Oligonucleotide-based therapy for neurodegenerative diseases.

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Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Oligonucleotide-based therapy for neurodegenerative diseases Iddo Magenn, Eran Hornsteinn Department of Molecular Genetics, Faculty of Biochemistry, Weizmann Institute of Science, Rehovot, Israel

art i cle i nfo

ab st rac t

Article history:

Molecular genetics insight into the pathogenesis of several neurodegenerative diseases,

Accepted 5 April 2014

such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, encourage direct interference with the activity of neurotoxic genes or the

Keywords:

molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are

Antisense oligonucleotides

recently emerging as an efficient strategy for drug development and these can be employed

Delivery

as new treatments of neurodegenerative states. Here we review advances in this field in

RNA interference

recent years which suggest an encouraging assessment that oligonucleotide technologies

siRNA

for targeting of RNAs will enable the development of new therapies and will contribute to

microRNA

preservation of brain integrity.

Neurodegeneration

1.

This article is part of a Special Issue entitled RNA Metabolism 2013.

Introduction

Oligonucleotide-based therapy covers a range of methods for modifying gene expression, which have the potential to revolutionize the development of therapeutics and biomedical practice. The uniting idea underlying oligonucleotidebased therapies is to interfere with gene expression at the post-transcriptional RNA level through Watson–Crick base pairing. In this review, we describe ways to change the molecular genetics status of the nervous system, and discuss the approaches by which synthetic oligonucleotides may be employed for brain and spinal cord therapy. We describe the specific challenges associated with RNA delivery into the central nervous system (CNS) by referring to strategies that are under investigation for a number of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's

& 2014 Published by Elsevier B.V.

disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). In many neurodegenerative diseases, the expression of some proteins is up-regulated as primary or secondary event in the molecular pathogenesis and often abnormal proteins possess aggregation tendency that is thought to play a pivotal role in the pathogenesis. Thus, mutated proteins or even accumulation and aggregation of wild-type proteins may be toxic and can contribute to disease onset or progression. In other cases RNA-based intervention can rescue the expression of a protein that is not well spliced or translated. Many of the strategies to manipulate the expression of specific genes at the RNA level have proven very promising in animal preclinical models and in the most advanced examples initial evidence for the relevance of oligonucleotide-based approaches already exist from clinical trials in human patients.

n

Corresponding authors. E-mail addresses: [email protected] (I. Magen), [email protected] (E. Hornstein).

http://dx.doi.org/10.1016/j.brainres.2014.04.005 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Magen, I., Hornstein, E., Oligonucleotide-based therapy for neurodegenerative diseases. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.04.005

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2. Biochemical properties of different types of therapeutic oligonucleotides The biochemical properties of oligonucleotide-based technologies divide the many chemical variants that can be generated through synthetic production into two primary categories: double stranded RNA (dsRNA) that generates RNAi activity through RISC-dependent silencing and single-stranded antisense oligonucleotides (ASOs). These two primary types of nucleic-acid molecule affect downstream targets by different mechanisms that are described in the following reviews (Bennett and Swayze, 2010; Davidson and McCray, 2011; Goodchild, 2011; Kole et al., 2012) and in the text below.

2.1.

Single stranded—antisense oligonucleotides

Antisense oligonucleotides, ASOs, is the more established family that is utilized for RNA-based therapy. These singlestranded oligos are able to either knockdown RNA expression or to modify splicing. Hence, ASOs can reduce or increase the expression of specific proteins, depending on context and biochemistry. ASOs mechanism of action depends on steric blocking of translation, inhibition of splicing or recruitment

of RNase H, and in this sense are essentially different from dsRNAs that impart RISC-dependent silencing. The phosphodiester bonds at the backbone of ribonucleic acid can be modified in order to change the chain biochemical properties. For example, in phosphorothioate nucleotides (PS), one oxygen atom is substituted by sulfur, thus conferring nuclease resistance. PS nucleotides also contribute to the pharmacokinetic profile by improving serum binding and enable RNase H digestion of target RNA. Thiophosphoramidate modifications or isosteres are also used as means for further improving ASOs nuclease resistance. Other non-natural backbones may be used to replace the ribose nucleic acid backbone with peptide nucleic acids (PNAs) or phosphorodiamidate morpholino oligonucleotides (PMOs). “Locked-nucleic acids” (LNAs), are bicyclic nucleic acids that tether the 20 -O to the 40 -C via a methylene bridge. LNAs inhibit nuclease activity and offer a superior binding affinity (Lennox and Behlke, 2011; Petersen et al., 2002; Petersen and Wengel, 2003). Additional insight into the therapeutic use and chemistry of oligonucleotides is reviewed in (Bennett and Swayze, 2010; Lennox and Behlke, 2011). Different nucleosides with distinctive properties can be assembled in a single synthetic oligo strand. Gapmer is a broadly used ASO design, which includes two nucleaseresistance sequences, upstream and downstream of a central

