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Expert Opinion on Drug Delivery

ISSN: 1742-5247 (Print) 1744-7593 (Online) Journal homepage: http://www.tandfonline.com/loi/iedd20

Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (): from culture studies to clinical trials

Alesya A. Fokina, Boris P. Chelobanov, Masayuki Fujii & Dmitry A. Stetsenko

To cite this article: Alesya A. Fokina, Boris P. Chelobanov, Masayuki Fujii & Dmitry A. Stetsenko (2016): Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes): from studies to clinical trials, Expert Opinion on Drug Delivery, DOI: 10.1080/17425247.2017.1266326

To link to this article: http://dx.doi.org/10.1080/17425247.2017.1266326

Accepted author version posted online: 28 Nov 2016.

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Download by: [Kinki University] Date: 04 December 2016, At: 21:08 Publisher: Taylor & Francis

Journal: Expert Opinion on Drug Delivery

DOI: 10.1080/17425247.2017.1266326

Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes): from cell culture studies to clinical trials

Alesya A. Fokina*1, Boris P. Chelobanov,1 Masayuki Fujii2, Dmitry A. Stetsenko1

1 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian

Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia

2 Department of Biological & Environmental Chemistry, Kindai University 820-8555, 11-6

Kayanomori, Iizuka, Fukuoka, Japan

* Correspondent author:

Alesya A. Fokina

Email: [email protected]

Abstract

Introduction. Development of efficient in vivo delivery systems remains a major challenge en route to clinical application of antisense technology, including RNA-cleaving such as deoxyribozymes (DNAzymes). The mechanisms of uptake and trafficking are clearly dependent on cell type and the type of oligonucleotide analogue. It appears likely that each particular disease target would pose its own specific requirements for a delivery method.

Areas covered. In this review we will discuss the available options for DNAzyme delivery in vitro and in vivo, outline various exogenous and endogenous strategies that have been, or are still being, developed and ascertain their applicability with emphasis on those methods that are currently being used in clinical trials.

Expert opinion. The available information suggests that a practical system for in vivo delivery has to be biodegradable, as to minimize concerns over long-term toxicity, it should not accumulate in the organism. Extracellular vesicles may offer the most organic way for drug delivery especially as they can be fused with artificial liposomes to produce hybrid . Chemical modification of DNAzymes holds great potential to apply oligonucleotide analogs that would not only be resistant to digestion, but also able to penetrate cells without external delivery agents.

Keywords: antisense oligonucleotide, cell uptake, extracellular and intracellular barriers, downregulation of expression, exogenous and endogenous delivery systems

Article highlights box

• DNAzymes are artificial DNA molecules that could be designed for cleaving natural RNA

targets to achieve therapeutic effect from downregulation of specific .

• The main obstacle towards clinical applications of DNAzymes seems to be insufficient

cellular uptake, which necessitates the need for delivery systems to ensure their successful

use for either in cell culture or in vivo.

• DNAzymes may be delivered into cells either endogenously or exogenously. Endogenous

systems were designed to produce DNAzyme molecules inside cells using reverse

. More common is exogenous delivery, for which a number of delivery systems have been developed such as liposomes or oil-based emulsions, inorganic nanoparticles,

polymers or , or systems based on conjugating DNAzymes to transporter

molecules such as peptides or structural motifs.

• Many of the exogenous delivery systems have been used to promote cell uptake of

DNAzymes in cell culture, much less have been tried in vivo and very few have been

employed so far for clinical trials. This emphasizes the need for simpler, less expensive and

non-toxic approaches to DNAzyme delivery, which would be sufficiently effective to be

applicable for clinical trials.

• Chemical modification could be very promising for designing DNAzymes, which are stable

in biological media, have better catalytic properties and, potentially, enhanced cell uptake in

the absence of external delivery agents.

Abbreviations: antisense oligonucleotide (ASO), cell-penetrating peptide (CPP), Clustered

Regularly Interspaced Short Palindromic Repeats (CRISPR), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methanesulfonate (DOTAP), External Guide Sequence (EGS), Food and Drug Administration

(FDA), latent membrane (LMP1), Moloney murine leukemia (MoMuLV), nuclear export signal (NES), nuclear localization signal (NLS), phosphordiamidate (PMO), polyamidoamine (PAMAM), polyethylene glycol (PEG), poly-lactic-co-glycolic acid (PLGA), polypropyleneimine dendrimer (PPI), primer binding site

(PBS), reticuloendothelial system (RES), reverse transcriptase (RT), RNA interference (RNAi), respiratory syncytial virus (RSV), scavenger receptor (ScR), splice correcting oligonucleotides

(SCO), submicron emulsion (SME), transferrin (Tf).

1. Introduction

The idea of using antisense oligonucleotides as potential therapeutics has been put forward by

Paul Zamecnik more than 30 years ago [1]. However, we still see just a handful of FDA- approved oligonucleotide drugs available on the market. At the same time a broad spectrum of known target , from which expression is responsible for the onset and development of vast array of diseases opens attractive opportunities to develop oligonucleotide-mediated gene silencing for some of these diseases. The main reason for such an apparent imbalance between the present low utility and potentially wide applications of oligonucleotide therapeutics, apart from toxicity and side effects, is rooted in the problem of their in vivo delivery to the precise places within the organism where those compounds will be able to exert their specific activity. In order to penetrate into cells oligonucleotides must successfully overcome both extracellular and intracellular barriers, which have been described, e.g. in the reviews of Juliano et al. [2-4]:

• Extracellular barriers

- Nuclease degradation

- Kidney clearance

- Reticuloendothelial system (RES) uptake

• Intracellular barriers

- Cell uptake

- Endosomal sequestering and endosomal escape

- Intracellular trafficking

Although the precise mechanisms of oligonucleotide uptake by cells remain still largely unclear

[5], many researchers have focused their efforts on the development of different ways for effective oligonucleotide delivery by trying to overcome the hurdles listed above. Chemical modifications in the sugar- backbone of oligonucleotides or their encapsulation into different nanostructures can prevent oligonucleotide degradation by [6]. Such chemical modifications may also be useful for improvement of pharmacokinetic properties of biomacromolecules including oligonucleotides. For example, a covalent attachment of polyethylene glycol (PEG) chains is a universal concept to increase molecular size and reduce elimination of oligonucleotides by RES and kidneys from the blood stream [7].

