Those affected by DS present with a host of characteristic phenotypes and health complications that impede regular functioning, outlined in Table 1. These individuals have an increased risk of heart disease, early onset Alzheimer’s disease, leukemia and testicular cancer (Hill et al., 2003; Hasle et al., 2016). This not only disturbs the individual’s quality of life, but also puts a strain on family, carers and the health care system (Hill et al., 2003). While individual phenotypes vary, certain characteristics such as ID, hypotonia, craniofacial dysmorphology and the histopathology of Alzheimer’s disease, are present to some degree in all cases of DS. Understanding the key cognitive components of DS is crucial in comprehending the unique challenges of developing effective interventions that can improve their overall wellbeing.

Individuals with DS account for approximately 10–20% of the intellectually disabled population, with some form of impairment present in nearly all cases of DS (Torr et al., 2010). This is the result of overexpressed genes altering neural development and functioning. Individuals with DS generally have an intelligence quotient (IQ) that ranges from 20 to 80. Their IQ being its highest when they are children and gradually declines as the individual ages (Carr, 1988; Anneren and Edman, 1993; Chapman and Hesketh, 2000). This is believed to be a consequence of the slow rate of neural development when compared to unaffected children (Carr, 1988; Chapman and Hesketh, 2000). By adulthood, an individual with DS is likely to have an IQ of 25 to 55, equating their mental age to be approximately 7–8 years old (Pennington et al., 2003).

The specific cognitive shortfalls associated with DS include deficits in memory, language, speech, hearing, processing speed and numerous aspects of executive function (Silverman, 2007; Lanfranchi et al., 2010). In early childhood, ID is not as prominent, with steady cognitive decline being observed from late childhood into early adulthood, with a further decline in middle to late adulthood, typically associated with early onset Alzheimer’s disease (Grieco et al., 2015). Interestingly, there is a discrepancy between cognitive tasks, with verbal-based tasks being performed worse than non-verbal-based tasks (Abbeduto et al., 2001; Couzens et al., 2011; Channell et al., 2014). Language skills until age 5 are generally within the expected developmental range, however, steadily decline into adulthood (Guralnick, 2002). These language shortfalls have been hypothesised to be a secondary consequence of the deficit in verbal working memory more specifically, in the phonological loop located in the temporal lobe (Chapman, 2006; Vicari and Carlesimo, 2006). Additionally, those with DS have been shown to exhibit numerous deficits in executive function, including impairments of attention, inhibition, processing speed, multi-tasking, self-monitoring, working memory and organisation (Grieco et al., 2015). These executive function skills are processed in the frontal lobes and have a secondary impact on one’s learning ability. Furthermore, learning and memory deficits are believed to result from errors in the encoding and retrieval of memories, which are processed in the temporal lobe and the hippocampus (Carlesimo et al., 1997). While there is some debate in the literature over the underlying mechanisms that result in these cognitive deficits, there is largely an agreement that the individual deficits in each cognitive area are largely of primary origin, with some top-down cascade also influencing cognitive functioning (Grieco et al., 2015). This conclusion has been supported by studies that identify alterations in brain structure in similar brain regions.

Deficits in cognition are almost always the result of abnormal brain structure, with global cortical volume loss often being correlated with cognitive decline (Jack et al., 2008; Soria-Pastor et al., 2008; Calabrese et al., 2011; Mcdonald et al., 2012). Consistently, the brains of individuals with DS are smaller when compared to healthy individuals, with a study on children finding a 13.3% reduction in total brain volume (Rachidi and Lopes, 2011; Śmigielska-Kuzia et al., 2011). This has been observed to increase to 20% in adults with DS (Kemper, 1991). Interestingly, this global reduction in brain volume has not been observed in trisomic mouse models (Contestabile et al., 2010). Some suggest that these findings indicate that volume is not directly related to cognitive impairment. However, we postulate that this may be a result of the difference in neuronal complexities between mice and humans, resulting in more severe and widespread disability in humans due to the complexity of our neuronal development when compared to that of mice. Global cortical volume loss often results from a catastrophic cascade of errors that leads to drastically reduced neuronal cell numbers. With that said, early DS mouse models exhibited increased ventricle size and brachycephaly (Guedj et al., 2012). These phenotypes typically indicate some form of cortical atrophy, which may be attributed to improper neurodevelopment or neuronal cell death that has also been observed (Arbones et al., 2019).

