Gene Therapy and Nucleic Acid Therapeutics

Nucleic acid therapeutics have become an integral part of the landscape of modern therapeutics with the uprise of the newly emerged vaccines for COVID-19. Besides the well-known mRNA vaccines, the number of approved nucleic acid therapeutics steadily increases, and the rapid development of gene-editing therapeutics in clinical trials demonstrates the huge potential of gene therapy. The use of exogenous nucleic acids is an attractive way to achieve highly specific therapeutic effects, but it is also a challenging task. Among the main obstacles for nucleic acid therapeutics are unfavorable physiochemical properties, immune activation, and the fact that nucleic acids are prone to degradation by nucleases.

PNA – a Nucleic Acid with Unique Properties

Nucleic acids are polymer or oligomer chains of monomers called nucleotides. Those nucleotides are composed of a pentose sugar, a negatively charged phosphate group, and a nucleobase. Chained together through phosphodiester linkages, those nucleotides form a so-called sugar phosphate backbone, to which the nucleobases are attached. The most well-known representatives of nucleic acids are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These biopolymers exist in all living organisms and are an essential part of life. They store genetic information and play key roles in its transmission and expression. [1] [2]

Approximately three decades ago, researchers started to experiment with artificial DNA-like structures called xeno nucleic acids (XNAs). XNAs store the same genetic information as natural nucleic acids and can interact with them but are held together by a different backbone. As this can cause drastic changes in physical and chemical properties, it was hypothesized that xeno-nucleic acids could improve the fast-growing field of nucleic acid therapy. [2] Among the xeno nucleic acid family, peptide nucleic acid (PNA) stands out as one of the most promising candidates for a broad range of applications. While most modified nucleic acids retain the negative charge of the backbone, PNA is made of a charge-neutral backbone. This distinction makes PNA stand out as a DNA-analogue with unique properties. In PNA, the sugar-phosphate backbone is replaced with a pseudopeptide backbone derived from aminoethylglycine (aeg). Although this change is quite dramatic, does it not compromise the ability of PNA to interact with DNA or RNA. In fact, PNA even was among the first molecules to demonstrate that the sugar-phosphate structure is no requirement for base recognition. The peptide-like backbone leads to very favorable conformational properties for DNA hybridization while retaining high flexibility. This allows PNA to form exceptionally strong and sequence-specific bonds with natural DNA and RNA through standard Watson-Crick pairing. Remarkably, the affinity of the pairing even exceeds the specificity of DNA itself, as a single Watson-Crick base pair mismatch can decrease the melting temperature of the DNA-PNA complex by 8–20 °C. [2]

The most significant distinction between PNA and natural nucleic acids is the absence of negative charges on the backbone. Because of this, no electrostatic repulsion is observed in PNA/DNA hybrids, which contributes to the high binding stability. It also accounts for the fact that PNA hybridizes almost independently of salt concentration. This property can be exploited to target DNA sequences that are involved in secondary structures and are susceptible to high ionic strengths. Another advantage of the artificial aeg backbone is that it is not recognized by proteases or nucleases, which causes PNA to be highly resistant to degradation in biological systems. [3] [4]

Applications of PNA

Unsurprisingly, these remarkable properties have sparked great interest among the scientific community. In the last decades, PNA has been employed in a variety of applications, including the use as an antigen, antisense, antibacterial, anticancer, and antiviral agent. The high metabolic stability paired with its high affinity and sequence specificity also makes PNA an excellent tool for probes and diagnostics. The ability to bind to double-stranded DNA and RNA even allows PNA to target structured nucleic acids. Therefore, fluorescent-labelled PNA probes are extensively used in nucleic acid detection and quantitation, such as staining by fluorescence in situ hybridization (FISH) or array hybridization. In regular FISH, shorter probes can be difficult to detect due to insufficient hybridization, while longer probes can cause non-specific background noise. PNA FISH mitigates these problems due to its superior selectivity. [5], [6]

Synthesis of PNA

Because of the peptide-like nature of the PNA backbone, solid phase synthesis (SPS) – which was originally developed for peptide synthesis – is often used for PNA. The strategy relies on anchoring the growing PNA-chain on a solid resin support. Analogous to solid-phase peptide synthesis (SPPS), the monomers are modified with specific protecting groups. While the primary amine of the monomers is masked with a temporary protecting group, the reactive amino groups of the nucleobases are masked with permanent protecting groups. After a monomer is attached to the resin, the temporary protecting group is removed, followed by the addition of the next monomer via the formation of a peptide bond. This process can be repeated until the desired sequence is made. At the end of the chain elongation, the permanent protecting groups are cleaved simultaneously with the removal from the solid support. A schematic overview of PNA SPS is shown in the depiction below.

