Peptide Nucleic Acids (PNAs) for Gene Editing
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What is the gene editing?
Over three decades ago, a breakthrough in gene editing—then known as gene targeting—was made when it became possible to build nucleases to produce site-specific DNA double-strand breaks (DSBs), which could increase the rate of homologous recombination (HR) by a factor of over a thousand. After that, gene editing went from being a specialized method to being used in every field of scientific research. Genome editing involves using the body's own mechanisms for fixing DNA damage. Many different types of nucleases allow for the targeted introduction of DNA breaks into the genome. To achieve DNA sequence specificity, the initial platforms used rational protein engineering of homing endonucleases or DNA-binding proteins like zinc fingers or TAL effectors. The Fok1 endonuclease's endonucleolytic DNA cleavage domain is fused with zinc fingers or TAL effectors to form ZFNs or TALENs. To achieve catalytic activity from the two Fok1 domains, which are in fact a dimer due to the endonucleolytic domain's dimerization, two ZFNs or TALENs must be engineered to target neighboring genomic locations. Modern nuclease platforms have their roots in the CRISPR-Cas9 immune system in bacteria. One such platform uses a 100-nucleotide (nt) single guide RNA (sgRNA) to guide the Cas9 endonuclease [the Cas9-gRNA ribonucleoprotein (RNP) complex] to a specific location in the genome. Once the sgRNA has hybridized with the target DNA, Cas9 is activated to cut the chromosomal target. Due to Cas9's high activity and specificity, along with the ease, cheap cost, and rapidity of sgRNA design and engineering, this nuclease platform has become the gold standard for genome editing, allowing for the study of fine-tuned genetic alterations and the development of preclinical gene correction treatments.
Why use the PNA in the field of gene editing?
Because of its exceptional features that improve efficiency, stability, and specificity, peptide nucleic acid (PNA) is gaining more and more attention as a possible tool for gene editing. To complement DNA or RNA sequences, PNAs bind more strongly than conventional nucleic acid probes. Because of this, certain genetic sequences may be targeted more effectively. Crucial for preserving genomic integrity during gene editing, the enhanced specificity reduces the likelihood of accidental alterations in non-target regions. Because they cannot be hydrolyzed by nucleases, PNAs remain stable in natural settings. Because of their increased stability, they may remain in cells for longer, which improves their efficacy in gene editing applications. They can be designed to target various types of nucleic acids (DNA, RNA) and can be adapted for use in different organisms, from bacteria to plants to animals.
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Mechanisms of PNA-mediated gene editing
The gene editing has the ability to permanently alter genomic DNA, and triplex-forming PNAs are just that. The binding of homopyridine PNAs to homopurine regions of DNA can occur in accordance with either the Watson-Crick or Hoogsteen base pairing rules. To make this procedure more efficient, homopyrimidine PNAs are bonded together using a chemical linker to generate a bis-PNA. One bis-PNA strand is engineered to bind via a Hoogsteen motif, with a pseudoisocytosine (J) in place of a cytosine (C) to avoid self-complementarity. Because of this change, one strand can join in an anti-parallel fashion using Watson-Crick base pairing, whereas the other strand can connect in a parallel orientation relative to the DNA strand using Hoogsteen base pairing. The ensuing triplex causes DNA helical distortion, which can trigger DNA repair processes and lead to "donor" DNA recombination with native DNA around the PNA binding site.
A supFG1 reporter gene in mouse cells was used to show that bis-PNAs may cause mutagenesis at or around the PNA recognition site. After that, PNA-DNA hybrids could guide site-specific recombination by utilizing bis-PNAs coupled to a short donor DNA fragment. This method relied on the PNA molecule for two reasons: first, it formed a triplex with the targeted DNA; and second, it positioned the donor DNA fragment where it could be recombinated most effectively. To show that they could effect site-specific sequence alterations inside the reporter gene at levels greater than previous triplex-forming oligonucleotide (TFO)-DNA counterparts, Rogers's team used a mutant pSupFG1 plasmid in human cell-free extracts. In addition, they demonstrated that bis-PNAs can only induce gene editing when the nucleotide excision repair factor XPA recognizes the PNA/DNA/PNA complex. Only then can XPA begin to repair the altered helix "lesion" and facilitate recombination with the donor DNA. Remarkably, the recombination was substantially greater when the donor DNA and bis-PNA were intermingled instead of covalently attached.