Table 1 – Selected clinical trials with RNA-based therapy in neurodegenerative diseases. Disease (alphabetical)

Target

Delivery system

Company (drug name)

ClinicalTrials. gov identifier (s)

Age-related macular degeneration (AMD)

DNA damageinducible transcript 4

Naked siRNA

Quark Pharmaceuticals (PF-04523655)

Amyotrophic lateral sclerosis (ALS)

SOD1

Naked ASO

Dystrophin, exon 51

Naked ASO (morpholino)

Isis Pharmaceuticals (SOD1Rx) Sarepta therapeutics (AVI-4658/PMO)

Dystrophin, exon 51

Naked ASO

GlaxoSmithKline (PRO051/GSK2402968)

Dystrophin, exons 44, 45, 53

Naked ASO

TTR

Naked ASO

TTR

LNP-formulated GalNacconjugated siRNA

Integrin alpha (4) beta1 CASP2

Naked ASO

SMN2

Naked ASO

Prosensa therapeutics (PRO044, PRO045, PRO053) Isis Pharmaceuticals (ISIS-TTRRx) Alnylam Pharmaceuticals (Patisaran ALN-TTR02) Isis Pharmaceuticals (ATL/TV-1102) Quark Pharmaceuticals (QPI-1007) Isis Pharmaceuticals (ISIS-SMNRx)

NCT00725686 NCT01445899 NCT00713518 NCT00701181 NCT01041222 Miller et al. (2013) NCT00159250 Cirak et al. (2012) NCT00844597 NCT01396239 NCT01540409 NCT01128855 NCT01910649 NCT01254019 NCT01153932 NCT01480245 NCT01462292 NCT01037309 NCT01826474 NCT01957059 NCT01737398

Duchenne muscular dystrophy (DMD)

Familial amyloid polyneuropathy

Multiple sclerosis Optic atrophy, non-arteritic anterior ischaemic optic neuropathy Spinal muscular atrophy (SMA)

Naked siRNA

NCT01960348

– NCT01064505 NCT01839656 NCT01780246 NCT02052791 NCT01703988 NCT01494701


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10 nucleotide sequence that is RNaseH-sensitive. The RNase H-sensitive ‘gap’ enables cleavage of duplexed mRNA:ASO, and is the source for the design nickname, whereas the flanking sequence provide stability (Teplova et al., 1999). Other types of ASOs depend on steric-blocking for inhibiting the translation of target mRNAs and do not induce RNase H-mediated cleavage activity (Bennett and Swayze, 2010; Lennox and Behlke, 2011). A couple of ASO drugs have been already approved by the U.S. Food and Drug Administration (FDA), including Fomivirsen (Vitravene) that was an antiviral ASO for repression of cytomegalovirus mRNA translation. It gained FDA approval for intraocular treatment for cytomegalovirus retinitis in immunosuppressed patients and was discontinued later due to commercial considerations. Another ASO drug is the systemically-delivered Kynamro (Mipomersen), which is indicated for treatment of familial hypercholesterolemia. Kynamro targets the apolipoprotein B-100 mRNA, a critical component of atherogenic lipid particles (Crooke and Geary, 2013). Several other drugs that have reached advanced clinical trials are listed in (Tse, 2013) and in Table 1.

2.2.

Modifying splicing by oligonucleotides

Splice-modifying ASOs can repair defective pre-RNA splicing, or regulate the presence of disease-related splice variant proteins. Splice-modifying ASOs are thought to be a valuable therapeutic option primarily because substantial numbers of disease-causing mutations dysregulate splicing. Accordingly, several splice-modifying ASOs reached advanced clinical trials (phase III) for modifying Dystrophin splicing in Duchenne muscular dystrophy, a severe muscle wasting disorder (Cirak et al., 2012; Koo and Wood, 2013). Splice-modifying ASOs are also developed for modifying SMN2 splicing in spinal muscular atrophy, a pediatric motor neuron disease (Hua et al., 2010; Rigo et al., 2012) and for Tau splicing in frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17) (Kalbfuss et al., 2001).

2.3.

RNAi and RISC-dependent silencing

RNA interference (RNAi) is an endogenous silencing mechanism in which short RNA molecules degrade specific mRNA molecules. The discovery of RNAi by Fire et al. (1998), earned them the Nobel Prize in physiology or medicine in 2006. RNAi serves as a defense mechanism against viral infection (Lu et al., 2005; Wilkins et al., 2005) and is broadly employed in regulation of expression at a posttranscriptional level. The largest group of genes that drive silencing activity in metazoan is called microRNAs (miRNA). miRNAs are genome-encoded hairpins that give rise to short, 22 nt, single stranded RNA with capacity to target dozens of targets (Fabian et al., 2010). Mature miRNAs are processed from a primary transcript known as a pri-miRNA by the microprocessor complex, which consists of an RNase III enzyme Drosha and a dsRNA-binding protein Dgcr8. A dsRNA hairpin of 70 nt, called pre-miRNA is then exported from the nucleus into the cytoplasm. In the cytoplasm, the pre-miRNA is bound and processed by Dicer, which resides at the heart of the RISC-loading complex. Dicer preferentially selects one ‘guide’ strand for loading onto the RNA induced silencing complex