Unfortunately, the synthesis of PNA is not as straightforward as that of peptides; specific conditions and coupling reagents are necessary to achieve the desired yield and purity. Furthermore, due to chain aggregation and difficult couplings, PNA SPS is usually limited to shorter sequences of up to 15–25 bases. The classic approach for PNA SPS utilizes the Boc- and Z-protecting groups; however, the harsh conditions during synthesis and the need to utilize HF led to the exploration of new synthetic approaches. The main strategy nowadays revolves around fluorenylmethoxycarbonyl (Fmoc) as a temporary and benzhydryloxycarbonyl (Bhoc) as a permanent protecting group—an approach that is very similar to modern SPPS. The biggest drawback of this strategy are the basic conditions that are used to deprotect the temporary Fmoc protecting group (usually 20% piperidine in DMF). This can cause deletions via ketopiperazine formation and transacylation, leading to a branched chain. Because of this, a multitude of new protection strategies are currently being investigated. [7]

Aggregation is a major problem of PNA synthesis. The tendency to aggregate is mainly observed in purine-rich sequences, especially in those containing many Guanine units. Combined with the poor solubility of monomers, which leads to inefficient couplings, PNA SPS is limited to rather short sequences. Longer chains often result in poor solubility and get increasingly difficult to purify and characterize. Luckily, due to the high binding affinity to DNA, short oligomers of approximately 15 bases already show excellent specificity and often even shorter sequences suffice for target recognition. To overcome aggregation, low-loading resins are preferably used for synthesis. Additionally, more hydrophilic polyethylene-glycol-based resins, such as TentaGel or ChemMatrix, can be used to minimize aggregation during synthesis. [7]

Modifications

Despite the remarkable properties of PNA, the fact that so far only one PNA drug is in clinical trials shows that making a viable PNA therapeutic is a difficult task. While PNA generally offers unique properties, does it have some shortcomings? Limited solubility in aqueous media can be observed as the lack of electrostatic repulsion favors folding in compact structures and aggregation. [8] Another problem of PNA is poor cellular uptake as well as unfavorable pharmacokinetics. Unmodified PNAs are not taken up by cells in vitro and are cleared rapidly (10–30 min in mice) through the kidneys after intravenous injection. [4]

To address these issues, chemical modifications to the backbone and the conjugation of PNA to other biomolecules have been researched extensively. A possible strategy to increase solubility is the addition of lysine at the C-terminus of the PNA. The additional positive charge helps to decrease aggregation and improves water solubility but does not negatively impact the hybridization properties of the PNA. To overcome the challenge of poor cellular uptake, it is possible to use nuclear microinjection and electroporation to incorporate PNA probes into cells. However, these methods have limited applicability due to various reasons. The most promising approach to delivering PNA to target tissue is the conjugation of PNA with delivery-enhancing compounds such as cell-penetrating peptide (CPP). The uptake of those PNA-CPP conjugates is, however, limited by endosomal entrapment, which remains an unsolved issue until today. [4]

Another strategy to improve the biological and physicochemical properties of PNA relies on backbone modifications. The classic aminoethylglycine backbone is highly versatile and allows several sites for chemical modification, most notably the α- and γ-positions. α-modified PNAs derived from Arginine show increased stability of PNA/DNA duplexes and improved cellular uptake without compromising sequence selectivity; however, they are often outperformed by γ-modified PNAs. The introduction of a chiral center in the γ-position preorganizes the PNA backbone in a right-handed helical structure (for structures derived from (S)-enantiomers), which leads to even stronger duplex formation with complementary DNA. The most promising modifications for medicinal chemistry on the γ-position are mini-PEG (polyethyleneglycol) and guanidine. The γ-mini-PEG modification greatly increases solubility without causing cytotoxicity while also showing improved nucleic acid binding compared to regular aeg PNA. Due to the superior hybridization properties, γ-mini-PEG PNAs can invade any sequence of double-stranded DNA with standard Watson-Crick base pairing for target recognition. Guanidine side chains improve cellular uptake, solubility, and hybridization properties. [4]

Conclusion

PNAs are an important class of nucleic acids with unique properties. They hybridize to complementary DNA/RNA with high specificity and affinity. Combined with their high metabolic stability and salt tolerance, they make an excellent tool for biodiagnostics and are used in applications across several different scientific fields. For in vitro applications where cellular uptake is not a problem, PNA is often used as the gold standard. If the target locations are cells or tissue, chemical modifications are needed in order to enable successful administration. The increased complexity of modifications along with the difficulty of synthesizing modified PNA monomers has caused the development of PNA therapeutics to lag behind applications as a diagnostic tool. However, at the same time, these difficulties have created new opportunities, as they have led to the development of modified PNAs with improved cellular uptake, selectivity, and solubility. Although PNA has been studied extensively, more research is needed in this exciting area to unlock the huge therapeutic potential that it has to offer.

Bibliography

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[5] A. Gupta et al. J. Biotech., 2017, 259, 148–159.

[6] T. Vilaivan, Beilstein J. Org. Chem., 2018, 14, 253–281.

[7] F. Albericio, B. de la Torre et al., Chem. Soc. Rev., 2023, 52, 2764.

[8] V. Monga, G. Singh, Bioorganic Chemistry, 2023, 141, 106860.