Bioweapons from XNA, a Nuclease Resistant Synthetic Genetic Polymer that can Supplant DNA

INTRODUCTION

The arrival of novel life forms via synthetic biology (biological engineering) brought about atypical problems: (1) uncertainty of behaviour and interaction of artificial life forms with existing life forms as well as the ecosystem as a whole, (2) safety concerns for humans, and (3) containment issues (Acevedo-Rocha & Budisa, 2016; Schmidt, 2010; Tucker & Zilinskas, 2006). In an attempt to address all such concerns, XNAs – xeno nucleic acids based information storage - were designed as an alternative to the genuine deoxyribose or ribose based nucleic acids that form DNA (Fiers et al., 2016; Marliere, 2009; RJ, 2015; Diafa & Hollenstein, 2015). Organisms with XNA content cannot grow in nature as artificial synthetic nutrients must be supplied for sustained activity (Herdewijn & Marliere, 2009). In XNAs, alterations are made to the sugar of natural nucleobases by replacing it with another sugar or non-sugar moiety to form xeno nucleotides; alternatively, modifications can also be made to the nucleobase (C5 in pyrimidine or N7 in purine) or the backbone (Diafa & Hollenstein, 2015; Johnson, 2015). For instance, hexitol nucleic acid (HNA) contains a hexose instead of the traditional deoxyribose/ribose. Modifications to the nucleobase can alter strength and specificity of binding whereas modifications to the backbone can greatly increase resistance to nucleases (Morihiro, Kasahara, & Obika, 2017). There are a multitude of xeno nucleic acids amongst which 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), homochiral DNA (hDNA), xylonucleic acid (XyNA), deoxy-xylonucleic acids (dXyNA), arabinonucleic acid (ANA), 2′- deoxy-2′-fluoroarabinonucleic acid (FANA), 2′-deoxy-2′-fluororibonucleic acid (FRNA). Threose nucleic acid (TNA), hexitol nucleic acid (HNA), and glycol nucleic acid (GNA) can form double helices (Schmidt, 2010). Much like DNA, XNA can be utilised to pack information that can be duplicated using mutated forms of naturally existing polymerases (Steele & Gold, 2012). Natural polymerases stringently exclude xeno nucleic acids. However, Steele and Gold (2012) isolated functional polymerases for xeno nucleic acids by placing xeno nucleotides, primers, and plasmids coding for the engineered polymerases. The formation of synthetic genetic polymers (XNA) using mutated polymerases are verified using primer extension assays. Screening and identification of engineered polymerases that can ligates more than a few xeno nucleic acids are then optimized via directed evolution and molecular tweaking (Chaput, Yu, & Zhang, 2012). Replication of XNA as well as synthesis of XNA from DNA as well as reverse transcription of DNA from XNA can be carried out using said engineered polymerases (V. B. Pinheiro et al., 2012); engineered polymerases are commercially available for αS, CyDNA, fDNA, RNA, 2’F, 2’Ome, 2’Seme, 2’N3, ANA, FANA, HNA, CeNA, TNA, and LNA (Vitor BPinheiro & Holliger, 2012). Bst DNA polymerase (in the presence of MgCl2+) has been shown to extend 60% of a TNA template into DNA (Dunn & Chaput, 2016). In fact, with reverse transcription from XNA to DNA, information can be decoded and proteins synthesized from designed synthetic genetic polymers. The various XNAs have specific binding patterns. XNAs can base pair with both DNA and RNA. (R)-GNA and hDNA, on the other hand, do not pair with DNA or RNA, giving them a unique property – one that can enter natural cells without interfering; xylonucleic acid (XyNA) and deoxy-xylonucleic acids (dXyNA) can bind both RNA poly A strands and DNA at once leading to the formation of a triplex (Anosova et al., 2016). Synthetic xeno polymers can be utilised to store and propagate the genetic code, as drugs, sensors, catalysts, and to regulate gene expression. This paper outlines the positive and negative aspects of the synthetic system; I argue in this paper that the synthetic system can also be utilized as a bioweapon – to design drugs that are anywhere from mildly toxic to deadly.

