Peptide Nucleic Acid (PNA) for Antisense Therapy

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What is the antisense therapy?

Antisense therapy is a molecular strategy that seeks to disrupt gene expression through the use of synthetic oligonucleotides specifically engineered to attach to mRNA transcripts. This binding obstructs the translation of the target protein, hence silencing the gene linked to a certain illness. Antisense oligonucleotides (ASOs) are short, synthesized nucleic acid strands that are complementary to certain mRNA sequences. Upon introduction into cells, they associate with their target mRNA by base pairing. Phosphorothioate oligonucleotides represent the prevailing benchmark for antisense treatment, exhibiting satisfactory physical and chemical characteristics together with notable resistance to nucleases. Recently, novel generations of phosphorothioate oligonucleotides including 2'-modified nucleoside building blocks have been designed to augment RNA binding affinity and mitigate indirect harmful effects. Antisense therapies are, after years of challenges, nearing the realization of their potential in clinical settings.

The antisense approach-the basic principle. (Jansen B., et al., 2002)

Why can peptide nucleic acid (PNA) be used in antisense therapy?

The metabolic stability and high binding affinity of PNA positioned this synthetic oligomer as a leading contender for antisense and antigene treatment. Extensive research over nearly two decades has demonstrated that, unlike ribose-based antisense agents, PNA hybridization to mRNA does not provoke an RNase H or dicer response; however, PNAs function as steric blockers that impede the splicing of target mRNA or translation by binding to the initiation site. Although cellular permeability is a challenge, especially for systemic administration, it was established years ago that PNA modified with four lysines at the C-terminus effectively corrected faulty splicing in a transgenic mouse, so confirming their therapeutic potential. A recent study demonstrated the effective application of a GPNA to inhibit the expression of EGFR, a critical driver in non-small-cell lung cancer, in a murine model. Although these cases are promising, the lack of further research indicates that the complete therapeutic potential of PNAs remains unexploited. A recent invention demonstrated that a PNA oligomer engineered to form a triplex with dsRNA effectively inhibited the translation of the targeted mRNA.

Mechanism of action of antisense PNA

Splicing modulation or translation disruption are the mechanisms by which antisense PNA exerts its effects. In the nucleus, PNA acts by modulating splicing, and in the cytoplasm, it targets mRNA and noncoding RNA (ncRNA) through translational arrest or block, depending on the target location.

Splicing modulation: It is necessary to splice the converted pre-mRNA into the proper mature mRNA after DNA transcription.3, 5 Proteins and tiny nuclear RNA in the spliceosome, conserved sequences in the splice junctions, and enhancer and silencer sequences in both the introns and the exons coordinate the process of normal RNA splicing, which involves the exclusion of nonfunctional introns and the joining of exons. Inhibiting or modifying RNA splicing to cause intron inclusion, exon exclusion (skipping), or inhibition can be one way that PNA silences gene expression. The therapeutic potential of splicing modulation is demonstrated by the fact that PNA can prevent splicing machinery from incorrectly deleting specific sequences in pre-mRNA. This leads to the correct protein being produced, the correct exons being included in the mRNA sequence, and the possible amelioration of certain genetic diseases. Instead of allowing the spliceosome and splicing factors to reach the transcript sites on the pre-mRNA, as exon inclusion does, exon skipping fixes broken reading frames and makes shorter but otherwise functional alternative proteins. Nonfunctional, destabilizable messenger RNAs (mRNAs) caused by faulty splicing in particular diseases can be corrected with PNA by inserting introns. As a consequence of steric hindrance caused by PNA binding to the pre-mRNA, intron inclusion, exon skipping, and exon inclusion might occur.

Translational block and translational arrest: The binding affinity of PNA for mRNA-targeted regions is inversely proportional to the steric hindrance it causes; higher binding affinities lead to better hybridization and, ultimately, translational blockage or arrest. Three mechanisms exist by which PNA can halt or halt translation: RNA transfer to the cytoplasm, ribosome assembly, and elongation. With mRNA, PNA may create duplexes or triplexes. Duplex-forming PNA blocks translation by targeting the AUG start codon region or the 5′ untranslated region (5′UTR) of mRNA and limiting ribosome assembly. By binding to the coding region of mRNA, duplex-forming or triplex-forming PNA halts translation by blocking elongation. The attractiveness of using steric hindrance to impede or pause translational machinery is enhanced by the fact that PNA may be readily delivered to relevant regions of mRNA. The sections under "antisense applications as anti-pathogenic agents" and "antisense applications in neurodegenerative diseases" contain other examples of PNA causing translational block and translational arrest.

Diagram illustrating PNA's action mechanisms. (MacLelland V., et al., 2023)

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Antisense PNA as the anti-pathogenic agents

In its role as an anti-pathogenic agent, PNA has the ability to target and control the activity of a wide array of genes that either contribute to or are intrinsic to an infectious agent's activity. The inability of a pathogen to multiply and survive within its host is caused by the disruption of essential cellular activities caused by the creation of a PNA sequence that can target a bacterial or viral agent or essential supporting proteins.