Across the literature, the primary regions affected in mice are the cerebellum and the hippocampus. Some mouse models have detected a cerebellar volume loss of up to 12% and in models that do not exhibit a volume loss, a lower density of neuronal cells is still observed (Baxter et al., 2000; Olson et al., 2004a). A well-characterised mouse model for DS is the Ts65Dn mouse which is trisomic for approximately two-thirds of the genes orthologous to human chromosome 21 (Hsa21). Interestingly, in Ts65Dn mice, there is no alteration in hippocampal volume at 7 months of age and no alteration in neuronal density at one month of age (Lorenzi and Reeves, 2006; Olson et al., 2007). However, at 16–17 months old, neuronal density has been found to be significantly lower in the cornu ammonis (CA) 1 region of the hippocampus and synapse density has also been found to be reduced in the dentate gyrus, CA1 and CA3 when compared to diploid mice (Kurt et al., 2004; Lorenzi and Reeves, 2006). This is similar to what is observed in humans, where these regions have been reported to have a reduced volume of the brainstem, frontal, temporal and parietal lobes. It is suggested that this is indicative of a decreased formation of new neurons during development and increased atrophy in adult life. An overview of the common neuronal features of DS in humans can be found in Figure 2. Additionally, in this figure we have indicated similar phenotypes observed in the murine models previously mentioned.

Studies on humans with DS have noted similar abnormalities to the murine models listed in comparable brain regions. Evidence of improper cortical development from DS-affected brains have been observed at the cellular level with abnormal dendrite formation, impaired neurogenesis, lower neurotransmitter counts, and reduced synaptic proteins (Vacca et al., 2019). Notably, dendritic abnormalities in the hippocampus and the dentate gyrus appear to be the most pervasive, with murine models exhibiting reduced synaptic density, altered dendritic arbours, and altered dendritic spines (Contestabile et al., 2010). Numerous other alterations in neurotransmitter systems, cellular mechanisms, and degradation due to age-associated dementia are outlined in the literature. However, these are outside of the scope of this review, but have been reviewed by Arbones et al. (2019) and Vacca et al. (2019). The previously mentioned alterations in brain morphology are common hallmarks of many forms of ID and provide the morphological basis for the poor cognition observed in DS individuals. Consistent neurobiological abnormalities had sparked interest in the possibility of a critical region of chromosome 21 that may be present in all cases of DS, providing a rationale for the main phenotypes and potential therapeutic target.

As there exists both a complete and partial trisomy of chromosome 21 in DS populations, the concept of a DSCR has been debated amongst the literature. Korenberg et al. (1990) hypothesised a DSCR that extended from the start of q21.2 to the end of chromosome 21. This was heavily based on earlier literature that pinpointed a crucial area on the distal end of chromosome 21. This was later refined by Delabar et al. (1993) using genotype–phenotype analysis of 10 people with partial trisomy 21 to the region D21S55 (~37.8 Mb) to MX1 (~41.7 Mb). They hypothesised that this region is responsible for 19 of the 33 phenotypes they assessed. Another paper by Korenberg et al. (1994) emphasised the importance of genes outside the D21S55 region and, through their own molecular and phenotypic analysis, defined a region with a proximal boundary of D21S17 (~35.9 Mb) and a distal boundary at MX1. All DCSRs mentioned can be seen in Figure 3 (Korenberg et al., 1990; Delabar et al., 1993; Korenberg et al., 1994; Yamamoto et al., 2011; Schnabel et al., 2018).