In order to enhance cell uptake of negatively charged oligonucleotides many different types of delivery systems have been developed (Fig. 1) including strategies that potentiate the escape of oligonucleotides from [8]. Successful in vivo oligonucleotide delivery depends on the method of oligonucleotide administration into the organism such as systemic or local injection or inhalation. At present, it looks like it would be necessary to tailor an individual method of administration for a particular disease target.

Oligonucleotides with potential therapeutic properties can be conveniently divided into several types depending on their . Some oligonucleotide therapeutics are designed based onto the mechanisms of action that involve cellular enzymatic machinery such as:

• RNA interference (RNAi) (depends on RISC complex) [9]

• EGS (External Guide Sequence) technology (involves RNase P) [10]

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing system (requires Cas9 ) [11]

• RNase H recruiting antisense oligonucleotides (ASO) [12].

There are other potential oligonucleotide therapeutics, for which the mechanism of action may not require the use of cellular components and thus act independently through binding to their targets. These include:

• Non-RNase H-dependent ASO that produce physical arrest of mRNA and inhibit gene expression at translational level [13]

• Blockmirs that upregulate gene expression [14]

• Antagomirs that bind target miRNA and inhibit their function [12]

• Splice-correcting oligonucleotides (SCO) [15] • RNA-cleaving oligonucleotides such as or deoxyribozymes

(DNAzymes) [16]

• [17]

• Oligonucleotide decoys [18].

In this review we shall focus on the methods for cellular delivery of single-stranded (ss)DNA oligonucleotides, in particular, DNAzymes, which recently have successfully entered clinical trials [16]. We would like to emphasise that the methods described may also apply to other types of therapeutic oligonucleotides such as ASO, SCO, aptamers or oligonucleotide decoys. Thus, for oligonucleotides, in which the mechanism of action does not depend on harnessing cell machinery, the development of delivery systems and choice of administration methods must take into account where and how such a therapeutic oligonucleotide will work and also which type of a therapeutic oligonucleotide, exogenous or endogenous, is going to be employed.

2. 10-23 DNAzyme

Catalytic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes, or DNAzymes) represent a special type of antisense oligonucleotide inhibitors of gene expression at the post- transcriptional level, which are able to induce sequence-specific hydrolysis of the mRNA of the target gene. The most widely used representative of the type is 10-23 DNAzyme. It has been obtained by SELEX (Selection of by Exponential Enrichment) procedure in 1997 in the lab of G. F. Joyce [19]. A typical 10-23 DNAzyme consists of catalytic core of 15 flanked on both ends by substrate-recognition domains (Fig. 1). The

DNAzyme catalyses hydrolysis of phosphodiester bonds in RNA between an unpaired purine and a base-paired pyrimidine in the presence of Mg2+ . Although such an RNA-cleaving agent will have potential limitations in target site selection and efficacy of RNA cleavage at low physiological concentration of Mg2+, its ease of synthesis and flexibility of design has prompted widespread application of 10-23 DNAzymes for targeting a number of therapeutically relevant RNA targets both in vitro and in vivo [16]. Several preclinical studies on different animal models have indicated that such DNAzymes may have clinical utility as specific inhibitors of gene expression. Favourable properties of 10-23 DNAzymes such as enhanced biological stability compared to other oligodeoxyribonucleotides and usually insignificant side effects in vivo due to their high specificity and lack of immunogenicity, have opened the way for DNAzymes to enter clinical trials [16]. Currently, the main limitation that obstructs extensive application of

DNAzymes as well as other antisense molecules is their generally poor cellular uptake that necessitates the design of special delivery systems.

3. Endogenous DNAzyme delivery

Although in most cases researchers tend to use endogenous RNA like siRNA or antisense RNA, which are relatively easy to produce endogenously inside cells, e.g. by employing viral vectors, several methods have been developed using specially constructed that have been able to produce single-stranded DNAzyme molecules [20-26] or circular DNAzymes [27, 28]. In most cases DNAzyme expression vectors were delivered into eukaryotic cells by with commercially available cationic DOTAP or ® 2000 [20, 23] or transfection reagents such as GenePORTER 2 [22] or Gene-Juice [25]. In the work reported by Sugiyama et al. cells were infected with lentiviral DNAzyme [26]. To achieve DNAzyme expression in bacterial cells, the plasmids encoding DNAzymes were delivered by [27, 28] or through bacterial expression vector where DNAzyme expression were integrated [24].

In order to produce ssDNA the reverse transcription step was introduced with reverse transcriptase (RT) that has been either encoded in the vector or just present in the cells in that particular case. For example, a plasmid has been designed to express Moloney murine leukemia virus (MoMuLV) RT [22, 23]. Such plasmids also contain sequences of interest or DNAzyme sequences and special elements recognized by RT to initiate or terminate reverse transcription to produce ssDNA (Fig. 2). For ssDNA synthesis RT expressed in cells uses endogenous tRNAPro as a primer, which binds to the primer binding site (PBS) on the 3’-end of the RNA transcript.

After reverse transcription, ssDNA is released when mRNA template is degraded either by endogenous RNase H or by RNase H activity of RT.

Another research group used HIV-1 RT for endogenous synthesis of a DNAzyme [26]. In this case, the constructed plasmid did not express the HIV-1 RT but contained a structural domain to initiate and terminate reverse transcription by HIV-1 RT. Thus, ssDNA has been expressed in

SupT1 cells after infection with HIV-1 NL4-3. Notably, such an endogenously produced

DNAzyme inhibited HIV-1 replication without the emergence of escape .

A different approach to obtain an endogenous DNAzyme has been designed [27, 28] in which the authors have constructed circular DNAzymes (Fig. 3) by cloning a DNAzyme into an ssDNA phage vector. Such a phage single strand expression system replicated circular DNAzymes in and, as it has been shown [27], exhibited high activity in suppressing β-lactamase and bacterial growth. Although DNAzymes delivered in such a way have been able to inhibit gene expression in cells, it remains to be seen if the constructs will be safe for use in animals and humans. For example, for retroviral vectors it was shown that they continue to express native viral and provoke immediate and late inflammatory responses [29].

As these methods became available only recently, they have not yet been widely used to deliver ssDNA into cells. Thus, one may conclude that endogenous in vivo DNAzyme delivery is still in its infancy at the moment.

3. Systems for exogenous DNAzyme delivery

Traditionally, delivery of ssDNA into cells takes place exogenously through oligonucleotide complexes with lipids, nanoparticles etc. and via conjugation with transporter molecules. We shall discuss those methods in detail below.

3.1. and oil based formulations

Amongst all the lipid-based delivery systems, perhaps, the most popular would be various kinds of liposomes or lipid-containing vesicles.

