The polyQ extensions in mHTT is inherited in an autosomal dominant manner and leads to a gain-of-function, whereby the mutant induces a toxic effect regardless of the presence of the normal HTT. The number of repeats inherited leads to somatic instability of the region, which causes the expansion of the polyQ repeat domain over time, which in turn aggravates the severity of the mutation (Kennedy et al., 2003). Additionally, a number of single nucleotide polymorphisims (SNPs) are linked to the CAG expansion, suggesting its involvement in the mutation (Warby et al., 2009; Carroll et al., 2011).

Even though HTT is expressed ubiquitously in the body, mHTT has the biggest cytotoxic effect in nerve cells, predominantly in the striatum. This could be due to the differential transcription of HTT mRNA in cells of neuronal and non-neuronal origin, where nerve cells have more HTT mRNA present in the cell nucleus, suggesting higher protein levels favouring aggregation (Didiot et al., 2018). Secondly, nerve cells show to be more prone to CAG region instability leading to its expansion, compared to non-neuronal glia cells (Kennedy and Shelbourne, 2000; Shelbourne et al., 2007). The third possible reason is the different processes regulating protein aggregation in specific parts of the brain, which lead to the accumulation of mHTT aggregates only in the striatum and not in the other brain parts, as shown in aged mice (Yang et al., 2020).

The development of HD is driven by the expansion of the polyQ region of HTT. However it is not the whole protein that is the involved in the pathophysiology, but only the N-terminal fragments, predominantly the ones encoded by exon 1, are identified as the cytotoxic variants (Mangiarini et al., 1996; Barbaro et al., 2015). The toxic variants are referred to as mHTT. mHTT aggregates and gives rise to nuclear and cytoplasmic inclusions (Lunkes et al., 2002; Landles et al., 2010), and its overexpression in mice brains leads to age-dependent accumulation in the striatum leading to the HD-like phenotypes (Yang et al., 2020). The N-terminal fragments were identified in human HD brains post mortem (Lunkes et al., 2002).

The N-terminal fragment can be generated through aberrant splicing of mRNA or through protease-mediated cleavage of the protein, and both mechanisms are affected by the pathogenic expansion of the polyQ region. The expanded polyQ domain leads to the incomplete pre-mRNA splicing of exon 1 to exon 2 (Neueder et al., 2018). The resulting mRNA has a stop codon after the exon 1, which in turn leads to the translation of only the exon 1 fragment (Sathasivam et al., 2012). HTT protein cleavage by caspase and calpain proteases occurs both in healthy and in HD brains (Kim et al., 2001). However, cleavage at specific sites of mHTT leads to the generation of cytotoxic fragments and development of HD (Gafni et al., 2004; Graham et al., 2006). Additionally, a HD-specific SNP, which introduces a missense mutation, alters the post-translational modifications of the protein and can induce different cleavage of the protein, leading not only to generating the toxic mHtt forms, but also an aggregating form of wild-type HTT (wtHTT) (Martin et al., 2018).

The pathogenic exon 1 of the mHTT protein consists of three domains, including the polyQ sequence flanked by N- and C-terminal domains (Figures 2A,B). The polyQ domain is a conformationally versatile peptide which can take on different structures, such as intrinsically disordered, an α-helical conformation near the N-terminal domain, or a β-sheet confirmation found in mHTT aggregates (Boatz et al., 2020; Urbanek et al., 2020). These polyQ conformations are highly influenced by conformational features of the flanking N- and C-

Terminal regions (Urbanek et al., 2020). The N-terminal region (HTT^NT) is a 17 amino acid long sequence and predominantly takes on the form of a compact α-helical coil structure (Thakur et al., 2009). The coiled HTT^NT acts as an anchor tethering the protein to intracellular membranes and its compact structure resists aggregation (Thakur et al., 2009; Tao et al., 2019). However, the polyQ sequence can influence the HTT^NT conformation, whereby the expansion of the polyQ region induces HTT^NT unfolding and promoting its self-aggregation (Thakur et al., 2009; Boatz et al., 2020). The C-terminus is a proline-rich domain (PRD), which adopts an intrinsically disordered confirmation with polyproline II helices (Boatz et al., 2020). In contrast to HTT^NT, the PRD sequence has a suppressive effect on the polyQ protein aggregation potential (Bhattacharyya et al., 2006).