In along with repairing mutations that cause illness, one group has started exploiting PNAs' gene-editing capabilities to insert stop codons that inhibit HIV-1 infection. R5 HIV-1 tropic infection cannot arise in patients whose CCR5 genes have a naturally occurring 32 bp deletion (CCR5-delta32). This is because these patients generate shortened CCR5 proteins. Schleifman's team used this genotype and its associated phenotype to drive the delivery of CCR5-targeting PNAs along with a donor DNA fragment engineered to insert a stop codon into human CD34+ hematopoietic progenitor cells. With this method, Schleifman's team successfully edited genes (roughly translated to "knockout") in 2.46 percent of the cells they tested. After transplanting these modified cells into mice, researchers found that the gene alteration reduced viral load and remained in the body for more than four months after engraftment.
Gene editing by triplex-forming oligonucleotides. (Quijano E., et al., 2017)
Guidelines for triplex PNA design for gene editing. (Economos N G., et al., 2020)
In vivo gene editing using PNA-nanoparticles (NPs)
To change the CCR5 and β-globin genes in NOD-scid IL2rγnull mice, polymeric nanoparticles containing donor DNA and PNA were initially injected intravenously in vivo. Coatings of cell penetrating peptides generated from the Drosophila antennapedia peptide (AP) or the HIV-1 transactivating protein (TAT) were applied to either unmodified or modified PLGA nanoparticles. In vitro experiments with CD34+ cells demonstrated that PLGA NPs treated with either TAT or AP increased donor DNA recombination relative to unmodified particles. Mouse engrafted with human hematopoietic cells showed gene change in the bone marrow, spleen, thymus, small intestine, and lung following intravenous administration of either unmodified or TAT-NPs loaded with PNA and donor DNA that targeted CCR5. This would suggest that PLGA NPs are dispersed all over the mouse body before entering human cells and modifying genes. When compared to treating mice with bare PNA and donor DNA, which resulted in a little amount of gene alteration, using NPs offered a clear benefit in vivo. This finding agrees with other research showing that intravenous administration leads to the quick clearance of naked oligos. Curiously, the advantage of TAT-coated NPs over unmodified NPs did not materialize in vivo, in contrast to the in vitro findings. Individual colony sequencing using human hematopoietic progenitors obtained from treated spleens suggested that the level of alteration of some cell types, specifically myeloid colony-forming cells, might be significantly greater, ranging from 14% to 19%, according to deep sequencing results.
The capacity of mononuclear cells isolated from treated mice's bone marrow and spleen to develop into myeloid and erythroid lineages was unaffected by intravenous PNA/DNA NP therapy, as demonstrated in vitro. By transferring bone marrow from mice that had been treated with intravenous PNA/DNA NPs to mice that had not, the targeted CCR5 modification could be found in the recipient mouse's bone marrow 10 weeks after the transplant, proving that the treatment could modify HSCs in situ. In order to demonstrate the adaptability of this method, the scientists used intravenous PNA/DNA NP injections to alter the human β-globin gene in both eGFP reporter mice and mice reconstituted with human hematopoietic cells. The study that has been discussed above is groundbreaking because it shows that hematopoietic progenitor cells may be modified in vivo using PNA-NPs, and it also uses a chimeric mouse to directly edit human cells at precise sites.