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(RISC), while asymmetrically discarding the other, ‘passenger’ strand. RISC is a cytoplasmic silencing apparatus that elicits mRNA decay and translational inhibition, based on interacting with a particular ‘guide’ RNA, reviewed in Gregory et al. (2006). Because an initial step for the programming of RISC depends on its loading with a ‘guide’ RNA, vectors that can drive the expression of short hairpin RNAs (shRNAs), which are also identified by Dicer, are equally loaded onto RISC and enable targeting of mRNAs with desired experimental or medicinal preference. In addition, Dicer is able to identify and process synthetic dsRNA substrate as well, composed of two annealed oligonucleotides or a synthetic pre-miRNA stem and loop (hairpin) structure. Accordingly, artificial dsRNAs, known as short interfering RNA (siRNAs), are commonly used to provoke RNAi activity inside cells. This property enables experimental and medicinal silencing to rely on either synthetic oligonucleotides (siRNAs) or hairpins which are expressed from viral or plasmid vectors (shRNAs). Since unmodified dsRNAs are susceptible to cleavage by endogenous nucleases in vivo, chemical modifications are utilized to reduce dsRNAs nuclease vulnerability (Davidson and McCray, 2011). 20 -O-methyl base modification is a prevailing chemical solution (Choung et al., 2006; Elmen et al., 2005; Grunweller et al., 2003), that increases the oligo stability and contributes also to avoiding RNA-triggered immune response (Hornung et al., 2005; Judge et al., 2005). An added benefit of incorporation of 20 -O-methyl bases is in reducing off-target effects (Elbashir et al., 2002). Hence, it is not surprising that 20 -O-methyl bases are broadly employed in dsRNA synthesis. While complete substitution of nucleic acids with 20 -O-methyl inactivates RNAi function and is therefore counterproductive, a pattern of alternating 20 -O-methyl bases is comparable with the activity of unmodified RNA, and is quite stable in serum (Choung et al., 2006; Czauderna et al., 2003; Peek and Behlke, 2007). Conjugation of peptides to the dsRNA is also developed, to increase cell membrane permeability (Ifediba et al., 2010). RISC-dependent silencing requires double-stranded RNA, because effective RISC-loading requires identification of both the guide and passenger strands by Dicer. When the passenger strand is chemically modified, its off-target potential is dramatically reduced (Zhang et al., 2012). However, the challenges associated with distribution of dsRNAs into mammalian tissues and their limited bio-availability led to the development of a single-stranded siRNA (ss-siRNA), which exhibit improved penetration profile into cells. A ss-siRNA that targets HTT was synthesized by Isis Pharmaceuticals (Yu et al., 2012). That ss-siRNA was stabilized by extensive modifications, using phosphonate group, 20 -O-methyl, 20 -fluoro and phosphorothioate groups, while still maintaining at least partial RISC loading capacity. These ss-siRNAs were able to inhibit expression of the mutant HTT protein (Yu et al., 2012), but despite the success of this approach, compromised RISC loading required very high oligonucleotide doses, emphasizing the need for further optimization. Therefore, ss-siRNAs are intriguing but will require additional developments to improve silencing efficacy.

2.4.

miRNA as targets for therapy

RNAi utilizes the endogenous pathways that are naturally used by miRNAs.