The formation of mHTT aggregates is self-initiated and is dependent on the polyQ region length, protein concentration and time (Boatz et al., 2020; Scherzinger et al., 1999). The mHTT protein is able to spontaneously aggregate and form different structures such as monomers, oligomers and fibril-rich inclusion bodies (IB) (Olshina et al., 2010; Wagner et al., 2018). The oligomeric aggregates are small, soluble, and are found in the nucleus and in the cytoplasm of the cell (Kim et al., 2016; Yang et al., 2020). The IB, on the other hand, are insoluble protein aggregates, found in both the cytoplasm and in the nucleus (Bäuerlein et al., 2017). IB are mostly spherical and made of fibrils (Bäuerlein et al., 2017), which are long rod-like structures of tightly packed mHTT proteins (Boatz et al., 2020). The mHTT fibrils are amyloid-like structures where the mHTT polyQ region forms a tightly packed, highly rigid and dehydrated anti-parallel β-sheet core flanked by mHTT N- and C-terminal domains (Boatz et al., 2020) (Figure 2C). The molecular architecture of the deposits share a number of features with aggregates found in Alzheimer’s and Parkinson’s disease (Boatz et al., 2020).

Notably, these structural data are derived from artificial systems. In other neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease the shape of fibrils obtained from primary material differ in structure from fibrils produced in vitro (Scheres et al., 2020; Schweighauser et al., 2020). It remains to be seen how closely artificial HTT structures resemble fibrils in vivo.

The process of HTT aggregation has an initial a lag phase, followed by an exponential growth phase and then plateau. The aggregation of mHTT is initiated by primary nucleation where a spontaneous aggregation nucleus induces the conformation change of other proteins promoting their addition to the aggregate, through templated aggregation (Wagner et al., 2018). The size of the nucleus can range from a monomer to a oligomer (Chen et al., 2002; Thakur et al., 2009). The conformational changes of the N-terminal domain of mHTT, caused by the expansion of the polyQ region, accelerate nucleation and aggregate formation (Thakur et al., 2009; Boatz et al., 2020).

Aggregates expand through the interactions between the polyQ repeat regions, which adopt a β-sheet conformations (Boatz et al., 2020). These aggregates elongate into fibrils, speeding up the growth process, by recruiting new monomers. Elongating fibrils can induce fibril-dependent nucleation, leading to the formation of fibril branches (Wagner et al., 2018). Fibrillary branching is seen in HTT aggregates, but not in other amyloid fibrils (Boatz et al., 2020). Interestingly, the elongating fibrils can also recruit wtHTT (Chen et al., 2001; Slepko et al., 2006), which naturally do not form fibrillary structures.

HTT oligomers and IBs greatly impact cell function by attracting and sequestering a number of proteins involved in processes of energy production, protein trafficking, RNA post-translational modifications and cell death (Ratovitski et al., 2012; Kim et al., 2016; Ramdzan et al., 2017). This altering of key cellular functions leads to the complete dysregulation of the cell, causing it to gradually slow-down and ultimately resulting in cell death.

In cell culture models, mammalian soluble mHTT oligomers can interact with up to 800 different proteins (Kim et al., 2016). The protein interaction is dependent on the length of the polyQ repeat of mHTT and the time of the mHTT expression (Kim et al., 2016). The proteins, which interact with the oligomers, are important in key cellular functions of RNA-binding, biogenesis, transcription, translation, cytoskeletal organisation, mitochondrial function, and vesicle transport (Ravikumar et al., 2004; Beal, 2005; Kim et al., 2016). The mHTT fibrils and IBs have a different range of interacting proteins than the mHTT oligomers, where IB interact with about 80 proteins (Kim et al., 2016). This is probably due to their more rigid and tightly packed structure and insolubility. The IB are enriched with proteins associated with chaperones and other proteolysis proteins, as the IB show a high concentration of ubiquitin (Tan et al., 2008; Kim et al., 2016; Ramdzan et al., 2017). The proteins interacting with the IB often contain prion-like sequences, which can be easily recruited to the inclusions after their formation (Ramdzan et al., 2017). Electron tomography reveals in artificial systems that IBs also come in contact with intracellular membranes, mostly the endoplasmic reticulum (ER) membrane. The IB and ER membrane interaction leads to the “freezing” of the ER membrane dynamics at the site of interaction. This IB and ER contact results in the reorganisation of the proteins at the membrane and thus impairing the proper function of the ER (Bäuerlein et al., 2017). It is not clear, however, whether interactions of IBs with the ER membrane observed in artificial systems has functional relevance.