APPLICATIONS

Antisense XNA oligonucleotides (ASO)

Nucleic acids can themselves be utilised as a drug for remedying diseases; however, (1) degradation of unprotected molecules by nucleases, and (2) poor binding to target molecules as well as large dose requirements were previously an obstacle (Morihiro et al., 2017). Contrary to DNA, XNA is nuclease resistant and as such can parade through cellular machinery undetected, and unscathed. For instance, antisense oligonucleotides target mRNA and microRNA sequence (Morihiro et al., 2017). Binding of antisense oligonucleotide (ASO) to its target sequence initiates degradation; in fact, binding of antisense XNA oligonucleotide to its target blocks translation thereby modifying the protein content of the cellular system. Viruses such as HIV, influenza virus, herpes simplex virus, cytomegalovirus, Epstein Barr virus, hepatitis B virus and human papilloma virus are effective targets of ASOs (Bai, You, Bo, & Wang, 2013). For instance, hepatitis C virus (HCV) causes chronic liver disease which typically results in liver cirrhosis; antisense oligonucleotide ISIS 6547 showed a dose-dependent inhibitory effect on HCV; in this case, the location of the viral DNA compared to the location of RNAse H was also a determining factor (Zhang et al., 1999). Further, antisense oligonucleotides UL36ANTI against the herpes simplex virus showed reduction of 99% at a concentration of 0.8uM in vitro (Pari, Field, & Smith, 1995). The latter implies that deadly viruses can also be controlled using ASOs; all viruses are categorized as either DNA or RNA based viruses that become deadly due to their capacity to replicate and spread. Given the success of ASOs with many a virus, it is conceivable that even deadly viruses – such as the Marburg virus, Ebola virus, and Lassa virus - can be controlled using ASOs. Moreover, ASOs are currently being utilised to treat cancer as well. In fact, there are a number of ASOs - Custirsen (OGX-011), EGFR antisense DNA, Apatorsen (OGX-427), ISIS-STAT3Rx (ISIS 481464/ AZD9150), ISIS-ARRx (AZD5312), Trabedersen (AP 12009), EZN-2968, and LErafAON-ETU (Moreno & Pego, 2014) - in clinical trials for the treatment of various cancer. However, there are countless uses for ASOs. For instance, ghrelin is an appetite stimulator typically associated with obesity; ASOs that target ghrelin can in theory lead to lowered level of ghrelin and therefore weight.

In fact, a number of drugs – Vitravene, Kynamro, and Miravisen (Morihiro et al., 2017) - are based on XNA technology. Vitravene (formivirsen sodium, 6.6 mg), a 21-mer phosphorothioate oligonucleotide (5'-GCG TTT GCT CTT CTT CTT GCG-3'), was developed as an antiviral for cytomegalovirus (CMV) retinitis, a condition that can present itself in patients with AIDS (acquired immunodeficiency syndrome) (U.S. Food and Drug Administration, 2002). One to two hours post-injection, degradation of the 21-mer oligonucleotide by exonucleases to a 20-mer oligonucleotide commences; seven days post-injection, 59% of the 21-mer oligonucleotides were shortened by one to two nucleotides (19-mer or 20-mer) (U.S. Food and Drug Administration, 2002). Due to small subject samples, the efficacy and safety of Vitravene could not be established by the FDA. Oddly enough, the 21-mer oligonucleotide sequence is uniquely complimentary to the virus (human mRNA are not complimentary to the 21-mer oligonucleotide). However, at large doses, multiple side effects were observed in treated patients; interaction with intracellular proteins and serum results in toxic side effects and immune reactions (Bai et al., 2013). Kynamro (mipomersen sodium injection, 200 mg), a 20-mer phosphorothioate oligonucleotide (5′-GCCUCAGTCTGCTTC GCACC-3′), is an apolipoprotein B (apoB) inhibitor that is used to treat homozygous familial hypercholesterolemia (HoFH) (Geary, Baker, & Crooke, 2015). A gapmer design was utilised with a 5’ and 3’ wing of O-MOE nucleosides (5 at the 5’ wing, 5 at the 3’ wing) and 2-deoxynucleosides. Binding of 20-mer ASO to human mRNA results in its degradation by RNAse H1. The 20-mer ASO has a half-life of one to two months (Drug Bank, 2007); degradation commences in the 2-deoxynucleosides region via endonucleases and subsequently exonucleases. Kynamro is accompanied by a backbox warning of hepatotoxicity with serious side effects such as fatigue, nausea, headaches, flu-like symptoms, and reactions to injection (Drug Bank, 2007). Miravisen is an miRNA blocker for patients with hepatitis C virus (HCV); effect of antimiR-122 was noted to be dose-dependent as well as long- lasting (Serino, Sallustio, & Schena, 2016). Research conducted on Vitravene, Kynamro and Miravisen suggest that there is variation in resistance to degradation by nucleases, in half-lives, and efficacy based on the nucleotides as well as the design of the oligonucleotides. Alternatively, drug cocktails are also a possibility.