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Since alterations in miRNAs expression levels were found in numerous neurodegenerative diseases (Abe and Bonini, 2013; Junn and Mouradian, 2012), miRNAs are emerging as key players in the process of neurodegeneration, and as potential targets for therapeutic intervention. For example, miRNAs are at the focus of research by multiple groups in research of the frontotemporal dementia—ALS Spectrum (Droppelmann et al., 2014; Gascon and Gao, 2014). miRNA mimetics, or ‘mimics’, may be introduced as miRNA replacement, a strategy for compensating for a pathologically-decreased miRNA levels. Replacement is based on either vectors for the expression of a miRNA gene or on synthetic mimics, which are dsRNAs and may differ from siRNAs only in their sequence. Using miRNA mimics therapy might have nonetheless a unique limitation: as opposed to ASOs and siRNAs that are designed by user/pharmacist, miRNAs are endogenous genes and their targets are sculptured to possess cis-binding sites by evolution. Therefore, manipulating miRNAs levels is forced to affect the many natural targets simultaneously. Likewise, miRNA inhibition is used in the case of aberrantly-high miRNA expression. ‘Anti-miRNA oligonucleotides’ (AMOs) are single-stranded ASOs, which suppress miRNA function and depend on Watson–Crick base complementation to tightly bind and inactivate the miRNA. AMOs are designed to possess nuclease stability and increased binding affinity to the miRNA. AMOs are thought to function by binding to miRNAs inside the RISC (Lennox and Behlke, 2011). Locked-nucleic acid (LNA) chemistry is probably the most successful technology used today for manufacturing AMOs (Murphy et al., 2013; Stenvang et al., 2012). Recently, AMOs targeting specific miRNAs were tested in SOD1 G93A model of ALS. Different type of AMOs distribute throughout brain and spinal cord and knockdown specific miRNA activity. 20 -O-Methoxyethyl and 20 -fluoro sugar-modified AMO against miR-155 was continuously administrated by osmotic pump intracerebroventricularly (ICV). The functional efficacy of that AMOs was shown by de-repression of targets of the miR155. The impact of miR-155 knockdown on disease duration was dramatic, although the mechanism required additional investigations (Koval et al., 2013). Likewise, LNA antagomirs harboring 30 -cholesterol-conjugated LNA appear to knockdown specifically miR-29a in the CNS. However, miR-29 KD did not modify disease progression in a significant manner (Nolan et al., 2014), maybe because of functional redundancy with other miR-29 family members. The seed is the denominator of miRNA family members and define their common set of targets (Lewis et al., 2003; Obad et al., 2011). To inhibit entire families of miRNAs, and hence to effectively de-repress their common set of targets, short AMOs were developed, matching only the ‘seed’ sequence, a 7–8 nt long segment at the miRNA 50 end that complements sequences on target mRNA. The most advanced anti-miRNA drug under development is Miravirsen, an LNA AMO that is generated by ‘Santaris’ for chronic viral hepatitis C therapy. Miravirsen sequesters mature miR-122, exhibits prolonged safety profile and is effective in reducing HCV RNA levels in phase II clinical trial (Janssen et al., 2013). An alternative way for the inhibition of miRNA function is to utilize sponge RNAs. Sponge RNAs contain complementary

binding sites to the miRNA of interest and act as competitive inhibitors for the miRNA interaction with targets (Ebert and Sharp, 2010). Sponge RNAs specifically interfere with the activity of whole families of miRNAs because the sponge molecule functions as decoy target to all miRNAs that share the same ‘seed’ sequence. As a result of miRNA sequestration by sponges, the miRNA downstream targets are de-repressed and upregulated. Sponge efficiency depends on miRNA affinity and on sponge:miRNA stoichiometry. Sponges, that perfectly complement a miRNA sequence, were also studied, but they appear to be degraded by Ago2 in the RISC and thus to hold weaker inhibitory activity (Bonci et al., 2008; Care et al., 2007; Ebert et al., 2007; Ebert and Sharp, 2010; Gentner et al., 2009; Haraguchi et al., 2009; Sayed et al., 2008; Scherr et al., 2007). Sponges are broadly used as fast and effective tool for experimental miRNA knockdown and may be exploited in the future for miRNA knockdown therapy, if expressed from viral vectors. However currently sponge technology is very young and is rapidly developing.

2.5. Long intergenic non-coding RNAs and cis-natural antisense transcripts Long intergenic non-coding (linc) RNAs of 4200 nucleotides make a big family of noncoding RNAs, whose function remains to be fully elucidated (Ernst and Morton, 2013; Guttman and Rinn, 2012). One class of lincRNAs are cisnatural antisense transcripts (NATs), a group of RNAs that have transcript complementarity to RNA transcripts encoded by the same genes coding for the NATs (Osato et al., 2007). LincRNAs, and particularly NATs are upregulated in some neurodegenerative diseases and can be a target for drug therapy. For instance, a NAT that regulates the expression of the Huntingtin (HTT) gene was recently found in brains of HD patients (Chung et al., 2011). NATs that enhance the stability of BACE1 mRNA by competing with miRNA on binding sites are upregulated in AD and perhaps contribute to upregulation of the beta-amyloid-generating enzyme (Faghihi et al., 2008, 2010). Thus using oligonucleotides that inhibit NATs (AntagoNATs) (Wahlestedt, 2013), or inhibition of lincRNAs (Margueron and Reinberg, 2011), may benefit patients with neurodegenerative diseases.

3. Platforms for oligonucleotide formulation into the central nervous system Antisense-oligonucleotides can be delivered to the brain “naked”, i.e., without any surrounding delivery system. Nonetheless, oligonucleotide formulation improves delivery into the central nervous system (CNS) (Southwell et al., 2012). In this context it is important to make the distinctions between single-stranded oligonucleotides and RNAs and doublestranded RNAs, whose spontanoues delivery in ‘naked’ form is inefficient. dsRNAs fail to penetrate into cells, and are rapidly cleared from circulation (Bumcrot et al., 2006). Therefore, formulation of dsRNAs for systemic delivery is required for therapeutic applications (Whitehead et al., 2009).



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3.1.

Aptamer formulation

Aptamers are chemically synthesized oligonucleotides or peptides that fit and bind to a specific target molecules and serve as carriers (Keefe et al., 2010; Proske et al., 2005). In this context, Pegaptanib (Macugen) is a Pfizer/Eyetech aptamer drug that was approved for therapeutic use for treatment of macular degeneration by the US FDA in 2004 as inhibitor of VEGF interaction with its receptors (Gragoudas et al., 2004).