In addition to the intracellular effect of mHTT in cells that express it, mHTT is able to spread its aggregating potential to neighbouring cells. The mHTT monomers and aggregates can spread through the late endosomal/lysosomal secretory pathway and via cell-cell contacts through tunnelling nanotubes (Costanzo et al., 2013; Trajkovic et al., 2017), and are also found in the cerebral spinal fluid of patients (Tan et al., 2015). The spread of mHTT leads to the induction of mHTT aggregation in neighbouring cells and leads to the spread of aggregates into other parts of the brain (Babcock and Ganetzky, 2015). Interestingly, the spread of mHTT can also nucleate wtHTT aggregation (Isas et al., 2017). The uptake of mHTT aggregates by healthy cells, not expressing mHTT, increases the percentage of wtHTT dimers compared to monomers, which can lead to wtHTT aggregation (Sameni et al., 2020). Therefore, the spread of mHTT between cells is able to infect healthy cells which do not have the mHTT gene (Jeon et al., 2016) and lead to their functional derailment.

A factor that enhances the cytotoxicity of mHTT is the reduced functionality of the protein quality control and turnover machinery in case of HD. The half life time of wtHTT in Q19 cells is 57 h, while that of mHTT in Q68 cells is reduced to 28 h (Wu et al., 2016). In healthy tissue, protein turnover is controlled by molecular chaperones, the ubiquitin proteasome system (UPS) and lysosomal proteolysis/autophagy systems, such as chaperone-assisted autophagy (CASA) and chaperone-mediated autophagy (CMA). One of the signals enhancing the clearance of wtHTT is its phosphorylation (Thompson et al., 2009). However, in HD, the HTT protein turnover is decreased. The expanded polyQ tract slows down the degradation rate of mHTT, by reducing mHTT phosphorylation (Thompson et al., 2009) and impairing its lysosomal uptake via macroautophagy (Qi et al., 2012). The cellular defence mechanisms attempt to compensate for the reduction in mHTT degradation, by upregulating the CMA pathway. However, over time, the efficiency decreases, which leads to accumulation of mHTT allowing for its aggregation (Koga et al., 2011). Additionally, the UPS system efficiency is reduced, as different components of the system are sequestered by different mHTT aggregate forms (Tan et al., 2008; Kim et al., 2016; Ramdzan et al., 2017). These defects in the protein turnover machinery favour the accumulation of aggregating proteins; all of this only aggravates cellular dysfunction leading to its death.

The current understanding of the molecular basis of HD, allows for the identification of the toxic agent, mHTT, and the different forms of aggregation that it adopts. The awareness of how the different structures induce cell damage leading to cell death provides a promising platform to propose mechanism that could be used to treat the development of HD.

ASOs are short, synthetic, single-stranded oligonucleotide analogues. They are 16–22 nucleotides long and bind predominantly to pre-mRNA through Watson-Crick binding (Southwell et al., 2018). Depending on the site of binding to the mRNA, ASOs can evoke a number of cellular responses which can prevent protein expression (Rinaldi and Wood, 2018) (Figure 3). ASOs binding can cause the degradation of mHTT pre-mRNA by the RNase H1 endonuclease, which recognizes the DNA and RNA duplex (Southwell et al., 2018). Another response is the ASO mediated translational arrest, where the bound oligonucleotide stalls the translational machinery on the mRNA (Gagnon et al., 2010). A third potential outcome is the altering of the pre-mRNA splicing, where ASOs can mask splicing sequences and lead to the expression of an altered and non-toxic protein (Evers et al., 2014).

The design of ASOs can target a number of different sequences that are specific to the mHTT pre-mRNA, allowing for the selective targeting of mHTT production. ASOs can be designed to selectively target the expanded polyQ repeat region of the mHTT mRNA, without affecting the wtHTT mRNA levels (Gagnon et al., 2010). Another ASO target can be one of the 50 identified viable HD-specific SNPs; since ASOs target the pre-mRNA, the presence of both, exons and introns in the sequence increases the number of possible SNP target sites (Carroll et al., 2011). The fine tuning of ASO design to the targeted SNP is vital, as different ASO-SNP combinations demonstrate varied tolerability and efficiency in knocking down protein production (Southwell et al., 2014).