Antisense XNA oligonucleotides, however, can also be used as a bioweapon. These nuclease- resistant synthetic polymers can hinder any target sequence among which vital sequences. A number of monogenic autosomal or X-linked recessive diseases are due to a loss of function of a single gene; antisense XNA oligonucleotides as such can be utilised to emulate any of the said monogenic diseases. For instance, mitochondrial genes are partitioned between mitochondrial and nuclear DNA. Energy production is dependent upon the respiratory chain which is composed of 5 complexes (complexes I-V) comprising 100 proteins of which 87 are coded for by nuclear DNA (Rotig, 2003). Mitochondrial failure is associated with cellular death, and subsequent shutdown of the entire system. For example, ATPAF2, a mitochondrial gene on nuclear DNA, codes for ATP synthase. Inhibition of ATPAF2 results in low (< 30%) levels of ATP synthase; in natural cases, patients present with 3-methylglutaconic aciduria, lactic acidosis, hyperammonemia, hypertrophic cardiomyopathy and neonatal-onset hypotonia with death ensuing a few months thereafter (OMIM, 1999). An ASO against ATPAF2 would in theory have the capacity to mimic the disease given that the cascade of events are dependent upon the single gene. In another instance, the onset of cancer is dependent upon two sets of genes – oncogenes and tumour suppressor genes. For example, retinoblastoma is a form of cancer that develops due to a loss of function mutation in both copies of the Rb tumour suppressor gene (Cooper, 2000). Theoretically speaking, retinoblastoma can be induced using ASOs. Given that cancer in itself is dependent upon a few features such as (1) replicative immortality, (2) avoiding immune response, (3) avoiding growth suppression, (4) continued proliferation, (5) avoiding apoptosis, and (6) angiogenesis, the disease itself is not induced. However, it can be mimicked for a short period. In such cases, the half-life of the ASO determines the degree of impact. Further, β-thalassaemia (HBB), tay-sachs disease (Hexosaminidase A), gaucher disease (GBA), Meckel-gruber syndrome (MKS1), congenital adrenal hyperplasia (CYP21A2), severe combined immunodeficiency (ADA), cystic fibrosis (CFTR), spinal muscular atrophy (SMN1), recessive blindness (TRPM1), congenital adrenal hyperplasia (CYP21A2), a-antitrypsin deficiency (SERPINA1), and mucopolysaccharidoses are all autosomal recessive diseases that can be triggered. The effect of the ASO is only as powerful as its target’s significance. For instance, an ASO against the Hexosaminidase A gene would deplete the hexosaminidase A protein population leading to the accumulation of ganglisides in the central nervous system. In this case, the extent of the effect will be directly proportional to the half-life of the ASO. The potential uses of ASOs are therefore multifaceted. Xeno nucleic acids display distinct pharmacokinetic properties implying that different XNAs behave differently in any given environment. This variation in the pharmacokinetic properties of the xeno nucleic acids gives much leeway in terms of half-lives, and resistance to nucleases; in other words, the use of distinct xeno nucleic acids for any one particular ASO can generate molecules with variation in both half-lives and resistance to nucleases.

