of use of Vitamin A dietary supplementation in the subset with RPE65 deficiency [21]. Repeated systemic administration of tauroursodeoxycholic acid has shown to decrease endoplasmic reticulum stress and apoptosis in mice deficient in
Polydactyly, mental retardation, hypogonadism, obesity, renal failure, diabetes mellitus Cardiomyopathy, obesity, hearing loss, multiple endocrine dysfunctions Neuro-degeneration, seizures, multiple types with variable presentations, early brain death in infantile variants Renal failure from nephronophthisis called oculo-renal syndrome, cystic-renal disease sets in Cone shaped epiphysis of phalanges, abnormal femoral epiphysis known as cono-renal syndrome, ataxia, renal failure Steatorrhea, ataxia, spino-cerebellar degeneration, acanthocytosis, abnormal fat absorption Developmental delay, cerebellar agenesis, abnormal respiratory patterns, also called cerebello-oculo-renal syndrome
Peroxisomal disorders Infantile Refsum disease
Adrenoleuko-dystrophy Zellweger disease Lhermitte-Duclos syndrome
(Leopard spot retinopathy) Inborn error of phytanic acid metabolism, myelin loss, hearing loss, hepatomegaly, hypotonia, dysmorphism Myelin loss, neuro-degeneration, X-linked disorder, adrenal insufficiency, dementia Also called cerebro-hepato-renal syndrome, cranio-facial anomalies, hepatomegaly, renal cysts, hypotonia Dysplastic gangliocytoma of cerebellum, can be a part of Cowden syndrome, megalencephaly, hemangioma, hydrocephalus
Indian J Pediatr Table 3
Common genotypes and associated phenotypes Chromosome [11, 26]
Protein [11]
Function [2, 10, 26]
Prevalence [26]
Phenotype [26]
GUCY2D
17p13.3
Guanylate cyclase
Phototransduction
6–21%
RPE65
1p31
RPE protein 65
Retinoid cycle
3–16%
Marked initial poor vision, normal appearing fundus with peripheral granular changes Night blindness initially followed by visual decay later.
CRX
19q13
Cone rod homeobox
Transcription factor
1–3%
Severe low vision in early life, clumping of pigments with macular pseudo-coloboma
AIPL-1
17p13.1
Aryl hydrocarbon interacting protein
Cell cycle progression
5–10%
Variable light sensitivity followed by poor vision. Evolving RP like picture
CRB-1
1q31
Crumbs like protein
Photoreceptor morphogenesis
9–13%
CEP290
12q21.32
Centrosomal protein 290
Ciliary function
20%
Nanophthalmos may occur. RP like fundus with coloboma/Coats like lesions. Poor vision with atrophic spots. Striking tapetal reflex is visible.
enzymes responsible for LCA [22]. As studies have concluded a role of thyroid hormone in photoreceptor viability, the role of suppressing deiodinase iodothyronine has been suggested in preventing retinal degeneration [23]. Intravitreal and topical delivery of iopanoic acid, an inhibitor of deiodinase iodothyronine has been shown to improve cone survival in mice LCA [23]. For patients with poor vision, low vision aids and social rehabilitation is an integral part of health care [2].
Discovery of LCA Genes The genetic heterogeneity of LCA has been questioned as early as 1963 due to intra-familial variations [13, 24]. Till now at least 23 genes have been implicated in the western literature along with more than 400 mutations [10, 11, 13]. The genetic mutations can be variable, as seen by a recent Chinese study describing 4 new genetic defects [25]. The common genotypes along with phenotypic variations have been tabulated in Table 3. The inheritance of LCA genes is thought to be complex, with impact of many modifier genes [10, 11]. This concept has been used to prove the variable expressivity of phenotype in CRB1 mutant mice [11]. Triallelic inheritance has been suggested in some syndromes with LCA phenotype, which means that a third mutation in an independent gene is affecting the expressivity. It has been shown that LCA patients have higher chances of having a third allele and that this can also affect severity of phenotype [11]. An Arabic study has suggested that the LCA genes are fully penetrant based on a perfect relationship between mutation and disease [27]. This makes LCA a good target for gene therapy.