Preclinical tests performed in vitro and in vivo in mice and non-human primate models suggest that ASOs are able to reduce mHTT in a dose-dependent manner, both in the nucleus and the cytoplasm (Didiot et al., 2018). This ASO-mediated mRNA suppression can reverse the disease course, by rescuing the HD caused motor dysfunction and brain atrophy (Kordasiewicz et al., 2012) and by attenuating the psychiatric and cognitive impairments (Southwell et al., 2018).

The size of the ASO molecules prevent them from passing the blood-brain barrier, therefore ASOs need to be delivered by intrathecal delivery, straight to the cerebrospinal fluid (CSF) (Southwell et al., 2018). Once in the CSF, ASOs efficiently pass through nerve cell membranes, have a long half-life and are effective in low doses (Southwell et al., 2018). Injections into the brain, however, are invasive neurosurgical procedures with considerable risks. Modifications of ASOs increase their metabolic stability and affinity to the pre-mRNA target, greatly enhancing their drug-like properties. These alterations target the phosphate as well as the sugar groups of the molecule (Dhuri et al., 2020). Current advances in ASO-based therapies aim to improve their delivery through the blood-brain barrier by designing new ASO structures, such as the tricyclo-DNA ASO molecules (Imbert et al., 2019), or by encompassing ASOs into nanocarriers (Min et al., 2020).

RNAi based technologies can be used as therapeutic approaches to reduce mHTT levels, as they use the endogenous RNAi mechanism that mediates post-transcriptional gene silencing. RNAi is facilitated by a small interfering RNA (siRNA), generated by endoribonuclease processing of a double stranded RNA (dsRNA). The dsRNA is processed to generate the siRNA guide strand, and releases the complementary passenger strand, which is discarded. The siRNA then combines with the RNA-induced silencing complex (RISC), associate with the target mRNA, and induces its degradation (Boudreau et al., 2009). This endogenous RNAi pathway can be highjacked by introducing an artificial siRNA, to target and induce mHTT mRNA degradation. The siRNA molecule can either be delivered into the cell directly as a single stranded siRNA molecule, or encoded on a DNA plasmid and expressed in a dsRNA short hairpin RNA (shRNA) or artificial microRNA (miRNA) scaffold (Figures 4A–C).

Introduction of an artificially generated siRNA into the cell is an approach to target mHTT mRNA (Figure 4A). The siRNA needs no further processing, therefore once it enters the cell it can directly induce mHTT mRNA degradation via the RNAi system. Due to the large and negatively charged structure, the siRNA molecule need to be chemically conjugated or modified to enhance its delivery into the nerve cells, prolong its efficacy and reduce its cytotoxicity (Alterman et al., 2019; DiFiglia et al., 2007; Liu et al., 2003). Since, siRNA cannot pass the blood-brain barrier, it needs to be administered by intracerebroventricular injection straight into the CSF, from where it reaches the nerve cells.

The artificial siRNA mediated RNAi approach, first tested in cell culture (Liu et al., 2003), is able to suppress the production of mHTT. In vivo studies in animal models, show that siRNA silences mHTT expression, leading to reduced sizes of IB, prolonged longevity of striatal nerve cells, and reduced HD associated motor function disturbances (Alterman et al., 2019; DiFiglia et al., 2007; Wang et al., 2005). Carefully designed siRNA can specifically target mHTT, without disturbing production and function of wtHTT; this specificity is achieved by targeting specific HD-associated SNPs or specific nucleotide inserts or deletions in the sequence (van Bilsen et al., 2008; Pfister et al., 2009; Zhang et al., 2009).

ShRNA scaffold-based expression is preferable to direct siRNA delivery, as it offers a longer-lasting suppression of mHTT, due to the stable expression of siRNA from the vector. Expression of shRNA is under the regulation of an expression promoter, which can induce high levels of shRNA, increasing the mHTT mRNA silencing efficiency (Harper et al., 2005). The siRNA expression from the shRNA scaffold requires the expression and processing of shRNA to generate siRNA, before it can induce mHTT mRNA suppression (Figure 4B). The vector is delivered into the nerve cells via striatal injection of a recombinant adeno-associated virus (rAAV) vector (Rodriguez-Lebron et al., 2005). The vectors allow the successful internalisation and replication of the siRNA encoding DNA vector (Burger et al., 2004).

