Huntington disease (HD) has the highest prevalence of all autosomal CAG repeat neurodegenerative diseases. The mutant huntingtin gene, which has an expansion in the CAG repeat region to more than 36 CAGs within exon 1 (1
), confers toxicity ascribed to the mutant protein, although mRNA contribution to the disease process has yet to be fully tested. Neuropathology is characterized by striatal and cortical neuronal atrophy (2
). Patients with HD usually develop involuntary movements, cognitive dysfunction, and behavioral changes in the fourth decade of life.
Despite the identification of mutant huntingtin as the genetic cause of HD in 1993 (1
), there is a high unmet need for disease-modifying therapy. Several therapeutic candidates with the potential to alter the underlying progression of disease have been tested in clinical trials (3
). These agents include neuroprotective strategies to counter the toxic cellular effects of mutant huntingtin protein and cellular replacement strategies to counter the loss of neurons in the striatum. Such strategies are indirect and function downstream of the effects of mutant huntingtin protein. At best, these approaches repair damage after it has occurred. Moreover, mutant huntingtin protein has multiple deleterious molecular and cellular consequences, each of which could be the basis of a therapeutic approach. However, targeting these individually might be insufficient for significant clinical benefit (Figure ). With the advent of oligonucleotide approaches to gene suppression such as RNAi and antisense oligonucleotides (ASOs), therapeutic strategies directly targeting the causative gene — mutant huntingtin — may be developed and tested. In theory, by reducing huntingtin mRNA levels, the synthesis of mutant huntingtin protein would be reduced, representing a strategy upstream in the pathogenetic process, potentially preventing cellular damage.
Figure 1 Multiple pathogenic mechanisms of mutant huntingtin include loss of BDNF neurotrophic support for striatal neurons, impaired axonal transport, altered vesicle recycling, mitochondrial dysfunction, increased autophagy, protein aggregation, and transcriptional (more ...)
Gene silencing through RNAi or ASO action should be a viable therapeutic strategy in the treatment of HD. Current oligonucleotide-based approaches have not allowed for the targeting of mutant alleles with CAG repeat expansions, such as mutant huntingtin, with high selectivity or potency in vitro (9
). Since wild-type huntingtin has numerous physiological activities in cells that are important for neuronal function, complete suppression of both mutant and wild-type huntingtin may not be desirable (11
), and allele-specific silencing of mutant huntingtin by targeting associated SNPs (12
) represents a promising alternative. Another potential therapeutic strategy comprises simultaneous partial (rather than complete) lowering of wild-type and mutant huntingtin expression, with the aim of reducing mutant huntingtin expression sufficiently for therapeutic benefit while preserving sufficient wild-type huntingtin expression for maintaining normal cellular physiology. Clearly, specific and potent allelic silencing, if achievable, would be ideally suited for therapy in autosomal dominant diseases such as HD.
Allele-specific silencing is predicated on experimentally verified functions of wild-type huntingtin. Huntingtin has multiple effects in development (15
) and neuronal physiology (15
), including regulation of transcription (16
), membrane dynamics (19
), mitochondrial efficiency (20
), BDNF transcription (15
), autophagy (23
), and endosomal recycling (ref. 7
and Figure ). Huntingtin knockout mice (Hdh–/–
) exhibit embryonic lethality by day 8.5, at a time preceding nervous system development (27
). Elimination of wild-type huntingtin in adult mice is associated with neuronal loss in several brain regions, especially cortex and striatum, motor deficits, behavioral changes, and shortened lifespan (11
). Overexpression of wild-type huntingtin improves survival in cultured neuron-derived cells (28
) and neuroblastoma cells expressing mutant huntingtin (30
). Loss of wild-type huntingtin increases caspase-3 activity and apoptosis (31
). During development, elimination of wild-type huntingtin reduces neuronal survival and produces mice with behavioral changes (32
). Overexpression of wild-type huntingtin in the YAC128 mouse model of HD reduces the extent of striatal neuropathology (33
Wild-type huntingtin is associated with vesicles in neurons (34
). Huntingtin in axons moves in anterograde and retrograde directions (35
), and reduction of wild-type protein levels is associated with reduced BDNF (36
) and mitochondrial trafficking in neurons (37
). Post-translational modification of wild-type huntingtin provides further regulation of axonal trafficking (38
). Wild-type huntingtin forms a complex with Rab11 and affects recycling of transferrin receptor and EAAC1, a receptor that controls uptake of cysteine in neurons (39
). Loss of wild-type huntingtin impairs vesicle recycling and increases production of reactive oxygen species (40
). Mutant huntingtin has similar effects in neurons as disrupting the Rab11 complex (39
To summarize, elimination of wild-type huntingtin has multiple harmful impacts on adult neurons: loss of pro-survival mechanisms, decreased levels of an essential growth factor (BDNF), and impaired axonal trafficking and endosomal recycling, leading to accumulation of toxic reactive oxygen species. Thus, silencing of the huntingtin gene must be done with the recognition that wild-type huntingtin expression should be maintained at safe levels.