Aptamers

Aptamers are 20 to 60-mer ssDNA or ssRNA oligonucleotides or polypeptides that have specific ligand binding property (Lakhin, Tarantul, & Gening, 2013). Aptamers can bind metal ions, small organic compounds, biological cofactors, metabolites, proteins, virus, bacteria, yeast, and mammalian cells (Kong & Byun, 2013). Aptamers have an unlimited shelf life, and no immunogenicity; aptamers are also nuclease resistant, chemically produced with little batch-to- batch variation, and show reversible denaturation. 3’ capping and modification of the sugar of the nucleotides confers protection against endonucleases and exonucleases (White, Sullenger, & Rusconi, 2000). SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a technique used to isolate aptamers that specifically bind a ligand (White et al., 2000; Shigdar et al., 2013; Song, Lee, & Ban, 2012; Kong & Byun, 2013; Meek, Rangel, & Heemstra, 2016; Diafa & Hollenstein, 2015). For instance, cell-SELEX is utilized to target a particular cell type based on cell surface proteins or cellular structure; cancer cells can be identified in such a manner typically by conjugating a fluorophore to an aptamer (targeting cell-surface proteins) permitting visualization of such things as disease progression. A pool of 1012-1015 random oligonucleotides with a 5’ and 3’ constant region and a variable region in the middle - known as the library is used as potential aptamers.

Selection of aptamer consists of:
(1) Combining oligonucleotide library with the target
(2) Isolation of oligonucleotide-target complex
(3) Isolation of oligonucleotides from oligonucleotide-target complex
(4) PCR amplification of aptamers
(5) Double stranded aptamer to single stranded aptamers
(6) Repeat

Aptamers with high affinity are typically isolated after 5 to 15 rounds of SELEX; however, length of the oligonucleotide is proportional to the number of rounds of SELEX required for selection of high affinity aptamers. Half-lives of aptamers depend on the modification made to the nucleic acid. Compared to unmodified RNA with a half-life of less than one second in vivo, 2’NH2, for instance, has a half-life of 367 hours or 15.3 days (Shigdar et al., 2013). Aptamers can be used as a therapeutic tool, to deliver drugs, to diagnose diseases, for bio-imaging, as an analytical reagent, to detect hazard, and to inspect food (Song et al., 2012; Taylor et al., 2016). Both in vitro, and in vivo applications exist; in vitro applications include biosensors (electrochemical aptasensor, fluorescence-based optical aptasensor, and colorimetric-based optical aptasensor amongst others). In particular, fluorescence-based optical aptasensors are based on a quencher-fluorophore pair bound to an aptamer; binding of target molecule to the aptamer results in a conformational change that disrupts the quencher-fluorophore pair permitting visualization. Disease progression and drug efficacy can be visualized in such a manner. Moreover, electrochemical aptasensor can detect the presence of anthrax spores (Bacillus anthracis spores); in vitro aptasensor (chemiluminescence-based, colorimetric, nanomaterial- based, fluorescence-based, electrochemical-based) can be used to detect cocaine (Mokhtarzadeh, Ezzati Nazhad Dolatabadi, Abnous, de la Guardia, & Ramezani, 2015). In vivo aptamer-based drugs can target cells like molecules. For instance, aptamers conjugated to drugs and targeting cell surface receptors are internalized rapidly via endocytosis; such a mechanism is capable of cell-type specific drug delivery such as small interfering RNAs, toxins, chemotherapeutic agents, anticancer drug-encapsulated polymers, liposome nanoparticles, radionuclides, a viral capsid, enzymes, nano-carriers, and photodynamic therapeutic agents (Zhou & Rossi, 2011). Typically, selection of aptamers against a cell-type can be achieved by isolating proteins present on its surface, and using such methods as affinity chromatography to isolate high affinity aptamers; selective uptake of aptamer permits delivery of drugs to specific cell-types. However, serum stable aptamers are required for in vivo applications. A number of commercial drugs are aptamers. Macugen (pegaptanib sodium, Valenat Pharmaceuticals) is an aptamer targeting VEGF (vascular endothelial growth factor); anti-HIV gp120 aptamer conjugated to a tat/rev siRNA was found to be an effective method of controlling HIV (Zhou & Rossi, 2011).