In vivo experiments in animal models on expressing shRNA-encoding siRNA against mHTT demonstrate that shRNA can prevent appearance of aggregates. Tests in mice demonstrate that shRNA expression reduces mHTT mRNA levels and IB formation, leading to improvements in the behavioural phenotype of HD (Harper et al., 2005; Rodriguez-Lebron et al., 2005). Experiments in rats indicate ability of shRNA to prevent the HD induced neurodegeneration of the striatum (Franich et al., 2008) and the possibility of targeting HD specific SNPs, thus increasing the shRNA specificity for mHTT (Drouet et al., 2014).

The use of an artificial miRNA scaffold for siRNA expression is similar to the shRNA-based approach (Figure 4C). Both generate a dsRNA molecule, both require rAAV vector delivery system and Intrastriatal injection, and are able to spread in the brain (Stanek et al., 2014). However, the artificial miRNA shows to be better tolerated in the mouse cerebellum, than the shRNA (Boudreau et al., 2009). Additional modifications of the miRNA scaffold structure can further increase its safety, by reducing unwanted off-target effects that may occur from the discarded passenger strand (Miniarikova et al., 2016).

In vivo experiments, which tested the ability and efficiency of artificial miRNA targeting the mHTT mRNA show that rAAV mediated expression of artificial miRNA can transduce over 80% of cells in the mice striatum (Stanek et al., 2014) and can also successfully spread in larger brains, such as that of a minipig (Evers et al., 2018). The expression of miRNA shows to be effective in lowering mHTT in different animal models with ranging brain sizes, from mice, rats, minipigs to sheep (Stanek et al., 2014; Evers et al., 2018; Pfister et al., 2018; Spronck et al., 2019). The lowering of mHTT using artificial miRNA appears to be safe in the putamen of rhesus macaque monkeys (McBride et al., 2011) and other in vivo experiments show no major adverse effects of artificial miRNA gene therapy.

A wealth of data indicates the potential of targeting the mHTT mRNA for preventing mHTT protein aggregation and subsequent development of HD. In fact, two molecules targeting mHTT mRNA are in phase I/IIa of clinical trials. First one is ASO IONIS-HTT_Rx that shows a dose-dependent ability to reduce mHTT in the CSF of adults with early HD (Tabrizi et al., 2019). Second, is an artificial miRNA molecule, AMT-130, developed by UniQure, for which the first clinical trials begun in 2020 (http://uniqure.com/gene-therapy/huntingtons-disease.php). These two molecules illustrate an unprecedented potential of mRNA targeting approaches to fight HD, even though there is still a long way to being accepted as therapeutics.

Despite promising results in animal experiments, RNA-based therapeutics for HD did not yet hit the clinics. Lack of experience with RNA-based drugs in the human brain means that tolerability and efficiency of a drug can vary between patients and the potential effects on the cellular machinery are not yet fully understood. The most significant limitation is, however, that RNA drugs do not pass the blood-brain barrier. Therapies can circumvent this mainly by two strategies, direct injection into the brain or CSF, or application by adenoviruses. Injections into the brain require a complex neurosurgical procedure. After application, it needs to be ensured that the RNA drug is spread through the tissue. While this is effective in animal models with ranging brain sizes, from mice, rats, minipigs to sheep (Stanek et al., 2014; Evers et al., 2018; Pfister et al., 2018; Spronck et al., 2019), a successful therapy needs to consider the mass of the human brain. The human brain is three orders of magnitude larger than that of the tested animals, making diffusion of a drug throughout the brain more challenging. Additionally, this application would need to be repeated regularly to ensure a constant suppression of the production of mHTT. Application of drugs via an adenovirus may reduce the invasiveness and they may offer long-term effects by installing a stable source for RNA production in nerve cells. However, once administered, the drug cannot be removed from the body and in case of adverse reaction, the drug can become a threat to the patient’s health.

UPS is one of the principal post-translational modification pathways, responsible for, amongst many, protein quality control, and maintenance of protein homeostasis. One of its main functions is targeting of misfolded proteins to proteasomal degradation. The UPS system relies on the post-translational conjugation of a small regulatory protein, ubiquitin, to the target protein. The ubiquitin tag can be a monomer or a polyubiquitin chain, consecutively attached to the lysine (K) residues of the previous ubiquitin. Depending on the lysine residue attachment, the ubiquitinated protein is destined for different degradation fates—K48 for UPS and K63 for autophagosome. The enzymatic cascade that leads to the covalent binding of ubiquitin molecules to the target protein involves the E1, E2, and E3 ligases which are responsible for activating, conjugating and ligating ubiquitin to the target protein, respectively (Hershko and Ciechanover, 1998) (Figure 5A).