PNA and donor DNA-mediated gene editing. (Ricciardi A S., et al., 2018)
Gene editing by PNA without a donor DNA
Dr. Carmen Bertoni has disclosed an alternative method of PNA-mediated gene editing that does not use donor DNA. Her research on Duchenne muscular dystrophy (DMD) involves gene editing by binding single-stranded PNAs (ssPNAs) to the relevant genetic mutation. This condition is marked by a lack of dystrophin protein. With the exception of one base mismatch that attempts to fix an A to T transversion in exon 10 of the dystrophin gene—a mutation that causes a cryptic splice at this location—the PNAs are either fully complementary or homologous to the coding strand of the target sequence. Myoblasts isolated from the DMD model mdx5cv mice were transfected with ssPNA using LipofectamineTM 2000. According to quantitative PCR (qPCR), gene editing using ssPNAs targeting the non-coding strand was calculated at around 3%, whereas ssPNAs targeting the coding strand resulted in approximately 7% correction. In the absence of mismatched nucleotides and full complementarity to the target gene mutation, control ssPNAs that were otherwise identical to the PNAs utilized for repair failed to provide any discernible correction. Additionally, the scientists noted that two weeks following treatment, dystrophin immunostaining confirmed that mdx5cv mice that were directly injected with naked ssPNAs in the tibialis anterior region had dystrophin-positive fibers. The in vivo correction efficiency for ssPNAs targeting the non-coding strand was estimated to be 2.8% and for ssPNAs targeting the coding strand to be 3.3% based on qPCR analysis of gDNA isolated from treated muscles. Additionally, dystrophin-positive fibers were seen four months following ssPNA therapy, suggesting that the genetic repair remains effective over the long run. Researchers showed that ssPNAs could fix the dystrophin mutation in mdx5cv mice muscle satellite cells (SCs), which are cells that can self-renew and differentiate into muscle fibers. Ex vivo qPCR analysis of gDNA extracted from SCs transfected with ssPNAs revealed a dystrophin gene correction of up to 2.1%. mdx/nude mice had SCs treated with ssPNA and implanted into their tibialis anterior. Dystrophin expression was seen in treated mice over a period of up to 24 weeks, in contrast to control ssPNA-treated animals who were transplanted with SCs but did not demonstrate dystrophin expression. Muscle morphology improved noticeably as the number of dystrophin-positive fibers increased, which is an intriguing finding. These findings point to the possibility that the PNA is not promoting site-specific recombination with donor DNA, but rather is functioning independently as a genetic information source for a polymerase. Additional research is needed to confirm if a PNA may serve as a substrate for a polymerase. It has been demonstrated that more than one mismatch in the PNA sequence can significantly decrease target sequence binding, hence this technique might only work for introducing a single base-pair mutation into the genome.
PNA for CRISPR-Cas9
Although CRISPR-Cas9 gene treatments have several promising uses, they still need refinement to make them more targeted. One problem that still prevents CRISPR from being used for treatments in humans is Cas9 cutting in non-target regions. Even when the difference between the wild-type and mutant alleles is as little as one base pair, treating inherited disorders can be difficult. To achieve a tenfold improvement in Cas9 selectivity, one study employs synthetic peptide nucleic acids (PNAs) that bind particular spacer sequences in the guide RNA (gRNA). Varying PNA lengths, binding positions, and degrees of homology with the gRNA are investigated. Both on-target/off-target and allele-specific situations are effectively enhanced by PNAs bound in the area distal to the protospacer adjacent motif (PAM) site, according to the results. Furthermore, Cas9 activity can be altered allele-specifically by intentionally disrupting the binding of PNAs to the gRNA at the PAM-proximal region. These developments bode well for overcoming existing constraints and increasing CRISPR's therapeutic potential.
Peptide nucleic acids (PNAs) were used to regulate Cas9 activity. (Carufe K E W., et al., 2024)
References
- Quijano E., et al., Focus: genome editing: therapeutic peptide nucleic acids: principles, limitations, and opportunities, The Yale journal of biology and medicine, 2017, 90(4): 583.
- Carufe K E W., et al., Peptide Nucleic Acid-Mediated Regulation of CRISPR-Cas9 Specificity, nucleic acid therapeutics, 2024.
- Ricciardi A S., et al., Peptide nucleic acids as a tool for site-specific gene editing, Molecules, 2018, 23(3): 632.
- Economos N G., et al., Peptide nucleic acids and gene editing: perspectives on structure and repair, Molecules, 2020, 25(3): 735.