Despite the fact that cationic lipids can be toxic to cells (for example, in the case of

DOTAP previous studies have reported the ability to induce inflammatory response elements such as TNF α, IL-12 and IFN-γ in an in vivo setting), - the DOTAP and DOPE cationic lipid carriers were used for intratumoral delivery of DNAzyme Dz13 in humans in a Phase I clinical trial study [30]. Recently, specific lipid formulations containing DOTAP and DOPE have been used for local DNAzyme delivery in vivo by Li et al. [31] during autologous vein bypass grafting and by Chan et al. through intravitreal injection [32]. In such formulation Dz13 was able to inhibit neontimal hyperplasia in rabbits [31] and, as was demonstrated [32], strongly decreased c-jun gene expression and c-jun-dependent genes in rabbits’ eyes. The use of cationic liposomes requires careful choice of lipid concentration, in order to show low toxicity and high efficiency of delivery. It has been demonstrated for both DOTAP (Roche) or Superfect (Qiagen) that their less than cytotoxic concentrations were insufficient for promoting DNAzyme complexation with a transfection agent and thus high level of delivery has not been achieved [33].

For dermal application of DNAzymes [34-36] the authors have used nanosize drug carrier systems such as multiple water-in-oil-in-water (W/O/W) emulsions, W/O emulsions, submicron emulsions or microemulsions due to their ability to interact with skin lipids and improve penetration of drugs into the skin. The tables with emulsions compositions is available at [35].

To protect the drug against degradation, a hydrophilic therapeutic agent may be incorporated into the inner aqueous phase of the carrier systems such as W/O/W emulsions. These emulsions were prepared using a two-step procedure. Firstly, the primary W/O emulsion was obtained. Secondly, the primary emulsion (40% w/w) was dispersed in the aqueous phase containing a hydrophilic emulsifier. The simple W/O emulsions were produced by adding the aqueous phase (65 mM

MgSO4 solution containing a DNAzyme) to the oily phase (15.8% w/w heavy paraffin oil, 4.0% w/w Sorbitan oleate and 0.2% w/w soya lecithin) followed by homogenisation step. Then a chilled primary emulsion was slowly added to the outer water phase (1.0% w/w Steareth 20 and

58.7% w/w preserved water), 0.3% w/w Sepineo® P600 was added as a thickening agent under gentle stirring. Drug release and skin uptake studies using various skin conditions and experimental setups revealed that such a multiple emulsion is a promising drug carrier system for topical application of DNAzymes to treat eczema skin conditions. Such a DNAzyme has been developed by Sterna Biologicals GmbH & Co. KG [37] and currently is going throw Phase IIa clinical trial. The same research group has developed submicron emulsions (SMEs) for topical application of DNAzyme Dz13 [38]. The SMEs were produced by high-energy emulsification.

The blend of emulsifiers for SMEs without preservatives was previously optimized by selecting the most thermo-stable droplet size. The authors not only used SME as a delivery system, but in addition they encapsulated the DNAzyme either into liposomes or chitosan polyplexes to protect it from nuclease digestion. A combination of SME with chitosan-DNAzyme polyplex has been the most successful strategy, which allowed for effective topical delivery of Dz13 and it has been validated at both the skin and cellular level by in vitro model systems. Although such a delivery system for Dz13 has been developed for topical treatment of actinic keratosis it would also be interesting to see if it would be suitable to treat skin carcinoma. During clinical trials the

DNAzyme has been encapsulated into DOTAP/DOPE liposomes and delivered via intratumoral injection. In this case the moderate efficiency of Dz13 has been explained by its low level of delivery into the tumor.

Currently, the methods for oligonucleotide delivery using lipid carrier systems are being developed quite extensively. Although some of those methods have not yet been applied to deliver DNAzymes, we would like to mention here some other lipid-based platforms, which maybe useful for in vivo delivery of oligonucleotide therapeutics. For example, hybrid nanoparticles have been obtained by mixing a polymer with a lipid matrix (reviewed in [39]).

Such a combination allows the construction of nanocarriers with desirable properties to fit a particular therapeutic application and provides a more sustained and localized release of the drug

[39]. Cochleates are lipid crystals formed upon the interaction of charged lipids and cations [40].

Оligonucleotides can be sequestered within the layers of those stable structures. When cochleates were exposed to low calcium concentrations inside the cells they would open and release their content into cytosol. In contrast to liposomes, cochleates are solid, hold on tightly to their medication payload and thus can be delivered through oral administration as a pill, provide multi-organ protection and enhanced safety as well as targeted delivery to the site of the infection or inflammation and demonstrate the ability to effectively penetrate into various tissues

[41, 42].

3.2. Nanoparticles

3.2.1. Delivery systems based on polymers and dendrimers

During the last few years, the number of papers on new delivery systems based on polymeric particles with low toxicity and high delivery efficiency has increased. Biodegradable polymeric nanoparticles represent an effective type of carrier for targeted in vivo delivery of drugs and vaccines [43]. Currently biodegradable nanoscale systems based on homo- and co-polymers of lactic and glycolic acids, chitosan or PAMAM dendrimers have been designed for oligonucleotide delivery. One of their main advantages is that these systems promote oligonucleotide activity for a prolonged time without frequently repeated administration. An important feature is the possibility of controlled release of oligonucleotides from polymeric microspheres. Release profiles for phosphorothioate-modified 10-23 DNAzymes from poly- lactic-co-glycolic acid (PLGA) microspheres were investigated [44]. The profiles for DNAzymes were described as biphasic, characterized by initial ‘burst’ of release over five days followed by a second phase of sustained release over the next 40 days. The oligonucleotides that are released during the initial ‘burst’ phase have evidently concentrated around the surface of the polymeric microsphere while during the slow phase the oligonucleotides that are liberated are encapsulated deeper in the interior. However, no cell culture results with PLGA-encapsulated DNAzymes have been published yet, so further development is required.

In the paper of Dass et al. [45] it was demonstrated for the first time that DNAzyme Dz13 can be delivered into cells by encapsulation into particles of natural biodegradable polymer chitosan, which has been achieved by a complex coacervation method. The Dz13–chitosan nanoparticles have been obtained with a median diameter of 350 nm, a highly positive surface charge and high encapsulation efficiency. Apoptotic promoted by Dz13 was enhanced through its encapsulation and delivery by chitosan nanoparticles into osteosarcoma SaOS-2 cells. The particles were efficient intracellular transporters as determined by scanning electron microscopy, which showed internalisation of the particles into -resembling vesicles within 48 h.