The UPS system has potential to efficiently reduce mHTT aggregation and development of HD, by the means of mHTT degradation. Even though the UPS system is not efficient in endogenously degrading mHTT oligomers and larger aggregates, artificial tagging of mHTT monomers could reduce the mHTT pool and thus slow down aggregation. This approach is employed by two molecular strategies, the proteolysis-targeting chimera (PROTAC) and Trim-Away.

The proteolysis-targeting chimera (PROTAC) is a small molecule that artificially targets a protein of interest (POI) to the UPS based proteolysis pathway (Sakamoto et al., 2001) (Figure 5B). PROTACs are bifunctional molecules, consisting of a warhead specifically targeting a to-be-degraded protein, and a unit connecting to an E3 ligase. This allows the PROTAC to ensure ubiquitination of the target protein and initiate degradation. The protein targeting domain can be a small molecule, mimicking a known inhibitor of the target protein (Qiu et al., 2021; Yan et al., 2021) or a peptide sequence, designed based on POI crystal structure and the known protein-protein interaction motifs (Jiang et al., 2018; Jin et al., 2020).

PROTAC mediates the interaction between POI and E3 ligase enzyme, leading to the K48 polyubiquitination of POI, targeting it for proteasomal degradation. During this process, PROTAC is not degraded and therefore one molecule can catalyse the ubiquitination of multiple POIs. The PROTAC approach is dependent on UPS; hence, it can only target soluble proteins with cytosolic domains. In the context of mHTT, this approach can target the soluble monomers and oligomers of mHTT. Specifically targeting oligomeric aggregation precursors would allow specific removal of the course of the toxic aggregation without interfering with the biological function of the monomer.

PROTAC molecules efficiently cross the cell membrane and experimental data suggests they can be administered orally or by intraperitoneal injection (Bondeson et al., 2015; Wei et al., 2021). However, due to the size of the PROTAC molecule ranging from 900 to 1,100 Da (Atilaw et al., 2021), it is a challenge to develop PROTACs that may pass through the blood-brain barrier to reach the HD nerve cells, to avoid intrathecal injection into the CSF as discussed for RNA-based drugs. This challenge may become more significant of the PROTAC uses a peptide-based domain as warhead, as this further increases the molecular weight, hence reducing its bioavailability (Jin et al., 2020).

The E3 ligase ligand recruits the E3 ligase to the PROTAC bound POI. Initially, these were peptide based short motifs that endogenously recruit the E3 ligase (Sakamoto et al., 2001; Schneekloth et al., 2004). However, the large size of the peptide renders the PROTAC less efficient in passing through the plasma membrane. The identification of small molecule ligands for a number of E3 ligases, including mouse double minute 2 homologue (MDM2) (Smith et al., 2008), cellular inhibitor of apoptosis protein 1 (cIAP1) (Itoh et al., 2010), cereblon (CRBN) (Winter et al., 2015), and Von Hippel-Lindau (VHL) (Zengerle et al., 2015), allows for the design of more efficient small-molecule PROTACs. Each E3 ligase has a specific range of target proteins; therefore, the possibility of choosing the E3 ligase recruited by PROTAC is crucial for the efficiency of the method (Bondeson et al., 2018). So far, only four E3 ligases are being used in PROTAC, however over 600 E3 ligases are encoded in the human genome, and the ability to use more of them would greatly widen the therapeutic potential of PROTAC to target many more proteins (Bondeson et al., 2018).

Warhead and E3 recruitment unit are connected by a linker. This linker moiety is critical for stability and functionality of the PROTAC. It is often based on alkyl, poly (ethylene glycol) (PEG) or extended glycol chains. These materials are synthetically available, have known flexibilities and their structure can be easily be fine-tuned by robust chemical methods (Troup et al., 2020). The flexibility, length and the attachment site of the linker to the two active domains affects PROTAC potency and efficacy. This is because the correct relative spatial orientation of the POI and the E3 ligase is vital for effective POI ubiquitination (Cyrus et al., 2011; Smith et al., 2019; Sun et al., 2019).

Up to date, over 40 protein targets are successfully degraded using PROTACs (Sun et al., 2019). Most of the targets are cancer related proteins (Qiu et al., 2021; Wei et al., 2021; Yan et al., 2021), with one PROTAC molecule, ARV-110, in a clinical trial (Poh, 2020). The knowledge of PROTAC design and efficiency can be translated into proof-of-concept studies targeting mHTT (Tomoshige et al., 2017, 2018).