Huntingtin is a large protein (~350 kDa) with many potential sites of protein interaction, including the polyglutamine region. In theory, expansion of the polyglutamine series can strengthen protein interactions or permit new interactions (41
). Immunoisolation and pull-down experiments have been used to create protein networks of huntingtin (42
). Wild-type huntingtin has many sites for proteolytic cleavage and may be targeted by caspases, calpains, and aspartyl proteases (44
). Mutant huntingtin shares these sites of proteolysis (45
). It is unclear if differences in cleavage rates contribute to toxicity of mutant huntingtin fragments through changes in folding, clearance of mutant huntingtin fragments, or the propensity for mutant huntingtin (and fragments) to aggregate or form aberrant protein interactions (48
). Thus, combinatorial changes by the mutant huntingtin and its fragments might underlie complexities in HD pathogenesis. Thus, directly silencing mutant huntingtin offers a most rational therapeutic strategy.
RNAi and ASOs are the two major approaches to therapeutic gene suppression (Figure ). They offer powerful solutions for selectively inhibiting disease targets, including those such as huntingtin that are difficult to modulate specifically with traditional pharmaceutical classes such as small molecules or proteins, due to protein size or intracellular location. Although small-molecule screens have been conducted, initially to prevent huntingtin aggregation (51
) and more recently to reverse pathogenic processes (52
), clinical testing has not yet been initiated with these small molecules. Furthermore, neither the function of the pathogenic protein nor its interacting partners needs to be known to implement either protein lowering approach; the only consideration is that excess protein drives pathology, as is the case with mutant huntingtin protein. A contribution of mutant mRNA to pathology would also be susceptible to gene silencing. Both therapeutic approaches require adequate in vivo delivery of drug; this critical aspect for drug development is discussed in detail later in this review.
siRNA, miRNA, and shRNA cellular pathways.
After entry into cells, synthetic siRNAs leverage the naturally occurring process of RNAi in a consistent and predictable manner by directing sequence-specific degradation of mRNA. Since siRNAs harness an endogenous catalytic mechanism, potent and selective siRNAs with picomolar EC50
s in vitro can usually be identified if the starting pool of siRNAs is sufficiently large. Endogenous miRNA can be derived from a stem-loop structure in which opposite RNA strands have complementarity (Figure ). miRNA precursor structures can be found in introns and give rise to endogenous miRNAs through a series of enzymatic steps (53
). The extent of complementarity between miRNAs and target mRNAs is quite variable. In theory, the lack of exact complementarity could lead to off-target miRNA effects. Recent evidence suggests that the miRNA silencing confers a slow degradation of target mRNA, in contrast to the abrupt cleavage found in RNAi (54
). ASOs are single-stranded oligodeoxynucleotides of approximately 15–25 nucleotides that suppress the synthesis of the targeted protein. After entering the cell and binding to the complementary mRNA, ASOs reduce gene expression by enabling the RNase-H–mediated degradation of the target mRNA or physically blocking translation of the target mRNA (55
With careful oligonucleotide sequence design and selection, these approaches can be highly specific for the target of interest (Table ), and appropriate chemical modifications can provide stability in vivo (56
). A preferred sequence design for potential therapeutics comprises alignment of mRNAs from rodent, monkey, and human and identification of all possible conserved target regions (58
). Typically, the starting pool comprises hundreds or thousands of possible sequences. For a large gene such as huntingtin, with a transcript size of ~13 kb, approximately 13,000 sequences can be initially considered as candidates, since the starting pool of sequences is obtained by tiling across the entire huntingtin transcript (Figure ). If sufficient homology exists across species, hundreds of sequences against conserved regions can be designed and screened in vitro for subsequent testing in preclinical models and potential clinical advancement. The possible target regions within the mRNA of interest are subjected to a BLAST-like analysis to select sequences that are unlikely to result in off-target silencing of genes that have partial homology to the gene of interest (59
). These selected sequences are evaluated empirically in vitro to identify the most potent siRNAs from dose-response studies in cell culture systems.
Comparison of oligonucleotide therapeutic modalities to suppress huntingtin
Oligonucleotide therapeutic approaches for lowering huntingtin.
RNAs can potentially stimulate innate immunity (60
). With use of appropriate chemically modified nucleotides (e.g., 2′-O-methyl substitutions), immune stimulation can be eliminated (Table ) without compromising silencing activity (61
). These same chemical modifications, together with modifications at the 3′ end such as phosphorothioate linkages, provide the added benefit of stability against nucleases that are present in biological fluids (57
). Thus, with appropriate design, selection, and chemical modification, potent and specific oligonucleotides can be identified for in vivo studies and potential clinical testing.