The Dz13–chitosan nanoparticles were stable at room temperature for up to one month.

Subsequent in vivo analysis following intratibial and intramuscular injection of the particles revealed no possible toxic side effects to surrounding tissues. Radiography and histology have also demonstrated undisturbed bone structure and muscular morphology in mice administered intratibially or intramuscularly with Dz13–chitosan nanoparticles.

As was shown [46] for two different clinically-relevant disease models using a clinically adoptable dosing regimen, Dz13–chitosan nanoparticles were efficacious against osteosarcoma, reducing significantly the tumor volume and osteolysis. No toxicity against other bone-dwelling cells was observed with the formulation and no side-effects were noted in vivo in lymphatic and reticuloendothelial tissues proximal and distal to the administration site.

In the following paper [47] the same authors have studied a combined encapsulation into chitosan particles of clinically-used anticancer agent doxorubicin (Dox) and Dz13. A formulation of Dz13–Dox nanoparticles (DDNPs) was achieved with high (>91%) loading of both compounds, which consisted of individual 50 nm particles forming aggregates as large as

500 nm with a large positive zeta potential. The DDNPs could be stored at various temperatures for a week without loss of activity but were prone to degradation in serum. DDNPs inhibited osteosarcoma growth more effectively than a separate treatment with Dz13 or Dox chitosan nanoparticles as well as Dox administered intraperitoneally. Apart from inhibiting tumor growth,

DDNPs protected the affected bone from substantial destruction by aggressive tumor invasion and reduced the incidence of to the lungs without causing adverse effects in mice.

One can conclude that chitosan nanoparticles could be considered as a promising delivery system for antisense oligonucleotides, in particular for their therapeutic application in vivo. Chitosan is an inexpensive and readily available material and in addition, due to the presence of reactive surface groups it can potentially be modified with various molecules that can ensure its delivery into target organs.

Dendrimers are regular, highly branched synthetic macromolecules that have been employed for oligonucleotide delivery [48]. Amongst all the dendritic vectors applied so far for knocking down gene expression, PAMAM dendrimers and their derivatives currently dominate the area

[48]. A new biodegradable polyamidoamine (PAMAM) dendrimer e-PAM-R modified with arginine via an in vivo cleavable ester bond has been used as a non-viral vector for DNAzyme delivery into cells [49]. The e-PAM-R vector was expected to have advantages for gene delivery such as enhanced transfection efficiency through introducing arginine for better DNA packing and fast biodegradation to lower cytotoxicity. Indeed it was shown that the generation 4 (G4) e-

PAM-R–DNAzyme complex at a mass ratio 2:1 exhibited much higher transfection efficiency and significantly higher suppression of TEL/AML 1 protein in 293 cells than either lipoplexes prepared with Lipofectamine® or a conventional G4 PAMAM-R dendrimer (optimal mass ratio

4) modified with arginine attached through the amide bond. Thus, introduction of biodegradability was deemed quite important for DNAzyme delivery [48, 49]. Although G3 or

G4 e-PAM-R dendrimers showed cell viability at the highest concentration, which may represent nearly non-toxic status for in vivo application, careful in vivo experiments in animals are required to draw unequivocal conclusions about their potential for clinical application. Modified polypropyleneimine (PPI) dendrimers were also used to deliver 10-23 DNAzymes

[50]. It has been suggested that only terminal functional groups on the surface of a PPI dendrimer rather than those in the interior are responsible for its toxicity [51]. Amino- functionalized PPI dendrimers are poorly degraded in water and are quite toxic, which is a major disadvantage for a transfection agent. Several PPI dendrimers of generations G2, G4 or G5 were prepared in which both surface and internal amino groups were modified to reduce toxicity and increase water solubility. Triethylene glycol gallate or acetyl groups were employed for the surface primary amino groups whilst the internal tertiary amino groups were quaternized via methylation with MeI or MeCl to create a positively charged microenvironment. The constructs were soluble in water and stable under physiological conditions. Out of all the constructs studied,

G4 PEG(MeI) dendrimer showed the best complexation with a 33 nt DNAzyme with a slight excess of the dendrimer. All the modified dendrimers exhibited low toxicity in the cultures of

MCF7, Malme-3M, HT29 and K562-C1000 cells. The highest level of transfection of the

DNAzyme in vitro into A2780 ovarian sarcoma cells was achieved with G4 (MeI) and G4

(MeCl) dendrimers. In those cases, the level of cytotoxicity was ca. 15%. The authors have argued that the efficiency of transfection in the case of the modified PPI dendrimers was comparable to commercially available transfection agent DOTAP (Roche). For in vivo transfection G4 PEG(MeI) dendrimers were employed because acetylated PPI dendrimers formed insoluble precipitates with DNAzymes at the concentrations used for in vivo experiments. Preliminary data on NMRI mice showed accumulation of DNAzyme-dendrimer particles in tumor tissue with both nuclear and cytosolic intracellular localization.

A β-cyclodextrin-based polymeric particle system for the delivery of a 10-23 DNAzyme targeted to c-myc mRNA has been described [52] and similar systems were previously employed to deliver plasmids. Their main component is a relatively small cyclodextrin-containing polycation with up to 5 cyclodextrin units and a total of 10 positive charges per polymeric chain, which are modified by two imidazole residues (CDP-Imid) that is able to form complexes (polyplexes) with nucleic acids through electrostatic interactions. Positively charged outer surface of polyplexes could interact with negatively charged proteoglycans on the cell surface and promote particle internalization via endocytosis. Imidazole modification also helps endosomal escape thus increasing overall efficiency of the delivery. Incorporation of certain weak bases with pKa in the physiological range results in enhanced transfection efficiency [53]. Polyplexes should be relatively small in size and protected from potential aggregation and non-specific interactions with blood plasma components and non-target cells. To solve those problems, polyplexes were modified with polyethylene glycol (PEG) adamantane derivatives (AD-PEG and AD-Glu-Glu-

PEG-Gal), which form a protective layer on the surface of polyplexes via van der Vaals contacts between adamantane residues and the interior of cyclodextrin cavities. It was shown that

PEGylated polyplexes possess low toxicity and exhibit ca. 98% penetration into human carcinoma cells A2780, HT-29 and HeLa in contrast to free DNAzymes. Target-specific delivery of DNAzymes into tumor cells was achieved by modifying adamantane-modified polyplexes by transferrin. Transferrin (Tf) is an iron-containing protein that interacts with the transferrin receptor, which is overexpressed in tumor and other fast proliferating cells and thus helps targeting those types of cells by the polyplexes. Intravenous injection of DNAzymes in the form of their Tf-AD-PEG polyplexes into mice of the NMRI line bearing xenografts of HT-29 intestinal cancer cells has resulted in up to 24 h accumulation of the particles in the tumor, liver and kidneys in contrast to ‘naked’ DNAzymes. Intracellular distribution of the particles was observed only in the case of tumor tissue and only for the Tf-modified polyplexes. However, the authors have only studied the delivery and have not demonstrated biological effects such as decrease of tumor size in the case of DNAzymes delivered by polyplexes.