The efficiency of mHTT degradation by PROTAC was tested in an in vitro cell culture system (Tomoshige et al., 2017, 2018). The PROTACs target mHTT soluble oligomers through small molecule probes, benzothiazole-aniline (BTA) and phenyldiazenyl benzothiazole (PDB), and lead to POI ubiquitination by the cIAP1 E3 ligase. The PROTACs successfully reduce mHTT levels in primary cell cultures derived from HD patients in a dose-dependent manner. In this study, however, the tested PROTACs are unable to differentiate between mHTT and wtHTT, as the levels of the latter are also reduced during treatment (Tomoshige et al., 2017, 2018). This could be a potential downside as the effect that reducing wtHTT levels has on the cell is still unsure. Nevertheless, this preliminary evidence of targeted mHTT degradation provides a promising platform for the development of PROTAC as a HD therapeutic.

Trim-Away is another method of rapid POI degradation, which utilizes the UPS system (Figure 5C). The approach relies on the antibody based recognition of POI, and binding of the cytoplasmic antibody receptor and simultaneously E3 ubiquitin ligase, TRIM21 (Clift et al., 2017). The TRIM21 E3 ligase is produced in different cell types and tissue, and efficiently attaches to the Fc of the antibody. This binding leads to the autoubiquitination of the TRIM21, and this signal induces proteasome-based degradation of the whole protein complex. Both mHTT and wtHTT proteins have been tested as POIs for the Trim-Away approach (Clift et al., 2017). Microinjection of the designed POI targeting antibody resulted in the selective degradation of mHTT. This method was designed with a purpose of generating protein knockdowns used in protein function research. However, Trim-Away has not yet been successfully applied to develop new therapeutic approaches involving protein degradation.

A drawback of this method as a potential therapeutic is the required delivery of an extracellular antibody into the nerve cells. Methods such as injection, transfection and electroporation are not feasible in a living brain. Due to their large size, antibodies are unable to pass the brain-blood barrier or cell membrane and so require a new functional delivery method that would allow their entry into the brain and the nerve cell.

An alternative protein degradation system is the autophagy-lysosomal pathway, which sequesters and breaks down cytoplasmic components via the lysosome (Shaid et al., 2013). Approaches based on this pathway have the potential to target larger mHTT aggregates that cannot be degraded by the UPS system. Endogenously, the autophagy process is initiated by the formation of a phagophore, a lunate-shaped membrane (Figure 6A). The phagophore elongates and engulfs proteins and organelles designated for degradation, finally closing off to form the autophagosome vesicle. The vesicle then fuses with a lysosome, to form the autolysosome, where the lysosomal enzymes degrade the sequestered proteins and organelles. This system is able to degrade proteins in both a selective and non-selective manner, where cellular queues and specific K63 ubiquitin tagging can lead to targeted protein degradation (Shaid et al., 2013).

Autophagy based techniques that operate through the removal of proteins through targeting them to the autophagosome, described below, are the autophagosome-tethering compound (ATTEC) and autophagy-targeting chimera (AUTAC). The method based on the endo-lysosomal degradation pathway is a lysosome-targeting chimera (LYTAC) and another method is using the CMA pathway; in both cases proteins are degraded by targeting them straight to the lysosome.

The autophagosome-tethering compound (ATTEC) is a direct strategy to target POI for degradation via the autophagosome. ATTEC mediates the tethering of POI to LC3, one of the main autophagy proteins found in the phagophore and later autophagosome (Li et al., 2019) (Figure 6B). Once sequestered in the autophagosome, the POI is destined for degradation. A proof-of-concept study identified molecules that efficiently interact with both mHTT and LC3 (Li et al., 2019). The tests conducted in HD patient primary cell lines, Drosophila and mouse models with human mHTT, revealed that the molecules can specifically target the mHTT to the autophagosome, leading to mHTT degradation and its reduced levels. The identified molecules do not show any major adverse effect on the cellular function, and interestingly, do not affect the global autophagy function. Additionally, some of the identified molecules can pass through the blood-brain barrier, and can function at low concentrations. Even though the tests so far are based on in vitro cell culture models and in vivo tests, the preliminary results showing successful and specific mHTT degradation indicate a promising therapeutic potential of ATTEC for HD and other proteopathy diseases (Li et al., 2019).