3.2.

Encapsulation of RNAs in micro- and nano-particles

Lipid nanoparticles (LNPs) are currently the most effective in vivo system for oligonucleotides delivery. Formation of oligonucleotide-containing LNPs is made possible by electrostatic interactions between positively charged, cationic LNPs and negatively-charged oligonucleotides. Positively charged particles are further thought to penetrate through negatively charged cellular membranes. Given the relatively small particle size, at the nanometer–micrometer range, LNPs are able to penetrate the BBB. Traditionally, micrometer-sized vesicles are sonicated to produce smaller particles. The precision of this methodology is limited and it produces variability in LNP size. The ability to optimize the diameter of the LNPs is of great importance, as LNPs too big may not gain access to the brain, while LNPs too small may be cleared by the kidney (Longmire et al., 2008). Recent microfluidic mixing techniques enable precision in LNP synthesis, to gain defined diameter, controlled siRNA charge ratio (i.e., the ratio between siRNA charge and total lipid charge) and effective silencing of neuronal gene expression (Belliveau et al., 2012; Rungta et al., 2013). Microfluidic mixing techniques are expected to improve gene silencing efficiency. Higher order complexes or employing peptide conjugates are considered even more effective for delivery (Davidson and McCray, 2011). Peptides may serve as ligands for membrane-bound receptors or as antibodies against cellular epitopes to increase cell-type specificity and enhance uptake after systemic delivery (Cardoso et al., 2008; Kumar et al., 2007; Pulford et al., 2010; Uno et al., 2011). Targeting specific cellular compartments is another intriguing direction. For example, many neurodegenerative diseases display defects in mitochondrial metabolism (Ahari et al., 2007; Grasbon-Frodl et al., 1999; Kasraie et al., 2008; Zsurka and Kunz, 2013). Therefore mitochondriotropic liposomes, which possess preferential avidity to mitochondria have the exciting potential for organelle-specific regulation, thus minimizing the risk of side effects (Biswas et al., 2011; Boddapati et al., 2005; Wang et al., 2011; Weissig et al., 2006). Oligosaccharide-based, non-lipid, nanoparticles are an alternative to lipid formulations. In one example, betacyclodextrins were recently reported to deliver siRNA into the brain in vivo, knocking down Huntingtin (HTT) and alleviating motor deficits in a mouse model of HD (Godinho et al., 2013). Polyethylenimine (PEI) is another accepted delivery carrier for oligonucleotides that was shown to effectively deliver siRNAs to neurons, in vivo, on its own (Tan et al., 2005; Zintchenko et al., 2008), or to direct miRNA delivery into the brain, when conjugated to a glycoprotein,

5

which provided affinity to acetylcholine receptors (Hwang do et al., 2011; Kumar et al., 2007).

3.3. Exosomes as delivery platform for packing oligonucleotides Exosomes are cell-derived nano-vesicles present in biological fluids such as blood, urine and the cerebrospinal fluid (CSF) (Keller et al., 2006; van der Pol et al., 2012). Exosomes are released from the cells when multivesicular bodies fuse with the plasma membrane (Booth et al., 2006). Exosomes are known to harbor endogenous miRNAs and protect them from degradation in cell-free extracellular fluids. Furthermore, because exosomes are purified from dendritic cells isolated from the same animal, they do not initiate immune response. Therefore, exosome clinical applications may benefit from very low immunogenicity, trans-BBB delivering profile, and long extracellular stability. Valadi and colleagues were first to report the exchange of mRNA and miRNAs between cells via exosomes (Valadi et al., 2007), which was later demonstrated also in the CNS (Morel et al., 2013). Exosomes that were fused to rabies viral glycoprotein delivered siRNA into mouse brains for knockdown of beta-site APP-cleaving enzyme 1, BACE1 in AD mouse model (Alvarez-Erviti et al., 2011). These early steps suggest exosomes as novel and promising technology and may pave the way to potential clinical studies. For a comprehensive review, see (Kalani et al., 2014).

3.4. Viral vectors and gene therapy for the expression of siRNAs A standing alternative to oligonucleotide-based therapy is based on the use of viral vectors for the expression of RNAs of choice. Viral expression vectors provide stable in vivo expression in mammalian tissues and are hence commonly used as transfer vectors in the development of clinical applications. Recombinant Viruses are able to introduce an shRNA for gene knockdown, as in the case of SOD1 knockdown that was clinically beneficial and delayed disease progression in an ALS model (Wang et al., 2013). However, viral gene therapy for neurodegeneration is not within the scope of this review.

4.

Routes of delivery into the CNS

4.1.