Since its conception, the idea of a DSCR has been heavily debated, with some reviews and more recent literature finding their own definitions of the region, such as in the review by Rachidi and Lopes (2008). This led to the question of whether it is valid to conclude that such a region exists at all. Recent research has failed to support the prevalence of a DSCR, as per the original Korenberg et al. (1990) hypothesis. Two critical studies on mice had shown that the triplication of the DSCR alone does not result in the characteristic facial or neurological DS phenotypes (Olson et al., 2004b, 2007). The physical phenotypes observed when comparing DS mice (Ts65Dn) and triplicated DSCR-only mice (Ts1Rhr) presented an opposite phenotypic pattern, where Ts65Dn mice were smaller while Ts1Rhr mice were larger (Olson et al., 2004b). Additionally, neurological characteristics like cerebellar volume, hippocampal size and function were either not as severe or protected in Ts1Rhr mice, suggesting that the DSCR proposed in the original hypothesis does not fully recapitulate the main DS phenotypes (Olson et al., 2007). However, some caution is advised when drawing conclusions based on this study as the orthologous region of Hsa21 in murine models is located across three separate chromosomes, potentially adding some variability. Additionally, animal models may require multiple copies to replicate the human phenotype, such as in experiments involving SOD1 replicating motor neuron disease (Ferraiuolo et al., 2007; Nardo et al., 2016). Future studies should be conducted with more copies of the DSCR to see if phenotypes differ. Additionally, the researchers bred trisomic Ts65Dn mice with monosomic DSCR Ms1Rhr mice, producing offspring that were trisomic for chromosome 21 with a normalised analogous DSCR (Olson et al., 2007). These DSCR-normalised mice were found to perform similarly in the Morris water maze when compared to a control euploid mouse (Olson et al., 2007). These researchers agree that the DSCR hypothesis has been disproved, however, they concluded that the genes within the DSCR are necessary but not sufficient to produce a learning deficit in DS (Olson et al., 2007). Most importantly, this suggested that a return to normal gene expression in DSCR genes may induce an improvement in cognitive functioning. Contradicting these studies, other researchers concluded that the DSCR is sufficient to produce the DS phenotype, coming to this conclusion as Ts1Rhr mice were found to significantly differ on 20 of 48 characteristics when compared to control ‘2 N’ mice (Belichenko et al., 2009). These included altered long-term potentiation effects, dendritic spine enlargement and density in the fascia dentata, among others (Belichenko et al., 2009). While they concluded that the triplicated region present in Ts1Rhr mice is sufficient to reproduce the DS phenotype, they also mention that differing combinations of single or multiple gene dosage effects may give rise to different phenotypes. This is very similar to a more recent ‘gene dosage effect’ hypothesis proposed by Moreau et al. (2021). This hypothesis suggests that DS results from an imbalance in gene dosage, leading to the overexpression of specific causative genes, which can alter interactions between other genes in the genome. Unlike the DSCR hypothesis, which attributes the DS phenotypes solely to the genes within a specific region. The gene-dosage effect hypothesis considers a broader range of genetic interactions across the entire genome that are responsible for the DS phenotypes. We consider this to be more plausible than the original DSCR hypothesis. However, the gene-dosage hypothesis requires more sophisticated methods to identify the most relevant genes involved in DS.

Pelleri et al. (2016) conducted a thorough review which detailed the score for association with DS of numerous individuals with partial trisomy 21 to identify a highly restricted DSCR (HR-DSCR). This region is defined by a triplication of genes present in all DS cases and absent in all non-DS cases. They identified the HR-DSCR region as genes with a prevalence score over 97. However, it contained genes only homologous to the chimpanzee genome that have not been thoroughly researched. We suggest that it would be more useful to direct future exploration toward genes just outside this HR-DSCR that are homologous to more common disease models. The small region with a score over 90 includes nearly the entire q22.13 segment (Pelleri et al., 2016). A high score in this study indicates an increased probability of association with DS. This region includes seven protein coding genes: DYRK1A, DSCR3, TTC3, PIGP, RIPPLY3, KCNJ15, KCNJ6, and DSCR4. Of these the former 5 are expressed in the adult brain and are potential targets for DS treatment. Given the wealth of literature and its potential implications for various diseases, our review will focus on the gene DYRK1A. From the Pelleri et al. (2016) study, this gene has a prevalence score of 91 out of 100 for its association with DS. Additionally, DYRK1A has been consistently overexpressed in DS human and mouse models and has been found to play a vital role in neural function, processing and development (Guimera et al., 1999; Dowjat et al., 2007). Interestingly, a recovery in the developmental cognitive deficit was reported after a partial rescue of DYRK1A in DS mice. Researchers utilised mice with a gene trap vector inserted in intron 4 resulting in disruption of the 321 amino acid kinase domain resulting in a haploinsufficiency of DYRK1A and was referred to as Dyrk1a^m1 (Jiang et al., 2015). They bred this mouse with their DS mouse model, Dp (16)1, to generate a DS mouse with a normalised DYRK1A expression, Dp (16)1/Dyrk1a^m1 (Jiang et al., 2015). These mice exhibited performance improvements in T-maze and contextual fear-conditioning tests when compared to Dp (16)1 mice (Jiang et al., 2015). Thereby supporting the potential causative role of DYRK1A in the cognitive phenotype and potential for recovery if the DYRK1A gene is normalised. All the chromosomal segments for the murine models are presented in Figure 3.