A recent approach that targets proteins for degradation via autophagosomes, is the autophagy-targeting chimera (AUTAC) based system (Takahashi et al., 2019) (Figure 6C). The principle of targeting proteins is similar to that of ATTEC; however, the structure and function of AUTAC resembles that of PROTAC, as both have a POI targeting domain and a degradation tag, joined by a linker moiety and both induce POI ubiquitination. The AUTAC molecule has a guanine derivate domain, which upon binding to POI mimics a protein post-translational modification of S-guanylation. The tag is recognized by the ubiquitination system and leads to the K63 polyubiquitination of the POI. The selective autophagy pathway, via the p62 reporter of autophagy, in turn recognizes this modification and sequesters POI in the autophagosome and destines it for degradation.

The AUTAC approach has not yet been tested on HTT; however, in vitro proof-of-concept studies show that this technique can successfully degrade cytosolic proteins as well as larger structures, such as mitochondria. The ability to degrade structures of different sizes makes AUTAC an interesting approach for degrading larger mHTT aggregates. Further in vitro and in vivo studies, together with the knowledge gained from PROTAC design, can make the AUTAC molecule a very promising HD therapeutic method.

A different cellular pathway, based on lysosomal degradation, considered for targeted protein degradation is CMA (Figure 7). CMA targeted degradation is based on the chaperone-dependent selection of protein targeted for degradation, and its direct translocation across the lysosomal membrane. An adapter molecule facilitates the interaction between POI and the chaperone complex, HSC70 and co-chaperones. The translocation through the lysosome membrane occurs through interaction of HSC70 with a transmembrane lysosomal protein, lysosome-associated membrane protein 2 (LAMP2A). This binding induces LAMP2A multimerisation and simultaneously the chaperone complex unfolds the POI. The POI then passes through the formed LAMP2A channel, and into the lysosome, where it is degraded (Xilouri and Stefanis, 2015).

A proof-of-concept study has demonstrated feasibility of the CMA based approach of targeted mHTT degradation (Bauer et al., 2010). The molecule used to induce mHTT degradation, called RHQ, had a peptide sequence that interacts with the polyQ tract, and a HSC70 binding motif. In vitro studies show that RHQ is able to distinguish between the polyQ size difference between mHTT and wtHTT, and selectively induce mHTT monomer degradation. The in vivo experiments indicate the efficiency of RHQ in degrading mHTT in.

The mice striatum and reversing the HD symptoms, by improving metabolic and neurobehavioral outcomes. A shortcoming of this approach is the peptide structure of the molecule, which reduces the efficiency of passing through the plasma membrane, and requires its delivery in vivo via the rAAV approach. Nevertheless, the CMA based approach presents yet another potential therapeutic solution for targeting protein aggregation in HD, by targeting mHTT to the lysosome.

The previously presented methods focus on the degradation of cytoplasmic mHTT monomers and aggregates. However, mHTT is also found in the extracellular space, where it induces the spread of mHTT aggregation in the brain tissue. This form of cytotoxic mHTT can potentially be targeted using the lysosome-targeting chimera (LYTAC) technology, which exploits the endosomal/lysosomal pathway and triggers the degradation of extracellular and membrane bound proteins (Banik et al., 2020) (Figure 8). The approach is based on tethering the POI to a lysosome targeting receptor (LTR) via the LYTAC molecule. The POI attaches to LYTAC by a targeting sequence, which is either an antibody (Ahn et al., 2021; Banik et al., 2020) or a small synthetic peptide (Ahn et al., 2021). This interaction of POI and LTR induces the internalisation of the complex by endocytosis. Upon the change in pH of the endosome, the POI and LYTAC is released from the LTR, which is then recycled back to the membrane. The POI and LYTAC are degraded by proteases, after the endosome and lysosome fusion.

There are no studies of applying this method to degrade mHTT, but two proof-of-concept studies show that this method successfully degrades both, membrane bound and extracellular proteins, both in vitro and in vivo (Ahn et al., 2021; Banik et al., 2020). Additionally, the targeting of POI has been shown in both, a tissue-specific and non-specific manner. This can be achieved by the specialised targeting of a LTR by the LYTAC LTR targeting motif (Ahn et al., 2021; Banik et al., 2020). Together, these features of LYTAC demonstrate unprecedented potential for targeting extracellular mHTT in the brain and the central nervous system. Being able to degrade free flowing mHTT can reduce its potential of infecting new cells and nucleating mHTT and wtHTT aggregation, thus slowing down the spread of HD in the brain.