Systemic intravenous administration

Intravenous route is probably the most feasible for considering new therapies for human diseases. Accordingly, the broadest clinical experience with ASO drugs to date is with systemic exposure. However, the entrance of most molecules into the CNS is limited by the blood–brain barrier (BBB), a highly selective permeability barrier of brain microvascular endothelial cells. The BBB separates the circulating blood from the brain tissue and regulates the CNS chemical microenvironment, preventing the spontaneous diffusion of many macro-molecules from the bloodstream into the CNS. For these reasons, systemic ASO delivery is inefficient and necessitates doses that are 100-fold higher than required by direct intra-CNS delivery, in order to approach similar tissue


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concentrations (Banks et al., 2001; Erickson et al., 2012). Such high doses may further result in increased risk of toxicity, such as hepatotoxicity (Swayze et al., 2007).

4.2.

Directed CNS delivery to the cerebrospinal fluid

The biodistribution of ASOs and siRNA can be significantly influenced by the route of delivery and systemic administration often results in accumulation in liver or other organs at the expense of CNS bioavailability. This is an important reason for localized delivery, which also reduces loss and toxicity to other organs. The cerebrospinal fluid (CSF) is a unique biofluid that is produced by the choroid plexus and circulates throughout the CNS. Delivery of the RNA into the cerebrospinal fluid (CSF) via the lateral ventricles results in efficient distribution of the infused RNA throughout the CNS including the spinal cord (Kordasiewicz et al., 2012; Passini et al., 2011; Smith et al., 2006). The intrathecal (IT) space (Watson et al., 2006) is another route for drug delivery into the CSF, which results in efficient distribution at therapeutic doses throughout the CNS. IT delivery can be employed in mice but is considered increasingly more accessible in larger animal species (Kordasiewicz et al., 2012; Locatelli et al., 2007; Morel et al., 2013; Smith et al., 2006). Under some conditions, slow pressurized infusion, socalled convection-enhanced delivery, considerably improves the distribution of siRNAs in the brain (Querbes et al., 2009; Stiles et al., 2012). In human patients, plausibly the route of choice into the CSF delivery is IT, as it is well tolerated, and routinely used in the clinic for drug delivery.

4.3.

Noninvasive intranasal administration

Delivery into the brain by the intranasal route is mediated via the olfactory and trigeminal nerves, which initiate in the nasal cavity at olfactory neuroepithelium and terminate in the brain (Pardeshi and Belgamwar, 2013). Intranasal administration is the by far least invasive route for the delivery of drugs into the CNS and was already proven feasible in clinical trials with insulin for AD patients (Claxton et al., 2013; Freiherr et al., 2013; Stein et al., 2011). Furthermore, antimiR 206 was successfully delivered into the brains of Tg2576 AD transgenic mice (Lee et al., 2012). Naked dsRNAs exhibit poor intranasal delivery (DeVincenzo et al., 2008), emphasizing the demand for careful formulation. With one formula that is based on polyethylene glycol–polycaprolactone-nanomicelles conjugated to the cell-penetrating peptide Tat (MPEG-PCL-Tat), intranasal delivery of siRNAs into the brain was superior to intravenous route (Kanazawa et al., 2013). Because RVG-targeted exosomes were already proven to be useful in carrying siRNA to the brain via intravenous injection (Alvarez-Erviti et al., 2011) and since intranasal delivery of exosomes is beneficial in delivering small molecules (Zhuang et al., 2011), exosome-encapsulated RNAs in nasal sprays are worthwhile exploring as alternative as safe minimallyinvasive delivery route.

5. Oligonucleotide therapies in neurodegenerative diseases Below we provide an updated summary of oligonucleutide therapeutic modalities for neurodegenerative diseases. Another important review on this topic may be found in (Southwell et al., 2012).

5.1.

Alzheimer's disease (AD)

Alzheimer's disease (AD) is the most common form of dementia. In AD, deposition of insoluble beta-amyloid (Aβ) plaques and neurofibrillary tangles results in progressive cortical atrophy. The finding of reduced synthesis of the neurotransmitter acetylcholine in AD (Francis et al., 1999), drove the development of drugs that inhibit the expression of the metabolizing enzyme, acetylcholinesterase (AChE). Consistently, ASO therapy against AChE decreased AChE activity and improved cognition in a mouse AD model (Chauhan and Siegel, 2007; Fu et al., 2005). ASOs which are directed to target AChE and other AD-causing genes, impinge on the same pathways that are currently targeted by AChE small molecule inhibitors, and therefore may be conceivable candidates for clinical development. Likewise, ASOs directed at the mutated beta-site of APP, were injected to transgenic mouse model of AD, Tg2576, that harbors mutation in the APP gene (Chauhan and Siegel, 2007). β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is an enzyme involved in the processing of amyloid precursor protein, which gives rise to beta amyloid (Aβ). Knockdown of beta-secretase (BACE1) transcript by continuous infusion of LNA-modified siRNAs downregulated BACE1 and reduced insoluble Aβ levels in AD transgenic mice (Tg19959) (Modarresi et al., 2011). Exosome-mediated delivery of siRNA is also considered as delivery platform for siRNA-based therapy for AD (Alvarez-Erviti et al., 2011). In the context of modifying miRNA expression, miR-206 and miR-34c are both upregulated in AD brains. miR-206 inhibits brain-derived neurotrophic factor (BDNF) and miR206 knockdown restored BDNF levels in the Tg2576 model of AD (Lee et al., 2012). Accordingly, anti-miR-34c improved memory in double transgenic model of AD APPPS1-21 (Zovoilis et al., 2011). BACE1 is targeted by miR-107 (Wang et al., 2008).