The minimal inhibitory doses of CPP-PNA conjugates have been established, and several possible gene targets in pathogenic microbes have been discovered. So far, PNAs that target mRNA segments around the start codon have shown the most efficacy against microbes. Proteins are downregulated in these cases because PNA antisense pathways use steric hindrance to obstruct translation. The inhibition of bacterial cellular growth can be achieved by targeting the messenger RNA (mRNA) of genes that are crucial to the bacteria's survival. The acpP gene encodes the fatty acid biosynthesis ACP protein, which is one of the most well-established in vivo targets of antisense PNA. Bacterial fatty acid production relies on the ACP protein, which donates activated fatty acid groups. As a result, growth inhibition and normal cell functions leading to death are caused by acpP depletion. Using a BALB/c mouse model, Tan's group examined whether acpP targeting antisense PNA might limit the development of E. coli when placed intraperitoneally (i.p.). Notably, a mutant strain of Escherichia coli (SM105/101) was utilized for this study. This strain has a membrane that is less saturated with lipid A, making it more permeable to molecules with a large molecular weight, like PNA. After inducing peritonitis by intra-abdominal injection of E. coli, mice were given either PNA or PNA-(KFF)3K. After 7 days of PNA treatment, mice infected with E. coli showed a complete recovery, as the bacterial load was significantly reduced, proinflammatory serum cytokine production was decreased (TNF-α, IL-1β, IL-6, and IL-12), and the infection could not be fatalized.

Antisense PNA targets the sequence in acpP mRNA. (Popella L., et al. 2021)

The antisense PNA as the Anti-microbial agents. (MacLelland V., et al., 2023)

Antisense PNA as the anti-viral agents

Anti-pathogenic effects of PNAs targeting viruses like HIV and hepatitis B virus (HBV) are promising. Despite the availability of a vaccination to prevent HBV infection, the disease continues to be a major concern for public health, as infected individuals are more likely to develop chronic and severe liver ailments. There is a significant incidence of rebound infection following therapy with existing HBV treatments since they mainly elicit a virostatic effect. The effectiveness of antisense PNA targeting the epsilon signal in duck herpes simplex virus (DHBV) was studied by Ndeboko et al. This signal is essential for viral reverse transcriptase activity and is involved in hepadnavirus encapsidation. Primary duck hepatocytes (PDH) were used in vitro for analysis, whereas ducklings were used in vivo for the same purpose. In vitro, anti-epsilon PNA reduced HBV reverse transcriptase activity early in the process, resulting in a dose-dependent anti-viral impact. The results of the in vitro tests were confirmed by in vivo investigations utilizing HBV-infected primary duck hepatocyte cultures. These studies demonstrated that anti-epsilon PNA reduced viral DNA by 30% compared to untreated controls. It was also shown that (D-Arg)8 and Decanoyl-(D-Arg)8 could suppress the replication of HDBV when the anti-viral efficacy of several solo CPPs was examined. Choosing the right CPP may help with specificity of inhibition, and this shows that CPPs have a special capacity to boost the activity of antisense PNA.

Antisense PNA as the anti-cancer agents

Cancer is one of several human diseases in which many ncRNAs have important functions. The aberrant overexpression or involvement of certain miRNAs and lncRNAs in tumor initiation and development has led to their proposal as therapeutic targets in diverse human carcinomas. These putative indicators can be targeted specifically to tumors using PNA, an anti-cancer drug. So far, there is evidence that PNA can be used to treat a wide variety of cancers, including glioblastoma (GBM), colorectal cancer (CRC), ovarian cancer, breast cancer, lymphomas, cervical cancer, lung cancer, and many more.

Initially, the study found that PNA nanoparticle (NP) inhibited miR-21 and upregulated PTEN in vitro. After incubating U87 cells with PNA NP for 48 hours, qRT-PCR findings demonstrated a 40-60% reduction of miR-21. By monitoring PTEN levels—a tumor suppressor that is dormant in GBM and a predicted target of miR-21—we were able to assess the impact of miR-21 suppression. At the maximum dosage, we found a 30–40% decrease in cell viability and a 2- to 3-fold increase in PTEN mRNA levels. Then, we compared TMZ monotherapy with PNA NP plus TMZ treatment to see how the suppression of miR-21 affected the susceptibility of tumor cells to TMZ. The results of the viability experiment showed that the cell viability was much lower in the group that received both TMZ and the adjuvant. In addition, the PLA-HPG-CHO NP showed more synergy with TMZ at lower treatment dosages than the PLA-HPG NP, which might lead to a decrease in TMZ dosage when administered concurrently.

In a groundbreaking study, researchers showed that a peptide with a low pH-induced transmembrane structure (pHLIP) coupled with anti-miR PNA could block the oncogenic miR-155 in a lymphoma mouse model by selectively targeting the tumor microenvironment and then crossing plasma membranes in the acidic conditions present in solid tumors.

References

1. Jansen B., et al., Antisense therapy for cancer—the time of truth, The lancet oncology, 2002, 3(11): 672-683.
2. MacLelland V., et al., Therapeutic and diagnostic applications of antisense peptide nucleic acids, Molecular Therapy-Nucleic Acids, 2023.
3. Saarbach J., et al., Peptide nucleic acid (PNA) and its applications in chemical biology, diagnostics, and therapeutics, Current opinion in chemical biology, 2019, 52: 112-124.
4. Wu J., et al., Recent advances in peptide nucleic acid for cancer bionanotechnology, Acta Pharmacologica Sinica, 2017, 38(6): 798-805.
5. Popella L., et al., Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics, Nucleic acids research, 2021, 49(8): 4705-4724.