3.2.2. Inorganic nanoparticles

Inorganic nanoparticles have also been employed for DNAzyme transfection [54-58].

Tack et al. [54] have concluded that DNAzyme delivery by is possible through the use of cell targeting ligands such as transferrin (Tf). Optimized colloidal gold probes were prepared by adsorption of Tf-conjugated cationic polymers polyethylenimine (PEI) or polylysine

(PL) onto the nanodispersion followed by complexation with the DNAzymes. The Tf-PL probes were less efficient and more toxic than the Tf-PEI probes. The best were obtained with gold particles loaded at low pH and with low DNAzyme concentrations and in the absence of the polyvinylpyrrolidone (PVP) stabilizer. Best delivery rates were 56% of total transfection and 36% if only live cells were counted. It is necessary to note that the authors did not clarify the reasons for this toxicity. No data on biological activity of DNAzymes in transferrin receptor- expressing HT29 cells were reported as the authors did not continue their studies in this direction.

A related study in which gold nanoparticles have been harnessed for DNAzyme delivery into cells has also been described [55]. In this case the researchers have prepared multivalent 10-23

DNAzyme-gold (DzNP) conjugates varying DNAzyme density or orientation, linker length or composition in order to clarify the role of steric environment and gold surface chemistry on the catalytic activity of the DNAzyme. The catalytic proficiency was modulated by steric packing and proximity of the catalytic loop to the gold surface. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease digestion, readily enter mammalian cells and catalytically downregulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.

A conjugate of 10-23 DNAzyme with dextran-coated magnetic iron oxide nanoparticles (MNs) modified with polyarginine lipopeptide myristoyl-ARRRRRRRC (MPAP) has been proposed for treatment of hepatitis C by targeting its NS3 RNA [56]. MPAP acted as a cell-penetrating peptide (CPP) and enhanced the efficiency of cell uptake. Both DNAzyme and MPAP were attached via biodegradable disulfide linkage. Dz-MPAP-MNs do not provoke undesired immune responses in Huh-7 cells in vitro and have a higher knockdown efficacy than the ‘naked’

DNAzyme transfected with Lipofectamine 2000. The nanoparticles were able to accumulate exlusively in liver when mice were injected through their tail veins. Notably, in the absence of a

CPP the Dz-MNs had poorer cell uptake into Huh-7 cells containing the HCV replicon RNA.

However, the authors did not perform control experiments on the delivery of Dz-MPAP conjugate in the absence of dextran-coated MNs, so the nanoparticle role in uptake is unclear.

Chen et al. [57] have demonstrated downregulation of protein expression and antitumor effects of DNAzyme Dz1 targeted to latent membrane protein (LMP1) RNA of Epstein-Barr virus, when delivered by arginine-modified hydroxyapatite nanoparticles (Arg-nHAP) both in

CNE1-LMP1 cells in culture and in a CNE1-LMP1 xenograft model in nude mice. While the transfection efficiency was approximately the same as for commercial transfection agents, cell viability in the case of Arg-nHAP was slightly better (ca. 80%) than for Fugene HD and

Lipofectamine 2000, which have shown approximately 65% and 50% cell viability, respectively, suggesting that Arg-nHAP was less cytotoxic. Using specific inhibitors, cell uptake of the Arg- nHAP-Dz1 complex was shown to be mediated by energy-dependent endocytosis pathway.

Furthermore, effective intracellular delivery and nuclear localization of the complex was confirmed by confocal microscopy. These results suggest that Arg-nHAP may be an efficient vector for oligonucleotide therapeutics although further studies are required to address such issues as in vivo stability, and toxicity of the nanoparticles before making firm conclusion on their clinical potential.

A series of 10-23 DNAzymes targeted to different segments of influenza A virus RNA were electrostatically bound to titanium oxide nanoparticles coated with polylysine [58]. The resulting nanocomposites were shown by confocal microscopy to be readily taken up MDCK cells. The

DNAzymes bound to TiO2 nanoparticles were still able to cleave the RNA in vitro albeit with reduced efficiency than the parent DNAzymes. Whether such TiO2 nanocomposites will be active in cell culture or in vivo remains to be seen.

4. Chemical modifications of DNAzyme protecting from nuclease degradation and improving its cell delivery

A way for improving cell uptake of oligonucleotides in the absence of any external transfection agent is through their conjugation with specific transporter molecules such as cell- penetrating peptides (CPP), escort aptamers or receptor ligands. For 10-23 DNAzymes their conjugates with peptides, amines and cholesterol have been tried.

Kubo et al. [59-61] have prepared 10-23 DNAzyme conjugates with various peptides including signal peptides and amines attached to the 5’-end of the DNAzyme via an amide bond.

These modifications hardly affected catalytic activity of the DNAzyme and in some cases even improved RNA cleavage due to increased affinity of the DNAzyme for the RNA target or possibly making the cleavage of the more favourable in the active conformation of DNAzyme-RNA complex. It was shown that the 5’-conjugated DNAzymes were more resistant to DNase I than unmodified DNAzymes whereas peptide conjugates were more stable than amine conjugates. Fluorescent DNAzyme conjugates with NES (nuclear export signal) or NLS (nuclear localization signal) peptides were used to show that the uptake of

DNAzyme-peptide conjugates into Jurkat cells was higher than for unconjugated DNAzyme, and that intracellular distribution of the conjugates depended on the nature of the peptide. For example, in the case of SV-40 large T-antigen NLS conjugate the DNAzyme appeared predominantly in the nucleus while for the HIV-1 REV NES peptide DNAzyme localization was largely cytoplasmic. Suppression of BCR/ABL chimeric gene by DNAzyme-peptide conjugates were largely enhanced in human leukemia cells, the NES conjugate was more effective as compared to the NLS conjugate [62].