5.2.

Parkinson's disease (PD)

Parkinson's disease (PD) is a neurodegenerative disease that primarily results from a loss of dopaminergic cells in midbrain's substantia nigra. Early in the course of the disease, the most obvious symptoms are movement-related, including resting tremor, muscle rigidity, bradykinesia and postural instability. Specific mutations or copy number variations in the gene encoding for alpha-synuclein are sufficient to cause familial PD (Chartier-Harlin et al., 2004; Ibanez et al., 2004; Kruger et al., 1998; Polymeropoulos et al., 1997). Thus, alphasynuclein may be an obvious candidate for knockdown, either through RNAi or ASO.



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5.3.

Huntington's disease (HD)

Huntington's disease (HD) is a dominant autosomal inherited disease, characterized by progressive deterioration of voluntary motor control and by psychiatric and cognitive disturbances. Expansion of a nucleotide triplet repeat stretch within the Huntingtin gene (HTT) results in a mutant form of the protein, which is neurotoxic. Due to the purely-genetic nature of the disease, it is an excellent candidate for RNAbased therapy. However, a major challenge in RNA-based therapy for HD is a selective targeting of the mutant HTT. Methoxyethyl (MOE)-modified ASO was able to selectively inhibit only the mutated HTT allele, in vivo, without any sign of toxicity (Carroll et al., 2011), delayed disease progression and mediated a sustained reversal of disease phenotype (Kordasiewicz et al., 2012). Similar ASO effectively lowered Huntingtin in many brain regions of nonhuman primates (Kordasiewicz et al., 2012). Similarly, siRNAs are effectively used for repression of HTT and is beneficial in this disease (DiFiglia et al., 2007). In a parallel approach that was described in a series of studies from the Davidson lab, recombinant adeno-associated virus is employed for silencing HTT by shRNA in both mice and rhesus monkeys, with no apparent toxicity or inflammatory responses (Boudreau et al., 2011; McBride et al., 2008; McBride et al., 2011). Several other diseases are caused by similar molecular mechanism of expanded polyglutamine repeats. One ASO prototype against the polyglutamine repeats, discriminates between wild-type and mutant genes on the basis of repeat length and offers a therapy by repressing expanded CAG tract in spinocerebellar ataxia 1, in spinocerebellar ataxia 3/Machado-Joseph disease and in dentatorubral–pallidoluysian atrophy (Evers et al., 2011; Hu et al., 2009).

5.4.

Amyotrophic lateral sclerosis (ALS)

ALS is a progressive fatal neurodegenerative disease of motoneurons in the spinal cord and brain. At the final disease stage, respiratory muscle dysfunction leads to suffocation resulting in death. There is no known cure or effective treatment for the disease. About 5000 people are diagnosed with ALS in the US each year, and global population of ALS patients is estimated at 400,000 (Kiernan et al., 2011). The progression of ALS is rapid, with most patients surviving only a few years following diagnosis; the only approved treatment for ALS, Rilutek (Riluzole), shows only marginal effect on disease progression. Studies over the last 20 years have uncovered ALS-causing mutations in several genes such as SOD1, TDP-43 (TARDBP), FUS (TLS) and C9ORF72 (Chen et al., 2004; DeJesus-Hernandez et al., 2011; Greenway et al., 2006; Kabashi et al., 2011; Kiernan et al., 2011; Kwiatkowski et al., 2009; Lagier-Tourenne and Cleveland, 2009; Renton et al., 2011; Rosen et al., 1993; Sreedharan et al., 2008; Vance et al., 2009). The most established antisense approach to treat ALS in recent years employs ASOs for the knocking down of SOD1, a protein that is sufficient to cause the disease in human when mutated. Indeed, administration of the ASO SODr146192 reduced both SOD1 protein and mRNA levels throughout

7

the brain and spinal cord in the SOD1G93A model of ALS in rats. When treatment is initiated near onset, this ASO-based therapy is sufficient to slow down disease progression (Smith et al., 2006). Accordingly, the levels of SOD1 in the CSF were found to be useful as pharmacodynamic marker for monitoring the ASO therapy protocol (Winer et al., 2013). In humans, initial phase I clinical study by ISIS therapeutics, using an ASO against SOD1, ISIS-333611 was well-tolerated and was detectable in the CSF (Miller et al., 2013). Similar approaches for the knockdown on other aberrant gene products will probably be evaluated in cases whereby pathogenesis can be partially overcome by reduced expression levels of the mutant gene, including in the case of C9ORF72. Additional siRNAmediated knockdown of SOD1 were extensively reviewed elsewhere (Nizzardo et al., 2012).