The conclusion of much of the literature appears to be that a strictly defined DSCR does not exist, and while there are certain genes that appear necessary to produce the phenotypes of DS, these are not sufficient when viewed in isolation. Therefore, we also agree that the restrictive DSCR approach is not adequate to explain the phenotypic outcomes of DS. Importantly, the recovery of the widely researched gene DYRK1A has shown to alleviate the severity of cognitive phenotypes in DS models, making the regulation of these genes a promising potential therapeutic strategy.

In humans, chromosome 21 contains over 300 genes, with only one-third found to be dose-sensitive and are hypothesised to be the primary genes associated with the DS phenotype (Yahya-Graison et al., 2007; Becker et al., 2014). DYRK1A is one of these dose-sensitive protein-coding genes that, if over or under-expressed, can affect essential cellular development and functional roles depending on the pattern of expression. Duchon and Herault (2016) postulated that the formation of active DYRK1A protein complexes may be the cause of the dose sensitivity (Duchon and Herault, 2016). Whereby the dosage sensitive protein forms a tripartite complex with two partners (Veitia et al., 2008; Duchon and Herault, 2016). This hypothesis was corroborated by co-immunoprecipitation studies that identified DYRK1A complexes that formed with cytoskeleton filamentous actin, neurofilaments and tubulin (Kaczmarski et al., 2014). However, the symptoms observed when DYRK1A is under-expressed differ from those when it is over-expressed, contrary to what this tripartite model may suggest (Arque et al., 2013; Raveau et al., 2018). Additionally, numerous DS studies and transgenic models overexpressing DYRK1A have found that DYRK1A protein activity is increased, contradicting this model. These findings suggest that even if a tripartite system exists, it likely does not act alone (Ryoo et al., 2007; Wegiel et al., 2011; Kim et al., 2016). Therefore, the DYRK1A system is likely to be more complex, with other mechanisms influencing symptomology. Further research may elucidate this underlying mechanism, as this is speculation at the time of writing.

The dose-dependent nature of DYRK1A has made it an attractive target gene and protein for therapeutic intervention, resulting in the development and discovery of numerous pharmacological therapies. Given the extensive range of DYRK1A inhibitors currently available, we will briefly outline the most notable ones, with a primary focus on those aimed at alleviating neurological deficits.

The mechanistic action of an ASO is largely dependent on its chemistry and the region of mRNA in which it is designed to anneal, which can be split into two groups: occupancy-mediated degradation and occupancy-only mechanisms, also known as steric interference. Depending upon the base modifications, phosphorothioate (PS) ASOs can be designed to exploit both groups of mechanisms, while PMOs do not support occupancy-mediated degradation.

As of April 2023, there have been a total of 13 antisense oligonucleotide therapies approved by the FDA, outlined in Table 3. These have received approval to treat previously untreatable genetic-based diseases like spinal muscular atrophy, Duchenne muscular dystrophy and familial amyloid polyneuropathy (Shadid et al., 2021). In this review we will not spend the time to explore the various FDA approved ASOs as this is outside the scope.

Developing treatments for neurological conditions remain some of the most challenging but these conditions have become a major focus for researchers in the field of oligonucleotide therapy. Antisense oligonucleotide researchers and clinicians treating neurological disorders are faced with the ongoing challenges of CNS drug development. Currently, there are no effective modalities of reaching the CNS without invasive methods. For example, spinal muscular atrophy treatment has seen success using ASOs, however drug delivery involves an invasive injection into the spinal canal.