3’-Cholesterol-DNAzyme conjugates were used to inhibit expression of M. tuberculosis gene ICL [63] and gene N of Respiratory syncytial virus (RSV) [64]. However, the authors did not evaluate cellular uptake of these conjugates and in the latter case they did not compare inhibitory effects of conjugated and unmodified DNAzymes [64]. Inhibition efficacy in the case of cholesterol conjugate of anti-ICL DNAzyme was comparable to the unmodified counterpart

[63]. It has been concluded that this modification did not improve DNAzyme penetration into human promonocytic cells THP-1 infected with M. tuberculosis [63].

Cell uptake of a DNAzyme that was targeted to Tat-Rev RNA of HIV-1 and had a polyguanine tract dG10 at the 3'-end was studied [65]. It was shown that such a DNAzyme was able to go into THP-1 cells in the absence of a transfection agent and decrease the level of Tat

RNA by nearly 100%. Analogously, DNAzymes with a polyguanine tract dG10 at the 3'-end with or without phosphorothioate modifications were used to cleave JEV RNA [66]. It has been demonstrated that the G-rich oligonucleotides are involved in the formation of G-tetrads that can be recognized by scavenger receptors (ScRs). Addition of a continuous stretch of 10 deoxyguanosine residues (poly-dG10) to the 3’-end of a DNAzyme has resulted in the enhanced delivery into the cells expressing ScRs.

Chemical modifications may improve DNAzyme delivery by offering protection from nuclease digestion and thus increasing the time for DNAzyme circulation in vivo. We have summarized herein the currently available modifications that improve biological stability of DNAzymes and the positions for those modifications, which in accordance with literature data have only minor negative influence on DNAzyme catalytic proficiency or even improve it (Fig. 4). To summarize, one should say that amongst all the DNAzyme properties such as RNA cleavage efficiency, stability in biological media and cell uptake, the last two represent by far the most promising parameters for their successful in vivo application as antisense therapeutics. Evidence in favour of this have been described [66-68], in which phosphorothioate-modified DNAzymes with more than tenfold lower catalytic activity compared to unmodified analogues, but more resistant to degradation and with better uptake were able to inhibit gene expression in cell culture and in vivo whereas their parent DNAzymes were inactive. The nuclease-resistant DNAzymes containing phosphorothioate linkages were efficiently taken up by mouse neuronal and glial cells. In murine neuroblastoma cells phosphorothioate-modified DNAzymes were taken up approximately sixfold better than the phosphodiester DNAzymes.

Analogously, a DNAzyme targeted to β1-integrin mRNA containing 2’-O-methylribonucleotides in the substrate-binding domains showed higher activity in mice than the corresponding siRNA although the latter was more active in cell culture [69]. The authors have clearly attributed this to higher biological stability of the DNAzyme.

Apart from phosphorotioate linkages and 2’-O-methylribonucleotides, other popular modifications are LNA and 3’-3’ “inverted” nucleotides at the 3’-end of a DNAzyme

[70, 71]. Replacement of native phosphodiester bonds with N3’-P5’ phosphoramidate linkages

(Fig. 4) also increased DNAzyme stability in biological media [72]. Introduction of modifications into the catalytic core makes DNAzymes more resistant towards

[68, 70, 73]. Mapping of the functional phosphate groups in the catalytic core of a DNAzyme has also been performed [74]. The main pathway of oligonucleotide degradation in biological fluids is their digestion by 3’- [75, 76]. Indeed, the introduction of the 3’-3’ “inverted” onto the 3’-end protects the DNAzyme from degradation in serum increasing its half- life by more than tenfold. The same protecting effect is achieved by introducing two phosphorotioate linkages at both ends of substrate-binding domains of a DNAzyme [68, 70].

One of the reasons for the poor cell uptake of oligonucleotides is their negative charge. The paper by Lam et al. [77] has described a 2’-deoxyuridine derivative containing a guanidinium group, which was incorporated into DNAzyme substrate-binding domains. Although the presence of those modifications did not enhance RNA-cleaving activity, all the modified species were found to be catalytically competent and such results clearly demonstrate that 10-23

DNAzymes can be selectively endowed with functionalities that reduce their negative charge and can potentially enhance their cell uptake. However, the authors did not perform any cellular experiments with their new DNAzymes. In the last few years, oligonucleotide analogues with charge neutral backbone such as phosphordiamidate morpholino oligonucleotides (PMO) have become increasingly popular [78].

Recently, a new class of oligonucleotide analogues has been described, which substitute charge neutral phosphoryl guanidine groups for anionic phosphodiester groups while maintaining natural sugar residues [79]. DNAzymes with either PMO or phosphoryl guanidine modifications have not been described yet, but it would be very interesting to see how such groups will affect their catalytic activity and cellular uptake.

5. Methods for DNAzyme delivery used in clinical trials

As it was described in a previous review [16], DNAzymes have recently entered into clinical trials. We discuss herein the methods for DNAzyme delivery in humans (Table 1). Looking at the Table 1, it can be conclude that the DNAzymes, which have reached clinical trials are those with minimal chemical modifications. Two out of three Dzs, Dz13 [30] and ghd40 [80, 81] have an “inverted” residue at the 3’-end, and the remaining one (Dz1) was modified by only two phosphorothioate linkages at both 3’- and 5’-ends of its substrate-binding arms [82]. It looks like the authors of those studies were conscious about the cost of potential therapeutics. In the case of SB011, additional nuclease resistance is provided by the delivery system, which is the

W/O/W emulsion. It is also important to add that all listed DNAzyme formulations were applied topically (Table 1) and thus have required less modifications compared to those oligonucleotide drugs, which require delivery systemically into the blood stream. Although in one of the most recent papers it has been shown that even a ‘naked’ Dz without any transfection agent could be active in vivo [83] as a potential anticancer agent, we think that such a DNAzyme may need to be better equipped against serum nucleases and fast clearance from blood stream.

Administration routes and particularly delivery systems for DNAzyme formulations are very important in the case of topically aplied drugs. For example, three different therapeutics coming from the same anti-GATA-3 DNAzyme are being currently developed by Sterna Biologicals for the treatment of asthma (SB010), atopic dermatitis (SB011) and (SB012) depending on both administration route and DNAzyme formulation (Table 1).

DNAzyme delivery systems used in humans were generally simple such as liposomes and

W/O/W emulsions whereas in other cases DNAzymes were delivered just as saline or PBS solutions (Table 1). In the case of anti-cancer DNAzymes DZ1 and Dz13 moderate efficiency has been shown during clinical trials, which may be explained by inefficient delivery of

DNAzymes into the tumors.