5.5.

Spinal muscular atrophy

A well-documented example of the association between splicing and human disease is spinal muscular atrophy (SMA), a pediatric neurodegenerative disorder caused by mutations in the SMN1 gene. Human SMN2 acts as a disease modifier, because it is encoding for an identical protein that can partially compensate for the loss of SMN1. However SMN2 transcript is predominantly spliced to generate an isoform lacking exon 7, which results in a defective protein product. Upregulation of SMN2 by ASOs that force inclusion of exon 7 proved effective in animal models are now in phase II clinical trials (Hua et al., 2010; Rigo et al., 2012).

5.6.

Summary

The last years have seen great advances in the field of oligonucleotide therapy and suggest emerging avenues for medicinal interventions, including in neurodegeneration. Successful preclinical studies in animal models, which are summarized also in Table 2, provide reassurance to these strategies, however, clinical trials in human neurodegeneration states are still limited at present (Miller et al., 2013). As of 2014 two antisense drugs have been approved by the U.S. Food and Drug Administration, fomivirsen (Vitravene) and Mipomersen (Kynamro). The hopeful FDA approval of RNA-based therapy for CNS is still a vision for the future. Nonetheless, oligonucleotide therapy holds a great promise as a treatment for neurodegenerative diseases, since many of these diseases involve genetic abnormalities and since new platforms for enhanced delivery are continuously being developed. In our view these therapies have very good prospects to eventually provide desperately-needed remedy for neurodegenerative diseases.

Non-standard abbreviations (only those appearing more than once are listed here) (r) AAV Aβ ACh AChE AD

(recombinant) adeno-associated virus beta-amyloid acetylcholine acetylcholinesterase Alzheimer's disease


421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 Oligo

Delivery platform

Delivery route to CNS

References

AD

dsRNA for RISC-dependent silencing

Naked, LNA modifications Naked, transfection reagent Naked, chemical modifications Naked, chemical modifications Naked, chemical modifications Cyclodextrin particles Naked, chemical modifications Conjugation to RVG peptide Exosomes with RVG peptide Tf-lipoplexes α-Tocopherol conjugation Receptor-mediated lipid nanoparticles PEI complexation Naked, chemically modified ASOs or anti-miRs

Intracerebroventricular Intraparenchymal Intrathecal Intrathecal Intraparenchymal (Convection enhanced) Intraparenchymal Intraparenchymal (Convection enhanced) Intravenous Intravenous Intraparenchymal Intracerebroventricular Intraparenchymal & intracerebroventricular

Modarresi et al. (2011) Zovoilis et al. (2011) Locatelli et al. (2007) Morel et al. (2013) Stiles et al. (2012) Godinho et al. (2013) Querbes et al. (2009) Kumar et al. (2007) Alvarez-Erviti et al. (2011) Cardoso et al. (2008) Uno et al. (2011) Rungta et al. (2013)

Intrathecal ICV and IV ICV IV ICV IT Intraperitoneal and ICV ICV ICV Intramuscular Intraparenchymal IT and ICV IT ICV ICV Systemic (IV) ICV

Tan et al. (2005) Banks et al. (2001) Chauhan and Siegel (2007) Erickson et al. (2012) Fu et al. (2005) Miller et al. (2013)n Koval et al. (2013) Smith et al. (2006) Nolan et al. (2014) Cirak et al. (2012)n Carroll et al. (2011) Kordasiewicz et al. (2012) Passini et al. (2011) Hua et al. (2010) Lee et al. (2012) Hwang do et al. (2011) Yu et al. (2012)

ALS HD Not tested in a disease model but in normal animals

AD

ASO

ALS

DMD HD SMA AD Not a disease HD

n

Human trials.

Anti-miR ss-siRNA

PEI with RVG conjugation Naked, chemically modified siRNAs


Disease model

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Table 2 – Selected reports of oligonucleotide therapies into the CNS.


481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

ALS APP ASOs BACE1 BBB BDNF CNS CSF DMD dsRNA HD HTT ICV IT IV LNAs lincRNA LNPs miRNA NAT PD PEI PS RISC RVG siRNA SOD1 ssRNA

amyotrophic lateral sclerosis amyloid precursor protein antisense oligonucleotides beta-secretase 1 gene blood–brain barrier brain-derived neurotrophic factor central nervous system cerebrospinal fluid Duchenne muscular dystrophy double stranded RNA Huntington's disease Huntingtin gene intracerebroventricular intrathecal intravenous locked nucleic acids long intergenic non-coding RNA lipid nanoparticles microRNA natural antisense transcripts Parkinson's disease polyethyleneimine phosphorothioate RNA-induced silencing complex rabies viral glycoprotein small interfering RNA superoxide dismutase 1 single stranded RNA

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