Angelman syndrome

What is AS?

A report describing AS was published in 1965 by a British pediatrician Harry Angelman . AS is a genetic disorder causing neurodevelopmental disease with a conserved and recognizable set of symptoms. Most often, AS is characterized with serious intellectual disability (complete lack of speech and decreased cognitive performance), happy demeanor with easily provoked laughter, movement and balance disorders (ataxia). Among common symptoms are seizures, that start in early childhood; notably, AS shows a characteristic electroencephalographic pattern. Finally, children with AS have sleeping disorders and disrupted circadian rhythm. Less often symptoms include microcephaly and body dysmorphia, gastrointestinal tract disorders, and hypopigmentation [2]. Features, resembling autistic-spectrum disorders, could be observed in people with AS.
Although AS is considered rare, it’s prevalence is about 1/10,000-1/20,000. AS could be recognized at 6-12 months’ age, when babies start to exhibit lack of usual motor activities, like crawling. Hence, it is important to undergo genetic testing as soon as parents suspect AS: although the definitive cure is (so far) absent, different therapeutic approaches are available to increase the quality of life and mitigate symptoms

Mutation interfering with the expression of UBE3A gene, coding for ubiquitin protein ligase E3A, lead to the AS [4, 5]. In humans, the UBE3A gene is subject to genomic imprinting, a process that results in the gene being expressed in a parent-of-origin specific manner. For the region of chromosome 15 that includes UBE3A (15q11-q13), only the maternal copy of the gene is usually active in certain areas of the brain, while the paternal copy is typically silenced by a process involving DNA methylation and the imprinting center [6–8].

The silencing of the paternal UBE3A allele is controlled by an imprinting control region located upstream of the SNURF-SNRPN gene, within the same 15q11-q13 region. This region includes the Prader-Willi syndrome/Angelman syndrome (PWS/AS) imprinting center. The paternal allele of the imprinting center is methylated, a chemical modification of DNA that does not change the DNA sequence but affects gene expression. Methylation of the ICR on the paternal chromosome prevents the binding of transcription factors necessary for the expression of UBE3A, leading to its silencing. The silencing mechanism also involves the transcription of an antisense RNA from the SNURF-SNRPN locus on the paternal allele, which spans the UBE3A gene. This antisense RNA, UBE3A antisense transcript (UBE3A-ATS or SNHG14), is thought to interfere with the transcription of the paternal UBE3A allele. The exact mechanism by which UBE3A-ATS silences UBE3A is still under investigation, but it is believed that the transcript might block the progression of RNA polymerase during the transcription of UBE3A, prevent the proper splicing of UBE3A pre-mRNA, or recruit chromatin-modifying enzymes that repress transcription. The methylation of DNA and the action of antisense RNA are associated with changes in chromatin structure that further reinforce the silencing of UBE3A on the paternal allele. Histone modifications, such as deacetylation and methylation, lead to a more condensed chromatin state, making the DNA less accessible for transcription.

Deletion of the maternal copy of chromosome 15q11-q13. This is the most common cause, accounting for about 70% of cases. The deletion removes the active UBE3A gene along with neighboring genes, leading to the syndrome.

Mutation of the maternal UBE3A gene. In about 11% of cases, a mutation within the UBE3A gene itself prevents the production of functional UBE3A protein from the maternal allele.

Paternal uniparental disomy (UPD). A rare cause where the child inherits two copies of chromosome 15 from the father and none from the mother. Since the paternal UBE3A gene is normally silenced in the brain, this results in no active UBE3A gene expression in the child’s brain.

Imprinting defect. Also rare, this involves a failure in the process that normally silences the paternal copy of UBE3A in the brain, without a deletion or mutation in the gene itself. This can be due to deletions or mutations in the imprinting center.
The UBE3A gene encodes an E3 ubiquitin ligase that tags defective proteins for degradation. The absence or insufficiency of this enzyme in the brain disrupts the normal turnover of proteins, affecting neuronal function and development, and leading to the symptoms of Angelman Syndrome.

Gene therapy solutions for AS treatment and management

Currently, there are two main approaches to modify the phenotype of AS by modulating gene expression: independent expression of a functional copy of UBE3A and unsilencing of the paternal copy of UBE3A using antisense oligonucleotides (ASOs).15:33

I) Independent expression of a functional copy of UBE3A is a viable approach, since the coding RNA spans for only ca. 2,600 bp, being an optimal size for AAV vector capacity. Several reports describe expression of UBE3A using the strong constitutive promoter and AAV vector, that mitigated AS-symptoms in model mice [9]. An alternative approach included the isolation of hematopoietic stem and progenitor cells (HSPC). HSPCs were then introduced into culture and transfected with a lentiviral vector expressing UBE3A. After, the modified HSPC were engrafted to mice and successfully colonized the bone marrow. Further, microglial cells, derived from these HSPC would migrate to brain, starting to express UBE3A giving therapeutic effect. Described approach allowed to successfully restore AS-like phenotype. This approach has such advantages as the most long-term and stable expression platform.
In addition, CRISPR-Cas9 and CRISPR-Cas13 genome and transcriptome editing approaches were successfully tested and partially restored AS-associated phenotypes in murine models, holding promise for further clinical application.

As double-stranded RNA molecules are not normal for the cell and get digested with ribonuclease H, application of ASOs, complementary to the UBE3A-ATS, is a viable way to decrease the pool of the antisense RNA, and thus activate the expression of UBE3A paternal copy. Application of ASOs led to increase of UBE3A expression in cortex, cerebellum, and hippocampus of model-mice [11]. Treatment reversed memory-related abnormalities, although motor dysfunction was not reversed. In fact, application of ASO to treat and manage AS was approved for clinical trials by FDA.

Drug treatment solutions for AS treatment

A promising way to unsilenced paternal copy of UBE3A are topoisomerase I inhibitors – either naturally occurring or semisynthetic compounds of plant (mostly) origin, that are usually used for cancer treatment. One of those – FDA approved anticancer compound topothecan – efficiently inhibited TOP1 (brain-expressed topoisomerase), leading to the un-silencing of paternal derived UBE3A allele. Investigation of molecular mechanism of topotecan action in vitro allowed to assume that the latter does not change the methylation pattern or the activity of imprinting center, but reduced the level of anti-sense transcript expressed from a paternal-derived UBE3A allele. In vivo, topotecan was administrated by infusion into brain hemisphere or intratecal, giving a dose-dependent activation of paternal copy of UBE3A allele. The expression lasted even 12 weeks after the elimination of topotecan, giving hope for a lasting effec