Liposomes are very popular delivery system, according to data from clinicaltrials.gov.com. At least 1852 results show use of liposomes mainly for delivery of low molecular weight therapeutics such as doxorubucine or analogues. There are only very few results using such delivery systems for oligonucleotides in humans and DNAzyme Dz13 is among them.

Properties of liposomes including their in vivo toxicity depend on their structure and size [84]. In accordance with tsurface charge they may be divided into cationic, neutral or anionic liposomes.

It looks like the toxicity of cationic liposomes is mainly associated with their positive charge

[85]. For example, it has been shown that induction of inflammatory response in mice by

DOTAP-cholesterol cationic liposomes was due to their positive charge. Their genotoxicity also correlated with surface charge [85]. Delivery of Dz 13 has been carried out with cationic

DOTAP-DOPE liposomes as local intratumoral injection. In terms of potential genotoxic effect of liposomes such local delivery into tumor may have a positive effect as compared to systemic administration. Thus, cationic liposomes may be safe and well tolerated when delivered locally.

For systemic delivery it would be necessary to modify the liposomes to make them more efficient and less toxic. Among the potential cationic lipid-encapsulated oligonucleotide therapeutics, the current leader is Atu027 from Silence Therapeutics which consists of AtuRNAi targeting protein kinase N3 and AtuPLEX delivery agent based on cationic lipids (clinical trial

ID NCT01808638) [86]. EphA2 gene targeting using small interfering RNA (siRNA) delivery by neutral liposomes is now recruiting participants for Phase 1 clinical trials (clinical trial ID NCT01591356) [87]. Lipid nanoparticles are also popular delivery systems for siRNA. For example, lipid nanoparticles have been developed by Alnylam and Tekmira as delivery vehicles for their siRNA clinical candidates [88, 89]. Normally such delivery systems are developed by the companies, which are keen to move their siRNA-based drug to market. Special delivery platform LODER based on miniature biodegradable polymeric matrix has been developed for siRNA delivery by Silenseed company [90]. Cyclodextrin nanoparticles have also been used for siRNA delivery into humans (clinical trials ID NCT00689065) [91]. No additional delivery systems were mentioned when other oligonucleotide therapeutics such as antisense oligonucleotides, SCO or antagomirs were used in humans recently [12]. For the DNAzymes nearly the same situation applies. It looks like liposomes and other delivery platforms are mainly used to deliver siRNA in vivo, since siRNA in particular needs to be protected from nucleases in addition to improving their delivery into cells and preventing their possible immunogenicity.

Naked but chemically modified siRNA also has been used in humans [92].

Thus many methods for DNAzyme delivery are being currently developed. However, it seems that practical reseachers prefer to avoid complicated delivery systems due to potential toxicity issues and cost considerations.

6. Conclusions

The majority of DNAzyme delivery methods described herein have not become routine yet because the whole area of DNAzyme delivery only began to develop quite recently. For example, only in one paper by Lee et al. [93] have biodegradable arginine-modified PAMAM dendrimers been proposed for DNAzyme delivery into cells. Up to now, the most popular method of DNAzyme delivery is still cationic liposomes, despite their known immunogenicity and cytotoxicity. It seems that researchers in this area currently prefer to keep up with established procedures rather than take a risk of exploring brand new ideas. However, the concept of a chemically modified oligonucleotide that would not need any extra help to be taken up by cells is certainly very attractive. Ideally, such chemical modifications would need to be either commercially available or easy to introduce, they should not enhance toxicity or decrease catalytic activity. Finally, such modifications should improve DNAzyme stability in biological media and, perhaps, most importantly, ensure its efficient delivery into target cells, tissues or organs, preferably in the absence of additional transfection agents. For example, the phosphorothioate modification has been shown to be beneficial for DNAzyme cell uptake and, despite known off-target effects resulting from protein binding, phosphorothioate groups are still very popular for DNAzyme modification. Although phosphorothioates decrease catalytic activity, phosphorothioate-modified DNAzymes were more active in vivo due to increased biological stability and better cell uptake, although the data on toxicity of those DNAzymes are still lacking. Those chemical modifications that have not yet been tried in a DNAzyme context such as (PMO) [78], tricyclic DNA [94] or new phosphoryl guanidine oligonucleotides [79] hold their potential for the future. However, the DNAzymes currently undergoing clinical trials have very few chemical modifications such as phosphorothioate groups or a 3’-3’ ‘inverted’ residue at the 3’-end, and their delivery systems are still very traditional.

7. Expert opinion

Our opinion is that in the immediate future, for each specific disease target it may be necessary to adopt an individual method for oligonucleotide delivery, which has to be efficient and cost effective at the same time, also simple to use and non-toxic. We believe that such kind of a system should be designed on a biodegradable platform since in such a case it will not accumulate in the organism and thus cause no concerns over long-term toxicity. For example, chitosan is a cheap, non-toxic, positively charged and biodegradable compound, and as such, it may be looked at as a nearly ideal vehicle for oligonucleotide delivery in vivo [45-47].

Furthermore, specific moieties for addressable delivery could be attached to the primary amino groups of chitosan, for example, aptamers to cell proteins or cell-specific peptides. Another promising delivery vehicles are the exosomes, which are natural vesicles secreted by cells to function as chemical mediators of intercellular communication. Recently, exosomes have been shown to deliver siRNA into the mouse [95]. Exosomes have been regarded as the only practical tool available at the moment to deliver exogenous cargo into gametes and , which are naturally resistant to the uptake of foreign substances [96]. If we learn how to produce those extracellular vesicles in vitro, how to load them with molecular cargo and how to harness them for therapeutical use, this may result in a development of the most natural way of drug delivery. Here a recently published approach to fuse artificial liposomes with exosomes to produce hybrid vesicles may offer a breakthrough [97].

We also think that a promising way forward to solve the delivery problem may lay in a chemical modification of a DNAzyme that will enable it to be taken up by cells more readily in the absence of any external delivery agents. Examples of suitable chemical modification could be conjugation with a cell-penetrating peptide (CPP) or fusion with an or appending lipid tails. A step forward could be a combination of several chemical modifications known to be beneficial on their own, as many researchers tend to use one or, at most, two modifications at a time to minimize uncertainty. For example, if phosphorothioate groups decrease catalytic activity, they could be used together with such modifications that enhance activity such as 2’-O- methylribo- or LNA nucleotides. For this, new or seldom tried chemical modifications such as morpholinos (PMO) or phosphoryl guanidines may become applicable. Even the use of such well-known groups as phosphorothioates may be reexamined after an exciting new chemistry has been developed to produce stereo-regular oligonucleotide phosphorothioates [98]. Thus, chemical modification of DNAzymes may not only improve their resistance to nuclease digestion but also quite possibly ensure their targeted delivery after systemic or local administration. Such an approach will help oligonucleotides pass the extracellular and intracellular barriers and let researchers avoid complex and expensive delivery systems, which current DNAzyme clinical trials suggest are not preferred for use anyway.