Aptamers are highly useful in the design of bioweapons as they permit targeted delivery. In vivo aptamers can be deadly. For instance, an immune response requires both B-cells and T-cells; aptamers against CD3/CD4/CD8 (T-cells) and/or CD19/CD20 (B-cells) conjugated to toxins can specifically target and destroy cells required for proper immune function. Immune shutdown would mimic AIDS with opportunistic infections – such as candidiasis, coccidioidomycosis, cryptococcosis, cytomegalovirus diseases, encephalopathy, herpes simplex (HSV), histoplasmosis, isosporiasis, kaposi's sarcoma, lymphoma, tuberculosis, mycobacterium Pneumocystis carinii pneumonia, salmonella septicemia, toxoplasmosis of brain - leading to death (Vyas, 2015). Co-infection with a virus, bacteria or fungus can therefore be deadly. Aptamers can be cell-type specific, and as such, any organ can also be targeted. Cardiomyocytes possess cell-surface markers such as β1- and β2-adrenergic receptors (Forough, Scarcello, & Perkins, 2011). The heart therefore can be attacked using β1- and β2-adrenergic receptors as ligands and aptamers conjugated to toxins (aptamer:toxin). In particular, ricin, ricin A chain, pseudomonas exotoxin, diphtheria toxin, clostridium perfringens phospholipase C, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, cobra venom factor, gelonin, saporin, modeccin, viscumin and volkensin can be utilized as toxins that can be conjugated to aptamers (EP 1552002 A2, 2005). Necrosis of cardiomyocytes due to exposure to toxins results in heart dysfunction/failure (Tavernarakis, 2007). In this case, half-life of aptamer would be irrelevant; proper delivery of toxin to cardiomyocytes is dependent on the concentration of the solution. Heart failure and death should ensue shortly thereafter. The positive aspect of such a delivery system is that once the task has been achieved, no or very little trace will remain of the toxin; if the half-life of the aptamer is short, the body should be able to degrade the aptamer prior to death thereby mimicking a natural death. Erythrocytes, leucocytes, and platelets are derived from hematopoietic stem cells (HSCs). CD34 and AC133 are cell-surface marker found on hematopoietic stem cells (HSCs) (Guo, Lubbert, & Engelhardt, 2003; Handgretinger & Kuci, 2013). An aptamer:toxin conjugate against CD34 and/or AC133 would specifically target and kill hematopoietic stem cells resulting in pancytopenia and bone marrow failure (Chen, Lipovsky, Ellison, Calado, & Young, 2004). Pancytopenia, a medical emergency, results in anaemia, leucopenia, and/or thrombocytopenia. For instance, levetiracetam induced pancytopenia has been reported to severely decrease leucocyte, erythrocyte, and platelet levels; cessation of drug permitted normal levels to return (Alzahrani et al., 2015). In children (1 month to 16 years old), pancytopenia resulted in pallor, fever, petechial haemorrhages, bleeding (nose, gums and gastro intestinal tract), hepatomegaly, splenomegaly, and lymphadenopathy (Khan & Hasan, 2012). Renewal of erythrocytes, leucocytes, and platelets take 4 months, 24 hours, and 5 days respectively. As such, effects should begin 24 hours post-injection of aptamer:toxin conjugate; however, it can take as long as 4 months for death to ensue. Death would ensue as a result of depletion of erythrocytes. Alternatively, a CD235a aptamer:toxin conjugate would specifically target and eradicate erythrocytes resulting in severe anemia, cardiac failure and death. In fact, with cell surface markers, vital organs - such as the medulla oblongata, lungs, kidneys, and liver - can be targeted to induce conditions.

CONCLUSION

Xenonucleic acids (XNA), generated by modifying the sugar, base or phosphate of natural nucleotides, are deemed useful tools because they are nuclease resistant, lack immunogenicity, and are chemically synthesized in vitro. Xenonucleotides were created to ensure the containment of novel life forms and/or novel molecules; however, some xenonucleotides possess the ability to be assimilated into DNA helices. Natural DNA polymerase exclude xenonucleic acids (XNAs) for polymerization whereas synthetic or mutated XNA polymerases can synthesize short polymers. Although XNAs are currently used in anti-sense oligonucleotides, anti-gene oligonucleotides and aptamers amongst others, they are also very powerful bioweapons. Diseases can be triggered using antisense oligonucleotides, and aptamers. In fact, cells, and organs can be specifically targeted for destruction. The advantage of XNA as a bioweapon is that once the effect has been generated, the toxin is degraded. In other words, a toxin against any organ or gene can be delivered in such a manner to induce damage; following damage, the toxin is degraded, and no trace remains.