To provide some context, spinal muscular atrophy is the most common genetic cause of death in infants and is an inherited neurological disorder that leads to the atrophy of the alpha motor neurons (D'amico et al., 2011). This causes a degeneration of the bulbar and spinal muscles, in addition to respiratory muscles, which later result in respiratory failure. The 2′-Methoxyethyl (MOE) PS ASO Nusinersen was approved by the FDA in 2016. This ASO increased the amount of SMN protein via inhibition of the negative splicing factors of intron 7 in the SMN2 pre-mRNA, thereby promoting the inclusion of exon 7 (Wurster and Ludolph, 2018). Phase I, II, and III studies on infants found statistically significant improvements after treatment with Nusinersen, which ultimately led the drug from the bench to the clinic (Finkel et al., 2016, 2017a; Darras et al., 2019). However, much like other ASOs, the treatment had to be administered repeatedly; on days 0, 15, 29, 64, and then every 4 months (Finkel et al., 2017b). Secondly, Nusinersen is too large a molecule to cross the BBB. Therefore, the ASO had to be administered via invasive intrathecal injection. The crossing of the BBB is one of the largest challenges facing drug treatment of neurological disorders.

The BBB is the divider between the CNS and the periphery, acting as a mediator which protects the brain from toxic substances while allowing a steady supply of nutrients. Many advances in ASO chemistry have improved cellular uptake, although not many have been designed to overcome the crossing of the BBB. Current ASOs do not efficiently cross the BBB due to their charged nature and large size, with some studies finding that less than 1% of ASOs delivered peripherally reach the brain (Agrawal et al., 1991; Cossum et al., 1993; Banks et al., 2001; Farr et al., 2014). Multiple methods are being investigated to increase BBB penetrating efficiency. One method utilises receptor-mediated endocytosis which has been used to successfully deliver ASOs to the brains of parenchyma via nanoparticles (Pardridge, 2007; Kozlu et al., 2014). Another method utilised is a 5–30 amino acid long, positively-charged, cell-penetrating peptide which has shown successful distribution in the brain after crossing the BBB in mice (El-Andaloussi et al., 2005; Du et al., 2011). Adeno-associated viral vectors and encapsulating the gene therapies in exosomes have shown some promise in crossing the BBB (Stanimirovic et al., 2018). However, adeno-associated viral vectors have shown some levels of toxicity in recent studies on primates (Keiser et al., 2021). This leaves the only effective modality of delivery being direct administration to the CNS via intrathecal injection (Geary et al., 2015). While this has been an effective delivery method for many individuals, there is a risk of developing lumbar puncture syndrome (Cordts et al., 2020). A summary of the main delivery methods for ASOs can be found in Figure 7.

We have identified a few potential strategies to modulate the expression of DYRK1A through utilising ASOs. One possible method for addressing DS phenotypes would be to lower the expression of a dose-sensitive, over-expressed gene transcript via inducing exon skipping to disrupt the reading frame. Theoretically, this will result in nonsense-mediated decay of the transcript and thereby reduce the expression of the over-expressed target protein. For this treatment to be effective, one would need to be aware of the importance of dosing the ASO to not suppress DYRK1A expression in an excessive manner (most likely around 33% reduction—for gene dosage normalisation), as this could result in negative side effects that may appear similar to DYRK1A syndrome phenotypes. It is unlikely to be completely suppressed but there would need to be tests on optimising dosage using titrations which may prove challenging, but possible. Antisense oligonucleotides offer an exquisite method for specificity and sensitivity. The DYRK genes have enough genetic differences to design ASOs that only target DYRK1A (Aranda et al., 2011). Moreover, although various DYRK1A transcripts exist, all have a similar reading frame, thus targeting out of frame exons in one transcript should target all DYRK1A transcripts with a similar effect.

Ultimately, ASOs have a high potential to treat numerous neurological disorders with high specificity. With current advancements, particularly with the more recent PMO and peptide-conjugated PMO technologies, we have witnessed significant increases in the safety profile and efficacy. Ultimately, providing these an advantage when compared to other DYRK1A inhibitors. Current research is highly focused on applying this treatment strategy to many other disorders that have previously been deemed untreatable. Current research has attempted to utilise ASOs to treat myotonic dystrophies, Huntington’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease to name a few (Gao and Cooper, 2013; Hinrich et al., 2016; Aslesh and Yokota, 2020; Boros et al., 2022). As ASO therapies become more widely adopted, we should see a reduction in costs due to streamlining synthesis consistency and efficiency. Particularly, the possibility of class approval for ASOs will speed up the application process and reduce costs attributable to ongoing clinical safety trials (Aartsma-Rus and Krieg, 2017). Moreover, if ASOs can be applied to widespread disorders like DS, the increased demand should help distribute the cost, making them more affordable.