Acknowledgement

The authors thank Professor Richard Cosstick for critical reading of the manuscript.

Funding

This work was partly carried out with support from RSF grants 14-44-00068 (DA Stetsenko) and

15-15-00121 (AA Fokina and BP Chelobanov). Funding was also received from the Institute of

Chemical Biology and Fundamental Medicine SB RAS and Kindai University.

Declaration of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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RNA substrate 3’-N N NNNNNNNY R N NNNNNNN-5’

5’-N’N’N’N’N’N’N’N’N’R’ N’N’N’N’N’N’N’N’-3’ G1 A15 G2 G14 3 13 C 10-23 C 4 12 T DNAzyme A A5 A11 G6 C10 7 9 C T8 A

R = r(G, A); Y = r(C, U); R’ = d(G, A); N = r(A, G, C, U) N’ = d(A, G, C, T)

Fig. 1. The 10-23 DNAzyme. The arrow marks the point of cleavage.

DNAzyme delivery systems

Lipid and oil based Nanoparticles Transporter molecules systems or structural motifs

DOTAP/DOPE Cell-penetrating peptides liposomes Amines W/O/W, SME Polymer-based emulsions Cholesterol systems Inorganic nanoparticles Poly(dG) tail PLGA microspheres Colloidal gold β-Cyclodextrin Magnetic iron oxide Chitosan Hydroxylapatite PEI TiO2 PPI dendrimers

Arg-PAMAM dendrimers

Fig. 2. Exogenous DNAzyme delivery systems. RT

SOI pRSV/CMV pssXD/E AAAA SL PBS DNAzyme

Fig. 3. Schematic representation of endogenous DNAzyme production with pssXD/E vectors designed in [19, 20]: SOI (sequence of interest): encoded DNAzyme sequence; SL (stem loop): structural element for termination of reverse transcription; PBS (primer binding site) along with some flanking regions of the promoter that are essential for the reverse transcription initiation by MoMuLV RT; pRSV and pCMV: the Rous sarcoma virus (RSV) promoter and a stronger cytomegalovirus (CMV) promoter.

3’-ATTCTCATACTCA AAGTTGTAAAAGCCTAG-5’ 3’-AATTGAGCGGAAC AGCAACCCTTGGCCTAG-5’ G A G A G G G G C C C C A T A T A A A A C C G G C A C T A T

C-Dz7 C-Dz482

Fig. 4. Circular DNAzymes targeting β-lactamase mRNA [27]. RNA binding arms are underlined. 2'-OMe RNA (LNA)

3’-N N N N N N N N N Y R N N N N N N N N-5’ | | | | | | | | | | | | | | | | | | 5’-N’N’N’N’N’N’N’N’N’R’ N’N’N’N’N’N’N’N’-3’ 1 1 16 15 G2 p p A 2 p 15 14 G 3 p G 2’-deoxy 3 p 14 13 phosphorothioate C 4 p C p 13 (PS) 4 5 p 12 T p 10-23 12A 5 6 p 11 A p 11A 7 p 6 1 10 G p 8 9p C 7p p 9 3'-3' “inverted” 8 0 C T A backbone R = r(G, A); Y = r(C, U); R’ = d(G, A); N = r(A, G, C, U) N’ = d(A, G, C, T)

N3’→ P5’ phosphoramidate 2'R (2'-endo) 2'S (3'-endo) 2'-deoxy-2'-C-methylpyrimidines

Fig. 5. Chemical modifications that improve biological stability of 10-23 DNAzyme. Modifications in the substrate-binding domains: the terminal nucleotides of the substrate-binding domains can be changed to 2’-OMe RNA, LNA, 2’-deoxy phosphorothioate (PS) or N3'→P5' phosphoramidate backbone as well as capped on the 3’-end by a 3’-3’ “inverted” nucleotide [63, 65]. Modifications in the catalytic core: nucleotides shown in red and numbered from 1 to 15 belong in the catalytic core; those underlined highlight positions suitable for modification. A total of 6 deoxyribonucleotides at positions G2, C7, T8, A11, G14 and A15 simultaneously can be changed to their 2’-OMe RNA analogues [63, 64]; Up to 3 deoxyribonucleotides at positions T4, C7 and T8 simultaneously can be changed to 2’- deoxy-2’-C-methylpyrimidines in the following configuration: 4S,7R,8S and 4S,7S,8R [66]; Symbols p1 to p16 designate phosphate groups in the catalytic core, where p1 is related to the 5’- phosphate group of G1. Underlined p1, p3, p6-p8 and p14-p16 can be replaced with phosphorothioates (PS) [67]. The point of cleavage is marked by arrow.

Table 1. Methods for DNAzyme delivery in humans. DNAzyme and Chemical Delivery Method of Disease Phase (trial ID) Ref its RNA target modifications system administration Nodular 3’-3’ Inverted DOPE / Phase I Dz13 targeting Intratumoral basal-cell nucleoside at the DOTAP (ACTRN12613 [30] c-jun mRNA injection carcinoma 3’-end liposomes 000302752) Two DZ1 targeting Nasopharyn- phosphorothioate Epstein-Barr Saline Intratumoral Phase I/II geal groups at both [82] virus LMP1 solution injection (NCT01449942) carcinoma ends of substrate- mRNA binding arms Phase I (NCT01470911 SB010 NCT01554319 [80] Severe nebulized Phosphate- Inhalation NCT01577953) allergic solution of buffered using bronchial hgd40 targeting saline nebulizer asthma GATA-3 solution Phase IIa mRNA [81] (NCT01743768) 3’-3’ Inverted nucleoside at the SB011 emulsion containing 2% 3’-end Topical Atopic W/O/W Phase II hgd40 targeting application [37] dermatitis emulsion (NCT02079688) GATA-3 onto skin mRNA SB012 enema- Phosphate- applied hgd40 Rectal Ulcerative buffered Phases I/II targeting application by [37] colitis saline (NCT02129439) GATA-3 enema solution mRNA