From Mouse to Dog to Man The successful foray into gene therapy for LCA is perhaps the first convincing evidence for realistic genetic therapeutics in mankind. The first LCA gene (GUCY2D) was mapped to human chromosome 17 in 1996, and later was found to encode for retina specific guanylate cyclase [28]. RPE65, known as LCA2, had been mapped to human chromosome 1 and mouse chromosome 3 in 1994 but was proven to have a role in LCA in 1997 in 2 humans [29]. Subsequently, the revolutionary role of gene therapy with RPE65 was seen in a Briard dog in 2001, when the gene particle and adeno-associated virus (AAV) construct was injected in the sub-retinal space. Qualitative as well quantitative evaluations showed photopic behavior in the previously blind dog with null RPE65 mutations. This was followed up 4 y later by more positive results in a larger canine model, with stable results till 3 y after the injection [11]. Since then stable results have been seen in over 50 dogs. Positive results were further observed in murine models in 2008/9 [13]. Human Trials Based on the results in the canine model, 3 independent studies were commenced nearly simultaneously in 2008 on RPE65 deficient patients [13, 30–33]. Absence of systemic immunological response and serious adverse events was noted in all these 3 studies. One of these trials was conducted at the Children’s hospital of Philadelphia and its collaborating partners. Sub-retinal injection of AAV-RPE65 was delivered in 3 patients aged 19–26 y [30]. Visual improvement was noted on pupillometry, nystagmus charting, visual acuity, visual fields and mobility charting after 15 d. Only 1 adverse effect of macular hole development
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was noted. In a dose escalation phase 1 trial, the number of patients was increased to 12 by the same study group and encouraging results were seen [34]. One 8-y-old child gained light sensitivity similar to age matched controls. It was noted that earlier treatment had better chances of visual gain. At 3 y of follow-up of a cohort of 5 Italian patients, stable visual results were noted with maximal chance of visual gain in the earliest period of treatment by the same group of authors after gene therapy [35]. Following initial years of good results with initial injections, the injection was given in the 3 contralateral eyes of oldest of these study patients, and the therapy was again found to be safe and efficacious at 6 mo [36, 37]. University college of London and Moorfield’s eye hospital, following a pilot study on three young adult patients, given sub-retinal injections of recombinant AAV2-RPE65 complementary DNA under the control of a human promoter, found no statistical improvement in several visual parameters assessed. Some positive change on dark adaptometry and microperimetry was observed in one subject. While studying 12 additional participants in an open label trial, improvements were seen in retinal sensitivity in six patients for up to 3 y. This improvement was noted to peak at 6–12 mo and then gradually decline. Though efficacy remained doubtful, safety was established. The authors concluded that the results were different from those seen in dogs because of different species requirement of RPE65 [38]. The third of these trials was conducted at University of Florida and University of Pennsylvania on 3 patients aged 21–24 y [32, 33]. They noted that although response was good, it was imperfect with variable recovery kinetics. Safety was established as a phase 1 trial. The results were found to be consistent with stable increment in photoreceptor sensitivity at 1 y [39]. Based on these results, the Children’s hospital of Philadelphia and University of Iowa have launched a collaborative phase 3 study, starting in 2012 as BSafety and Efficacy Study in Subjects with Leber Congenital Amaurosis^ (NCT00999609). Primary outcome measure is mobility testing at 1 y. Thirty one patients over 3 y of age have been enrolled and study is estimated to be completed in 2029 [13]. The abstract of the early results of this study group was recently published, which showed significant improvement in mobility and visual sensitivity, but not visual acuity. Pharmaceuticals have, hence, announced their intent to acquire US-FDA approval for gene therapy for LCA after study completion. Gene therapy for LCA is still in its nascent stages and before embarking on planned large scale therapy we first need to identify which genes are responsible in the population, given that the genetic defects are ethnically different [25, 27]. The next step is choosing the ideal virus-vector. It should be the least immunogenic and at the same time the most potent virusgene combination [2, 40]. Thirdly, the best candidates would
be children young enough for tissue and visual cortex to respond to therapy and old enough for the growing eye ball to tolerate the vector implantation [2, 10, 11]. Whether it should be the better eye or the worse eye, unilateral or bilateral and the number/periodicity of inoculations are the questions for future evaluation.
Genetic Strategies and Eye Disease Four basic genetic manipulations include genetic replacement (replacing a defective gene), gene silencing (countering overexpression), gene addition (as in LCA) and gene correction [26, 41]. While viral vectors, by enabling cellular transduction have been successful [2], others like liposome and polymers have not been found beneficial. Gene therapy has been extended to other ocular disorders like retinal dystrophy (peripherin gene), Stargardt’s disease (ABCA4 gene-phase 1 trial), choroideremia, RP (MERTK gene), Usher syndrome, age related maculopathy (AAV based) etc. [41] Among all these, LCA has had the most success.
Conclusions Currently there is a better understanding of LCA as a heterogenic group of disorders and means of differentiating it from its complicated phenotypes. Sixty five percent of the LCA genes have been elucidated, and with availability of novel in situ PCR based technology, genetic analysis may become easier to obtain. The National Library of Medicine maintains a genetic registry for LCA which is now easily accessible. Further, foray into vector technology has improved over the previous vectors and new and more efficient vectors have been identified and tested. Like RPE65, other LCA genes such as GUCY2D and RPGRIP1 interventions have already been found to be successful in animal studies. Hence, promising management approaches in LCA are likely to move from bench to bedside in the coming decade.
Contributions All the authors contributed to concept and outline of the review. BT and PV wrote and critically revised the manuscript, BT and PB performed the review of literature. PV will act as guarantor for the paper. Compliance with Ethical Standards
Conflict of Interest None.
Source of Funding None.
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